A TEXT-BOOK

OF

PHYSIOLOGICAL CHEMISTET

BY

OLOF HAMMARSTEN

EMERITUS PBOFESSOR OF MEDICAL AND PHYSIOLOGICAL CIIEMISTRY~IN THB UNIVERSITY OF UP8ALA

WITH THE COLLABORATION OF

S. G. HEDIN

PROFESSOR OF MEDICAL AND PHYSIOLOGICAL CHEMISTRY IN THB UNIVERSITY OF TTPSALA

FROM THE A UTIIORS ENLARGED AND REVISED
EIGHTH GERMAN EDITION

BY

JOHN A. MANDEL, Sc.D.

PROFESSOR OF CHEMISTRY IN THE NEW YORK UNIVERSITY AND BBLLEVUE HOSPITAL MEDICAL COLLKGE

SEVENTH EDITION
TOTAL ISSUE, EL^VEX THOUSAND

NEW YORK

JOHN WILEY & SONS, INC.

LONDON: CHAPMAN & HALL, LIMITED

1914

Copyright, 1893, 1898, 1900, 1904, 1908, 1911, 1914,

BY

JOHN A. MANDEL

THE SCIENTIFIC PRESS

ROBERT ORUMMOND AND COMPANT

BROOKLYN, 71. V

PREFACE TO THE EIGHTH GERMAN EDITION

THE revision of this edition has been accomplished with the collabora-
tion of Professor S. G. Hedin, and the work has been divided so that
Hedin has revised Chapters I, III, VIII, XII and XVI, besides the Index
of Authors, while Hammarsten has revised Chapters II, IV, V, VI, VII,
IX, X, XI, XIII, XIV, XV, and XVII, besides the General Index.
The numerous recent developments in physiological chemistry have made a
thorough revision and reconstruction necessary in all the chapters, and in
order to prevent a noticeable increase in the size of the work, it was also
necessary to change the arrangement of the foot-notes more economically
The number of chapters in this edition is XVII instead of XVIII as in the
seventh edition, because for several reasons it was found advisable to
combine the first two chapters of the seventh edition into one chapter, and
at the same time certain parts of the first chapter have been incorporated
into other chapters, thus for example the oxidation processes have been
introduced into Chapter XVI (on respiration and oxidation). In general
the plan of the work remains unchanged.

OLOF HAMMARSTEN.

UPSALA, September, 1913.

iii

TRANSLATOR'S PREFACE TO THE SEVENTH
AMERICAN EDITION

WORKERS in Biochemistry are to be congratulated on the appearance
of a new edition of Hammarsten's " Physiologischen Chemie." At this
time, when so many new and important biochemical facts are being
published and when so many older theories and deductions are found
more or less erroneous, due to recent investigations using, new methods,
it is very fortunate that we have this complete and critical compilation
from the master hand of Professor Hammarsten, now in his 73d year.
We all owe him a great debt of gratitude for his painstaking work for so
many years.

I take great pleasure in expressing my indebtedness to my assistant,
Dr. A. 0. Gettler, for the help he has given me in revising the proof and for
making the Indexes.

JOHN A. MANDEL.
NEW YORK, June, 1914.

v

CONTENTS

CHAPTER I

PAGE

GENERAL AND PHYSICO-CHEMICAL *•

CHAPTER II
THE PROTEINS 77

CHAPTER III
THE CARBOHYDRATES 196

CHAPTER IV
ANIMAL FATS AND PHOSPHATIDES 232

CHAPTER V
THE BLOOD 250

CHAPTER VI
CHYLE, LYMPH, TRANSUDATES AND EXUDATES 345

CHAPTER VII
THE LIVER 381

CHAPTER VIII
DIGESTION , • 451

viii CONTENTS.

CHAPTER IX

PAGE

TISSUES OF THE CONNECTIVE SUBSTANCE 514

CHAPTER X
THE MUSCLES 565

CHAPTER XI
BRAIN AND NERVES 604

CHAPTER XII
ORGANS OP GENERATION 620

CHAPTER XIII
THE MILK 643

CHAPTER XIV
THE URINE 672

CHAPTER XV
THE SKIN AND ITS SECRETIONS 837

CHAPTER XVI
RESPIRATION AND OXIDATION 850

CHAPTER XVII
METABOLISM 878

INDEX TO AUTHORS 945

GENERAL INDEX 983

PHYSIOLOGICAL CHEMISTRY.

CHAPTER I.
GENERAL AND PHYSICO-CHEMICAL.

I. OSMOTIC PRESSURE.

WHE^ certain substances are placed in contact with water they
dissolve therein and finally a liquid is obtained which contains an equal
quantity of the dissolved substance in each unit volume. There exists
between the water and the soluble body a certain attractive force. Upon
this force depends also the so-called diffusion, which manifests itself when
two different solutions of the same or different substances are brought
into immediate contact with each other. The dissolved molecules and
the water intermingle with each other so that finally the dissolve.d
bodies are equally divided in the entire quantity of water. Imagine
a cane-sugar solution in contact with pure water; the equilibrium or the
homogeneity of the system can then be brought about in two ways;
namely, the sugar molecule can migrate in part into the water, and sec-
ondly, the water can pass into the solution. If the two fluids at the
beginning are in immediate contact with each other then the two proc-
esses take place simultaneously.

The conditions change when the two liquids are separated from
each other by a membrane, which allows of the passage of water but
not of the dissolved substance (In this case cane-sugar). In the presence
of such a so-called semipermeable membrane the equilibrium can only
be established by the water passing into the cane-sugar solution. Semi-
permeable membranes have been artificially prepared, and they also
occur in nature, or conditions exist which give results like those of the
membranes. To the first group belong TRAUBE'S so-called precipitation
membranes.1 Such a membrane, for example can be produced by care-
fully dropping a concentrated solution of copper sulphate into a dilute

jArch. f. (Anat. u.) Physiol., 1867, pages 87 and 129.

rA'ND PHYSICO-CHEMICAL.

solution of potassium ferrocyanide. Thereby the drop of copper sulphate
is surrounded by a membrane of copper ferrocyanide, which is imper-
vious to copper sulphate as well as to potassium ferrocyanide, but allows
water to pass. The drops retain their blue color in the yellow solution
but increase in volume, due to the taking up of water, until the tension of
the membrane prevents the further increase in size. If the difference in
concentration of the two solutions is great enough, the membrane is
ruptured by the pressure.

In order to give the copper-ferrocyanide membrane a greater rigidity,
PFEFFER has suggested forming the precipitate on a porous, rigid wall.1
For this purpose he makes use of a small, porous earthenware cell which,
after careful cleaning, is treated with copper sulphate and potassium
ferrocyanide so that the membrane is precipitated on the inner wall
of the cell. The membrane thus obtained is impervious to the cane-
sugar. If the cell is filled with a cane-sugar solution and then placed
in pure water, no sugar leaves the cell, while water passes into the cell,
and this continues until the opposite pressure produced presents the
further passage of water. If the cell is completely closed and in con-
nection with a manometer, then on the establishment of an equilibrium
the manometer indicates the force with which the inclosed solution
attracts water.

As the sugar is attracted with the same force by the water as the water
is by the sugar and also as the sugar cannot pass through the membrane
therefore the sugar exerts a pressure upon the membrane equal to the
pressure indicated by the manometer. This pressure is called the
osmotic pressure of the enclosed solution. For dilute cane-sugar solutions
PFEFFER'S determinations show that the osmotic pressure is approx-
imately proportional to the concentration and slowly rises with the
temperature.

Experiments with other semipermeable membranes have also been
carried out by DE VRIES, and these will be discussed on page 5. DE
VRIES' experiments have led to the following result: Solutions of analo-
gously constructed bodies having the same molecular concentration give the
same osmotic pressure.

VAN'T HOFF first called attention to the analogy which exists between
the laws of osmotic pressure of a dissolved substance and of gases,2
namely, that the osmotic pre$sure is proportional (or inversely propor-
tional to the volume of the solution) to the concentration, and corre-
sponds completely with BOYLE-MARIOTTE'S law on the relation between
the volume and pressure of gases. Also, that equimolecular solutions

1 Osmotische Untersuchungen, Leipzig, 1877.

2 Zeitschr. f. physik. Chem., 1, 481 (1887). t

OSMOTIC PRESSURE. 3

have the same_ osmotic pressure, corresponds to AVOGADRO'S law, that
equal volumes of different gases under the same pressure contain the
same number of molecules.

From PFEFFER'S results of the osmotic pressure of cane-sugar solu-
tions VAN'T HOFF has calculated that it is the same as the pressure exerted
by any gas of the same molecular concentration and temperature. In
general the following is true:

Dissolved bodies exert in solution the same osmotic pressure they would
exert if they were gases at the same temperature and in equal volume.

Recently MORSE, FRAZER and collaborators have brilliantly substan-
tiated the theory of VAN'T HOFF for solutions of cane-sugar and glucose,
by making use of PFEFFER'S method but using a very refined technique.1

From what has been given, the osmotic pressure of a solution, sepa-
rated from the surrounding pure solvent by a semipermeable membrane,
exerts its effects in two ways. First the pure solvent tries to enter
the solution and secondly the dissolved substance presses upon the
membrane with a force equal to the gas pressure. According to whether
we consider either one or the other of these ways, the osmotic pressure
of a solution can be considered as its ability to attract the solvent, or as
a pressure directed toward the outside. This last conception seems prob-
ably for the present to be the most acceptable, nevertheless, the fact that
the pure solvent enters through the unmovable semipermeable membrane
(as in PFEFFER'S experiments) is difficult of reconciliation with this mode
of explanation. Obviously, and for physiological purposes, it seems best
to make use of the former explanation, in which the osmotic pressure
is considered as a measure of the force with which a solution attracts
the solvent.

PFEFFER'S above-described method of ^directfe determining the
pressure can only be used in exceptional cases, first because the prepara-
tion of the semipermeable membrane is connected with difficulties, and
second, because there are only a few crystalline bodies for which imper-
meable membranes have been found. There are other quicker and easier
ways of determining the osmotic pressure.

Solutions of non-volatile substances boil at a higher temperature
than the pure solvent. This is due to the fact that the dissolved sub-
stance, because of the osmotic pressure, holds on to the solvent with
a certain force. As in boiling a part of the solvent is separated from
the dissolved body, and as the osmotic pressure can be considered as a
measure of the attractive power between the solvent and the dissolved
substance, then it is clear that solutions which are prepared with the
same solvent and have the same osmotic pressure (isosmotic solutions)

1 Amer. Chem. Journ., 37, 425, 558 (1907); 41, 1, 257 (1909).

4 GENERAL AND PHYSICO-CHEMICAL.

must also boil at the same temperature. The rise in the boiling-point
of a solution above the boiling-point of the solvent (elevation of the
boiling-point) is also, like the osmotic pressure, for dilute solutions pro-
portional to the concentration.

Solutions have a lower freezing-point than the pure solvent, and as
in dilute solutions the solvent can be frozen out from the dissolved body,
then isosmotic solutions have the same freezing-point. The depres-
sion of the freezing-point is also proportional to the concentration.

The determination of the elevation of the boiling-point for the esti-
mation of the osmotic pressure of animal fluids is applicable only in
exceptional cases, because on heating, precipitates often form. The
determination of the depression of the freezing-point has been found of
much greater use. This can be accomplished in an easy manner by aid of
the apparatus suggested by BECKMANN. In regard to the use of this
method we must refer to more complete works.1

The above rule that equimolecular solutions of different bodies have
the same osmotic pressure is only applicable to non-electrolytes. The
electrolytes (bases, acids, salts) show in aqueous solution a much greater
pressure (i.e., a much lower depression of the freezing-point) than equi-
molecular solutions of non-electrolytes. As is known, ARRHENIUS has
explained this lack of correspondence by the assumption that the mole-
cule of the electrolyte is divided or dissociated into so-called ions hav-
ing an opposed electric charge. An ion exerts upon the osmotic pressure
the same influence as the non-dissociated molecule. The larger the
number of dissociated molecules the more does the osmotic pressure
of the solution rise above the pressure of an equimolecular solution of a
non-dissociated body. The osmotic action of a dissociated body is equal to
that of a non-dissociated body which in a given volume contains as many
molecules as the dissociated body contains ions plus non-dissociated mole-
cules. If we assume that a is the degree of dissociation, i.e., the number of
the molecules that are dissociated, then 1— a is the number that is not
dissociated. If in the dissociation of a molecule n ions are formed
then the relation of the molecules present before the dissociation to the
ions + molecules present after the dissociation is 1:(1 — a+na) or
= l:(l-f-[tt— l]a). The expression (l+[w — l]a) is generally denoted by
the letter i, and can be directly determined by estimating the freezing-
point of a solution of known molecular concentration.

A gram-molecule aqueous solution (one that contains as many grams per
liter as the molecular weight of the substance) of any non-electrolyte freezes
at about - 1.86°, or, the depression of the freezing-point A is = 1.86°. For example,

1 Ostwald-Luther, Hand- und Hilfsbuch zur Ausfiihrung physik.-chemischer
Messung, 3 Aufl., 1910.

OSMOTIC PRESSURE. 5

if we find that A for a gram molecular solution of NaCl is 3.40° then we have
according to the above 1 : (l+[n — l]a) =1.86 : 3.40. In the dissociation of
NaCl two ions are formed, therefore n =2, and from the above equation the degree
of dissociation can be calculated, a =0.83. The degree of dissociation can also
be calculated from the electrical conductivity. Only the ions take part in the con-

/,...., •, ,. -, / conductivity \

duction of electricity, and the molecular conductivity I = — , -, —

J \ molecular concentration/

is proportional to the degree of dissociation. The dissociation increases with the
dilution and at infinite dilution all molecules are dissociated (a = l). If we desig-
nate with )uoo the limit value which the molecular conductivity approaches in
infinite dilution and with ^v the molecular conductivity at some definite dilution

»»p

v, then the degree of dissociation at this dilution is a = — :.

The positively charged ions are called cations, and the negatively
charged ones anions. Common for all acids are the positively charged
H-ions while the negatively charged OH-ions are common for all bases.

Osmotic Experiments with Plant Cells. We often meet the
word osmosis in literature without understanding exactly what is meant
thereby. As a rule diffusion streams are meant, which are modified
by means of the permeability conditions of an inclosing membrane.
We now know that the driving force, namely, the streaming, is brought
about by the differences in concentration, i.e., by difference in the osmotic
pressure on the two sides of the membrane.

After NAGELI found that certain plant cells, when they were treated
with a sufficiently concentrated solution of certain substances, changed
their appearance so that the protoplasm retracted,1 DE VRIES studied
this phenomenon further.2 He called it plasmolysis. The most important
substances for bringing about plasmolysis are the salts of the alkalies and
alkaline earths, varieties of sugars, polyatomic alcohols, and neutral amino-
acids. An indispensable condition for bringing about plasmolysis is that
the solution must not have any destructive action upon the cells. NAGELI
gave the correct interpretation of plasmolysis, which is that those bodies
which plasmolyze plant cells pass through the cell membrane of the cell, but
not through the protoplasmic layer which follows. Instead of this the sub-
stance attracts water from the inner parts of the cell. The cell contents
surrounded by protoplasm therefore diminish in volume and the protoplasm
recedes more or less from the cell membrane. From this it follows that
only those solutions whose power of attracting water is greater than that
of the cell contents can bring about plasmolysis. As the ability to attract
water (or the osmotic pressure) increases with concentration, there must
be a limit solution for every substance above which all higher concentra-
tions plasmolyze. The limit solution is called isotonic with the cells;

1 Pflanzenphysiol. Untersuch., 1855.

2 Sine Analyse der Turgorkraft, Jahresber. f. Wissensch. Botanik, 14, 427 (1884).

6 GENERAL AND PHYSICO-CHEMICAL.

weaker solutions are called hypotonic, and stronger hypertonic. DE
VBIES, with the aid of equal cells (cells of the epidermis of the lower
side of the leaf of the Tradescantia discolor) has, for various substances,
determined the concentration of this limit solution. It was found that
the limit solution of analogously constructed salts had the same molec-
ular concentration. Thus the alkali salts of the type NaCl (haloid
salts, nitrate, acetate) plasmolyzed at one molecular concentration and
the salts of the type Na2S04 (sulphate, oxalate, diphosphate, tartrate)
at another concentration. If the plasmolyzing power of a molecule
of the first group is equal to 3, then the molecule of the second group
equals 4. The concentration of the limit solution varied in DE VRIES'
experiments between the limits corresponding to a NaCl solution of
0.6-1.3 per cent.

As above mentioned, only those substances bring about plasmolysis
which cannot themselves pass through the protoplasm envelope of the
cell content, and these substances only in the case that the concentration
is sufficient. If a body is taken up by the protoplasm it produces no
plasmolysis, because its tendency to attract water has been satisfied
by its own passage into the cell. These substances do not produce
plasmolysis in any concentration. If a body slowly passes in, then
at first it causes plasmolysis, but this then ceases later. The plasmolytic
methods have been used by DE VRIES, and especially by OVERTON.*

Experiments with Blood Corpuscles. Over a hundred years ago
HEWSON observed that the blood corpuscles were destroyed in water,
and that salts in certain concentrations prevented destruction.2 HAM-
BURGER3 has carefully and systematically investigated the action of
salts of the alkalies and alkaline earths, and concludes that when blood
is mixed with certain volumes of solutions of different concentrations
of the same salt, all solutions whose concentration lie below a certain
limit cause the exudation of haemoglobin. On comparing the molec-
ular concentration of the limit solution of different salts it was found
that they bore the same relation to each other as the relative figures
found by DE VRIES for the molecular concentration of the plasmolytic
salt solutions. From this it probably follows that the protective action
of the salts upon the blood corpuscles depends upon the same reason
as the plasmolysis. This conclusion is also supported by the fact that
those substances which, according to DE VRIES, in proper concentration
cause plasmolysis in living plant cells, can also under similar conditions
prevent the exudation of haemoglobin. Those bodies, on the contrary,

1 Vierteljahrschr. d. Naturf. Gesellsch. zu Zurich, 40, 1 (1895); 41, 383 (1896).

2 Phil. Trans., 1773, p. 303.

' Arch. f. (Anat. u.) Physiol., 1888, p. 31; Zeitschr. f. Biol., 26, 414, (1889).

OSMOTIC PKESSURE. 7

which do not cause plasmolysis, act in aqueous solution in the same
manner upon the blood corpuscles as pure water. This has been espe-
cially shown by the investigations of GRYNS.1

Different investigators have attempted to perform plasmolytic
experiments with animal cells, but without any special result. With
the microscope one can often observe that the red blood corpuscles
shrink under the influence of a strong salt solution, but the limit solu-
tion when the shrinking just begins cannot be exactly determined because
the changes in volume are so very small. If we summate the changes
in volume of many corpuscles, which can be done by centrifuging the
blood mixture in a graduated tube, then very small changes can be detected.
Such determinations have been made by HEDiN,2 KOEPPE 3 and others.
It was found that the blood corpuscles swell in a weak salt solution,
shrink in a stronger solution, and there is a certain concentration which
does not change the volume. By determining the freezing-point HEDIN
found that this concentration for NaCl was nearly isosmotic with the
serum of the blood corpuscles used. The depression of the freezing-
point was about 0.56° and the concentration of the NaCl solution is
0.9 per cent, or about 0.15 normal.

The question as to the permeability of the blood corpuscles has been
investigated by HEDIN, using a method depending upon the following:4

The depression of the freezing-point of a solution is proportional to its con-
centration. A certain amount of the substance to be tested is dissolved in blood.
The serum of this treated blood freezes at a lower temperature than before the
salt was added. The depression of the freezing-point can be designated as a.
Now the same amount of substance is dissolved in serum using the same volume
of serum as blood was previously used. The depression of the freezing-point
of this serum can be designated as 6. From this it is evident that a =b if
the blood corpuscles take up just as much dissolved substance from the blood
as an equal volume of serum. If the blood corpuscles take up less than the serum

then a> b or-r > 1, and when they take up more than the serum then a <b or^ < 1.

The result T, in the calculation of which the change taking place in the volume

of the blood corpuscles on the addition of the substance must be considered,
gives immediately an approximate idea of the quantity of substance which has
passed into the blood corpuscles.

The results were as follows:

The salts of the fixed alkalies and alkaline earths, neutral amino-
acids, varieties of sugars as well as hexatomic and pentatomic alcohols
pass into the blood corpuscles only to a slight degree. Erythrite (tetra-

1 Pfluger's Arch., 63, 86 (1896).

2 Skand. Arch. f. PhysioL, 5, 207, 238, 377 (1895).

3 Arch. f. (Anat. u.) PhysioL, 1895, 154.

4 Pfluger's Arch., 68, 229 (1897); 70, 525 (1898).

8 GENERAL AND PHYSICO-CHEMICAL.

tomic alcohol), passes slowly, and glycerin (triatomic) also passes slowly,
but faster than erythrite. Ethylene glycol (diatomic alcohol) passes rather
rapidly, and the monatomic alcohols immediately divide themselves
equally in the serum and blood corpuscles. Ether, esters, aldehyde,
and acetone divide themselves so that the blood corpuscles contain more
than does an equal volume of serum. These bodies are equally absorbed
by the blood corpuscles. Ammonium salts with univalent anions pass
in quickly while with divalent or polyvalent anions the greater part
remains in the serum; still they pass in to a greater extent than do the
corresponding salts of the fixed alkalies.

OVERTON had previously arrived at the same results, using plant
cells and chiefly by making use of the plasmolytic method. Urea is prob-
ably more quickly taken up by the blood corpuscles than by plant cells,
and ammonium salts also seem to pass more easily into the blood cor-
puscles than into the plant cells.

In regard to other salts HEDIN'S results have been substantiated
by OKER-BLOM,1 by estimating the electrical conductivity of the blood.

It must also be stated that according to HEDIN, only those bodies
which do not pass, or pass slowly into the cells, can essentially alter
the volume of the cells. A close correspondence exists in this regard
between the plant and animal cells.

GURBER found that when blood corpuscles are repeatedly washed
with salt solution until the wash solution does not show any alkaline
reaction, and are then suspended in NaCl solution and treated with
C(>2, the alkaline reaction increased while the blood corpuscles became
richer in chlorine. No exchange of K or Na took place.2 GURBER
explains the experiment as follows: the carbonic acid set a small amount
of HC1 free from the salt, and this HC1 was taken up by the blood corpuscles.
The Na2COs formed at the same time gave the alkaline reaction to the
solution. KoEPPE3 as well as HAMBURGER and v. LiER4 claim, on the
contrary, that an exchange of HCOa-ions and Cl-ions takes place between
the blood corpuscles and the solution, and HAMBURGER and v. LIER
claim to have shown that the blood corpuscles are permeable only for
anions, while the cations do not pass in.

HAMBURGER5 and his collaborators have also found about the same
osmotic, phenomena with other free mobile cells such as leucocytes,
spermatozoa as with the red blood corpuscles. The osmotic relations
have also been tried with intact parts of organs, therefore with cells

1 Pfliiger's Arch., 81, 167 (1900).

2 Sitzungsber. d. med. phys. Gesellsch. zu Wiirzburg, 1895.
•Pfluger's Arch., 67, 189 (1897).

4 Arch. f. (Anat. u.) Physiol., 1902; 492.

* Osmotischer Druck und lonenlehre, Wiesbaden, 1902, 1, 401.

OSMOTIC PRESSURE. 9

in connection with other tissue constituents. By investigations on the
changes in the weight (instead of the volume changes in the above-mentioned
experiments with plant cells and blood corpuscles) which frog muscles
undergo in solutions, various experimenters, NASSE/ LoEB,2 and OVER-
TON,d have tried to prove the ability of muscle to take up various substances.
OVERTON found that as long as the irritability of the muscle was retained
the muscle took up the same bodies as the plant cells. The sarcolemma
is not responsible for the permeability, but the outer layers of the muscle
protoplasm are.

The skin of amphibians seems according to OVERTON to behave like the muscles4
in regard to permeability.

Theories of Admissibility. On what does the permeability or non-
permeability of membranes and of cells for certain bodies depend? The
discoverer of precipitation membranes, M. TRAUBE, considered the mem-
brane as a sort of molecular sieve. The relation of the size of the particles
passing and the width of the pores of the membrane is important.5 This
view cannot be contested. The copper ferrocyanide membrane may be
considered to act in this way and the non-permeability of most mem-
branes for colloid substances depends upon the fact that the pores are
too narrow for the particles.

The question as to the occurrence of a special outer limiting layer
of the cells is of interest for the understanding of the metabolism of the
cells as well as for the knowledge as to the manner in which the cells take
up and give out substances. In this connection it must be recalled that
in the protoplasm of certain cells we find an outer dense layer or a true
membrane which seems to consist of protein substances. Still, even in cells
in which no special outer limiting layer can be seen, the presence of such a
limiting layer must be admitted because of the permeability condi-
tions of these cells.

NERNSTG has shown, by special experiments, that the permeability
of a membrane for a certain substance is essentially dependent upon
the solvent power of the membrane for this substance. This question
which is very important for the study of the osmotic phenomenon in
living cells has been especially studied by OVERTON. 7 From the behavior

1 Pfliiger's Arch., 2, 114 (1869).

2 Ibid., 69, 1; 71, 457 (1898).

3 Ibid., 92, 115 (1902); 105, 176 (1904).

« Verhandl. d. phys. med. Gesellsch. zu Wiirzburg (N. F.), 36, 277 (1904).

6 Arch. f. Anat. Physiol. u. Med., 1867, 87.
6Zeitschr. f. physikal. Chem., 6, 37 (1890).

7 Vierteljahrsschr. d. Naturf. Gesellsch. in Zurich, 44 (1899) and Overton, Studien
iiber die Narkose, Jena, 1901.

10 GENERAL AND PHYSICO-CHEMICAL.

of living cells to dye-stuffs, as well as the special ease in which certain
substances, which are not soluble in water or only slightly so, but are readily
soluble in fats or fat-like bodies, pass into animal and plant protoplasms
has led OVERTON to the conclusion that the protoplasmic limiting layer
behaves like a substance layer having the solvent properties similar to the
fatty oils. According to OVERTON the protoplasmic layer is probably
impregnated with lipoids, i.e., bodies more or less similar to the fats
in regard to their solubilities and their solvent power upon certain sub-
stances. The lipoids do not form a chemically definable class of bodies.
Certain of them are still of an unknown constitution while others are
known, especially the lecithins (the phosphatides as a group) and the
cholesterin are to be especially mentioned on account of their great
importance.

The assumption that an accumulation of lipoids occurs, as a special
limiting layer, in the cells is not sufficiently founded and not generally
true at least for the animal cells. Still this assumption is not absolutely
necessary for a comprehension of the action of lipoids in the above
sense. Objections have been raised by a few investigators against
OVERTON'S theory, which has found general acceptance.1 Thus it fails
to explain all cases, although this was suggested by OVERTON himself,
for instance according to COHNHEIM, it does not explain the absorption
processes in the intestinal canal, and according to MOORE and ROAF it
cannot explain certain properties of the cells, namely the varied composi-
tion of the electrolytes within and outside of the cells, and the selective
taking up of certain soluble substances such as food products, drugs,
toxins and antitoxins by the cells. The investigations of the last men-
tioned experimenters are based essentially upon investigations of the
behavior of mineral substances, and they show that the above theory
offers certain difficulties in explaining the exceedingly important exchange
of mineral substances between the cells and the external fluid. Also the
fact that the cells are readily permeable for water is explained with diffi-
culty by OVERTON'S theory.

J. TRAUBE 2 especially has put forth objections to OVERTON'S theory.
According to him, the passage of a substance from a watery solution
into the cells, is in the first place due to its so-called solution tenacity in the
watery solution. This solution tenacity is according to TRAUBE the attrac-
tion between the solvent and the solute; and is not identical with the
osmotic pressure, but is measured by the surface tension of the solution.

1 See O. Cohnheim, Die Physiologic der Verdauung u. Ernahrung (1908). J. Loeb
in Oppenheimer's Handbuch der. Biochem. Bd. 2, 105. T. B. Robertson Journ. of
biol. Chem., 4 (1908). B. Moore and H. Roaf, Biochem. Journ., 3 (1908).

'Pfliiger's Archiv., 105, 541 (1904); 123, 419 (1908); 132, 511 (1910); 140, 109
(1911).

OSMOTIC PRESSURE. 11

It has been shown that those substances which are not taken up by the
cells at all or only slightly, do not lower the surface tension of the water
when dissolved therein. On the contrary, those substances which lower
the surface tension, pass into the cells. According to GIBBS those sub-
stances, which when dissolved in water lower the surface tension, occur
in greater concentration on the surface as compared with the interior.
Thus according to TRAUBE the solution tenacity is less the lower the
surface tension of the watery solution. Otherwise the direction of
movement of a substance in the boundary between two phases (watery
solution and cells) is determined by the relationship between the solution
tenacity of the substances in the two phases. However, the solution tenac-
ity of a substance can only be directly measured in the watery solution.
TRAUBE supports his theory upon different experiments in which mem-
bers of the same homologous series were dissolved in water in such con-
centration as to have the same surface tension and also showed the
same ability to pass into the cells. The disagreement in other cases can
be explained by the unknown solution tenacity in the cell phase. As we will
show below TRAUBE'S proposition calls to mind the accepted views as to
the origin of the adsorption phenomena or the taking up of dissolved sub-
stances by solid bodies. LOWE l has also found, in studying the taking
up of different dissolved substances by lipoids, that the process does not
take place as called for by OVERTON'S theory according to- HENRY'S law
of absorption but rather an adsorption.

Certain substances which are of the very greatest importance for life
processes and which probably are burned to a great extent within the cells,
have according to the above experiments only a limited ability to enter
the cells. These bodies are the sugars and the amino-acids. Also the
presence of salts within the cells is not easily understood in view of the
above experiments. In consideration of this it must be remarked that
the above described experiments on the permeability of animal cells have
been carried out with cells that were removed from their attachment to
the living animal. Although these cells are not considered as physio-
logically dead cells still it is very probable that certain life functions
have been arrested. It is readily conceivable that the oxidation processes,
whereby the organic substances taken up within the cells are trans-
formed into simpler products, are at least partly brought to a standstill
(see Chapter XVI). That, nevertheless, at least salts and sugar also
attract water in the living organism and therefore only pass into the
cells in small quantities follows from the experiments of HEIDENHAIN,
according to whom these substances are designated as lymph forming
agents of the second order (Chapter VI). This action is also explained

1 Bioch. Zeitschr., 42; 150, 190, 205, 207 (1912).

12 GENERAL AND PHYSICO-CHEMICAL.

by HEIDENHAIN as being dependent upon their power of abstracting
water from the tissues.

If we admit that the cells normally contain only small amounts of
sugar and amino-acids at any one time, then, if these substances are being
continuously burned within the cells, new quantities must constantly
be taken up and in this way gradually large quantities of the mentioned
substance would be taken up and burned. If the combustion is arrested
no new quantities are taken up. The fact that certain substances
are only taken up in small quantities at a time does not prove that they
are not burned within the cells.

According to MOORE and ROAF 1 the salts exist in the blood cor-
puscles in the form of " adsorpates;" these are adsorbed by the solid
constituents of the blood corpuscles. As we will see further on (page
27) an adsorbing substance can only take up a limited amount of another
substance. If, after the saturation limit is reached, more of the adsorbed
substance is added then practically no more is taken up. In this way
we can explain why the blood corpuscles only take up very little of the
salts added. The slight ability of the sugars and amino-acids to be
taken up can perhaps be explained in a similar manner.

Osmotic Pressure of Animal Fluids. As is apparent from the
above, a substance exerts upon living cells an entirely different influence,
depending upon whether the substance is able to pass into the cell or
not, and whether the substance which does not pass in has the ability
of attracting water or not. Therefore that part of the osmotic pres-
sure of body fluids which is caused by bodies not passing in is called
the effective osmotic pressure. In this manner therefore the salts of the
alkalies and alkaline earths and the sugars act. As sugar, as well as the
bodies which according to the just mentioned experiments are readily
taken up by the cells, occurs under ordinary conditions only in very
small amounts in the blood, and also as the proteins are practically with-
out influence upon the osmotic pressure, the normal osmotic pressure of
the blood is chiefly due to the salts. As the depression of the freezing-
point is almost the only method used for animal fluids, therefore ordinarily
the freezing-point depression (A) is given as a measure of the osmotic
pressure. For mammalian blood A is constant with the exception of slight
variations due to the food and perhaps also to other 'circumstances. It
is 0.560,2 which corresponds to a 0.90 per cent NaCl solution and to an
osmotic pressure of about 6J atmospheres. In lower animals A may
be slightly lower, for example, in the frog A = 0.46°. In invertebrate
sea animals the body fluid is equal to the osmotic pressure of the sur-

1 Bioch. Journ., 3, 55 (1908).

2 Hamburger, Osmotischer Durck u. lonenlehre, 1, 456.

COLLOIDS. 13

rounding sea water (A = 2.3°) and varies with the quantity of salt in the
water (BoiTAZZi). Jn lower fishes (Selachii) the osmotic pressure
of the blood is equal to the surrounding medium, and in higher fishes
(Teleostomi) lower (A = 1.0°) (BOTTAZZI). In Selachii the osmotic
pressure of the blood is chiefly due to urea (ScHROEDER).1

In sea fishes as well as fresh-water fishes, for example, the eel, a lower
osmotic pressure (A = 0.41°) is found when kept in fresh water than
when kept in sea water (A = 0.55°)2. In lower sea animals the osmotic
pressure is equal to the surrounding medium, while higher animals are
independent of the surroundings. HOBER calls attention to this condi-
tion and points out the analogy with the body heat of the various
animals.3

If we pass to other body fluids we must mention that the lymph
shows a somewhat higher osmotic pressure than the blood, and this is
due to the lymph taking up from the tissues metabolic products hav-
ing a low molecular weight.4 Milk and bile have the same osmotic
pressure as the blood,5 while saliva has a lower pressure.6 The urine
of man and mammalia generally has a much higher osmotic pressure
than the corresponding blood.7 For human urine A varies between 1.3
and 2.3°. After abundant drinking as well as under pathological con-
ditions (diabetes insipidus) the osmotic pressure of the urine can be lower
than the blood. In regard to the osmotic pressure of animal fluids under
normal and pathological conditions we refer to the work of KORANYI
and RicHTER.8

n. COLLOIDS.

The word colloid originated with GRAHAM, who included in this name
•different substances which did not have the property of diffusing through
.an animal membrane. In opposition to this GRAHAM called those
bodies which passed through a membrane, crystalloids, because they
were as a rule crystalline, a property which with few exceptions does
not belong to the colloids.9 GRAHAM included soluble silicic acid among

1 Bottazzi, Archives ital. de biol. 28, 61 (1897). Schroeder, Zeitschr. f. physiol.
Chem. 14, 576 (1890).

2Dekhuisen, Arch, ne'erland, 10, 121 (1905); Quinton, Compt. rend. soc. biol.,
57, 470, 513 (1904).

3 Physik. Chem. d. Zelle u. Gewebe, 3. Aufl. 353, (1911).

4Leathes, Journ. of Physiol., 19, 1 (1895).

6 Dresser, Arch. f. exp. Path. u. Pharm., 29, 303 (1892).

6Nolf, Traveaux du lab. de phys. de Liege, 6, 225 (1901).

7Kora"nyi, Zeitschr. f. klin. Med., 33, 1 (1897), 34, 1 (1898).

8 Physikalische Chemie und Medizin. Leipzig (1907).

9 Ann. d. Chem. u. Pharm., 121, 1 (1862) as well as Ann. de chim. et de Phys. (4),
3, 127 (1864).

14 GENERAL AND PHYSICO-CHEMICAL.

the colloids and also analogous forms of stannic acid, titanic acid,
molybdic acid and tungstic acid, aluminium hydroxide and analogous
metallic oxides, when they exist in the soluble form, and also starch, dex-
trins, the gums, caramel, tannin, albumin and gelatin.

Some colloids are characterized by the fact that under certain con-
ditions they solidify into a gelatinous form containing considerable water.
In the case where water is the solvent then GRAHAM called the soluble
form hydrosol and the gelatinous form hydrogel.

By diffusion through a membrane (called dialysis by GRAHAM) colloid sub-
stances can be separated from crystalloids. Colloidal silicic acid as well as
corresponding forms of certain other bodies are obtained by treating the soluble
alkali salt with hydrochloric acid, then removing the excess of hydrochloric acid
as well as of chlorides, by means of dialysis. Colloidal alumina was obtained by
GRAHAM by dissolving aluminium hydroxide in aluminium chloride. This last salt
was removed by dialysis and the hydroxide remained with more or less HC1 com-
bined in solution.

Various metallic sulphides can be obtained in colloidal solution. Such solu-
tions of AsaSs and Sb2S3 can be obtained by passing H2S into dilute solutions
of the respective metallic oxide,1 and colloidal CuS can be prepared by washing
the precipitated compound with water, by which treatment the[CuS finally becomes
soluble in water.2

The metals can be obtained as hydrosols, and indeed in two waj^s:

1. By treating a salt with various reducing agents (for example formaldehyde,
hydrosulphurous acid, hydrazine, hydroxylamine) the various metals are obtained
in colloidal solution.3 As the solutions thus obtained are often very unstable,
it has been found advisable to help their stability by the addition of (organic
colloids (gelatin). We will discuss the mode of action of these so-called pro-
tective colloids on page 23.

2. BREDIG 4 has discovered a method which makes possible the production
of pure metallic sols by the cathode spraying of metallic wires under water.
SVEDBERG 5 prevents the heating of the fluid in this spraying by using the
induction current. This makes the spraying also possible under organic fluids
and sols of the light metals have also been prepared. Practically sols of all
metals and metalloids can be prepared in this way.

Among those bodies wrhich can be obtained in the colloidal state
we have acids as well as bases, and the chemical elements are also known
as colloids, as well as bodies of more complex molecular structure like
the proteins and starches. The colloid bodies, therefore, have from a
chemical standpoint nothing in common. More likely the colloidal con-
dition is due to physical properties, and this follows from the researches
of GRAHAM. The crystalloids and the colloids are therefore not to
be considered as chemically different classes of bodies, but rather only
as different physical conditions of matter and the boundary between

1 H. Schulze, Journ. prakt. Chem. (N.F.), 25, 431 (1882), and 27, 320 (1883).

2 Spring, Ber. d. d'chem. Gesellsch., 16, 1142 (1883).

3 Miiller, Allg. Cheinie d. Kolloide. Leipzig (1907), 6.

4 AWganische Fermente. Leipzig (1901), 24.

5 Ber. d. d. chem. Gesellsch., 38, 3616 (1905); 39, 1705 (1906).

COLLOIDS. 15

these two conditions is often very indefinite. Certain chemically definable
classes of substances, such as proteins, occur only or chiefly in the col-
loidal condition while others, such as the inorganic salts, occur as crys-
talloids. Finally we find others that can occur in both forms, namely
the soaps (page 17). In short the difference between the crystalloid and
colloidal condition may be considered in that the crystalloids occur in
solution as molecules of medium size while the colloids are either very large
molecules, molecular aggregations or at least particles of a larger spacial
volume than the crystalloids. According to such a conception many
properties of the colloids can be explained.

In order to give a better review we will give a classification of the
colloids which seems, for the present, to be rather universally accepted.
This was first suggested by PERRiN1 and later accepted by HoBER,2
A. MiJLLER,3 and Wo. OsTWALD,4 although different authors use different
names for the two classes. The classifications of HARDY 5 and ZSIG-
MONDY6 have also much in common with the classification given below.

One of the two groups of colloids is called hydrophile colloids (emul-
sion colloids, emulsoides) because in the aqueous solution a certain rela-
tion still exists between the dissolved substance and the solvent which
is evident especially by a certain viscosity of the solution. The hydro-
phile colloids often gelatinize on cooling, the gel is again soluble in
water (reversible), and in general the hydrophile colloids are separated
from their solution by electrolytes with greater difficulty than the col-
loids of the second group. Bodies of the greatest importance for phys-
iological chemistry like the proteins, starch, giycogen, and soaps in
watery solution belong to the hydrophile colloids.

Contrary to the hydrophile colloids, the colloids of the colloidal metal
type are called suspension colloids (suspensoids) as they must be con-
sidered as suspended solid particles in a solvent and have no close
relation to the solvent. The viscosity of the solution does not differ
much from that of the pure solvent; besides this, the suspension col-
loids do not gelatinize, do not swell up, and are readily precipitated
by electrolytes. To this group belong the metallic sols, the colloidal
metallic sulphides, and certain typical suspensions obtained by dissolving
water-insoluble substances in another liquid (alcohol, acetone) and then
pouring this solution into a large volume of water. In this way the
substance is precipitated in a finely divided condition. Such suspensions

1 Journ. de Chimie phy., 3, 84 (1905).

2 Physik. Chem. d. Zelle u. Gewebe, 2 Aufl. (1906), 208.

3 Allg. Chemie d. Kolloide (1907), 187.

* Zeitschr. f. Chem. u. Ind. d. Koll., 1, 331 (1907),

5 Proc. Roy. Soc., 66, 95 (1899).

6 Zur Erkenntnis d. Koll. (1905), 16.

16 GENERAL AND PHYSICO-CHEMICAL.

behave in many respects like suspension colloids. Suspensions of mastic,1
colophony,2 and cholesterin3 belong to this class.

The hydrophile colloids stand closer to the crystalloids than do the
suspension colloids, and the transition between the crystalloids and the
hydrophile colloids is only gradual. At the boundary we find the pep-
tones and proteoses which belong to the proteins, but at the same time
dialyze rather well. On the other hand, we also have colloids which to a
certain extent form intermediary steps between the hydrophile colloids
and suspension colloids. Finally, there are also numerous intermediary
members between the suspension colloids and the finely divided substances
suspended in water (kaolin) .

Osmotic Pressure. *As above stated, the osmotic pressure of solu-
tions of crystalloids can be determined only in exceptional cases by
means of the semipermeable membrane, because it is very difficult to
prepare membranes which are impermeable for crystalloids. As pre-
viously stated, most membranes are impermeable for colloids, and the
osmotic pressure of the colloids can be best directly determined by the
aid of a membrane in a so-called osmometer. As shown by MOORE and
ROAF, in such an apparatus changes in pressure can be determined which
are not detectable by the determination of the freezing-point.4

Equimolecular solutions of various non-electrolytes give the same
osmotic pressure. From this it follows that when different non-elec-
trolytes exist in solutions with the same percentage concentration, the
osmotic tension of these solutions must be in inverse proportion to their
molecular weights. Certain colloids which will be discussed in another
connection (proteins, glycogen, etc.) must have a very large molecule.
From this it follows that these bodies must exert a very low osmotic
pressure. The proteins always contain a small amount of salts which
exist either in a sort of combination with the colloids or are to be con-
sidered as contaminations which are difficult to remove. For this reason
it has been repeatedly stated that these salts are responsible for the
small differences in the osmotic pressure. By carefully washing crys-
stalline proteins from serum and egg-white, REID was able to prepare
bodies which gave finally no osmotic pressure in the osmometer.5 In
opposition to this, MOORE and ROAF as well as LILLIE call attention to
the fact that the osmotic pressure of protein solutions is influenced by
the treatment which the protein received before the determination.

1 Zeitschr. f. physik. Cbem., 57, 47 (1906).

2 Ibid., 38, 385 (1901).

3 Bioch. Zeitschr., 7, 152 (1908).
« Bioch. Journ., 2. 34 (1906).

6 Journ. of Physiol., 31, 438 (1904).

COLLOIDS. 17

STARLING,1 MOORE and PARKER,2 MOORE and ROAFS and LiLLiE,4 using
protein preparations which had not been exposed to any strong treat-
ment before use (serum proteins, ovalbumin), as well as REID 5 (with
haemoglobin), have been able to detect a low osmotic pressure and
indeed by the aid of osmometric methods. According to STARLING,
the proteins of the serum correspond to a pressure of 30-40 mm. Hg.
and REIDG found a pressure of 3-4 mm. Hg. for a 1 per cent haemoglobin
solution.

The influence of added bodies upon the osmotic pressure has been tested by
LILLIE by adding the substance to be tested in the same percentage concentration
to the inner and outer fluids. It was found that non-electrolytes were without
action while acid and alkalies increased the osmotic pressure of gelatin solutions,
while salts lowered the pressure of gelatin as well as ovalbumin solutions. ADAM-
SON and ROAF 7 arrived at similar results in regard to alkalies and acids. Besides
this, LILLIE found that the osmotic pressure was dependent upon the past history
of the colloid. Warming as well as shaking the solutions seems to change the
aggregate condition, which returns very slowly or not at all. The changes
in the osmotic pressure produced by salts, LILLIE explains by a change in the
aggregate condition of the colloid, by the addition of salts it is brought closer
to its precipitation point and is probably united in large aggregations. In this
way the number of particles is diminished and, as this number must be important
for the osmotic pressure, this pressure is lowered. In agreement with this
the above mentioned influence of acids and alkalies upon the osmotic pressure of
gelatin can be explained by an increase in the particles.8

As we have seen above the determination of the elevation of the boil
ing-point or the depression of the freezing-point is the simplest way
for estimating the osmotic pressure of a crystalloid substance in solution.
If such determinations are made with a colloidal solution then unmeas-
urable results are found for the elevation of the boiling-point or the depres-
sion of the freezing-point. This indicates, as above stated, that the
molecules or the particles must be very large. F. KRAFT9 found no
elevation of the boiling-point for soaps in watery solution but obtained
values which correspond to the calculated molecular weights when the
soaps were dissolved in alcohol. Therefore the soaps are colloidal in
watery solution and crystalloidal bodies in alcoholic solution.

Filterability. Large particles suspended in a liquid can be removed
from the fluid by filtering. The finer the suspended particles are the

1 Journ. of Physiol., 19, 322 (1896).

2 Amer. Journ. of Physiol., 7, 261 (1902).

3 Bioch. Journ., 2, 34 (1906).

4 Amer. Journ. of Physiol., 20, 127 (1907).
« Journ. of Physiol., 33, 12 (1905).

6 Bioch. Journ., 3, 422 (1908).

7 Ibid.

«Pauli, Roll. Zeitschr., 7, 241 (1900).

9Ber. d. d. chem. Gesellsch., 29, 1328 (1896); 32, 1584 (1899).

18 GENERAL AND PHYSICO-CHEMICAL.

closer must the filter be Extensive experiments on the filtering of
colloids have been carried out by BECHHOLD.1 He used paper niters
which were impregnated with collodion dissolved in glacial acetic acid.
According to the concentration of the collodion solution filters of dif-
ferent porosity were obtained. The colloid solutions were pressed
through the filter by a pressure up to five atmospheres. It was shown
that all colloid solutions contained particles of various sizes. Never-
theless for every solution a filter could be prepared whose pores were
small enough to retain all the particles. In this manner BECHHOLD was
able to classify the colloids in a series according to the size of the smallest
particles. He found that in general the inorganic colloids (Prussian
blue, platinum, iron oxide, gold, silver) form larger particles than the
organic colloids (gelatin, haemoglobin, seralbumin, proteoses, dextrin).
Still it must be remarked that according to ZsiGMONDY2 the size of the
particles of the same colloid are larger in one preparation than in another
and that the size can change on keeping.

On filtering proteose solutions through filters of unequal thickness
BECHHOLD was able to show that the larger the particles of the proteoses,
the easier are they precipitable by ammonium sulphate.

Diffusion. We have already seen that the osmotic pressure of a
colloid solution is very small and also that the osmotic pressure of a solu-
tion is the cause for the diffusion of the particles, therefore it is evident
that the diffusion ability of colloids can only be very slight. This is
not only true for the free diffusion but also for the diffusion through a
membrane. Both of these was first studied by GRAHAM. The first was
found very slight but measurable in several cases while the fact that
the colloids did not diffuse through membranes (npn-dialy sable) was
given as the most constant difference between colloids and crystalloids.
Nevertheless, there does not exist any sharp boundary and dialysis depends
principally upon the size of the particles as well as upon the character of
the membrane.

Internal Friction. By the internal friction of a fluid we mean the
force which resists the displacement of the particles of the fluid among
one another. The internal friction is therefore an expression for the
great thickness or viscosity of the fluid.

For physiological purposes the internal friction is determined by
measuring the time which a given volume of the fluid requires to flow
through a capillary tube under a pressure of its own weight.

It is generally accepted that the internal friction of suspension col-

1 Zeitschr. f. physik. Chem., 60, 257 (1907).

2 Zur Erkenntnis d. Koll., (1905), 104 as well as Zeitschr. f. Elektrochem., 12,
631 (1906).

COLLOIDS. 19

loids is equal to that of the pure solvent or differs from it only slightly.
On the contrary hydrophile colloids are, in proper concentration, very
viscous which is probably the reason that they gelatinize under certain
circumstances. PAULI as well as PAULI and HANDOVSjcY1 have ^inves-
tigated strongly dialyzed serum in regard to its internal friction. The
addition of a little salt (to 0.05 normal) causes a lowering of the internal
friction below that of a pure albumin solution, while acids and alkalies
in small amounts cause a powerful rise in the viscosity.

Optical Properties. Colloidal solutions are opalescent by reflected
light, which depends upon the fact that the light is reflected by the sus-
pended particles. "The reflected light is partly polarized. This phenom-
enon, called TYNDALL'S phenomenon, depends upon the presence of
small particles in the liquid, and is considered as a test for colloid solu-
tions. Still there are colloid solutions (certain gold solutions, ZSIG-
MONDY), which do not give TYND ALL'S phenomenon, and on the other hand
we also have solutions of certain high molecular crystalloids (cane
sugar, rafnnose), which produce this phenomenon.2

With the aid of the ultramicroscorje of SIEDENTOPF and ZSIGMONDY,
it has been made possible to see the colloidal particles directly.3 In
this apparatus the colloidal particles are strongly illuminated by direct
light, so that no ray of light falls directly into the eye of the observer.
The particles are hereby made visible on account of the formation of
diffraction disks which are visible through the miscroscope. In colloidal
solutions where the particles are close together, a more or less intense,
homogeneous, polarized sphere of light is seen in the microscope where
the individual particles cannot be distinguished from each other. This
is possible on diluting the solution. Those particles which are only
made visible by dilution are ca\\ed_submicrons, while those that gradually
disappear on dilution are called amicrons.

The investigations of ZSIGMONDY and others upon the giowth of colloidal
metallic particles are also interesting. Thus the reduction of gold chloride by
formaldehyde, whereby colloidal gold is formed, is accelerated by the addition of
colloidal gold, and the added particles indeed grow at the cost of the newly
reduced gold.4 In a similar manner the reduction of silver nitrate with ammonia
and formaldehyde is helped by the addition of colloidal gold when the reduced
silver precipitates upon the gold particles.5 In such processes the "amicrons can
enlarge so that they can be observed by the ultramicroscope (submicrons) .

Koll. Zeitschr., 3, 5 (1908); Pauli and Handovsky, Biochem. Zeitschr.,
18, 340 (1909); 24, 239 (1910).

2 Lobry de Bruyn and Wolff, Rec. trav. chim. des Pays-Bas., 23, 155 (1904).

3 Zsigmondy, Colloids and the Ultramicroscope, translated by Alexander, New York,
1909.

4 Zsigmondy, Zeitschr. f. physik. Chem.. 56, 65 (1906).

5 Zsigmondy and Lottermoser, ibid., 56, 77 (1906).

20 GENERAL AND PHYSICO-CHEMICAL.

According to the manner of preparation the colloids may have particles of different
sizes. (See page 00.)

Submicrons havey also been detected in solutions of organic colloids. The
work of GATIN-GRUZEWSKA and BILTZ, * who used a specially pure glycogen,
must be especially mentioned. They found that the aqueous solution of glycogen
contained amicrons as well as easily recognizable submicrons, whose presence
was only evident by a homogeneous sphere of light, but on the addition of alcohol,
conglomerate into detectable submicrons.

Molecular Movement. R. Bno\\N2 first f6und that small particles
suspended in water showed a quivering motion, and this phenomenon
has been called, from its discoverer, Brownian molecular motion, although
the particles in no manner are to be considered' as molecules. This
phenomenon has been observed since then by many investigators in
fluids having suspended solid particles as well as in substances dissolved
in colloidal condition.

The Brownian movement is considered by some as a manifestation of a general
molecular movement of matter. According to this view it is comparable with
the supposed motion of gas molecules according to the kinetic theory of gases.
PERRIN as well as SVEDBERG 3 claim that the law of gases also holds for very
dilute colloidal solutions.

Electrical Transportation of Suspended Particles. A not too weak
electric current has the power of causing motion in small quantities
of fluid enclosed in a capillary tube or in a porous diaphragm. The
particles suspended in a fluid also wander under the influence of the
electric current, and indeed to the anode or cathode, according to the
nature of the fluid and the particles. This phenomenon is called cata-
phoresis. Such movements have also been found in colloidal solutions.
According to BiLTZ,4 in dialyzed aqueous solution, the colloidal metallic
hydroxides wander to the cathode, and the other colloids (metals,
metallic sulphides, acids) wander to the anode. The colloidal particles
in water are therefore probably electrically charged, hence the nega-
tively charged wander to the anode and the positively charged to the
cathode. Dialyzed protein solutions show according to older investiga-
tions no cataphoresis. The addition of acid or alkali gives to the pro-
tein a positive or negative charge respectively, hence an alkaline solu-
tion wanders to the anode and an acid solution to the cathode (HARDY,5
According to MiCHAELis7 the proteins in perfectly neutral

1 Pfliiger's Arch., 105, 115 (1904).

2Edinb. Phil. Journ., 5, 358 (1828); 8, 41 (1830).

3 Perrin, Colloid-chem. Beihefte, 1, 221 (1910). Svedberg, Roll, Zeitschr., 7, 1
(1910).

4 Ber d. d. chem. Gesellsch., 37, 1095 (1904).
* Journ. of Physiol., 24, 288 (1899).

6 Hofmeister's Beitrage, 7, 531 (1906).

7Biochem. Zeitschr., 16, 81 (1909); 19, 181 (1909); 24, 79; 27, (38; 28, 193;
29, 439 (1910); 33, 456 (1911); 41, 373 (1912).

COLLOIDS. 21

solution wander to the anode in the case when the experiment is so
carried out that the formation of acid or alkali is prevented at the anode
or cathode respectively. If the neutral protein solution is treated with
a trace of acetic acid then the particles wander to the cathode. With
a certain very slight degree of acidity the direction of the wandering of
the particles is reversed. With this reaction no wandering or a double-
sided wandering of the protein bodies can, be detected. This so-called
isoelectric point has been determined by MICHAELIS, RONA and their
collaborators for different protein substances.1 MICHAELIS and RONA
claim to have found in the isoelectric point the most favorable reaction
for the heat coagulation of the protein substances, while SORENSEN and
JURGENSEN consider the reaction which the pure protein substance
gives to pure water as the optimal precipitation reaction.2

According to GATiN-GRUZEWSKA3 pure glycogen wanders distinctly
to the anode.

Precipitation of the Colloids.

The colloids can be separated from their solutions in various ways.
Many colloidal solutions are so unstable that they flock out after a
time without the addition of anything (silicic acid, metallic hydroxides).
Certain colloids appear as flocculent precipitates on heating their solu-
tions (certain proteins, see Chapter II). Others solidify on cooling
from hot concentrated solutions, as semisolid forms, so-called jellies
or hydrogels, containing considerable water (glue, starch, agar).

On evaporating the hydrosols at ordinary temperature we obtain
a residue which ZSIGMONDY divides into reversible and irreversible col-
loids, according whether they are again soluble in water or not.4 Accord-
ing to this definition starch, dextrin, agar, gum, and proteins belong to the
reversible colloids while colloidal silicic acid, stannic acid, colloidal metallic
hydroxides and sulphides, and the pure colloidal metals belong to the
irreversible colloids. The former are relatively non-sensitive toward
the addition of electrolytes, while the latter flock out on the addition
of the smallest quantity of electrolyte, and indeed again in an irreversible
form. This classification stands in accord with what was given above
(page 15), as the reversible colloids coincide in a measure with the
hydrophile colloids and the irreversible with the suspension colloids.

Electrolyte Precipitation of Suspension Colloids. It must be
remarked that for every precipitating electrolyte a certain minimal con-

1 See page 74.

2 See Ergebnisse d. Physiologic, 12, 506 which also gives the literature.

3 Pfliiger's Arch., 403, 287 (1904).

4 Zur Erkenntnis d. Koll., page 21.

22 GENERAL AND PHYSICO-CHEMICAL.

centration is necessary to bring about flocking. In comparing the
precipitation ability of various electrolytes the concentration of that
solution which is just sufficient to cause a visible cloudiness is given in
miilimolls ( = HFVTT gram-molecule) per liter.

HARDY 1 has also found that colloids which wander to the anode
are chiefly flocked out by the cations of the precipitating electrolyte,
and colloids wandering to the» cathode are chiefly flocked out by the anions.
H. SCHULTZE 2 has proven that the precipitating ability is influenced
greatly by the valence of the precipitating ions, as the divalent ions act
much stronger than the monovalent and the trivalent are still more
active than the divalent. This rule has been substantiated by HARDY.S

This valence. rule becomes clear by the following experiment of FREUNDLicn.4
The figures give the lowest precipitation concentration expressed in miilimolls
per liter. The hydrosol was As2S3 (negative) and the valence of the cations is
applicable chiefly for the precipitating action.

K2SO4 MgCl2.. ..0.717

— ~~ .................. 65'6 MgS04 ............... 0.810

KC1. 49 5 CaCl2 ................ 0.649

KN03 .................. 50.0 SrCl2 ................. 0.635

NaCL. ..51.0 BaCl2 ................ 0.691

LiCl ....... ... .58.4 Ba(N03)2 ............. 0.687

?nC!2 ................ 0.685

30.1 U02(N03)2 ............ 0.642

on c A1C18 ................ 0.0932

A1(NO3)3 ............. 0.0982

The precipitating action of anions upon a positive hydrosol *(Fe[OH]3) is shown
in the following experiment of FREUNDLICH :

KCL. ..9.03 K2SO4.. ..0.204

KNO3 ................. 11.90 T12SO4 ............... 0.219

NaCl ................... 9.25 MgSO4 ............... 0.217

2^p ................... 9.64 K2Cr207 .............. 0. 194

FREUNDLICH has extended the valence rule by the fact that with a negative
sol, H ions, the ions of the heavy metals, as well as organic cations in weaker con-
centration, have a greater precipitating action than other cations; OH ions as well
as organic anions act against the precipitating action of the cations. The reverse
is shown with a positive sol; OH ions and organic anions of smaller precipitation
concentration than corresponds to their valence; H ions and organic cations
act against the precipitating properties of the anions.

Certain above-mentioned suspensions (mastic), as well as other particles
suspended in water, act the same as suspension colloids. SCHULZE 5 has found
that cloudiness due to clay particles on the addition of clarifying bodies (alum,
lime) give a voluminous deposition. SCHLOESSING 6 found that clay suspensions

1 Zeitschr. f. physik. Chem., 33, 385 (1900).

2 Journ. prakt. Chem. (2),. 25, 431 (1882).
8Proc. Roy. Soc., 66, 110 (1899).

4 Zeitschr. f. Chem. u. Ind. d. Roll., 1, 323 (1907).
6 Ann. Phys. (2), 129, 366 (1866).
6Compt. rend., 70, 1345 (1870).

COLLOIDS. 23

which do not settle after months are precipitated in 24-48 hours by a minimum
quantity of lime or magnesia. He also calls attention to the essential r61e which
the salts of sea water must play in the sedimentation of the cloudy fresh water
flowing into the sea (delta formation).

In consideration of the conditions just mentioned, under which the
suspension colloids are precipitated by electrolytes, the mutual precipita-
tion ability of suspension colloids is of considerable interest. Accord-
ing to what has been stated previously, the colloids are considered as carriers
of electricity, and it has been proved that the oppositely charged col-
loids can act precipitatingly upon each other. This rule was first pro-
posed by LINDER and PiCTON,1 and has subsequently been substantiated
by many investigators. BILTZ 2 has made especially systematic investiga-
tions on this subject and finds that colloids carrying the same kind of
charge do not precipitate each other. For the mutual complete precipita-
tion of opposed electrically charged colloids, a certain quantitative rela-
tion is necessary. On the action of two colloids with opposite charges in
variable quantities an optimum of the precipitation action is noticed; while
on overstepping the desirable precipitation conditions in both directions
no precipitation occurs at all.

In analogy with the mutual precipitation ability of the colloids, BILTZ believes
that the especial great ability of most salts of the heavy metals to precipitate
colloids lies in the hydrolytically split and colloid-dissolving metallic hydroxides.

Protective Colloids. Certain hydrophile colloids, which are precip-
itated with difficulty by electrolytes, have the power of protecting
suspension colloids against the precipitating action of electrolytes. MEYER
and LOTTERMOSSER 3 have found with silver hydrosol that the presence
of protein prevented the flocking out by electrolytes. ZsiGMONDY4
has investigated the relative action of the protective colloids and has
found considerable differences. The figure in milligrams of colloid which
is just insufficient to protect 10 cc. of gold solution (0.0053-0.0058
per cent) against the action of 1 cc. 10 per cent NaCl solution is called the
gold equivalent for the respective colloid. Gelatin offers the best pro-
tection, then comes isinglass, casein, ovalbumin, gum arabic, Irish moss,
dextrin, starch. The colloidal sulphides (As2Sa, Sb2S3, CdS) are also
protected in the same manner against the influence of electrolytes (A.
MULLER and ARTMANN 5) . Inorganic colloids may also act as protective

'Journ. chem. Soc., 71, 572 (1897).

2 Ber. d. d. chem. Gesellsch., 37, 1095 (1904).

3 Journ. prakt. Chem. (2), 56, 241 (1897).

4 Zeitschr. analyt. Chem., 40, 697 (1901).
5Oester. Chem. Ztg., 7, 149 (1904).

24 GENERAL AND PHYSICO-CHEMICAL.

colloids. Thus according to BILTZ 1 zirconium hydroxide protects gold
better than does gelatin.

By the addition of organic protective colloids, the inorganic colloids
which on evaporation otherwise become irreversible, are made reversible,
in that the dry residue is soluble in water again. On this depends the
use of the protective action in the preparation of permanent inorganic
hydrosols, and this is of importance in many cases.

According to BECHHOLD2 the filterability of suspension colloids through
collodion filters is increased by the addition of organic colloids. It is also
well known that certain finely divided substances (carbon) pass more
easily through a filter in the presence of protein than without protein.

The action of the protective colloids is ordinarily explained accord-
ing to the theory of QuiNCKE3 on the mutual surface tension of the
active bodies, and the process belongs accordingly to the adsorption
phenomenon which will be discussed later. According to this theory
the protective colloid under certain conditions spreads like an envelope
around the particles. In this wise the entire mass takes the properties
of the protective colloid and is therefore not precipitated by the elec-
trolyte any more than the protective colloid itself. In filtration the pro-
tective colloid acts to a certain extent like a lubricant. This theory of
colloid envelope has recently received support by experiments of MICHAELIS-
and PiNCUSSOHN.4 They found that when suspensions of indophenol
and mastic were mixed together the number of particles visible in the
ultramicroscope diminished; after mixing, the physical properties of
the indophenol (pseudofluorescence, positive cataphoresis) were not
evident.

Electrolyte Precipitation of Hydrophile Colloids. The salts of the
alkalies precipitate the suspension colloids even in low concentrations.
The alkali salts behave differently toward the hydrophile colloids. This
may in part be due to the fact that hydrophile colloids have much less
of a certain electric charge than the suspension colloids. For this reason
the hydrophile colloids are often precipitated from their solution by alkali
salts. For this purpose, firstly, certain concentrations are necessary;
secondly, the precipitates of the hydrophile colloids are again soluble
in water (reversible) in opposition to those of the suspension colloids.
In regard to the ability of different alkali salts to act precipitatingly
certain laws have been formulated, but they cannot be arranged in a
general rule.

1 Ber. d. d. chem. Gesellsch., 35, 4431 (1902).

2 Zeitschr. f. physik. Chem., 60, 301 (1907).

3 Ann. Phys. (3), 35, 580 (1888).

4 Bioch. Zeitschr., 2, 251 (1907).

COLLOIDS. 25

On comparing the concentration of various salts just sufficient for precipita-
tion, where at one time the same anion with different cations was tested and
another time the same cation with different anions, PAULI has arranged the cations
and anions in the following order in increasing precipitation ability:

CNS <I <Br <N03 <C1 <OCO.CH3 <HP04 <S04
NH4<K<Na<Li.

The protein used in these experiments was white of egg. According to PAULI
certain ions have a precipitating action and others a solvent action. The action
of a salt corresponds to the algebraic sum of the action of the ions.1 PAULI has
attempted to associate the precipitation ability of the salts in relation to their
action upon the coagulation temperature, but without any positive results.2

Nevertheless SPIRO 3 has shown that the kind of protein as well as its con-
centration are of importance for the precipitation action, and HOBEB 4 has recently
shown that the series I<Br<Cl<S04 and Li<Na<K<Rb<Cs is valid in
alkaline reaction, but that the series is reversed in acid reaction. In nearly
neutral reaction irregularities in the ion series occur which can be considered as
a transition series between the two just-mentioned series. That the reaction must
be of great importance in the precipitation of proteins seems very probable in
consideration of the fact that the proteins take a decided electric charge on the
addition of acid or alkali. In regard to the precipitation by salts of the heavy
metals, the hydrophile colloids do not seem to differ essentially from the suspension
colloids.6

On boiling a protein solution the protein suffers an irreversible change
and under certain circumstances flocks out. Boiled but not flocked
egg-white behaves with precipitating substances, like a suspension colloid.6
In regard to the precipitation of proteins see Chapter II.

Theories of Precipitation Phenomena.

At least for the suspension colloids there is no question that they
are flocked out by ions which carry an electric charge opposite to the
colloid particles, and also by other colloids having an opposite charge.
This fact follows from HARDY'S theory, according to which the flocking
out is a neutralization process in which the charge of the colloid is
just neutralized and the colloid therefore precipitates.7 The mixture
formed on precipitation has been shown to be electrically neutral (iso-
electric) as the precipitated particles show no cataphoresis. In this
manner it is easily understood that polyvalent ions have a stronger
precipitating action than monovalent, as the electrical charge in, for

* Hofmeister's Beitrage, 3, 225 (1902).

2 Pfliiger's Arch., 78, 315 (1899).

3 Spiro, Hofmeister's Beitrage, 4, 300 (1903).

4 Ibid., 11, 35 (1908).

5 Pauli, Ibid., 6, 233 (1905).

6 Hardy, Proc. Roy. Soc., 66, 110 (1900).

7 Zeitschr. f. physik. Chem., 33, 385 (1900).

26 GENERAL AND PHYSICO-CHEMICAL.

example, a trivalent ion is three times greater than in a monovalent ion.
Otherwise greater precipitation ability of polyvalent ions can also be
explained by a greater hydrolytic cleavage of the salts (page 23).

The mechanism of the precipitation of the isoelectric solution accepted
in HARDY'S theory is explained by BREDIG 1 as follows : At the boundary
between suspended particles and solvent a certain surface tension exists
which tries to diminish the total contact surface between the two media,
which can happen by the small particles uniting to form larger ones,
when nocking is brought about. The electrical charge of the particles
acts against the surface tension so that equally charged particles repel
each other. If the electrical charge is discharged, as takes place in the
isoelectric point, then the surface tension reaches its highest value and
the precipitation may occur.

The correctness of HARDY'S claim that precipitation occurs just in
the isoelectric fluid is disputed on special grounds by BILLITZER. He
believes that the ions have a much greater charge than the colloid par-
ticles. An ion collects the oppositely charged colloid particles around
itself, and during these neutralization processes it may occur, that the
entire complex may become so large as to become visual and on
account of the gravity it precipitates out.

In general it can be stated that the stability of a colloid is greater the smaller*
cet. par., the particles are; as the probability that the number of particles sufficient
for the precipitation is then less. With equal size of particles the stability of a
colloid is dependent upon the size of the charge which the particles carry. Too
weak and very strongly charged colloids are relatively more stable; the first
because of the large number which must collect around an ion when flocking takes
place and the second because the number of particles required for the neutrali-
zation is perhaps too small, so that the necessary size of the complex for precipita-
tion is not attained.2

The findings of LINDER and PiCTON3 that when colloidal As2Sa is
precipitated with BaCb the solution becomes acid, and a small quantity
of barium remains in the precipitate, corresponds to BILLITZER'S theory.
This quantity of barium cannot be removed by water, but can be replaced
by the corresponding cation by washing with a solution of another salt.
According to BILLITZER in the mutual precipitation of colloids a quan-
tity relation exists which is dependent upon the electrical charges4 (see
also page 22).

The fact that the precipitation of colloids is a manifestation of
processes which occur in a homogeneous medium, makes the understand-
ing of these especially difficult. If, as is generally accepted, we consider

1 Anorganische Fermente (1901), 15.

2Zeitschr. f. physik. Chem. Soc., 45, 327 (1904); 51, 129 (1905).

3 Journ. Chem. Soc., 67, 63 (1895).

4 Zeitschr. f. physik. Chem., 51, 141 (1905).

COLLOIDS. 27

the colloid solution as a homogeneous fluid of suspended solid or fluid
particles, then in the " solution " there occur at least two special con-
stituents, separated from each other — the colloid particles and the sol-
vent. This is expressed as follows: the system contains two phases.
The solvent is often more correctly called the dispersion means and the
colloid particles called the disperse phase. If to such a system a new
substance is added, then the reaction which follows, depends essentially
upon the division of the new substance between the two phases. In
regard to the possible division two cases will be presented:

1. The process can be similar to the division of a soluble substance
between two solvents. If a substance is brought in contact with tw^>
solvents at the same time, then it divides itself so that the relation
between the concentration in the two solvents remains the same but
independent of the total quantity of the dissolved substance. If the
quantity of substance in each 100 cc. of the two solutions 1 and 2 is

designated by cj. and 02, then it follows that — = k where k is a constant.1

C2

The first example where this la\v was shown to be correct was the divi-
sion of succinic acid between water and ether (BERTHELOT and JUNG-
FLEiscH2). This law was also shown to be true for the division of
a gas between a gaseous and a fluid phase,- i.e., for the absorption of a
gas in a fluid (HENRY'S law of absorption). The conditions for the cor-
rectness of this law are that the temperature remains the same in experi-
ments with different quantities of substance as well as that the substance
has the same molecular size in the two phases.

2. In those cases where finely divided solids take up dissolved sub-
stances or gases the division is generally not independent of the total
quantity of the dissolved substance or of the gas. This is often called
adsorption.3 For example, if we are dealing with the adsorption of a
dissolved substance by a finely divided solid occurring in a solution,
then a greater percentage is taken up from a dilute solution than from a
concentrated one. On increasing concentration the adsorbed fraction
becomes continuously less so that the absolute quantity taken up reaches
a maximum which corresponds to the greatest adsorption ability of the
solid body. ,

This is expressed by the formula — =/b, where c\ and c2 indicate the concentra-

C<i

tion of the solid body and in the solution; n and k are constants and indeed, n is

, Zeitschr. f. physik. Chem., 8, 110 (1891).

2 Ann. Chim. phys. (4), 26, 396 (1872).

3 It must be remarked that in the older literature oftentimes no difference was
made between adsorption, and absorption, in which case both processes were included
under the name absorption.

28 GENERAL AND PHYSICO-CHEMICAL.

always >1. (If n = l then the formula would be ~=k and we^would be dealing
with a so-called solid solution.)

APPLEYARD and WALKER1 have studied the adsorption of organic
acids from aqueous and alcoholic solutions by means of silk; the divi-
sion was found to correspond to the above formula for adsorption.
FREUNDLiCH2 has also carefully tested the adsorption of crystalloids
by carbon. From these experiments it was shown that the equilibrium
could be quickly attained from both sides, i.e., that the process was readily
reversible. The above-given formula was found sufficiently accurate
for the case where only the total quantity of the dissolved (to adsorb)
substance varied. The series in which the organic acids were adsorbed
by silk, as found by APPLEYARD and WALKER, were pratically the same
as with carbon. The influence of temperature was slight.

According to KtisTER,3 the combination between starch and iodine
is to be considered as an adsorption compound, and BILTZ 4 finds for the
division of As2Os between iron hydroxide (1) and water (2) the for-
mula—=0.631.

C2

The theoretical foundations for the adsorption phenomenon are
not especially clear. Generally the adsorption is considered as con-
nected with segregation and surface tension phenomenon. At the con-
tact surface between a solid body and solution a surface tension exists
which is considered as positive, i.e., this attempts to diminish the
contact surface. The surface energy used thereby tends to be a min-
imum potential energy. As the product from size of surface and surface
tension are the same, and as the first cannot change, the surface energy
can only be diminished by a reduction of the tension. If, therefore,
the tension is diminished by increasing the concentration of a sub-
stance dissolved in a fluid, then this substance tries to collect itself
at the surface in greater concentration than in other parts of the fluid
(OsTWALD,5 FREUNDLICH 6) . In regard to the surface tension of solid-
fluid we only know that it is positive, but can otherwise show great
differences (OsTWALD,7 HULETTS). According to this theory the facts
are that certain solid substances possess the ability of adsorbing dis-

1 Journ. Chem. Soc., 69, 1334 (1896).

2 Ueber die Adsorption in Losimgen, Leipzig (1906).
8 Ann. d. Chem. u. Pharm., 283, 360 (1894).

*Ber. d. d. chem. Gesellsch., 37, 3138 (1904).

« Lehrb. d. allg. Chem., 2. Aufl., 2. Bd., 3. Teil, 237 (1906).

8 Ueber Adsorption in Losungen, 50-51.

'Zeitschr. f. physik. Chem., 34, 495, 1900.

» Ibid., 37, 385 (1901).

COLLOIDS. 29

solved bodies, and for this reason the adsorbed substance lowers the
surface tension of the solid-fluid, and indeed, the more the greater con-
centration in which it occurs. That especially carbon and colloid sub-
stances are adsorption bodies lies in the fact that they have an especially
large surface due to their finely divided state or porosity, which there-
fore, cet. par., must give them a great surface energy.

That proteins, on precipitation, carry down other bodies with avidity
is well known; inorganic hydrogels also take up dissolved substances
with energy. The curves obtained for the latter process by VAN BEM-
MELEN l show a close analogy with the characteristic curves for the
adsorption compounds. It often occurs that the body taken up homo-
geneously saturates the hydrogel, in which case — = k, and a sort of

C2

solid solution is the result. In certain cases, undoubtedly, chemical
combinations with quite positive conditions are formed.

The precipitation of colloids by electrolytes has also been discussed
by FREUNDLiCH2 from the standpoint of the adsorption hypothesis.
Thus, for the precipitation ability of an electrolyte, the electric charge
of the precipitating ion comes first into consideration and secondly, the
ability of the precipitating colloid to adsorb the same. According to
MOORE and ROAFS the salts of the red corpuscles are retained as adsorp-
tion compounds (adsorpates) by the proteins.

Thus far only the adsorption of crystalloids has been considered.
Colloids are also taken up by solid substances or by other colloids. Still in
these cases the conditions are more complicated than in the above-
mentioned adsorption phenomena, as the combinations formed are in special
cases irreversible or gradually become irreversible. It is well known that
carbon takes up colloidal colored substances, and we have numerous exam-
ples of the combination of dissolved colloids with solid colloids in technology.
BiLTz4 has been able to show that many dyeing processes are to be
considered as adsorption phenomena, and later FREUNDLICH and LOSEV 5
have measured the adsorption of basic and acid pigments by carbon
and also by fibers (wool, silk, cotton), and have shown the correspondence
of the two processes. With the basic pigments, which were used as
salts, a splitting occurred into a pigment base, which was taken up by
the fibers as well as by carbon, and an acid which quantitatively remained
behind. This is similar to the cleavage which precipitating electrolytes
undergo in the precipitation of the suspension colloids (see page 26).

« Zeitschr. anorg. Chem., 23, 111, 321 (1900).

2 Zeitschr. f. Chem. u. Ind. d. Koll., 1, 321 (1907).

3 Bioch. Journ., 3, 55 (1908).

*Ber. d. d. chem. Gesellsch., 37, 1766 (1904); 38, 2963, 2973, 4143 (1905).
5 Zeitschr. f. physik. Chem., 59, 284 (1907).

30 GENERAL AND PHYSICO-CHEMICAL.

Tanning is also brought about by adsorption processes, as the, prepared skins
adsorb the tanning substance.1

The precipitation of portein by adding finely divided solids (carbon*
kaolin 2) or by suspended solids (mastic 3) precipitated in the liquid,
as well as the action of protective colloids as already mentioned are also
due to adsorption processes. The precipitation of protein, which occurs
on shaking the protein solution with liquids, in which the protein is
not soluble, is also to be considered as a surface tension action (RAMSDEN 4) .

BECHHOLD,5 in his above-mentioned experiments on the filtration of
colloids, has observed conditions which he considers as adsorption phe-
nomena. Under certain circumstances a colloid can prevent the filtra-
tion of another colloid. A filter which was permeable for colloidal As2Sa,
but retained colloidal Prussian blue, did not allow a clear mixture of
the two to pass through. The particles of As2S,3, were adsorbed by the
particles of Prussian blue, and could therefore not pass through the
filter.

Gels. We have often mentioned gels or jellies (page 14). Only
certain colloids can occur in the form of gels. Certain gels are spon-
tanteously formed in sufficiently concentrated solutions (silicic acid,
certain metallic hydroxides) and these do not redissolve in water. Other
gels, like gelatin and agar, are formed on cooling of the hot, concentrated
solutions, and are again soluble in water.

According to HARDY 6 the gel formation of gelatin is to be considered
as a segregation process whereby a separation into two fluids occurs,
one of which solidifies. The two phases are only differentiated by the
microscope, and the chemical testing of the theory fails because of the cir-
cumstances that the two phases cannot be analyzed separately. In opposi-
tion to this PAULI claims that the gel passes through all of the intermediary
steps into the corresponding sol and is therefore homogenous in the same
sense as these.7

When gels are freed from water by evaporation or in other ways,
they show a special ability to take up water, which is brought about
by different processes which are included in the ordinary term imbibition.
The views on this imbibition are indefinite. Surface phenomena play
a r61e here. According to VAN BEMMELEN 8 the water is not chemi-

1 See Zeitschr. f. Chem. u. Ind. d. Koll., 2, 257 (1908).

2 Bioch. Zeitschr., 5, 365, 1907.

3 Ibid., 2, 219 (1906); 3, 109 (1906).

4 Zeitschr. f . physik. Chem., 47, 343 (1904).
6 Ibid., 60, 299 (1907).

• Ibid., 33, 326 (1900).

'Bioch. Zeitschr., 18, 367 (1909).

8 Zeitschr. anorg. Chem., 13, 233 (1896); 20, 185 (1899).

COLLOIDS. 31

cally combined in definite proportions, but the quantity continually
changes with the temperature and the vapor pressure. On the other
hand, the imbibition stands in close relation to the osmotic pressure
which is evident, if we define the osmotic pressure of a substance as
its ability to attract water. The relation between imbibition and
osmotic pressure is still closer in those cases when the substance finally
is dissolved in water.

If a hydrogel is placed in a salt solution instead of in pure water,
the imbibition phenomena essentially change. This was first studied
by HoFMEiSTER,1 using gelatin plates. The process is rather com-
plicated, as salt is taken up by one side of the gelatin plate and water
by the other, and the taking up of water is influenced by the quantity
of salt taken up. It has also been found that when gelatin plates are
treated with solutions of increasing concentration of the same salt,
the taking up of salt increases at first with the salt concentration, then
becomes slower, and attempts to reach a maximum and then remains
almost stationary. As long as the taking up of salt increases, the quan-
tity of water passing into the gelatin also increases; when the salt fails
to pass then the water also ceases to pass. It has also been found that
the maximum of salt absorption for sulphate, tartrate and citrate can
be attained with much lower molecular concentrations than with chloride,
nitrate and bromide. From this it follows that the sulphate, tartrate
and citrate have a retarding action upon imbibition within certain limits
of concentration, while the chloride, nitrate and bromide have an
accelerating action.

PAULI 2 has investigated the influence of salt solutions upon the solid-
ification and melting-point of gelatin. If the salts are arranged in the
order of their ability to lower the solidification point of gelatin we
come to the series sulphate, citrate, tartrate, acetate (water), chloride,
chlorate, nitrate, bromide, iodide. This series corresponds well with
that of HOFMEISTER.

Acids and alkalies exert a special influence upon gelatin, as they
both, in very dilute solutions, strongly accelerate imbibition (SriRO,3
Wo. OsTWALD4). From the previously mentioned investigations of
LILLIE, on the osmotic tension of gelatin solutions, it was found that the
addition of acids and alkalies increased it (page 17).

Since GRAHAM'S fundamental experiments it was believed that col-
loidal sols could not diffuse into gels while crystalloids^ could pass just

1 Arch. f. exp. Pathol. u. Pharm., 28, 210 (1891).

2 Pfliiger's Arch., 71, 333 (1898).

3 Hofmeister's Beitrage, 5, 276 (1904).

4 Pfliiger's Arch., 108, 563 (1905).

32 GENERAL AND PHYSICO-CHEMICAL.

as quickly into gels as into pure water. Nevertheless, SPIRO 1 has observed
that dissolved ovalbumin as well as haemoglobin could pass into gelatin
plates. On the other hand K. MEYER 2 as well as BECHHOLD and ZEIGLER 3
have found that the distance passed by a crystalloid in gelatin may be
much shorter than in pure water. In such experiments no doubt adsorp-
tion processes must be considered.

m. CATALYSIS.

When two bodies which can act chemically upon each other are
brought together the reaction generally takes place so fast that it can-
not be measured. In other cases, by special means, we can observe
how the reaction gradually proceeds. When cane-sugar is inverted
by weak acid, the decrease in the rotation of the solution can be fol-
lowed with the polariscope; and when an ester is decomposed by alkali
the quantity of still free alkali can be determined by titration. The
quantity of substance measured in gram-molecule per liter (mole)
which is decomposed in the unit of time, is called the reaction velocity
of the system. The so-called law of mass action, as proposed by GULD-
BERG and WAAGE, states that the reaction velocity is every moment
proportional to the molecular concentration of the reacting bodies. A
mixture of alcohol and acetic acid is transformed into acetic ether and
water, especially in the presence of some mineral acid. If the molec-
ular concentration of the alcohol and acid be designated by CA and Cs,
then according to the law of mass action the reaction velocity is
Vi=ki.CA-Cs, where k\ indicates a constant which is independent of
the quantity of reacting substances and the time limit is so short that
the concentration can be considered as constant. This reaction, like
many others, is reversible, 4.e., two reactions occur simultaneously:
one between the alcohol and acetic acid, producing acetic ether and
water, and second, between acetic ether and water, re-forming alcohol
and acetic acid. This is expressed as follows :

The velocity of reaction when it passes from left to right is called
v\. If the velocity in the reverse reaction is called V2 and the molecular
concentration of the acetic ether and water is called CB and Cw,
then we obtain V2 = k2-CE'Cw. At the beginning when CE as well as

1 Hofmeister's Beitrage, 5, 294 (1904).

2 Ibid., 7, 393 (1905).

3 Zeitschr. f. physik. Chem., 56, 105 (1906).

CATALYSIS. 33

Cw = Q, the velocity of the ester formation is expressed by the formula
Vl = ki • CA • Cs ; afterward it is expressed by the difference vi—V2 or
ki'CA'Cs—k2'CE'Cw. Of the two reaction velocities v\ and V2 at the
begining v\ always diminishes while V2 increases. When k\ -CA'CS =
k2-CE'Cw is attained, then the, velocity of both reactions is the same;
no measurable decomposition occurs and the system is in equilibrium.
The equilibrium condition is the same irrespective of whether we start
from alcohol + acetic acid or from the corresponding quantity of acetic
ether + water. On equilibrium it is

or

K is called the equilibrium constant; as is apparent it can be determined
in two ways — either from the concentration of the reacting bodies when
equilibrium is present or from the velocity coefficient ki and £2 as deter-
mined in a manner given below.

In the above-mentioned transformation of alcohol and acetic acid
these two bodies are simultaneously used up. The reaction is therefore
called bimolecular, and a reaction is called mono-, bi-, tri-, etc., molecular
according to the number of the kinds of molecules which diminish their
concentration thereby.1

BERZELIUS 2 found that certain bodies by their mere presence, and
not by their affinity, have the power of awakening the dormant affinity
at a certain temperature, i.e., the power of starting a reaction. These
phenomena were called catalytic by BERZELIUS.

According to OSTWALDS catalysis is the acceleration (or retardation)
of a slow-proceeding chemical change by the presence of a foreign body.
That body which influences a reaction in this manner is called a catalyst.
It does not itself undergo any appreciable change by the reaction.

Catalytic reactions have been studied, especially by WiLHELMY.4
VAN'T HOFF,S OSTWALD,S ARRHENIUS 7 and BREDIG.S Of all other sub-
stances the acids and alkalies seem to act most catalytic. A well-known

llt is assumed here that of every kind of molecule one molecule of each takes
part in the reaction.

2Berzelius, Arsberattelse om framstegen i Fysik och Kemi., 13, p. 245 (1836).

3 Lehrb. d. allg. chem. 2. Aufl. II., 1, 515.

4 Poggendorff's Ann., 81, 413 (1850).

5 Etudes de dynam. chim. (1884).

6 Lehrb. d. allg. Chem., 2. Auf. II, 2, 199.

7 Zeitschr. f. physik. Chem., 4, 226 (1889).

8 Anorganische Fermente (1901); Bioch. Zeitschr., 6, 283 (1907).

34 GENERAL AND PHYSICO-CHEMICAL.

example is the inversion of cane-sugar by means of acid. This reac-
tion is monomolecular because only the cane sugar is consumed. If
the concentration of the cane-sugar at the beginning is C moles, and if
x moles are transformed in t time, then at that time there are (C—x)
moles remaining. If dx indicates the quantity- which is transformed

in dt time, then the reaction velocity «is — . According to the law

dt

of mass action this is at every moment proportional to the concentration
of the decomposing substance, -or

f -*•«?-*). , a)

For practical use this equation is integrated into the following:

fc = -nat. log. — - (2)

t (_/ x

If the theoretical considerations upon which this formula is based
are correct, then the x values determined by the polariscope after various
times must give the same figure for k. This is indeed the case.1 k is
called the velocity coefficient (also velocity constant or specific reaction
velocity). If in the equation (1) C—x or the concentration of the still

dx

undecomposed cane-sugar =1, then the equation becomes -r — k. from

dt

which it follows that k indicates the reaction velocity if the concentra-
tion of the substrate could be kept the entire time at = 1 .

In these experiments k retains the same value. If in different experi-
ments the quantity of catalyst (acid) varies, then the obtained value
for k is proportional to the concentration of the H ions. This is so
prominent that the catalytic action of acids is due to the H ions (Ait-
RHENIUS 2) . Still irregularities occur as the anions of acids as well as
of salts present can under certain circumstances influence the action of
H ions (see page 70).

FRANKEL 3 has recently studied the decomposition of diazoacetic ether under
the influence of different acids. The reaction is as follows:

N2 : HC.CO.O.C2H6-hH20=HO.CH2.CO.O.C2H5+N2.

1 See Poggend. Ann., 81, 413 and 499 (1850).

2 Zeitschr. f. physik. Chem., 4, 226 (1889).
8 Ibid., 60, 202 (1907).

CATALYSIS.

35

The progress of the reaction can be determined by measuring the nitrogen
set free. The following figures explain the results:

Acid.

Cone, of the
Acid in Mol.
per Liter.

Cy.

Cone, of the
H ions by
Electric
Conductivity.

K
Velocity
Coefficient.

K
CH

Nitric acid

0 001820

0 001820

0 0703

38 7

Picric acid

0.000909
0 000909

0.000909
0 000909

0.0346
0 0356

38.0
39.2

w-Nitrobenzoic acid

0.000364
0 009900

0.000364
0 . 001680

0.0140
0.0632

38.3
37.7

Fumaric acid

0 003640

0.001460

0.0571

39.1

Succinic acid

0.009090

0.000724

0.0285

38.5

Acetic acid

0.018200

0.000563

0.0218

38.7

V

As 77— for the different acids and different quantities of acid is the same, then
the velocity coefficient is here also proportional to the concentration of the H ions.

As the catalytic action of acids is caused by the H ions, so are the
catalytic properties of bases due to the OH ions. The first determined
case of this kind was the transformation of hyoscyamine into the stable
a tr opine.1

KOELICHEN 2 has studied a specially pretty case of the catalytic action of
OH ions in the decomposition of diacetonalcohol into acetone :

CH3.CO.CH2.C(CH3)2.OH=2CH3.CO.CH3.

The reaction is reversible, and from the following table it is seen that the
velocity constant for various concentrations of the same catalyst remains the same
as well as by using different bases.

Catalyst. Cone, of the

Catalyst.

Piperidine 0. 1090

Triethylamine 0.4900

Ammonia 0 . 5500

Tetraethylammonium / 0 . 0760
hydroxide j 0.0076

Sodium hydroxide < ' ~

Velocity
Constant.

0.038
0.036
0.038
0.037
0.037
0.036
0.035

By this a rule which VAN'T HOFF and OSTWALD 3 proved by thenno-
dynamic means, is substantiated, namely, that the equilibrium at con-
stant temperature does not change with the quantity and kind of catalyst
when the catalyst is not changed by the reaction.

Among other kinds of ions which act as catalysts we must mention (1) iodine
ions, which decompose H202 in proportion to their concentration,4 and (2) cyan-

1 Ber. d. d. chem. Gesellsch., 21, 2777 (1888).

2 Zeitschr. f. physik. Chem., 33, 129 (1900).

3 Van't Hoff, Vorlesimgen, 1, 211.

4 Walton, Zeitschr. f . physik. Chem., 47, 185, 1904.

36 GENERAL AND PHYSICO-CHEMICAL.

ions, which transform benzaldehyde into benzoin according to the following
equation:

2C6H5.COH = C6H5.CO.CH(OH).C6H

If those bodies which accelerate a reaction are to be considered as catalysts,
then certainly the solvents must belong to the catalytes. Attention must be
called to the enormous influence which the solvent can exert upon the velocity
of a reaction under otherwise equal conditions. Thus MENSCHUTKIN 2 found for
the reaction

(C2H5)3.N+C2H6.I = (C2H6)4.N.I.,
the following velocity in different solvents:

Hexane 0.00018

Heptane 0 . 000235

Xylene 0.00287

Benzene 0.00584

Ethyl alcohol 0.03660

Benzyl alcohol f. .0. 13300

Recently BREDIG and FAJANS 3 have been able to show that an optically active
solvent can help in the decomposition of optical antipodes to a varying extent.
Of the optical antipodes 'of campho-carboxylic acid, the d-form is 17 per cent
more quickly decomposed than the /-form, when they are dissolved in nicotine
or when nicotine is present, dissolved with the catalyte, while in an optically
indifferent solvent and without any nicotine the catalyte decomposes both forms
with equal rapidity. The reaction proceeds differently with or without catalyst,
and hence the catalyst brings about changes in reaction other than those of velocity.
It is apparent that this does not conform with OSTWALD'S definition of a catalyst
(page 33). It must be mentioned that BREDIG and FISKE have been able to
perform the asymmetric synthesis of benzaldehyde and hydrocyanic acid by means
of quinine and quinidine as catalysts (page 60).

Catalysis in Heterogeneous Systems. The above-treated catalytic
processes all occur in homogenous systems, i.e., the systems which by
mechanical means cannot be separated into different constituents. In
heterogeneous systems with phases which can be separated from each
other by mechanical means, catalytic reactions can also occur, and
indeed, in such cases the substances taking part [in the reaction and
the catalyst occur in different phases. Such a reaction is the union of
detonating gas, the synthesis of SOs (from 862 and 0), and the decom-
position of H202 by platinum. In case the system is two-phased, and
the reaction takes place only at the boundary between both phases, or
in the one we can differentiate two simple limits:

1. The accumulation of the bodies which are necessary for the
reaction at the proper place takes such a short time that in comparison

1 Stern, ibid., 50, 513 (1905).

*/6w2., 6,41 (1890).

8 Ber. d. d. chem. Gesellsch., 41, 752 (1908).

ENZYMES. 37

with the real chemical reaction it can be neglected. In these cases
the reaction velocity behaves similarly to a homogeneous system.1

2. The chemical reaction occurs at a rate which in comparison with
the time necessary for the accumulation can be neglected. In this
case the time necessary can be generally compared with a diffusion
process.2

The catalytic processes in heterogeneous systems have excited
interest since BREDIG 3 showed that the colloidal metals prepared by
him showed catalytic properties. The best-studied process is the decom-
position of H202 by colloidal platinum, gold, and other metals or oxides
(MnC>2, PbO2). Attention must be called to the small quantity of
catalyst sufficient to decompose H202. The action of 1 gram atom
platinum in 70 million liters of reaction mixture has been detected. The
decomposition of H2C>2 by platinum catalyst in nearly neutral or faintly
acid solution has been shown to be a monomolecular reaction.

Still certain differences occur from the conditions formed in the
homogeneous catalysis. At one time in certain experiments the value
for k rises considerably during the catalysis, and secondly, k is not
proportional to the ferment concentration, but rises more quickly than
this.

In connection with these experiments BREDIG has expressed the
view that an analogy exists between the catalytic processes of the inor-
ganic world and the enzyme action of the organic.

The following important facts give support to BREDIG' s view:

1. In both cases we are dealing with catalytic processes; the metallic sol and
the enzyme are active in very small quantities and during the reaction they do
not undergo any appreciable change.

2. In the decomposition of H202 by platinum sols or by the enzyme haemase,
the reaction is monomolecular. .

3. The action of metallic sols as well as enzymes is paralyzed by certain
poisons (HCN, H2S).

4. Both classes of bodies are colloid substances and possess an enormous
surface upon which their catalytic action depends.

According to NEiLSON,4 ethyl butyrate, salicin and amygdalin are decom-
posed by platinum black as well as by enzymes.

IV. ENZYMES.

Chemical Processes in Plants and Animals. It follows from the law
of the conservation of matter and of energy that living beings, plants
and animals, can produce neither new matter nor new energy. They

1 Goldschmidt, Zeitschr. f. physik. Chem., 31, 235 (1899).

2 Nernst and Brunner, ibid., 47, 52 and 56 (1904).

3 Anorganische Fermente, Leipzig, 42 (1901).

4 Amer. Journ. of Physiol., 10, 191 (1904); 15, 148 (1906).

38 GENERAL AND PHYSICO-CHEMICAL.

are only called upon to appropriate and assimilate material already exist-
ing and to transform it into new forms of energy.

Out of a few relatively simple combinations, especially carbon
dioxide and water, together with ammonium compounds or nitrates,
and a few mineral substances, which serve as its food, the plant builds
up the extremely complicated constituents of its organism — proteins,
carbohydrates, fats, resins, organic acids, etc. The chemical work
which is performed in the plant must, therefore, in the majority of cases,
consist in syntheses; but besides these, processes of reduction take
place to a great extent. The radiant energy of the sunlight induces
the green parts of the plant to split off oxygen from the carbon dioxide
and water and this reduction is generally considered as the starting-
point in the syntheses that follow. According to a hypothesis suggested
by A. BAEYER,1 formaldehyde is first produced, CO2+H20 = CH2O+O2,
which by condensation is transformed into sugar. From the sugar other
bodies can then be built up.

With the aid of the silent electric discharge W. LOEB 2 has succeeded
in obtaining from carbon dioxide and water, formaldehyde, and as a
product of polymerization, also glycolaldehyde, CH2OH.CHO, from
which sugar can be readily produced. Still the conditions under which
these bodies were formed cannot be applied to the conditions in the
plants. The investigations of USHER and PRISTLEYS are of greater
interest in that they show the formation of formaldehyde in the photo-
lytic decomposition of moist carbonic acid in the presence of chloro-
phyll. These investigations also do not seem to be entirely free from
exception. The conception as to the formation of sugar from formalde-
hyde is also often different from that explained by v. BAEYER'S idea,
and his view as to the assimilation of carbonic acid constitutes a hypoth-
esis which requires further proof. The essentials of this hypothesis,
namely, a formation of formaldehyde with a subsequent sugar formation
by condensation of the aldehyde groups, is still very generally accepted
as probably correct. Independent of the ways and means of how the
assimilation processes in the plants originate, it is obvious that the free,
radiant energy of the sun is hereby bound and stored in a new form, as
chemical energy, in the combinations formed by the syntheses.

In animal life the conditions are not the same. Animals are depend-
ent either directly, as the herbivora, or indirectly, as the carnivora,
upon plant-life, from which they derive the three chief groups of organic
nutritive matter — proteins, carbohydrates, and fats. These bodies,
of which the protein substances and fats form the chief mass of the

1 Ber. d. d. chem. Gesellsch., 3.

1 Zeitschr. f. Electrochem., 12.

1 Proc. Roy. Soc. London, 78, Series B.

ENZYMES. 39

animal body, undergo within the animal organism a cleavage and oxi-
dation, and yield as final products exactly the above-mentioned chief
components in the nutrition of plants, namely, carbon dioxide, water,
and ammonia derivatives, which are rich in oxygen and have little energy.
The chemical energy, which is partly represented by the free oxygen
and partly stored up in the above-mentioned more complex chemical
compounds, is transformed into other forms of energy, principally heat
and mechanical work. While in the plant we find chiefly reduction
processes and syntheses, which by the introduction of energy from
without produce complex compounds having a greater content of energy,
we find in the animal body the reverse of this, namely, cleavage and oxi-
dation processes, which, as we used to state, convert chemical tension
into living force.

This difference between animals and plants must not be overrated,
nor must we consider that there exists a sharp boundary line between
the two. This is not the case. There are not only lower plants, free
from chlorophyll, which in regard to chemical processes represent inter-
mediate steps between higher plants and animals, but the difference
existing between the higher plants and animals is more of a quantitative
than of a qualitative kind. Plants require oxygen as peremptorily as
do animals. Like the animal, the plant also, in the dark and by means
of those parts which are free from chlorophyll, takes up oxygen and
eliminates carbon dioxide, while in the light the oxidation processes going
on in the green parts are overshadowed or hidden beneath the more intense
reduction processes. As in the animal, we also find a heat production
in fermentation produced by plant organisms; and even in a few of the
higher plants — as the aroideoe when bearing fruit — a considerable develop-
ment of heat has been observed. On the other hand, in the animal
organism, besides oxidation and splitting, reduction processes and syn-
theses also take place. The contrast which seemingly exists between
animals and plants consists merely in that in the animal organism the
processes of oxidation and splitting are predominant, while in the plant
chiefly those of reduction and synthesis have thus far been studied.

WOHLER 1 in 1824 was the first to observe an example of the SYN-
THETICAL PROCESSES within the animal organism. He showed that
when benzoic acid is introduced into the stomach, it reappears as hippuric
acid in the urine after combining with glycocoll (aminoacetic acid).
Since the discovery of this synthesis, which may be expressed by the
following equation :

Benzoic acid Glycocoll Hippuric acid

1 Berzelius, Lehrb. d. Chemie, ubersetzt von Wohler, 4, p. 356, Abt. 1, Dresden
(1831).

40 GENERAL AND PHYSICO-CHEMICAL.

and which is ordinarily considered as a type of an entire series of syn-
theses occurring in the body, where water is eliminated, the number of
known syntheses in the animal kingdom has increased considerably.
Many of these syntheses have also been artificially produced outside
of the organism, and numerous examples of animal syntheses of which
the course is absolutely clear will be found in the following pages. Besides
these well-studied syntheses, there also occur in the animal body similar
processes unquestionably of the greatest importance to animal life, but
of which we know nothing with positiveness. We enumerate as examples
of this kind of synthesis the re-formation of the red-blood pigment (the
haemoglobin), the formation of the different proteins from simpler sub-
stances, and the production of fat from carbohydrates. This last-
mentioned process, the formation of fat from carbohydrates, is also an
example of reduction processes which occur to a considerable extent in
the animal body.

Certain reactions, which are either not reproduceable with dead ma-
terial or are only possible under conditions which destroy the cells, belong
to the chemical decompositions going on within the living organism.
Thus the synthesis of glycogen or of protein has not been accomplished
outside of the organism or without the aid of agents prepared by the
cells. On the other hand proteins and starches can be split into simpler
products without these agents, but for this purpose the action of acids or
alkalies of a concentration which would kill the cells is necessary. In
certain cases it is possible to bring about such reactions outside of the
organism without any injurious effect upon the cells. This is accomplished
by the aid of substances which are formed within the cells but have the
power of being active after they have left the cells. These substances
have been called enzymes or ferments.

Enzymotic Processes. We must now mention a group of reactions
which are more or less related to enzyme action.

In the first place the so-called hydrolytic cleavage processes in which
complex substances are divided into simpler substances with the simul-
taneous decomposition of water and the taking up of its constituents.
These processes are of the greatest importance in the digestion of the
food-stuffs and for making them of value but they are also important for
the metabolic processes in general. As examples of such cleavages
we will mention the division of proteins into simpler products, the trans-
formation of starch into sugar and the cleavage of neutral fats into the
corresponding fatty acid and glycerin:

Tristearin Glycerin Stearic Acid.

The importance of the hydrolytic cleavage processes for digestion
will be discussed in detail in Chapter VIII.

ENZYMES. 41

Other cleavage processes are certain so-called fermentation processes,
which are connected with the presence of living organisms, fungi and
bacteria of various kinds. Among these we include chiefly the alcoholic
fermentation and butyric acid fermentation of carbohydrates. Accord-
ing to the view based upon PASTEUR'S investigations it has been gen-
erally considered that these processes are phases of the life of these
organisms and the name organized ferments or ferments have been given
to such organisms, especially to the ordinary yeast fungus.

A ferment, according to this view, is a living organism. By the name
enzyme, as introduced by KUHNE, we mean a product of the chemical
processes in the cells, which is active without the life of the cell and which
can be separated from the cell. The decomposition of invert sugar into
carbon dioxide and alcohol in fermentation is considered as a fermentative
process closely connected with the life of the yeast fungus. The inver-
sion of cane-sugar previous to fermentation is on the contrary, an enzymotlc
process which is brought about by a body or mixture of bodies which
are formed in the fungus and which can be removed from the fungus
and are still active after the death of the fungus. Consequently fer-
ments and enzymes are capable of manifesting a different behavior toward
certain chemical reagents. Thus there exist a number of substances,
among which we may mention arsenious acid, phenol, toluene, salicylic
acid, boracic acid, sodium fluoride, chloroform, ether, and protoplasmic
poisons, which in certain concentration kill ferments, or at least retard
their action, but which do not noticeably impair the action of the enzymes.

The above view as to the difference between ferments and enzymes
has lately been essentially shaken by the researches of E. BUCHNER :
and his pupils. He has been able to obtain from beer-yeast, by grind-
ing and strong pressure, a cell-fluid rich in protein, and which when intro-
duced into a solution of a fermentable sugar caused a violent fermenta-
tion. The objections raised from several sides that the fluid expressed
still contained dissolved living cell substance has been so successfully
answered by BUCHNER and his collaborators that there is at present
no question that alcoholic fermentation is caused by a special enzyme or
mixture of enzymes called zymase, which is formed in the yeast-cell.

As from the yeast-cells so also from other lower organisms, indeed

1 E. Buchner, Ber. d. deutsch. chem. Gesellsch., 30 and 31; E. Buchner and Rapp,
ibid., 31, 32, 34; H. Buchner, Stizungsber. d. Gesellsch. f. Morphol. u. Physiol. in
Miinchen, 13 (1897), part 1, which also contains the discussion on this topic. See also
E. and H. Buchner and M. Hahn, Die Zymasegarung, Munchen (1903); Stavenhagen,
Ber. d. deutsch. chem. Gesellsch., 30; Albert and Buchner, ibid., 33; Buchner, ibid.,
33; Albert, ibid., 33; Albert, Buchner, and Rapp, ibid., 35; in regard to the opposed
views see Macfadyen, Morris, and Rowland, ibid., 33; Wroblewski, Centralbl. f . Physiol.,
13, and Journ. f. prakt. Chem. (N. F.), 64.

42 GENERAL AND PHYSICO-CHEMICAL.

from the lactic-acid bacilli and beer vinegar bacteria, it is possible to
separate the specific fermentative principle of these organisms from the
living organism and to bring about changes with the dead organism
(E. BUCHNER, and MEISENHEIMER and GAUNT, HERZOG1). The ques-
tion whether there exist ferment processes which, in PASTEUR'S sense,
are the result of the biological phenomena connected with the metab-
olism of the micro-organism and which we can directly identify with the
life processes, is very difficult to answer; hence for the present we have
no foundation for a sharp differentiation between the organized ferments
and enzymes. The metabolic processes of the living organisms which
we recognize as fermentation phenomena must as a rule be ascribed to
enzymes acting within the cell. If such processes are closely connected
with the life of the cell, then this is explained in part by the fact that
this special enzyme is produced only by living cells and in part by the
fact that it cannot be separated from the living cells or that it is readily
destroyed on the death of the cell. The names enzyme and ferment are
now generally used in the same sense.

Formerly the view was generally accepted that ANIMAL OXIDATION
takes place in the fluids, while to-day we are of the opinion, derived
from the investigations of PFLUGER and his pupils,2 that it is connected
with the form-elements and the tissues. The question as to how this
oxidation in the form-elements is induced and how it proceeds cannot
be answered with certainty. On the other hand, it is accepted that the
living protoplasm in some manner or other takes part, in which case the
oxidation processes must cease with the life of the cells while Ion the
other hand it has been found that certain oxidative processes can be
brought about by means of catalyts in the ordinary sense as well as by
enzymotic substances. If in the latter case the oxygen of the air is
directly transported to the oxidizable substance then we call this a direct
oxidation. Ordinarily the oxidative processes take place in the follow-
ing way. First a peroxide is formed by taking up oxygen, like in
the formation of hydrogen peroxide, H-O-O-H, which then transfers
oxygen to the oxidizable substance by aid of the mentioned substances.
In these cases the oxidation is indirect. All these oxidative processes
will be treated in detail in Chapter XVI. At the same time also other
enzymes will be discussed which decompose hydrogen peroxide, with
the setting free of oxygen, without oxidizing at the same time. These

XE. Buchner and J. Meisenheimer, Ber. d. d. chem. Gesellsch., 36; 634 (1903);
and Annal. d. Chem. u. Pharm., 349; with Gaunt, ibid., 349; Herzog, Zeitschr. f.
physiol. Chem., 37.

2 Pfliiger, Pfliiger's 'Archiv, 6 and 10; Finkler, ibid., 10 and 14; Oertmann, ibid.,
14 and 15; Hoppe-Seyler, ibid., 7.

ENZYMES. 43

have been called catalases. Also reduction processes will be mentioned
which seem to be brought about by enzymes.

Besides these processes just mentioned the following processes,
namely, autolysis and putrefaction, are to be considered as due to enzyme
action entirely or in part.

If an animal organ is kept in water at 37° C. under conditions so
that no micro-organisms are active then the organ gradually dissolves
in great part under the influence of the contained enzymes. This
process is called autodigestion or autolysis. The action of micro-
organisms can be prevented either by removing the organ under strictly
aseptic conditions or by allowing the digestion to take place in the
presence of antiseptic substances (toluene, chloroform, etc.). As the
animal organs consist chiefly of protein substances the autolysis con-
sists chiefly in the action of enzymes which dissolve proteins. Autol-
ysis was first observed and studied by SALKOWSKI and his pupils
with liver, muscle and supra-renal capsule.1 JACOBY then showed
that the enzymes active in autolysis do not orginate in the digest-
ive tract and are not identical with trypsin or pepsin.2 BIONDI found
that hydrochloric acid had a favorable influence upon the autolysis
of the liver while HEDIN and ROWLAND 3 observed that the organic
acids accelerate the autolysis of nearly all organs. This has been sub-
stantiated by several authors (WIENER, AniNKiN4). The findings of
LANE-CLAYPTON and SCHRYVERS that the autolysis of the liver and
kidneys begins only after a latent period of from two to four hours
when the post mortem formation of acid is at its height, substantiates
the influence of the acid reaction.

The autolysis is retarded to a great extent by an alkaline reaction.
This is shown by the experiments of SCHWIENIG with the liver as well
as those of HEDIN and ROWLAND with several other organs. HEDIN
has also shown by experiments with various organs that a preliminary
treatment with acetic acid markedly helps the autolysis in alkaline reac-
tion, which for the spleen at least is explained by a destruction through
the treatment of acetic acid of a substance which has an inhibiting action
in alkaline solution. Such an inhibiting substance destroyed by acetic

1 Zeitschr. f . klin. Med., 1890, Suppl., Schwiening, Virchow's Arch., 136, 444 (1894),
Biondi, ibid, 144, 373 (1896).

2 A complete summary of the literature of intracellular enzymes and autolysis may
be found in Jacoby, Ueber die Bedeutung der intrazellularen Fermente, etc., Ergeb-
nisse der Physiologie, Jahrg. 1, Abt. 1, 1902.

3 Zeitschr. f. physiol. chem., 32, 341, 531 (1901).

4 Wiener, Centralbl. f. Physiol., 19, 349 (1905); Arinkin, Zeitschr. f. physiol. Chem.,
53, 192 (1907).

6 Journ. of Physiol., 31, 169 (1904).

44 GENERAL AND PHYSICO-CHEMICAL.

acid is also found in serum.1 The serum also inhibits the autolysis of
the liver (BAER, LONGCOPE and others)2 and also the thymus under
certain circumstances (RnoDiN3).

Experience has shown that the post-mortem autolytic process may
also be influenced by many other bodies and indeed in various ways.
For example, according to HESS and SAXL, arsenious acid exerts a
retarding action on the first stages of autolysis, while phosphorus accel-
erates it. IZAR as well as LAQUEUR and ETTINGER 4 obtained with small
quantities of different arsenic preparations an acceleration of the autol-
ysis and with larger amounts a retardation. LAQUEUR 5 obtained a
retardation with oxygen and an acceleration with carbon dioxide.
ASCOLI and IZARG have thoroughly investigated the action of inorganic
colloids upon autolysis. Radium rays as well as radium emanations
accelerate post-mortem autolysis of normal as well as carcinoma tissues

(WOHLGEMUTH, NEUBERG, LOWENTHAL and EOELSTEIN7).

The products of the activity of the different enzymes dissolving pro-
teins in autolysis have been studied by HEDIN and his collaborators, by
studying the action of organ press juices upon protein added, or upon
the protein contained in the juice. The same cleavage products were
found as in the deep-seated cleavage of proteins in the digestive canal. 8
Similar investigations have also been carried on by LEVENE and JONES 9
who chiefly considered the decomposition of the nuclein substances.
The combined action of various enzymes in autolysis also explains to us
why, as especially shown by LEVENE and by JONES, the products obtained
by the hydrolytic cleavage of an organ by means of an acid are some-
what different from those products produced on autolysis. In autol-
ysis we are not only dealing with the cleavage of the proteins, but other
enzymotic processes also occur such as the splitting of fats and carbo-
hydrates, the splitting off of NH2 groups from amino-acids, oxidations,
reductions and perhaps also syntheses.

1 Hammarsten's Festschr., 1906.

2Baer, Arch. f. expt. Path. u. Pharm., 56, 68 (1906); Longcope, Journ. med.
Research, 13, 45 (1908).

3 Zeitschr. f. physiol. Chem., 75, 197 (1911).

4 Hess and Saxl, Zeitschr. f. expt. Path. u. Therapie, 5 (1908); Izar, Bioch. Zeitschr.,
21, 46 (1909); Laqueur and Ettinger, Zeitschr. f. physiol. Chem., 79, 1 (1912).

5 Zeitschr. f. physiol. Chem., 79, 82 (1912).

6 Bioch. Zeitschr., 17, 361 (1909).

7Wohlgemuth, Berl. klin. Wochenschr., 26, 704; Neuberg, Zeitschr. f. Krebsfor-
schung, 2, 171 (1904); Lowenthal and Edelstein, Bioch. Zeitschr., 14, 484 (1908).

8Leathes, Journ. of Physiol., 28, 360 (1902); Dakin, ibid., 30, 84 (1904); Hedin,
ibid., 30, 155 (1904); Cathcart, ibid., 32, 299 (1905).

9-Levene, Amer. Jour, of Physiol., 10, 11, 12 (1904); Jones, Zeitschr. f. physiol.
Chem., 42, 35 (1904).

ENZYMES. 45

It is at present impossible to state what part autolytic processes
take in life under physiological conditions, and we can have only con-
jectures on this subject. In the autolysis of a removed organ or of one
through which the blood is not flowing, the conditions in many ways are
quite different from the conditions in life. The products which appear
after weeks or months of autolysis, sometimes in very small quantities,
do not give any clue to the nature of the vital processes, and conclusions
must be drawn very carefully from these results.

For the present it is impossible to judge of the importance of the
enzymes active in autolysis for physiological conditions, but this does
not exclude the possibility that in normal cell life the enzymes play a
very important role. Numerous observations show this to be true, and
we tend more and more toward the view that the chemical transforma-
tions in the living cells are brought about by enzymes, and that these
latter are to be considered as the chemical tools of the cells (HOFMEISTEB
and others.1).

From this standpoint the enzymes are of especial interest because
to-day it is the general belief that nearly all chemical processes of great
importance do not occur in the animal fluids, but on the contrary in the
cells, which are the real chemical workshops of the organism. It is also
chiefly the cells, which by their more or less active efficiency regulate
the extent of the chemical processes and thereby also the intensity of
the general metabolism. The following will be given as special examples
of the action of such enzymes under pathological conditions. The
changes of the liver and blood in acute phosphorus intoxication and
in acute yellow atrophy of the liver, where we find, in the urine, the
enzymotic decomposition products of the proteins.2 Another example
is the solution of pneumonic infiltrations by the enzymes of the migrated
and inclosed leucocytes as studied by FR. MULLER,S and this is at the same
time an example of heterolysis, i.e., of a solution or a destruction in an
organ by enzymes not belonging therein but introduced from without.
An autolysis, although not very marked, occurs in those organs or parts
of organs which have not been normally nourished because of a dis-
turbance in the circulation, and they are gradually consumed by this
action. The part injured undergoes solution, while the healthy part
remains unattacked. By this solvent action as well as by the forma-
tion of bactericidal bodies, as observed by CoNRADi,4 and of antitoxins

1 F. Hofmeister, Die chemische Organisation der Zelle, Braunschweig, 1901.

2 Jacoby, Zeitschr. f. physiol. Chem., 30, 174 (1900).

3 Verhandl. d. naturforsch. Gesellsch. zu Basel, 1901. See also O. Simon, Deutsch
Arch. f. klin. Med., 1901.

4 Hofmeister's Beitrage, 1.

46 GENERAL AND PHYSICO-CHEMICAL.

«

(BLUM l) by means of autolysis, we can consider this autolysis as a
remedy and perhaps also as a protective agent for the animal body.
In this connection the investigations of BILLARD 2 must be mentioned
where the autolytic fluid from the pig liver was strongly antitoxic
toward viper poison, cobra poison, tetanus toxin and also toward
cocaine, curare and strychnin.

As above stated, the chemical processes in animals and plants do
not stand in opposition to each other; they offer differences indeed,
but still they are of the same kind from a qualitative standpoint. PFLU-
GER believes that there exists a blood-relation between all living cells
of the animal and vegetable kingdoms, and that they originate from the
same root. The animal body is a complexity of cells, hence study of the
chemical processes must not only be made upon higher plants, but also
upon unicellular organisms in order that we get a proper explanation
of the chemical processes in the animal organism. Although a bio-
chemical study of the micro-organisms is very important, we must bear
in mind also the important role played by such organisms in animal
life, chiefly as exciters of disease; hence the study of the conditions of
life of these micro-organisms and the chemical investigation of the prod-
ucts produced by them must be of infinite importance.

If in the autolysis of animal tissues micro-organisms are added and
if no antiseptic is present which prevents their development, then they
increase abundantly because of the favorable conditions for development.
At the same time the enzymes are also formed to a great extent and by
whose aid the exchange of matter takes place in the bacteria. It follows
that many chemical processes occur depending upon the kind of bacteria
present and which are foreign to bacteria-free autolysis. The entire
process has been called putrefaction. Among the products formed we
will mention the sulphureted hydrogen, indol and skatol which chiefly
give the odor to putrefying proteins. In regard to other putrefactive prod-
ucts we refer to Chapter VIII. Under ordinary circumstances compounds
of a basic nature may also be produced by putrefaction. To this class
belong the cadaver alkaloids called ptomaines, first found by SELMI in
human cadavers and then specially studied by BRIEGER and GAUTIER.S
Certain of these are poisonous, designated as toxines, while others are
non-poisonous. They all belong to the aliphatic compounds and gen-
erally do not contain oxygen. As an example of these basic substances

lHofmeister's Beitrage, 5, p. 142.

2Cornpt. rend. Soc. Biol., 70, 623 (1911).

3 Selmi, Sulle ptomaine od alcaloidi cadaverici e loro importanza in tossicologia,
Bologna, 1878; Ber. d. d. chem. Gesellsch., 11, Correspond, by H. Schiff; Brieger,
Ueber Ptomaine, Parts 1, 2, and 3, Berlin, 1885-1886; A. Gautier, Traite" de chimie
ppliqu6e a la fhysiologie, 2, 1873, and Compt. rend., 94.

ENZYMES. 47

we must mention the two diamines, cadaverine or pentamethylenediamine,
C5Hi4N2, and putrescine or tetramethylenediamine, C4Hi2N2, which
have awakened special interest because they occur in the contents of the
intestine and in the urine in certain pathological conditions, especially
in cholera and cystinuria.1 The putrefaction bases martitine, CgHigNs,
putrine, CnH^e^Os, and viridinine, CsH^^Os, isolated by ACKER-
MANN, also belong to this group. Of special interest is the bacterial
poison isolated by FAUST,2 called sepsine, CsH 14^62, which is the sub-
stance producing the characteristic toxic action of putrefactive masses.
Sepsine was prepared by FAUST as a crystalline sulphate which, on repeated
evaporation of its solution, was readily converted into cadaverine sulphate.

Of especially great interest are the toxines which are found in the
higher plants and animals, like the jequirity-bean and castor-seed, in
the poison of snakes and spiders, in blood-serum, etc., and particularly
those produced by pathogenic micro-organisms have an unmistakable
relation to the enzymes. A closer study of these various bodies, lysines,
agglutinines, toxines, etc., as well as of the anti toxines and the theory
of immunity, does not lie within the scope of this work, but on account
of the great importance of the subject it will be briefly discussed on
page 66.

Classification of the Enzymes. If we exclude those processes which
are the result of several enzymotic reactions (i.e. autolysis, putrefaction)
then the most important enzymotic processes studied so far are the fol-
lowing :

1. Hydrolytic cleavage processes.

2. Cleavages of another variety (fermentation).

3. Oxidations.

We have no general chemical reaction in the ordinary sense which
is common to all enzymes or ferments and each enzyme is characterized
by its action and by the conditions under which this action is developed.
As the action of an enzyme upon a substance, or related substances, or
groups is limited therefore these substances or groups are called the
substrate of the enzyme.

In regard to the terminology it must be remarked that an enzyme
is often named after the substrate (amylase, protease, lipase); in other
cases the kind of action determines the name (oxidase, reductase) and
finally one of the products produced by its action forms the basis for
the name (alcoholase) .

1 See Brieger, Berlin, klin. Wochenschr., 1887; Baumann and Udransky, Zeitschr.
f. physiol. Chem., 13 and 15; Brieger and Stadthagen, Berlin, klin. Wochenschr., 1889.

2 Faust, Arch. f. exp. Path. u. Pharm., 51; Ackermann, Zeitschr. f. physiol. Chem.,
54 and 57.

48 GENERAL AND PHYSICO-CHEMICAL.

Of the above mentioned enzymotic reactions the hydrolytic cleavage
processes have been best studied, and the general properties of the
enzymes which will be given apply chiefly to the hydrolytically splitting
enzymes. Among these the following are to be mentioned especially:

1. Enzymes which split fats and other esters with the formation of
the corresponding alcohol and acid. They are called Upases or esterases.

2. Enzymes which split complex carbohydrates with the formation
of simpler ones. To these belong:

a. Disaccharide splitting enzymes for instance saccharase (invertase,
invertin), maltase, lactase which act upon the corresponding disaccharide
saccharose (cane-sugar) maltose and lactose (milk sugar);

b. Polysaccharide splitting enzymes such as amylase, ptyaUn. The
name diastase is often used to designate all the enzymes of this group.
In close relation to these enzymes stand the glucoside splitting enzymes
which occur especially in higher plants and the best known of which is
amygdalase (emulsiri) occurring in the almond.

3. Enzymes which act upon the proteins or their related cleavage
products. Of these we have :

a. Peptidases and erepsin which split polypeptides or peptones;

b. Proteases which act upon proteins as substrate (pepsin, trypsin,
autolytic enzymes).

Among the hydrolytic enzymes of the animal kingdom we also
include the arginase, which splits arginine into urea and ornithin and the
histozym, which splits hippuric acid. The two following groups also
belong here, namely, the nucleases which split nucleic acids and which
will be discussed in Chapter II, and the coagulating enzymes, rennin
and thrombin, which are probably active as proteases. The deamidizing
enzymes which split off the NH2 group from amino combinations are,
at least in certain cases, to be classed as hydrolytic enzymes. This is
for example the case with the adenase and guanase which splits off ammonia
from the two bodies adenine and guanine converting them into hypoxan-
thine and xanthine respectively. The urease which splits urea also belongs
to this group.

General Properties of the Enzymes. When possible we make use
of watery solutions of enzymes in experimentation. In case they are
insoluble in water (certain Upases) we use them in the form of more or
less purified powders or together with the tissue where they are formed.
We have no general method for preparing enzyme solutions. In certain
cases they are contained in secretions (gastric and pancreatic enzymes) ;
in others they are prepared from the cells by crushing and pressing out
the cell juice (zymase, organ enzymes), and finally, most enzymes can
be extracted from the cells with water or glycerin, and as this last gives
permanent solutions it has found great use as an extraction medium.

ENZYMES. 49

The aqueous solutions can be kept at low temperatures for a long time
after the addition of toluene or chloroform.

In all these cases the enzymes are obtained strongly contaminated
with other bodies, especially by proteins. Only in exceptional cases is
it possible to free the enzyme solution from protein so that the solution
does not give the ordinary protein reactions. This is true for the solu-
tion of saccharase obtained from yeast by treatment with water; if
this is shaken with kaolin the protein is adsorbed by the kaolin while the
solution contains the enzymes.1

No enzyme has thus far been obtained in a perfectly pure form,
and the chemical constitution as well as structure is therefore unknown.
The enzymes probably belong to the colloids; if they themselves are not
colloids, they occur at least with colloids, from which they may be sepa-
rated only with difficulty, if at all. The enzymes are characterized by
the fact that they are readily taken up by finely divided substances
(inorganic precipitates, carbon, kaolin, infusorial earth and other col-
loids such as alumina, iron hydroxide, proteins2). This process may
act selectively, as from a solution certain enzymes can be taken up and
others not at all, or only to a slight extent (HEDiN,3 MICHAELIS and
EnRENREiCH4). The adsorption process is more or less irreversible
and differs in this from the adsorption of crystalloid substances. Still
the trypsin and rennin adsorbed by charcoal can be to a slight extent
expelled from the charcoal by means of other adsorbable substances such
as casein and albumin (HEDIN) .5 Rennin taken up by charcoal can to
a very slight extent be set free by the addition of glucose (HEDIN) and
saccharase adsorbed by charcoal can be set free by cane-sugar (ERIKS-
SON) .6 As we will learn below, the adsorbed enzyme is inactive. The
so-called shaking inactivation of enzymes or the loss in activity of enzymes,
which occurs on shaking their solution seems to be due to an adsorp-
tion of the enzyme when it is either taken up by the precipitate
formed on shaking (ABDERHALDEN and GUGGENHEIM) or is concentrated
at the surface between the solution and the froth (S. AND S. SCHMIDT-
NIELSEN) .7 These latter found the inactivation of rennin by shaking
was regained if the froth was allowed to subside.

All enzymes lose their specific action on sufficiently heating their

1 Michaelis, Bioch. Zeitschr., 7, 488 (1907).

2 Dauwe, Hofmeister's Beitrage, 6, 426 (1905).

3 Bioch. Journ., 2, 112 (1907).

* Bioch. Zeitschr., 10, 283 (1908).

5 Bioch. Journ., 2, 81 (1906); Zeitschr. f. physiol. Chem., 63, 143 (1909).
6Hedin, ibid., 63, 143 (1909); Ericksson, ibid., 72, 313 (1911).
7 Abderhalden and Guggenheim, Zeitschr. f. physiol. Chem., 54, 352 (1907); S. and
S. Schmidt-Nielsen, ibid., 68, 317 (1910) which also contains the literature.

50 GENERAL AND PHYSICO-CHEMICAL.

aqueous solutions, and even at ordinary -temperature the enzymes are
gradually decomposed. In general the enzymes lose their activity by
heating for a short time to 70° C. MADSEN and WALBUM have followed
this process at different temperatures and found that the decomposition
of trypsin, pepsin and rennin at given temperatures proceeds mono-
molecularly, i.e., that the velocity of reaction at every moment is pro-
portional to the concentration of the enzyme (page 34) .l The readiness
with which an enzyme is decomposed is nevertheless to a great extent
dependent upon the presence of other bodies (page 54).

Certain enzymes are also sensitive to light. According to SCHMIDT-
NIELSEN 2 rennin is injured by light and in particular, by the ultra-violet
rays. The experiments of JODLBAUER and TAPPEINERS with invertin
have led to the same results; the visible rays can also in some cases
(peroxidase, hsemase) in the presence of oxygen or certain fluorescent
substances exert an injurious action.4

According to SCHMIDT-NIELSEN 5 the weakening in the rennin under
the influence of light proceeds like a monomolecular reaction.

Experiments on the cataphoresis of enzymes have been made by
BIERRY, HENRY and SCHAEFFER 6 as well as by MICHAELIS. . These
investigators found that saccharase migrates to the anode. MICHAELIS 7
found that the migration direction of other enzymes was dependent upon
the reaction, namely in faintly acid reaction they moved to the cathode
and in faintly alkaline reaction to the anode. Recently PEKELHARING
and W. E. RINGER 8 have observed that the migration direction of pig
pepsin was very materially influenced by the addition of small amounts
of proteoses. From what was previously stated (page 20) the saccharase
must therefore have a negative charge. As MiCHAELis,9 has on the other
hand, found that the saccharase can be adsorbed by the positively
charged aluminium hydrate and not by the negatively charged kaolin,
he concludes that the formation of adsorption compounds, at least in
certain cases, depends upon an opposed electric charge of the two com-
ponents.

1 Arrhenius, Immunochemie, Leipzig, 1907, 58.

* Hofmeister's Beitrage, 5,355(1904); 8,481(1906); Zeitschr. f. physiol. Chem., 58,
233 (1908).

8 Arch. f. klin. Med., 87, 373 (1906).

4 Bioch. Zeitschr., 8, 61 and 84 (1908). See also Agulhon, Compt. rend., 163, 979
(1911).

6 Zeitschr. f. physiol. Chem., 58, 232 (1908).

6 Compt. rend. soc. biol., 63, 226 (1907).

7 Bioch. Zeitschr., 16, 81, 486; 17, 231 (1909).

8 Zeitschr. f. physiol. Chem., 75, 282 (1911).

9 Bioch. Zeitchr., 10, 299 (1908).

ENZYMES. 51

Like the colloids the enzymes only diffuse very slowly and the dif-
fusion through membranes does not occur in most cases; only certain
membranes such as collodion tubes allow certain enzymes to pass
through. The collodion tubes can be impregnated in such a way with
lecithin or cholesterin that the diffusion is very slight. The same
applies to the filtration through collodion membranes (BIERRY and
ScHAEFFER).1 It must not be forgotten in such experiments that the
membrane can adsorb a considerable part of the enzyme (BECHHOLD).2

Just as it is difficult to prepare an enzyme free from non-enzymotic
contaminations, so also is it difficult to exclude the possibility that a
so-called enzyme is not a mixture of several related enzymes. In fact
the several enzymotic processes proceed step by step, and it is possible
that the various steps are caused by different enzymes. Thus the
decomposition of protein into amino-acids, with proteoses, peptones,
and polypeptides as intermediary products, may be the result of the
activity of several enzymes which are active one after another or are
parallel with one another in activity. Erepsin does not attack genuine
proteins, but completes the decomposition which has been begun by
other enzymes (pepsin, trypsin).

The enzymes are formed within the living cells. In certain cases
the cells do not secrete the complete enzyme, but substances which are
transformed first outside of the cells into active enzymes. These pre-
liminary steps or mother substances of the enzymes have been called
proenzymes or zymogens. These under certain conditions are changed
into enzymes and in certain cases this is brought about by the inter-
action of special but not well known substances which have been called
kinases (see Chapters V and VIII). In other cases the transformation
of the zymogen into the active enzyme is brought about by well defined
chemical substances. Thus the proenzymes of pepsin and of rennin are
activated by acids (see below on the retardation of enzyme action and
also Chapter VIII).

In certain other cases the presence of bodies which resist temperature
and are dialyzable and therefore not enzymes, are necessary or helpful
besides the real organic enzyme. Thus the presence of an acid is neces-
sary for the action of pepsin and hydrocyanic acid, according to MENDEL
and BLOOD,3 favors to a high degree the action of papain (a plant pro-
tease). R. MAGNUS4 has been able to separate by dialysis, from a solu-
tion of liver-lipase, a body which is necessary for the action upon amyl

1 Compt. rend. soc. biol., 62, 723 (1907).

2 Zeitschr. f. physik. Chem., 60, 257 (1907).
s Journ. of biol. Chem., 8, 177 (1910).

4 Zeitschr. f. physiol. Chem., 42, 149 (1904).

52 GENERAL AND PHYSICO-CHEMICAL.

salicylate. Enzymes made inactive by dialysis can be activated again
by the addition of boiled enzyme or the concentrated dialysate. HARDEN
and YOUNG 1 on filtering yeast-press juice through earthenware filters
impregnated with gelatin, have found different constituents of the zymase
on the filter and in the filtrate. The true enzyme remains on the filter.
This alone is inactive, but becomes active when the other part which
has passed through the filter, and which is dialyzable and resistant to
temperature, is added. This part is consumed during fermentation
and therefore the enzyme becomes inactive. After the addition of, best,
boiled press-juice to this the fermentation begins again (see also Chapter
III). Certain of the just-mentioned substances which are resistant
to heat, whose presence are necessary for the action of certain enzymes,
are ordinarily called co-enzymes. As they are not to be classified with
the enzymes, they are more correctly called activators, as suggested by
EuLEE.2 Their action is probably different in different cases, and
differs also from the activating action of the kinases.

Many enzymes are secreted by the cells as such or as proenzymes.
They act outside of the cells in which they were formed, or they act after
having been transformed into the enzyme, and hence are called secre-
tion enzymes or extracellular enzymes. Besides these extracellular
enzymes we also have another group which acts within the cells, hence
are intracellular and therefore are called intracellular enzymes or endo-
enzymes. To this group belongs, beside the yeast zymase, numerous
enzymes such as oxidases and hydrolytic enzymes.

Formation and Secretion of Enzymes. The investigations of PAWLOW 3
and his pupils upon the formation and secretion of the enzymes active
in the alimentary tract are very important. According to these investiga-
tions the amount of secretion of the glands and the behavior of the enzymes
contained in the secretion are dependent upon the amount and com-
position of the food taken and in such a manner that the kinds and
amounts of enzymes are appropriate for the digestion of the food-
stuffs (see Chapter VIII). Similar results were also obtained by WEIN-
LAND4 who found that the pancreas does not normally contain any
lactase but did contain this enzyme after feeding the animal with
milk or milk sugar. This has been substantiated by BAiNBRiDGE.5
Analogous experiments have been made with salivary ptyalin by NEIL-

. Physiol. Soc., 32 (1904); Proc. Chem. Soc., 21, 189 (1905); Proc. Roy.
Soc., 77 (ser. B), 405 (1906); ibid., 78, 369 (1906).
2Zeitschr. f. physiol. Chem., 57, 92 (1908).

3 Arbeit der Verdauimgsdrusen, Wiesbaden, 1898, s. 51.

4 Zeitschr. f. Biol., 38, 607 (1899); 40, 386 (1900).
6 Journ. of Physiol., 31, 98 (1904).

ENZYMES. 53

SON and LEWIS 1 with the same results. On the other hand the cor-
rectness of these observations is disputed by BiERRY,2 PLIMMER,S WOHL-
GEMUTH 4 and POPIELSKI 5 as they could not find any accommodation.
MENDEL6 and his co-workers by careful investigations on certain enzymes
obtained from embryonal intestine and other embryonal tissues could
not find any marked difference between these enzymes and the enzymes
of the full grown animal. These results speak against the accepted influence
of the food and of the processes depending upon the taking up of food,
upon the formation of enzymes. Recently the investigations of LON-
DON 7 and his collaborators upon the influence of the food upon the
digestion juices have shown that the amount of juice secreted is dependent
upon the constitution of the food but not the ferment content of the
same. The observations of COHNHEIM 8 also speak against the view
that the kind and quantity of enzymes secreted in the intestinal tract
accommodate themselves to the digestion, as he found that the organism
secretes as much fluid (gastric juice, pancreatic juice and bile) when
already digested food is introduced into the stomach as when undigested
food is introduced. ARRHENITJS 9 has calculated from LONDON'S figures,
that the total amount of digestive juice secreted was proportional to the
quantity of food-stuffs. From experiments which EULER and his
collaborators have made upon the formation of inverting enzymes he
concludes that we have inverting enzymes whose formation is specific
by getting accustomed to the substrate, while the formation of others
is in no wise thus influenced.10

•

In this connection we will call -attention to the appearance of enzy-
motic substances in the blood after the subcutaneous or intravenous
(parenteral) introduction of certain food-stuffs. WEINLAND first showed
that the parenteral introduction of cane-sugar caused the appearance
in the serum of a cane-sugar splitting enzyme.11 ABDERHALDEN and
KAPFBERGER 12 have substantiated and developed these observations.
Bodies having a similar action also appear after the injection of milk

I Journ. of Biol. Chem., 4, 501 (1908).
2Compt. rend. soc. biol., 58, 701 (1905).

3 Journ. of PhysioL, 34, 93 (1906).

4 Bioch. Zeitschr., 9, 1 (1908).

5 Pfliiger's Arch., 127, 443 (1909).

6 Amer. Journ. of PhysioL, 20, 81, 97 (1907); 21, 64, 69, 85, 95 (1908).
' Zeitschr. f. physiol. Chem., 68, 366 (1910).

8/6id, 84, 419 (1913).

9 Ibid., 63, 323 (1909), see also London, ibid., 65, 189 (1910).

">Ibid., 70, 279; 76, 388; 78, 246; 79, 274; 80, 241 (1912).

II Zeitschr. f. Biol., 47, 279 (1905).

12 Zeitschr. f. physiol. Chem., 69, 23 (1910).

54 GENERAL AND PHYSICO-CHEMICAL.

sugar and of starch. ABDERHALDEN and his co-workers have shown
that the parenteral introduction of protein or peptone gives the blood
serum of the animal the power of splitting proteins, which power is
destroyed on heating to 60-65° C.1 The introduction of very large quan-
tities of sugar or proteins per os (over feeding) has the same effect as
the parenteral introduction. ABDERHALDEN considers the active sub-
stances thus obtained as enzymes. The question is still undecided whether
the substances introduced bring about a formation of the enzymes or
whether they only transport the already formed enzymes to the blood.

Heat Production. The question whether in the hydrolytic processes
with the aid of enzymes heat is given off or taken up has been attacked
in two different ways. GRAFE 2 could not find either any setting free or
taking up of heat in the digestion of protein in a RUBNER calorimeter.
On the other hand HARI 3 by determining the calorific values of albumin
before and after digestion came to about the same results. If we exclude
the work developed in the process then it follows that the energy supply
of the organism is not perceptibly changed by the hydrolytic cleavages
of the protein. The chief source of energy is to be sought in the oxida-
tion processes that follow the cleavages.

Modes of Action of the Enzymes. The enzymes do not suffer any
appreciable change during the reaction they perform, and insignificant
amounts of the enzyme are able to decompose relatively enormous amounts
of the substrate. For example, 1 part of saccharase can invert 100,000
parts of cane-sugar (O'SuLLiVAN and THOMPSON)4 and 1 part of rennin
can decompose more than 400,000 parts of casein (HAMMARSTEN) 5.
For these reasons the enzymes have for a long time been considered as
catalytic substances. Nevertheless the enzyme reactions always take
place in heterogeneous media where on one hand the enzyme exists as
colloid and on the other the substrate in many cases is a colloid (starch,
proteins). As above mentioned, the enzymotic decompositions are
often complicated by their taking place over several intermediary steps
to the final product. As indicated by several conditions, the enzymes
also, before they act upon the substrate, combine therewith in some way
or another. The fact that the action of an enzyme is dependent upon the
stereometric construction (page 61) of the substrate speaks essentially
for this view. The substrate also protects certain enzymes against destruc-

* Ibid., 61, 200; 62, 120, 243 (1909); 64, 100, 423, 426, 427; 66, 88; 69, 23 (1910);
71, 110, 367, 385 (1911). See also 77, 250 (1912).
2 Arch. f. Hygiene, 62, 216 (1907).
»Pfluger's Arch., 115, 11 (1906); 121, 459 (1908).
4 Journ. chem. Soc., 57, 926 (1890).
6 See Maly's Jahresber, 7.

ENZYMES. 55

live influences (heat, alkalies)1; According to this only that part of the
added enzyme which is combined with the substrate is active. In
judging of the rapidity of enzyme reactions the following must be con-
sidered:

1. The velocity with which the enzyme combines with the substrate.

2. The result of the division, i.e., how much of the added enzyme
is combined with the substrate.

3. The velocity of the chemical processes produced by the enzyme.

The velocity of the combination of the enzyme with the substrate (1) can at
least in many cases be ignored in consideration of the time necessary for the
chemical reaction (see page 37). This applies to those cases where the chemical
transformation in the presence of an excess of substrate at the beginning of the
processes remains the same in each successive time interval. The quantity of
enzyme combined with the substrate, does not, in these cases, increase with the
time, which would be the case if the time necessary for the combination is not in
comparison with that for the chemical reaction. Equal decomposition for equal
time at the beginning of the processes have been found for the following enzymes —
invertase,2 diastase,3 trypsin with casein,4 as substrate.

The second question, as to the division of the enzyme between different phases
we will discuss after we have spoken of the velocity of the real chemical reaction
(page 58) as well as the retardation of enzyme action (page 62).

In regard to the chemical reaction they proceed probably in a different
manner according to the kind of combination between the substrate and
the enzyme. In one case we can consider that the combination of the
enzyme with the substrate is of such a kind that both form a homogeneous
phase and that one serves as solvent for the other (page 27). In this
case the chemical reaction produced by the enzyme takes place in a
homogeneous medium. Secondly, we can consider the combination of
the substrate and enzyme as an adsorption combination (see page 27)
in which case the combination does not form a homogeneous phase and
the reaction differs more or less from one taking place in a homogeneous
system. Bearing this in mind it would be interesting to investigate
whether the facts found for enzymotic reactions correspond with
catalytic reactions in homogeneous media.

For these latter the following laws (see page 33) have been found:

1. When the quantity of catalyst remains constant, the reaction

1 O'Sullivan and Thompson, Journ. Chem. Soc., 57, 926 (1890); Bayliss and Starling,
Journ. of PhysioL, 30, 71 (1903); Hedin, ibid., 30, 173 (1903); 32, 474 (1905); Taylor,
Journ. of biol. Chem., 2, 90 (1906).

2 O'Sullivan and Thompson, Journ. Chem. Soc., 57, 926 (1890); Ducleau, Traite
de microbiologie II, 137; Brown, Trans. Chem. Soc., 81, 373 (1902); Armstrong,
Proc. Roy. Soc., 73, 500 (1904); Hudson, Journ. Amer. Chem. Soc., 30, 1160, 1564
(1908).

3 Brown and Gliddinning, Proc. Chem. Soc., 18, 43 (1902).
* Hedin, Journ. of Physiol., 32, 471 (1905).

56 GENERAL AND PHYSICO-CHEMICAL.

velocity for every moment is proportional to the concentration of the
body decomposed, which is shown by the velocity coefficient in the same
experiment being constant at different times.

2. The velocity coefficient or the reaction velocity with constant
concentration of substrate is proportional to the quantity of catalyst.

The first law has been shown for certain enzymes in case an excess
of enzyme is present, namely for saccharase,1 lactase 2 and trj^psin.3 It
was found that the decomposition in a certain time was proportional to
the substrate. In other cases the determination of the correctness of
the law was accomplished with difficulty. A part of the enzyme may
during an experiment be either destroyed or in other ways (combining
with the product) be put out of action; then reverse reactions may take
place (page 11) and finally in many cases our analytical methods are
incapable of obtaining comparative results for different decompositions,
as the reaction in many cases takes place step by step, or several reac-
tions occur at the same time.4 Only in a few cases with especially
simple reactions have constant values been found for the velocity
coefficient at the beginning, as long as the quantity of reaction product
was small and the active quantity of enzyme remained unchanged
according to the formula (see page II).5

Recently HUDSON 6 has found constant values for k for the entire
process of inversion of cane-sugar by saccharase in a faintly acid reac-
tion. The reason for the different results of earlier investigators 7 is
due, in part, according to HUDSON, to the fact that the multirotation
of the glucose formed was not considered by these experimenters before
the extent of inversion was determined polariscopically. In the cleavage
of salicin by emulsin HUDSON and PAINE 8 obtained constant values
for k in the entire process.

1 Brown, Proc. Chem. Soc., 18, 14 (1902).
* Armstrong, Proc. Roy. Soc., 73, 500 (1904).

3 Hedin, Journ. of Physiol., 32, 475 (1905).

4 Hedin, Zeitschr. f. physiol. Chem., 57, 468 (1908).

6Senter, Zeitschr. f. physik. Chem., 44, 257 (1903); Issajew, ibid., 42, 102; 44,
546; Euler, Hofmeister's Beitrage, 7, 1 (1906); Dietz, Zeitschr. f. physiol. Chem.,
52, 301 (1907); Taylor, Journ. of biol. Chem., 2, 93 (1906); Nicloux, Compt. rend.
soc. biol., 56, 840 (1904); Rona, Bioch. Zeitschr., 33, 413 (1911); 39, 21 (1912); Euler,
Zeitschr. f. physiol. Chem., 51, 213 (1907).

6 Journ. Amer. Chem. Soc., 30, 1160, 1564 (1908).

7 See Henri, Zeitschr. f. physik. Chem., 39, 194 (1901) also A. J. Brown, Trans.
Chem. Soc., 81, 373 (1902).

8 Journ. Amer. Chem. Soc., 31, 1242 (1909).

ENZYMES. 57

The second law for catalytic reactions which we have formulated,
that with constant quantities of substrate the reaction velocity is pro-
portional to the quantity of enzyme, has been shown in certain cases
where the substrate was in excess (practically constant quantity) namely
with kephir lactase,1 trypsin with casein as substrate.2 In the just-
mentioned monomolecular enzyme reactions the velocity coefficient in
a few cases was found proportional to the quantity of enzyme (catalase
from blood,3 erepsin with glycyl-glycine as substrate,4 pancreatic lipase 5)
and in others not (catalase from Boletus scaber, lipase from pig fat).6
It has been shown for several enzymotic reactions that with the same
substrate the same decomposition can be obtained if the time of action
varies in inverse proportion to the added quantity of enzyme. If p is
the quantity of enzyme and t the time of action, then the decomposition
is the same in all tests where p.t is the same figure. This rule has been
found true for the following enzymes: saccharase (O'SuLLiVAN and
THOMPSON as well as HUDSON 7), pepsin (SjOQViST8), rennin (especially
FuLD9), peptone-splitting enzyme (VERNON 10), fibrin ferment of snake
poison (MARTIN n), trypsin (HEDIN 12), pepsin, rennin, trypsin, pyocy-
aneus protease (MADSEN 13). On the action of trypsin upon casein this
law has been shown correct for different stages in the reaction.14 This
indicates that the progress of the entire reaction remains the same with
different quantities of enzyme, only that the time for the same decom-
position is inversely as the quantity of enzyme. As clearly shown by
HEDIN, this indicates that the velocity coefficient is proportional to the
quantity of enzyme which is called for by the second law. If we start
with the above-mentioned assumption that only that enzyme is active
which is combined, then it follows from the proportionality between the
velocity coefficient and the quantity of enzyme, that always the same
fraction of the enzyme is combined with the substrate, or that the divi-
sion of the enzyme remains independent of the quantity.

Armstrong, Proc. Roy. Soc., 73, 500 (1904).

2 Hedin, Journ. of PhysioL, 32, 471 (1905).

3 Senter, Zeitschr. f . physik. Chem., 44, 257 (1903).
4Euler, Zeitschr. f. physiol. Chem., 51, 213 (1907).

5 Kastle and Loevenhart, Amer. Chem. Journ., 24, 491 (1900).
6Euler, Hofmeister's Beitrage, 7, 1 (1906).

7 Trans. Chem. Soc., 57, 926, 1890; Journ. Amer. Chem. Soc., 30, 1160, 1564 (1908).

8 Skand. Arch. f. Physiol., 5, 358 (1895).

9 Hofmesietr's Beitrage, 2, 169 (1902).

10 Journ. of Physiol., 30, 334 (1903).

11 Ibid., 32, 207 (1905).

12 Ibid., 32, 468 (1905); 34, 370 (1906).

13 Arrhenius, Immunochemie, Leipzig, 1907, 46.
"Zeitschr. f. physio!. Chem., 57, 478 (1908).

58 GENERAL AND PHYSICO-CHEMICAL.

In determining the quantity of enzyme the so-called SCHUTZ'S rule plays
an important part. In its newest form this is, that the decomposition is pro-
portional to the square root of the quantity of enzyme and the time, or decom-
position =k\/pt where fc is a constant, p the quantity of enzyme and t the time
of the action. This was first _shown by SCHUTZ 1 for pepsin and also,
in this form, decomposition =k\p as the time (t) was constant. The form
decomposition = k \/pt was given by SCHUTZ, and HuppERT.2 According to
PAWLOW this rule also applies to trypsin digestion.3 SCHUTZ 7s rule is good for
a certain stage of digestion only and it indicates that the extent of the validity
must be very dependent upon the method used for the determination of the
decomposition as the different digestion products are determined by different
methods. It must also be remarked that within the entire domain where
SCHUTZ'S rule is applicable the same value for pt must correspond to the same
decomposition, and necessarily the above-discussed enzyme-time rule must also
be valid. SCHUTZ'S rule has also been proved for the action of gastric and pan-
creatic lipase.4 According to ARRHENIUS 5 the validity of the rule can be explained
by the assumption that the enzyme combines with the reaction products so that
the active mass of enzyme changes in inverse proportion to the auantity of
reaction products.

K Reversibility of Enzyme Action and Enzymotic Syntheses. Many
catalytic processes have been shown to be reversible, i.e., the same
catalyst can influence the reaction in different directions according to
the concentration of the substances present. Thus far we have only
spoken of enzymotic cleavages; according to the above it is to be expected
that synthetical processes can also be produced by enzymes.

The first example of such a reaction was given by CROFT-HILL .6
He treated a 40 per cent glucose solution with maltase at 30° C. for a
very long time and concluded from the change in rotation and reducing
power that some maltose was formed from the glucose. EMMERLING 7
showed afterward that a synthesis of maltose did not occur, but rather
an isomeric carbohydrate, isomaltose was formed, which is not split by
maltase. According to ARMSTRONG 8 emulsin splits isomaltose, but
not maltose, and therefore it can synthesize maltose from glucose. A
similar reaction had previously been shown by E. FISCHER and ARM-
STRONG,9 that kefir-lactase produced isolactose and not lactose from
galactose and glucose. According to CREMER 10 yeast-press juice has
the power of forming glycogen from glucose or fructose.

1 Zeitschr. f. physiol. Chem., 9, 577 (1885).
'Pfliiger's Arch., 80, 470 (1900).

8 Arbeit der Verdauungsdrusen, Wiesbaden, 1898, 33.

4 Stade, Hofmeister's Beitrage, 3, 318 (1903); Engel, ibid., 7, 77 (1906), see Fromme,
ibid., 7, 77, (1906).

6 Immunochemie, 1907, 43.

6 Journ. of chem. Soc., 73, 634 (1898).

7 Ber. d. d. chem. Gesellsch., 34, 600 and 2207 (1901). .
"Proc. Roy. Soc. (ser. B), 76, 592, (1905)

9 Ber. d. d. chem. Gesellsch., 35, 3151, (1902).

., 32, 2062(1899).

ENZYMES. 59

A. DANILEWSKI first made the observation that concentrated solutions
of peptic cleavage products of protein substances separates an insol-
uble substance under the influence of rennin. This phenomenon has
since been observed by various investigators and the precipitate has
been called plastein by SAWJALOW l and coagulose by LAWROW.2 The same
phenomenon is also obtained by other proteolytic enzymes.3 The plas-
teins are considered by various investigators as synthetically formed
protein. The best proof for this view has been given by HENRIQUES
and GJALBACK. They show with the formol titration (see Chapter II)
that the nitrogen titratable by formol diminishes in the reaction and
they also found that the nitrogen precipitatable by tannic acid was
increased in the plastein formation. In a later work these authors
find that peptic cleavage products from proteins show a plastein for-
mation when under the influence of pepsin-hydrochloric acid in con-
centrated solution while in dilute solution the cleavage goes further and
they conclude from this that the process is reversible. Even protein
which has been partly split by acid or alkali shows a plastein formation
with pepsin-hydrochloric acid.4

The behavior of amygdalin and its cleavage products with enzymes
requires special mention. The cleavage takes place step by step as
follows :

. (1)

Amygdalin Mandelic acid nitrileglucoaide Glucose

. (2)

Mandelic acid nitrileglucoside Mandelic acid nitrile Glucose

..... (3)

Mandelic acid nitrile Benzaldehyde Hydrocyanic acid

The entire process with the formation of the end products sugar,
benzaldehyde and hydrocyanic acid takes place under the influence of
emulsin from almonds. The first part of the process can be especially
brought about by the influence of yeast (FISCHER) 5 and the second and
third parts under the influence of prunase from the leaves of Pruneae.6 Of

iZeitschr. f. physiol. Chem., 54, 119 (1907).

*Ibid., 61, 1; 53, 1 (1907); 56, 343 (1908); 60, 520 (1909). v

3 Kurajeff, Hofmeister's Beitrage, 4, 476 (1904); Niirnberg, ibid., 4, 543 (1904).

4 Zeitschr. f. physiol. Chem., 71, 485 (1911); 81, 439 (1912).

5 Ber. d. d. chem. Gesellsch., 28, 1508 (1896).

«H. E. Armstrong, E. F. Armstrong and Horton, Proc. Roy. Soc., 85, 359, 363,
370 (1912).

60 GENERAL AND PHYSICO-CHEMICAL.

the three above given reactions 1 and 3 can be reversed by enzymes and
indeed 1 even by using yeast (EMMERLiNG)1 and 3 with emulsin (ROSEN-
THALER 2). In the last instance the reaction is asymmetric in that
the d-form of the mandelic acid nitrile is formed. The asymmetric C
atom is marked in the above formula. Subsequently ROSENTHALER was
able to divide the emulsin into a splitting component (5-emulsin) and
a synthetical form (o--emulsin)3. In connection with the views on the
structure and mode of action of enzymes it is of special interest that
recently BREDIG and FISKE 4 have been able to prepare the two optical
antipodes of mandelic acid nitrile from benzaldehyde and hydrocyanic
acid by means of optically active catalysts. By using quinine as
catalyst the dextro-rotatory nitrile was formed and by quinidine (iso-
meric with quinine but opposed in regard to rotation power) the laevo-
rotatory nitrile was formed. This indicates that possibly the enzymes
also have an asymmetric structure. The synthetic formation of gluco-
sides by the aid of emulsin has also been performed by VAN'T HoFF.5

An undoubted synthesis of fat and other ester-like combinations
of fatty acids is also known. KASTLE and LOEVENHARTG have shown
the formation of ethyl butyrate from ethyl alcohol and butyric acid
under the influence of a pancreas enzyme. In an analogous manner
HANRiOT7 obtained monobutyrin from butyric acid and glycerin with
blood serum. POTTEVIN 8 by means of a pancreas enzyme transformed
oleic acid and glycerin into mono- and triolein as well as oleic acid esters
with monatomic alcohols. The synthetical action of the pancreas has
been closely studied by DiETZ.9

The enzyme used by DIETZ was insoluble in water, and its action was tested
with t-amyl alcohol and w-butyric acid or the corresponding ester. It was shown
that the reaction took place in the insoluble phase (enzyme). From the formula
alcohol +acid<=±ester -f- water, it follows that when the molecular concentrations
of alcohol, acid, ester and water are designated CA, Cs, CE, Cw, the reaction velocity

of the ester formation for a homogeneous system is -T^ki.CA.Cs—kz.CE.Cw (see

dx
page 32), which equation can be simplified to -^ =ki.Cs — kz.Cs as the alcohol

and water were in excess and their concentration considered as constant and
included in the constants ki and &2. At equilibrium we have kiCs=k2Cs or

1 Ber. d. d. chem. Gesellsch., 34, 3810 (1901).

2 Bioch. Zeitschr., 14, 238 (1908).
8 Ibid., 17,257 (1909).

4 Bioch. Zeitchr., 46, 7 (1912).

6 Sitzungsber. preuss. Akad. Wiss., 1909, s. 1065; 1910 s. 963.

• Amer. Chem. Journ., 24, 491 (1900).

7Compt. rend., 132, 212 (1901).

•/We*., 136, 1152 (1903), 138, 378 (1903); Ann. Inst. Past., 20, 901 (1906).

•Zeitschr. f. physiol., Chem., 52, 279 (1907).

ENZYMES. 61

p = -^ =K (pageB32). It follows that the same equilibrium is attained irrespective

JCz LS

of whether we start with alcohol-f acid, or ester +H20. The equilibrium is also
independent of the antecedents as well as the quantity of enzyme.

On comparing the equilibrium constants (K) which are obtained with dif-
ferent quantities of ester or acid, it is shown that in the above equation V€E
must be introduced instead of CE in order to obtain constant values for K. In
the saponification of the ester the reaction velocity is proportional to V(7#,
and not CE. According to DIETZ this is due to the fact that the system is a
heterogeneous one, and that only that part of the ester which is absorbed by
the solid phase (enzyme) takes part in the reaction. The velocity constant of
the ester formation is shown to be proportional to the quantity of enzyme.

According to what was stated above (page 35), the equilibrium in a reversible
reaction must be independent of the nature of the catalyst. This was not the
case in DIETZ'S experiments. With picric acid as the catalyst a different equilib-
rium was obtained than with the pancreas enzyme. With the acid as catalyst
the equilibrium was moved toward the direction of the ester. While this action
is not understood it may perhaps be explained by the fact that the system in one
case was homogeneous and in the other case heterogeneous.

Similar observations that the enzymotic end-condition can be different from
the stabile end-condition of the same system have previously been made by
TAMMANN,1 but in these cases generally so-called false equilibrium existed, which
for example, by the addition of more enzyme changed, so that the cleavage pro-
ceeds further. These false equilibria are generally caused by the enzyme being
destroyed or put out of action in other ways.

Among the enzymotic-ester syntheses we must also include) he or-
mation of carbohydrate phosphoric acid ester in fermenting sugar solu-
tions in the presence of soluble phosphates, as first observed by HARDEN
and YouNG.2 These will be discussed in detail in Chapter III.

It is seen that enzymotic syntheses are known. From this it fol-
lows that the questionable enzyme reactions are to be considered as
reversible. In certain cases another substance which cannot be split
by the enzyme is formed while in other cases the opposite direction of the
reaction can be detected by means of various constituents of the same
enzyme solution.

Specificity of Enzyme Action. It has been known for a long time
that a great difference exists in regard to the action of enzymes in the
sense that different enzymes act only upon certain classes of bodies (pro-
teins, carbohydrates, fats). Then there also exist differences in the
manner in which different enzymes of the same group influence different
members of the same class (maltase, lactase, saccharase). Finally, it is
possible for one enzyme to attack one of two optical antipodes and the
other not at all, or only to a slight degree. That optical antipodes
are burned with unequal facility in the organism was shown by E. FISCHER,
and that of the numerous aldohexoses only three, d-glucose, d-mannose

1 Zeitschr. f. physiol. Chem., 16, 271 (1892).

2 Proc. Roy. Soc. B, 1908 p. 209.

62 GENERAL AND PHYSICO-CHEMICAL.

and d-galactose, and of the ketohexoses only one, d-fructose are fer-
mentable; and then that the synthetically prepared stereoisomeric
glucosides behave differently with the enzymes. Thus of two isomeric
glucosides, one methyl-d-glucoside, the (a) was attacked by yeast and
the other (/3) only by emulsin, while the corresponding methyl-Z-glucosides
were not split by either of these enzymes. The corresponding glucoside
obtained from galactose behaves in a similar manner.1 On the behavior
of amygdalin to various enzymes see page 59. In connection with
these observations FISCHER presents the theory that for the action of
an enzyme a certain correspondence in stereometric structure of the
enzyme and substrate must exist; the enzyme must fit the substrate
somewhat like a key fitting a lock.

Then followed similar observations of DAKiN,2 who found that racemic
mandelic acid ester, on incomplete hydrolysis by liver press-juice, yielded
a strongly dextrorotatory acid, while the ester remaining was levorotatory.
The dextrorotatory ester was more quickly hydrolyzed than the levoro-
tatory ester. Finally, we must mention the investigations of FISCHER
and AsDERHALDEN3 on the cleavage of polypeptides by pancreas
press-juice. From abundant material they concluded that those polypep-
tides which consist entirely of the optical forms of amino-acids occurring
in nature are hydrolyzed and the others not. If in a racemic form besides
a polypeptide consisting of natural amino-acids, another occurs also,
then only the first is hydrolyzed. Besides this, other factors are also
of importance. Thus Z-leucyl-glycine is not hydrolyzed, although both
constituents occur in nature. The size of the molecule seems also to be
of importance, as mono-, di- and triglycyl-glycine are not split, while
tetraglycyl-glycine is. See also Chapter VIII.

Retardation of Enzyme Action. There are several reasons for the
assumption that the hydrolytic enzymes are only active after they have
combined with the substrate. From this it follows that those substances
which prevent the formation of such combination may cause the retarda-
tion of enzyme action. For this reason the enzyme action is retarded
by such substances which adsorb the enzyme (page 49). HEDiN4 has
made experiments on the retarding action of charcoal upon the action
of trypsin upon casein, and the action of rennin upon milk and it was
shown that the retardation was more pronounced if the powder and
enzyme were allowed to act upon each other before the substrate was
added than if this was present from the beginning. This fact indicates

1 Zeitschr. f. physiol. Chem., 26, 60 (1898) (collection of Fischer's works).

2 Journ. of Physiol., 30, 253 (1903); 32, 199 (1905).

3 Zeitschr. f. physiol. Chem., 46, 52 (1905); 61, 264 (1907).

*Bioch. Journ., 1, 484; 2, 81 (1906); Zeitschr. f. physiol. Chem., 50, 497 (1907);
60, 143 (1909). See also Jahnson-Blohm, ibid., 82, 178 (1912).

ENZYMES. 63

that the adsorption process is only reversible with great difficulty or
that the enzyme to a certain extent is fastened to the charcoal. That
the substrate influences the formation of adsorption combination is
shown by the fact that the substrate is also adsorbed by the charcoal.
A small part of the adsorbed enzyme can indeed be subsequently
displaced on the charcoal by other adsorbable substances and in this way
become active again. As various substrates are unequally adsorbed
by charcoal the retardation is, therefore, also different in degree. The
retardation of the saccharase action by charcoal is the same as for the
retardation of the trypsin or rennin action (ERIKSSON x).

The action of several enzymes is retarded by normal serum. This
was first observed by HAMMARSTEN and RODEN 2 for the action of rennin.
Besides this certain constituents of the serum as well as other protein
containing fluids have a retarding action and in many such cases the
order of the addition of the bodies is important. The retardation by
charcoal corresponds to this retardation in several ways and this has
led HEDIN 3 to the assumption that the retardation in both cases is brought
about by a colloidal reaction (adsorption) between the enzyme and a
solid or colloid phase. The facts correspond to this assumption namely
that during the action of the retarding substance upon the enzyme
the amount of water present is without importance for the final result
of retardation. Such a retardation by normal serum or fluids con-
taining protein has been observed in the following cases: retardation of
trypsin digestion of casein by native seralbumin,4 retardation of the action
of rennin by neutral serum and by white of egg,5 and the action of sac-
charase by serum.6 Besides this HEDIN 7 found a similar retardation by
seralbumin upon the digestion of casein by means of the a-protease of the
spleen. The retardation by normal serum or seralbumin has been shown
in the cases investigated not to be a specific kind, i.e., a given enzyme
is retarded about to the same extent regardless from what species of
animal it was prepared.

A specific retardation due to kind have been observed in the following
cases :

1. The antienzyme obtained by immunization (see page 66) retards
in those cases tested, only or chiefly the enzyme used in the imrnuniza-

., 72,313 (1911).
2 Upsala lakarefor. forh., 22, 546 (1887).

3Bioch. Journ., 1, 484 (1906); Zeitschr. f. physiol. Chem., 60, 364 (1909); Brgebn.
d. Physiol., 9, 433 (1910).

4 Journ. of Physiol., 32, 390 (1905); Bioch. Journ., 1, 474 (1906).

6 Zeitschr. f. physiol., 60, 85, 364; 63, 143 (1909).
e Ibid., 72,313 (1911).

7 Hammarsten's Festschr., 1906.

64 GENERAL AND PHYSICO-CHEMICAL.

tion. HILDEBRANDT 1 first produced an anti-enzyme toward emulsin;
and MORGENROTH 2 obtained in a similar manner an anti-rennin in goats'
serum; BORDET and GENGOU 3 immunized against fibrin ferment, SACHS' 4
against pepsin, SCHUTZE as well as BERTARELLI 5 against various
plant lipases, SCHUTZE 6 against lactase, PRETI as well as SCHUTZE and
BRAUN 7 against diastase, K. MEYER 8 against the proteases of bacillus
prodigiosus and bacillus pyocyaneus.

2. The retarding body of the rennin enzyme which was obtained
by treating a neutral infusion of the mucous membrane with dilute
ammonia and neutralizing, has been recently shown by HEDiN9 to
chiefly retard the enzyme of the same species (see Chapter VIII). In
these cases the importance of the order of treatment was also evident.

Most of the retarding substances contained in the serum lose their
retarding power on sufficiently heating them. This also occurs in cer-
tain cases by treatment with acid. Thus normal horse serum as
well as egg-white lose their ability to retard rennin by treatment
with very dilute hydrochloric acid and for this reason rennin which
has been inactivated by serum or egg-white can be set free again
by the use of hydrochloric acid (HEDIN) 10. Native seralbumin loses
its power of attaching itself to trypsin by treatment with dilute
acetic acid.

Certain proteins which are digested with difficulty retard the diges-
tion of more readily digestible ones without the order-phenomenon being
observed. In such cases the total digestion is probably diminished
because the more difficultly digested protein as substrate attracts a
part of the enzyme. As the order-phenomenon does not exist, the enzyme
is taken up in a complete and readily reversible manner (enzyme devia-
tion HEDiN).11 It is easily understood that the retardation must be
less effective than in those cases where the enzyme is attached to the
retarding substance. The tryptic digestion of casein in the presence
of seralbumin, treated with acid, is diminished by enzyme deviation as
well as the digestion of readily split proteins is retarded by egg-white

1 Virchow's Arch., 131, 33 (1893).

2Centralbl. f. Bakt., 26, 349 (1899); 27, 357 (1900).

3 Ann. inst. Past. 15, 129 (1901).

4 Fortschr. d. Med., 20, 593 (1901).

5 Deutsch. med. Wochenschr., 1904; Centralbl. f. Bakt., 40, 231 (1905).

6 Zeitschr. f. Hyg., 48, 457 (1904).

7Bioch. Zeitschr., 4, 6 (1907); Zeitschr. exp. Pathol. u. Therap., 6, 307 (1909).
«Bioch. Zeitschr., 32, 280 (1911).

• Zeitschr. f. physiol. Chem., 72, 187; 74, 242; 76, 355 (1911).
« Zeitschr. f. physiol. Chem., 60, 85, 364 (1909).
a., 52, 412(1907).

ENZYMES. 65

which is. difficult to digest (DELEZENNE and POZERSKI/
COMPEL and HENRI,3 HEDiN4).

At this time we must also mention the retarding action which the
proteolytic primary cleavage products (proteoses, peptones) exert upon
digestion. These products are further split; a part of the enzyme is
combined with the products and in this way prevented from dissolv-
ing new protein (HEDIN) .5 The retarding power of proteoses and pep-
tones upon rennin action is probably similar to the above.6

Finally, the end products of enzymotic activity i.e., bodies which
cannot be further split by the enzyme, have also a retarding action on
the enzyme action. That the inversion of cane-sugar is retarded by
invert sugar has been claimed by many (HENRI,7 A. J. BROWN,S BAREN-
DRECHT,9 ARMSTRONG10), and indeed BARENDRECHT claims that glucose
as well as fructose has a retarding action, and that galactose has an
even stronger retarding action than the direct cleavage products of cane-
sugar. H. E. and E. F. ARMSTONNGU found that saccharase, maltase and
lactase are retarded by just those varieties of sugar which are produced
by their activity. The accumulation of the amylolytic cleavage prod-
ucts have according to SH. LEA,12 a retarding action upon saliva.

The retarding action of amino-acids upon the decomposition of glycyl-
Z-tyrosine by yeast-press juice has recently been studied by ABDERHALDEN
and GiGON.13 They found that cleavage of peptides is retarded by
those optically active amino-acids which occur in the proteins. This
result is remarkable in consideration of the observations of FISCHER
and ABDERHALDEN that only those polypeptides were split by pancreatic
juice which are composed of natural optically active amino-acids (page 62).

The retardation of the action of papain by egg protein and by serum, which
is prevented by heating or action of hydrochloric acid, as shown by the investiga-
tions of DELEZENNE, MOUTON and POZERSKI as well as by JONESCU and SACHS 14
is a peculiar behavior.

. rend. soc. biol., 55, 935 (1603).

2 Journ. of Physiol., 31, 495 (1904).

3 Compt. rend. soc. biol., 58, 457 (1906).

4 Zeitschr. f. physiol. Chem., 52, 422 (1907).
* Zeitschr. f. physiol. Chem., 52, 422 (1907).

6 Ibid., 46, 307.

7 Zeitschr. f . physik. Chem., 39, 194 (1901).

8 Journ. Chem. Soc., 81, 382 (1902).

9 Zeitschr. f. physik. Chem., 49, 456 (1904).

10 Proc. Roy. Soc. (ser. B), 73, 516 (1904).

11 Ibid., 79, 360 (1907).

12 Journ. of Physiol., 1911.

13 Zeitschr. f. physiol. Chem., 53, 251 (1907).

14 Delezenne, Mouton and Pozerski, Compt. rend., 142; Jonescu, Bioch. Zeitschr.,
2; Sachs, Zeitschr. f. physiol. Chem., 51, 488 (1907).

66 GENERAL AND PHYSICO-CHEMICAL.

In consideration of what has been said (page 58) about enzymotic
syntheses it seems very possible in the retardation of enzymotic cleavages
by means of cleavage products that we are dealing with synthetic
processes where the cleavage products supply the material. This is espe-
cially shown by the above-mentioned investigations of ROSENTHALER
on emulsin that the retarding action of benzaldehyde or of hydrocyanic
acid upon emulsin action, as shown by TAMMANN/ is explainable by
syntheses. LicnwiTZ2 considers the interaction of the products as a
reversible paralyzation of the enzyme.

Appendix : Antigens and Anti-bodies. In connection with the retar-
dation of enzyme action we can also call attention to other similar proc-
esses. Under the name antigen we include those substances which,
when injected into animals, cause the formation of bodies in the organ-
ism with which they can in some way or another react. The process
is called immunization and the bodies formed are called anti-bodies or
in certain cases immune bodies. Generally these anti-bodies are specific
in the sense that they only react with the corresponding antigen. The
chemical constitution of the antigen as well as of the anti-body is not
known; they belong perhaps to the colloids, or at least they occur asso-
ciated with colloids.

The antigens are either substances soluble in water or occur as
constituents of the cells. We will first discuss the antigens soluble in
water.

To this group belong, in the first place, certain poisonous substances
of animal or plant origin (toxins), for example, snake poisons, bacterial
poisons, ricin (from the seeds of Ricinus communis), also enzymes as well
as certain proteins without special action. The reaction with the anti-
bodies (which are obtained in the blood serum of animals) manifests
itself with the poisons by the suppression of the poisonous action, with
the enzymes by retardation of the enzyme action, and with certain pro-
teins by formation of a precipitate which contains the antigen as well
as the anti-body. Anti-bodies of this last type are called precipitins.

The longest known (due to the epoch-making investigations of v.
BEHRiNG3) and best studied are those anti-bodies which are produced
by toxins and which neutralize the action of the toxins upon the animal
organism (antitoxins). According to the older view this takes place
by some sort of an action of the anti-body upon the cells sensitive to the
toxins. After it was shown that the toxins could also be neutralized
in vitro by the anti-bodies, it is now generally accepted that the neu-

1 Ibid., 16, 271 (1892).
*Ibid., 78, 128 (1912).
'Deutsch. med. Wochenschr., 1892; Zeitschr. f. Hygiene, 12 (1892).

ENZYMES. 67

tralization is brought about by some sort of a combination between the
toxin and the anti-body. The views are very contradictory in regard
to the nature of this combination and the manner in which it is formed.

The oldest theory, which has contributed much to our knowledge
of these conditions, is that of P. EHRLICH, whom we must thank for the
method of measuring the quantity of toxin by injection into an ani-
mal. The quantity of toxin which is just sufficient to kill a guinea-pig
of given weight in a certain time is selected as the unit. According to
the so-called side-chain theory of EHRLICH 1 the toxins firstly have a so-
called haptophore group, by means of which the toxin can attach itself
to a certain cell, and secondly, a so-called toxophore group, by which
the toxin exerts its poisonous action. The formation of anti-body
after the injection of the toxins EHRLICH explains by the fact that those
cells which are attacked by the toxins are supplied with so-called recep-
tors, which just fit the haptophore group of the toxins; the toxins are
thus anchored on the questionable cells and can then begin their action
by aid of the toxophore group. By the attachment of the receptors, the
cells are induced to produce new receptors, and indeed, so many recep-
tors are produced that they are thrown off and appear free in the blood
plasma. The receptors circulating in the blood are the anti-bodies.
As these are able to combine with the toxins they can protect against
the toxin those cells which are supplied with the same receptor under
whose influence they were found. The toxophore group of the toxins
can gradually be destroyed on keeping. A toxin so changed can be
continuously anchored to cell-receptors and in this way form anti-bodies,
but cannot produce any poisonous action. A toxin without toxophore
groups is called a toxoid by EHRLICH. It follows that the toxoids can
combine with the anti-bodies.

According to EHRLICH, on the neutralization of a toxin a chemical
combination takes place between the toxin and the anti-body, and so
much of this combination is formed that either the toxin or the anti-
body is completely consumed. Now the bacterial poisons are not
simple bodies, but mixtures of several poisons of different toxicity and
different avidity toward the anti-bodies. Generally the most poison-
ous is first neutralized, but it also occurs that a less poisonous or indeed
a non-poisonous body is first combined with the anti-body (proto-toxoids)
or that non-poisonous bodies are combined parallel with the true toxins
(syntoxoids) . The less poisonous or non-toxic bodies first combined
after the binding of the true toxins are called toxons (also epitoxoids).
According to the relative quantity and the avidity of the different con-
stituents of the toxic solution, the addition of a certain quantity of anti-
body can produce entirely different results.

1 See Michaelis, Die Bindungsgesetze von Toxin und Antitoxin, Berlin, 1905.

68 . GENERAL AND PHYSICO-CHEMICAL.

ARRHENIUS opposes EHRLICH'S theory that the combination between
toxin and anti-body is of a chemical nature, but claims, that their for-
mation does not proceed until one of the components has been used up.
An equilibrium is established between the free toxin and the free anti-
body on one side and the combination of the two on the other, which
the law of mass action requires according to the formula:

C . C = K . CN (page 32).

toxin anti-body toxin +anti-body

For tetanolysin (a substance obtained from tetanus cultures, which dissolves
red-blood corpuscles) and its anti-body, as well as for diphtheria toxin and the
corresponding anti-body, n=2 was found, i.e., in the combination of a molecule
of toxin with a molecule anti-body two molecules toxin-antitoxin combination
was formed.

The toxic action which a mixture of toxin and anti-body exerts
depends upon the quantity of toxin which, according to the above
formula, must always remain free.1 According to this theory the toxin
is a unit poison, as ARRHENIUS 2 now admits with EHRLICH, that the
poison is gradually transformed into a non-toxic or only slightly toxic
substance whichjhas the same ability to combine with antitoxin as the
toxin itself.

EHRLICH'S theory, as well as that of ARRHENIUS admits of a chem-
ical combination between the antigen and the anti-body. According
to EHRLICH besides this the substrate (or the cells sensitive to the anti-
gen) combines with the antigen, which is not conformable with the theory
of ARRHENIUS.

The combination toxin-anti-body is first gradually produced, and
then it is taken up from all sides so that the toxin is fastened to the
anti-body by a secondary process (exception, cobra poison). The com-
bination toxin-antitoxin is not reversible in the ordinary sense. This
is most easily shown by the fact that to- a certain limit more toxin is
neutralized according to the time allowed to elapse before the quantity
of toxin remaining free is determined by injection into an animal or in
other ways.3 In certain cases it is possible to obtain the toxin again in
an active form from the toxin-antitoxin combination, and indeed by
treatment with very dilute hydrochloric acid (MORGENROTH 4) . See
also page 64 on the setting free of rennin from its combination with
normal serum and with egg-white. HEDiN5 has also been able to obtain

1 Zeitschr. f. physik. Chem., 44, 7 (1903).

2 Immunochemie, Leipzig, 1907, 132.

3 Martin and Cherry, Proc. Roy. Soc., 1898, 420.

4]Berl. klin. Wochenschr., 1905, No. 5; Festschr. z. Eroffnung d. pathol. Instit.
Berlin, 1906; Virchow's Arch., 190, 371 (1907).
6 Zeitschr. f. physiol. Chem., 77, 229 (1912).

ENZYMES. 69

the rennin again in an active form, from the combination of the rennin
with anti-rennin obtained by immunization by treatment with hydro-
chloric acid and then neutralizing.

Recently a third manner of considering the toxin-antitoxin reaction
has been presented which is based on the fact that the reaction takes
place in a heterogeneous system. According to this the reaction is con-
sidered as an adsorption process, and in support of this assumption, sev-
eral examples can be given where finely divided solids or colloid sub-
stances take up toxins or enzymes, in an irreversible manner (NERNST,1
BiLTz,2 LANDSTEINER 3) .

In reference to the formed antigens we must call attention to the fol-
lowing :

If certain cells, for example, bacteria, blood-corpuscles, and sperma-
tozoa are injected into animals, then anti-bodies are formed which have
been called immune bodies (also amboceptors or sensibilizators) . By
themselves the immune bodies are inactive, but form with complements,
substances occurring in normal serum, so-called cytotoxins, which destroy
the kind of cells active in their formation. These cytotoxins are called
bacteriolysins, hamolysins, etc., according to the kind of cells used.
The immune bodies are specific in that they together with the com-
plement only attack those cells from which they are formed and
they are also stable against heat; the complements can act together
with different immune bodies and are very unstable, as they are gen-
erally destroyed by heating to 56° C. for one-half hour. Other anti-
bodies, produced under the influence of injected cells, show their action
by flocking together and agglutinating the cells set free in their forma-
tion. These anti-bodies are called agglutinins.

In regard to the immune bodies, EHRLICH believes that they com-
bine with those cells under whose influence they have been formed
and also with the complements. They serve to fasten (amboceptors)
the complement, which produces the real poisonous action, to the cells.
The immune bodies correspond therefore to the haptophore groups of
the toxins and the complements of the toxophores. According to
BORDET the immune bodies act upon the cells in the way that the latter
are sensitive toward the complements (sensibilizators).

If a certain immune serum is heated to 56° then, according to what
has been given, the complement is destroyed and the serum 'now con-
tains only the amboceptor of the original cyto-toxin and this amboceptor
can be made active again by the addition of normal serum (complement).

1 Zeitschr. f. Elektrochem., 10, 379 (1904).

2 Ber. d. d. Chem. Gesellsch., 37, 3147 (1904); Beitr. z. exp. Therapie, 1, 30 (1905).

3 Zeitschr. f. Chem. u. Ind. d. Roll, 3, 221 (1907); Bioch. Zeitschr., 15, 33 (1908).

70 GENERAL AND PHYSICO-CHEMICAL.

If, therefore, an antigen of the corresponding immune serum be heated
to 56° (amboceptor) and mixed with sufficient amount of normal serum
(complement), then the complement is bound up so that when subsequently
serum-free red-blood corpuscles and a certain quantity of immune serum,
obtained by immunization with these and after losing its complement
by heating to 56°, are added, no solution of the red-blood corpuscles
(haemolysis) takes place. If in the first mixture either the antigen or the
corresponding amboceptors are absent then the complement is not
combined and a haemolysis occurs because the complement cannot unite
with the haemolytic amboceptors added. In this manner it has been
attempted to determine the presence of an antigen or of amboceptors
which fit with the antigen (method of complement deviation).

The protective substances formed by immunization can protect the
organism against many fatal doses of the antigen and this protective
power can be brought about by the parenteral introduction of the immune
serum of another organism. The immunity is called active when the
organism obtains the antigen and itself produces the corresponding
protective substance. On the contrary the immunity is called passive
if the organism receives the anti-body formed in another living being
by active immunization.

During immunization under certain circumstances it is observed
that a condition of super-sensitiveness toward the antigen exists. This
super-sensitiveness occurs only toward the antigen used and is therefore
specific. The same has been observed in using the soluble as well as the
formed antigens. This mysterious phenomenon has been called anaphyl-
axis.

V. IONS AND SALT ACTION.

We have previously mentioned various processes which depend upon
the influence of ions. To these belong the precipitation of suspension
colloids by electrolytes as well as different catalytic processes. That
in the last case we are dealing with the action of ions is proven by the
fact that the velocity coefficient is proportional to the concentration
of a certain kind of ion. Nevertheless it has been shown, that the
velocity coefficient in the inversion of cane-sugar, by acid, is only propor-
tional to the H ions when dilute acids are used. With greater concen-
tration disturbances occur which can be ascribed to the action of the
negative ions of the acids. The catalytic processes can be influenced
by salts in a similar manner (salt action).

The enzyme action has shown itself proportional to the quantity of enzyme
in certain cases. EULER l has attempted to show a correspondence between ion-

^eitschr. f. physik. Chem., 36, 641 (1901).

IONS AND SALT ACTION. 71

action and enzyme action by the assumption that the enzymes cause an increase
in those ions, which could cause the reaction without the presence of the enzyme.
On the other hand J. LOEB x believes that the enzymes can also be electrolytically
dissociated and that their action depends on the amount of ions. Thus pepsin
is a weak base which forms a salt with the hydrochloric acid added and that this
salt is more strongly dissociated than the base ; for this reason the action of pepsin
is increased by acid.

Many enzymotic processes are influenced by the presence of salts
of the alkalies or alkaline earths. According to BIERRY, GIAJA and HENRI
as well as PRETI 2 pancreatic juice dialyzed for a long time has no action
upon starch, but becomes active again on adding NaCl or other salts.
According to WoHLGEMUTH3 the diastatic power of saliva is increased
ten-fold by the addition of NaCl. The anions are the active part in both
cases (see page 52 on co-enzymes). The strong retarding action which
NaFl exerts upon the enzymotic cleavage of esters is also to be men-
tioned (LOEVENHART and PIERCE, AMBERG and LoEVENHART4).

Other actions of salts are also ascribed to ion-action. To these
belong the experiments of DRESSER 5 according to which mercury salts,
which are relatively strongly dissociated, have a poisonous action upon
organic formations (yeast, frog heart), while potassium-mercury hypo-
sulphite was nearly non-toxic. As the last-mentioned salt contains
very few free Hg ions the posionous action of the first salt is ascribed to
the ions. PAUL and KRONIG 6 have arrived at similar results by inves-
tigating the poisonous action of mercury salts upon spores. They found
that K2Cy4Hg, which hardly contains any Hg ions, is much less poison-
ous than an equivalent solution of HgCy2- The same conditions were
observed by MAILLARD 7 for copper salts.

This leads us to the question as to the importance of water and the
mineral bodies, which are of just as great moment for the life of the
cells and their metabolism as the organic constituents. In regard to
the water this follows from the fact that the animal body consists of
about two-thirds water. If we also recall that water is of the
greatest importance for the normal physical condition of the tissues,
that the solution of numerous bodies and the dissociation of chemical
compounds, that all flow of juices, all exchange of material, all supply
of food, all growth or destruction and all removal of destructive prod-

1 Bioch. Zeitschr., 19, 534 (1909).

2Compt. rend. soc. biol., 60, 479 (1906); 62, 432, (1907); Bioch. Zeitschr., 4, 1
(1907); 40,357 (1912).

3 Bioch. Zeitschr., 9, 1 (1908).

4 Journ. of Biol. Chem., 2, 397 (1907); 4, 149 (1908).

5 Arch. exp. Pathol. u. Pharm., 32, 456 (1893).

6 Zeitschr. f. physik. Chem., 31, 411 (1896).
7Compt. rend. soc. biol., 50, 1210 (1898).

72 GENERAL AND PHYSICO-CHEMICAL.

ucts, are connected with the presence of water, and that besides this
the water by its evaporation is an important regulator of temperature,
it is evident that water must be a necessity of life.

The mineral substances found habitually in the cells of higher plants
and of animals are potassium, sodium, calcium, magnesium, iron, phos-
phoric acid, sulphuric add, chlorine, and perhaps also iodine (JUSTUS).1
Besides, in certain cells or organs we also find manganese, lithium, barium,
silicium, fluorine, bromine, and arsenic.

* We are chiefly indebted to LIEBIG for showing that the mineral
bodies are as important for the normal constitution of the organs and
tissues, as well as for the normal performance of the processes of life,
as the organic constituents of the body. v The importance of the mineral
constituents is evident from the fact that we know no animal tissue
and no animal fluid which is free from, mineral bodies, and also from
the fact that certain tissues or tissue" elements contain chiefly certain
mineral bodies and not others. In regard to the alkali compounds this
division is, in general, as follows: The sodium compounds occur chiefly
in the fluids, while the potassium compounds occur especially in the
form-elements. C6rresponding to this, the cells contain chiefly potas-
sium as phosphate, while they are less rich in sodium and chlorine com-
pounds. * The fundamental experiments of FoRSTER2 have shown us that
inorganic salts, as constituents of the food are necessary for the animal

organism^

We have already called attention to the importance for every organ-
ism of the salts for the production of a rather constant osmotic pressure.
That the importance of the salts is not limited to the maintenance
of the osmotic pressure follows from the fact that different salt solutions
of the same osmotic pressure are not of the same value for the main-
tenance of the functional powers on extirpated organs. Since S.
RINGER 3 showed that various organic structures retained their best
functional activity in a solution which contained NaCl, CaCk. and
KC1 at the same time, various investigators, have given the most suit-
able composition of such solutions. For the transfusion fluid for the
mammalian heart LOCKE 4 suggests the following composition; NaCl

1 Justus, Virchow's Arch., 170, 176 and 190. In regard to arsenic see the works
of Gautier, Compt. rend., 129, 130, 131, 139; Bertrand. ibid., 134; Segale, Zeitschr.
f. physiol. Chem., 42; Kunkel, ibid., 44. In regard to the barium see Schultze and
Thierfelder, Sitzungsber. d. Gesellsch. naturforsch. Freunde, 1905, No. 1, and in
regard to lithium see Hermann, Pflliger's Arch., 109; and in regard to manganese see
Bradley, Journ. of Biol. Chem., 3.

2 Zeitschr. f. Biol., 9, 297 (1873); 12, 464 (1877).

3 Journ. of Physiol., 6, 154, 361 (1885); 7,118(1886); 16, 1, 17, 23 (1895); 18,
425 (1896).

4 Centralbl. f. Physiol., 14, 672 (1900).

IONS AND SALT ACTION. 73

0.9-1 per cent, CaCl2 0.02-0.024 per cent, KC1 0.02-0.042 per cent,
NaHCO3 0.01-0.03 per cent. Each of the salts NaCl, GaCl2 and KC1
individually has a poisonous action upon the organ but this action is
counteracted by the presence of the two other salts (antagonistic salt
action).

This neutralizing action of salts has been studied during recent
years especially by J. LOEB and his collaborators. As general results
it has been found that the most favorable quantity relations of the
three salts NaCl, KC1 and CaCl2 for the maintenance of life is the same
as exists in blood.. Especially interesting are the experiments with the
Fundulus heteroclitus, a genus of killifish. This fish, it is remarkable, can
also live in distilled water and is therefore within wide limits, not depend-
ent upon the osmotic pressure of the surrounding medium. For this
reason it is specially suited for the study of the poisonous action of salts
or mixture of salts. KC1 in concentrations in which it exists in sea water
acts as a poison upon these fishes, if it is alone in solution. The same
is true for NaCl. On the contrary these fishes live for an indefinite
time in a pure CaCl2 solution in a concentration similar to sea water.
One mol. KC1 can be very nearly de-toxicated by 17 mol. NaCl or by 8J mol.
Na2S04. \ mol. K2S04 is just as poisonous as 1 mol. KC1. The toxicity
of the potassium salts is therefore dependent upon the K ions and the
de-toxicating substance on the Na ion. CaCl2 de-toxicates a KC1 solu-
tion even when ^ mol. CaCl2 to 1 mol. KC1 is present. SrCl2 shows
almost as great a de-toxicating action as CaCl2. NaCl in concentra-
tions, in which it occurs in sea-water can only be incompletely de-toxi-
cated by KC1; only by the addition of CaCl2 can the complete de-
toxication be brought about. The poisonous action of acids upon
Fundulus can be arrested by neutral salts.1 Fundulus can accommodate
themselves to a rise in temperature; a rise in temperature can be more
easily endured when the concentration of the surrounding medium is raised
at the same time (LOEB and WASTENEYS). Can fishes also accommodate
themselves to an abnormal concentration of the surroundings as long
as the rise in concentration takes place gradually? In both cases the
accommodation, according to LoEB,2 depends upon a slow proceeding
process, possibly a tanning of the surface of the animal.

The fertilized eggs of the Fundulus develop, according to LOEB, just
as well in water free from salt as in sea-water. If the fertilized eggs are
placed in a NaCl solution of the same osmotic pressure as the sea-water

iBioch. Zeichr., 31, 450; 32, 155, 308; 33, 480. 489 (1911); 39, 167; 43, 181

(1912).

2Loeb and Wasteneys, Joura. of exp. Zool., 18, 543 (1913) and Loeb, Bioch.

Zeitschr., 53, 391 (1913). ...... ..

74 GENERAL AND PHYSICO-CHEMICAL.

they die; the toxicity of the NaCl solution can be arrested by small
quantities of almost any salt with polyvalent cations. Not only the salts
of the alkaline earths, but also those of the heavy metals (for instance
zinc sulphate or lead acetate) can neutralize the toxicity of the NaCl in
proper concentration.1 The eggs can develop in solutions which kill the
completed fish.

Y The antagonistic action of salts upon organic structures depends,
according to LOEB, upon the fact that the salts mixed in proper propor-
tions causes a " tanning " of the protoplasmic surface of the cells whereby
the cells become impermeable for certain destructive substances to which
the salts also belong/ The fertilized eggs of Fundulus can be tanned
by NaCl -fa heavy metal but not the completed fish.2 Many observa-
tions indicate that the egg is more permeable after fertilization than
before.3

Appendix :\ Determination of the Reaction of a Solution. The
reaction of the solution, in which a chemical reaction takes place, plays
an important r61e in many cases. As the acid or alkaline reaction of a
solution depends upon the amount of H or OH ions it is often of import-
ance to be able to determine the concentration of these ions in solution.
These cannot be determined by titration with alkali or acid in the pres-
ence of organic salts. In this titration the existing equilibrium in the
solution is disturbed and therefore also other decompositions occur
besides the neutralization of H or OH ions. The quantity of alkali or
acid used does not therefore correspond to the original concentration
of H or OH ions.

According to the law of mass action there exists, between the H and
OH ions formed by the dissociation of the water on the one hand and
the concentration of the non-dissociated molecules on the other, the
following equation

where CH, COH represents the concentration of the H and OH ions,
CHZO the non-dissociated water molecules and KI a constant. As CH*O
can only be considered as constant in certain dilute solutions we have
CH-COH = K, where K is called the dissociation constant of the water.
As K is a constant it follows that the figures for CH and COH can be
calculated, if the other is known. As it is more convenient to determine
CH than COH> therefore CH is also ordinarily determined for solutions

1 Pfluger's Arch., 88, 68 (1901).

2 Science, 34, 653 (1911).

8Lillie, Amer. Journ. of Physiol., 27, 289 (1911); McClendon, ibid., 27, 240;
Science, 32, 122, 317; Lyon and Shackell, ibid., 32, 249 (1910).

IONS AND SALT ACTION. 75

with alkaline reaction. Complete investigations on this subject have
been carried out by SORENSEN.1 He found the value io~14>14 for K
at 18° C. CH is determined in either of two ways. The best method,
the electromotive, is based upon the electromotive force of gas chains,
as developed by NERNST.;2 namely, if platinum foil covered with
platinum black is introduced into a watery solution and this saturated
with hydrogen, then a difference of electrical potential is produced between
the platinum and the solution and this potential is theoretically propor-
tional to the concentration of the hydrogen ions in the solution. We
cannot give any further detail as to this theory or to the performance
of the measurement of the difference in potential.3 If the concentration
of the hydrogen ions CH is expressed in gram ions per liter by the figure
10~p, then according to the suggestion of SORENSON the name hydrogen
ion exponent and the symbol pn is used for the numerical value of the
exponents of this potence. The relationship between pH and the electro-
motive force TT at the contact between the platinum and the solution
can be expressed graphically by a straight line; hence it follows that if
TT is known then pH can be very easily found (the exponential line).

The other method used by SORENSEN 4 for the determination of CH
is a colorimetric method and depends on the use of indicators. After
much investigation 20 indicators are recommended, of which certain ones
require strictly fixed methods of use. As soon as more than a qual-
itative approximation is required then the shade of color produced by
the indicator must be compared with a shade of color produced by the
same indicator in a solution of known concentration of H ions. Such
standard solutions which allow of a variation in the concentration of the
H ions at one's pleasure have been given by SORENSEN, and the original
article gives a table of curve's from the corresponding value for p& which
can be read off, when the composition of a standard solution is known. The
figure PH for the standard solutions is determined by aid of the electro-
motive method. Standard solutions are selected so that they serve as
natural protectors against too sudden changes in pa (so called buffer)5.

As above stated the dissociation constant according to SORENSEN
for water is 10~1414 at 18° C. or CH- COH = 10~14-14. In neutral reac-
tion CH = COH and therefore CH = 10~7'07 or pn = 7.Q7. Smaller values
for pn correspond to acid and greater values to alkaline reaction.

HASSELBACH6 has suggested a modification of SORENSEN'S method

1 Bioch. Zeitschr., 21, 131 (1909) also Ergebn. d. Physiol. Vol. 11.

2 Zeitschr. f. physik. Chem., 4, 129 (1889).

3 In regard to the determination see the work of Sorensen cited on page 74.

4 Sorensen, Enzymstudien, Bioch. Zeitschr., 21, 253.
* Ibid., 167.

6 Bioch. Zeitschr., 30, 317 (1910).

76 GENERAL AND PHYSICO-CHEMICAL.

for the electrometric determination of reaction in fluids containing
carbon dioxide. By the aid of this method HASSELBACH and LUNDS-
GAARD l have made determinations of the reaction of the blood. From
these it follows that at a temperature of 38.5° C. where the value for
pH = 6.78 corresponds to the neutral reaction the figure obtained for PH
with defibrinated ox-blood was 7.36 showing therefore a slight alkaline
reaction. The influence of the respiratory variation in the CO2 tension
upon the H ion concentration of the blood is of a measurable size.
The total blood, has a greater H ion concentration than the serum at
equal CCb tension but less than the blood corpuscles. For human blood
saturated with C02 under 40 mm. tension at 38° C. LUNDSGAAKD 2
found pH = 7.19. MICHAELIS and DAVIDOFFS found the average values
of normal venous blood for pn = 7.35 at 37.5° C.

1 Biochem. Zeitschr., 38, 77 (1911).
2/fod., 41, 2641(1912).
*Ibid., 46, 131 (1912).

CHAPTER II.
THE PROTEIN SUBSTANCES.

THE chief mass of the organic constituents of animal tissues consists
of amorphous nitrogenized, very complex bodies of high molecular weight.
These bodies, which are either proteins in a special sense or bodies nearly
related thereto, take first rank among the organic constituents of the
animal body on account of their great abundance. For this reason they
are classed together in a special group which has received the name
protein group (from Trpcorcvw, I am the first, or take the first . (place) .
The bodies belonging to these several groups are called protein sub-
stances, although in a few cases the protein bodies in a special sense are
designated by the same name.

The several protein substances 1 contain carbon, hydrogen, nitrogen, and
oxygen. The majority contain also sulphur, a few phosphorus, and a
few also iron. Copper, chlorine, iodine, and bromine have been found
in some few cases. On heating the protein substances they gradually
decompose, producing a strong odor of burned horn or wool. At the same
time they produce inflammable gases, water, carbon dioxide, ammonia,
and nitrogenized bases, besides many other substances, and leave a large
quantity of carbon. On deep hydrolytic cleavage they yield abundance
of a-monamino-acids of various kinds as decomposition products.

The nitrogen occurs in the protein bodies in various forms, and this
is also revealed in the division of the nitrogen among the cleavage prod-
ucts. On boiling with dilute mineral acids we obtain (1) so-called amide
nitrogen, which is readily split off as ammonia; (2) a guanidine residue
which is combined with diaminovaleric acid as arginine, and which
has also been called the urea-forming group; (3) basic nitrogen or diamino-
acid nitrogen, or hexone bases nitrogen, which is precipitated by phos-
photungstic acid as basic products (to which also the guanidine residue
of arginine belongs); (4) monamino-acid nitrogen; and (5) the nitrogen

1 See " Eiweisskorper," Ladenburg's Handworterbuch der Chemie, 3, 534-589,
which gives a complete summary of the literature of protein substances up to 1885.
The more recent literature may be found in O. Cohnheim, Chemie der Eiweisskorper,
Braunschweig, 1911. See also Oppenheimer's Handbuch der Biochem. der Menschen
und der Tiere, 1908.

77

78 THE PROTEIN SUBSTANCES.

in variable amounts which appears as humus-like melanoidins, which
seem to be of only secondary formation as products of elaboration.

The quantitative division of the total nitrogen between the above
five groups is different in the various protein substances, and more-
over cannot be given with certainty, because of the above-mentioned
melanoidin formation and the errors in the methods used.1 The follow-
ing gives at least an approximate idea of this division.2 The loosely
combined so-called amide nitrogen seems to be entirely absent in the
protamines. In the gelatins we find 1-2 per cent, and 5-10 per cent
in other animal protein substances,3 in certain plant proteins, the
prolamines (see page 108), 13-25 per cent of the total nitrogen is amide
nitrogen. The guanidine nitrogen may amount in the protamines to
22-44 per cent of the total nitrogen, in the histones to 12-13 per cent,
in the gelatins about 8 per cent, and in the other protein bodies about
2-5 per cent. As basic nitrogen precipitable by phosphotungstic acid
(including the guanidine residue) we find 35-88 per cent in the protamines,
35-42.5 per cent in the histones, 15-30 per cent in the other animal pro-
tein substances. In the prolamines 3-6 per cent of the total nitrogen is
found as products precipitable by phosphotungstic acid but in plant
globulin (globulin of the wheat) indeed 37 per cent. The chief quantity
of the nitrogen, 55-76 per cent, occurs, with the exception of the pro-
tamines, as the monamino-acid groups. The results for the melanoidin
nitrogen vary so considerably that they will not be mentioned.

Recently D. v. SLYKE 4 has perfected a method which is based upon the
deamidation of the amino-acids by HN02 (see below) and which allows of a still
more detailed differentiation of the nitrogen partition. In this method the
nitrogen of the ammonia, the melanines, the cystine, arginine, histidine, proline and
oxyproline besides one-half of the tryphtophane nitrogen as well as the nitrogen
of the remaining amino-acids can be specially determined.

From recent as well as older observations it follows as chief result
that the nitrogen in the proteins occurs in such combinations so that
on hydrolysis with acids, its chief amount splits off in the form of amino-
acids.

1 See the work of Hausmann, Zeitschr. f. physiol. Chem., 27 and 29; Henderson,
ibid., 27; Kossel and Kutscher, ibid., 30; Kutscher, ibid., 31, 38; Hart, ibid., 33;
Giimbel, Hofmeister's Beitrage, 5; Rothera, ibid.

2 See the works given in footnote 1 and Blum, Zeitschr. f. physiol. Chem., 30;
Kossel, Ber. d. d. chem. Gesellsch., 34, 3214; Hofmeister, Ergebnisse der Physiol.,
Jahrg. I, Abt. 1, 759, which also contains the literature; Osborne and Harris, Journ.
Amer. Chem. Soc., 25; and Giimbel, I.e.

3 Skraup and v. Hardt-Stremayr, Monatsh. f. Chem., 29, found lower results than
other investigators and they found also that about two-thirds of the amide nitrogen
was readily split off and one-third slowly.

4 Ber. d. d. chem. Gesellsch, 43 and 44 and Journ. of biol. Chem., 9, 10 and 12.

NITROGEN DISTRIBUTION. 79

By the action of nitrous acid upon proteins at least a partial deamidation
takes place and so-called desamino proteins are obtained. The nitrogen expelled
originated from the NH2 groups according to the formula RNH2+HN02 = ROH-j-
N2+H20. The amount of such nitrogen is generally only small, 1-2 per cent,
and for this reason it has been accepted that such groups only occur in small
amounts in the proteins. This is probably true for a large number of proteins
but not for all and as example of these we will recall that KOSSEL and CAMERON 1
have shown that those protamiries which contain no other hexone base besides
arginine although they have NH2 groups at the ends in the guanidine residue
HN.CNH.NH2 of the numerous arginine groups, do not yield any nitrogen on
using v. SLYKE'S method while those protamines containing lysine do. We must
be very careful in drawing certain conclusions from the results obtained by the
action of nitrous acid upon proteins.

The nitrous acid can develop nitrogen from the NH2 groups of the acid amides
as well as from the NH2 groups of the amino-acids. On the contrary no nitrogen
is evolved in v. SLYKE'S method from the guanidin groups and from the peptid
combinations containing imid groups (see below). This is also the reason, as
remarked above, why those protamines containing only arginine do not yield
any nitrogen while those protamines which also contain lysine where there exist
free NH2 groups do give off nitrogen. On hydrolyzing these deamidized pro-
tamines and also other deamidized proteins we therefore do not obtain any lysin
as shown by SKRAUP and collaborators and by LEVITES for certain proteins.
The quantity of monamino-acid nitrogen is therefore in such cases found to be
increased.

According to OSBORNE, LEAVENWORTH and BRAUTLECHT,2 who worked
with plant proteins, the splitting off of NH3 on the acid hydrolysis of the proteins
was very similar to the splitting off of NH3 from the acid amide asparagine, so
that the binding of NH2 groups on the carboxyl groups seems very probable.
The quantity of NH3 split off in the hydrolysis ran parallel with the amount
of asparagine and glutamic acid present and the quantity of NH3, split off by
hydrolysis with alkali corresponded nearly to the sum of the ammonia that was
split off by acid hydrolysis and one-half of the arginine nitrogen. According to
these investigators the NH2 groups occur chiefly as acid-amide combinations.

A part of the nitrogen in the proteins occurs from the above, undoubt-
edly as NH2 groups; the extent of this part, which is different in different
proteins cannot be positively given. The chief mass of the nitrogen
in the proteins, although other forms of binding occur, exists as imide-
like combinations of amino-acids united together and this will be com-
pletely developed in the following pages.

The sulphur occurs in the different proteins in very different amounts.
Certain of them, such as the protamines and apparently also certain

1 In regard to the action of nitrous acid upon proteins, their deamidation and cleav-
age products see C. Paal, Ber. d. d. Chem. Gesellsch., 29; H. Schiff, ibid., 1354; O.
Loew, Chemiker Ztg., 1896 and O. Nasse, Pfluger's Arch., 6; Treves and Salomone,
Bioch. Zeitschr, 7; Skraup, Monatsh. f. Chem., 27 and 28, with Hoernes, ibid., 27,
with Kaas, Annal. d. Chem. u. Pharm., 351; Lampel, Monatsh. f. Chem., 28; Traxl,
ibid., 29; Levites, Zeitschr. f. physiol. Chem., 43, and Bioch. Zeitschr., 20; D. v.
Slyke, foot-note 4, page 78; Kossel and Cameron, Zeitechr. f. physiol. Chem., 76; Kossel
and F. Weiss, ibid., 78.

2 Amer. Journ. of Physiol., 23.

80 THE PROTEIN SUBSTANCES.

bacterial proteids,1 are free from sulphur; some, such as gelatin and
elastin, are very poor in sulphur, while others, especially horn sub-
stances, are relatively rich in sulphur. On hydrolytic cleavage with
mineral acids, the sulphur of the protein substances is regularly, at
least in part, split off as cystine (K. MORNER) or, with bodies poorer in
sulphur, as cysteine (EMBDEN), but this, according to MORNER and PATTEN,
is a secondary formation. From certain protein substances a-thiolactic
acid (SUTER, FRIEDMANN, FRANKEL), which MORNER claims is also pro-
duced secondarily, mercaptans and sulphureted hydrogen (SIEBER and
SCHOUBENKO, RUBNER), and a body having the odor of ethyl sulphide
(DRECHSEL) have been obtained.2

A part of the sulphur separates as potassium or sodium sulphide on
boiling with caustic potash or soda, and may be detected by lead acetate
and quantitatively determined (FLEITMANN, DANILEWSKY, KRUGER,
FR. SCHULZ, OSBORNE, K. MoRNER3). What remains can be detected
only after fusing with potassium nitrate and sodium carbonate and
testing for sulphates. The ratio between the sulphur split off by alkali
and that not split off is different in various proteins. No conclusions
can be drawn from this in regard to the number of forms of combination
which the sulphur has in the protein molecule. As shown by K. MORNER,
only about three-fourths of the sulphur in cystine can be split off by
alkali, and the same is true for the cystine-yielding complex of the pro-
tein substances. If the quantity of lead-blackening sulphur in a pro-
tein body be multiplied by f, we obtain the quantity corresponding to
the cystine sulphur in the body. By such calculation MORNER found
in certain bodies, such as horn substance, seralbumin and serglobulin,
that the quantity of cystine sulphur and total sulphur were identical,
and therefore we have no reason for considering the sulphur in these
bodies as existing in more than one form of combination. In other
proteins, such as fibrinogen and ovalbumin, on the contrary, only one-
half or one-third of the sulphur appeared as cystine sulphur.

Just as in the products of acid hydrolysis of proteins we know of
two forms of oxygen bondage, the hydroxyl form OH and the carbonyl

1 See Nencki and Schaffer, Journ. f . prakt. Chem. (N. F.), 20, and M. Nencki, Ber.
d. d. chem. Gesellsch., 17.

2 K. Morner, Zeitschr. f. physiol. Chem., 28, 34, and 42; Patten, ibid., 39; Embden,
ibid., 32; Suter, ibid., 20; Friedmann, Hofmeister's Beitrage, 3; Sieber and Schou-
benko, Archiv d. sciences biol. de St. Petersbourg, 1; Rubner, Arch. f. Hygiene, 19;
Drechsel, Centralbl. f. Physiol., 10, 529; Frankel, Sitzungsber. d. Wien. Akad., 112,
TI b, 1903.

3 Fleitmann, Annal. der Chem. und Pharm., 66; Danilewsky, Zeitschr. f. physiol.
Chem., 7; Kriiger's, Pfluger's Archiv, 43; F. Schulz, Zeitschr. f. physiol. Chem., 25;
Osborne, Connecticut Agric. Expt. Station Report 1900; Morner, 1. c.

HYDROLYSES OF PROTEINS. 81

form in CONH; so according to TREAT B. JOHNSON * two analogous forms
of sulphur bondage exist in the proteins, namely the mercaptan form
SH as in cystine and the form NH.CH.CS.NH corresponding to the oxygen
binding in the polypeptids (see page 86). He has in fact also prepared
thio-polypeptides from glycocoll and these were analogous to the corre-
sponding glycin polypeptids (see page 88) and like certain proteins
gave H2S on acid hydrolysis.

The constitution of the protein bodies is still unknown, although
the great advances made in the last few years have brought us very much
closer to the elucidation of the question. In studying the constitution
of the protein bodies they have been broken up in various ways into
simpler portions, and the methods used for this purpose have been of
different kinds. In such decompositions, for which only purified proteins
are to be used, first large atomic complexes — proteoses and peptones —
are obtained which still have protein characteristics, and these then
suffer further decomposition until finally we obtain simpler, generally
crystalline, or at least well-characterized, end products.

As to the products obtained by hydrolytic cleavage with mineral acids,
important investigations have been carried out by numerous older and
more recent experimenters.2 Besides certain acids, which will be men-
tioned later and which occur in few cases only, we obtain the following:
monamino-acids such as glycocoll, alanine, aminovaleric acid, leu-
cine, isoleucine, serine, aspartic and glutamic acids, cysteine and its disul-
phide cystine, phenylalanine, tyrosine, pyrollidine — and oxypyrollidine
carboxylic acid, tryptophane and also the three hexone bases, histidine,
arginine and lysine, the two latter being diamino-acids. Besides these
also ammonia, sulphureted hydrogen, ethyl sulphide and melanoidins,
which latter seem to be secondary products, have been obtained.

On the hydrolysis with alkalies we obtain, after a preliminary forma-
tion of intermediary steps which will be discussed later, chiefly the same
cleavage products as in acid hydrolysis but with the exception that in
the alkali hydrolysis a considerable part of the amino-acids become
racemerized and therefore appear in optically inactive form while in the
acid hydrolysis chiefly optically active acids are obtained. Because
of the action of the alkali a part may suffer further decomposition which
leads to the formation of simpler cleavage products and ammonia.

On fusing proteins with caustic alkali, ammonia, methyl mercaptan and other
volatile products are evolved and other products are produced such as leucine,

1 Journ. of biol. Chem., 9.

2 In regard to the literature see O. Cohnheim, Chemie der Eiweisskorper, Braun-
schweig, 1911, and F. Hofmeister, Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 759,
1902; E. Fischer, Untersuchungen iiber Aminosauren, Polypeptide und Proteine (1899-
1906), Berlin, 1906. See also special references.

82 THE PROTEIN SUBSTANCES.

from which then volatile fatty acids such as acetic acid, valeric acid and also
butyric acid are formed, also tyrosine from which latter phenol is formed and
indol and skatol.

Most proteins are split by proteolytic enzymes in the same manner
as on hydrolysis with acids or alkalies, but more or less completely depend-
ent upon the kind of enzymes. In the first place proteoses and peptones
(see below) are formed, then also polypeptids and amino-acids of
various kinds, in certain cases also oxyphenylethylamine, diamines, and
a little ammonia and other bodies.

A great many substances are produced in the putrefaction of pro-
teins. First the same bodies as are formed in the decomposition by
means of proteolytic enzymes are produced, and then a further decom-
position occurs with the formation besides ammonia, carbon dioxide
and hydrogen, of a large number of bodies belonging in part to the aliphatic
and in part to the aromatic and heterocyclic series.

To the aliphatic series belong volatile fatty acids and as shown
by NEUBERG 1 and collaborators not only fatty acids of the normal chain
but also with branched chains, also optically active acids, also succinic
acid, methane, methyl mercaptan and others. To this series belongs
also the two putrefaction bases cadaverine and putrescine, produced from
the diamino acids, and also the so-called ptomaines or cadaver alkaloids
which may originate, at least in part, from other tissue constituents
and not from proteins.

The putrefactive products of the aromatic and heterocyclic series
originate from the corresponding amino-acids. From tyrosine the
aromatic oxy-acids such as p-oxyphenyl-propionic acid, the p-cresol,
phenol and oxyphenylethylamine are formed. The phenylalanine is the
mother substance of the phenylpropionic acid, the phenylacetic acid and
the phenylethylamine. Indolpropionic acid, indolacetic acid, skatol
and indol originate from the tryptophane (indolaminopropionic acid) ; the
imidazolpropionic acid and imidazolethylamine originate from the histi-
dine.2

By the moderate action of chlorine, bromine, or iodine upon proteins,
these halogens enter into more or less firm combination with the proteins
and according to the method of procedure we can prepare derivatives
having different but constant amounts of halogens. The proteins are
so changed that they do not split off sulphur on treatment with alkali,
nor do they respond to MILLON'S or ADAMKIEWICZ-HOPKINS reaction.
Side processes, oxidations and cleavages may also take place here. The
most striking fact seems to be a substitution of hydrogen by iodine in the

1 Bioch. Zeitschr., 37, where the earlier works of Neuberg are cited.

2 Ackermann, Zeitschr. f. physiol. Chem., 65.

OXIDATION OF PROTEINS. 83

aromatic nucleus of tyrosine and also perhaps in the indol nucleus of
tryptophane and the imidazol nucleus of histidine.1 Halogen proteins
occur, as will be shown later, in the animal kingdom, especially in the
albuminoid group and indeed iodized tyrosine (3-5 di-iodotyrosine)
has been isolated.

By the oxidation of protein by means of potassium permanganate, MALY
obtained an acid, oxyprotosulphonic acid, C 51.21, H 6.89, N 14.59 S 1.77, 0 23.24
per cent, which is not a cleavage product, but an oxidation product in which
the group SH is changed into SOi.OH. This acid does not give the proper color
reaction with MILLON'S reagent, yields no tyrosine or indol, but gives benzene
on fusing with alkali. On continued oxidation MALY obtained another acid,
peroxyproteic acid, which gives the biuret reaction, but is not precipitated by
most protein precipitants. The oxy protein obtained by SCHULZ on the oxida-
tion of protein by hydrogen peroxide is closely related to oxyprotosulphonic
acid in composition and general characteristics, but contains lead-blackening
sulphur and gives MILLON'S reaction. The oxyprotein is claimed to be a pure
oxidation product, while in the production of oxyprotosulphonic acid SCHULZ
claims that a cleavage takes place. According to BURACZEWSKI and KRAUZE
the oxyprotosulphonic acid is a mixture of several substances. According to
the investigations of v. FURTH 2 there exist at least three different peroxyproteic
acids (from casein) which differ from each other by a different division of the
nitrogen in the molecule. On treatment with baryta- water we find that they
split off basic complexes and oxalic-acid groups, and new bodies, the desamino-
proteic acids, which give the biuret reaction, are produced. These later acids, which
on hydrolysis give benzoic acid but no diamino-acids, may be further oxidized,
which is not true of the peroxyproteic acids, and yield a new group of acids, the
kyroproteic acids, which give the biuret reaction, hold about one-half of their
nitrogen (11.08 per cent total nitrogen) in acid-amicie-like combination, but'
yield neither basic products nor benzoic acid.

On the oxidation of gelatin or protein with permanganate we also obtain
oxaminic acid, oxamide, oxalic acid, oxaluric-acid amide, succinic acid, several
volatile fatty acids, and guanidine, which was first shown by LOSSEN as an oxida-
tion product.3

On the oxidation of gelatin by ferrous sulphate and hydrogen peroxide
BLUMENTHAL and NEUBERG have obtained acetone as a product, and ORGLER
the same from ovalbumin. The action of ozone upon casein has been studied

1 In regard to the action of halogens upon proteins see Loew, Journ. f. prakt.
Chem. (N. F.), 31; Blum, Munch, med. Wochenschr., 1896; Blum and Vaubel, Journ.
f. prakt. Chem. (N. F.), 57; Liebrecht, Ber. d. deutsch. chem. Gesellsch., 30; Hop-
kins and Brook, Journ. of Physiol., 22; Hopkins and Pinkus, Ber. d. deutsch. chem.
Gesellsch., 31; Hofmeister, Zeitschr. f. physiol. Chem., 24; Kurajeff, ibid., 26; Oswald,
Hofmeister's Beitrage, 3; C. H. L. Schmidt, Zeitschr. f. physiol. Chem., 35, 36, 37;
Neuberg, Biochem. Zeitschr., 6: Pauly and Gundermann, Ber. d. d. chem. Gesellsch.,
41, 43; Krzemecki, Chem. Centralbl., 1912; Pauly, Zeitschr. f. physiol. Chem., 76.

2 Maly, Sitzungsber, d. k. Akad. d. Wissensch., Wien, 91 and 97. Also Monatshefte
f. Chem., 6 and 9. See also Bondzynski and Zoja, Zeitschr. f. physiol. Chem., 19;
Bernert, ibid., 26; Schulz, ibid., 29; Buraczewski and Krauze, ibid, 76; v. Fiirth, Hof-
meister's Beitrage, 6.

3 Lessen, Annal. d. Chem. u. Pharm., 201; Kutscher, Zeitschr. f. physiol. Chem.,
32; Zickgraf, ibid., 41; Seemann, ibid., 44; Kutscher and Schenck, Ber. d. d. chem.
Gesellsch., 37 and 38.

84 THE PROTEIN SUBSTANCES.

by HARRIES and LANGHELD l and the action of chlorine by HABERMANN and
EHRENFELD and PANZER. 2

Nitric acid gives various yellow products, which turn reddish-brown in
alkaline solution. Of these we must especially mention the so-called xantho-
protein, besides nitrated proteoses and peptones. The xanthoprotein does not
yield any tyrosine on acid hydrolysis and it does not give the Millon or the lead-
blackening reactions. Among the cleavage products v. FURTH 2 has obtained a
melanoidin substance, xanthomelanoidin.

On the nitration of the protamines (see below) KOSSEL 4 and co-workers have
obtained nitroprotamines which give nitroarginine on hydrolysis which shows that
the nitro groups have entered the guanidine groups of the arginine.

By the dry distillation of proteins we obtain a large number of decomposition
products having a disagreeable burned odor, and a porous glistening mass of carbon
containing nitrogen is left as a residue. The products of distillation are partly
an alkaline liquid which contains ammonium carbonate and acetate, ammonium
sulphide, ammonium cyanide, an inflammable oil, and other bodies, and a brown
oil which contains hydrocarbons, nitrogenized bases belonging to the aniline and
pyridine series, and a number of unknown substances.

The occurrence of protein substances which contain a carbohydrate
group has been known for a long time. The nature of this carbohydrate,
which can be split off by acid and which may amount to as much as 35
per cent, has been explained chiefly by the investigations of FRIEDRICH
MuLLER5 and his students. They have shown that it is always an
amino-sugar, and generally glucosamine and perhaps galactosamine
as Jan exception. That so-called true proteins also yield a carbohydrate
on hydrolytic cleavage was first shown by PAVY, using ovalbumin. The
continued investigations of FR. MULLER, and others have demonstrated
that in these cases the carbohydrate is also glucosamine. A carbohy-
drate complex, although sometimes only to a very slight amount, has
been detected in other proteins, ovoglobulin, serglobulin, seralbumin,
peaglobulin, albumin of the graminese, yolk-proteid, and fibrin. In
other proteins, on the contrary, such, as edestin (of the hemp-seed) and
casein, myosin, pure fibrinogen, and ovovitellin, carbohydrates have
been sought for with negative results. All proteins hence do not contain
a carbohydrate group, and future investigators must therefore decide
whether the carbohydrate groups belong positively to the protein com-

1 Blumenthal and Neuberg, Deutsch. med. Wochenschr., 1901; Orgler, Hofmeister's
Beitrage, 1; Harries and Langheld, Zeitschr. f. physiol. Chem., 51.

2 Habermann and Ehrenfeld, Zeitschr. f. physiol. Chem., 32; Panzer, ibid., 33
and 34.

3 See Maly's Jahresber, 30, p. 24.

4 Kossel and Kennaway, Zeitschr. f. physiol. Chem., 72, with E. Wechsler, ibid.,
78 and with F. Weiss, ibid., 78.

6 In regard to the literature on this subject see the work of Fr. Miiller, Zeitschr.
f. Biologic, 42, and Langstein, Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 63, Zeitschr.
f. physiol. Chem., 41, and Hofmeister's Beitrage, 6. See also Abderhalden, Bergell,
and Dorpinghaus, Zeitschr. f. physiol. Chem., 41.

CARBON NUCLEI. 85

plex or whether they are united with the protein only as impurities. Sev-
eral observations 1 show that in working with crystalline proteins a con-
tamination with ot'her protein substances is unfortunately not excluded,
and this must not be lost sight of, especially as the quantity of carbohy-
drates obtained is often very small. In this connection we must call
attention to the findings of OSBORNE and collaborators that on recrystalliz-
ing ovalbumin six times they found that the glucosamine content was
reduced to 1.23 per cent while other investigators give 7-8-15 per cent.
Under these circumstances we are not warranted in considering the
carbohydrate groups as belonging to the carbon nucleus produced on
the destruction of the real protein complex.

The previously mentioned methods used in studying the structure
of the protein substances are not of the same value, but they in part
substantiate each other. Of these we must mention the hydrolysis
by means of boiling dilute mineral acids, or by proteolytic enzymes,
as the best methods for obtaining the carbon nuclei in the protein mole-
cule. The most important of the carbon nuclei obtained are as follows:

I. The Nuclei belonging to the Aliphatic Series.

A. Sulphur free, but containing nitrogen: 1. A guanidine residue (combined
with ornithine as arginine). 2. Monobasic monamino-acids: Glycocoll, alanine,
valine (amino valeric acid), leucine, and isoleucine. 3. Bibasic monamino-
acids: Aspartic acid and glutamic acid. 4. Oxy monamino-acids: serine oxy-
aminosuccinic acid and oxyaminosuberic acid. 5. Monobasic diamino-acids:
Diaminoacetic acid, ornithine (from arginine) and lysine. 6. Oxy diamino-acids:
Oxydiaminosuberic acid, oxydiaminosebacic acid, diaminotrioxydodecanoic acid,
caseanic and caseinic acids.

B. Sulphurized: Cysteine and its sulphide cystine, thiolactic acid (mercaptans,
and ethyl sulphide).

n. The Nuclei belonging to the Carbocyclic Series.

Phenylalanine and tyrosine.

III. The Nuclei belonging to the Heterocyclic Series.
Proline, oxyproline, tryptophane and histidine.

In regard to these carbon nuclei it must be remarked that they are
not all found in every protein body thus far investigated, and also that
one and the same cleavage product, such, for example, as glycocoll,
leucine, tryosine, etc., is obtained in very variable amounts from differ-
ent protein substances.

It is very difficult to say to what extent all the above-mentioned
carbon nuclei exist in the protein molecule. It is not inconceivable

1 Osborne, D. B. Jones and Leavenworth, Amer. Journ. of Physiol., 24.

86 THE PROTEIN SUBSTANCES.

that in the hydrolysis certain carbon nuclei may be secondarily formed
from others. Even if we. admit the above, still it is undoubtedly true
that the chief cleavage products of the protein substances are amino-
acids. EMIL FISCHER has shown that the amino-acids have the property
of readily grouping together when water is split off and the amide group
of one amino-acid unites with the carboxyl group of the other. In
accord with this behavior we can, as HOFMEISTER 1 and others have
explained, but which was first proved by the epoch-making investiga-
tions of EMIL FISCHER, consider the proteins as chiefly formed by the
condensation of amino-acids, where the amino-acids are united to each
other by means of imino-groups according to the following scheme:

— NH.CH.CO— NH.CH.CO - NH.CH.CO— NH.CH.CO-

C4H9 CH2.C6H4(OH) CH2.COOH

(Leucine) (Tyrosine) (Aspartic acid) (Lysine)

Such chaining of animo-acids is for the synthesis of protein-like bodies
of the very greatest importance. The older statements of GRIMAUX,
SCHUTZENBERGER and PICKERING on the artificial preparation of pro-
tein-like substances where these investigators were able to prepare sub-
stances, which in many properties are similar to the proteins, from various
amino-acids either alone or mixed with other bodies such as biuret, alloxan,
xanthine, or ammonia. Of special interest are the investigations of
CURTIUS and his collaborators, in which they were able to prepare syn-
thetically the so-called biuret base (triglycyl-glycine ethyl ester) and sub-
sequently many other bodies which were related to the proteins. The
most important work on the chaining of amino-acids has been per-
formed by E. FISCHER 2 and his pupils but especially by ABDERHALDEN.
They have prepared a large number of complex bodies called polypeptides
by FISCHER, which according to whether they contain two or more
amino-acid groups united together, are called di-, tri-, tetrapeptides,
etc. As examples of polypeptides we will mention — dipeptides: glycyl-
tyrosine, alanylglycine, leucylglycine, leucylcystine, prolylphenylalanine,

1 " Ueber den Bau des Eiweissmolekiils." Gesellsch. deutsch. Naturforscher und
Aertze, Verhandl. 1902, and Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 759.

2 See Pickering, King's College, London, Physiol. Lab. Collect. Papers, 1897, which
also cites Grimaux's work; also Journ. of Physiol., 18, and Proceed. Roy. Soc., 60,
1897; Schiitzenberger, Compt. rend., 106 and 112; Curtius, Journ. f. prakt. Chem.
(N. F.), 26 and 70, and Ber. d. d. chem. Gesellsch., 37; Fischer and collaborators,
Untersuchungen iiber Aminosauren, Polypeptide und Proteiine (1899-1906) Berlin 1906
and Ber. d. d. chem. Gesellsch., 39, 40, 41, 42, and Annal. d. Chem. u. Pharm., 354,
357, 363, 365, 369, 375; see also Abderhalden, Ber. d. d. chem., Gesellsch, 40 to 43 and
Zeitschr. f. physiol. Chem., 72, 75, 77.

POLYPEPTIDES. 87

leucylhistidine; tripeptides: di-glycylglycine, alanylglycyltyrosine, leu-
cyltryptophylglutamic acid; tetrapeptides: glycylglutamyldiglycine, dileu-
cylglycylglycine; pentapeptides : tetraglycylgly cine and leucyltriglycylgly-
cine; hexa- and heptapeptides: leucyltetraglycylglycine and leucylpentagly-
cylglycine. The most complex polypeptide thus far prepared is an
octadecapeptide with 15 glycocoll and 3 leucine residues namely: Weucyl-
triglycyl-Z-leucyltriglycyl-/-leucyl-octaglycylglycine =

NH2CH(C4H9)CO.[NHCH2CO]3.NHCH(C4H9)CO.
[NHCH2CO]3.NHCH(C4H9)CO.[NHCH2CO]8.NHCH2COOH.

with the supposition that the amino-acids are here also combined together
in the imide binding.

The large number of amino-acids isolated from the proteins make
a large number of bindings possible. The number of possible combina-
tions is still further increased by the fact that all the amino-acids with
the exception of glycocoll contain at least one asymmetric carbon atom,
and this leads to the possible formation of stereochemically different
peptides. Thus in order to give a simple example, from two optically
active amino-acids, four different isomeric forms of dipeptides may occur,
namely (if we designate the optical antipodes by d- and 1-) dd, II, dl and Id.
Of these forms two can form a racemic dipeptide, thus d-alanyl-d-leucine+
Z-alanyl-Z-leucine and d-alanyl-Meucine+/-alanyl-d-leucine. As the pro-
teins are optically active and on hydrolysis yield chiefly optically active
amino-acids, those polypeptides which can be built up from the natural
amino-acids of the proteins are of special importance in the study of the
constitution of the proteins.

Most of the artificial polypeptides are constructed from monamino-
mono-carboxylic acids, but polypeptides have also been prepared which
contain diamino-acids or amino-dicarboxylic acids, and in this way the
number of possible polypeptides becomes still greater. With an aminodi-
carboxylic acid such as aspartic acid, other amino-acids can be bound
with one carboxyl group or with both, but also, if we start with aspara-
gine, they can be anchored with the amide group. If we start from the
acid amides we can also obtain a peptide which still contains the CONH2
group and on total hydrolysis yields NHs, like most proteins. A poly-
peptide of this kind is the tripeptide, glycl-/-asparaginyl-Z-leucine prepared
by E. FISCHER and KOENIGS.

NH2CH2CO.NHCHCO.NHCH(C4H9)COOH

CH2CONH2

In consideration of the form of binding of the sulphur in the proteins
it is interesting to consider the preparation of thiopolypeptids as performed

88 THE PROTEIN SUBSTANCES.

by TREAT B. JOHNSON.1 He has prepared the following: thioglycylglycin-
thioamide NH2CH2CS.NHCH2CSNH2 which is analogous to glycylgly-
cinamide, NH2CH2CO.NHCH2CONH2 and also dithiopiperazine

CH2.CSX

HN/ >NH.

XCS.CH2

Polypeptides of higher amino-fatty acids such as a-aminolauryla-
lanine, a-aminolaurylleucine and others have been prepared by HOP-
WOOD and WEiZMANN.2 These peptids are different from the so-called
lipopeptids prepared by BONDI and his collaborators3 which are not
chains of only amino-acids but combinations between a high fatty acid,
such as lauric- or palmitic acid and an amino-acid (glycocoll or alanine)
or a dipeptide (lauryl-alanylglycine) .

Methylated polypeptides such as methyl- and dimethylleucylglycine (E.
FISCHER and GLUUD) and betaindiglycylglycine

(CH3)2N. CH2CO.NHCH2CO.NHCH2CO

(ABDERHALDEN and KAUTZSCH) are also known.4 Amides of amino-acids and
dipeptides have been prepared by BERGELL 6 and his co-workers.

The methods used by E. FISCHER in the synthetical preparation of
polypeptides are chiefly as follows:

The first dipeptide prepared by him, glycylglycine, he obtained from glycocoll
ethyl ester which in water is transformed into a diketopiperazine, glycine anhy-
dride, according to the following equation:

/CH2.C(X

2(NH2CH2CO.O.C2H5) = 2C2H5OH+NH< >NH.

XCO.CH/

By the action of dilute alkali upon this anhydride with the taking up of water the
glycylglycine NH2CH2CO.NHCH2COOH is formed, and according to this prin-
ciple other dipeptides can also be prepared.

Another method which has much greater application consists in the anchoring
of an amino-acid to a halogen of an acid radical, for example, by the action of
brompropionyl bromide or chloride upon glycocoll according to the following
equation:

CH3CHBrCOCl+NH2CH2COOH=HCl+CH3CHBrCO.NHCH2COOH

1 Journ. of biol. Chem., 9.

2 See Chem. Centralbl., 1911.

3 Bioch. Zeitschr., 17 and 23; see also Abderhalden and C. Funk, Zeitschr. f. physiol.
Chem., 65.

4 Fischer and Gluud, Annal. d. Chem. u. Pharm., 369; Abderhalden and Kautzsch,
Zeitschr. f. physiol. Chem., 72 and 75.

• Ibid., 64, 65 and 67.

POLYPEPTIDES. 89

(brompropionyl glycine). On subsequent treatment with ammonia the halogen
(Br) is replaced by NH2 and the dipeptide alanylglycine

CH3CHNH2CO.NHCH2COOH+NH4Br

is obtained. By the second action of brompropionylchloride and then treatment
with NH3 we introduce a new alanyl group and the tripeptide alanyl-alanyl
glycine is prepared. By the action of a halogen derivative of an acid radical
another amino-acid residue can be introduced, and the chain of amino groups
can be thus extended.

I The prolongation of the chain on the other side, namely, at the carboxyl,
FISCHER has accomplished by chlorination of the amino-acids by special treatment
with phosphorus pentachloride. The carboxyl is thus transformed into COO,
while the acid at the same time fixes a molecule of HC1, for example CHaCHNH^Ci

COC1

Just as in the case of the carboxyl group of an amino-acid, so also can a poly-
peptide or its halogen acyl combination be chlorinated and then combined with
a new amino-acid, or a new peptide. As an example, FISCHER, from a-brom-
isocapronyldiglycyl glycine, first prepared a-bromisocapronyldiglycylglycyl chlo-
ride, and then with diglycylglycine he obtained the heptapeptide leucyl-
pentaglycylglycine,

C4H9CH(NH2)CO.(NHCH2CO)6.NHCH2COOH.

For the various combinations of the optically active amino-acids to poly-
peptides it was important to possess methods of preparation of these amino-acids,
and for this purpose FISCHER in many cases used the so-called WALDEN'S reversion.
This consists in that one optically active amino-acid, for example the Z-form, is
transformed into the corresponding halogen fatty acid by the action of nitrosyl
bromide, yielding the optical antipode the d-form. By the action of ammonia
the d-amino-acid is now obtained which in the above-mentioned manner can be
retransformed into the /-form. Thus from d-leucine we first obtain Z-bromiso-
caproic acid and then by the action of ammonia /-leucine and in the preparation
of the polypeptides the same occurs. Thus, for example, if by reversion d-leucine
is changed first into Z-bromisocapronyl chloride, if this last is combined with
Meucine, then we obtain the dipeptide Z-leucyl-/-leucine. On combination with
diglycylglycine the tetrapeptide /-leucyl-diglycyl glycine is produced. WALDEN'S
reversion does not take place with all amino-acids; other methods can also be
used to obtain the optical antipodes, such as the preparation of the alkaloidal
salts of the benzoyl or formyl combinations of the racemic amino-acids.

The /3-naphthalinsulpho combination of the polypeptides and peptones may
serve, as FISCHER, ABDERHALDEN and FUNK * have shown, in explaining the
structure of these bodies. By the action of /3-naphthaline sulphochloride the
NH2 groups existing at the beginning of the chain in the amino-acids react there-
with and on subsequent total hydrolysis this naphthaline-sulpho combination
remains unsplit. Thus for instance we can differentiate between glycylalanine
and alanylglycine because after hydrolysis in the first case we obtain naphthalin-
sulphoglycine and alanine and in the second naphthalin-sulphoalanine and
glycocoll (glycine). Tyrosine may, depending upon whether the NH2 as well
as the OH groups are free or not or if only one is available, yield di- or mononaph-
thalinsulpho-derivatives and in this way we can also draw conclusions as to the
structure of tyrosine containing peptides.

The previously mentioned deamidation method of van Slyke (page 78) where
oxyacids are formed by the action of HNO2 upon the NH2 groups can also give

1 E. Fischer and Abderhalden, Ber. d. d. Chem. Gesellsch., 40; Abderhalden and
C. Funk, Zeitschr. f. physiol. Chem., 64.

90 THE PROTEIN SUBSTANCES.

certain conclusions as to the structure of the peptides by comparing the hydro-
lytic products before and after deamidation.

A comparison of the artificially prepared polypeptides with the pro-
teins, and especially with the cleavage products of these last, the so-called
preoteoses and peptones, is of great interest in several respects, especially
in connection with certain reactions. For instance there are several
polypeptides which give the biuret reaction which is characteristic of
the proteins in general, and also several (polypeptides containing tyrosine),
which give MILLON'S reaction (see further on). The above-mentioned
octadecapeptide is precipitated by phosphotungstic acid, tannin and
ammonium sulphate; we also know tri- and pentapeptides containing
tyrosine, which are very similar in properties to the proteoses.
p The behavior of the polypeptides with proteolytic enzymes is of
great interest. As this interesting question will be thoroughly treated
in other chapters (I and VIII) it is sufficient here to recall that the
possibility that polypeptides as well as proteins are hydrolyzed by the
same enzymes, yielding amino-acids, is a weighty proof of the prob-
ability that in the proteins the amino-acid chains are of the same kind
as in the polypeptides.

A very important support for such a view is found in the occurrence
of polypeptides among the cleavage products of proteins, a find which
to a certain extent forms the reverse of the above-mentioned syntheses.
Such polypeptides are chiefly di- but also tri- and tetrapeptides. They
have been obtained in the hydrolytic products of silk waste, silk fibroin
and elastin (FISCHER, ABDERHALDEN) , gelatin (LEVENE, WALLACE and
BEATTY) and of gliadin (OSBORNE and CLApp)1. Of .special interest in
this connection are those polypeptides which like glycyl-d-alanine,
d-alanyl-glycine, glycyl-Z-tyrosine, /-prolyl-Z-phenylalanine and d-alanyl-
glycyl-/-tyrosine, are identical with the corresponding synthetically pre-
pared polypeptides or at least very closely related.

We have therefore conclusive reasons for the assumption that in the
proteins, peptide bindings chiefly occur, i.e., a combination of the a-
amino-acids by means of the imide binding. It is also possible that
other linking may occur, and FISCHER has also given expression to such
a possibility. Besides the above-mentioned imide binding another kind
must also without doubt exist in the proteins, namely, the anchoring
of the urea-forming group (the guanidine residue) with the ornithin
(diamino-valeric acid) by the imide binding. This imide linking is

1 Fischer and Abderhalden, Sitz. Ber. d. d. Berl. Akad. d. Wissensch, 30, and Ber.
d. d. Chem. Gesellsch., 39, 40; Abderhalden, Zeitschr. f. physiol. Chem., 62, 63 and 72;
Levene and Wallace, ibid., 47, with Beatty, Ber. d. d. Chem. Gesellsch., 39, and Bioch.
Zeitschr., 4; Osborne and Clapp, Amer. Journ. of Physiol., 18.

CLASSIFICATION. 91

not, like the a-amino-acids, broken by trypsin, but rather by an enzyme
arginase, discovered by KOSSEL and DAKiN.1

If the proteins are considered as consisting chiefly of peptide-like
complexes consisting of amino-acids united and containing also several
NH2 groups at the ends, it is readily understood that the proteins are
amphoteric electrolytes, like the amino-acids, which form salts with bases
as well as with acids and undergo hydrolytic dissociation. As we also
accept the theory that the protein molecule contains a large number of
COOH as well as NH2 groups, it follows that the proteins may be poly-
basic acids as well as polyacidic bases. The different proteins act in
this regard somewhat differently, thus the protamines are strongly basic
while casein behaves strikingly acid, and others take a certain mean
position. It is unfortunately impossible to base a classification of the
proteins upon this behavior, as well as upon chemical constitution.
The general properties, such as solubility and precipitation properties,
are too uncertain to aid us, and especially as in the investigations of
proteins we, as a rule, cannot decide whether we are dealing with a pure
or with a contaminated substance, namely, with mixtures. Experience
has shown that the solubility and precipitation properties of the proteins
are strongly influenced by the presence of other bodies, and under such
circumstances a proper classification, as demanded by science, is impos-
sible. On the other hand, a classification is important, and as the
ones used up to the present time were based upon the solubility and
precipitation properties, we give the following schematic summary of the
chief groups of protein bodies:

I. Simple Proteins.
A. TRUE ALBUMINOUS BODIES OR PROTEIDS.

Seralbumin,
Lactalbumit
Fibrinogen,

Albumins. .

Lactalbumin, and others.

Globulins c 7 , 7 .

berglobuhns, and others.

Phosphoproteins (Nucleoalbu-

mins),

Ovovitettin,

Casein, and others.

(Coagulated proteins.)

Histones.

(Protamines?)

1 Zeitschr. f. physiol. Chem., 41.

92 THE PROTEIN SUBSTANCES.

B. ALBUMINOIDS OR ALBUMOIDS.
Keratins.
Elastin.

Collagen and glutin.
Reticulin.

(Fibroin, Sericin, Coilin, Cornein, Spongin, Byssus, and others.)
C. CLEAVAGE PRODUCTS OF TRUE ALBUMINOUS BODIES.

Alkali and Acid Albuminates.
Proteoses, Peptones, Polypeptides.
(Amino-acids.)

n. Compound Proteins.

I Mudn substances.

Glycoproteins

Nucleoproteins.

f HcBmoglobin,
Chromoproteins ............... \ TT

[ Hcemocyamn.

As there are two classifications recognized by English-speaking scien-
tists we will give the classifications adopted by the American Physio-
logical Society and the American Society of Biological Chemists and
also the British Medical Association.

Classification adopted by the American Physiological Society and
the American Society of Biological Chemists :

I. Simple Proteins.

A. Albumins.

B. Globulins.

C. Glutelins.

D. Prolamins (Alcohol-soluble proteins).

E. Albuminoids.

F. Histones.

G. Protamines.

n. Conjugated Proteins.

A. Nucleoproteins.

B. Glycoproteins.

C. Phosphoproteins.

D. Haemoglobins.

E. Lecithoproteins.

CLASSIFICATION. 93

HI. Derived Proteins.
1. PRIMARY PROTEIN DERIVATIVES.

A. Proteans.

B. Metaproteins.

C. Coagulated proteins.

2. SECONDARY PROTEIN DERIVATIVES.

A. Proteoses.

B. Peptones.

C. Peptides.

Classification of proteins adopted by the British Medical Association:

I. Simple Proteins.

1. Protamines.

2. Histones.

3. Albumins.

4. Globulins.

5. Glutelins.

6. Alcohol-soluble proteins.

7. Scleroproteins.

8. Phosphoproteins.

II. Conjugated Proteins.

1. Glucoproteins.

2. Nucleoproteins.

3. Chromoproteins.

in. Products of Protein Hydrolysis.

1. Infraproteins.

2. Proteoses.

3. Peptones.

4. Polypeptides.

To this summary must be added that we often find in the investiga-
tions of animal fluids and tissues protein substances which do not fall
in with the above schemes, or are classified only with difficulty. At the
same time it must be remarked that bodies will be found which seem
to rank between the different groups, hence it is very difficult to sharply
divide these groups.

94 THE PROTEIN SUBSTANCES.

I. Simple Proteins.

A. True Albuminous Bodies.

The albuminous bodies are never-failing constituents of the animal
and vegetable organisms. They are especially found in the animal
body, where they form the solid constituents of the muscles and of the
blood-serum, and they are so generally distributed that there are only
a few animal secretions and excretions, such as the tears, the perspira-
tion, and perhaps the urine, in which they are entirely absent or occur
only in traces.

All albuminous bodies contain carbon, hydrogen, nitrogen, oxygen
and sulphur;1 a few contain also phosphorus. Iron is generally found
in traces in their ash. The elementary composition of the different
albuminous bodies varies a little, but the variations are within relatively
close limits. For the better-studied animal albuminous bodies the
following composition of the ash-free substance has been found:

C 50.5 —54.6 per cent

H 6.5 — 7.3 "

N 15.0 —17.6 "

S 0.5 — 2.2 "

P 0.42— 0.85 "

O 21.50—23.50 "

The animal proteids are odorless, tasteless, and ordinarily amorphous.
The crystalloid spherules (Dotterplattcheri) occurring in the eggs of certain
fishes and amphibians, do not consist of pure proteids, but of albuminous
bodies containing large amounts of lecithin, which seem to be combined
with mineral substances. Crystalline proteids2 have been prepared
from the seeds of various plants, and crystallized animal proteids (see
seralbumin and ovalbumin, Chapters V and XII) can be readily pre-
pared. In the dry condition the proteids appear as white powders,
or when in thin layers as yellowish, hard, transparent plates. A few
are soluble in water, others only soluble in salt or faintly alkaline or acid
solutions, while others are _ insoluble in these solvents. Solutions of
proteids are optically active and turn the plane of polarized light to the
left. All proteids when burned leave an ash, and it is therefore ques-

1 See foot-note 1, p. 80.

2 See Maschke, Journ. f. prakt. Chem., 74; Drechsel, ibid. (N. F.), 19; Griibler,
iUd. (N. F.), 23; Ritthausen, ibid. (N. F.), 25; Schmiedeberg, Zeitschr. f. physiol.
Chem., 1; Weyl, 1; ibid., 1.

PROPERTIES. 95

tionable whether there exists any proteid body which is soluble in water
without the aid of mineral substances. Nevertheless it has not beer,
thus far successfully proved that a native proteid body can be prepared
perfectly free from mineral substances without changing its constitution
or its properties.1

As previously stated, the albuminous bodies are amphoteric elec-
trolytes, and are polyacidic bases as well as polybasic acids. The base-
and acid-combining powers of various proteids have been the subject of
numerous investigations which cannot be given in short. In regard to
various methods used in such investigations as well as to the dissociation
of protein salts we refer especially to the work of T. B. ROBERTSON .2

The proteids can be salted out from their neutral solutions by neutral
salts (NaCl, Na2S(>4, MgSCU, [NEUbSO^ and many others) in sufficient
concentrations. By this salting out the properties remain unchanged
and the process is reversible, as on diminishing the concentration of the
salt the precipitate redissolves. The various proteids act in an entirely
different manner toward the same salt, and also for one and the same
proteid the behavior toward different neutral salts is different, as some
cause a precipitate, while others on the contrary do not precipitate.

The behavior of various proteids with one and the same salt, such
as MgS04 or (NH^SCU, is often made use of in the isolation of the
proteid, and special methods of separation are based upon fractional
precipitation. It has been shown that these methods may lead to great
errors, and give good results only under special conditions.3

The conditions are different from those of salting out, when the pro-
teid solution is precipitated by salts of the heavy metals. Here the
precipitates (often called metallic albuminates) are not true combina-
tions in constant proportions, but are rather to be considered as loose
adsorption compounds of the proteid with the salt.4 These reactions
are irreversible in so far that dilution with water or removal of the salt
by means of dialysis does not restore the unchanged proteid. On the
other hand the precipitate, at least in certain cases may be redissolved
in an excess of the salt solution or of the proteid solution, and in this
sense the process is a reversible one.

1 See E. Harnack, Ber. d. d. chem. Gesellsch., 22, 23, 25, and 31; Werigo, Pfliiger's
Archiv, 48; Biilow, ibid., 58; Schulz, Die Grosse des Eiweissmolekiils, Jena, 1903.

2 Ergeb. d. Physiol. 10; Journ. of physical Chem., 14, 15, and Journ. of biol. Chem.,
9.

3 SeeCohnheim, Chemie der Eiweisskorper, 3. Aufl., 1911; Pinkus, Journ. of Physiol.,
27; Pauli, Hofmeister's Beitrage, 3, p. 225; Halsam, Journ. of Physiol., 32.

4 See Galeotti, Zeitschr. f. physiol. Chem., 40, 42, 44, and 48 and Bonamartini and
Lombardi, ibid., 58. See also the opposed views of Lippich, ibid, 74.

96 THE PROTEIN SUBSTANCES.

The precipitation of proteids and also other soluble proteins by salts
stands in close relation to their colloidal nature, and in this connec-
tion we refer to what has been said in Chapter I. The proteids do not
as a rule diffuse through animal membranes, or only to a very slight
extent, and hence have in most cases a pronounced colloidal nature in
GRAHAM'S sense. They belong to the hydrophile colloids; their solu-
tions show properties in common with those of typical colloids and also
true solutions. Certain of them, especially the peptones and a few
proteoses, which will be discussed later, seem to occupy an intermediate
position, as their solutions are characterized by a lesser viscosity and
greater diffusibility and nitration ability, are not readily precipitable
by alcohol or coagulable by heat, and are only partially precipitable
by salts.

The solutions (or suspensions) of proteids in water, the proteid hydro-
sols, are converted by various means into proteid hydrogels. Of these
means we must specially mention the following: flocking out with salts,
precipitation with alcohol, gelatinization of a gelatin solution on cool-
ing, and coagulation by the action of enzymes or heat.

Those proteids which occur, according to the common views, pre-
formed in the animal fluids and tissues, and which have been isolated
from these by indifferent chemical means without losing their original
properties, are called native proteids. New modifications having other
properties can be obtained from the native proteids by heating, by the
action of various chemical reagents such as acids, alkalies, alcohol, and
others, as well as by proteolytic enzymes. These new proteids are called
modified (" denaturierte ") proteids, to differentiate them from the native
proteids.

The precipitation with alcohol is a reversible reaction, as the pre-
cipitate redissolves on subsequent dilution with water. The proteids
are changed by the action of alcohol, some readily and quickly, others
with difficulty and very slowly; the proteid then becomes insoluble in
water and is modified.

On heating a solution of a native proteid it is modified at a different
temperature for each different proteid. With proper reaction and other
favorable conditions, for instance in the presence of neutral salts, most
proteids can in this way be precipitated in a solid form as coagulated
proteid. The hydrosol is converted into hydrogel, but as a modification
takes place, this process is irreversible. The temperature at which
coagulation occurs is a variable one for the same protein under different
conditions of the experiment. The various temperatures at which
coagulation of different proteids occurs in neutral solutions containing
salt have in many cases given us good means for detecting and separating
proteids. The views in regard to the use of these means are somewhat

PROPERTIES. 97

divided 1 and the same applies to the question as to manner of heat
coagulation and the conditions under which it takes place.

The heat coagulation of a protein solution is dependent upon the hydrogen
ion concentration of the solution. According to MICHAELIS and co-workers
the optimum of the hydrogen ion concentration falls in the coagulation of a
protein solution (precipitation of the modified protein) with the isoelectric point
of the solution and the optimum of the flocking is not changed in regard to the
hydrogen ion concentration by changes in the protein concentration. According
to SORENSEN and JuRGENSEN,2 on the contrary the optimal hydrogen ion con-
centration is the same as contained in a solution of the pure protein in pure
water caused by the electrolytic dissociation of this protein and is therefore
independent of the protein concentration. This hydrogen ion concentration,
according to these workers diminishes during the heat coagulation which they
consider as a proof of the diminution in the protein concentration of the solution.

A modification can be brought about also by the action of acids,
alkalies, or salts of the heavy metals, in certain cases by water alone,
and also by the action of alcohol, chloroform (SALKOWSKI), and ether,
by violent shaking (RAMSDEN3), etc.

An adsorption of proteids by a suspension colloid such as silicic acid,
colloidal ferric hydroxide and kaolin, can easily take place, and indeed the
proteid of a solution can be removed by the use of colloidal ferric hydrox-
ide or shaking with kaolin (RoNA and MICHAELIS 4) . That the proteids
can serve as preventives in the precipitation of suspension colloids has
been mentioned in Chapter I. In the same manner a mastic suspension
is protected from the precipitating action of an electrolyte by an excess of
a proteid solution, while the reverse may be brought about, namely, a
proteid solution can be precipitated by a large quantity of mastic emulsion
in the presence of a proportionately small amount of electrolyte. The
method for the removal of proteid from solutions, as suggested by
MICHAELIS and RoNA,5 is based upon this behavior.

We have already discussed in Chapter I the electric charge of the
proteins under various conditions and the migration of these in electric
fields of currents.

1 See Halliburton, Journ. of PhysioL, 5 and 11; Corin and Berard, Bull, de 1'Acad-
roy. de Belg., 15; Haycraft and Duggan, Brit. Med. Journ., 1890, and Proc. Roy.
Soc. Edin., 1889; Corin and Ansiaux, Bull, de PAcad. roy. de Belg., 21; L. Fredericq,
Centralbl. f. Physiol., 3; Haycraft, ibid., 4; Hewlett, Journ. of PhysioL, 13; Duclaux,
Annal. Institut Pasteur, 7. In regard to the relationship of the neutral salts to the
heat coagulation of albumins see also Starke, Sitzungsber. d. Gesellsch. f. Morph. u.
Physiol. in Munchen, 1897; Pauli, Pfliiger's Arch., 78.

2 See Ergeb. d. PhysioL, 12 which contains the pertinent literature.

3 See Salkowski, Zeitschr. f. physiol. Chem., 31; Fr. Kriiger, Zeitschr. f. Biologie,
41; Loew and Aso, Bull. Coll. Agric., Tokio, 4; Ramsden, Zeitschr. f. physik. Chem.,
47 and Arch. f. (anat. u.) PhysioL, 1894.

4 Biochem. Zeitschr., 5.

6 Biochem. Zeitschr., 2, 3 and 4.

98 THE PROTEIN SUBSTANCES.

The determination of the molecular weight of the proteids has been
attempted by various methods which are more or less uncertain.1 There
is no doubt that the molecular weight of the proteids is very high, but
the statements about the size vary considerably. For the true proteids
thus far investigated, values ranging from 4000 — 6000 — 10,000 have
been found.

The general reactions for the proteids are very numerous, . but only
the most important will be given here. To facilitate the study of these,
they have been divided into the two following groups. It must be
remarked that the precipitation reactions are not only applicable for
the soluble true proteids but also, more or less, for other soluble proteins
in general. The color reactions are applicable to all soluble or insoluble
proteins with few exceptions, which will be mentioned later.

Precipitation Reactions of the Proteid Bodies.

1. Coagulation Test. An alkaline proteid solution does not coagulate
on boiling, and a neutral solution only partly and incompletely; the reac-
tion must therefore be acid for coagulation. The neutral liquid is first
boiled and then the proper amount of acid added carefully. A flocculent
precipitate is formed, and with proper technique the filtrate should be
water-clear. If dilute acetic acid be used for this test, the liquid must
first be boiled and then 1, 2, or 3 drops of acid added to each 10-15 cc.,
depending on the amount of proteid present, and boiled before the addi-
tion of each drop. If dilute nitrio acid (25 per cent) be used, then to
10-15 cc. of the previously boiled liquid ")5-20 drops of the acid must
be added. If too little nitric acid be adde^f, a soluble combination of the
acid and proteid is formed, which is precipitated by more acid. A pro-
teid solution containing a small amount of salts must first be treated with
about 1 per cent NaCl, since the heating test may fail, especially on using
acetic acid, in the presence of only a slight amount of proteid.

2. Precipitation by Alcohol. The solution must not be alkaline,
but must be either neutral or faintly acid. It must, at the same time,
contain sufficient quantity of neutral salts.

3. Neutral Salts, such as Na2SC>4 or NaCl, when added to saturation
precipitate certain proteids but not others. Ammonium sulphate when
dissolved to saturation in the liquid is considered as the general pre-
cipitant for proteids. In the presence of free acetic or hydrochloric
acid the above-mentioned salts, NaCl or Na2S04, in sufficient con-
centration, are also general precipitants for the proteids.

4. Precipitation by Metallic Salts such as copper sulphate, ferric
chloride, neutral and basic lead acetate (in small amounts), mercuric

1 See especially F. N. Schulz, Die Grosse des Eiweissmoleciile, Jena, 1903.

COLOR REACTIONS. 99

chloride and others. On this is based the use of proteids as antidotes
in poisoning with metallic salts.

5. Precipitation by Mineral Adds at Ordinary Temperatures. The
proteids are precipitated by the three ordinary mineral acids in proper
amounts, but not by orthophosphoric acid. If nitric acid be placed in a
test-tube and the proteid solution be allowed to flow gently thereon, a
white opaque ring of precipitated proteid will form where the two liquids
meet (HELLER'S albumin test).

6. Precipitation by the so-called Alkaloid Reagents. To these belong
the precipitation by metaphpsphoric acid and by hydroferrocyanic acid,
which is carried out by the aid of potassium ferrocyanide in a liquid
containing acetic acid; precipitation by phosphotungstic acid or phos-
phomolybdic acid in the presence of free mineral acids; precipitation
by potassium-mercuric iodide or potassium-bismuth iodide in solutions
acidified with hydrochloric acid; precipitation by tannic acid in acetic
acid solutions. The absence of neutral salts or the presence of free
mineral acids may prevent the appearance of the precipitate, but after
the addition of a sufficient quantity of sodium acetate the precipitate
will in both cases appear; precipitation by picric acid in solutions acid-
ified by organic acids. Proteids are also precipitated by trichloracetic
acid in 2-5 per cent solutions, by phenol, salicyl sulphonic acid, nucleic
acid, taurocholic acid and by chondroitin sulphuric add in acid solutions.

Color Reactions for Proteid Bodies.

1. Millon's Reaction.1 A solution of mercury in nitric acid contain-
ing some nitrous acid gives a precipitate with proteid solutions which
at the ordinary temperature is slowly, but at the boiling-point more
quickly, colored red; and the solution may also be colored a feeble
or bright red. Solid albuminous bodies, when treated by this reagent,
give the same coloration. This reaction is due to the tyrosine and is
also given by other monohydroxyl benzene derivatives. According to
O. NASSE 2 it is best to use a solution of mercuric acetate which is treated
with a few drops of a 1 per cent solution of potassium or sodium nitrite;
previous to use a few drops of acetic acid are added.

2. Xanthoprotdc Reaction. With strong nitric acid the albuminous
bodies give, on heating to boiling, yellow flakes or a yellow solution.

1 The reagent is prepared in the following way: 1 pt. mercury is dissolved in 2 pts.
nitric acid (of sp.gr. 1.42), first cold and then warmed. After complete solution of
the mercury add 1 volume of the solution to 2 volumes of water. Allow this to stand
a few hours and decant the supernatant liquid.

2 See O. Nasse, Sitzungsb. d. Naturforsch. Gesellsch. zu Halle, 1879, and Pfluger's
Arch., 83; see also Vaubel and Blum, Journ. f. prakt. Chem. (N. F.), 57.

100 THE PROTEIN SUBSTANCES.

After making alkaline with ammonia or alkalies the color becomes orange-
yellow, due to the nitroderivatives of the benzene and indol groups.

3. Adamkiewicz' s Reaction. If a little proteid is added to a mixture
of 1 vol. concentrated sulphuric acid and 2 vols. glacial acetic acid a
reddish-violet color is obtained slowly at ordinary temperatures, but
more quickly on heating. According to HOPKINS and COLE 1 this reaction
takes place only on using glacial acetic acid containing glyoxylic acid.
According to them it is better to use a solution of glyoxylic acid, which
can be readily prepared by adding sodium amalgam to a concentrated
solution of oxalic acid and filtering after the discharge of the gas. A
dilute aqueous solution of the acid or some of the solid acid is added to the
proteid solution and sulphuric acid allowed to flow down the side of the
test-tube, when the reddish-violet color will appear at the point of con-
tact of the two liquids or on shaking the mixture. This color reaction,
which is generally called the ADAMKIEWICZ-HOPKINS reaction depends
upon the tryptophane and therefore gelatin (which does not contain
any tryptophane) does not give this reaction.

As further color reactions we will mention: 4. Biuret Test. If a
proteid solution be first treated with caustic potash or soda and if then
a dilute copper-sulphate solution be added drop by drop, first a reddish
then a reddish-violet, and lastly a violet-blue, color is obtained. 5. Pro-
teids are soluble on heating with concentrated hydrochloric acid, produc-
ing a violet color, and when they are previously boiled with alcohol and
then washed with ether (LIEBERMANN 2) they give a beautiful blue
solution. This blue color is due, according to CoLE,3 to a contamination
of the ether with glyoxylic acid, which reacts with the tryptophane
groups split off by the hydrochloric acid. The violet color obtained
with proteins not purified with ether is also considered as a tryptophane
reaction with the furfurol (oxymethylfurfurol) formed from the hexose
containing protein by the action of the concentrated hydrochloric
acid. Reaction 6 with concentrated sulphuric acid and sugar (in small
quantities) is explained in the same way. The beautiful red coloration
is connected with the formation of furfurol from the sugar. 7. With
p-dimethylaminobenzaldehyde and concentrated sulphuric acid thepro-
teids give a beautiful reddish- violet or deep- violet coloration (O. NEU-
BAUER and E. RoHDE4). Other . aldehydes also give color reactions
by virtue of the tryptophane group in proteins. Other reactions are 8;
ARNOLD'S reaction 5 is a purple-violet coloration which the proteins give

1 Proceed. Roy. Soc., 68.

2 Centralbl. f. d. med. Wissensch., 1887.
8 Journ. of Physiol., 30.

4 Zeitsche. f. physiol. Chem., 44.

6 Arnold, Zeitschr. f . physiol. Chem., 70.

PKOTEIN REACTIONS. 101

with sodium nitroprusside and ammonia. This reaction is not given by
all proteins and is due to the cystine groups. 9, ABDERHALDEN and
SCHMIDT'S reaction with triketohydrindenhydrate which gives a blue
coloration on boiling. The triketohydrindenhydrate (also called " Nin-
hydrin") reacts with all compounds which have an amino group in
the a-position to the carboxyl, is according to ABDERHALDEN and
SCHMIDT 1 an excellent reagent for the detection of dialyzable amino-
acids and non-biuret giving amino-acid derivatives. They have been
able to detect by this reagent such non-biuret giving substances in the
dialysate on the dialysis of different animal fluids. They have also
determined the delicacy of this reagent with different amino-acids.

The biuret reaction is not only given by protein substances, but also by many
other bodies. According to H. SCHIFF 2 this reaction occurs with those bodies
containing amino groups, CONH2, CSNH2, C(NH)NH2 or also CH2NH2, united
either directly by their carbon atoms or by means of a third carbon or nitrogen
atom. As examples of such bodies we can mention several diamines or amino-
amides, such as oximide, biuret, glycinamide, a- and /3-aminobutyramide, aspartic-
acid amide, etc., although we are not certain as to the conditions necessary for the
bringing about of this reaction. The biuret reaction alone is therefore no proof
as to the protein nature of a substance — for example, urobilin gives a very similar
color reaction — and a protein substance can still retain its protein nature, as by
the action of nitrous acid or by a splitting off of ammonia, although it does not
give the biuret reaction.

The delicacy of the various reagents differs for the different proteids,
and on this account it is impossible to give the degree of delicacy for
each reaction for all proteids. Of the precipitation reactions, HELLER'S
test (if we eliminate the peptones and certain proteoses) is recommended
in the first place for its delicacy, though it is not the most delicate reac-
tion, and because it can be performed so easily. Among the precipita-
tion reactions, that with basic lead acetate (when carefully and exactly
executed) and with alcohol and the reactions given under 6, are the most
delicate. The color reactions 1 to 4 show great delicacy in the order in
which they are given.3

No proteid reaction is in itself characteristic, and, therefore, in testing
for proteids one reaction is not sufficient, but a number of precipitation
and color reactions must be employed.

For the quantitative estimation of coagulable proteids the determina-
tion by boiling with acetic acid can be performed with advantage, for
by operating carefully, it gives exact results. Treat the proteid solution
with a 1-2 per cent common-salt solution, or if the solution contains
large amounts of proteid dilute with the proper quantity of the above
salt solution, and then carefully neutralize with acetic acid. Now deter-

1 Zeitschr. f. physiol. Chem., 72 and 85.

2 Ber. d. d. chem. Gesellsch., 29 and 30.

3 In regard to the precipitation and color reactions of proteids with aniline dyes
see Heidenhain, Pfliiger's Arch., 90, 96.

102 THE PROTEIN SUBSTANCES.

mine the quantity of acetic acid necessary to completely precipitate
the proteids in small measured portions of the neutralized liquid which
have previously been heated on the water-bath, so that the nitrate does
not respond to HELLER'T test. Now warm a larger weighed or meas-
ured quantity of the liquid on the water-bath, and add gradually the
required quantity of acetic acid, with constant stirring, and continue
heating for some time. Filter, wash with water, extract with alcohol
and then with ether, dry, weigh, incinerate, and weigh again. With
proper work the nitrate should not give HELLER'S test. This method
serves in most cases, and especially so in cases where other bodies are
to be quantitatively estimated in the nitrate.

In many cases good results may be obtained by precipitating all the
proteid with tannic acid and determining the nitrogen in the washed
precipitate by means of KJELDAHL'S method. On multiplying the quan-
tity of nitrogen found by 6.25 we obtain the quantity of proteid. Many
other methods for the quantitative estimation of proteins have been
suggested.

The removal of proteids from a solution may in most cases be per-
formed by boiling with acetic acid. Small amounts of proteid which
remain in the nitrates may be separated by boiling with freshly pre-
cipitated lead carbonate or with ferric acetate, as described by HOF-
MEiSTER.1 If the liquid cannot be boiled, the proteid may be precipi-
tated by the very careful addition of lead acetate, or by the addition
of alcohol. If the liquid contains substances which are precipitated
by alcohol, such as glycogen, then the proteid may be removed by tri-
chloracetic acid as suggested by OBERMAYER and FRANKEL.2 Recently
MICHAELIS and RONA have suggested a method for the removal of proteids
by using kaolin, colloidal ferric hydrate or a mastic emulsion. The
principle of these methods has already been given on page 97 and in
regard to the practical execution of the method we refer to the works
there cited.

In the precipitation of proteid as well as the quantitative estimation by means
of heat, it must be borne in mind, as shown by SPIRO,S that several nitrogenous
substances, such as piperidine, pyridine, urea, etc., disturb the coagulation of the
proteids.

Synopsis of the Most Important Properties of the Different Groups of

Albuminous Bodies.

As it is not possible to base the classification of the different proteid
groups according to their constitution, we are obliged to make use of
their different solubilities and precipitation properties in their general
characterization. As there exist no sharp differences between the various
groups in this regard it is impossible to draw a sharp line between them.

Albumins. These bodies are soluble in water in neutral reaction and
are not precipitated by the addition of a little acid or alkali. They are

1 Zeitschr. f. physiol. Chem., 2 and 4.

2Obermayer, Wien. med. Jahrb., 1888; Frankel, Pfluger's Arch., 52 and 55.

3 Zeitschr. f. physiol. Chem., 30.

GLOBULINS. 103

precipitated by the addition of large quantities of mineral acids or metallic
salts. Their solution in water coagulates on boiling in the presence of
neutral salts, but a weak saline solution does not. If NaCl or MgS04
is added to saturation to a neutral solution in water at the normal tem-
perature or at 30° C. no precipitate is formed; but if acetic acid is added
to this saturated solution the albumins readily separate. When ammonium
sulphate is added to one-half saturation the albumin solutions are not
precipitated at ordinary temperatures. Of all the native proteids the
albumins are the richest in sulphur, containing from 1.6 per cent to 2.2
per cent. So far as they have been investigated they do not yield any
glycocoll on acid hydrolysis.

Globulins. These substances are, as a rule, insoluble in water, but
dissolve in dilute neutral salt solutions. The globulins are precipitated
unchanged from these solutions by sufficient dilution with water, and
on heating they coagulate. The globulins dissolve in water on the addi-
tion of very little acid or alkali, and on neutralizing the solvent they
precipitate again. The solution in a minimum amount of alkali is pre-
oipitated by carbon dioxide, but the precipitate may in certain cases be
redissolved by an excess of the precipitant. The neutral solutions of the
globulins containing salts are partly or completely precipitated on satura-
tion with NaCl or MgSO4 in substance at normal temperatures, depending
upon the kind of globulin. The globulins are completely precipitated
by half-saturating with ammonium sulphate. The globulins contain an
average amount of sulphur generally not below 1 per cent. As a differ-
ence between the albumins and globulins the latter yield glycocoll among
the hydrolytic cleavage products, and according to OBERMAYER and
WILLHEIM x they contain fewer NH2 groups at the end of the chain, as
determined by formol titration, as compared to the total number of N-
atoms.

A sharp line cannot be drawn between the albumins and globulins
from their properties and this is shown from the researches of MoLL,2
which show that by the action of dilute alkalies and warmth upon
seralbumin it attains the properties of serglobulin. It is evident that
we are here dealing with a change of the external properties of the
albumins to a greater similarity to those of the globulins, and not with
a true transformation of the albumin, which is free from glycocoll, into
globulin which contains glycocoll. The same follows from the observa-
tions of others.3 This is an instructive example of the subordinate impor-
tance the solubility and precipitation properties have in the differentia-
tion of various groups of proteids.

1 Bioch. Zeitschr., 38.

2 Moll, Hofmeister's Beitrage, 4 and 7; also Breinl, Arch. f. exp. Path. u. Pharm., 65.
•Obermayer and Willheim, 1. c.; R. Gibson, Journ. of biol. Chem., 12.

104 THE PROTEIN SUBSTANCES.

It is just as difficult to draw a sharp line between the globulins and
albuminates as it is between the globulins and albumins. Several globu-
lins are very readily changed by the action of very little acid, as also by
standing under water when in a precipitated condition, into albuminates,
and then become insoluble in neutral salt solutions. OssoRNE,1 who has
closely studied this property in connection with edestin (from hemp-seed) ,
considers the globulin, " globan," which has been made insoluble in salt
solution, as an intermediate step in the formation of the albuminate which is
produced by the hydrolytic action of the H ions of water or of the acid.

Phosphoproteins are a group of phosphorized proteids which occur
extensively in the animal and plant kingdoms and which include the
nucleoalbwnins and the little-studied lecithalbumins.

Nucleoalbumins. These proteids behave like rather strong acidsr
are nearly insoluble in water, but dissolve easily with the aid of a little
alkali and, in the entire absence of phosphatides, contain also
phosphorus. Certain of the nucleoalbumins, resemble the globulins by
their solubility and precipitation properties. Others resemble the
albuminates, but differ from both of these groups by containing phos-
phorus. They stand close to the nucleoproteins by their content of
phosphorus, but differ from these in not yielding any purine bases on
cleavage. It has not yet been found possible to obtain from the neucleo-
albumins any proteid-free pseudonucleic acids corresponding to the
nucleic acids, but only acids rich in phosphorus, which always give the
proteid reactions.2 For this reason the nucleoalbumins cannot be classed
as compound proteins. In peptic digestion a proteid rich in phosphorus
can be split off from most nucleoalbumins, and this has been called
para- or pseudonuclein. The claim made that the pseudonuclein is a
combination of proteid with metaphosphoric acid has been shown to be
incorrect by the investigations of GIERTZ.S

! The separation of pseudonuclein in peptic digestion is no doubt characteristic
of the nucleoalbumin group, but the non-appearance of the pseudonuclein pre-
cipitate does not entirely exclude the presence of a nucleoalbumin. The extent
of such a formation is dependent upon the intensity of the pepsin digestion, the
degree of acidity, and the relation between the nucleoalbumins and the digestive
fluids. The separation of a pseudonuclein may, as shown by SALKOWSKI,
not occur even in the digestion of ordinary casein, and WR6BLEWSKI and others4
did not obtain any pseudonuclein at all in the digestion of the casein from human
milk. The most essential characteristic of this group of proteids is that they con-
tain phosphorus, and that the purine bases are absent in their cleavage products.

1 Zeitschr. f. physiol. Chem., 33.

2 Levene and Alsberg, ibid., 31; Salkowski, ibid., 32; Levene, ibid., 32; A. Reh,
Hofmeister's Beitrage, 11; Dietrich, Bioch. Zeitschr., 22.

3 Giertz, Zeitschr. f. physiol. Chem., 28.

4Salkow8ki, Pfliiger's Arch., 63; Wr6blewski, Beitrage zur Kenntnis des Frauen-
kaseins, Inaug.-Diss., Bern, 1894.

LECITHALBUMINS. 105

The nucleoalbumins are often confounded with nucleoproteins and
also with phosphorized glucoproteins. From the first class, they differ
by not yielding any purine bases when boiled with acids, and from the
second group by not yielding any reducing substance on the same treat-
ment. The best studied member of this group is the casein of milk,
which will be discussed in detail in chapter XIII.

NEUBERG and POLLAK l have artifically prepared phosphoproteins by the
action of phosphorus oxychloride upon an alkaline solution of lactalbumin or blood
globulin. The product obtained from lactalbumin was rather close to casein
in regard to composition and other properties.

Lecithalbumins. In the preparation of certain protein substances,
products are often obtained containing lecithin, and this lecithin (see
the phosphatides, Chapter IV) can be removed only with difficulty or in-
completely by a mixture of alcohol and ether. Ovovitellin (Chapter XII)
is such a protein body containing considerable lecithin, and HOPPE-SEYLER
considers it a combination of proteid and lecithin. Similar substances
occur in fish-eggs. These last lecithalbumins often have the solubilities
of the globulins and are readily soluble in dilute salt solutions. The
behavior of the nucleoalbumin of the eggs of the perch shows how easily
this solubility may be changed. This nucleoalbumin, which contains con-
siderable amounts of lecithin, is readily soluble in dilute NaCl solution,
but at ordinary temperatures it is changed by 0.1 per cent HC1 almost
instantaneously and without splitting off lecithin, so that it becomes in-
soluble in dilute salt solutions (HAMMARSTEN) . LIEBERMANN 2 has obtained
proteids containing lecithin as an insoluble residue on the peptic digestion
of the mucous membrane of the stomach, liver, kidneys, lungs, and spleen*
He. considers them as combinations of proteid and lecithin and calls them
lecithalbumins. Further investigation of these bodies is desirable. £$

MAYER and TERROINE 3 have shown that from lecithin emulsified in water and
a dialyzed solution of ovalbumin or dialyzed blood serum a precipitate can be
obtained which has some similarity to the lecithalbumins, but which in other
respects is so strikingly different that we are not justified in calling this pre-
cipitate lecithalbumin.

Nothing characteristic has thus far been found which differentiates
this group from others in the quantity of amino-acids split off on hydrol-
ysis. The members of this group differ essentially among themselves,
e.g., vitellin yields glycocoll while casein does not.

In order to give a review of the three above-mentioned groups of pro-
teids we give (page 106) a tabulation of the amounts of the amino-acids

1 Ber. d. d. chem. Gesellsch., 43 and Bioch. Zeitschr., 26.

2 Hoppe-Seyler, Med. chem. Untersuch., 1868; also Zeitschr. f. phyeiol. Chem., 13,
479; Hammarsten, Skand. Arch. f. Physiol., 17; Liebermann, Pfliiger's Archiv, 50
and 54.

3 Compt. rend. soc. biol., 62.

106

THE PROTEIN SUBSTANCES.

obtained on cleavage, but we must bear in mind that the figures, because
of the difficulty in the quantitative estimation, are not quite exact, but
must be considered as minimum values. As a representative of the glob-
ulin group we give fibrin, which is a coagulated globulin; and as repre-
sentative of the phosphoprotein group, besides casein also ovovitellin,
although not quite pure. The results are based on 100 parts of the
substances.

The proteins occurring in the plant kingdom either in the seeds or
tuberes belong chiefly to the globulins, which correspond essentially in
properties to the animal globulins. Besides these many other less
abundant proteins occur, which like the albumins are soluble in water,
while toward certain salts they behave like globulins. It is not clear
whether the phosphorized plant proteids contain their phosphorus as
impurities or whether they are the same as the animal phosphoproteins.
In seeds there occur also proteins, which are not represented in the animal
kingdom and of these we must especially mention the prolamines. They
are soluble in alcohol and besides this they are characterized by not
yielding any lysine on hydrolysis.

Lact-
albumin.1

Ser-
albumin.1

Ov-
albumin.

Ser-
globulins.2

Fibrin.*

Casein.'

Vitellin.M

Glycocoll

0 0

0 0

0.0

3.5

3 0

0 00

1 I8

Alanine

2 5

2 7

2.2

2.2

3.6

1 50

0 759

Valine

0.9

2.5

1.0

7 20

2 408

Leucine

19 4

20 0

10 7

18 7

15 0

9 35

11 O8

Isoleucine

1 4310

Serine

0 6

0 8

0 50

Aspartic acid

1 0

3 1

2 2

2 5

2 0

1 39

2 139

Glutarnic acid.

10 1

7 7

9 1

8 5

10 4

15 55

12 95*

Cystine
Phenylalanine
Tyrosine ,
Proline

2.4

0.85
4.0

2.53
3.1
2.1
1.04

0.33
5.17
1.77
3.56

1.513
3.8
2.5
2 8

1.173
2.5
3.5
3 6

0.07
3.2*0
4.50
6 70

2^88
3.379
4 189

Oxyproline

0 23

Tryptophane
Histidine

3.0711

—

1.71

—

—

1.50
2 50

1 909

Arginine

4 91

3 O6

3 81

7 459

Lysine

3 76

4 O6

5 95

4 819

Ammonia

1 34

1 60

1 259

M

1 Abderhalden and H. Pribram, Zeitschr. f. physiol. Chem., 21.

2 Abderhalden, Lehrb. d. physiol. Chem., 1909.
8 K. Morner, Zeitschr. f. physiol. Chem., 34.

4 Osborne and co-workers, Amer. Journ. of Physiol, 24.

6 Abderhalden and Voitinovici, Zeitschr. f. physiol. Chem., 52.

8 Kutscher, Endprodukte der Trypsin Verdauung, Habit. Schrift., Marburg, 1899.

7 Osborne and Guest, Journ. of biol. Chem., 9.

8 Abderhalden and Hunter, Zeitschr. f. physiol. Chem., 48.

9 Osborne and D. B. Jones, Amer. Journ. of Physiol., 24.

10 Levene and D. v. Slyke, Journ. of biol. Chem., 6.

11 Colorimetric determinations by Fasal, Bioch., Zeitschr, 44.

COAGULATED PROTEINS.

107

In the tabulation of the hydrolytic products of plant proteins we
give edestin, of the hemp-seed, and legumin of the pea as examples of
globulins. The other three, hordein of barley, gliadin of wheat and zein
from corn belong to the prolamine group.

Edestin.1

Legumin.4

Hordein.6

Gliadin.*

Zein.w

Glycocoll

3.8

0.38

0.0

0.689

0.0

Alanine

3.6

2.08

0.43

2.0

9.79

Valine

5 62

1 O6

0 13

3 34

1.88

Leucine . . .

20 9

8 0

5.67

6 62

19.55

Serine ....

0.33

0.53

0.13

1.02

Aspartic acid

4.5

5.3

__

0.58

1.71

Glutamic acid

18. 743

13.8

43 497

43.66

26.17

Cystine

0.25

0.45

Phenvlalanine

2.4

3.75

5.03

2.35

6.55

Tyrosine

2.1

1.55

1.67

1.20

3. 55-10. 111

Proline

1 7

3 22

13 73

13 22

9 04

Oxyproliii6

2 0

Tryptophanc

0 3812

__._

1.00

Histidinc

1.1

2.42

1 28

0 61

0 82

Arsjinine . .

11.7

10.12

2.16

3.16

1 55

Lysin6 ....

1.0

4.29

0.00

0 00

0 00

Ammonia

1.49

4.87

5.22

3.61

Coagulated Proteins. Proteins may be converted into the coagu-
lated condition by different means: by heating, by the action of alcohol,
especially in the presence of neutral salts, by chloroform, ether, and
metallic salts, and by the prolonged shaking of their solutions and in
certain cases, as in the conversion of fibrinogen into fibrin (Chapter V),
by the action of an enzyme. The nature of the processes which take
place during coagulation is unknown. The coagulated albuminous
bodies are insoluble in water, in neutral salt solutions, and dilute acids
or alkalies, at normal temperature. They are dissolved and converted
into albuminates by the action of dilute acids or alkalies, especially
on heating. /

Coagulated proteins also seem to occur in animal tissues. We find,
at least in many organs such as the liver and other glands, proteins

1 Abderhalden, Zeitschr. f. physiol. Chem., 37 and 40.

2 Levene and D. v. Slyke, Journ. of biol. Chem., 6.

3 Osborne and Biddle Amer. Journ. of Physiol, 26.

4 Osborne and Clapp, Journ. of biol. Chem. 3.
6 Abderhalden and Babkin, ibid., 47.

6 Osborne and Clapp, Amer. Journ. of Physiol., 19.

7 Osborne and Jones, ibid., 26.

8 Osborne and Guest, Journ. of biol. Chem., 9.

9 Abderhalden and Samuely, Zeitschr. f. physiol. Chem., 44.

10 Osborne, Jones and Clapp, Amer. Journ. of Physiol., 26.

11 Kutscher, Zeitschr. f. physiol. Chem., 38.

12 Fasal, Bioch. Zeitschr., 44.

108 THE PROTEIN SUBSTANCES.

which are not soluble in water, dilute salt solutions, or very dilute alkalies,
and only dissolve after being modified by strong alkalies.

Histones are basic proteins which stand to a certain extent between
the strongly basic protamines (see below) and the true proteins. Their
content of nitrogen varies between 16.5 and 19.8 per cent, and in certain
instances is not higher than in other proteins, especially vegetable pro-
teins. According to KOSSEL and KUTSCHER and LAWROW they are,
on the contrary, richer in basic nitrogen, and especially yield more arginine
than other proteins. KOSSEL first isolated a peculiar protein substance
from the red corpuscles of goose blood which was precipitated by ammonia,
and because of its similarity in certain regards to the peptones (in the
old sense) he called it histone. At the present time a number of very
different bodies are described as histones, such as those obtained from
nucleohistone (LILIENFELD), from haemoglobin (globin according to
;SCHULZ), from mackerel spermatozoa (scombron according to BANG),
from the codfish (gadushistone according to KOSSEL and KUTSCHER),
from the burbot (lotahistone, EHRSTROM), and from the sea-urchin
(arbacin, MATHEWS), although probably not all are true histones,
especially the above mentioned globin.1

Sulphur has been found in those histones in which it has been tested
for, but they do not, at least not all, give the lead-blackening test with
alkali and lead acetate. They give the biuret test, but as a rule only
a, faint MILLON'S reaction. The goose-blood histone first studied by
KOSSEL gives the three following reactions: First, the neutral salt-
free solution does not coagulate on boiling; second, gives a precipitate
with ammonia which is insoluble in an excess of the precipitant; third,
gives a precipitate with nitric acid which disappears on heating and
reappears on cooling.

The different histones behave differently with these three reactions,
and hence they are not specific. On the other hand, all histones seem
to be precipitated from neutral solution by alkaloid reagents, and they
also produce precipitates in protein solutions. These two reactions are
likewise not specific for the histones, as the protamines have a similar
behavior. The histones differ from the protamines by having a much
lower content of basic nitrogen, and also probably by always containing
sulphur. True proteins, as OssoRNE's2 edestan, also give these two
reactions; therefore it is impossible by qualitative tests alone to identify

1 Kossel, Zeitschr. f. physiol. Chem., 8, and Sitzungbers. der Gesellsch. zur Beford.
-d. ges Naturwiss. zu Marburg, 1897; Kossel and Kutscher, ibid., 1900, and Zeitschr.
f. physiol. Chem., 31; Lawrow, ibid., 28, and Ber. d. d. chem. Gesellsch., 34; Lilienfeld,
Zeitschr. f. physiol. Chem., 18; Schulz, ibid., 24; Bang, ibid., 27; Ehrstrom, ibid.,
32; Mathews, ibid, 23.

2 Zeitschr. f. physiol. Chem., 33.

HISTONES AND PROTAMINES. 109

a substance as a histone with positiveness. The large content of basic
nitrogen and of arginine is not a sure point of difference between histones
and other bodies. Histone yields little more than 40 per cent basic
nitrogen, while a heteroproteose yields about the same, namely, 39
per cent. Histone yields 14-15.5 per cent arginine (gadushistone),
and the lotahistone only 12 per cent. The vegetable proteid excelsin
is just as rich in arginine, namely, 14.14 per cent (OSBORNE and CLAPP1).
The characteristics of the histones, according to KOSSEL, are the above-
given reactions and the high amount of hexone bases, especially arginine.
The arginine nitrogen amounts to about 25 per cent of the total nitrogen,
the lysine N = 7-8.5 per cent and the histidine N = 1.8-4.5 per cent. No
proteids, with the exception of certain protamines, are known for the
present, which contain as much arginine and lysine as the histones. On
hydrolytic cleavage the histones, like other proteins, but unlike- the pro-
tamines, yield a large number of monamino-acids. ABDERHALDEN and
RoNA2 obtained from thymus histone the following: leucine 11.8,
alanine 3.46, glycocoll 0.50, proline 1.46, phenylalanine 2.20, tyrosine
5.20, and glutamic acid 0.53 per cent.

On pepsin digestion the histones, according to KOSSEL and PRINGLE 3
yield so-called histone-peptone, which also contains 25 per cent of the
total nitrogen as arginine nitrogen. This histone-peptone differs from
the protamines in not giving a precipitate with proteid in neutral or
ammoniacal solution, but is precipitated in neutral reactions by sodium
picrate. This property is used in its isolation.

According to KOSSEL the histones are probably intermediate bodies
between the protamines and protein bodies on the demolition of the
latter, and if this be true, then it is not to be expected that a sharp dif-
ferentiation exists between histone and proteid, and for this reason it
is hardly possible for the present to give a precise definition for the
histones.

Protamines. In close relation to the proteins stands a group of
substances, the protamines, discovered by MIESCHER, which are desig-
nated by KOSSEL as the simplest proteins or as the nucleus of the pro-
tein bodies. Thus far they have been found only in combination with
nucleic acids in fish spermatozoa,4 and the investigations of KOSSEL and
WEISSS have shown that the material from which the protamines are

1 Amer. Journ. of Physiol, 19 and 23.

2 Zeitschr. f . physiol. Chem., 41.
» Ibid., 49.

4 Nelson, Arch. f. exp. Path. u. Pharm., 59, has recently shown that the body
Called by him thymamine and prepared from the thymus glands, is a protamine, still
he has not given sufficient evidence of the protamine nature of the substance.

6 Zeitschr. f. physiol. Chem., 52.

110 THE PROTEIN SUBSTANCES.

formed, at least in the salmon, is the muscle proteid. The question has
been raised whether the protamines are true proteids or not, and whether
it would not be more correct to consider them as cleavage products of
proteid, or as fractions thereof. According to the generally accepted
view we will treat them as true proteids.

Protamine was discovered by MIESCHER l in salmon spermatozoa.
Later KOSSEL and his pupils isolated and studied similar bases from the
spermatozoa of herring, sturgeon, mackerel, and other fishes. As all
these bases are not identical, KOSSEL uses the name protamines to designate
the group, and calls the individual protamines according to their origin
salmine, clupeine, scombrine, sturine, cyprinine, cydopterine, crenilabrine
etc.

They differ materially from the proteins by the fact that they yield
chiefly diamino-acids (always abundant arginine) as cleavage products,
and only a small amount of monamino-acids. They are strongly basic
substances rich in nitrogen (about 30 per cent or more) and have high
molecular weight.

The percentage composition of these bodies has not been satisfactorily
determined. As probable formulae we have for salmine C32H54Nis04
(MIESCHER, SCHMIDEBERG, NELSON), or CsoHsyHiTOe (KOSSEL and GOTO),
for clupeine CsoH^NuCg, and for sturine CseHegHigO? (KOSSEL) or
Ca^TiNiTOg (GOTO), or according to MALENUCK C27H55Hi307 for
sturine from Accipenser Guldenstadtii. On boiling with dilute mineral
acids as also by tryptic digestion, the protamines first yield peptone-
like substances called protones, from which simple products (amino-acids)
are derived on further cleavage. All protamines yield arginine, the four
protamines salmine, clupeine, cyclopterine, and sturine, yielding 87.4,
82.2, 62.5, and 58.2 per cent respectively. In the three protamines sal-
mine, clupeine and scombrine the arginine nitrogen, according to KOSSEL
and PRiNGLE2, amounts to about 89 per cent of the total nitrogen.
Sturine yields besides this the two hexone bases lysine, 12 percent, and
histidine, 12.9 per cent. Histidine has not been found in any other pro-

1 In regard to protamines, see Miescher, Histochemische und Physiologische
Arbeiten, Leipzig, 1897; Piccard, Ber. d. deutsch. chem. Gesellsch., 7; Schmiedeberg,
Arch. f. exp. Path. u. Pharm., 37; Kossel, Zeitschr. f. physiol. Chem., 22 (Ueber die
basischen Stoffe des Zellkerns), 25, 165 and 190, 26, 40, 44, and 69; and Sitzungsber.
der Gesellsch. zur Beford. der ges. Naturwiss. zu Marburg, 1897; Berl. klin. Woch-
enschr., 1904; Kossel and Mathews, Zeitschr. f. physiol. Chem., 23 and 25; Kossel
and Kutscher, ibid., 31; Goto, ibid., 37; Kurajeff, ibid., 32; Morkowin, ibid., 28;
Kossel and Dakin, ibid., 40, 41, and 44; Maleniick, ibid., 57; Pringle, ibid., 49; Ken-
naway, ibid., 72; Cameron, ibid., 76; F. Weiss, 59, 60 and 78; Nelson, Arch. f. exp.
Path. u. Pharm., 59.

2 Zeitschr. f. physiol. Chem., 53.

PKOTAMINES.

Ill

tamine. The carp protamine, cyprinine, occurs in two different modi-
fications, namely, a- and /3-cyprinine. The a-cyprinine yields only little
arginine, 4.9 per cent, but the lysine content is pronounced, 28.8 per cent.
Of the total nitrogen 30.3 per cent exists as lysine. KOSSEL and DAKIN
have obtained from salmine the following cleavage products, namely,
arginine 87.4, serine 7.8, aminovaleric acid 4.3, and a-pyrrolidine-car-
boxylic (proline) acid 11 per cent, and according to them the salmine
contains about 10 mol. arginine, 2 mol. serine, 1 mol. aminovaleric acid,
and 2 mol. proline. Scombrine contains only arginine, alanine, and pro-
line. According to KOSSEL, every protamine contains only 2 or 3 mon-
amino-acids (clupeine contains 4) and for every 2 molecules of arginine
only 1 molecule of monamino-acid occurs. The above-mentioned pro-
tones (of the salmine group) are symmetrically constituted diarginides
with a monamino-acid — for example diarginylserine, diarginylproline etc.,
— and these diarginides are united together forming the protamine.
Thus according to KOSSEL in clupeine we can accept the presence of
diarginylalanine, diarginylserine, diarginylproline, and diarginylvaline
(KOSSEL and PRINGLE).

The following summary according to KOSSEL gives a view of the
cleavage products of the protamines thus far investigated:

Scorn-
brine.

Salmine.

Clupeine.

Sturine.

Cyclop-
terine.

a-Cyp-
nmne.

0-Cyp-
rinine.

Creni-
labrine.

Alanine

1

0

+

+

?

?

?

?

Serine

0

+

+

0

?

?

?

?

Valine

o

_1_

+

o

?

4-

4-

?

Leucine
Arginine
Lysine

0

+

0

0

1

0

0

+

0

+
+

-)-

?

+

o

?

+

4-

?

+
-f

?

4-

4-

Histidine . . .

0

0

0

-(-

o

0

o

0

Proline

-)-

+

o

?

?

?

?

Tyrosine
Tryptophane

0
0

0
0

0
0

0
0

+
+

0
0

0
0

+
0

Solutions of these bases in water are alkaline and have the property
of giving precipitates with ammoniacal solutions of proteins or primary
proteoses, but the researches of HUNTER 1 show that these precipitates
are not histones, as generally considered. The salts with mineral acids
are soluble in water, but insoluble in alcohol and ether. They are more
or less readily precipitated by neutral salts (NaCl). Among the salts
of the protamines, the sulphate, picrate, and the double-platinum chloride
are the most important, and are used in the preparation of the protamines.
The protamines are, like the proteins, levogyrate; but by the action of
alkali the rotation is reduced or made to disappear, which according

Zeitschr. f. physiol. Chem. 53.

112 THE PROTEIN SUBSTANCES.

to KOSSEL and WEISS l depends at least in part, to a racemisation of the
hexone bases, especially arginine within the protamine molecule. They
give the biuret test beautifully, but with the exception of cyclopterine,
jS-cyprinine and crenilabrine do not give MILLON'S reaction. The pro-
tamine salts are precipitated in neutral or even faintly alkaline solutions
by phosphotungstic acid, picric acid, chromic acid, and alkali ferrocy-
anides.

The protamines are prepared, according to KOSSEL, by extracting
the heads of the spermatozoa, which have previously been extracted
with alcohol and ether, with dilute sulphuric acid (1-2 per cent), filtering,
and precipitating with 4 vols. of alcohol. The sulphate may be purified
by repeated solution in water and precipitation with alcohol, and if
necessary, conversion into the picrate. For more details see the works
of KOSSEL and MALENUCK. The double-platinum salt is best suited
for analysis and can be obtained, according to GOTO, by precipitating
the methyl-alcohol solution of the protamine hydrochloride with plat-
inum chloride. MIESCHER also precipitates the base as a double-plat-
inum salt.

B. Albuminoids or Albumoids.

Under this name we collect into a special group all those protein
bodies which cannot be placed in either of the other groups. Most and
best studied of the bodies belonging to this group are important con-
stituents of the animal skeleton or the cutaneous structure. Some are
hardened secretions, and all occur as a rule in an insoluble state in the
organism, and they are distinguished in most cases by a pronounced
resistance to reagents which dissolve proteins, or to chemical reagents
in general, and it is due to these external properties that they are put
in a special group. From a purely chemical standpoint there is no
reason why they should be separated from the true proteids in a special
group. Most of the bodies belonging to the albuminoids have been
given on page 92.

The Keratins. Keratin is the chief constituent of the horny struc-
ture of the epidermis, of hair, wool, of the nails, hoofs, horns, feathers,
of tortoise shell, etc., etc. Keratin is also found as neurokeratin (KUHNE)
in the brain and nerves. The shell membrane of the hen's egg seems
also to consist of keratin, and according to NEUMEISTER 2 the organic
matrix of the eggshells of various vertebrate animals belongs in most
cases to the keratin group.

1 Zeitschr. f. physiol. Chem., 59, 60, and 78.

2 Ktihne and Ewald, Verb. d. naturhistor.-med. Vereins zu Heidelberg (N. F.); 1;
also Kiihne and Chittenden, Zeitschr. f. Biologic, 26; Neumeister, ibid., 31.

KERATINS. 113

It seems that there exist a number of keratins, and these form a special
group of bodies. This fact, together with the difficulty in isolating the
keratin from the tissues in a pure condition without a partial decom-
position, is sufficient explanation for the variation in the elementary
composition given below. As examples the analyses of a few tissues
rich in keratin and of keratins are given:1

Human hair

C
43.72

H
6.34

N
15.06

3
4.95

29

0
.93

(RUTHERFORD

Nail
Neurokeratin. . . .
Neurokeratin. . . .

51.00
56.11-58.45
56.61

6.94
7.26-8.02
7.45

17.51
11.46-14.32
14.17

2.80
1.63-2.24
2 27

21

.75

and HAWK)
(MULDER)
(KUHNE)
(ARQIRIS)

Horn (average) . .
Tortoise shell. . . .
Shell membrane. .
Egg membrane . .

50.86
54.89
49.78
53.92

6.94
6.56
6.64
7.33

ie.77

16.43
15.08

3.20
2.22
4.25
1.44

19
22

'.56
.50

(HORBACZEWSKI
(MULDER)
(LINDVALL)
(PREGL)

(Scyllium)

\* •*"•****/

MoHR2 has determined the quantity of sulphur in various keratin
substances. Sulphur is in great part in loose combination, and it is
removed principally by the action of alkalies (as sulphides), or indeed in
part by boiling with water. Combs of lead after long usage become black,
and this is due to the action of the sulphur of the hair. On heating keratin
with water in sealed tubes to a temperature of 150° C. or higher, it dis-
solves with the elimination of sulphureted hydrogen or mercaptan
(BAUER), and the solution contains proteose-like substances (KRUKEN-
BERG) called atmidkeratin and atmidkeratose by BAUER.3 Keratin is
dissolved by alkalies, especially on warming, producing besides alkali
sulphides also proteose substances.

Besides the well-known cleavage products such as leucine, tyrosine,
aspartic acid, glutamic acid, arginine, and lysine, FISCHER and DORPING-
HAUS,4 have found glycocoll, alanine, valine, proline, serine, phenyla-
lanine, and pyrrolidone-carboxylic acid (secondary from glutamic acid)
among the cleavage products of horn substances. EMMERLING claims
to have found cystine as a sulphurized cleavage product, but K. MORNER

1 Rutherford and Hawk, Journ. of biol. Chem., 3; Mulder, Versuch einer allgem.
physiol. Chem., Braunschweig, 1844-51; Kiihne, Zeitschr. f. Biologic, 26; Horbaczew-
ski, see Drechsel in Ladenburg's Handworterbuch. d. Chem., 3; Lindvall, Maly's
Jahresbericht, 1881; Argiris, Zeitschr. f. physiol. Chem., 54; Pregl., ibid., 56.

2 Zeitschr. f. physiol. Chem., 20.

3 Krukenberg, Untersuch. tiber d. chem. Bau d. Eiweisskorper, Sitzungsber. d.
Janaischen Gesellsch. f. Med. u. Naturwissensch., 1886; Bauer, Zeitschr. f. physiol.
Chem., 35.

4 Zeitschr. f. physiol. Chem., 36, which contains also the older literature.

114 THE PROTEIN SUBSTANCES.

was the first to prove positively the abundant occurrence of cystine in
the cleavage products. MORNER obtained from ox horn, human hair,
and the shell-membrane of the hen's egg 6.8, 13.92, and 7.62 per cent
cystine calculated on the basis of the dry substance. BUCHTALA l obtained
the following amounts of cystine from the respective keratin forma-
tions, namely, 12.98-14.53 per cent from human hair, 5.15 per cent from
nails, 7.98 per cent from horsehair, 3.20 per cent from horse hoofs, 7.27
per cent from ox hair, 5.37 per cent from ox hoofs, 7.22 from pig bristles,
2.17 per cent from pig hoofs, 6.30 per cent from goose feathers, 2.14
per cent from chicken spurs, 1.88 per cent from the epidermis scales of
chicken feet and 4.7 per cent from elephant epidermis. From the amount
of sulphur split off by alkali, MORNER concludes that, at least in ox
horn and human hair, all the sulphur exists as cystine. GALiMARD2
was able to get only a qualitative test for cystine in the keratin of the
adder eggs. SUTER, MORNER, and FRiEDMANN3 have obtained^a-thio-
lactic acid as a hydrolytic cleavage product of the keratin substances.
The last-mentioned investigator was also able to detect thioglycolic acid
in the cleavage products of wool.

The shell membrane of the hen's egg, and the eggshells of amphibians
and certain fishes are, as above mentioned, ordinarily classified as kera-
tins. These bodies among themselves, as well as on comparison with
other keratins, show a marked difference in properties, this being very
evident from the tabulation on page 115.

The large quantity of cystine in the keratins is considered as espe-
cially characteristic, and they differ in this regard from the other proteins.
The shell membrane of the hen's egg behaves like a keratin in regard to the
large amount of cystine contained, but differs essentially by the absence
of tyrosine. It is remarkable that the egg membrane of the Selachii,
which biologically is analogous with ovokeratin, differs from the typical
keratins by the absence of cystine, while it contains, on the contrary,
large amounts of tyrosine. The typical keratins differ among them-
selves in regard to composition, thus the keratin from the sheep hoofs
contains 2 per cent phenylalanine, while this amino-acid is absent in the
keratin of hair and feathers. It is difficult to say whether or not this
is due to a difference in the purity of the bodies or not. The keratins
investigated chemically, thus far, do not form a sufficient characteristic
group.

1 Morner, ibid., 34 and 42; Emmerling, Ref. in Chemiker Zeitung, 1894; Buchtala,
Zeitschr. f. physiol. Chem., 52, 69, and 78.

2 Chem. Centralbl. II, 1905.

3 Suter, Zeitschr. f. physiol. Chem., 20; Morner, ibid., 42; Friedmann, Hofmeister's
Beitrage, 2.

KERATINS.

115

Keratin
from
Horse-
hair.i

Keratin
from
Sheep
Wool.*

Keratin
from
Goose
Feathers5

Keratin
from
Sheep
Horn. <

Shell
Mem-
brane
of the
Hen's
egg.6

Egg
Mem-
brane
of Scyl-
lium
stellare*

Tortoise
Shell of.
Chelone
imbri-
cata*

Glycocoll

4 7

0 58

2.6

0.45

3 9

2 6

19 36

Alanine

1 5

4.40

1.8

1.6

3.5

3.2

2 95

Valine

0 9

2.80

0.5

4.5

1.1

5 23

Leucine

7.1

11.5

8.0

15.3

7.4

5.8

3 26

Serine.

0.6

0.1

0.4

1.1

Aspartio acid

0 3

2 3

1 1

2.5

1.1

2 3

Glutamic acid 10
Cystine

3.7

7 982

12.9
7.3

2.3

17.2
7.5

8.1
7.627

7.2
?

5 19

Phenylalanine

0 0

0.0

1.9

3.3

1 08

Tyrosine

3.2

2.9

3.6

3.6

0.0

10.6

13 59

Proline.

3.4

4.4

3.5

3.7

4.0

4.4

Histidine

0.613

1.7

Arginine

4.453

^_

2.7

3.2

Lysine

1 123

0.2

3 7

Bodies occur in the animal kingdom which form to a certain extent
intermediate substances between coagulated protein and keratin. C.
TH. MORNER n has detected such a body (album&id) in the tracheal car-
tilage which forms a net-like trabecular tissue. This substance appears
to be related to the keratins on account of its solubilities and the quan-
tity of the sulphur (lead-blackening) it contains, while according to its
solubility in gastric juice it must stand close to the proteins. Another
substance, nearly like keratin, is the horny layer in the gizzard of birds.
According to J. HEDENIU^ this substance is insoluble in gastric or pan-
creatic juice, and acts quite like keratin. According to K. B. HOFMANN
and PREGL,12 who call this substance koilin, it does not yield any cystine
on hydrolysis, or at least not a determinable quantity. According to
others the quantity of cystine is very small. BUCHTALA 13 obtained only

1 Abderhalden and Wells, Zeitschr. f . physiol. Chem., 46.

2 Buchtala, ibid., 52.
* Argiris, ibid., 54.

4 Abderhalden and Voitinovici, ibid., 52.
6 Abderhalden and Le Count, ibid., 46.

6 Abderhalden and Ebstein, ibid , 48.

7 Korner, ibid., 34 and 42.

8 Pregl, ibid., 56.

9 Buchtala, ibid., 74.

10 Abderhalden and Fuchs, Zeitschr. f . physiol. Chem., 57, have shown that the
same variety of keratin, on ageing of the horn structure, becomes somewhat poorer
in glutamic acid.

11 See Maly's Jahresber., 18.

12 Hedenius, Skand. Arch. f. Physiol., 3; Hofmann and Pregl, Zeitschr. f. physiol.,
Chem., 52.

13 Zeitschr. f . physiol. Chem., 69.

116 THE PROTEIN SUBSTANCES.

a little more than 0.5 per cent pure crystalline cystine and on account
of .the low cystine content as well as for other Reasons the koilin differs
from the keratins.

Keratin is amorphous or takes the form of the tissues from which
it was prepared. It is insoluble in water, alcohol, or ether. On heating
with water to 150-200° C. it dissolves. It also dissolves gradually in
caustic alkalies, especially on heating. It is not dissolved by artificial
gastric juice or by trypsin solutions. Keratin gives the xanthoproteic
reaction, as well as the reaction with MILLON'S reagent, although the
latter is not always typical.

In the preparation of keratin a finely divided horny structure is
treated first with boiling water, then consecutively with diluted acid,
pepsin-hydrochloric acid, and alkaline trypsin solution, and, lastly, with
water, alcohol, and ether.

Elastin occurs in the connective tissue of higher animals, sometimes
in such large quantities that it forms a special tissue. It occurs most
abundantly in the cervical ligament (ligamentum nuchse).

Elastin used to be generally considered as a sulphur-free substance.
According to the investigations of CHITTENDEN and HART, it is a question
whether or not elastin contains sulphur, as it may have been removed by
the action of the alkali in its preparation. H. SCHWARZ has been able
by another method, to prepare an elastin containing sulphur, from the
aorta, and this sulphur can be removed by the action of alkalies, without
changing the properties of the elastin; and ZOJA, HEDIN, BERGH,
and RICHARDS and GIES * have found that elastin contains sulphur. The
most trustworthy analyses of elastin from the cervical ligament (Nos.
1 and 2) and from the aorta (No. 3) have given the following results,
which compare well with each other:

c H N s o

1. 54.32 6.99 16.75 .... 21.94 (HORBACZEWSKI 2)

2. 54.24 7.27 16.70 21.79 (CHITTENDEN and HART)

3. 53.95 7.03 16.67 0.38 (H. SCHWARZ)

ZOJA found 0.276 per cent sulphur and 16.96 per cent nitrogen in
elastin. HEDIN and BERGH found different quantities of nitrogen in
aorta-elastin, depending upon whether HORBACZEWSKI'S or SCHWARZ'S
method wras used in its preparation. In the first case they found 15.44
per cent nitrogen and 0.55 per cent sulphur, and in the other 14.67 per

1 Chittenden and Hart, Zeitschr. f. Biologie/ 25; Schwarz, Zeitschr. f. physiol.
Chem., 18; Zoja, ibid., 23; Bergh, ibid., 25; Hedin, ibid.; Richards and Gies, Amer.
Journ. of Physiol., 7.

2 Zeitschr. f. physiol. Chem., 6.

ELASTIN. 117

cent nitrogen and 0.66 per cent sulphur. RICHARDS and GIES found
0.14 per cent sulphur and 16.87 per cent nitrogen in elastin. The ques-
tion whether elastin is a unit body still remains open.

The quantity of hydrolytic cleavage products are given in the table
on page 125. It is sufficient to here call attention to the fact that no
aspartic acid and only very little glutamic acid have been found. The
hexone bases have been obtained, but only in very small amounts, so
that the basic nitrogen represents only 3.34 per cent of the total nitro-
gen (RICHARDS and GIES) . From an elastin proteose, WECHSLER l
obtained 1.86 per cent arginine, 0.5 per cent, histidine and 2.48 per cent
lysine.

Indol and skatol have not been found on the putrefaction of elastin,2
but SCHWARZ, on the contrary, obtained indol, skatol, benzene, and
phenols on fusing aorta-elastin with caustic potash. On heating with
water in closed vessels, on boiling with dilute acids, or by the action of
proteolytic enzymes, the elastin dissolves and splits into two chief prod-
ucts, called by HORBACZEWSKI hemielastin and elastinpeptone. Accord-
ing to CHITTENDEN and HART, these products correspond to two proteoses
designated by them protoelastose and deuteroelastose. The first is soluble
in cold water and separates out on heating, and its solution is precipi-
tated by mineral acid as well as by acetic acid and potassium ferrocyanide.
The aqueous solution of the other does not become cloudy on heating,
and is not precipitated by the above-mentioned reagents.

Pure elastin when dry is a yellowish-white powder; in the moist
state it appears like yellowish-white threads or membranes. It is insol-
uble in water, alcohol, or ether, and shows a resistance toward the action
of chemical reagents. It is not dissolved by strong caustic alkalies at
the ordinary temperature and only slowly at the boiling temperature.
It is very slowly attacked by cold concentrated sulphuric acid, but it
is relatively easily dissolved on warming with strong nitric acid. Elastins
of different origin act differently with cold concentrated hydrochloric
acid; for instance, elastin from the aorta dissolves readily therein, while
elastin from the ligamentum nuchse, at least from old animals, dissolves
with difficulty. Elastin is more readily dissolved by warm concen-
trated hydrochloric acid. It responds to the xanthoproteic reaction,
and to that with MILLON'S reagent, but not to the ADAMKIEWICZ-
HOPKINS reaction.

On account of its great resistance to chemical reagents, elastin may
be prepared (best from the ligamentum nuchae) in the following way:
First boil with water, then with 1 per cent caustic potash, then again

1 Zeitschr. f. physiol. Chem., 67.

2 See Walchli, Journ. f. prakt. Chem. (N. F.), 17.

118

THE PROTEIN SUBSTANCES.

with water, and lastly with acetic acid. The residue is treated with cold
5 per cent hydrochloric acid for twenty-four hours, carefully washed
with water, boiled again with water, and then treated with alcohol and
ether.

In regard to the methods used by SCHWARZ and by RICHARDS and GIES, which
are somewhat different, we refer to the original publications.

Collagen, or gelatin-forming substance, occurs very extensively in
vertebrates. The flesh of cephalopods is also said to contain collagen.1
Collagen is the chief constituent of the fibrils of the connective tissue and
(as ossein) of the organic substances of the bony structure. It also occurs
in the cartilaginous tissues as chief constituent; but it is here mixed
with other substances, producing what was formerly called chondrigen.
Collagen from different tissues has not quite the same composition, and
probably there are several varieties of collagen.

By continued boiling with water (more easily in the presence of a
little acid) collagen is converted into gelatin. HOFMEISTER 2 found that
gelatin on being heated to 130° C. is again transformed into collagen;
and this last may be considered as the anhydride of gelatin. Collagen
and gelatin have about the same composition.3

Collagen

Gelatin (commercial) . . .
Gelatin from tendons. . .
Gelatin from ligaments. .
Fish glue (isinglass) ....

50.75
49.38
50.11
50.49
48.69

H

6.47
6.80
6.56
6.71
6.76

N

17.86
17.97
17.81
17.90
17.68

24.92

0.70 25.13
0.26 25.26
0.57 24.33

(HOFMEISTER)
(CHITTENDEN)
(VAN NAME)
(RICHARDS and GIES)
(FAUST)

Gelatins of different origin show a somewhat variable composition,
which seems to indicate the occurrence of different collagens. It is diffi-
cult to say whether the variable content of sulphur is due to a contami-
nation with a substance rich in sulphur or to a splitting off of loosely
combined sulphur during the purification. C. MORNER* has prepared
a typical gelatin containing only 0.2 per cent of sulphur by a method
which eliminated any possible changes due to reagents.

SADIKOFF 5 has prepared gelatins by various methods from tendons and
from cartilage. Those from tendons, some of which were prepared after pre-
vious tryptic digestion, some after treatment with 0.25 per cent caustic potash,
and some after treatment with sodium hydroxide and then carbonate, showed

1 Hoppe-Seyler, Physiol. Chem., p. 97.

2 Zeitschr. f. physiol. Chem., 2.

3Hofmeister, 1. c.; Chittenden and Solley, Journ. of Physiol., 12; van Name,
Journ. of Exper. Med., 2; Richards and Gies, Amer. Journ. of Physiol., 8; Faust,
Arch. f. exp. Path. u. Pharm., 41.

4 Zeitschr. f. physiol. Chem., 28.

5 Ibid., 39 and 41.

COLLAGENS. 119

somewhat different physical properties among each other, but had about the same
elementary composition, with 0.34-0.53 per cent sulphur. SADIKOFF seems to
think that the gelatins prepared up to this time were perhaps not unit bodies
but were possibly mixtures. The bodies prepared by SADIKOFF from cartilage
he calls gluteins, because they were essentially different from the other gelatins
or glutins. They were poorer in carbon and nitrogen, 17.17 to 17.87 per cent,
but somewhat richer in sulphur, 0.53-0.718 per cent, than the tendon glutin.
The gluteins differ also from the glutins in that on boiling with a mineral acid
they have a faint reducing action, and also in that they give a color reaction
with phloroglucin-hydrochloric acid which is probably due to contamination. The
glutins differ from the gluteins by a different behavior with certain salts.

The decomposition products of the collagens are the same as those of
the gelatins and will be found in the table on page 125. Of special
mention is the fact that gelatin contains no tyrosine and tryptophane
but does yield considerable glycocoll. This latter substance has, because
of its sweet taste, been called gelatin sugar. SKRAUP 1 has obtained on
the hydrolytic cleavage of gelatin a crystalline acid having the formula
Ci2H25N50io, which he calls glutinic add. Gelatin yields considerable
basic nitrogen, according to HAUSMANN,2 35.83 per cent of the total
nitrogen. It also yields considerable arginine (9.3 per cent), lysine 5-6
per cent, but only little histidine (0.4 per cent) . The aromatic group in
gelatin is therefore, as directly shown by FISCHER and also by SriRO,3
represented by phenylalanine.

Collagen is insoluble in water, salt solutions, and dilute acids and
alkalies, but it swells up in dilute acids. By continued boiling with
water it is converted into gelatin. Various collagens are converted into
gelatin with varying readiness; the formation of gelatin occurs also
from difficultly soluble collagens by continuous boiling with water.
Collagen is dissolved by the gastric juice and also by the pancreatic
juice (trypsin solution) when it has previously been treated with acid
or heated with water above 70° C.4 By the action of ferrous sulphate,
corrosive sublimate, or tannic acid, collagen shrinks greatly. Collagen
treated by these bodies does not putrefy, and tannic acid is therefore of
great importance in the preparation of leather.

Gelatin or glutin is colorless, amorphous, and transparent in thin
layers. It swells in cold water without dissolving. It dissolves in warm
water, forming a sticky liquid, which solidifies on cooling when sufficiently
concentrated. As PAULI and RONA 5 have shown, various bodies may
have a different influence upon the gelatinization-point of a gelatin

1 Monatshefte f. Chem., 26.

2 Zeitschr. f. physiol. Chem., 27.

3 Fischer, Levene and Aders, Zeitschr. f. physiol. Chem., 35; Spiro, Hofmeister's
Beitrage, 1.

4 Kuhne and Ewald, Verb. d. Naturhist. Med. Vereins in Heidelberg, 1877, 1.
6 Hofmeister's Beitrage, 2.

120 THE PROTEIN SUBSTANCES.

solution; thus certain substances such as sulphates, citrates, acetates,
and glycerin may accelerate, while the chlorides, chlorates, bromides,
alcohol, and urea retard, this power.

Gelatin solutions are not precipitated on boiling, or by mineral
acids, acetic acid, alum, basic lead acetate, or metallic salts in general. A
gelatin solution acidified with acetic acid may be * precipitated by potas-
sium ferrocyanide on carefully adding the reagent. Gelatin solutions
are precipitated by tannic acid in the presence of salt, and according to
TRUNKEL 1 completely if the gelatin and tannic acid are in the propor-
tion 1 : 0.7. According to him the precipitation is not due to a chemical
combination but to an adsorption phenomenon. Solutions of gelatin
in water are also precipitated by acetic acid and common salt in sub-
stance; mercuric chloride in the presence of HC1 and NaCl; by meta-
phosphoric acid and phosphomolybdic acid in the presence of acid;
and lastly also by alcohol, especially when neutral salts are present.
Gelatin solutions do not diffuse. Gelatin gives the biuret reaction,
but not ADAMKIEWICZ-HOPKINS reaction. It gives MILLON'S reaction
and the xanthoproteic reaction so faintly that they probably occur from
impurities consisting of proteids. According to C. MORNER, pure gelatin
gives a beautiful MILLON'S reaction, if not too much reagent is added.
In the other case no reaction or only a faint one is obtained.

By continued boiling with water gelatin is converted into a non-
gelatinizing modification called /3-glutin by NASSE. According to NASSE.
and KRUGER the specific rotatory power is hereby reduced from — 167.5°
to about— 1360.2 According to TRUNKEL, who has especially studied
the rotation behavior of gelatin, the rotation of /3-glutin is less than the
ordinary a-glutin. On prolonged boiling with water, especially in the
presence of dilute acids, also in the gastric or tryptic digestion, the gelatin
is transformed into gelatin proteoses, so-called gelatoses and gelatin
peptones, which diffuse more or less readily.

According to HOFMEISTER two new substances, semiglutin and hemicollin,
are formed. The former is insoluble in alcohol of 70-80 per cent and is precipitated
by platinum chloride. The latter, which is not precipitated by platinum chloride,
is soluble in alcohol. CHITTENDEN and SOLLEY 3 have obtained in the peptic
and tryptic digestion a proto- and a deuter o-gelatose, besides a true peptone. The
elementary composition of these gelatoses does not essentially differ from that of
the gelatin.

PAAL 4 has prepared gelatin-peptone hydrochlorides from gelatin by the
action of dilute hydrochloric acid. These salts are partly soluble in ethyl and

1 Bioch. Zeitschr., 26.

2 Nasse and Kriiger, Maly's Jahresber., 19, p. 29. In regard to the rotation of
/3-glutin. see Framm, Pfliiger's Arch., 68; Trunkel, 1. c.

3 Hofmeister, 1. c.; Chittenden and Solley, 1. c.

4 Ber. d. deutsch. chem. Gesellsch., 25.

RETICULIN. 121

/

methyl alcohol, and partly insoluble therein. The peptones obtained from
these salts contain less carbon and more hydrogen than the gelatin from which
they originated, showing that hydration has taken place. The molecular weight
of the gelatin peptone as determined by PAAL, by RAOULT'S cryoscopic method,
was 200 to 352, while that for gelatin was 878 to 950. The gelatin peptones
isolated by SIEGFRIED and his pupils which will be discussed below, are of great
interest.

Collagen (contaminated with mucoid) may be obtained from bones by
extracting them with hydrochloric acid (which dissolves the earthy
phosphates) and then carefully washing the acid out with water. It
may be obtained from tendons by extracting with lime-water or dilute
alkali (which dissolve the proteids and mucin), and then thoroughly
washing with water. Gelatin is obtained by boiling collagen with water.
The finest commercial gelatin always contains a little proteid, which
may be removed by allowing the finely divided gelatin to swell up in
water and thoroughly extracting with large quantities of fresh water.
Then dissolve in warm water and precipitate with alcohol.

Collagen may also be purified from proteids, as suggested by VAN
NAME, by digesting with an alkaline trypsin solution or by extracting
the gelatin for many days with 1-5 p. m. caustic potash, as suggested
by C. MORNER. The typical properties of gelatin are not changed by
this.

Chondrin or cartilage gelatin is only a mixture of gelatin with the specific
constituents of the cartilage and their transformation products.

Reticulin. The reticular tissues of the lymphatic glands contain a
variety of fibers which have also been found, by MALL in the spleen, intes-
tinal mucosa,~ liver, kidneys, and lungs. These fibers consist of a special
substance, reticulin, investigated by SiEGFRiED.1

Reticulin has the following composition: C 52.88; H 6.97; N 15.63;
S 1.88; P 0.34; ash 2.27 per cent. The phosphorus occurs in organic
combination. It yields no tyrosine on cleavage with hydrochloric acid.
It yields, on the contrary, sulphureted hydrogen, ammonia, lysine,
arginine, and valine. On continued boiling with water, or more readily
with dilute alkalies, reticulin is converted into a body which is precipitated
by acetic acid, and at the same time phosphorus is split off. , ;

Reticulin is insoluble in water, alcohol, ether, lime-water, sodium
carbonate, and dilute mineral acids. It is dissolved, after several weeks,
on standing with caustic soda at the ordinary temperature. Pepsin-
hydrochloric acid or trypsin does not dissolve it. Reticulin responds
to the biuret, xanthoproteic, and ADAMKIEWICZ-HOPKINS reactions,
but not to MILLON'S reagent.

1 Mall, Abhandl. d. math.-phys. Klasse d. Kgl. sachs. Gesellsch. d. Wiss., 1891;
Siegfried, Ueber die chem. Eigensch. der retikulirten Gewebe, Habil.-Schrift, Leipzig,
1892.

122

THE PROTEIN SUBSTANCES.

According to TEBB reticulin is only a somewhat changed, impure collagen
but this is disputed by SIEGFRIED. l

It may be prepared as follows, according to SIEGFRIED: Digest intes-
tinal mucosa with trypsin and alkali. Wash the residue, extract with
ether, and digest again with trypsin and then treat with alcohol and ether.
On careful boiling with water the collagen present either as contamina-
tion or as a combination with recticulin is removed. The thoroughly
boiled residue consists of reticulin.

Ichthylepidin is an organic compound, so-called by C. MORNER,* which occurs
with collagen in fish-scales and forms about one-fifth of their organic substance.
This compound, with 15.9 per cent nitrogen and 1.1 per cent sulphur, stands on
account of its properties rather close to elastin. It is insoluble in cold and hot
water, as well as in dilute acids and alkalies at the ordinary temperature. On
boiling with these it dissolves. Pepsin-hydrochloric acid, as well as an alkaline
trypsin solution, also dissolves it. It responds beautifully to MILLON'S reagent,
the xanthoproteic reaction, and the biuret test. At least a part of the sulphur
is split off by the action of alkali. Ichthylepidin stands very close to elastin
in regard to its solubilities; but it differs essentially in composition as it is markedly
poorer in glycocoll, but much richer in proline and glutamic acid (ABDERHALDEN
and VOITINOVTCI 3).

As skeletins, KRUKENBERG 4 has designated a number of nitrogenized
substances which form the skeletal tissue of various classes of inverte-
brates. These substances are chitin, spongin, conchiolin, byssus, cornein,
and crude silk (fibroin and sericin). Of these, chitin does not belong to
the protein substances, and silk is hardly to be classed as a skeletin.
Only those so-called skeletins will be discussed that actually belong to
the protein group, and chitin will be discussed in another chapter.

The elementary composition of certain of the bodies belonging to
this group is as follows : 5

Conchiolin
Sponsrin.

(from the shells of pinna) . .

. 52
46

C

.70
50

6
6

H
.54
30

I
16.
16

* S
60 0.85
20 0.50

(WETZEL)
(CROOKEWITT)

ft

. 48

75

6

35

16

40

(POSSELT)

Cornein

48

%

5

00

16

81

(KRUKENBERG)

Fibroin. . .

48

23

6

?,7

18

31

(CRAMER)

< <

. 48

30

6

50

10

20

(VIGNON)

Sericin

44

32

f>

18

18

30

(CRAMER)

< <

. 44

50

6

32

17

14

(BONDI)

1 Tebb, Journ. of Physiol., 27; Siegfried, ibid., 28.

2 Zeitschr. f . physiol. Chem., 24 and 37. See also Green and Tower, ibid., 3u.

3 Zeitschr. f . physiol. Chem., 52, p. 368.

4 Grundziige einer vergl. Physiol. d. thier. Geriistsubst. Heidelberg, 1885.

5 Krukenberg, Ber. d. d. chem. Gesellsch., 17 and 18, and Zeitschr. f. Biologic, 22;
Croockewitt, Annal. d. Chem. u. Pharm., 48; Posselt, ibid., 45; Cramer, Journ. f.
prakt. Chem., 96; Vignon, Compt. rend., 115; Wetzel, Zeitschr. f. physiol. Chem., 29
and Centralbl. f. Physiol., 13, 113; Bondi, Zeitschr. f. physiol. Chem., 34.

CORNEIN. 123

Spongin forms the chief mass of the ordinary sponge. It dissolves with
difficulty in concentrated mineral acids but dissolves with readiness in caustic
alkalies. It does not give the MILLON reaction or ADAMKIEWICZ'S. It gives
no gelatin. On hydrolysis spongin yields considerable glycocoll 13.9 per cent,
glutamic acid 18.1 per cent, leucine 7.5 per cent, proline 6.3 per cent, lysine
3-4 per cent, and arginine 5-6 per cent.1 Tyrosine and phenylalanine could
not be detected. After HUNDESHAGEN had shown the occurrence of iodine
and bromine in organic combination in different sponges and designated the albu-
moid containing iodine, iodospongin, HARNACK 2 later isolated from the ordinary
sponge, by cleavage with mineral acids, an iodospongin which contained about
9 per cent iodine and 4.5 per cent sulphur. STRAUSS 3 has obtained sponginoses
of various kinds from spongin by dilute acids. The heterosponginose contained
the greater part of the iodine and sulphur, while the deuterosponginose contained
the carbohydrate groups. Iodospongin is considered as a derivative of the
heterosponginose. Conchiolin is found in the shells of mussels and snails and
also in the eggshells of these animals. It yields, according to WETZEL,* glycocoll,
leucine, and abundance of tyrosine. The quantity of diamino-nitrogen amounts
to 8.7 per cent and the amide nitrogen 3.47 per cent (from the shell of pinna).
The Byssus contains a substance, closely related to conchiolin, which is soluble
with difficulty. According to ABDERHALDEN 5 it yields considerable glycocoll
and tyrosine and also alanine, aspartic acid and very large amounts of proline.

Cornein is the name given to the substance of the axial system of
certain Anthozoa. The substance occurring in the groups of Gorgonia
and Antipathes has been called gorgonin by C. MORNER 6 and differs from
the pennatulin of the Pennatulideae by the latter being readily soluble
in pepsin-hydrochloric acid. The cleavage products have not been care-
fully studied; one of the crystalline products, called cornicrystalline by
KRTTKENBERG, is nothing but iodine crystals, as shown by MORNER.
After DRECHSEL 7 found nearly 8 per cent iodine in the dry substance of
the axial system of the Gorgonia Cavolini, C. MORNER showed that in
the Anthozoa in general the organic skeletal substance contains halogens
in organic combination. Iodine was found in all varieties, and indeed
in amounts from traces up to 7 per cent. Bromine was found, with the
exception of two Antipathes, in amounts of 0.25 to 4 per cent, while
chlorine, which was never absent, occurred as a few tenths per cent.
The halogens occur in the organic skeletal substance as gorgonin and
pennatulin.

DRECHSEL obtained leucine, tyrosine, lysine, ammonia and an iodized
amino-acid, iodogorgonic acid, as cleavage products of gorgonin. This last

1Abderhalden and Strauss, Zeitschr. f. physiol. Chem., 48; Kossel and Kutscher,
ibid., 31, 205.

2 Zeitschr. f. physiol. Chem., 24; Hundeshagen, Maly's Jahresber., 25, 394; see
also L. Scott, Biochem. Zeitscbr., 1.

' Biochem. Centralbl., 3.

4 Zeitschr. f. physiol. Chem. 29, and Centralbl. f. Physiol., 13, 113.

6 Zeitschr. f. physiol. Chem., 55.

6 Zeitschr. f. physiol. Chem., 51 and 55.

» Zeitschr. f . Biol., 33.

124 THE PROTEIN SUBSTANCES.

is identical with 3-5 di-iodo-tyrosine, HOI2C6H2.CH2.CHNH2COOH,
synthetically prepared by WHEELER and JAMIESON.1 On acid cleavage
of gorgonin, HENZE 2 obtained the three hexone bases, abundant tyrosine
and very little leucine. On cleavage with barium hydroxide he obtained
only lysine, besides tyrosine and glycocoll in larger amounts.

Fibroin and sericin are the two chief constituents of raw silk. By
the action of boiling water the sericin (silk gelatin) dissolves and can be
obtained by a method suggested by BoNDi,3 while the more difficultly
soluble fibroin remains undissolved in the shape of the original fiber.
The sericin, whose sufficiently concentrated hot solution gelatinizes on
cooling, is precipitated by mineral acids, several metallic salts, and by
acetic acid and potassium ferrocyanide. The spider silk investigated
by FISCHER 4 yielded fibroin but not sericin.

ABDERHALDEN and his collaborators5 have investigated a great
number of varieties of silk and found sericin in varying amounts (15 to
28 per cent). The composition of the various kinds of silk is char-
acterized, especially, by a varying amount of glycocoll and in this regard
we can differentiate between two chief groups. The one group is, like
the Italian silk, very rich in glycocoll while the other group, like the
Tussah silk, contains a much smaller quantity of glycocoll.

Sericin, whose proper concentrated warm solution gelatinizes on
cooling, is precipitated by mineral acids and several metallic salts and
by acetic acid and potassium ferrocyanide. In regard to the products
of hydrolysis it differs very essentially from fibroin by being much poorer
in glycocoll, alanine and tyrosine.

Fibroin is soluble in concentrated acids and alkalies and reprecipitable
(in a modified form) on neutralization. It gives the biuret test and
MILLON'S and ADAMKIEWICZ-HOPKIN'S reactions, the last but faintly.
Fibroin has an especially great interest because of the hydrolyses per-
formed by FISCHER and his co-workers, and especially by the finding of
the previously mentioned polypeptides by these workers. Of the cleavage
products which characterize fibroin we must mention the large amount
of glycocoll, alanine and tyrosine, and the very small amounts of hexone
bases, besides the almost complete absence of monamino-dicarboxylic
acids. The quantity of the hydrolytic cleavage products of the three
silk substances, in so far as they have been investigated, are given in the
following table, which also includes the results for elastin, gelatin, and

1 Wheeler and Jamieson, Amer. Chem. Journ., 23; \7heeler, ibid.,

2Henze, Zeitschr. f. physiol. Chem., 38 and 51.

8 Zeitschr. f. physiol Chem., 34.

4 Ibid., 53.

6 See Zeitschr. f. physiol. Chem., 59, 61, 62, 64, 71, 74, 80.

ALBUMINATES

125

koilin. The fibroin A came from ordinary silk ; fibroin B and the sericin
originated from Indian Tussah silk.

Elastin.i

Gelatin. »

Koilin.*

Fibroin A1

Fibroin B •

Sericin.7

SpiderSilk*

Glycocoll
Alanine

25.75
6 6

19.25
3.0

1.2

5.8

36.0

21.0

9.5
24 0

1.5

9 8

35.13
23 4

Valine
Leucine

1.0
21.1

9.23

13.2

1.5

1.5

4.8

1.76

Serine

0.4

1.6

2.0

5.4

Aspartic acid

;

1 23

2 3

2 5

2 8

Glutamic acid
Cystine

0.8

16. 83

5.2

0 710

—

1.0

1.8

11.70

Phenylalanine
Tyrosine
Proline

3.9
0.34
1.7

l.O3

7.7

2.3
5.4
5.5

1.5
10.5

0.6
9.2
1.0

0.3
1.0
3.0

8.20
3 68

Oxyproline

6.4

,

Histidine

0 4

0 035

)

Arginine
Lysine

0.3

9.3
5 6

3.605
1 645

1.0

—

—

[5.249

C. Cleavage Products of Simple Proteins.

On the hydrolysis of proteins by the aid of acids, alkalies or by
enzymes, cleavage products are obtained which represent various inter-
mediary steps between the native proteins on one side and the simple
cleavage products, the amino-acids, on the other side. Among these
products we have for a long time known two chief groups which still
retain, to a high degree, their protein character, namely, the albuminates
and the proteoses (and peptones).

1. Albuminates.

Alkali and Acid Albuminates. The native proteins are modified
by the action of sufficiently strong acids or alkalies. By the action of
alkalies all native albuminous bodies are converted, with the elimina-
tion of nitrogen, or by the action of stronger alkali, with the extraction
of sulphur also, into a new modification, called alkali albuminate. If
caustic alkali in substance or in strong solution be allowed to act on a

1 Cited from Abderhalden's Lehrbuch d. physiol. Chem., 1909.
2Cohnheim, Chemie d. Eiweisskorper 3 d. Aufl.

3 Skraup and Biehler, Monatsh. f. Chem., 30.

4 K. B. Hoffmann and Pergl. Zeitschr. f. physiol. Chem., 52.
6 v. Knaffl-Lenz, ibid., 52.

6 Abderhalden and Spack, ibid., 62.
7Strauch, ibid., 71.

8 E. Fischer, ibid., 53.

9 Calculated as arginine.

10 This figure is somewhat uncertain.

126 THE PROTEIN SUBSTANCES.

concentrated proteid solution, such as blood-serum or egg-albumin, the
alkali album inate may be obtained as a solid jelly which dissolves in
water on heating, and which is called " LIEBERKUHN'S solid alkali
albuminate." By the action of dilute caustic alkali solutions on dilute
proteid solutions we have alkali albuminates formed slowly at the ordinary
temperature, but more rapidly on heating. These solutions may vary
with the nature of the proteid acted upon, and also with the intensity
of the action of the alkali, but still they have certain reactions in common.

If proteid is dissolved in an excess of concentrated hydrochloric acid,
or if we digest a proteid solution acidified with 1-2 p. m. hydrochloric
acid in the thermostat, or digest the proteid for a short time with
pepsin-hydrochloric acid, we obtain new modifications of proteid which
may show somewhat varying properties, but have certain reactions in
common. These modifications, which may be obtained in a solid gelat-
inous condition on sufficient concentration, are called acid albuminates
or acid albumins, and sometimes syntonin, though we perfer to apply
the term syntonin to the acid albuminate, which is obtained by extract-
ing muscles with hydrochloric acid of 1 p. m.

The alkali and acid albuminates have the following reactions in
common: They are almost insoluble in water and dilute common-salt
solution (see page 104), but they dissolve readily in water on the addi-
tion of a very small quantity of acid or alkali. Such a solution as nearly
neutral as possible does not coagulate on boiling but is precipitated at
the normal temperature on neutralizing the solvent by an alkali or an
acid. A solution of an alkali or acid albuminate in acid is easily pre-
cipitated on saturating with NaCl, but a solution in alkali is precipitated
with difficulty or not at all, according to the amount of alkali it contains.
Mineral acids in excess precipitate solutions of acid as well as alkali
albuminates. The nearly neutral solutions of these bodies are also pre-
cipitated by many metallic salts.

Notwithstanding this agreement in the reactions, the acid and alkali
albuminates are essentially different, for by dissolving an alkali albumi-
nate in some acid no acid, albuminate solution is obtained, nor is an alkali
albuminate formed on dissolving an acid albuminate in water by the
aid of a little alkali. In the first case we obtain a combination of the
alkali albuminate and the acid, soluble in water, and in the other case a
soluble combination of the acid albuminate with the alkali added. The
chemical process in the modification of proteids with an acid is essentially
different from the modification with an alkali, hence the products are
of a different kind. The alkali albuminates are relatively strong acids.
They may be dissolved in water with the aid of CaCOs, with the elimina-
tion of CO2, which does not occur with typical acid albuminates, and
they show in opposition to the acid albuminates also other variations

PROTEOSES AND PEPTONES. 127

which stand in connection with their strongly marked acid nature. Dilute
solutions of alkalies act more energetically on proteids than do acids
of corresponding concentration. In the first case a part of the nitro-
gen and often also the sulphur, is split off, and from this property we may
obtain an alkali albuminate by the action of an alkali upon an acid albu-
minate; but we cannot obtain an acid albuminate by the reverse reac-
tion (K. MoRNER1). This does not exclude the possibility that, by
a more severe acid treatment, products can be obtained which perhaps
correspond to those products obtained by a more mild alkali treatment.

The preparation of the albuminates has been given above. The
corresponding albuminate obtained by the action of alkalies or acids
upon a proteid solution may be precipitated by neutralizing with acid
or alkali. The washed precipitate is dissolved in water by the aid of a
little alkali or acid, and again precipitated by neutralizing the solvent.
If this precipitate, which has been washed in water, is treated with
alcohol and ether, the albuminate will be obtained in a pure form.

In the preparation of acid as well as of alkali albuminates, proteoses and the
closely related albuminates are formed. The " alkali albumose " obtained by
MA AS 2 belongs to this class. The lysalbinic acid and protalbinic acid obtained
by PAAL 8 from ovalbumin are likewise alkali albuminates. These have been
carefully studied by SKRAUP and his co-workers.4

Desam.inoalbuminic acid is an alkali albuminate which SCHMIEDEBERG 6 obtained
by the action of such weak alkali that a part of the nitrogen was evolved but the
quantity of sulphur remained the same. The proteid combination obtained by
BLUM6 by the action of formol on proteid and called by him protogen, has similarities
with the alkali albuminates in regard to solubilities and precipitation, but is not
identical therewith.

2. Proteoses and Peptones.

Peptones were formerly designated as the final products of the decom-
position of protein bodies by means of proteolytic enzymes in so far as
these final products are still true proteins, while the intermediate prod-
ucts produced in the peptonization of proteins, in so far as they are
not substances similar to albuminates, were designated as proteoses
(albumoses, or propeptones) . Proteoses and peptones may also be
produced by the hydrolytic decomposition of the proteins with acids

1 Pfltiger's Arch., 17.

2 Zeitschr. f. physiol. Chem., 30.

3 Ber. d. d. chem. Gesellsch., 35.

4 Hummelberger, Lampel and Woeber, Monatsh. f . Chem., 30.
6 Arch. f. exp. Path. u. Pharm., 39.

6 Blum, Zeitschr. f. physiol. Chem., 22. The older investigations of Loew may
be found in Maly's Jahresber., 1888. On the action of formaldehyde see also Benedi-
centi, Arch. f. (Anat. u.) Physiol., 1897; S. Schwarz, Zeitschr, f. physiol. Chem., 30;
Bliss and Novy, Journ. of Exper. Med., 4,

128 THE PROTEIN SUBSTANCES.

or alkalies, and by the putrefaction of the same. They may also be
formed in very small quantities, as by-products, in the investigations
of animal fluids and tissues, and the question as to the extent to which
these exist preformed under physiological conditions requires very
careful investigation.

Between the peptone, which represents the final cleavage product,
and the proteose, which stands closest to the original protein, we have
undoubtedly a series of intermediate products. Under such circum-
stances it is a difficult problem to try to draw a sharp line between the
peptone and the proteose group, and it is just as difficult to define our
conception of peptones and proteoses in an exact and satisfactory manner.

In the past we used to consider the peptones as the end products
in the hydrolysis, they still being true proteins, but we must call atten-
tion to the fact that since that time we have learned of polypeptide-
like cleavage products of the proteins, and also that polypeptides have
been prepared synthetically. With this in mind it is not possible to
say what we understand by the conception true proteid, and also that
possibly there exists a large number of intermediary steps between the
original modified proteid and the simplest cleavage products. There
is no doubt that those bodies which have been called proteoses and
peptones are chiefly mixtures; and the question has been proposed by
ABDERHALDEN 1 whether it is not best to drop the conception of pro-
teoses and to call all products precipitable by ammonium sulphate, etc.,
and previously described as proteoses, peptones.

Although there is much in favor of such a proposition, still on account
of the great importance which the conception of the proteoses has gen-
erally received, it is probably too early to drop the question of proteoses
entirely from a text-book, and we will therefore, as in the past editions,
discuss the historical development of the proteoses and peptones in
the ordinary sense.

The proteoses (or albumoses) used to be considered as those protein
bodies whose neutral or faintly acid solutions do not coagulate on boil-
ing and which, to distinguish them from peptones, were characterized
chiefly by the following properties : The watery solutions are precipitated
at the ordinary temperature by nitric acid, as well as by acetic acid and
potassium ferrocyanide, and this precipitate has the peculiarity of dis-
appearing on heating and reappearing on cooling. If a proteose solu-
tion is saturated with NaCl in substance, the proteose is partly pre-
cipitated in neutral solutions, but on the addition of acid saturated
with salt it is more completely precipitated. This precipitate, which
dissolves on warming, is a combination of the proteose with the acid.

1 Oppenheimer's Handb. der Biochem., Bd. 1, 1908.

PROTEOSES AND PEPTONES. 129

We formerly designated as peptones those protein bodies which are
readily soluble in water and which are not coagulated by heat, whose
solutions are precipitated neither by nitric acid, nor by acetic acid and
potassium ferrocyanide, nor by NaCl and acid.

The reactions and properties which the proteoses and peptones have
in common were formerly considered as the following: They all
give the color reactions of the proteins, but with the biuret test they give
a more beautiful red color than the ordinary proteins. They are pre-
cipitated by ammoniacal lead acetate, by mercuric chloride, tannic, phos-
photungstic, and phosphomolybdic acids, by potassium-mercuric iodide
and hydrochloric acid, and also by picric acid. They are precipitated
but not coagulated by alcohol, that is, the precipitate obtained is soluble
in water even after being in contact with alcohol for a long time. The
proteoses and peptones also have a greater diffusive power than native
proteins, and the diffusive power is greater the nearer the questionable
substance stands to the final product, the now so-called true peptone.

These old views have gradually undergone an essential change. After
HEYNSIUS' 1 observation that ammonium sulphate was a general pre-
cipitant for proteins, and for peptones in the old sense, KUHNE and his
pupils2 proposed this salt as a means of separating proteoses and pep-
tones. Those products of digestion which separate on saturating their
solution with ammonium sulphate, or can indeed be salted out at all,
are considered, by KUHNE and also by most of the modern investigators,
as proteoses, while those which remain in solution are called peptones
or true peptones. These true peptones are formed in relatively large
amounts in pancreatic digestion, while in pepsin digestion they are formed
only in small quantities or after prolonged digestion.

According to SCHUTZENBERGER and KUHNE 3 the proteins yielded
two chief groups of new protein bodies when decomposed by dilute
mineral acids or with proteolytic enzymes; of these the anti group shows
a greater resistance to further action of the acid and enzyme than the
other namely, the hemi group. These two groups are, according to
KUHNE, united in the different proteoses, even though in various relative
amounts, and each proteose contains the anti as well as the hemi group.
The same is true for the peptone obtained in pepsin digestion, hence he
calls it amphopeptone. In tryptic digestion a cleavage of the ampho-

1 Pfliiger's Archiv, 34.

2 See Kiihne, Verhandl. d. naturhistor. Vereins zu Heidelberg (N. F.), 3; J. Wenz,
Zeitschr. f. Biologie, 22; Kiihne and Chittenden, Zeitschr. f. Biologic, 22; R. Neu-
meister, ibid., 23; Kiihne, ibid., 29.

3 Schiitzenberger, Bull, de la Soc. chimique de Paris, 23; Kiihne, Verhandl. d.
naturhist. Vereins zu Heidelberg (N. F.), 1, and Ktthne and Chittenden, Zeitschr. f.
Biologie, 19. See also Paal, Ber. d. deutsch. chem. Gesellsch., 27.

130 THE PROTEIN SUBSTANCES.

peptone takes place into antipeptone and hemipcptone. Of these two
peptones the hemipeptone is further split into amino-acids and other
bodies while the antipeptone is not attacked. By the sufficiently
energetic action of trypsin only one peptone remains to the last — the so-
called antipeptone.

KUHNE and his pupils, who have conducted extensive investiga-
tions on the proteoses and peptones, classify the various proteoses accord-
ing to their different solubilities and precipitation properties. In the
pepsin digestion of fibrin l they obtained the following proteoses : (a)
Heteroproteose, insoluble in water but soluble in dilute salt solution;
(6) Protoproteose, soluble in salt solution and water. These two pro-
teoses are precipitated by NaCl in neutral solutions, but not completely.
Heteroproteose may, by being in contact with water for a long time
or by drying, be converted into a modification, called (c) Dysproteose,
which is insoluble in dilute salt solutions, (d) Deuteroproteose is a pro-
teose which is' soluble in water and dilute salt solution and which is
incompletely precipitated from acid solution by saturating with NaCl,
and is not precipitated from neutral solutions.

The proteoses obtained from different protein bodies do not seem to be
identical, but differ in their behavior to precipitants. Special names have been
given to these various proteoses according to the mother-protein, namely, albu-
moses, globuloses, vitelloses, caseoses, myosinoses, elastoses, etc. These various
proteoses are further distinguished, as proto-, hetero-, and deuterocaseose, for
example. CHITTENDEN 2 has suggested the common name proteoses for the prod-
ucts formed intermediary between the proteins and peptones in the digestion of
animal and vegetable proteins. We have made use of it in this sense in pref-
erence to the word albumose (which is used in the German and by some other
writers), but which will be used in this book as indicating the intermediary
products in the hydrolysis of albumins and not as a general term. Certain
proteoses have also been obtained in a crystalline state (SCHROTTER).

NEUMEiSTER3 designates as atmidalbumose that body which is obtained by
the action of superheated steam on fibrin. At the same time he also obtained a
substance called atmidalbumin, which stands between the albuminates and the
proteoses.

Of the soluble proteoses NEUMEISTER designates the protoproteose
and heteroproteose as primary proteoses, while the deuteroproteoses,
which are closely allied to the peptones, he calls secondary proteoses. As
essential differences between the primary and secondary proteoses he

1 See Kiihne and Chittenden, Zeitschr. f. Biologie, 20.

2 Kiihne and Chittenden, Zeitschr. f. Biologie, 22 and 26; Neumeister, ibid., 23;
Chittenden and Hartwell, Journ. of Physiol., 11 and 12; Chittenden and Painter,
Studies from the Laboratory, etc., Yale University, 2, New Haven, 1887; Chittenden,
ibid., 3; Sebelien, Chem. Centralblatt, 1890; Chittenden and Goodwin, Journ. of
Physiol., 12.

3 Zeitschr. f. Biologie, 26. See also Chittenden and Meara, Journ. of Physiol.,
15, and Salkowski, Zeitschr. f. Biologie, 34 and 37.

PROTEOSES AND PEPTONES. 131

suggests the following.1 The primary proteoses are precipitated by
nitric acid in salt-free solutions, while the secondary proteoses are pre-
cipitated only in salt solutions, and certain deuteroproteoses, such as
deuterovitellose and deuteromyosinose, are precipitated by nitric acid
only in solutions saturated with NaCl. The primary proteoses are pre-
cipitated from neutral solutions by copper-sulphate solution (2:100),
and by NaCl in substance, while the secondary proteoses are not. The
primary proteoses are completely precipitated from a solution saturated
with NaCl by the addition of acetic acid saturated with salt, while the
secondary proteoses are only partly precipitated. The primary proteoses
are readily precipitated by acetic acid and potassium ferrocyanide, while
the secondary are only incompletely precipitated after some time. The
primary proteoses are also, according to PiCK,2 completely precipitated
by ammonium sulphate (added to one-half saturation), while the second-
ary proteoses remain in solution.

The true peptones, as they were formerly considered to be, are exceed-
ingly hygroscopic, and if perfectly dry, sizzle like phosphoric anhydride
when treated with a little water. They are exceedingly soluble in water,
diffuse more readily than the proteoses, and are not precipitated by
ammonium sulphate. In contradistinction to the proteoses, the true
peptones are not precipitated by nitric acid (even in solutions saturated
with salt), by sodium chloride and acetic acid saturated with salt,
potassium ferrocyanide and acetic acid, picric acid, trichloracetic
acid, potassium-mercuric iodide, and hydrochloric acid. They are
precipitated by phosphotungstic acid, phosphomolybdic acid, corrosive
sublimate (in the absence of neutral salts), absolute alcohol, and tannic
acid, but the precipitate may redissolve on the addition of an excess of
the precipitant. As an important difference between amphopeptone and
antipeptone we must also mention that the former gives MILLON'S
reaction, while the antipeptone does not.

In regard to the precipitation by alcohol we must call attention to the observa-
tions of FRANKEL that not only are the acid combinations of peptone (PAAL)
soluble in alcohol, but also the free peptone, and FRANKEL has even suggested a
method of preparation based on this behavior. SCHROTTER 3 has also prepared
crystalline proteoses which were soluble in hot alcohol, especially methyl alcohol.

The views on the hydrolytic cleavage products of peptic and tryptic
digestion which were accepted until a few years ago have recently been
considerably modified in several points.

1 Neumeister, Zeitschr. f. Biologic, 24 and 26.
2Zeitschr. f. physiol. Chem.; 24.

3Frankel, Zur Kenntnis der Zerfallsprodukte des Eiweisses bei peptischer und
tryptiecher Verdauung, Wien, 1896; Schrotter, Monatshefte f. Chem., 14 and 16.

132 THE PROTEIN SUBSTANCES.

The older view that in peptic digestion only proteoses and peptones,
but no simpler cleavage products, are formed, has been shown not to be
true. The works of ZUNZ, PFAUNDLER, SALASKIN, LAWROW, LANG-
STEIN/ and others have shown that by very lengthy digestion amino-
acids may in part be formed and also other products such as oxyphenyl-
ethylamine, tetra- and pentamethylenediamine. The biuret reaction
does not disappear and the above mentioned products seem to be formed
only under special conditions. In ordinary, not too lengthy pepsin,
digestion, it is generally admitted that no amino acids are formed but
only proteoses and peptones.

In connection with the above-mentioned experimental results it must be
remarked that not all the products found, for example, the oxyphenylethylamine
and the diamines, are produced by the action of pepsin, but rather by the action of
other enzymes. In certain cases, undoubtedly, impure pepsin was used, or indeed
autodigestion of the stomach was carried on, and the action of other enzymes
was not excluded. In other cases the digestion with pepsin and considerable
acid (even 1 per cent H2S04) was continued for a very long time, indeed for an
entire year, without controlling the influence of the acid alone upon the proteoses.

KUHNE'S view that in tryptic digestion (pancreatic digestion) a
peptone, so-called antipeptone, always remains which cannot be further
split is not strictly true. By sufficiently long autodigestion of the pan-
creas, KuTSCHER2 was able to obtain, as final products, a mixture of
digestion products which failed to respond to the biuret test, and the same
results have been obtained by others. An antipeptone in the old sense,
i.e., a digestion product which is resistant to tryptic digestion but which
still gives the biuret test, is* without question not always obtained as end
product in trypsin digestion. On the contrary as FISCHER and ABDER-
HALDEN3 have shown, polypeptide-like bodies are produced in tryp-
tic digestion (and the same is probably true also for peptic digestion)
which do not give the biuret test, i.e., ".abiuret" products, and which
are resistant to further tryptic digestion but yield amino-acids on
hydrolysis with acids. This behavior stands in close relation to the
observation that in tryptic digestion certain amino-acids, such for example,
as tyrosine, tryptophane and leucine are split off earlier and more readily
than the others of the protein molecule.

Antipeptone, which is only attacked with great difficulty by trypsin
has in fact been isolated by SIEGFRIED (see below) and although the

1 Zunz, Zeitschr. f. physiol. Chem., 28, and Hofmeister's Beitrage, 2; Pfaundler,
Zeitschr. f. physiol. Chem., 30; Salaskin, ibid., 32; Salaskin and Kowalewsky, ibid.,
38; Lawrow, ibid., 33; Langstein, Hofmeister's Beitrage, 1 and 2.

2 Zeitschr. f. physiol. Chem., 25, 26, 28, and ,Die Endprodukte der Trypsinver-
dauung; Habilitationsschrift Strassburg, 1899.

3 Zeitschr. f. physiol. Chem., 39.

PROTEOSES AND PEPTONES. 133

views of KUHNE are not in all points correct still the fact remains that
under certain circumstances the protein can be split into fractions, of which
the hemi group is further easily decomposed by enzyme action while the
other, the anti group, is very much more resistant to such action. It
also seems as if the first group is characterized by a larger content of
tyrosine, tryptophane and the latter by its content of glycocoll, phenyl-
alanine and proline.

By the use of the methods specially worked out by the HOFMEISTER
school, of fractionally salting out with ammonium sulphate or zinc sul-
phate or also by SIEGFRIED'S iron-alum method, numerous attempts to
separate the various proteoses and peptones have been made.1 Not
only have we learned by these methods of a larger number of proteoses,
but our older conception of the products formed primarily has been
materially modified. Immediately at the commencement of diges-
tion, even in peptic digestion, a splitting of the protein molecule into
several complexes takes place. In opposition to the view of HuppERT,2 that
the proteoses, in pepsin digestion, are always derived from the primarily
formed acid albuminate, PICK and ZTJNZ have shown that several pro-
teoses, as well as acid albuminate, appear as primary products at the
commencement of the digestion. According to GOLDSCHMIDT 3 a splitting
off of proteoses and the formation of acid albuminate takes place simul-
taneously by the action of dilute acids alone. Besides the proteoses
we also have, according to ZUNZ and PFAUNDLER, even at the beginning,
other primary bodies, which cannot be salted out and which do not
give the biuret reaction, but are in part precipitated by phosphotungstic
acid. These little-known products seem to be intermediate between
the peptones and the amino-acids, and they correspond probably to
the polypeptide bodies obtained by FISCHER and ABDERHALDEN in tryptic
digestion.

By fractional precipitation of WITTE'S peptone with ammonium sulphate
PICK has obtained various chief fractions of proteoses. The first contains the
proto- and heteroprpteoses whose precipitation limit lies at 24-42 per cent satu-
ration with ammonium sulphate solution, i.e., the presence of 24-42 cc. of the
saturated ammonium sulphate solution in 100 cc. of the liquid. Then follows
a fraction A at 54-62 per cent saturation, then a third fraction B, with 70-95
per cent saturation, and finally fraction C, which precipitates from the saturated
solution on acidification with sulphuric acid saturated with the salt.

1 Umber, Zeitschr. f. physiol. Chem., 25; Alexander, ibid., 25; Pfaundler, ibid.,
30; Zunz, ibid., 28, and Hofmeister's Beitrage, 2; Pick, ibid., 2, and Zeitschr. f. physioL
Chem., 24 and 28; Siegfried, see footnote 3, p. 136.

2 Schiitz and Huppert, Pfliiger's Arch., 80.

* F. Goldschmidt, Ueber die Einwirkung von Sauren auf Eiweisstoffe, Inaug.-
Diss. Strassburg, 1898.

134 THE PROTEIN SUBSTANCES.

The hetero and protoproteoses are not, according to our present
views, the only primary proteoses. In the proteose fraction obtained
on saturating with ammonium sulphate in neutral liquids, \vhich should
contain secondary proteoses only, primary proteoses such as the gluco-
proteose (PICK), which contains a carbohydrate group and the so-called
synproteose (HOFMEISTER l) occur. It is no longer sufficient to consider
an unequal ability to be salted out, as an essential difference between
the primary and secondary proteoses.

There is no doubt that there exists a large number of so-called pro-
teoses having various precipitation properties, and different other prop-
erties and new differences appear, while working with them according to
different methods. For example RONA and MiCHAELis2 find that cer-
tain proteoses are precipitated by mastic emulsion while others are not.
Those that are precipitatable by mastic, can all be salted out, while all
those that can be salted out are not all precipitated by mastic. The
hetero- and protoproteoses act, according to ZuNZ3 like strong protec-
tion colloids toward colloidal gold, which is not the case with the others,
and also, according to this worker, the so-called proteoses are more readily
precipitated by chondroitin-sulphuric acid and acetic acid than the so-
called secondary proteoses. According to HUNTER 4 only the primary
proteoses are precipitated by protamines while the secondary are not.
It is also possible that numerous intermediary members exist between
those proteoses which stand close to the original protein and those that
are further removed. The difficulties in isolation and purification of
these different members are so very great that the proteoses thus far
isolated must not be considered as chemical individuals. Under these
circumstances the above-mentioned differentiation and classification
of the various proteoses is of little value and a more detailed discus-
sion of the properties of the various proteoses thus far isolated is with-
out interest.

It would be of great interest if certain differences in the chemical
structure of the different proteoses could be determined with certainty.
Such differences are claimed to have been found in certain cases. Thus
HART has found that the heteroproteose (from muscle syntonin) was
considerably richer in arginine and poorer in histidine than the proto-
proteose, and PICK has also found marked differences between the hetero-
and protoproteose from fibrin. The hetero-proteose yields very little

1 Ueber Bau und Gruppirung der Eiweisskorper, Ergebnisse der Physiol., Jahrg. I,
Abt. 1, 783.

2 Biochem. Zeitschr., 3.

3 Arch, internat. d. Physiol., 1 and 5, and Bull. Soc. Scienc. med. et natur. Brux-
elles, 64.

4 Journ. of Physiol., 37.

PROTEOSES AND PEPTONES. 135

tyrosine and indol but abundant leucine and glycocoll, and about 39
per cent of the total nitrogen in a basic form. The protoproteose,
according to PICK, on the contrary yields considerable tyrosine and indol,
only little leucine but no glycocoll, and contains only about 25 per cent
basic nitrogen. FRIEDMANN, HART, and LEVENE have obtained very
similar results in regard to the quantity of basic nitrogen in the two-pro-
teoses, although LEVENE as well as ADLER l did not find the same results
as PICK in regard to the amounts of monamino-acids in the two proteoses.
The work of LEVENE, v. SLYKE and BIRCHARD 2 show, in many important
points, a decided contradiction to the statements of PICK and these
divergent results may possibly be explained by the fact that they were
not working with pure substances, but rather with mixtures.

According to PICK the heteroproteose is also more resistant toward
tryptic digestion than the protoproteose, a behavior which coincides
with RUHNE'S view of a resistant atomic complex, an anti group, in the
protein bodies. KUHNE and CHITTENDENS regularly obtained on the
tryptic digestion of heteroproteose a separation of so-called antialbumid,
a body which is attacked with great difficulty in tryptic digestion, but
which separates as a jelly-like mass and which is richer in carbon (57.5-
58.09 per cent), but poorer in nitrogen (12.61-13.94 per cent), than the
original protein. The occurrence of such resistant complexes in diges-
tion has also been repeatedly observed.

This antialbumid later attracted increased interest, because as
first found by D ANILE WSKY and later other investigators have shown,
that solutions of rennin, gastric juice, pancreatic juice, and papain cause
a similar coagulum in not too dilute proteose solutions. These coagula,
called plasteines (coagulum by rennin) by SAWJALOW, and coaguloses
(coagulum by papain) by KiJRAjEFF,4 are similar in many respects to
antialbumid, having a higher content of carbon (57-60 per cent) and
nitrogen (13-14.6 per cent). In other cases the quantity of carbon as
well as nitrogen is lower (LAWROW).

We cannot for the present make any positive statement as to the
importance and mode of formation of the coaguloses or plasteins. It

1 Hart, Zeitschr. f. physiol. Chem., 33; Pick, ibid., 28; Friedmann, ibid., 29; Levene,
Journ. of Biol. Chem., 1; R. Adler, Die Heteroalbumose und Protalbumose des Fibrins
Dissert. Leipzig, 1907.

2 Journ. of Biol. Chem., 8 and 10.

3 Zeitschr. f. Biol., 19 and 20.

4 The works of Danilewsky and Okunew are cited and reviewed in the following
Sawjalow, Pfliiger's Arch., 85, and Centralbl. f. Physiol., 16; and Zeitschr. f. physiol.
Chem., 54; Lawrow and Salaskin, Zeitschr. f. physiol. Chem., 36; Lawrow, ibid., 51,
53, 56 and 60; Kurajeff, Hofmeister's Beitrage, 1 and 2; see also Sacharow, Biochem.
Centralbl., 1, 233; Levene and v. Slyke, Biochem. Zeitschr., 13.

136 THE PROTEIN SUBSTANCES.

is rather generally admitted that they are formed by a synthesis, a view
which has received support by the investigations of V. HENRIQUES and
GjALDBAK.1 According to SAWJALOW a plastein is not formed from a
proteose alone, but always from a mixture of proteoses. LAWKOW claims
that they may be produced from proteoses as well as from polypeptide
substances, and correspondingly we must differentiate between the coag-
uloses or coagulosogens from the proteose group coaproteoses, and from
the polypeptide group or coapeptides. The latter yield on hydrolysis
chiefly monamino-acids, while the first yield also basic nitrogenous prod-
ucts. Perhaps the plasteinogen investigated by BAYER2 which differs
essentially from the true proteid in its elementary composition as well
as from other coaguloses, belongs to the coapeptides.

The different behavior on saturating their solution with ammonium
sulphate has been generally used, as above remarked, for years to dif-
ferentiate between the proteoses and peptones. Those precipitable
by this salt were called proteoses, and those not were called peptones.
This method of division, which never had sufficient support and which
was perfectly arbitrary, cannot be considered at the present time. We
know now, thanks to the works of EMIL FISCHER and his co-workers,
that there are polypeptides either prepared artificially or found among
the cleavage products of the proteins, which are precipitated by ammo-
nium sulphate. At the present it is generally conceded that the peptones
in the ordinary sense are only a mixture of different bodies. The chief
step in these investigations must be the isolation from this mixture
of unit bodies with definite chemical characteristics. Of such bodies,
besides the polypeptides previously mentioned and studied by FISCHER
and others, we must mention the products isolated by SIEGFRIED and
his pupils.3

These so-called peptones are in part peptic-peptones and partly
tryptic-peptones, and some are prepared from proteid (fibrin) and others
from gelatin. The tryptic fibrin-peptones are antipeptones in KUHNE'S
sense because they are very resistant to the further action of trypsin.
They are according to NEUMANN simultaneously bibasic acids and mono-
acidic bases. They give the biuret reaction, but not MILLON'S reaction;
they contain no tyrosine and yield on hydrolysis, arginine, lysine, glutamic
acid, and it seems also aspartic acid. A pepsin-glutin peptone isolated
by SIEGFRIED and SCHMITZ yielded arginine, lysine, glutamic acid, gly-

1 Zeitschr. f. physiol. Chem., 71 and 81.

2 Hofmeister's Beitrage, 4; see also L. Rosenfeld, ibid., 9; J. Lukomnik, ibid., 9
and F. Micheli, Biochem. Centralbl., 6, p. 562.

3 The works of Siegfried and his pupils, Fr. Miiller, Borkel, Miihle, Kriiger, Scheer-
messer, Neumann, H. Schmitz, may be found in Arch. f. (Anat. u.) Physiol., 1894 and
Zeitschr. f. physiol. Chem., 21, 41, 43, 45, 48, 50, and 65 and Pfliiger's Arch. 136.

PROTEOSES AND PEPTONES. 137

cocoll and besides these also leucine and proline although not in quan-
tities that could be determined. Of the total nitrogen they found
19.7 per cent arginine, 9.1 per cent lysine, 49.2 per cent glycocoll, 9.3
percent glutamic acid and 12.7 per cent proline and leucine together.
SIEGFRIED has given proof in several ways as to the purity and unity
of the peptones isolated by him.

In another manner, namely by fractional precipitation with metallic salts,
especially with mercuric-potassium iodide and the preparation of phenyliso-
cyanate compounds, HOFMEISTEK and his pupils STOOKEY, RAPER and ROGO-
ZINSKI i have isolated peptones or polypeptide-like bodies from blood proteid.
One of these, called arginine-histidine peptone, yielded arginine and histidine as
basic hydrolytic products while another yielded chiefly lysine^as basic product
and hence was called lysine-peptone.

From glutin-peptone, SIEGFRIED, on wanning with hydrochloric
acid, obtained a base, C2iH39N908, which can also be directly obtained
from gelatin. This he calls a kyrin, because it is to be considered a$ a
basic protein nucleus, and he calls this special one glutokyrin. The
glutokyrin gives the biuret reaction and is considered as a basic peptone.
On complete hydrolytic cleavage it yields arginine, lysine, glutamic
acid, and glycocoll. Of the total nitrogen two-thirds belong to the
bases and one-third to the amino-acids. Recently he with O. PILZ
on further hydrolysis has prepared a /3-glutokyrin, which only yielded
arginine, lysine and glutamic acid. Similar basic nuclei, protokyrins,
have recently been obtained by SIEGFRIED 2 from fibrin and casein, using
the same method. Caseinokyrin gives a non-crystalline sulphate, but
a crystalline phosphotungstate. The free caseinokyrin has an alkaline
reaction, gives the biuret test, and its composition corresponds to the
formula C23H47N90s. It yields arginine, lysine, and glutamic acid on
cleavage. The basic nitrogen amounts to about 85 per cent of the total
nitrogen, and caseinokyrin, behaves in this respect like a protamine.

Among the known cleavage products of proteins, arginine is the only
one which, up to the present, is never absent, and for this reason we
designate as proteins only those atomic complexes which contain, besides
chained monamino-acids, also arginine, or, more simply, show the prev-
iously mentioned imide bindings. Hence caseinokyrine, wrhich yields
only arginine, lysine and glutamic acid, and scombrin, which yields
only arginine, proline, and alanine, are the simplest known proteins.

Scombrin belongs to the previously mentioned group of protamines
which, according to KossEL,3 are formed by a successive cleavage of the

1 Hofmeister's Beitrage, 7, 9, and 11.

2Kgl. Sachs. Ges. d. Wiss., Math.-Phys. Klasse, 1903, and Zeitschr. f. physiol
Chem., 43, with Pilz., ibid., 58.
3 Zeitschr. f. physiol. Chem., 44.

138 THE PROTEIN SUBSTANCES.

typical protein. The occurrence of basic protokyrins in the hydrolytic
cleavage of genuine proteins like gelatin has given valuable support to
KOSSEL'S theory as to a basic nucleus in the protein bodies.

On account of the cleavage taking place in digestion, the digestive
products should have a lower molecular weight than the original protein.
This is really the case as shown by molecular weight determinations.
As these determinations have been made upon impure substances or
mixtures, the results 1 obtained are only of little value. The same is
true for the elementary analysis of the proteoses and peptones.2

In the preparation and separation of various proteoses and peptones
all precipitable protein is always removed first by neutralization and
then by boiling. The proteoses may then be separated from the pep-
tones by means of ammonium sulphate according to KUHNE'S method,
and divided into different fractions according to the method of PICK
and the HOFMEISTER school. The separation and preparation of pure
hetero- and protoproteoses can be best performed by the method sug-
gested by PICK, but this method, as well as that with ammonium sulphate,
gives good results only when the precautions suggested by HASLAMS
are carefully followed. We can here only refer to the cited works of
KUHNE and co-workers, of E. ZUNZ and especially those of the HOF-
MEISTER and the SIEGFRIED schools. In regard to the literature on the
detection of proteoses and peptones in animal fluids we refer to Chapters
V and XIV.

If we wish to detect the presence of so-called true peptone, by means
of the biuret reaction in a solution saturated with ammonium sulphate,
we add a slight excess of a concentrated solution of caustic soda and
cool, and then add a two per cent solution of copper sulphate drop by
drop, after the sodium sulphate has separated out.

In the quantitative estimation of proteoses and peptones we make
use of the nitrogen estimation, the biuret test (colorimetric), and the
polarization method. These methods do not give exact results.

The polypeptides have had their most important properties dis-
cussed on pages 85-91, and of the cleavage products of the proteins only
the amino-acids remain to be discussed.

1 Sabanejew, Ber. d. d. chem. Gesellsch., 26, 385; Paal, ibid., 27, 1827; Sjoqvist,
Skand. Arch. f. PhysioL, 5.

2 Elementary analyses of proteoses and peptones will be found in the works of
Kiihne and Chittenden and their pupils, cited in footnote 2, p. 130; also by Herth,
Zeitschr. f. physiol. Chem., 1, and Monatshefte f. Chem., 5; Maly, Pfliiger's Arch.
9, 20; Henninger, Compt. rend., 86; Schrotter, 1. c., Paal, 1. c.

3 Journ. of Physiol., 32 and 36.

GLYCOCOLL. 139

3. The Amino-acids.1

|o I

Glycocoll (amino-acetic acid), C2H5NO2 = • _ !""' > also called gty-

COOrl

cine or gelatin sugar, is found in the muscles of the invertebrates,
but has chief interest as a hydrolytic decomposition product of protein
bodies, especially fibroin, spider-silk elastin, gelatin, and spongin, as
well as of hippuric acid and glycocholic acid. i

Glycocoll forms colorless, often large, hard rhombic crystals or four-
sided prisms. The crystals have a sweet taste and dissolve readily in
cold water (4.3 parts). Glycocoll is insoluble in alcohol and ether and
dissolves with difficulty in warm alcohol. Like the amino-acids in gen-
eral it combines with acids and alkalies. With the latter compounds
we must mention those with copper and silver. Glycocoll dissolves
cuDric hydroxide in alkaline liquids, but does not reduce at boiling heat.
A boiling-hot solution of glycocoll dissolves freshly precipitated cupric
hydroxide, forming a blue solution, which in proper concentration deposits
blue needles of copper-glycocoll on cooling. The compound with hydro-
chloric acid is readily soluble in water but less soluble in alcohol.

SORENSEN 2 finds that phosphotungstic acid does not precipitate
glycocoll from dilute solutions but only from concentrated ones. By the
action of gaseous HC1 upon glycocoll in absolute alcohol, beautiful
crystals are obtained of the hydrochloride of glycocoll-ethyl ester, which
melts at 144° C. and from which the glycocoll-ethyl ester can be obtained
by the method suggested by E. FISCHER 3 for the separation of glycocoll
from the other amino-acids. On shaking with benzoyl chloride and
caustic soda, hippuric acid is formed, and this is also made use of in
different ways in detecting and isolating glycocoll (Cn. FISCHER, GON-
NERMANN, SpiRO4). The j3-naphthalene-sulpho-glycine with a melt-
ing-point of 159°, the 4-nitro-tolulene-2-sulpho-glycine, melting at 180°,
and the a-naphthylisocyanate compound melting at 190.5-191.5° are
also of importance. On putrefaction methane is probably produced
from glycocoll.

Glycocoll can be best prepared from hippuric acid by boiling it with
4 parts dilute sulphuric acid (1:6) for ten to twelve hours. After cooling

1 In regard to the division of the amino-acids among the three chief groups of
organic compounds we refer to pages 85-86.

2 Meddelelser, fraa Carlsberg-laboratoriet, 6, 1905.

3 Ber. d. d. chem. Gesellsch., 34.

4Ch. Fischer, Zeitschr, f. physiol. Chem., 19; Spiro, ibid., 28; Gonnermann,
Pfliiger's Arch., 59.

140 THE PROTEIN SUBSTANCES.

the benzole acid is removed, the filtrate concentrated, the remaining
benzoic acid removed by extracting with ether, the sulphuric acid pre-
cipitated by BaCOs, and the filtrate evaporated to the point of crys-
tallization. (In regard to its preparation from protein substances see
below.)

CH3
d-Alanine(a-aminopropionic acid), CsH7N02 = CH(NH2). The d-alanine

COOH

is obtained in relatively small amounts from the true proteids, but in
larger quantities from the albuminoids, especially from fibroin, spider-
silk and elastin.

d-alanine has been prepared from /-serine by E. FISCHER and K.
RASKE,1 and FISCHER has also obtained it from racemic alanine by split-
ting the benzoyl combination, or from Z-alanine by splitting with yeast
by WALDEN'S reversion.

1^ Alanine generally crystallizes in needles or oblique rhombic columns.
It is very readily soluble in water, having a sweetish taste, and dissolves
cupric hydroxide on boiling, producing a deep blue solution of a crystalliza-
ble copper salt. Alanine is insoluble in absolute alcohol. The rota-
tion of alanine at 20° C. in aqueous solution is (a)D=-J-2.7° and for a
solution in hydrochloric acid (9-10 per cent solution) is (a)j}=+10.3°.

The |3-naphthalene-sulpho-d-alanine melts, when dry, at about 123°
and sinters at 117° C. The phenylisocyanate melts at 168° and the
a-naphthylisocyanate alanine melts at 198°. On putrefaction alanine
yields propionic acid.

CH3CH3

V

/-ITT

d-Valine (a-amino-valeric acid), C5HnN02 = ^T-r/ATT-r x ^ v

L/H(JNri2), nas been

COOH

detected several times among the cleavage products of protein sub-
stances, although only in small quantities. KOSSEL and DAKIN obtained
4.3 per cent valine from salmine, and E. FISCHER and DoRpiNGHAiis2
5.7 per cent from horn substance. The largest quantity has been obtained
from casein and edestin, namely, 7.20 and 5.6 per cent respectively.
Because of the difficulty in separating valine from the two leucines3
the figures given are somewhat uncertain. The acid isolated by H.
and E. SALKOWSKi4 from putrefying proteid or gelatin seems to have
been 5-amino-n-valeric acid.

1 Ber. d. d. chem. Gesellsch., 40. ,

2 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41; Fischer and Dorpinghaus,
ibid., 36.

8 See Levene and v. Slyke, Journ. of Biol. Chem., 6.
4 Ber. d. d. chem. Gesellsch., 16 and 31.

LEUCINE. 141

d-valine can be obtained as microscopic crystalline leaves. It is
rather readily soluble in water and the solution has a faint sweetish taste
and at the same time somewhat bitter. The solution has a rotation
of (a)D=+6.42°. The hydrochloric acid solution (20 per cent) shows,
according to FISCHER, a rotation of (o)D= +28.8°. The copper salt,
which forms leaves which are rather soluble in water, is very easily soluble
in methyl alcohol (SCHULZ and WiNTERSTEiN1).

The phenylisocyanate melts at 147°, and on boiling with 20 per cent
hydrochloric acid for a short time, it is changed into d-phenylisopropyl
hydantoin, which melts at 131-133° C.

On putrefaction valine yields isobutylamine and iso valeric acid.
/-Leucine (aminocaproic acid, or, more correctly, a-aminoisobutylacetic
CH3CH3
\X
CH

acid), CeHi3N02= CH2 , is produced from protein substances in

CH(NH2)

COOH

their hydrolytic cleavage by proteolytic enzymes, by boiling with dilute
acids or alkalies or by fusing with alkali hydroxides, and by putrefaction.
There are also observations that indicate that in the hydrolysis besides
the ordinary leucine perhaps also normal leucine may be formed (HECKEL
and SAMEC2).

Because of the ease with which leucine (and tyrosine) are formed
in the decomposition of protein substances, it is difficult to decide pos-
itively whether these bodies when found in the tissues are constituents
of the living body or are to be considered only as decomposition products
formed after death. Leucine, it seems, has been found as a normal
constituent of the pancreas and its secretion, in the spleen, thymus, and
lymph glands, in the thyroid gland, in the salivary glands, in the kidneys
and in the liver. It also occurs in the wool of sheep, in dirt from the
skin (inactive epidermis), and between the toes, and its decomposition
products have the disagreeable odor of the perspiration of the feet.
It is found pathologically in atheromatous cysts, ichthyosis scales, pus,
blood, liver, and urine (in diseases of the liver and in phosphorus poison-
ing) . Leucine often occurs in invertebrates and also in the plant king-
dom. On hydrolytic cleavage various protein substances yield different
amounts of leucine, as shown in the tables given on pages 106, 107, 115 and
125. From the figures, there given, we call attention to the following:
EELENMEYER and SCHOFFER obtained 36-45 per cent leucine from the
cervical ligament, E. FISCHER and ABDERHALDEN 20 per cent from hsemo-

1 Zeitschr. f. physiol. Chem., 35.

2 Heckel, Monatsh. f. Chem., 29; Samec, ibid., 29.

142 THE PROTEIN SUBSTANCES.

giobin, and FISCHER and DORPINGHATJS 18.3 per cent from horn sub-
stance.1

The leucine obtained by cleavage of protein substances is generally
Z-leucine, which is levorotatory in water solution and dextrorotatory
in acid solution. The leucine prepared synthetically by HtiFNER2
from isovaleraldehyde, ammonia, and hydrocyanic acid is optically
inactive. Inactive leucine may also be prepared, by the cleavage of pro-
teins with baryta at 160-180° C., because of a ready racemation. The
d-Z-leucine may be split into the two components by various means, espe-
cially by the preparation of the formal combination.3

On oxidation the leucines yield the corresponding oxyacids (leucinic
acids). Leucine is decomposed on heating, evolving carbon dioxide,
ammonia, and amylamine. On heating with alkalies, as also in putre-
faction, it yields valeric acid and ammonia. On putrefaction it yields
isoamylamine and isocaproic acid.

Leucine crystallizes when pure in shining, white, very thin plates,
usually forming round knobs or balls, either appearing like hyaline, or
with alternating light and dark concentric la}'ers which consist of radial
groups of crystals. By slow heating, leucine melts and sublimes into
white woolly flakes, which are similar to sublimed zinc oxide. At the
same time an odor of amylamine is developed. Quickly heated in a
closed capillary tube, it melts with decomposition at 293-295°.

Leucine, as obtained from animal fluids and tissues is always impure,
and is very easily soluble in water and rather easily in alcohol. Pure
leucine is soluble with difficulty. Pure I- and d-leucine dissolve in 40-
46 parts water, more readily in hot alcohol, but with difficulty in cold
alcohol. The eW-leucine is much less soluble. According to HABER-
MANN and EnRENFELD4 100 parts of boiling glacial acetic acid dissolve
29.23 parts of leucine. The specific rotation of /-leucine, dissolved in
hydrochloric acid (20 per cent solution) is (a)D = +15.6° according to
FISCHER and WARBURG. In aqueous solution it is (a)D= — 10.40°,
according to F. EHRLICH and WENDEL.5

The solution of leucine in water is not, as a rule, precipitated by
metallic salts. The boiling-hot solution may, however, be precipitated
by a boiling-hot solution of copper acetate, and this fact is made use of
in separating leucine from other substances. If the solution of leucine

1 Erlenmeyer and Schoffer, cited from Maly, Chem. d. Verdauungssafte, in Her-
mann's Handb. d. Physiol., 5, Theil 2, p. 209; Fischer and his collaborators, ibid., 36.

2 Journ. f. prakt. Chem. (N. F.), 1.

3 Fischer and Warburg, Ber. d. d. chem. Gesellsch., 38.

4 Zeitschr. f. physiol. Chem., 37.

5 Fischer and Warburg, Ber. d. d. chem. Gesellsch., 38; E. Ehrlich and Wendel,
Biochem. Zeitschr., 8.

ISOLEUCINE. 143

is boiled with sugar of lead and then ammonia be added to the cooled
solution, shining crystalline leaves of leucine-lead oxide separate. Leucine
dissolves cupric hydroxide, but does not reduce on boiling.

Leucine is readily soluble in alkalies and acids. It gives crystalline
compounds with mineral acids. If leucine hydrochloride is boiled with
alcohol containing 3-4 per cent HC1, long narrow crystalline prisms of
leucine-ethyl-ester hydrochloride, melting at 134° C., are formed. The
picrate of the leucine ester melts at 128°. The phenylisocyanate of
cW-leucine melts at 165° and its anhydride at 125° C. The a-naphthyl-
isocyanate leucine melts at 163.5°, the naphthalene-sulpho-Z-leucine
at 68° C.

Leucine is recognized under the microscope by the appearance of balls
or knobs, by its action when heated (sublimation test), and by its
compounds, especially the hydrochloride and picrate of the ethyl ester
and the phenylisocyanate compound of the racemic leucine obtained
on heating with baryta water, the a-naphthylisocyanate compound and
the /3-naphthalene-sulpho-leucine. According to the method suggested
by LIPPICH x the leucine can be transformed into isobutylhydantoin,
having a melting-point of 205°, by boiling with an excess of urea and
baryta water. For the preparation and separation of leucine from the
other amino-acids of the leucine fraction special methods have been
suggested by F. EHRLICH and WENDEL, LEVENE and v. SLYKE.2

Leucinimide, C12H22N202 = ' V ' ^ > was first obtained by RITT-

.04x19

HAUSEN in the hydrolytic cleavage products on boiling proteins with acids, and
subsequently by R. COHN. SALASKIN 3 obtained it in the peptic and tryptic
digestion jf haemoglobin. As an anhydride of leucine (2.5-diacipiperazine) it
is probably formed by a secondary change, from leucine.

It crystallizes in long needles and sublimes readily. The melting-point has
not been found constant in the different cases. The leucinimide (3.6-diisobutyl-
2.5-diacipiperazine) prepared synthetically by E. FISCHER 4 from leucine-ethyl
ester melted at 271° C.

2-Isoleucine (/3-methyl-ethyl-a-amino-propionic acid),

CH3C2H5
\/

COOH

1 Ber. d. d. chem. Gesellsch., 39.

2 F. Ehrlich and Wendel, 1. c.; Levene and v. Slyke, Journ. of Biol. Chem., 6.

3 Ritthausen, Die Eiweisskorper der Getreidearten, etc., Bonn, 1872; R. Cohn,
Zeitschr. f. physiol. Chem., 22 and 29; Salaskin, ibid., 32.

4 Ber. d. d. chem. Gesellsch., 34.

144 THE PROTEIN SUBSTANCES.

is an isomer of leucine discovered by F. EHRLICH/ who first isolated
it from the mother-liquor after removing the sugar from beet-sugar
molasses. He also found it in the hydrolysis of several proteins, and
recently it has been found by others among the products of hydrolysis
of the proteins. The largest amount thus far found was 2.6 per cent
by LEVENE, v. SLYKE and BIRCHARD 2 in a heteroproteose. It seems
to be associated regularly with ordinary leucine, forming mixed crystals,
which give an impression of a chemical combination and which are dif-
ficult to separate. On this account the earlier claims as to the quantity
of leucine are somewhat uncertain, as they always refer to leucine
containing isoleucine.

The constitution of isoleucine has been explained by EHRLICH through
its relation to d-amyl alcohol. Just as according to F. EHRLICH valine
yields the isobutyl alcohol in alcoholic fermentation so isoleucine yields
d-amyl alcohol in the fermentation of sugar with yeast. On the other
hand, it can also be obtained, in a manner analogous to the synthesis
of leucine, from d-amyl alcohol (as a mixture of isoleucine and alloiso-
leucine, the latter is levogyrate and has a different stereometric configura-
tion from the isoleucine). The synthesis of isoleucine has been accom-
plished in other ways by several investigators.3 On putrefaction d-ca-
proic acid and d-valeric acid have been obtained from isoleucine.4

Isoleucine crystallizes in leaves or rods and plates of the rhombic
form. It is more soluble in water than leucine (1:25.8). Its solutions
have a bitter taste and are astringent. It is dextro-rotatory in aqueous
as well as in acid solution. In aqueous solution it has a specific rotation
of (a)D = -}-9.740 and in 20 per cent hydrochloric acid (a)D = +36.8°.
Like valine its copper salt is readily soluble in methyl alcohol. The
benzoyl combination melts at 116-117°, the benzene sulphoisoleucine
at 149-150°, the phenylisocyanate combination at 119-120°, and the
naphthylisocyanate combination at 178° C.

In the leucine fraction, from the amino-acids contained in nerve
substance, ABDERHALDEN and WEIL5 have obtained a new amino-acid,
C6HisN02 which is isomeric with leucine and which seems to be d-a-
amino-n-caproic acid and called d-caprine by them. When crystallized
from water it forms six-sided plates which unite to tufts having a faint
sweet taste. At 280° (uncorrected) it softens and at 285° (uncorrected)

1 Felix Ehrlich, Ber. d. d. chem. Gesellsch., 37.

2 Journ. of Biol. Chem., 8.

'Ehrlich, Ber. d. d. chem. Gesellsch., 40 and 41; Brasch and Friedmann, Hof-
meister's Beitrage, 11; Bouveault and Locquin, Compt. rend., 141, and Bull. soc.
chim. (3), 35; Locquin, Bull. soc. chim. (4), 1.

4 C. Neuberg, Bioch. Zeitschr., 37.

6 Zeitschr. f . physiol. Chem., 81 and 84.

SERINE. 145

it sublimes. Its solubility in water is 1.5:100; at 20° in aqueous so-
lution («)D+6.53° and in 20 per cent hydrochloric acid+14.1°. It gives
a copper salt crystallizing in needles.

CH2(OH)

Z-Serine (a-amino-/3-oxypropionic acid), C3H7N03 = CH(NH2), was

COOH

obtained by FISCHER and his collaborators as a cleavage product of
several proteins, generally only in small quantities. The largest quan-
tity, 6.6 per cent, was obtained by FISCHER and SKITA from sericine;
KOSSEL and DAKIN l obtained a still larger amount from salmine, namely
7.8 per cent. The racemic serine is the one generally obtained. From
fibroin FISCHER 2 obtained a mixture of active and inactive serine anhy-
dride from which he finally prepared Z-serine by hydrolysis. Serine has
also been found by G. EMBDEN and TACHAU 3 in fresh perspiration.

Synthetically cW-serine has been prepared by FISCHER and LEUCHS
from ammonia, hydrocyanic acid and glycol aldehyde, and also in other
ways by others.4 FISCHER and JACOBS 5 have prepared Z-serine from
d-/-serine by the preparation of the alkaloid salt of the p-nitro-benzoyl
combination. On reduction serine is transformed into alanine, and on
oxidation with nitrous acid it yields glyceric acid. The relation of serine
to alanine, lactic acid and glyceric acid is evident from the following for-
mulae:

CH2(OH) CH3 CH3 CH2(OH)
CH(NH2) CH(NH2) CH(OH) CH(OH)
COOH • COOH COOH COOH

Serine Alanine Lactic acid Glyceric acid

The Z-serine crystallizes in thin leaves or crusts. It is rather readily
soluble in water; the d-Z-serine is soluble in 23 parts water at 20° C.
The solution of /-serine has a sweet taste with an insipid after taste.
The specific rotation in aqueous solution at 20° C. is (a)D= — 6.83°
and the hydrochloric acid solution at 25° C. is (a)D = + 14.45°. The
/3-napthalene-sulpho-serine melts at 220° C. when anhydrous. The
Z-serine anhydride, which is identical with that obtained from fibroin,
forms thin, colorless needles which melt at 247° with decomposition.
Its specific rotation in aqueous solution at 25° C. (a)D= —67.46°.

1 Fischer and Skita, Zeitschr. f. physiol. Chem., 35; Kossel and Dakin, ibid., 41.

2 Ber. d. d. chem. Gesellsch., 40.

3 Bioch. Zeitschr., 28.

4 Fischer and Leuchs, Ber. d. d. chem. Gesellsch, 35; Erlenmeyer and Stoop, ibid.,
35; Leuchs and Geiger, ibid., 39.

5 Ber. d. d. chem. Gesellsch., 39.

146 THE PROTEIN SUBSTANCES.

Isoserine (/3-amino-a-oxypropionic acid) has been prepared by ELLINGER
from diamino-propionic hydrobromide and silver nitrite, and by NEUBERG and
SILBERMANN from the hydrochloric acid combination of diamino-propionic acid.
Other syntheses have been made by NEUBERG and MAYER and by NEUBERG
and AscHER.1

COOH
/-Aspartic acid (aminosuccinic acid), C4H7N04 = ATJ , has been

COOH

obtained on the cleavage of protein substances by proteolytic enzymes as
well as by boiling them with dilute mineral acids in comparatively small
quantities. This acid also occurs in secretions of sea-snails (HENZE2)
and is very widely diffused in the vegetable kingdom as the amide
Asparagine (aminosuccinic-acid amide), which seems to be of the
greatest importance in the development and formation of the proteins
in plants. d-Z-Aspartic acid has been prepared synthetically from fumaric
acid and alcoholic ammonia. On putrefaction of aspartic acid, propionic
acid and succinic acid are formed.

Z-Aspartic acid dissolves in 256 parts water at 10° C. and in 18.6 parts
boiling water, and on cooling crystallizes as rhombic prisms, and its
4 per cent solution acidified with HC1 has the rotation (a)D = +25.7°;
in alkaMne solution the acid is levo-rotatory. It forms with copper
oxide a crystalline compound which is soluble in boiling-hot wrater and
nearly insoluble in cold water, and wrhich may be used in the prepara-
tion of the pure acid from a mixture with other bodies.

The benzoyl-Z-aspartic acid melts at 184-185°. For identification
we make use of the analysis of the free acid and the copper salt, as well
as of the specific rotation.

COOH,
CH(NH2)

d-Glutamic acid (a-aminoglutaric acid), C5H9N04 = CH2 , is

CH2
COOH

obtained from the protein substances under the same conditions as the
other monamino-acids (see tables on pages 106, 107, 115 and 125) and
from the peptones (SIEGFRIED). It is absent in the protamines and in the
varieties of silk, it occurs only in small amounts writh the exception of spi-
der's web. HLASIWETZ and HABERMANN obtained 29 per cent from casein
by cleavage with hydrochloric acid, while KUTSCHER could obtain only
1.8 per cent glutamic acid by cleavage with sulphuric acid. Other

1 Ellinger, Ber. d. d. chem. Gesellsch., 37; Neuberg and Silbermann, ibid., 37J
Neuberg and Mayer, Biochem., Zeitschr 3; Neuberg and Ascher, ibid., 6.

2 Ber. d. d. chem. Gesellsch., 34.

GLUTAMIC ACID. 147

investigators such as ABDERHALDEN and FUNK and SKRAUP and TURK
have shown that the same quantities of glutamic acid can be obtained
by the use of the two mineral acids. SKRAUP and TURK obtained on the
hydrolysis of casein 20.3-22.3 per cent glutamic acid hydrochloride
corresponding to about 17 per cent glutamic acid. ABDERHALDEN and
SASAKI 1 obtained 13.6 per cent glutamic acid from meat syntonin. It
occurs most abundantly in the plant proteins where the quantity may
be more than 40 per cent. LEVENE and MANDEL2 have obtained a strik-
ingly large quantity of glutamic acid, namely 25 per cent, from a nucleo-
protein of the spleen.

On heating glutamic acid to 180-190° it is converted into pyrrolidon-
carboxylic acid, which latter can be re transformed into glutamic acid
by HC1 gas; therefore, a formation of pyrrolidon-carboxylic acid at the
same time, or in place of glutamic acid, in the hydrclyses, is not excluded.

On putrefaction glutamic acid gives 7-aminobutyric acid, n-butyric
acid and succinic acid.

d-Glutamic acid crystallizes in rhombic tetrahedra or octahedra or
in small leaves. It dissolves in 100 parts water at 16° C., and the solu-
tion has an acid taste with a peculiar after-taste. It is insoluble in
alcohol and in ether.

In water it has a rotation of (a)D = +12.04°. Strong acids increase
the rotation, and a 5 per cent solution of glutamic acid containing 9 per
cent HC1 has a rotation (a)D=+31.7°, while that obtained by heating
with barium hydroxide is optically inactive. d-Glutamic acid forms
a beautifully crystalline combination with hydrochloric acid, which is
almost insoluble in concentrated hydrochloric acid. This compound
is used in the isolation of glutamic acid. On boiling with cupric hydroxide
a beautiful crystalline copper salt, which is soluble with difficulty, is
obtained.3 The benzoyl-d-glutamic acid melts at 130-132° C. The
hydrochloride, the a-naphthylisocyanate of glutamic acid, which melts
at 236-237° C., the analysis of the free acid, and the specific rotation
are used in its detection.

As previously stated monamino-oxydicarboxylic acids have also
been found among the cleavage products of the proteins. To these belong
the following:

That oxyaminosuccinic acid, CnH7NOi occurs among the hydrolytic cleavage
products of proteids has been shown to be probable by SKRAUP. This acid has

1 Hlasiwetz and Habermann, Annal. d. chem. u. Pharm., 159; Kutscher, Zeitschr.,
f. physiol. Chem., 28; Abderhalden and Funk, ibid., 53; with Sasaki, ibid., 51; Skraup
and Turk, Monatsch. f. Chem., 30.

2 Bioch. Zeitschr, 5.

3 Several salts of glutamic acid have been prepared and studied by Abderhalden
and Kautzsch, Zeitschr. f. physiol. Chem., 64, 68, and 78.

.148 THE PROTEIN SUBSTANCES.

been prepared synthetically by NEUBERG and SILBERMANN from diaminosuccinic
acid and barium nitrite in sulphuric acid solution. Oxyaminosuberic acid,
C8Hi6N06, has been detected by WOHLGEMUTH 1 in the cleavage products of a
liver nucleoprotein.

Z-Cystine, C6Hi2N2S204 (a-diamino-/3-dithiolactolic acid), the disulphide

CH2— S— S— CH2
of cysteine (a-ammo-|3-thiolactic acid), CH(NH2) CH(NH2), was first

COOH COOH

obtained, with certainty, as a cleavage product of protein substances by
K. MORNER, and then also by EMBDEN. KULZ 2 obtained it once as a
product of tryptic digestion of fibrin. The quantities found by MORNER
and BUCHTALA in the various proteins are given in the tables on pages
106, 107, 115 and 125.

According to NEUBERG and MAYERS two kinds of cystine occur in nature,
namely, stone-cystine, designated /3-cystine. and protein-cystine, called a-cystine

CH2NH2 CH2NH,
Stone-cystine is the disulphide of /3-amino-a-thiolactic acid, CH — S — S — CH

COOH COOH

The protein-cystine has been chiefly obtained from the protein substance,
but also from calculi, while the stone-cystine has been obtained from urinary
calculi only.

Many objections have been raised from many sides as to the correctness of
this assumption. ROTHERA could not find any difference between the stone-
cystine and the cystine prepared from hair, and FISCHER and SUZUKI, and recently
also ABDERHALDEN,4 arrived at similar results, which seems to place the exist-
ence of stone-cystine in doubt. The occurrence of two structurally isomeric
cystines is not improbable, from certain observations of MORNER, but FRIEDMANN
and BAERS have shown that these observations do not lead to this assumption
and at the present time we cannot admit of the occurrence of two different cystines*

Cystine probably occurs normally as traces in the urine. In rare
cases, in cystinuria, it occurs in larger quantities in the urine, the sediment
or in calculi. Traces have also been found in the ox-kidney, in the liver
of the horse and dolphin, and in the liver of a drunkard. ABDER-
HALDEN 6 has found cystine in the urine and also abundantly in the
organs (spleen) in a case of parental cystine diathesis.

The constitution of cystine has been explained by FRIEDMANN. 7 and

, Zeitschr. f. physiol. Chem., 42; Neuberg and Silbermann, ibid., 44;
Wohlgemuth, ibid., 44.

2 K. Morner, ibid., 28, 34, and 42; Embden, ibid., 32; Kiilz, Zeitschr. f. Biologie,
27.

8 Zeitschr. f. physiol. Chem., 44.

4Rothera, Journ. of Physiol., 32; Fischer and Suzuki, Zeitschr. f. physiol. Chem.,
45; Abderhalden, ibid., 51.

5 Friedmann, Hofmeister's Beitrage, 3. With Baer, ibid., 8.

« Zeitschr. f . physiol. Chem., 38.

7 Hofmeister's Beitrage, 3.

CYSTINE. 149

he has also established the relation between cystine and taurinc. Cys-
tine is the disulphide of cysteine, which is a-amino-/3-thiolactic acid.
From cysteine bv oxidation FRIEDMANN obtained cysteinic acid,

CH2SO2OH
C3H7NS05 = CH(NK2), from which taurine CH2(S02OH) is produced'by

COOH CH2(NH2)

splitting off C02.

Cystine has also been prepared synthetically in several ways. For
example, FISCHER and RASKE 1 have prepared cystine from a-amino
/3-chlorpropionic acid (obtained from Z-serine) by the action of barium
hydrosulphide and a subsequent oxidation in the air.

/-Cystine crystallizes in thin, colorless, hexagonal plates. It is not
soluble in water, alcohol, ether, or acetic acid, but dissolves in mineral
acids and oxalic acid. It is also soluble in alkalies and ammonia, but
not in ammonium carbonate. Cystine is optically active, being levorota-
tory. MORNER found it to be (O:)D= —224.3°. On heating with hydro-
chloric acid it can, according to MORNER, be changed into a modifica-
tion crystallizing in needles and with a weaker levorotatory power, or
indeed dextrorotatory, composed of a mixture of the two optically active
cystines. On heating with HC1 to 165° for 12-15 hours NEUBERG and
MAYER obtained inactive cystine. By fungus fermentation with Asper-
gillus niger they obtained dextrorotatory cystine. Cystine has no
melting-point but slowly decomposes at 258-261°. On boiling cystine
with caustic alkali it decomposes and yields alkali sulphide, which can
be detected by lead acetate or sodium nitroprusside. According to MOR-
NER 2 75 per cent of the total sulphur is separated. Cystine treated
with tin and hydrochloric acid develops only a little sulphureted
hydrogen, and is converted into cysteine. Cystine yields sulphureted
hydrogen and methyl mercaptan on putrefaction.

On heating upon platinum-foil cystine ignites and burns with a bluish-
green flame, with the generation of a peculiar sharp odor. When warmed
with nitric acid it dissolves with decomposition, and leaves on evapora-
tion a reddish-brown residue, which does not give the murexid test.

Cystine is gradually precipitated from its sulphuric acid solution by
phosphotungstic acid. Cystine forms crystalline salts with mineral
acids and with bases. For isolating and separating cystine the precipita-
tion with mercuric acetate is especially suited. The benzoyl cystine
(BAUMANN and GOLDMANNS) melts at 180-181°; the phenylisocyanate
compound at 160°. On boiling wTith 25 per cent hydrochloric acid this

1 See Erlenmeyer and Stoop, Ber. d. d. chem. Gesellsch., 36; Gabriel, ibid., 38; Fischer
and Raske, ibid., 41.

2 Zeitschr. f. physiol. Chem., 34.

3 Ibid., 12.

150 THE PROTEIN SUBSTANCES.

compound passes to the anhydride, which is a hydantoin melting at
119° C. By the action of potassium cyanide MAUTHNER l obtained
a-amino-/3-suphocyanpropionic acid, CH2(SCN).CH(NH2)COOH.

Stone-cystine, according to NEUBERG and MAYER, differs in many respects
from the ordinary cystine, among which the following may be mentioned: The
optically active stone-cystine crystallizes in needles, the specific rotation is
(«)D = -206°; it melts at 190-192° with marked swelling up. The benzoyl
compound melts at 157-159°; the phenylcyanate compound melts at 170-172°,
and it is not changed on boiling with hydrochloric acid,

In the detection and identification of cystine we make use of the
crystalline form, the behavior on heating on platinum-foil, and the sul-
phur reaction after boiling with alkali. As to its preparation from protein
substances see K. MORNER and FOLIN 2. In regard to the detection
of cystine in the urine see Chapter XIV.

CH2.SH

Cysteine (a-amino-/3-thiolactic acid), C3H7NS02=CH(NH2), is formed from

COOH

cystine by reduction with tin and hydrochloric acid. It is also produced in the
cleavage of protein substances not as EMBDEN believes as a primary formation
but according to MORNER and PATTEN 3 as a secondary formation. Cysteine
can be easily converted into cystine by oxidation.

According to V. ARNOLD4 cysteine occurs as a constituent of the press-juice
or extracts of various animal organs. He has found it especially in the hair and
he considers it as a primary cell constituent.

Toward alkalies and lead acetate it acts like cystine. With sodium nitro-
prusside and alkali it gives a deep purple-red coloration; with ferric chloride
the solution gives an indigo-blue coloration which quickly disappears.

CH/

Thiolactic acid (a-thiolactic acid), C3H6S02=CH(SH), has been found once

COOH

as a cleavage product of ox-horn by BAUMANN and SUTER. MORNER, FRIED-
MANN and BAER obtained it from cystine. It has been shown by FRIEDMANN
that this acid is a regular cleavage product of keratin substances, and that it
can also be obtained from the proteins. FRANKEL 5 obtained the acid from
haemoglobin. The pyroracemic acid obtained by MORNER as a decomposition
product from several protein substances originates, according to MORNER, only
in part from the cystine.

Taurine (aminoethylsulphonic acid), C2H7NS03 = TTx , has

dlovfeUo.vJrl)

not been obtained as a cleavage product of protein suostances; still its

1 Zeitschr. f. physiol. Chem., 78.

2 Morner, Zeitschr. f. physiol. Chem., 34; Folin, Journ. of Biol. Chem., 8.

3 See foot-note 2, page 80.

4 Zeitschr. f. physiol. Chem., 70.

5 Morner, Zeitschr. f. physiol. Chem., 42; Suter, Zeitschr. f. physiol. Chem., 20;
Friedmann, Hofmeister's Beitrage, 3; with Baer, ibid., 8; Frankel, Sitzungsber. d.
Wien. Akad., 112, II, b, 1903.

TAURINE. 151

origin from proteins has been shown by FRIEDMANN by the close rela-
tion that taurine bears to cysteine; and this is the reason why it is
treated here in connection with the amino-acids.

Taurine is especially known as a cleavage product of taurocholic
acid, and may occur to a slight extent in the intestinal contents. Taurine
has also been found in the lungs and kidneys of oxen and in the blood
and muscles of cold-blooded animals.

- Taurine crystallizes in colorless, often in large, shining, 4- or 6-sided
prisms. It dissolves in 15-16 parts of water at ordinary temperatures,
but rather more easily in warm water. It is insoluble in absolute alcohol
and ether; in cold alcohol it dissolves slightly, but better when warm.
Taurine yields acetic and sulphurous acids, but no alkali sulphides,
on boiling with strong caustic alkali. The content of sulphur can be
determined as sulphuric acid after fusing with saltpeter and soda.
Taurine combines with metallic oxides. The combination with mercuric
oxide is white, insoluble, and is formed when a solution of taurine is boiled
with freshly precipitated mercuric oxide (J. LANG J). This compound
may be used in detecting the presence of taurine. Taurine is not pre-
cipitated by metallic salts.

The preparation of taurine from ox-bile is very simple. The bile
is boiled a few hours with hydrochloric acid. The nitrate from the
dyslysin and choloidic acid is concentrated well on the water-bath, and
filtered hot so as to remove the common salt and other substances which
have separated. The solution is evaporated to dryness and the residue
dissolved in 5 per cent hydrochloric acid, and precipitated with 10 vols.
95 per cent alcohol. The crystals are readily purified by recrystalliza-
tion from water.

The acid alcoholic solution can be used for the preparation of glycocoll. After
the evaporation of the alcohol, the residue is dissolved in water, treated with a
solution of lead hydroxide, filtered, the lead removed by H2S, and the filtrate
strongly concentrated. The crystals which separate are dissolved and decolor-
ized by animal charcoal and the solution then evaporated to crystallization.

Though taurine shows no positive reactions, it is chiefly identified
by its crystalline form, by its solubility in water and insolubility in
alcohol, by its combination with mercuric oxide, by its non-precipitability
by metallic salts, and above all by its sulphur content.

Z-Phenylalanine (phenyl-a-aminopropionic acid),

C6H5
CH2.

COOH

See Maly's Jahresber, 6.

152 THE PROTEIN SUBSTANCES.

was first found by E. SCHULZE and BARBIERI l in etiolated lupin sprouts.
It is produced in the acid cleavage of protein substances in quantities
rarely above 5-6 per cent. It has been prepared synthetically in several
ways by ERLENMEYER, JR., SORENSEN and E. FISCHER, WHEELER and

HOFFMAN.2

The Z-phenylalanine crystallizes in small, shining leaves or fine needles
which are rather difficultly soluble in cold water but readily soluble in
hot water. The solution has a faint bitter taste. A 5-per cent solution
acidified with hydrochloric acid or sulphuric acid is precipitated by
phosphotungstic acid, while a more dilute solution is not precipitated.
On putrefaction, phenylalanine yields phenylacetic acid. On heat-
ing with potassium dichromate and sulphuric acid (25 per cent) an odor
of phenylacetaldehyde is produced and benzoic acid is formed. In
aqueous solution it has a rotation of (a)D = — 35.1°. The phenyliso-
cyanate-1-phenylalanine melts at about 182° C.

Z-Tyrosine (p-oxyphenyl-a-aminopropionic acid),

C6H4(OH)

COOH

is produced from most protein substances under the same conditions
as leucine, which it habitually accompanies. The largest quantity of
tyrosine obtained from animal proteins was about 10-13 per cent (see
tables, pages 106, 107, 115 and 125): In gelatin and a few keratins
tyrosine is absent. It is especially found with leucine, in large quantities,
in old cheese (Tvpos), from which it derives its name. Tyrosine has not
been found with certainty in perfectly fresh organs. It occurs in the
intestine during the digestion of protein substances, and it has about
the same physiological and pathological importance as leucine.

Tyrosine was prepared by ERLENMEYER and LIPP from p-amino-
phenylalanine by the action of nitrous acid, and according to another
method by ERLENMEYER and HALSEY.S On fusing with caustic alkali
it yields p-oxybenzoic acid, acetic acid, and ammonia. On putrefaction
it may yield oxyphenylethylamine, oxyphenylpropionic acid, oxyphenyl-
acetic acid, p-cresol and phenol.

1 Ber. d. d. chem. Gesellsch., 14, and Zeitschr. f. physiol. Chem., 12.

2 Erlenmeyer, Annal. d. Chem. u. Pharm., 275; Sorensen, Zeitschr. f. physiol.
Chem., 44; E. Fischer, Ber. d. d. chem. Gesellsch., 37; Wheeler and Hoffman, Amer.
Chem. Journ., 45.

3 Erlenmeyer and Lipp, Ber. d. d. chem. Gesellsch., 15; Erlenmeyer and Halsey,
ibid., 30.

TYROSINE. 153

Naturally occurring tyrosine and that obtained by the cleavage of
protein substances by acids or enzymes, is generally Z-tyrosine, while
that obtained by decomposition with baryta-water or prepared syn-
thetically is inactive, v. LIPPMANN l has obtained d-tyrosine from
beet-sprouts. The statements as to specific rotation of tyrosine are
somewhat variable. For tyrosine from proteins E. FISCHER has found
a rotation of (C*)D=~ 12.56 to 13.2° for the hydrochloric acid solution,
while SCHULZE and WINTERSTEIN 2 obtained higher results using tyrosine
from plants, namely, (a)D=— 16.2°.

Tyrosine in a very impure state occurs in the form of balls similar
to leucine. The purified tyrosine, on the contrary, appears as colorless,
silky, fine needles which are often grouped into tufts or balls. It is diffi-
cultly soluble in water, being dissolved by 2454 parts of water at 20° C«,
and 154 parts boiling water, separating, however, as tufts of needles on
cooling. It dissolves more easily in the presence of alkalies, ammonia,
or a mineral acid. It is difficultly soluble in acetic acid. Crystals of
tyrosine separate from an ammoniacal solution on the spontaneous
evaporation of the ammonia. One hundred parts glacial acetic acid
dissolve on boiling only 0.18 part tyrosine, and by this means, especially
on adding an equal volume of alcohol before boiling, the leucine can be
quantitatively separated from the tyrosine (HABERM ANN and EHREN-
FELD 3) . The Z-tyrosine-ethyl-ester crystallizes in colorless prisms which
melt at 108-109° C. The naphthylisocyanate-Z-tyrosine melts at 205-
206°. Tyrosine can be oxidized with the formation of dark-colored
products by various plant as well as animal oxidases, so-called tyro-
sinases (see Chapters XV and XVI). In alcoholic fermentation of sugar
the tyrosine present at the same time is transformed according to F.
EnRLiCH4 into tyrosol (p-oxyphenylethyl alcohol), CgHioC^. Tyrosin is
identified by its crystalline form and by the following reactions:

PIRIA'S Test. Tyrosine is dissolved in concentrated sulphuric acid
by the aid of heat, by which tyrosine-sulphuric acid is formed; it is
allowed to cool, diluted with water, neutralized by BaCOa, and filtered.
On the addition of a solution of ferric chloride the filtrate gives a beautiful
violet color. This reaction is disturbed by the presence of free mineral
acids and by the addition of too much ferric chloride.

HOFMANN'S Test. If some water is poured on a small quantity of
tyrosine in a test-tube and a few drops of MILLON'S reagent added and

1 Ber. d. d. chem. Gesellsch., 17.

2 See Hoppe-Sey ler-Thierf elder, Handb. d. physiol. u. pathol. chem. Analyse, 8.
Aufl., 1909. Also E. Fischer, Ber. d. d. chem. Gesellsch., 32; Schulze and Winter-
stein, Zeitschr. f. physiol. Chem., 45.

3 Zeitschr. f. physiol. Chem., 37.

4 Ber. d. d. chem. Gesellsch., 44.

154 THE PROTEIN SUBSTANCES.

then the mixture boiled for some time, the liquid becomes a beautiful
red and then yields a red precipitate.

DENIGES' Test, modified by C. MORNER, is performed as follows:
To a few cubic centimeters of a solution consisting of 1 vol. formaline,
45 vols. water, and 55 vols. concentrated sulphuric acid add a little
tyrosine in substance or in solution and heat to boiling. A beautiful
permanent green coloration is obtained.

FOLIN and DENIS'S test. The reagent consists of a solution containing
10 per cent sodium tungstate, 2 per cent phosphomolybdic acid and
10 per cent phosphoric acid. In performing the test mix 1-2 cc. of the
reagent with an equal volume of the tyrosine solution and then add 3-10
cc. saturated sodium carbonate solution when a beautiful blue color
results. Its delicacy is 1:1000000. The reagent can also be used for the
colorimetric quantitative estimation of tyrosine in proteins. According
to ABDERHALDEN and FUCHS and to ABDERHALDEN1 the reagent suggested
by FOLIN and DENIS for tyrosine also gives a blue coloration with trypto-
phane, oxytryptophane and Z-oxyproline and the value of this reagent for
quantitative tyrosine determinations requires further testing.

H2C— CH2
I I

Z-Proline (a-pyrolidine carboxylic acid), C5H9N02 = H2C CH.COOH,

NH

was first obtained by E. FISCHER and then by FISCHER and collabora-
tors from several proteins as a primary cleavage product (ABDER-
HALDEN and KAUTZSCH2). The proline here obtained was generally
the laevo-rotatory modification. The largest quantity of proline was
secured from the vegetable proteins hordein and gliadin, namely, 13.7
per cent and 13.2 per cent, and also from gelatin, 7.7 per cent (see table
pages 106, 107, 115 and 125). KOSSEL and DAKINS obtained 11 per cent
from salmine. Proline also occurs in scombrine and clupeine, but not in
sturine, which, according to KOSSEL, seems to contradict the view as
to the common origin of orni thine and proline.

SoRENSEN,4 by means of a general method of preparing a-amino-
acids synthetically, has prepared a-amino-5-oxy valeric acid from phthali-
midemalonic ester and has obtained proline from this by evaporating with

1 Denig£s, Compt. rend., 130; C. Th. Morner, Zeitsohr. f. physiol. Chem., 37; Folin
and Denis, Journ. of Biol. Chem., 12; Abderhalden and Fuchs, Zeitschr. f. physiol.
<}hem., 83 and Abderhalden, ibid., 85.

i 2 E. Fischer, Zeitschr. f. physiol. Chem., 33 and 35. See also footnote 2, p. 86,
and Abderhalden and Kautzsch, Zeitschr. f. physiol. Chem., 78.

3 Zeitschr. f. physiol. Chem., 41.

4 Zeitschr. f. physiol. Chem., 44; with A. C. Anderson, ibid., 56.

TRYPTOPHANE. 155

hydrochloric acid, at the same time splitting off water. Recently he
has suggested another method which yields good results. Other syn-
theses of proline have also been performed by E. FISCHER and WILL-
STATTER.1 By the reduction of the ethyl ester of pyrrolidon carboxylic
acid (see glutamic acid) E. FISCHER and BOEHNER 2 have obtained racemic
a-proline. On putrefaction proline yields 6-amino-valeric acid and n-
valeric acid (NEUBERG and AcKERMANN3).

/-Proline crystallizes in flat needles. It is readily soluble in water
and alcohol. The solution has a sweet taste; the specific rotation at
20° C. is (a)D= —77.40°. The solution acidified with sulphuric acid is
precipitated by phosphotungstic acid. In the detection of this acid
we make use of the copper salt, the anhydride of the phenylisocyanate
compound (melting-point 144°), and the picrate. The inactive acid and
its compounds show somewhat different properties.

Oxyproline (oxy-a-pyrolidine carboxylic acid), CsHgNOa. This acid,
whose constitution is not understood was first obtained by E. FISCHER
on the hydrolysis of casein and of gelatin. It dissolves readily in water;
has a specific rotation of («)D= —81.04°, and the solution has a sweet
taste. Oxyproline crystallizes in beautiful colorless plates and gives
a readily soluble copper salt. The constitution of natural oxyproline
has recently been explained by LEUCHS and BREWSTER.4 They find
that the natural oxyproline is a 7-oxy-derivative -of pyrrolidine-a-carbox-
ylic acid. LEUCHS found the specific rotation of Z-oxyproline to be
(a)D=_76° at 20° C.

/-Tryptophane (indol-a-aminopropionic acid),

C.CH2.CH(NH2)COOH

NH

is*one of the cleavage products of the proteins formed in tryptic diges-
tion and other deep decompositions of the proteins, such as putrefaction,
cleavage with baryta-water or sulphuric acid. It gives a reddish-violet
product with chlorine or bromine which is called proteinochrome. NENCKI 5
considered tryptophane, which name is generally given to this acid,
as the mother-substance of various animal pigments.

1 Ber. d. d. chem.. Gesellsch., 33.

2 Ber. d. d. Chem., Gesellsch., 44.

* Neuberg, Bioch. Zeitschr, 37; Ackermann, Zeitschr. of Biol., &7-.

4 Fischer, Ber. d. d. chem. Gesellsch., 35 and 36; Leuchs and Brewster, Ber. d. d.
Chem., Gesellsch., 46.

5 In regard to tryptophane, see Stadelmann, Zeitschr. f. Biologie, 26; Neumeister,
ibid., 26; Nencki, Ber. d. d. chem. Gesellsch./*28; Beitler, ibid., M; Kurajeff, Zeitschr.
f. physiol. Chem., 26; Klug, Pfliiger's Arch.,. 86.

156 THE PROTEIN SUBSTANCES.

Tryptophane was first prepared in a pure form by HOPKINS and
CoLE,1 and they considered it as skatolaminoacetic acid. After ELLIN-
GER showed that skatolcarbonic acid (SALKOWSKI) and skatolacetic
acid (NENCKI) were indolacetic acid and indolpropionic acid respectively,
and after the synthesis of d-Z-tryptophane by ELLINGER and FLAM AND,2
the nature of this substance as indolaminopropionic acid was established.

By condensation of /3-indplaldehyde with hippuric acid ELLINGER and FLAMAND
prepared the azlactone (lactimide) :

N

.C6H5+2H20.

CO— O

On boiling with dilute caustic soda, with the taking up of water, the sodium
salt of indoxyl-a-henzoylaminoacrylic acid,

C8H6N.CH : C.NH.COC6H&
COONa

is obtained, from which by reduction and splitting off of the benzoyl group by
the action of sodium alcoholate the tryptophane is obtained :

C8H6N.CH : C.NH.COC6H6

| +H2+H20 =C8H6N.CH2.CH.NH2-r-C6H5COOH.

COOH COOH

The trytophane formed in digestion is /-tryptophane, which is laevoro-
tatory in aqueous solution (HOPKINS and COLE). Racemic cW-trypto-
phane has also been obtained by digestion in certain cases by ALLERS
and NEUBERG, this is probably formed from the Z-tryptophane (ABDER-
HALDEN and L. BAUMANN3), which very readily undergoes racemization.
f Tryptophane crystallizes in silky rhombic or six-sided leaves. It
does not have a sharp melting-point, and according to the rapidity of heat-
ing melts at 252°, 273° and 289°, according to various authorities.
Tryptophane is readily soluble in hot water, difficultly soluble in cold
water, and only slightly soluble in alcohol. The solution of d-l-trypto-
phane has a faintly sweetish taste, and /-tryptophane a faintly bitter taste.
The statements as to the optical behavior of tryptophane differ some-
what, which, according to ABDERHALDEN, is probably due to the readiness
with which it undergoes racemization. According to ABDERHALDEN
and L. BAUMANN,4 at 20° C. the aqueous solution has a rotation of

1 Journ. of Physiol., 27.

2Ellinger, Ber. d. d. Chem. Gesellsch., 37 and 38. With Flamand, ibid., 40, and
Zeitschr. f. physiol. Chem., 55.

3 R. Allers, Biochem. Zeitschr., 6; C. Neuberg, ibid., 6; Abderhalden and Baumann,
Zeitschr. f. physiol. Chem., 55. (Literature on the specific rotation.)

4 See Abderhalden and Baumann, Zeitschr. f. physiol. Chem., 55 (literature).

INDOL AND SKATOL. 157

(a)D= -30.33°. HOPKINS and COLE give (a)D=-33° for the watery

N N N

solution. It is dextrorotatory in — or — NaOH as well as in — HCL

12 1

Tryptophane yields indol and skatol when sufficiently heated. It
gives the ADAMKIEWICZ-HOPKINS x reaction and a rose-red color on the
addition of chlorine or bromine water (tryptophane reaction). The
brom-tryptophane is readily soluble in amyl alcohol or acetic ether
and on shaking with these solvents the reaction is more delicate.2 If
a pine stick previously moistened with hydrochloric acid and washed
with water is introduced into a concentrated tryptophane solution,
it becomes purple (pyrrole reaction) on drying. The melting-points
of the benzoylsulphotryptophane, the |8-naphthalenesulphotryptophane
and the naphthylisocyanatetryptophane are according to ELLINGER
and FLAMAND,3 185°, 180° and 158° C. respectively. Several compounds
of tryptophane have been prepared by ABDERHALDEN and KEMPE.4
Among these we will mention the tryptophane chloride hydrochloride,
because it is used as the starting material for the synthesis of trypto-
phane polypeptides. In the alcoholic fermentation of sugar, as found
by F. EHRLICH 5 the tryptophane present is transformed into tryptophol
(j3-indoxylethyl alcohol).

In regard to the rather complicated method for preparing trypto-
phane we must refer to the original work of HOPKINS and COLE, of
NEUBERG, and of ABDERHALDEN and KEMPE. FASAL6 has suggested a
quantitative colorimetric method for estimating tryptophane based
upon the ADAMKIEWICZ-HOPKINS reaction.

As shown by HOPKINS and COLE,? tryptophane on anaerobic putre-
faction yields indolpropionic acid and indolacetic acid, and indol and
skatol on aerobic putrefaction. Among these putrefactive products the
indol and skatol will be specially discussed.

CH

Indol, C8HyN = C6H4< y>CH, and Skatol, or ^-METHYLINDOL,
NH

1 In regard to this reaction see also Dakin, Journ. of Biol. Chem. 2, and O. Rosen-
heim, Biochem. Journ., 1.

2 Neuberg, Bioch. Zeitschr., 24.
'I.e.

4 Zeitschr. f. physiol. Chem., 52, and Ber. d. d. chem. Gesellsch., 40.

5 Ber. d. d. chem., Gesellsch., 45.

8 Hopkins and Cole, Journ. of Physiol., 27 and 29; Neuberg and Popowsky, Biochem.
Zeitschr., 2; Abderhalden and Kempe, Zeitschr. f. physiol. Chem., 52; Fasal, Bioch.
Zeitschr., 44.

7 Journ. of Physiol., 29.

158 THE PROTEIN SUBSTANCES.

C.CH3

are formed in variable quantities from pro-

NH

tein compounds under different conditions. Hence they occur habitually
in the human intestinal canal, and, after oxidation into indoxyl and
skatoxyl respectively, pass, at least partly, into the urine as the cor-
responding ethereal sulphuric acids, and also as glucuronic acids.

Indol and skatol crystallize in shining leaves, and their melting-
points are 52° and 95° C. respectively. Indol has a peculiar excremen-
titious odor, while skatol has an intense fetid odor. Both bodies are easily
volatilized by steam, skatol more easily than indol. They may both be
removed from the watery distillate by ether. Skatol is the more insoluble
of the two in boiling water. Both are easily soluble in alcohol and give
with picric acid a compound crystallizing in red needles. If a mixture
of the two picrates be distilled with ammonia, they both pass over with-
out decomposition; while if they are distilled with caustic soda, the indol
but not the skatol is decomposed. The watery solution of indol gives
with fuming nitric acid a red liquid and then a red precipitate of nitroso-
indol nitrate (NENCKi1). It is better first to add two or three drops
of nitric acid and then a 2-per cent solution of potassium nitrite, drop
by drop (SALKOWSKI 2) . Skatol does not give this reaction. An alcoholic
solution of indol treated with hydrochloric acid colors a pine chip cherry-
red. Skatol does not give this reaction. Indol gives a deep reddish-violet
color with sodium nitroprusside and alkali (LEGAL'S reaction). On
acidifying with hydrochloric acid or acetic acid the color becomes pure
blue. Skatol does not act the same. The alkaline solution is yellow
and becomes violet on acidifying with acetic acid and boiling. With a
few drops of a 4-per cent formaline solution and concentrated sulphuric
acid indol gives a beautiful violet color while skatol gives a yellow or
brown color (KoNDO3). On warming skatol with sulphuric acid a
beautiful purple-red coloration is obtained (CIAMICIAN and MAGNANINI 4) .
According to SASAKI skatol, in methyl alcohol free from aldehyde,
gives with concentrated sulphuric acid containing ferric salt a violet-
red ring at the juncture of the two liquids. Indol and tryptophane
do not give this reaction. DENIGES has carefully studied the behavior
of these two bodies with EHRLICH'S reagent, dimethylaminobenzaldehyde,
or with cinnamic aldehyde and vanillin. Comparative investigations on

1 Ber. d. d. deutsch. chem. Gesellsch., 8, 727, and ibid., 722 and 1517.

2 Zeitschr. f. physiol. Chem., 8, 447. In regard to newer reactions for indol and
skatol, see Steensma, ibid., 47, and Deniges, Compt. rend. soc. biol., 64.

3 Zeitschr. f. physiol. Chem., 48.

4 Ber. d. d. chem. Gesellsch., 21, 1928.

HISTIDINE. 159

the behavior of indol and skatol with the aromatic aldehydes have been
carried out by BLUMENTHAL.1

For the detection of indol and skatol in, and their preparation from,
faeces and putrefying mixtures, the main points of the usual method are
as follows: The mixture is distilled after acidifying with acetic acid;
the distillate is then treated with alkali (to combine with any phenols
which may be present) and again distilled. From this second distillate
the two bodies, after the addition of hydrochloric acid, are precipitated
by picric acid. The precipitated picrate is then distilled with ammonia.
The two bodies are obtained from the distillate by repeated shaking
with ether and evaporation of the several ethereal extracts. The residue,
containing indol and skatol, is dissolved in a very small quantity of
absolute alcohol and treated with 8-10 vols. of water. Skatol is precip-
itated, but not the indol. The further treatment necessary for their
separation and purification will be found in other works.2

Skatosine, CioHi6N202, is a base first obtained by BAUM in the pancreas auto-
digestion and later studied by SWAIN. It develops an indol- or skatol-like odor
on fusing with potassium hydroxide. LANGSTEIN 3 obtained a substance which is
perhaps identical with skatosine, in the very lengthy peptic digestion of blood
proteins. *

Z-Histidine, C6H9N302, is /3-imidazol-a-aminopropionic 4
CH-NHV

C

acid, =CH2

CH(NH2) .

COOH

Histidine was first discovered by KOSSEL in the cleavage products
of sturine. It was found at the same time by HEDIN in the cleavage
products of proteins by acid hydrolysis, and by KUTSCHER among the
products of tryptic digestion, and finally also as a cleavage product of
many different animal and plant protein substances. It does not occur
in the protamines, with the exception of sturine. Of the protein bodies
globin (from horse-haemoglobin) seems to be richest in histidine, as

1 Sasaki, Bioch. Zeitschr. 23, 29; Deniges, Compt. rend. soc. biol., 64; Blumenthal,
Bioch. Zeitschr., 19.

2 For quantitative, colorimetric determinations of indol in feces see Einhorn and
Hiibner, Salkowski's Festschrift, Berlin, 1904; C. A. Herter and Foster, Journ. of
biol. Chem., 2.

3 Baum, Hofmesister's Beitrage, 3; Swain, ibid.', Langstein, see Hofmeister, Ueber
Bau und Gruppierung der Eiweisskorper, in Ergebnisse der Physiologic, I, Abt. 1,
1902.

4 See Pauly, Zeitschr. f. physiol. Chem., 42; Knoop and Windaus, Hofmeister's
Beitrage, 7 and 8; Knoop, ibid., 10; Ackermann, Zeitschr. f. physiol. Chem., 65.

160 THE PROTEIN SUBSTANCES.

ABDERHALDEN found 10.96 per cent. It also occurs in germinating plants

(E. SCHULZE !).

Histidine has been prepared synthetically by PvMAN.2 4 (5) chlormethyl
glyoxalin yields with sodium chlormalonic ester the glyoxalinmethylchlormalonic
ester.

CH.NHv

|| ^CH , which on

C N'

CH2.CC1(C02.C2H5)2

hydrolysis gives cW-a-chlor-/3-glyoxalin-4 (5) propionic acid,

CH— NHv

JOS

C N

CH2CHC1.COOH

This latter treated with NH3 yields d-Miistidine, which is changed into the active
forms by means of tartaric acid.

In the anaerobic putrefaction of histidine, /3-imidazolylethylamine
and imidazolylpropionic acid are formed (ACKERMANN 3).

Histidine crystallizes in colorless needles and plates and is readily
soluble in water, but less soluble in alcohol, and has an alkaline reaction.
It is precipitated by phosphotungstic acid, but this precipitate is soluble
in an excess of the precipitant (FRANKEL). With silver nitrate alone
the aqueous solution is not precipitated; on the careful addition of
ammonia or baryta-water an amorphous precipitate, which is readily
soluble in an excess of ammonia, is obtained. Histidine can be pre-
cipitated by mercuric chloride, or, still better, by the sulphate acidified
with sulphuric acid, and can in this way be separated from the other
diamino-acids (KossEL and PATTEN). The hydrochloride crystallizes in
beautiful plates (BAUER), dissolves rather readily in water, but is insolu-
ble in alcohol and ether. With hydrochloric acid and methyl alcohol
it gives the dihydrochloride of histidine methyl ester, which melts at
196°. Histidine is laevorotatory, (a)D= —39.74°, while its solution in
hydrochloric acid is dextrorotatory. On warming it gives the biuret test
(HERZOG), and it also gives WEIDEL'S reaction if performed as sug-
gested by FISCHER (see Xanthine, Chapter V) (FRANKEL4). On adding

1 Kossel, Zeitschr. f. physiol. Chem., 22; Hedin, ibid., Kutscher, ibid., 25; Wetzel,
ibid., 26; Lawrow, ibid., 28, and Ber. d. d. chem. Gesellsch., 34; Kossel and Kutscher,
Zeitschr. f. physiol. Chem., 31; Hart, ibid., 33; Abderhalden, ibid., 37; Schulze, ibid.,
24 and 28.

2 Cited from Chem. Centralbl., 1911, 2, p. 760.

3 Zeitschr. f. physiol. Chem., 65.

4 Kossel and Patten, Zeitschr. f. physiol. Chem., 38; Bauer, ibid., 22; Herzog, ibid.,
37; Frankel, Sitz.-Ber. d. Wien. Akad., 112, II. B., 1903, and Hofmeister's Beitrage, 8.

ARGININE. 161

sufficient bromine water and warming, a reddish coloration ensues
which turns deep wine-red, later becoming cloudy, due to the forma-
tion of dark amorphous particles (F. KNOOP 1). It gives a very beautiful
diazo-reaction with diazobenzenesulphonic acid, in solutions made alkaline
with sodium carbonate, which according to PAULY is deep cherry-red
in dilutions of 1:20000 and still markedly red in 1:100000 (tyrosine
gives a similar reaction).

Several salts of histidine are known; H. PAULY 2 has especially
studied the iodized derivatives of histidine and imidazole.

On feeding cW-histidine to rabbits ABDERHALDEN and WEIL 3 obtained
from the urine d-histidine which was crystalline, was as sweet as sugar
and showed a specific rotation (a)D= -f-40.15° at 20° C.

Histidine is sometimes classified in a group, with the two diamino-
acids, arginine and lysine which KOSSEL has called the hexone bases,

d-Arginine ( 5-guanido-a-aminovaleric acid),

(CH2)2 ,

CH(NH2)

COOH

first discovered by SCHULZE and STEIGER in etiolated lupin- and pumpkin-
sprouts, has later been found in other germinating plants, in tubers and
roots. GULEWITSCH has found arginine in the ox-spleen, and TOTANI
and KATSUYAMA have found it in ox-testicles. It was first found
by HEDIN as a cleavage product of horn substance, gelatin, and several
proteins, and then by KOSSEL and his pupils as a general cleavage prod-
uct of protein substances as a class. The greatest quantity was obtained
from the protamines; but the histones and certain plant proteins,
edestin and the protein from pine seeds and especially excelsin (14.14
per cent), also yield abundant arginine. Arginine also occurs among
the products of tryptic digestion (KOSSEL and KuTSCHER4).

On boiling with baryta-water, as well as by the action of an enzyme,
arginase, discovered bv KOSSEL and DAKIN,S arginine yields urea and
ornithine.

1 Hofmeister's Beitrage, 11.

2 Ber. d. d. chem. Gesellsch., 43.

3 Zeitschr. f. physiol. Chem., 77.

4 Schulze and Steiger, Zeitschr. f. physiol. Chem., 11; Schulze and Castoro, ibid., 41;
Gulewitsch, ibid., 30; Totani and Katsuyama, ibid., 64; Hedin, ibid., 20 and 21; Kossel
and Kutscher, ibid., 22, 25, 26.

5 Zeitschr. f. physiol. Chem., 41, and Dakin, Journ. of biol. Chem., 3.

162 THE PKOTEIN SUBSTANCES.

Arginine has been prepared synthetically from ornithine (a-5-diamino-
valeric acid) and cyanamide by SCHULZE and WINTERSTEIN. Recently
SORENSEN and HOYRUP l have prepared d-Z-arginine from ornithuric
acid. The a-monobenzoyl ornithine obtained by splitting ornithuric

N

acid with — barium hydrate yields a-benzoylamino-5-guanido-valeric
5

acid with cyanamide and this on boiling with hydrochloric acid gave
5-guanido-a-aminovaleric acid (d-Z-arginine).

Arginine crystallizes in rosette-like tufts, plates, or thin prisms, is readily
soluble in water with alkaline reaction and almost insoluble in alcohol.
With several acids and metallic salts it forms crystalline salts and double
salts respectively. Its acidified watery solution is precipitated by phos-
photungstic acid. The most important salts are the copper-nitrate
(C6Hi4N4O2)2.Cu(N03)2+3H20 and the silver salts

CeHuN^HNOa+AgNOa

(the more readily soluble) and CeHuN-iC^.AgNOs+fEbO (the more
difficultly soluble), and its compound with picrolonic acid (STEUDEL 2).

Arginine is dextrorotatory. For arginine-chloride in watery solu-
tion with excess of hydrochloric acid, GuLEwrrscn3 found (a)D= +21.25°
at 20° C. The arginine obtained by KUTSCHER in the tryptic digestion
of fibrin was racemic arginine. As found by KOSSEL and WEISS (see
page 112) arginine or more properly the ornithine is very easily
racemerized within the protein molecule by the action of alkali. The
racemic arginine can, as RIESSER 4 has shown, during cleavage by means
of arginase, yield /-arginine, which is an asymmetric change. In^putre-
faction arginine yields ornithine, guanidine, putrescine and 5-amino-
valeric acid.

yNH2
Agmatine (guanidobutylamine), CsHi4N4=HN.C<f

\NH.CH2(CH2)2.CH2NH2,

is a base obtained by KOSSEL in the hydrolysis of herring sperm, and later by
KUTSCHER and ENGELAND 5 from ergot. KOSSEL has also obtained it synthetically
from cyanamide and tetramethylendiamine, and in this manner proved its con-
stitution. It is produced from arginine by splitting off C02 and bears the ^same
relation to arginine that putrescine does to ornithine and cadeverine does to
lysine (see below). Agmatine gives several crystalline salts as described by
KOSSEL. It is precipitated by phosphotungstic acid.

1 Schulze and Winterstein, Ber. d. d. chem. Gesellsch., 32 and Zeitschr. f. physiol-
Chem., 34; Sorensen and Hoyrup, Ber. d. d. chem. Gesellsch, 43 and Zeitschr. f. physiol-
Chem., 76.

2 Zeitschr. f. physiol. Chem., 37 and 44.

3 Ibid., 27.

4 Kutscher, Zeitschr. f. physiol. Chem., 28 and 32; Riesser, ibid., 49.

6 Kossel, ibid., 66 and 68; Engeland and Kutscher, Ceritralbl. f. Physiol., 24, 479.

ORNITHINE AND LYSINE. 163

CHi.(NH«)

/AlTT \

d-Ornithine (a-3-diaminovaleric acid), CiHitNaQi* >* /XTTT is not a primary

COOH

cleavage product of proteins, but is formed from arginine on boiling with baryta-
water. JAFFE,1 who first discovered this body, obtained it as a cleavage product
from ornithuric acid, which is found in the urine of hens fed with benzoic acid.
The ornithine which E. FISCHER and later SORENSEN,* have prepared syn-
thetically yields, as shown by ELLINGER, putrescine (tetramethylenediamine),
C4H8(NH2)2, on putrefaction. A. LOEWY and NEUBERGS have shown that
ornithine is split into putrescine and C02 in the organism of cystinuria patients.

Ornithine is a non-crystalline substance which dissolves in water, giving an
alkaline reaction, and yields several crystalline salts. It is precipitated by
phosphotungstic acid and several metallic salts, but not by silver nitrate and
.baryta- water (differing from arginine). Ornithine hydrochloride is dextrorotatory;
the synthetically prepared one is inactive. On shaking ornithine with benzoyl
chloride and caustic soda it is converted into dibenzoylornithine (ornithuric
acid). On splitting artificially prepared racemic ornithuric acid SORENSEN has
shown that the naturally occurring ornithuric acid is identical with the dextro-
rotatory a-5-dibenzoyldiaminovaleric acid. Salts and derivatives of ornithine
have been described by KOSSEL and his collaborators 4 and they have given a
method for its isolation from mixtures.

Diaminoacetic acid, C2H6N202=CH(NH2)2COOH was obtained by DRECHSEL 5
as a cleavage product of casein by boiling with tin and hydrochloric acid. It
crystallizes in prisms and gives a monobenzoyl compound which is not very soluble
in cold water and is almost insoluble in alcohol, and can be used in the isolation
of the acid.

CH2(NH2)

d-Lysine (a-€-diaminocaproicacid), C6Hi4N202= T/T )> was first

COOH

obtained by DRECHSEL as a cleavage product of casein. Later he and
his pupils, as well as KOSSEL and others, found it among the cleavage
products of various proteins. It has not been detected in some vegetable
proteins such as the prolamines (page 106). E. SCHULZE found lysine
in germinating plants of the Lupinus luteus, and WINTERSTEIN found
it in ripe cheese. It has been obtained in largest amounts (28.8 per cent)
by KOSSEL and DAKIN from the protamine a-cyprinine. From a gliadin
which was not contaminated and which they considered as a unit substance
although obtained from different fractions having different solubilities
in alcohol, OSBORNE and LEAVENWORTH 6 found a small amount of lysine

1 Ber. d. d. chem. Gesellsch., 10 and 11.

2 Fischer, Ber. d. d. chem. Gesellsch, 34; Sorensen, Zeitschr. f. physiol. Chem., 44.

3 Ellinger, Zeitschr. f. physiol. Chem., 29; Loewy and Neuberg, ibid., 43.

4 Kossel and Weiss, Zeitschr. f. physiol. Chem., 68.

5 Ber. d. k. sachs. Gesellsch. d. Wiss., 44

6Drechsel, Arch. f. (Anat. u.) Physiol., 1891, and Ber. d. d. chem. Gesellsch., 25;
Siegfried, Arch. f. (Anat. u.) Physiol., 1891, and Ber. d. d. chem. Gesellsch., 24; Hedin,
Zeitschr. f. physiol. Chem., 21; Kossel, ibid., 25; Kossel and Mathews, ibid., 25; Kossel

164 THE PROTEIN SUBSTANCES.

(0.07 and 0.15 per cent in two different fractions). The generally
accepted view that lysine is completely absent in gliadin is still doubtful.
They could not detect lysine in zein by the same method.

Lysine has been synthetically prepared by E. FISCHER and WEiGERT.1
This lysine was racemic, while that prepared from protein is always
optically active and dextrorotatory. The rotation depends upon the
concentration and degree of acidity; for the hydrochloride a rotation of
(«)D = + 14° to 17.25° has been found. On heating with barium hydroxide
it is converted into the racemic modification. According to ELLINGER
lysine yields cadaverine (pentamethylenediamine), CsHio(NH2)2, on
putrefaction, and this base is formed from the lysine in the organism
of those with cystinuria and at the same time C02 is split off (A. LOEWY
and NEUBERG).2

Lysine is readily soluble in water but is not crystallizable. The aque-
ous solution is precipitated by phosphotungstic acid, but not by silver
nitrate and baryta-water (differing from arginine and histidine). It
gives two hydrochlorides with hydrochloric acid, and with platinum
chloride a chloroplatinate which is precipitable by alcohol and has the
composition C6Hi4N2O2.H2PtCl6+C2H50H. It gives two silver salts
with AgNOa; one has the formula AgNOs+CeHu^Cb and the other
AgNO3+C6Hi4N202.HN03. With benzoyl chloride and alkali, lysine
forms an acid, lysuric add, CeHi2(C7H50)2N2O2 (DRECHSEL), which
is homologous with ornithuric acid, and whose difficultly soluble acid
barium salt may be used in the separation of lysine.3 The rather
insoluble picrate, which is precipitated from a not too dilute solution
of the hydrochloride by sodium picrate, may also be used in the detec-
tion of lysine.

KUTSCHEB and LOHMANN 4 have found a lysine having somewhat different
properties in the final products of pancreas autolysis.

In the preparation of the so-called hexone bases we can first precipitate
all the bases by phosphotungstic acid, when the monamino-acids remain in
solution. The precipitate is then decomposed in boiling water by barium
hydroxide and the bases obtained as silver compounds from this filtrate.
In regard to further details and the methods of separating the various

and Kutscher, ibid., 31; Kutscher, ibid., 29; Schulze, ibid., 28; Winterstein, cited in
Schulze and Winterstein, Ergebnisse der Physiologie, I, Abt. 1, 1902; Kossel and
Dakin, Zeitschr. f. physiol. Chem., 40; Osborne and Leavenworth, Journ. of biol.
Chem., 14

1 Ber. d. d. chem Gesellsch., 35.

2 See footnote 3, p. 163.

3 Drechsel, Ber. d. d. chem. Gesellsch., 28; see also C. Willdenow, Zeitschr. f . physiol.
Chem., 25.

4 Zeitschr. f. physiol. Chem., 41.

DIAMINO-ACIDS.

165

bases we will refer to STEUDEL in ABDERHALDEN'S Handbuch der biochem-
ischen Arbeitsmethoden, Bd. 2, II, s. 498.

We give below a tabulation of the amounts of the three hexone bases
found in certain protein substances (in weight per cent) :

Arginine.

Sturine1 58.2

Cyprinine (a)6 4.9

Other protamines l 62 . 5 — 87 . 4

Histones * 14.36—15.52

Casein 2 4.70— 4.84

Syntonin (from meat) 2 5 . 06

Heterosyntonose 2 8 . 53

Protosyntonose 2 4 . 55

Edestin3... 11.0—14.07

Proteid from conjferae seeds 3

Gluten casein l

Gluten proteins

10.9—11.3
4.4

2.75— 3.13
Gelatin"1 and 2. . ... 7.62 — 9.3

Elastin

0.3

Lyaine.

Histidine.

12.0

12.9

28.8

0.0

0.0

0.0

7.7 —8.3

1.21—2.34

1.92—5.80

2.53—2.59

3.26

2.66

3.08—7.03

0.37—1.12

3.08

3.35

1.3

1.17

0.25—0.79

0.62—0.73

2.15

1.16

0.0

0.43—1.53

2.49— 3.0

0.40

-j-

0.027

Of the oxydiamino-acids found on the hydrolysis of proteins we will
mention the following:

Oxydiaminosebacic acid, (?) CioH2oN205, has been isolated by WOHLGEMUTH 6
from a nucleoprotein of the liver. The free acid was obtained as small white
plates. It is soluble with difficulty in hot water, insoluble in cold water and
in alcohol. It was optically inactive in hydrochloric acid. The beautifully
crystalline phenylcyanate compound had a melting-point of 206°.

Dioxydiaminosuberic acid, CgHieNuOe, has been obtained by SKRAUP 7 on the
hydrolysis of casein with hydrochloric acid. The copper salt crystallizes hi
beautiful deep bluish-violet rosettes which are composed of long, irregular, right-
angled plates. It is quite soluble in cold water. The free acid crystallizes in
fern-like formations. Besides this acid SKRAUP obtained two other acids which
he calls caseanic acid, CgHie^Or, and caseinic acid, daEUNsOe. The caseanic
acid crystallizes, melts at 190-191°, is tribasic, and is probably an oxydiamino-
acid. The caseinic acid is dibasic and occurs in two modifications. The one
which melts at 228° is faintly dextrorotatory; the other modification melts
at 245° and is optically inactive. Both crystallize, but the inactive form does
not yield well-defined crystals. Caseinic acid seems also to be an oxydiamino-
acid.

Diaminotrioxydodecanoic acid, CiaH^^Os, is an acid obtained by FISCHER and
ABDERHALDEN 8 on the hydrolysis of casein and seems to stand close to SKRAUP'S
caseinic acid, but differs from it in its optical properties. This acid is faintly
levorotatory : (<*)D = about —9°. It crystallizes in plates, which grow into rosettes

1 Kossel and Kutscher, Zeitschr. f. physiol. Chem., 31.

2 Hart, ibid., 33.

3 Schulze and Winterstein, ibid., 33; see also Kossel, Ber. d. d. chem. Gesellsch.,
34, 3236.

4 Kossel and Kutscher, Zeitschr. f. physiol. Chem., 25, and Richards and Gies,
Amer. Journ. of Physiol., 7.

6 Kossel and Dakin, Zeitschr. f. physiol. Chem., 40.

6 Ber. d. d. chem. Gesellsch., 37, and Zeitschr. f. physiol. Chem., 44.

7 Zeitschr. f. physiol. Chem., 42.

8 Zeitschr. f . physiol. Chem., 42.

166 THE PROTEIN SUBSTANCES.

or spherical aggregations. It has a faint bitter taste, gives a crystalline hydro-
chloride which is slightly soluble in strong hydrochloric acid, and gives a crys-
talline copper salt.

After describing the different amino-acids it remains for us to call
attention to certain general reactions of the amino-acids.

By the action of formaldehyde the amino groups are changed into
methylene groups according to the scheme:

R.CH.NH2 R.CH.N : CH2

+H<30H= | +H20.

COOH COOH

The amino-acids behave like neutral bodies while the methylene
combinations are acids and on this behavior is based SORENSEN'S l
formoltitration which serves for the estimation of amino-acids in the
urine (Chapter XIV) as well as to follow the progress of proteolysis.
As the proteolysis progresses and imide bindings are loosened a large
number of atomic complexes with free NH2 and COOH groups are set
free. If now the NH2 groups are fixed as methylene groups by the addi-
tion of formol, the complex behaves like acids and the number of their

N

COOH groups can be determined by titration with — barium or sodium

5

hydroxide solution, using phenolphthalein or thymolphthalein as in-
dicator. With the presumption that for every COOH group set free
there existed a free NH2 group the extent of the proteolysis can also
be expressed in milligrams N by multiplying the number of cubic centi-

N

meters — alkali used by 2.8.
o

SIEGFRIED has found that amino-acids in the presence of alkali or
alkaline earths de-ionize carbon dioxide and form salts of the type of
the carbamino salts, SIEGFRIED'S " carbamino-reaction." For example
glycocoll in the presence of lime yields with carbon dioxide, calcium
carbamino-acetic acid, CH2.NH.COO

I I •

COO Ca

If the nitrogen is determined and at the same time the combined carbon
dioxide estimated by means of the calcium carbonate split off on boil-

r^o
ing the filtered solution, then the quotient — — - gives the number of N

atoms for every molecule C02 taken up. This quotient is equal to 1
for glycocoll and the aliphatic amino-acids because these go over quan-

1 Sorensen, Bioch. Zeitschr., 7; with Jessen Hansen, ibid., 7; with V. Henriques,
Zeitschr. f. physiol. Chem., 63 and 64; Henriques and Gjaldbak, Ibid., 67 and 75.

COMPOUND PROTEINS. 167

titatively into carbamino-acids. With the diamino-acid arginine, which
contains 4 nitrogen atoms, it is on the contrary only one-fourth because
this acid reacts with only one amino group, that of the a-amino valeric
acid chain.

The reaction which has been developed and extensively used by
SIEGFRIED l and his pupils is of great value in the characterization of pep-
tones, kyrines, and proteoses, for the separation and fractional pre-
cipitation and for the determination of their constitution. The binding
of the carbon dioxide as carbamino-salts seems also in many ways
to be of physiological importance, as for example, the solubility of cal-
cium carbonate in alkaline fluids and for the carbon dioxide binding in
blood, etc.

The amino-acids can by methylation form betaines, for example,
trimethy 1 gly cocoll or betaine CH2 — N (CHa) 3 . Betaine occurs abundantly

I I

CO — -O

in the plant kingdom. In the animal kingdom such bodies have been
found under physiological conditions in cold blooded animals and they
belong to those groups of bodies which have been called " aporrhegmas"
by ACKERMANN and KuTSCHER.2 As " aporrhegmas " they designate
all those fractions of amino-acids from the protein, which can be pro-
duced from the proteins in a physiological manner and indeed in the life
of animals as well as the plants. These bodies are essentially the same
as have been observed in the putrefaction of the amino-acids and which
have been specially 'mentioned with every amino-acid described.

The behavior of the amino-acids in yeast fermentation will be dis-
cussed in Chapter III.

In regard to the methods for separating and preparing, in a pure form,
the various amino-acids and other products of protein hydrolysis which
have not been given in the preceding pages, we must refer to ABDERHAL-
DEN'S Handbuch der biochemischen Arbeitsmethoden, 1909-1910 Bd. 2.

II. Compound Proteins.3

We designate as compound proteins those bodies which yield, on
cleavage, proteins (with their decomposition products) and other bodies
such as carbohydrates, nucleic acids, or pigments.

The compound proteins known at present can be divided into three
groups: glycoproteins, nudeoproteins and chromoproteins. Of these the

1 In regard to the literature see Siegfried in Ergebnisse d. Physiol. Bd. 9.

2 Zeitschr. f. physiol. Chem., 69. See also Engeland, ibid., 69.

3 Hoppe-Seyler has given the name proteide to these compound proteids, but as
this term is misleading in English we do not use it in English classifications in this

168 THE PKOTEIN SUBSTANCES.

last-mentioned group (haemoglobin and haemocyanine) will be discussed
in a subsequent chapter (Chapter V on the blood).

A. Glycoproteins (glucoproteins).

Glycoproteins l are those compound proteins which on decomposi-
tion yield a protein on the one side, and a carbohydrate or derivatives
of this on the other, but no purine bodies. Some glycoproteids are free
from phosphorus (mucin substances, chondroproteins, and hyalogens),
and some contain phosphorus (phosphoglycoproteins).

The glycoproteins free from phosphorus may, as regards the nature
of the carbohydrate groups split off, be divided into two chief groups,
the mucin substances and the chondroproteins. The first yield on hydrolytic
cleavage an amino-sugar, which has been shown to be glucosamine in
all but a few exceptions.2 In the chondroproteins, on the contrary, the
protein is united to chondroitin-sulphuric acid.

1. Mucin Substances.

Compared with the simple proteins the mucin substances are poorer
in nitrogen and as a rule also have considerably less carbon. The carbo-
hydrate complex, whose nature has been shown by the investigations
cf FR. MuLLER3 and his pupils, occurs, so it seems, in the mucin sub-
stances as a polysaccharide related to chitosan, which on hydrolytic
cleavage yields glucosamine (chitosamine), and, at least in most cases,
acetic acid also. The mucin substances differ very markedly among
themselves, hence we divide them into two groups, the mucins and the
mucoids.

The true mucins are characterized by the fact that their natural

1 Abderhalden (Lehrb. d. physiol. Chem., 1909, p. 191) has proposed dropping the
name glycoproteids entirely and to consider these bodies as simple proteins, because
it has not been shown that the carbohydrate groups occupy the same relationship
to the protein component that the hsemin or the nucleic acid bears to the haemo-
globin or the nucleoprotein molecule. It is possible that this proposition, which is
not applicable to the entire group (including the proteins containing chondroitin-
sulphuric acid) but applies only to the mucin group, will be found in the future to be
correct. It is the opinion of HAMMARSTEN that it is better to wait for further research
in this direction before we drop the generally accepted nomenclature and the usual
subdivisions of the proteins.

2 See Schulz and Ditthorn, Zeitschr. f. physiol. Chem., 29; A. v. Ekenstein and
Blanksma, Chem. Centralbl., 1907, 2. When both carbohydrate groups are simul-
taneously combined in one body, then probably we are not dealing with a chemical
individual, but rather with a mixture.

3 See Fr. Miiller, Zeitschr. f. Biologic, 42, which contains all the pertinent litera-
ture, and also L. Langstein, Die Bildung von Kohlenhydraten aus Eiweiss, Ergebnisse
der Physiologic, Jahr. I, Abt. 1.

MUCINS. 169

solutions, or solutions prepared by the aid of a trace of alkali, are mucilagi-
nous,' ropy, and give a precipitate with acetic acid which is insoluble in
excess of acid or soluble only with great difficulty. The mucoids do not
show these physical properties, and have other solubilities and precipit-
ation properties. As we have intermediate steps between different pro-
tein bodies, so also we have such between true mucins and mucoids, and
a sharp line cannot be drawn between these two groups.

It is just as difficult at present to draw a sharp line between the pro-
teins and the mucins or mucoids, since we have been able to split off
carbohydrate complexes from several proteins, and as proteins have
been isolated from white of egg which yield more or less glucosamine.
The very variable amounts of glucosamine obtained under various con-
ditions from the crystalline ovalbumin seem to indicate that we are
dealing with a contamination with a glycoprotein.

True mucins are secreted by the larger mucous glands, by certain
mucous membranes, and by the skin of snails and other animals. True
mucin also occurs in the navel-cord. Sometimes, as in snails and in
the membrane of the frog-egg (GIACOSA) and perch-eggs (HAMMARSTEN 1),
a mother-substance of mucin, a mucinogen, has been found which may
be converted into mucin by alkalies. Mucoid substances are found in
certain cysts, in the cornea, the crystalline lens, white of egg, and in
certain ascitic fluids. The so-called tendon-mucin, which, according
to the investigations of LEVENE and of CUTTER, and GiES,2 contains
chondroi tin-sulphuric acid or a related substance, cannot be classified
as a mucin, but must, like the chondromucoid and the osseomucoid,
be classified as chondroprotein. As the mucin question has not been
sufficiently studied, it is at the present time impossible to give any positive
statements in regard to the occurrence of mucins and mucoids, especially
as without doubt in many cases non-mucinous substances have been
described as mucins.

True Mucins. Thus far we have been able to obtain only a few
mucins in a pure and unchanged condition, because of the reagents used.
The elementary analyses of these mucins have given the following results :

C H N S
Mucin from mucous membrane (air-
passages) 48.26 6.91 10.70 1.40 (Fn. MULLER 3)

Mucin from submaxillary 48 . 84 6 . 80 12 . 32 0 . 84 (HAMMARSTEN 3)

Mucin from snail 50.32 6.84 13.65 1 .75 (HAMMARSTEN 3)

Synovial mucin 51 . 05 6 . 53 13.01 1 . 34 (v. HOLST 4)

1 Giacosa, Zeitschr. f. physiol. Chem., 7; Hammarsten, Pfliiger's Archiv., 36, and
Skand, Arch. f. Physiol., 17.

2 Levene, Zeitschr. f. physiol. Chem., 31; Cutter and Gies, Amer. Journ. of Physiol., 6.

3 Fr. Miiller, Zeitschr. f. Biologic, 42; Hammarsten, Zeitschr. f. physiol. Chem.. 12,
and Pfliiger's Arch., 36.

4 Zeitschr. f. physiol. Chem., 43.

170 THE PROTEIN SUBSTANCES.

MULLER obtained 35 per cent glucosamine from mucous-membrane
mucin and 23.5 per cent from the submaxillary mucin.

On boiling mucin with dilute mineral acids, acid aibuminate and
bodies similar to proteoses are obtained, besides a reducing substance
which is not free glucosamine (STEUDEL1). By the action of strong
acids upon mucins or mucoids OTORI 2 obtained several of the cleavage
products of the proteins, such as leucine, tyrosine, glycocoll, glutamic
acid, oxalic acid, guanidine, arginine, lysine, and humus substances,
and also carbohydrate cleavage products, such as levulinic acid. Cer-
tain mucins, as the submaxillary mucin, are easily changed by very
dilute alkalies, as lime-water, while others, such as tendon-mucin, are
not affected. If a strong caustic-alkali solution, such as a 5-per cent
KOH solution, is allowed to act on submaxillary mucin, we obtain alkali
aibuminate, bodies similar to proteoses and peptones and one or more
substances of an acid reaction which have strong reducing powers.

On peptic digestion proteoses and peptone-like bodies, still con-
taining the carbohydrate group, are produced. On tryptic digestion
still simpler cleavage products are formed, namely, leucine, tyrosine,
and tryptophane (POSNER and GiES3). The glucosamine, so far as we
know, is not split off by proteolytic enzymes, but only after strong
hydrolysis with acids.

In one or another respect the various mucins act somewhat dissimilarly.
For example, the snail and sputum mucins are insoluble in dilute hydro-
chloric acid of 1-2 p. m., while the mucin of the submaxillary gland and
the navel-cord is soluble. The former become flaky with acetic acid,
while the submaxillary mucin is precipitated in more or less fibrous,
tough masses. Still all the mucins have certain reactions in common.

In the dry state mucin forms a white or yellowish-gray powder. When
moist it forms, on the contrary, flakes or yellowish-white tough lumps
or masses. The mucins are acid in reaction. They give the color reac-
tions of the proteins. They are not soluble in water, but may give a
neutral solution with water with the aid of small amounts of alkali. Such
a solution does not coagulate on boiling, but acetic acid gives at the
normal temperature a precipitate which is nearly insoluble in an excess
of the precipitant. If 5-10 per cent NaCl be added to a mucin solution,
it can be carefully acidified with acetic acid without giving a pre-
cipitate. Such acidified solutions are copiously precipitated by tan-
nic acid; with potassium ferrocyanide they give no precipitate, but on
sufficient concentration they become thick or viscous. A neutral solu-
tion of alkali mucin is precipitated by alcohol in the presence of neutral

1 Zeitschr. f. physiol. Chem., 34.

2 Ibid., 42 and 43.

* Amer. Journ. of Phyaiol., 11.

HYALOGENS. 171

salts; it is also precipitated by several metallic salts. If mucin is heated
on the water-bath with dilute hydrochloric acid of about 2 per cent,
the liquid gradually becomes a yellowish or dark brown, and reduces
copper salts in alkaline solutions.

The mucin most readily obtained in large quantities is the submax-
illary mucin, which may be prepared in the following way: The filtered
watery extract of the gland, free from form-elements and as colorless
as possible, is treated with 25 per cent hydrochloric acid, so that the
liquid contains 1.5 p. m. HC1. On the addition of the acid the mucin
is immediately precipitated, but dissolves on stirring. If this acid liquid
is immediately diluted with 2-3 vols. of water, the mucin separates and
may be purified by redissolving in 1-5 p. m. acid, and diluting with
water and washing therewith. The mucin of the navel-cord may be
prepared in the same way. As a rule the mucins can be prepared by
precipitation with acetic acid and repeated solution in dilute lime-water
or alkali, and reprecipitation with acetic acid. Finally they are treated
with alcohol and ether. In the preparation of sputum mucin the method
is very complicated (Fit. MULLER).

Mucoids or Mucinoids. In this group we must include those non-
phosphorized glycoproteins which are neither true mucins nor chondro-
proteids, although they show among themselves such differences in
behavior that they can be divided into several subgroups of mucoids.
To the mucoids belong pseudomutin, the probably related body colloid,
ovomucoid, and other bodies, which on account of their differences will be
best treated individually in their respective chapters.

Hyalogens. Under this name KRUKENBERG l has designated a number of
different bodies, which are characterized by the following: By the action of
alkalies they change, with the splitting off of sulphur and some nitrogen, into
soluble nitrogenized products called by him hyalines, and which yield a pure car-
bohydrate by further decomposition. We find that very heterogeneous sub-
stances are included in this group. Certain of these hyalogens seem undoubtedly
to be glycoproteins. Neossin 2 of the Chinese edible swallow's-nest, membranin 3
of DESCEMET'S membrane and of the capsule of the crystalline lens, and spiro-
graphin 4 of the skeletal tissue of the worm Spirographis, seem to act as such.
Others, on the contrary, such as hyalin 5 of the walls of hydatid cysts, and onu-
phin* from the tubes of Onuphis tubicola, do not seem to be compound proteins.
The so-called mucin of the holothuria, 7 and chondrosin 8 of the sponge, Chondrosia

1 Verb. d. physik.-med. Gesellsch. zu Wiirzburg, 1883; also Zeitschr. f. Biologie, 22.

2 Krukenberg, Zeitschr. f . Biologie, 22.

3 C. Th. Morner, Zeitschr. f. physiol. Chem., 18.

4 Krukenberg, Wiirzburg, Verhandl., 1883; also Zeitschr. f. Biologie, 22.

6 A. Liicke, Virchow's Arch., 19; also Krukenberg, Vergleichende physiol. Stud.,
Series 1 and 2, 1881.

6 Sch'miedeberg, Mitth. aus d. zool. Stat. zu Neapel, 3, 1882.

7 Hilger, Pfliiger's Archiv, 3.

8 Krukenberg, Zeitschr. f . Biologie, 22.

172 THE PROTEIN SUBSTANCES.

reniformis, and others may also be classed with the hyalogens. As the various
bodies designated by KRUNKENBERG as hyalogens are very dissimilar, it is not
of much advantage to arrange these in special groups.

2. Chondroproteins.

Chondroproteins are those glycoproteins which as primary cleav-
age products yield protein and an ethereal sulphuric acid, the chondroitin-
sulphuric add. Chondromucoid, occurring in cartilage, is the best example
of this group. Amyloid occurring under pathological conditions also
belongs to this group. On account of the property of chondroitin-sul-
phuric acid of precipitating protein, it is also possible that under certain
circumstances combinations of this acid with protein may be precipitated
from the urine and be considered as Chondroproteins.

The chondromucoid, the so-called tendon-mucin, and the osseomucoid
have greatest interest as constituents of cartilage, of the connective
tissues, and the bones, and on this account these bodies and their cleavage
product, chondroitin-sulphuric acid, will be treated in a following chap-
ter (IX). On the contrary, amyloid, which has always been considered
in connection with the protein substances, will be described here.

Amyloid, so called by, VIRCHOW, is a protein substance appear-
ing under pathological conditions in the internal organs, such as the spleen,
liver and kidneys, as infiltrations; and in serous membranes as granules
with concentric layers. It probably also occurs as a constituent of
certain prostate calculi. The chondroprotein occurring under physio-
logical conditions in the walls of the arteries is, perhaps, according to
KRAWKOW, very closely related to the amyloid substance, but not iden-
tical with it, as shown by NEUBERG.1

Recently O. HANSSEN has studied the mechanically isolated amy-
loid obtained from the so-called " sago kernels " of an amyloid spleen,
and could not detect any conjugated sulphuric acid in it. According to
his investigations true amyloid is not a chondroprotein. MAYEDA 2
has also prepared an amyloid substance free from chondroitin-sulphuric
acid. On the other hand, HANSSEN has found that amyloid organs
(liver and spleen) are much richer in sulphuric acid that splits off than
normal organs, and it is not improbable that the amyloid formation goes
hand in hand with the formation of a chondroprotein.

The amyloid prepared by KRAWKOW and NEUBERG had about the
same composition: C 49.0-50.1; H 7-7.2; N 14-14.1, and S 1.8-2.8

1 Krawkow, Arch. f. exp. Path. u. Pharm., 40, which contains the literature; Neu-
berg, Verhandl. d. d. Pathol. Gesellsch., 1904.

2 Hanssen, Bioch. Zeitschr., 13; Mayeda, Zeitschr. f. physiol. Chem., 58.

AMYLOID. 173

per cent. The aorta amyloid of man and of the horse contained respect-
ively C 49.6 and 50.5; H 7.2; N 14.4 and 13.8; S 2.3 and 2.5 per cent.
As we cannot tell whether the amyloid analyzed was pure or not the
results are of questionable value.

According to older investigations amyloid splits, by the action of
alkali, into protein and chondroi tin-sulphuric acid (see Chapter IX),
and according to KRAWKOW it is therefore a firm, perhaps ester-like
combination of this acid with protein. The protein, from the investiga-
tions of NEUBERG, is of a basic nature and most comparable to the
histones. The investigations of MAYEDA do not coincide with this view
as the amyloid protein obtained by him did not behave like a histone.
Its content of hexone bases was not greater than that of the proteins
of the normal organs and this amyloid protein did not yield any his-
tone-peptone. To all appearances, different investigators have worked
with different substances and it is possible that in the amyloid degenerated
organs partly chondroproteins and partly amyloid proteins may occur,
both of which give the color reactions.

Amyloid is an amorphous white substance, insoluble in water, alcohol,
ether, dilute hydrochloric and acetic acids. It is soluble in concen-
trated hydrochloric acid or caustic alkali with decomposition. On boil-
ing with dilute hydrochloric acid it yields sulphuric acid and a reducing
substance. It is not dissolved by gastric juice, according to KRAWKOW,
which agrees with most of the older reports. It is nevertheless changed
so that it is soluble in dilute ammonia, while the typical amyloid is
insoluble therein. NEUBERG finds on the contrary that amyloid (from
liver) is digested by pepsin as well as by trypsin, although more slowly
than fibrin, and that it is also destroyed in autolysis, so that in life an
absorption is possible. The amyloid from the " sago " spleen studied
by HANSSEN showed the same behavior with gastric juice as KRAWKOW
found, while trypsin, as well as autolysis for months, was without action.
MAYEDA'S amyloid was gradually dissolved by gastric juice.

Amyloid gives the xanthoproteic reaction and the reactions of MIL-
LON and ADAMKIEWICZ-HOPKINS. Its most important property is its
behavior with certain coloring matters. It is colored reddish-brown
or a dingy violet by iodine; a violet or blue by iodine and sulphuric
acid; red by methylaniline iodide, especially on the addition of acetic
acid; and red also by aniline green. Of these color reactions those with
aniline dyes are the most important. The iodine reaction appears less
constant and is greatly dependent upon the physical condition of the
amyloid. The color reactions are due to the presence of the chondroitin-
sulphuric acid component, but this stands in opposition to the behavior
of the intact amyloid obtained by HANSSEN from the " sago " spleen
and the amyloprotein of MAYEDA.

174 THE PROTEIN SUBSTANCES.

In preparing amyloid, extract the finely divided organs with very
dilute ammonia. The undissolved amyloid in the residue, if it does not
resist pepsin digestion, can be directly extracted by dilute barium hydrate
solution and then precipitated from the filtrate by hydrochloric acid.
Otherwise the above mentioned residue is digested for several days with
pepsin. The digestion residue is dissolved in dilute ammonia, filtered,
the amyloid precipitated by dilute hydrochloric acid, the precipitate
dissolved in baryta-water, when the nucleins remain behind, the barium
filtrate precipitated with hydrochloric acid and purified, if necessary
by repeated solution in ammonia and precipitating with hydrochloric
acid, washing and treating with alcohol and ether.

Phosphoglycoproteins. This group includes the phosphorized glycoproteins.
They yield no purine bases (nuclein bases) as cleavage products. They are not
nucleoproteins and therefore they must not be mistaken for them. On pepsin
digestion they may, like certain nucleoalbumins, yield pseudonuclein, but they
differ from the nucleoalbumins in that they yield a reducing substance on boil-
ing with dilute acid. They differ from the nucleoproteins, which also yield reduc-
ing carbohydrates, in, as above stated, not yielding any purine bases.

Only two phosphorized glycoproteins are known at the present time, namely,
ichthulin, occurring in carp eggs and studied by WALTER,1 and which was con-
sidered as a vitellin for a time. Ichthulin has the following composition: C 53.52;
H 7.71; N 15.64; S 0.41; P 0.43; Fe 0.10 per cent. In regard to solubilities it
is similar to a globulin. WALTER has prepared a reducing substance from the
pseudonuclein of ichthulin which gave a highly crystalline compound with
phenylhydrazine.

Another phosphoglycoprotein is helicoproteid, obtained by HAMMARSTEN 2
from the glands of the snail Helix pomatia. It has the following composition:
C 46.99; H 6.78; N 6.08; S 0.62; P 0.47 per cent. It is converted into a gummy,
levorotatory carbohydrate, called animal sinistrin, by the action of alkalies.
On boiling with an acid it yields a dextrorotatory reducing substance.

The compound protein found by SHULTZ and DITTHORN 3 in the spawn of
the frog probably belongs to this group, but instead of glucosamine it gives
galactosamine on cleavage.

B. Nucleoproteins.

By this name we designate those compound proteins which yield
protein and nucleic acid on cleavage. The nucleoproteins seem to be
widely diffused in the animal body. They occur chiefly in the cell-
nuclei, but they also often occur in the protoplasm. They may pass
into the animal fluids on the destruction of the cells, hence nucleopro-
teins have also been found in blood serum and other fluids.

The nucleoproteins may be considered as combinations of a pro-
tein with a side chain, which KOSSEL calls the prosthetic group. This
side chain, which contains the phosphorus, may be split off as nucleic
acid on treatment with alkali. The protein may be of different kinds.

1 Zeitschr. f. physiol. Chem., 15.

2 Hammarsten, Pfliiger's Arch., 36.

3 Zeitschr. f. physiol. Chem., 29.

NUCLEOPROTEINS. 175

In certain cases this is histone, and the combinations between nucleic
acid and protamines are also sometimes classified as nucleoproteins.
The combination between protamine and nucleic acid is, it seems, a
salt-like combination, and entirely different from the combination of the
proteins with nucleic acid in the nucleoproteins. The following facts,
given in connection with the nucleoproteins, do not apply to the nucleo-
protamines. The nucleoproteins differ not only according to the protein
component they contain, but also as to the nucleic acids, which vary
among themselves. There are essentially different nucleic acids, some
among which contain a pentose carbohydrate while others contain a
hexose carbohydrate. The nucleic acids also differ in regard to the
amount of purine and pyrimidine bases they contain (see below).

The native nucleoproteins contain a variable, but not a high percentage
of phosphorus, which in most of the nucleoproteins investigated, ranges
between 0.5 and 1.6 per cent. They also regularly contain iron, and in
Octopodes, HENZE l has observed an iron-free nucleoprotein with 0.96
per cent copper. The nucleoproteins behave like wreak acids, especially
those having considerable protein in the molecule. They therefore
give the ordinary protein reactions and behave in this regard like the
proteins. The nucleoproteins prepared from organs rich in cell nuclei
seem to be characterized by containing more phosphorus and having a
stronger acid character. All nucleoproteins are bodies that are insoluble
in water, but whose alkali combination is soluble in water. From
such a solution the nucleoprotein can be precipitated by acetic acid, and
in an excess of the acid, the precipitate dissolves with more or less
difficulty and in some cases not at all. It dissolves, on the contrary, in
very dilute hydrochloric acid. In this respect nucleoproteins are similar
to the nucleoalbumins and the mucin substances, but differ from these
two groups in that they yield purine bases on hydrolysis. According
to PLIMMER and SCOTT 2 the nucleoproteins differ from the nucleo-albu-
mins by the fact that with sodium hydroxide in 1 per cent solution the
nucleoalbumins split off phosphoric acid while the nucleoproteins do not.
The nucleoproteins give the color reactions of the proteins, but those
which have been investigated are dextrorotatory and not laevorotatory
(GAMGEE and JONES 3).

The nucleoproteins are readily modified. The alkali combination
soluble in water suffers a decomposition on heating its solution, when
as neutral as possible, and coagulated protein separates while a protein
rich in phosphorus and poor in protein with strong acid character remains

1 Zeitschr. f. physiol. Chem., 55.

2 Plimmer and Scott, cited in Biochem. Centralbl., 8, p. 109.

3 Hofmeister's Beitrage, 4.

176 THE PEOTEIN SUBSTANCES.

in solution. By the action of weak acids and by gastric juice a similar
cleavage takes place, whereby the protein split off goes into solution
while the nucleoprotein rich in phosphorus, so-called nudein (MIESCHER,
HOPPE-SEYLER x) or true nudein, remains undissolved. As the nuclei n
is probably nothing but a partly modified nucleoprotein poorer in pro-
tein, having a composition varying with the intensity of the cleavage,
it seems unnecessary to give the name nuclein thereto. On the other
hand, the nucleins have other properties than the nucleoproteins, and
as the nucleins bear the same relation to the nucleoproteins that the
pseudonuclein does to the nucleoalbumins, we will here give a short
description of the nucleins as well as the pseudo- or paranucleins.

Nucleins or true nucleins are formed, as above stated, from nucleo-
proteins in their peptic digestion or by treatment with dilute acids. It
must be remarked that the nucleins are not entirely resistant toward
gastric juice, and also that at least one nucleoprotein, namely, the one
obtained from the pancreas, completely dissolves, leaving no nuclein
residue on treatment with gastric juice (UMBER, MiLROY2). The
nucleins are rich in phosphorus, containing in the neighborhood of 5 per
cent. According to LiEBERMANN,3 metaphosphoric acid can be split
off from true nucleins (yeast nuclein). The nucleins are decomposed
into protein and nucleic acid by caustic alkali, and as different nucleic
acids exist, so also there exist different nucleins. As previously stated
proteins may be precipitated in acid solutions by nucleic acids, and in
this way, as shown by MILROY, combinations of nucleic acid and pro-
teins may be prepared which behave quite like true nucleins. All nucleins
yield purine bases (so-called nuclein bases) on boiling with dilute acids.
They act like rather strong acids.

The nucleins are colorless, amorphous and insoluble or only slightly
soluble in water. They are insoluble in alcohol and ether. They are
more or less readily dissolved by dilute alkalies. The nucleins give the
biuret test and MILLON'S reaction. They show a great affinity for many
dyes, especially the basic ones, and take these up with avidity from watery
or alcoholic solutions. On burning they yield an acid residue which
is very difficult to incinerate and which contains metaphosphoric acid.
On fusion with saltpeter and soda the nucleins yield alkali phosphates.

To prepare nucleins from cells or tissues, first remove the chief mass
•of proteins by artificial digestion with pepsin-hydrochloric acid, lixiviate
the residue with very dilute ammonia, filter, and precipitate with hydro-
chloric acid. The precipitate is further digested with gastric juice,

1 Hoppe-Seyler, Med. chem. Unters., 452.

2 Umber, Zeitschr. f. klin. Med., 34; Milroy, Zeitschr. f. physiol. Chem., 22.

3 Pfliiger's Arch., 47.

PSEUDONUCLE1NS. 177

washed and purified by alternately dissolving in very faintly alkaline
water and reprecipitating with an acid, washing with water, and treating
with alcohol and ether. A nuclein may be prepared more simply by the
digestion of a nucleoprotein. In the detection of nucleins we make use
of the above-described method, testing for phosphorus in the product
after fusing with saltpeter and soda. Naturally the phosphates and
phosphatides must first be removed by treatment with acid, alcohol,
and ether, respectively. No exact methods are known for the quanti-
tative estimation of nucleins in organs or tissues.

Pseudonucleins or PARANUCLEINS. These bodies are obtained as
an insoluble residue on the digestion of certain nucleoalbumins or phospho-
glycoproteins with pepsin-hydrochloric acid. Attention is called to
the fact that the pseudonuclein may be dissolved by the presence of too
much acid or by a too energetic peptic digestion. If the relation between
the degree of acidity and the quantity of substance is not properly selected,
the formation of pseudonucleins may be entirely overlooked in the
digestion of certain nucleoalbumins. Pseudonucleins contain phosphorus,
which, as shown by LIEBERMANN/ is split off as metaphosphoric acid
by mineral acids.

The pseudonucleins are amorphous bodies insoluble in water, alcohol,
and ether, but readily soluble in dilute alkalies and barium hydroxide
solution. They are readily split by barium hydroxide solution with the
splitting off of phosphoric acid, and according to GIERTZ 2 they differ
in this regard from the true nucleins, which are neither dissolved nor
decomposed by baryta. They are not soluble in very dilute acids, and
may be precipitated from their solution in dilute alkalies by adding
acid. They give the protein reactions very strongly, but do not yield
purine bases.

In preparing a pseudonuclein, dissolve the mother-substance in hydro-
chloric acid of 1-2 p. m., filter if necessary, add pepsin solution, and
allow the mixture to stand at the temperature of the body for about
twenty-four hours. The precipitate is filtered off, washed with water,
and purified by alternately dissolving in very faintly alkaline water and
reprecipitating with acid.

Cleavage Products of the Nucleoproteins.
1. The Nucleic Acids.

All nucleic acids are rich in phosphorus and yield phosphoric acid,
purine bases and a carbohydrate or carbohydrate derivative as cleavage
products; most of them also contain pyrimidine bases. The older

1 Ber. d. d. chem. Gesellsch., 21, and Centralbl. f. d. med. Wissensch., 1889.

2 Zeitschr. f. physiol. Chem., 28.

178 THE PROTEIN SUBSTANCES.

statements as to the occurrence of more than two purine bases in a nucleic
acid are not correct and depend upon the fact that the two purine bases
xanthine and hypoxanthine can be secondarily formed from guanine and
adenine. There is no doubt that the most thoroughly studied nucleic
acids, such as the thymus-nucleic acids, the closely related or perhaps
identical acids of the salmon sperm (salmo-nucleic acid), of the herring
sperm and burbot sperm, and of the pancreas, do not contain more than
two purine bases, namely, guanine and adenine.

Of the known nucleic acids we have two, the guanylic acid and inosinic
acid, which contain only one purine base, namely, guanine and hypoxan-
thine, respectively. These two acids do not contain any pyrimidine
bases, which are found thus far in all carefully investigated nucleic acids.
The occurrence of pyrimidine bases is somewhat different in the various
nucleic acids. In one group of animal nucleic acids (thymonucleic acids)
thymine, cytosine and uracil are found, the uracil being produced second-
arily from the cytosine. The plant nucleic acids (the triticonucleic acid
and the yeast nucleic acid, which may perhaps be identical with it) do
not contain any thymine and yields as nitrogenous cleavage products
besides the two purine bases only cytosine and uracil.

All nucleic acids, as above stated, contain a carbohydrate group.
In the plant nucleic acids and in two animal ones, the guanylic and inosinic
acids, the carbohydrate is a pentose. In the remaining animal nucleic
acids it is on the contrary a hexose or at least a hexacarbohydrate.

The nature of this hexacarbohydrate has not been determined and
the nature of the pentoses occurring in the nucleic acids is also a disputed
point. Based upon the investigations of NEUBERG we have considered
the pentose of guanylic acid and of inosinic acid as Z-xylose. The correct-
ness of this view is disputed by others. According to LEVENE and JACOBS
the pentose of all nucleic acids containing pentose, is d-ribose. HAISER
and WENZEL who for a time considered the pentose of inosinic acid as
d-xylose are now of the view that it is probably d-ribose. The view of
LEVENE and JACOBS, that the pentose of the guanylic acid is d-ribose has
received important support by the investigations of SCHULZE and TRIER
on the identity of the plant guaninpentoside vernine with the guanin-
pentoside (see below) prepared by LEVENE and JACOBS. Still we have
no explanation why NEUBERG and REWALD 1 obtained only Z-xylose
from the pancreas on the hydrolysis of the entire organ, and LEVENE
and JACOBS on the contrary only d-ribose.

1 Neuberg and Brahn, Bioch. Zeitschr., 5; see also Ber. d. d. chem. Gesellsch
41 and 42; LEVENE and JACOBS, Ber. d. d. chem. Gesellsch., 42 and 43; Haiser and
Wenzel, Monatsh. f. Chem., 31; Schulze and Trier, Zeitschr. f. physiol. Chem., 70;
Rewald, Ber. d. d. chem. Gesellsch., 42.

NUCLEIC ACIDS. 179

All nucleic acids contain phosphoric acid. The relation between phos-
phorus and nitrogen is as 1 : 4 in the inosinic acid and as 1 : 5 in the guanylic
acid. In the thymus- and the salmo-nucleic acids the relation accord-
ing to SCHMIEDEBERG is 4:14 and according to STEUDEL 4:15. In the
triticonucleic acid, OSBORNE and HARRIS found the relation 4:16; in the
yeast nucleic acid, LEVENE and JACOBS found it was equal to 4:15.

According to the number of bases contained in the nucleic acids
we can differentiate between the simple nucleic acid with only one base
and the complex nucleic acids with several bases. LEVENE and MANDEL l
have called the first (inosinic acid, guanylic acid) nudeotides or mono-
nucleotides arid the last poly nudeotides.

The properties and the constitution of the nucleic acids, as far as we
know them, have been determined essentially by the work of KOSSEL
and his pupils, by SCHMIEDEBERG, STEUDEL and LEVENE 2 and their
collaborators.

On complete acid hydrolysis the nucleic acids are split into the three
above mentioned components, phosphoric acid, carbohydrate and bases.
The purine bases are more readily split off than the pyrimidine bases
and on careful acid hydrolysis of thymus nucleic acid, a new acid, the
thyminic acid of STEUDEL and BRIGL is obtained. This acid is very
similar to the thyminic acid of KOSSEL and NEUMANN 3 with the barium
salt, Ci6H23NsP2Oi2Ba, and the nudeotinphosphoric acid of SCHMIEDE-
BERG. This acid differs probably from the original nucleic acid only
by the absence of purine bases. By the action of strong nitric acid in
the cold we can, according to the method suggested by STEUDEL, ' split
off the purine bases while nearly all the phosphoric acid remains in organic
combination with the carbohydrate complexes.

The hydrolyses of pentose containing nucleic acids as carried out by
LEVENE and JACOBS in neutral, or, if the pyrimidine complexes of the
plant nucleic acid were being studied, in ammoniacal reaction, by heating
to high temperatures in the autoclave or in sealed tubes, are of special
interest. In these cases the binding with the phosphoric acid was rup-

1 Ber. d. d. chem. Gesellsch., 41.

2 The work of Kossel and his pupils on the nucleic acids can be found in: Arch,
f. (Anat. u.) Physiol. 1892, 1893 and 1894; Sitz. Ber. d. Berl. Akad. d. Wiss., 18, 1894;
Centralbl. f. d. med. Wiss. 1893; Ber. d. d. chem. Gesellsch., 26 and 27; Zeitschr. f.
physiol. Chem., 23 and 38; see also Neumann, Arch. f. (Anat. u.) Physiol., 1898 and 1899
Suppl.; Miescher, Hoppe-Seyler's Med. chem. Unters., p. 441 and Arch. f. exp. Path,
u. Pharm., 37; Schmiedeberg, ibid., 37, 43, and 57; Altman, Arch. f. (Anat. u.) Physiol.,
1889; Steudel, ibid., 42, 43, 46, 49, 50, 52, 53, 55, 56, 70, 77; Ascoli, Zeitschr. f. physiol.
Chem., 28 and 31; Levene, ibid., 32, 37, 38, 39, 43, 45; Levene and Mandel. ibid. 46,
47, 49, 50; Inouye and Kotake, ibid., 46; Levene and Jacobs, Ber. d. d. chem.
Gesellsch., 42, 43, 44; with La Forge, ibid., 43 and 45.

3 Steudel and Brigl, Zeitschr. f. physiol. Chem., 70; Kossel and Neumann, ibid., 22.

180 THE PROTEIN SUBSTANCES.

tured while the binding between the pentose and purine bases remained
intact. In this manner they obtained pentosides, i.e., glucoside-like com-
bination between pentose and a purine base. These pentosides have
also been called nucleosides and such a nucleoside was the inosine,
which was first found by HAISER and WENZEL 1 and which is the pen-
toside of inosinic acid and is a combination of hypoxanthine with pentose
(d-ribose). The other three nucleosides adenosine, guanosine and xantho-
sine have been prepared by LEVENE and JACOBS.

The nucleosides are crystalline bodies which give crystalline combina-
tions. Of special interest is guanosine because it is identical with the base
vernine, occurring in the plant kingdom and discovered by SCHULZE 2
and because of the identity of the pentose occurring in both has been
positively proved. The guanosine has also been found by LEVENE and
JACOBS3 in the pancreas. On acid hydrolysis every nucleoside splits
into purine base and pentose. By the action of nitrite and glacial acetic
acid the guanosine is transformed into xanthosine and the adenosine
into inosine.

MANDEL and DUNHAM have prepared, from acetone-yeast, a crystalline
adenine-hexose compound corresponding to the pentoside but whose
relation to the cleavage products of nucleic acids is not known. From
thymus nucleic acid LEVENE and JACOBS 4 have later isolated a guanine
hexoside.

The pyrimidine complexes corresponding to the nucleosides also
contain (in the plant nucleic acids) pentose, according to LEVENE and
LA FORGE 5 but in much firmer bondage. This is the reason why they
give only a faint orcin reaction, are much more resistant to enzymes
than the purine nucleosides and give off furfurol only very slowly on
distilling with hydrochloric acid. Still they contain pentose and pyrimi-
dine bases in equimolecular proportions. The pyrimidine complexes are
called cytidine and uridine, the first containing cytosirie and the second
uracil. Uridine is crystalline; the cytidine has not been obtained in a crys-
talline form but it gives several crystalline salts. The uridine is claimed
to exist pre-formed in the yeast nucleic acid and not produced secondarily
from the cytidine.

Based upon the investigations carried out by STEUDEL, LEVENE and
JACOBS we can for the present represent the structure of the nucleic acids
in the following way:

1 Monatsh. f. Chem., 29.

2 E. Schulze and Bosshard, Zeitschr. f. physiol. Chem., 10; with Trier, ibid., 70.

3 Bioch. Zeitschr., 28.

4 Mandel and Dunham, Journ. of biol. Chem., 11; Levene and Jacobs, ibid., 12.
6 Ber. d. d. chem. Gesellsch., 45.

NUCLEIC ACIDS. 181

The simple nucleic acids are ester-like combinations between phosphoric
acid and a purine base-pentoside.

The complex nuclei acids are complex molecules each composed of
four simple nucleic acids (nucleotides) . In regard to the complex nucleic
acids we differentiate between two groups.

The acids of the thymonucleic acid group are, according to STEUDEL,
tetrabasic phosphoric acid ester which corresponding to each phosphorus
atom, contains a hexose group and one of the four bases, guanine,
adenine, cystosine and thymine. From the name of this group we infer
that these acids contain thymine.

The plant nucleic acid group differs from the preceding by the follow-
ing. They do not contain any thymine but uracil instead. They do not
contain any hexose but do contain pentose. In the acids of this group
for each atom of phosphor we have 1 mol. pentose and on each the
purine and pyrimidine bases are combined.

It must be remarked that the complex nucleic acids have not been
prepared from isolated component proteins but generally from organs,
namely perhaps from a mixture of different nucleoproteins and that for
this reason we do not know whether these acids are chemical individuals
or only a mixture of closely related simple nucleic acids. On the other
hand it is also possible that the simple nucleic acids originate from more
complex nucleic acid by cleavage because such cleavages are in fact
known. Such an assumption does not apply at least for the guanylic
acid from the pancreas as it is obtained from a compound protein with
only one base, namely guanine.

All nucleic acids are amorphous, white, and have an acid reaction.
They are readily soluble in ammoniacal or alkaline water. They also
dissolve in concentrated acetic acid and form insoluble salts with copper
chloride and salts of the heavy metals, and as a rule insoluble basic
salts with the alkaline earths. Their solubility in water is very different.
Inosinic acid, for example, is very readily soluble in cold water while
#-guanylic acid is soluble with difficulty. The complex nucleic acids
are also soluble with difficulty in cold water. The solution of their
alkali combination is not as a rule precipitated by acetic acid but is
precipitated by a slight excess of hydrochloric acid, especially in the pres-
ence of alcohol. The nucleic acids soluble in dilute acids give in such
solution a precipitate with proteins, which are considered as nucleins. All
nucleic acids are insoluble in alcohol and ether. They do not give either
the biuret test or MILLON'S reaction. The nucleic acids are optically
active and, with the exception of inosinic acid (GAMGEE and JONES)
and of guanylic acid (LEVENE and JACOBS 1), are dextro-rotatory.

1 Gamgee and Jones, Proc. Roy. Soc., 72; Levene and Jacobs, Journ. of biol.
Chem., 12.

182 THE PROTEIN SUBSTANCES.

The proteolytic enzymes, such as pepsin and trypsin, decompose the
nucleoproteins more or less; the nucleic acids are apparently not
split by these enzymes or at least not as far as phosphoric acid and
purine bases. Such a cleavage can, on the contrary, be brought about
by erepsin (NAKAYAMA) or by other closely allied enzymes found in
various organs which have been called nudeases. Micro-organisms can also
bring about a more or less deep cleavage of the nucleic acids (ScniT-

TENHELM and SCHROTER1).

LEVENE and MEDIGRECEANU 2 differentiate between three kinds of
nucleases namely, nucleinases, nucleotidases and nucleosidases. The
nudeinases, which are found in the pancreatic juice and all organs
investigated, but not in gastric juice, acts only upon the complex nucleic
acids and splits them into nucleoticles. The nucleotidases, which, with
the exception of the gastric and pancreatic juices, occurs all over and
especially in the intestinal mucosa, split the simple nucleic acids (mono-
nucleotides) into phosphoric acid and the corresponding nucleoside
(purine pentoside). The nucleosidases, which are not found in the gastric,
pancreatic or intestinal juices, nor in the blood or the pancreas but in
other organs, split the nucleosides into purine base and pentose. It is
unknown how the cleavage of the pyrimidine and hexose complexes
of the nucleic acids is brought about.

According to W. JONES 3 the purine bases of the nucleic acids can be
deamidized without being previously split off as free base from the acid.
Thus the pig-pancreas contains an adenosin-deamidase which deami-
dizes the still combined adenine. On the contrary the same organ also
contains a guanase which deamidizes the free guanine but does not
contain a guanosine deamidase. The pig liver, in which only traces of
guanase occur, contain on the contrary a guanosine-deamidase. Recent
investigations of SCHITTENHELM and K. WIENER 4 show that we must
also admit of nucleoside-deamidases besides purine deamidases.

Inosinic Acid, CioHia^POg was first isolated by LIEBIG from the
flesh of certain animals and then closely studied by HAISER. It is obtained
from beef extracts, and according to the investigations of NEUBERG and
BRAHN, FR. BAUER, and LEVENE and JACOBS it is a simple nucleic acid.5

1 Nakayama, Zeitschr. f. physiol. Chem., 41; Iwanoff, ibid., 39; Fr. Sachs, " 1st die
Nuklease mit dem Trypsin ideritisch? " Inaug. -Dissert, Heidelberg, 1905; Schitten-
helm and Schroter, f. physiol. Zeitschr. Chem., 41.

2 Journ. of biol. Chem., 9.

3 Journ. of biol. Chem., 9.

4 Zeitschr. f. physiol. Chem., 77.

5 Liebig, Annal. d. chem. u. Pharm., 62; Haiser, Monatsh. f. chem., 16; Neuberg
and Brahn, Biochem. Zeitschr., 5 and Ber. d. d. chem. Gesellsch., 41, p. 3376; Bauer
Hofmeister's Beitrage, 10; Levene and Jacobs, Ber. d. d. chem. Gesellsch., 41, p. 2703.

INOSINIC AND GUANYLIC ACIDS. 183

On hydrolysis it yields phosphoric acid, hypoxanthine and pentose,
according to the equation:

The pentose, whose somewhat disputed nature has been discussed
on page 178, is combined with hypoxanthine in a glucoside-like com-
bination forming the pentoside inosine, which, according to LEVENE and
JACOBS, is combined with the phosphoric acid, like an ester by means
of the 6-carbon atom of the pentose (ribose).

Inosinic acid is amorphous, syrupy, readily soluble in water and pre-
cipi table by alcohol. It is lae vo-rotatory ; for the Ba salt containing
hydrochloric acid NEUBERG and BRAHN found (a)D=— 18.5° at 16° C.
It gives several crystalline salts among which the barium salt, which is
soluble with difficulty in water, must be mentioned.

In regard to the preparation of this acid we must refer to the works
of HAISER, NEUBERG and BRAHN, LEVENE and JACOBS mentioned in
footnote 5, page 182.

Guanylic acid. This acid, which was first prepared by BANG from
the pancreas has also been found by JONES and ROWNTREE in the spleen
and by LEVENE and MANDEL 1 in the liver. As cleavage products it yields
guanine, pentose and phosphoric acid and therefore its simplest formula
is CioHuNsPOg. This formula is accepted also by STEUDEL and BRIGL
and by LEVENE and JACOBS, while BANG basing his views on the results
of elementary analysis gives the formula C44He6N2oP4O34. Accord-
ing to this formula the acid would contain besides, guanine, pentose and
phosphoric acid also an unknown residue, C4Hio02, and according to BANG
is not a simple nucleic acid but would occupy an intermediary position
between the inosinic acid and the thymus nucleic acid. In opposition
to this it must be remarked that LEVENE and JACOBS 2 have recently
prepared the crystalline brucine salt of the acid and the analysis of this
salt as well as the barium salt substantiates the first mentioned, simple
formula. In regard to the pentose of guanylic acid see page 178.

The acid first described by BANG, the /3-acid, is soluble with great diffi-
culty in cold water and rather readily soluble in boiling water. It is easily
precipitated by acetic acid from the solution of the alkali combination
in water. The /3-acid may, according to BANG, be derived from another
guanylic acid, the a-guanylic acid, by the action of the alkali. The

1Bang, Zeitschr. f. physiol. Chem., 26; with Raaschou, Hofmeister's Beitrage, 4;
Jones and Rowntree, Journ. of biol. chem., 4; Levene and Mandel, Biochem. Zeitschr.
10.

2Steudel and Brigl, Zeitschr. f. physiol. Chem., 68; Bang, ibid., 69 and Bioch.
Zeitschr., 26; Levene and Jacobs, Journ. of biol. Chem., 12.

184 THE PROTEIN SUBSTANCES.

a-guanylic acid is readily soluble, even in cold water, and it is also similar
to thymus nucleic acid in other respects. It is precipitated from the
solution of its salts by hydrochloric acid but not by acetic acid, and its
solutions precipitate proteins. STEUDEL and BRIGL believe that the
/3-acid is a potassium salt and that the a-acid is the actual acid, but this
view BANG disputes. LEVENE and JACOBS found that the acid con-
taminated with alkali does not gelatinize while the pure acid does. The
specific rotation of the latter was («)D= —1.27° at 25° C.

In regard to the preparation of guanylic acid we refer, to the work
of BANG, LEVENE and JACOBS. 1

Thymonucleic Acids. A. NEUMANN has isolated two nucleic acids,
a- and jS-thymus nucleic acid, from the thymus gland. The a-acid is
soluble with difficulty, and in proper concentration gives a sodium salt
which gelatinizes in proper concentration, and a barium salt which is
precipitated by barium acetate in substance (KOSTYTSCHEW). The
barium salt of the /3-acid is not precipitated by barium acetate. The
a-acid is designated as anhydric by SCHMIEDEBERG,2 and the /3-acid as
hydrate, and the first can be transformed into the second by heating.
This transformation, according to KOSTYTSCHEW, is a decomposition
whereby two-thirds of the purine bases are split off.

According to SCHMIEDEBERG the thymus nucleic acid is identical
with the salmo-micleic acid (from salmon sperm), and also according
to STEUDEL probably with the acid from the herring sperm. Other
nucleic acids, at least those very closely related to this nucleic acid, have
been prepared from the sperm of the burbot (Lota vulgaris) by ALSBERG,
of the sturgeon (NOLL) and of the sea-urchin (MATHEWS), also from
ox-sperm, brain, spleen (LEVENE), pancreas (LEVENE, v. FURTH and
JERUSALEM, STEUDEL), mammary glands and kidneys (LEVENE and MAN-
DEL 3) and from other organs.

At the present time we arc not agreed as to the formula for the most
carefully studied thymonucleic acids (from the thymus, herring and sal-
mon sperms) . According to the numerous analyses of SCHMIEDEBERG and
his collaborators for every 4 atoms of phosphorus there occur 14 atoms
of nitrogen. The relationship of C to P was 40 to 4 and the relationship of
C to N in 12 out of 15 preparations was 40 to 14, and only in 3 prepara-
tions 40 to 15. From these facts SCHMIEDEBERG has given the acid

1 See footnotes 1 and 2, p. 183.

2 A. Neumann, Arch. f. (Anat. u.) physiol, 1898 and 1899; Kostytschew, Zeitschr,
f. physiol. Chem., 39; Schmiedeberg, 1. c.

3 Alsberg, Arch. f. exp. Path. u. Pharm., 51; Noll, Zeitschr. f. physiol. Chem., 25;
Mathews, ibid., 23; v. Fiirth and Jerusalem Hofmeister's Birtrage, 10 and 11; Steudel,.
Zeitschr. f. physiol. Chem., 53; Levene and Mandel, ibid., 46, 47. See also footnote 2,
p. 179.

PLANT NUCLEIC ACIDS. 185

the formula C4oH56Ni4Oi6.2P205. According to STEUDEL for every 4
atoms of phosphorus we have 15 atoms nitrogen and from this he has
calculated the formula C43HeiNi5P4O34+9H2O for the acid containing
water.

The probable constitution of the thymo-nucleic acids has been previ-
ously indicated and as positively known cleavage products we have at least
phosphoric acid, a hexose carbohydrate, guanine, adenine, thymine and
cytosine.

The thymo-nucleic acids have the reactions as given for the complex
nucleic acids. They are amorphous, dextro-rotatory, and soluble in cold
water with difficulty. They form soluble salts with alkalies and the acid
is precipitated from these solutions by mineral acid but not by acetic
acid. Tannic acid alone does not cause a precipitate but does in the
presence of sodium acetate. Proteins precipitate their solutions contain-
ing acetic acid. The two special thymo-nucleic acids differ from each
other by the different behavior of their salts (see above).

The preparation of the nucleic acids is based in the first place always
upon the cleavage of the nucleoprotein into protein and nucleic acid by
the action of alkali and then separating the nucleic acids from the
protein. The operations necessary for purifying the nucleic acids from
proteins are very complicated and we must refer to the works of

SCHMIEDEBERG, NEUMANN, LEVENE, and Others.1

Plant Nucleic Acids. The two best known acids of this group are
the yeast nucleic acid and the triticonucleic acid isolated from the
wheat embryo. The identity of these two acids, as suggested by OSBORNE
and HARRIS has become more and more probable. According to KOWA-
LEWSKY 2 the yeast nucleic acid contain only adenine, guanine and cytosine,
the uracil is only formed secondarily from the cytosine. The yeast nucleic
acid may perhaps be a triphosphoric acid with three molecules of pentose
each with a molecule of adenine, guanine and cytosine.

This view stands in opposition to the observations of LEVENE and
JACOBS 3 that the yeast nucleic acid contains one molecule of pentose
combined with adenine and guanine, and besides this it contains two
pyrimidinehexose complexes, cytidine and uridine.

The triticonucleic acid yields also, as OSBORNE and HEYL, WHEELER
and JOHNSON and recently LEVENE and LA FORGE 4 have shown, the same
hydrolytic products as the yeast nucleic acid and both contain d-ribose.

1 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 43 and 57; Herlant, ibid., 44; Neu-
mann, Arch. f. (Anat. u.) Physiol. 1899 Supplb.; Levene, Zeitschr. f. physiol. Chem.,
32 and 45; Kostytschew, 1. c.

2 Osborne and Harris, Zeitschr. f. physiol. Chem., 36; Kowalewsky, ibid., 69.

3 Ber. d. d. chem. Gesellsch., 44.

4 Osborne and Heyl, Amer. Journ. of Physiol., 21; Wheeler and Johnson, Amer.
Chem. Journ., 29; Levene and La Forge, Ber. d. d. Chem., Gesellsch., 43.

186 THE PROTEIN SUBSTANCES.

The somewhat different results found on the elementary analysis of these
two acids do not seem to be of very great importance and we have strong
evidence for the identity of these acids. OSBORNE and his collaborators
found the formula C^HeiNieP^si for triticonucleic acid.

The plant nucleic acids have the general reactions of the complex
nucleic acids but can be precipitated by an excess of acetic acid. They
are dextro-rotatory.

In regard to their preparation we refer to the works of KOSSEL, OSBORNE
and HARRIS and to LEVENE and co-workers.1

Plasminic acid is an acid which was prepared by ASCOLI and KOSSEL 2 by
the action of alkali upon yeast. It contains iron, and is soluble in very dilute
hydrochloric acid (1 p. m.). It is still a question whether it is a mixture or a
chemical individual.

2. Purine Bases.

The cleavage products obtained from the nucleic acids, the nudein
bases, which are also called alloxuric bases by KOSSEL and KRUGER, are
members of the larger group of purines, to which also belongs the uric
acid which is a substance occurring in the animal body. The constitu-
tion of these bodies has been explained by E. FiscHER,3 and he has
prepared many of the bodies synthetically. They can all be derived from
the synthetically prepared purine, CsHiN^ which has the formula given
below and which may be considered as a combination of a pyrimidine
ring with an imidazole ring.

HC— NH
HC C— NHV HC CH || >CH

II II >CH || || HC N

N— C W N-CH

Purine Pyrimidine Imidazole

The different purine bodies are derived therefrom by the substitution of the
various hydrogen atoms by hydroxyl, amide, or alkyl groups. In order to signify
the different positions of substitution FISCHER has proposed to number the nine
members of the purine nucleus in the following way :

I I
2C 5C— N7

C8.

3N— C— N9
4

1 See footnote 2, p. 179, and footnote 3 and 4, p. 185.

2Ascoli, Zeitschr. f. physiol. Chem., 28.

3 See E. Fischer, Untersuchungen in der Puringruppe (1882-1906) Berlin, 1907.

PURINE BASES.

187

For example, uric acid

HN— CO
id, OC C— NH

II

, is 2, 6, 8-trioxypurine; adenine,

'

HN— CO

C— N.

CH3, is

HN— C— NH
N=C.HN2

HC C — NHV , is 6-aminopurine, and heteroxanthine, OC
II II >CH I

N-€ N' HN-

7-methyl-2, 6-dioxypurine, etc.

The starting-point used by FISCHER for the synthetical preparation of the

purine bases was 2, 6, 8-trichlorpurine, which is obtained, with 8-oxy-2, 6-dichlor-

purine as an intermediary product, from potassium urate and phosphorus oxychlo-

ride.

The purine bodies or alloxuric bodies, found in the animal body or its
excreta are as follows: Uric acid, xanthine, heteroxanthine, \-meihylxan-
thine, paraxanthine, guanine, epiguanine, hypoxanthine, episarkine,
adenine. The bodies theobromine, theophylline, and caffeine, occurring in
the vegetable kingdom, stand in close relation to this group.

The composition of the purine bodies most important from a physio-
logical standpoint is as follows:

Xanthine
1-methylxan thine,
Heteroxanthine,
Theophylline,
Paraxanthine,
Theobromine
Caffeine,
Hypoxanthine,
Guanine
Epiguanine,
Adenine
Episarkine,

.C6H4N402
C6H6N4O2

:::::::::::::::::::::• '2

1 -methyl

, 6-dioxypurine
6-oxypurine

CeH6N4O2

7 tt y

1, 3-dimethyl

C7H8N4O2. .

C7H8N4O2

3, 7- "

C8H10N4O2

1, 3, 7-trimethyl

C5H4N4O

C6H5N6O

2-ami

no "
. 6-aminonurine

C6H7N5O.

7-methyl ' ' '

C6H6N5

C4H6N30(?)

After SALOMON l had shown the occurrence of xanthine bodies in
young cells, the importance of the purine bases as decomposition prod-
ucts of cell nuclei and of nucleins was shown by the pioneering researches
of KOSSEL, who discovered adenine and theophylline. In those tissues
in which, as in the glands, the cells have kept their original state, the
purine bases are not found free, but in combination with other atomic
groups (nucleic acids). In such tissues, on the contrary, as in muscles,
which are poor in cell nuclei, the purine bases are found in the free state.
Since the purine bases, as suggested by KOSSEL, stand in close relation-
ship to the cell nucleus, it is easy to understand why the quantity of
these bodies is so greatly increased when large quantities of nucleated

Sitzungsber. d. Bot. Verein der Provinz Brandenburg, 1880.

188 THE PROTEIN SUBSTANCES.

cells appear in such places as were before endowed in a relatively poor
manner. As an example of this, the blood, in leucaemia, is extremely rich
in leucocytes. In such blood KOSSEL 1 found 1.04 p. m. purine bases,
against only traces in the normal blood. That these bases are also inter-
mediate steps in the formation of uric acid in the animal organism is
probable, and will be shown later (see Chapter XIV).

Only a few of the purine bases have been found in the urine or in the
muscles. Only four bases — xanthine, guanine, hypoxanthine, and ade-
nine — have been obtained, thus far, as cleavage products of nucleins,
and these do not always have a primary origin. In regard to the purine
bodies from other substances we refer the reader to their respective
chapters. Only the above four bodies, the real nuclein bases, will be
considered at this time.

Of these four bodies, xanthine and guanine form one special group
and hypoxanthine and adenine another. By the action of nitrous acid
guanine is converted into xanthine and adenine into hypoxanthine.

C5H4N40.NH+HN02 =

Guanine Xanthine

Adenine Hypoxanthine

Similar transformation, where xanthine and hypoxanthine are pro-
duced secondarily, may also occur in the hydrolysis of nucleic acids as
well as in putrefaction and by the action of special enzymes. The
researches of SCHITTENHELM, LEVENE, JONES, PARTRIDGE, WINTERNITZ,
and BURIAN have shown that in various organs deamination enzymes,
such as guanase and adenase, occur, which convert guanine and adenine
into xanthine and hypoxanthine respectively, and also oxidases which
oxidize hypoxanthine into xanthine and this then into uric acid. This
formation of uric acid from the purine bases, which will be discussed in
detail in a following chapter (XIV), is of very great interest.

The nuclein bases form crystalline salts with mineral acids, which,
with the exception of the adenine salts, are decomposed by water. They
are easily dissolved by alkalies, while with ammonia their action is some-
what different. They are all precipitated from acid solution by phos-
photungstic acid; they also separate as silver compounds on addition
of ammonia and ammoniacal silver-nitrate solution. These precipitates
are soluble in boiling nitric acid of 1.1 specific gravity. All purine bodies
are also precipitated by FEHLING'S solution (see Chapter III) in the pres-
ence of a reducing substance such as hydroxylamine (DRECHSEL and
BALKE). Copper sulphate and sodium bisulphite may also be used to

1 Zeitschr. f. physiol. Chem.. 7.

XANTHINE. 189

advantage in their precipitation (KRUGER)1. This behavior of the
purine bases serves just as well as the behavior with the silver solution
for their precipitation and preparation.

HN— CO

I I
Xanthine, C5H4N402, = OC C— NH v (2, 6-dioxypurine) , is found

I II >CH

HN-C — W

in several cellular organs. It occurs in small quantities as a physio-
logical constituent of urine, and it occasionally has been found as a urinary
sediment, or calculus. It was first observed in such a stone by MARCET.
Xanthine is found in larger amounts in a few varieties of guano (Jarvis
guano) .

Xanthine can be prepared, according to E. FISCHER, by boiling uric
acid with 25 per cent hydrochloric acid or, according to SuNDViK,2 by
heating uric acid with anhydrous oxalic acid in glycerin to about 200° C.

Xanthine is amorphous, or forms granular masses of crystals, or may
also, according to HoRBACZEWSKi,3 separate as masses of shining, thin,
large rhombic plates with 1 mol. water of crystallization. It is very
slightly soluble in water, in 14,151-14,600 parts at 16° C., and in 1300-
1500 parts at 100° C. (ALMEN4). It is insoluble in alcohol or ether, but
is readily dissolved by alkalies and with difficulty by dilute acids. With
hydrochloric acid it gives a crystalline, difficultly soluble combination.
With very little caustic soda it gives a readily crystallizable compound,
which is easily dissolved by an excess of alkali. Xanthine dissolved in
ammonia gives with silver nitrate an insoluble, gelatinous precipitate
of silver xanthine. This precipitate is dissolved by hot nitric acid, and
by this means an easily soluble crystalline double compound is formed.
Xanthine in aqueous solution is precipitated on boiling with copper
acetate. At ordinary temperatures xanthine is precipitated by mercuric
chloride and by ammoniacal basic lead acetate. It is not precipitated
by basic lead acetate alone.

When evaporated to dryness in a porcelain dish with nitric acid,
xanthine gives a yellow residue, which turns, on the addition of caustic
soda, first red, and after heating, purple-red. If we place some chlorinated
lime with some caustic soda in a porcelain dish and add the xanthine

1 Balke, Zur Kenntnis der Xanthinkorper, Inaug.-Diss. Leipzig, 1893 ; Kriiger
Zeitschr. f. physiol. Chem., 18.

2E. Fischer, Ber. d. d. chem. Gesellsch, 43; Sundvik, Zeitschr. f. physiol.Chem.
76. In regard to the synthesis of xanthine and other purines see E. Fischer, footnote
3, p. 186.

s Zeitschr. f. physiol. Chem., 23.

4 Journ. f. prakt. Chem., 96.

190 THE PROTEIN SUBSTANCES.

to this mixture, at first a dark green and then quickly a brownish halo
forms around the xanthine grains and finally disappears (HOPPE-SEYLER).
If xanthine is warmed in a small vessel on the water-bath with chlorine-
water and a trace of nit.ic ?cid, and evaporated to dryness, and the
residue is then exposed under a bell-jar to the vapors of ammonia, a
red or purple-violet color is produced (WEIDEL'S reaction). E. FISCHER 1
has modified WEIDEL'S reaction in the following way: He boils the xan-
thine in a test-tube with chlorine-water or with hydrochloric acid and a
little potassium chlorate, then evaporates the liquid carefully, and moistens
the dry residue with ammonia.

HN— CO

I I
Guanine, C5H6N5O, = H2N.C C— NHX (2-amino-6-oxypurine) .

N— C— W

Guanine is found in all organs rich in cells. It is further found in the
muscles (in very small amounts), in the scales and in the air-bladder of
certain fishes, as iridescent crystals of guanine-lime; in the retinal epithe-
lium of fishes, in guano, and in the excrement of spiders it is found as
chief constituent. It also occurs in human and pig urine. Under patholog-
ical conditions it has been found in leucsemic blood, and in the muscles,
ligaments, and articulations of pigs with guanine-gout.

Guanine is a colorless, ordinarily amorphous powder, which may be
obtained as small crystals by allowing its solution, in concentrated
ammonia, to evaporate spontaneously. According to HORBACZEWSKI it may
under certain conditions appear in crystals similar to creatinine-zinc chlor-
ide. It is insoluble in water, alcohol, and ether. It is rather easily dissolved
by mineral acids and readily by alkalies, but it dissolves with great
difficulty in ammonia. According to WuLFF2 100 cc. of cold ammonia
solution containing 1, 3, or 5 per cent NHs dissolve 9, 15, or 19 milli-
grams of guanine respectively. The solubility is relatively increased
in hot ammonia solution. The hydrochloride readily crystallizes, and
has been recommended by KossEL3 for the microscopical detection of
guanine, on account of its behavior toward polarized light. The sul-
phate contains 2 molecules of water of crystallization, which is completely
expelled on heating to 120° C., and this fact, as well as the fact that
guanine yields guanidine on decomposition with chlorine-water, differ-
entiates it from 6-amino-2-oxypurine, which is considered as an oxida-
tion product of adenine and possibly occurs as a chemical metabolic

!Ber d deutsch. chem. Gesellsch., 30, 2236.
J Zeitschr f. physiol. Chem., 17.

3 Ueber die chem. Zusammensetz. der Zelle, Verh. d. physiol. Gesellsch. zu Berlin
1890-91, Nos. 5 and 6.

HYPOXANTHINE. 191

product (E. FISCHER). The 6-amino-2-oxypurine sulphate contains
only 1 molecule of water of crystallization, which is not expelled at 120° C.
Very dilute guanine solutions are precipitated by both picric acid and
metaphosphoric acid. These precipitates may be used in the quantita-
tive estimation of guanine. The silver compound dissolves with difficulty
in boiling nitric acid, and on cooling the double compound crystallizes
out readily. Guanine acts like xanthine in the nitric-acid test, but gives
with alkalies on heating a more bluish-violet color. A warm solution
of guanine hydrochloride gives with a cold saturated solution of picric
acid a yellow precipitate consisting of silky needles (CAPRANICA). With
a concentrated solution of potassium bichromate a guanine solution
gives a crystalline, orange-red precipitate, and with a concentrated
solution of potassium ferricyanide a yellowish-brown, crystalline pre-
cipitate (CAPRANICA). It also gives a compound with picrolonic acid
(LEVENE l). Guanine gives WEIDEL'S Reaction.

HN— CO

Hypoxanthine, SARKINE, C5H4N4O,=HC C — NHV =(6-oxypurine).

II II >CH

N-C— N f-

This body has been found in all cellular organs and in meat extracts,
and as a cleavage product of inosinic acid. It is especially abundant in
the sperm of the salmon and carp. Hypoxanthine occurs also in the mar-
row and hi very small quantities in normal urine, and, as it seems, also in
milk. It is found in rather considerable quantities in the blood and urine
in leucaemia.

Hypoxanthine can be obtained according to SuNDViK's2 method
from uric acid or xanthine by heating with a formate or more simply
by heating with chloroform in alkaline solution.

Hypoxanthine forms very small, colorless, crystalline needles. It
dissolves with difficulty in cold water, but the claims concerning
solubility therein are very contradictory.3 It dissolves more readily
in boiling water, in about 70-80 parts. It is almost insoluble in alcohol,
but is dissolved by acids and alkalies. The compound with hydrochloric
acid is crystalline, and is more soluble than the corresponding xanthine
derivative. It is easily soluble in ammonia. The silver compound
dissolves with difficulty in boiling nitric acid. On cooling, a mixture
of two hypoxanthine silver-nitrate compounds possessing an inconstant
composition separates out. On treating this mixture with ammonia
and an excess of silver nitrate and heating, a silver hypoxanthine is

1 Capranica, Zeitschr. f. physiol. Chem., 4; Levene, Bioch. Zeitschr., 4.

2 1. c. and Skand, Arch. f. Physiol., 25.

3 See E. Fischer, Ber. d. deutsch. chem. Gesellsch., 30.

192 THE PROTEIN SUBSTANCES.

formed, which when dried at 120° C. has a constant composition,
2(C5H2Ag2N40)H20, and is used in the quantitative estimation of
hypoxanthine. Hypoxanthine picrate is soluble with difficulty, but
if a boiling-hot solution of it is treated with a neutral or only faintly
acid solution of silver nitrate the hypoxanthine is almost quantitatively
precipitated as the compound CsHsAgN^.Ce^CNC^sOH. Hypo-
xanthine does not yield an insoluble compound with metaphosphoric
acid. When treated, like xanthine, with nitric acid, it yields, an almost
colorless residue which, on warming with alkali, does not turn red. Hypo-
xanthine does not give WEIDEL'S reaction. After the action of hydro-
chloric acid and zinc upon a hypoxanthine solution, followed by the
addition of an excess of alkali a ruby-red color develops, followed by
a brownish-red color (KOSSEL). According to E. FISCHER 1 a red
coloration occurs even in the acid solution.
N— C.NH2

Adenine, C5H5N5, = HC C — NH\ (6-aminopurine), was first found
II II >CH

N— C— N '

by KOSSEL 2 in the pancreas. It occurs in all nucleated cells, but in
greatest quantities in the sperm of the carp and in the thymus. Adenine
has also been found in lucsemic urine (STADTHAGEN 3) . It may be obtained
in large quantities from tea-leaves.

Adenine crystallizes with 3 molecules of water of crystallisation in
long needles which gradually become opaque in the air, but much more
rapidly when warmed. If the crystals are warmed slowly with a quan-
tity of water insufficient for solution, they suddenly become cloudy at
53° C., a characteristic reaction for adenine. It dissolves in 1086 parts
cold water, but is easily soluble in warm. It is insoluble in ether, but
somewhat soluble in hot alcohol and easily so in acids and alkalies. It
is more easily soluble in ammonia solution than guanine, but less soluble
than hypoxanthine. The silver compound of adenine is soluble with
difficulty in warm nitric acid, and deposits on cooling as a crystalline
mixture of adenine silver nitrates. With picric acid adenine forms a
compound, CsHsNs.Ce^CNC^sOH, which is very insoluble but
separates more readily than the hypoxanthine picrate, and can be
used in the quantitative estimation of adenine. We also have an adenine
mercury-picrate. Metaphosphoric acid with adenine gives a precipitate
which dissolves in an excess of the acid if the solution is not too dilute.
Adenine hydrochloride gives with gold chloride a double compound

1 Kossel, Zeitschr. f. physiol. Chem., 12, 252; E. Fischer, 1. c.

2 See Zeitschr. f. physiol. Chem., 10 and 12.
1 Virchow's Arch., 109.

PYRIMIDINE BASES. 193

which consists in part of leaf-shaped aggregations and in part of cubical
or prismatic crystals, often with rounded corners. This compound is
used in the microscopic detection of adenine. With the nitric-acid test
and with WEIDEL'S reaction adenine acts in the same way as hypoxan-
thine. The same is true, for its behavior with hydrochloric acid and
zinc with subsequent addition of alkali.

The procedure for the preparation and detection of the four above-
described purine bases is as follows: Boiling with 0.5-1 vol. per cent
sulphuric acid, saturating with baryta-water, removing the excess of
barium with CCb, precipitating all the bases as silver compounds
from the strongly ammoniacal filtrate and dissolving them in boiling
nitric acid when the xanthine compound remains in solution on cooling
while the combination with the other three bases precipitate. The silver
xanthine may be precipitated from the filtrate by the addition of ammonia
and the xanthine set free by means of sulphureted hydrogen. The
three above-mentioned silver-nitrate compounds are decomposed by sul-
phureted hydrogen and the guanine separated from the adenine and
hypoxanthine by treatment while hot with ammonia, in which the
guanine is soluble with difficulty. When the above filtrate containing the
adenine and hypoxanthine, which has been, if necessary, freed from
ammonia by evaporation, is allowed to cool, the adenine separates,
while the hypoxanthine remains in solution. According to BALKE l —
we can advantageously precipitate the purine bases with copper sulphate
and hydroxylamine. Details for the above methods may be found in
complete hand-books. The same procedures are followed in the quan-
titative estimation of the purine bases in animal organs.2

3. Pyrimidine Bases.
These bodies are closely related to the purines, and pyrimidme,

I I

CH, may be considered as the mother substance thereof.

I! II

N— CH

The pyrimidine bases contained in the nucleic acids are cytosine, uracil
and thymine.

N— CNH2

I II
Cytosine, C4H5NsO, = OC CH (6 amino-2 oxypyrimidine), was first

I!

HN— CH

prepared by KOSSEL and NEUMANN from thymus nucleic acid, and then
by KOSSEL and STEUDEL and others from various nucleic acids. WHEELER

*See footnote 1, p. 190.

2 See Burian and Hall, Zeitschr. f. physiol. Chem., 38; Kossel ibid., 5-8, Bruhns,
ibid., 14; His and Hagen, ibid., 30.

194 THE PROTEIN SUBSTANCES.

and JOHNSON l have also prepared it synthetically. It is transformed
into uracil by the action of nitrous acid.

The free base is soluble with difficulty in water (129 parts) and crystal-
lizes in thin leaves with a mother-of-pearl luster. It is insoluble in ether
and soluble with difficulty in alcohol. The double compound with platinum
chloride, the crystalline picrate, the nitrate, and the picrolonate are
of importance in the detection of cytosine. This base is precipitated
by phosphotungstic acid and by silver nitrate in the presence of an
excess of barium hydroxide, which fact is of importance in the detection
of cytosine (KUTSCHER). The double bismuth-potassium iodide gives
a brick-red precipitate. Cytosine gives the murexid reaction with
chlorine-water and ammonia (see Chapter XIV), and also the reaction
described by WHEELER and JOHNSON under uracil. In regard to
preuaration see KOSSEL and STEUDEL 2 and also KUTSCHER.S
HN— CO

I I
UracU, C4H4N2O2, = OC CH (2, 6-dioxypyrimidine), was first

I II
HN— CH

obtained by ASCOLI and KOSSEL from yeast nucleic acid and later from
various complex nucleic acids, perhaps secondarily from the cytosine as a
cleavage product. The synthetical preparation was first accomplished by
E. FISCHER and RoEDER.4

Uracil crystallizes in needles which cluster in rosettes. On careful
heating it sublimes in part undecomposed, but develops red vapors and
decomposes in part. It is readily soluble in hot water, but less so in cold
water, and nearly insoluble in alcohol and in ether. Uracil is readily
soluble in ammonia. It is precipitated by mercuric nitrate, but not by
phosphotungstic acid. It is precipitated by silver nitrate only on the
careful addition of ammonia or baryta-water. Uracil gives the WEIDEL
reaction and the following reaction described by WHEELER and JOHN-
SON .5 The uracil solution is treated with bromine-water until it is per-
manently cloudy and then treated with baryta-water, when a purple or
violet-colored precipitate appears almost immediately. The coloration

1 Amer. chem. Journ., 29.

2 Zeitschr. f . physiol. Chem., 37 and 38.

3 Ibid., 38. As it is not excluded, but rather probable according to Wheeler, that
besides thymine also other related pyrimidine bases such as isocytosine, 6-amino
pyrimidine and 6-oxpyrimidine can be formed in the hydrolytic cleavage of the nucleic
acids, Wheeler has prepared salts and compounds of these bodies and described them
as a matter of comparison, Journ. of biol. Chem., 3.

4 Ascoli, Zeitschr. f. physiol. Chem., 31; Kossel and Steudel, ibid., 37; Levene,
ibid., 38, 39; Levene and Mandel, ibid., 49; E. Fischer and Roeder, Ber. d. d. chem.
Gesellsch., 34.

6 Journ. of biol. Chem., 3.

THYMINE. 195

varies with the dilution. This reaction which, as remarked above, is also
given by cytosine, depends upon the fact that dibromoxyhydrouracil
is first formed, and from this, by the action of the barium hydroxide,
first isodialuric and then dialuric acid is produced, both of which give
the coloration. In regard to the preparation of uracil see KOSSEL and

STEUDEL.1

HN— CO

I I
Thymine, C5H6N2O2, = OC C.CH3 (5-methyluracil). This body,

HN— CH

which is identical with nucleosin obtained by SCHMIEDEBERG from sal-
mo-nucleic acid, was first prepared by KOSSEL and NEUMANN from
thymus-nucleic acid, and then by other investigators, especially LEVENE
and MANDEL, from other animal nucleic acids. FISCHER and ROEDER and
later GERNGROSS 2 have prepared it synthetically.

Thymine crystallizes in small leaves grouped in stellar or dendriform
clusters or, rarely, in short needles (GULEWITSCH 3) . It deflagrates at
318° C. and melts at about 321° and sublimes. It is soluble with diffi-
culty in cold water, more soluble in hot water, and insoluble in alcohol.
It behaves like uracil toward ammonia or baryta-water and silver nitrate.
Thymine is precipitated by phosphotungstic acid, especially when impure.
Bromine-water is decolorized by thymine, producing bromthymine.
For its detection we make use of the sublimation, the behavior toward
silver nitrate, and its elementary analysis.

MEYERS 4 has prepared compounds of pyrimidine bases with several metals
such as K, Na, Pb, Hg and he considers it incorrect to call the pyrimidine bodies

In regard to the methods of preparation see KOSSEL and NEUMANN
and W. JoNES,5

The purine and pyrimidine bodies stand in close chemical and phys-
iological relation to each other and for this reason the question has
been repeatedly raised whether the pyrimidine bases might not be formed,
at least in part, from the purine bases by the action of acids. Thus
far no conclusive investigations have been made supporting this view,
while on the contrary the investigations of STEUDEL 6 seem to contradict
such a view.

1 Zeitschr. f. physiol. Chem., 37.

2 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 37; Kossel and Neumann, Ber.
d. d. chem. Gesellsch., 26 and 27; Mandel and Levene, Zeitschr. f. physiol. Chem., 46
47, 49, 50; E. Fischer and Roeder, ibid., 34; Gerngross, ibid., 38.

3 Zeitschr. f. physiol. Chem., 27.

4 Journ. of biol. Chem., 7.

5 Kossel and Neumann, 1. c., and W. Jones, Zeitschr. f. physiol. Chem., 29, 461.

6 Zeitschr. f. physiol. Chem., 51 and 53 (against Burian).

CHAPTER III.
THE CARBOHYDRATES.

WE designate by this name bodies which are especially abundant
in the plant kingdom. As the protein bodies form the chief portion
of the solids in animal tissues, so the carbohydrates form the chief por-
tion of the dry substance of the plant structure. They occur in the
animal kingdom only in proportionately small quantities, either free or
in combination with more complex molecules, forming compound pro-
teins. Carbohydrates are of extraordinarily great importance as food
for both man and animals.

The carbohydrates contain only carbon, hydrogen, and oxygen. The
last two elements occur, as a rule, in the same proportion as they do in
water, namely, 2:1, and this is the reason why the name carbohydrates
has been given to them. This name is not quite pertinent, if strictly
considered, because we not only have bodies, such as acetic acid and
lactic acid, which are not carbohydrates and still have their oxygen and
hydrogen in the same proportion as in water, but we also have a sugar
(the methyl pentoses, CeH^Os) which has these two elements in another
proportion. At one time it was thought possible to characterize as
carbohydrates those bodies which contained 6 atoms of carbon, or a
multiple, in the molecule, but this is not' considered tenable at the present
time. We have true carbohydrates containing less than 6, and also those
containing 7, 8, and 9 carbon atoms in the molecule.

The carbohydrates have no properties or characteristics in general
which differentiate them from other bodies; on the contrary, the various
carbohydrates are in many cases very different in their external prop-
erties. Under these circumstances it is very difficult to give a positive
definition for the carbohydrates.

From a chemical standpoint we can say that all carbohydrates are
aldehyde or ketone derivatives of polyhydric alcohols. The simplest
carbohydrates, the simple sugars or monosaccharides, are either alde-
hyde or ketone derivatives of such alcohols, and the more complex
carbohydrates seem to be derived from these by the formation of anhy-
drides. It is a fact that the more complex carbohydrates yield two
or even more molecules of the simple sugars when made to undergo
hydrolytic splitting.

196

MONOSACCHARIDES. 197

Correspondingly the carbohydrates can be divided into three chief
groups, namely, 1. Simple sugars or monosaccharides, 2. Complex sugars
or disaccharides, trisaccharides and crystalline polysaccharides, and
3. Non-crystalline or colloid polysaccharides. Of these groups the mono-
saccharides, disaccharides and colloid polysaccharides are of special
physiological importance.

Our knowledge of the carbohydrates and their structural relation-
ships has been very much extended by the pioneering investigations of
KILLIANI 1 and especially those of E. FiscHER.2

As the carbohydrates occur chiefly in the plant kingdom it is naturally
not the place here to give a complete discussion of the numerous carbo-
hydrates known up to the present time. According to the plan of this
work it is only possible to give a short review of those carbohydrates
which occur in the animal kingdom or are of special importance as food
for man and animals.

1. Monosaccharides.

All varieties of sugars are characterized by the termination " ose,"
to which a root is added signifying their origin or other relations. Accord-
ing to the number of carbon atoms contained in the molecule the mono-
saccharides are divided into, trioses, tetroses, pentoses, hexoses, heptoses}
and so on.

All monosaccharides are either aldehydes or ketones of polyhydric
alcohols. The former are termed aldoses and the latter ketoses. Ordinary
glucose is an aldose, while ordinary fruit sugar (fructose) is a ketose.
The difference may be shown by the structural formulae of these two
varieties of sugar:

Glucose = CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO;
Fructose = CH2(OH).CH(OH).CH(OH).CH(OH).CO.CH2(OH).

A difference is also observed on oxidation. The aldoses can be con-
verted into oxyacids having the same quantity of carbon, while the ketoses
yield acids having less carbon. On mild oxidation the aldoses yield
monobasic oxyacids, and dibasic acids on more energetic oxidation. Thus
ordinary glucose yields gluconic acid in the first case and saccharic
acid in the second.

1 Ber. d. deutsch. chem. Gesellsch., 18, 19, and 20.

2 See E. Fischer's lecture, Synthesen in der Zuckergruppe, Ber. d. deutsch. chem.
Gesellsch., 23, 2114. Excellent works on carbohydrates are Tollen's Kurzes Hand-
buch der Kohlehydrate, Breslau, 2 (1895), and 1, 2. Auflage, 1898, which gives a
complete review of the literature, and E. O. v. Lippmann, Die Chemie der Zucker-
arten, Braunschweig, 1904.

198

THE CARBOHYDKATES.

Gluconicacid = CH2(OH).[CH(OH)]4.COOH;
Saccharic acid = COOH.[CH(OH)]4.COOH.

The monocarboxylic acids are easily transformed into their anhydrides
(lactones), and these latter are of special interest because, as shown by
FISCHER, they can be changed into the corresponding aldehyde, i.e.,
the corresponding aldose, by nascent hydrogen.

The monosaccharides are converted into the corresponding poly-
hydric alcohols by nascent hydrogen. Thus ARABINOSE, which is a
pentose, CsHioOs, is transformed into the pentatomic alcohol, ARABITE,
The three hexoses, GLUCOSE, MANNOSE, and GALACTOSE,
, are transformed into the corresponding three hexites, SORBITE,
MANNITE, and DULCITE, CeHuOe- The ketoses, on the contrary, due
to their constitution, yield a mixture of two alcohols on the same treat-
ment. From d-fructose, for example, we obtain a mixture of d-sorbite
and Z-mannite. On careful oxidation of the polyhydric alcohols the cor-
responding sugar can be prepared.

Numerous isomers occur among the monosaccharides, and especially
in the hexose group. In certain cases, as, for instance, in glucose and
fructose, we are dealing with a different constitution (aldoses and ketoses),
but in most cases we have stereoisomerism due to the presence of asym-
metric carbon atoms.

As the monosaccharides from the trioses upward contain asymmetric
carbon atoms they occur as optically active bodies in an /-, d,- and racemic
form, r or d-l form, which is a mixture or a combination of the I- and
d-form in equal parts. As the number of asymmetric carbon atoms
increases so does the number of possible stereoisomeric forms enlarge,.
As the number according to VAN'T HOFF is 2n, where n represents the num-
ber of asymmetric carbon atoms, then for the aldo-hexose, which con-
tains 4 asymmetric carbon atoms, 24 = 16 stereo-chemically different forms
can exist. In fact, of these, 12 have been prepared and their geometric
structure has been explained and for which FISCHER has given configura-
tion formulae.

As these relations are readily conceived we will, for example, give only the
configuration formulae for the most important pentoses and hexoses occurring
in the animal body.

COH
HOCH

COH
HCOH

HCOH

HOCH

HCOH

HOCH

CH2OH

i-Arabinoae

CH2OH

J-Arabinose

COH

COH

HOCH

HCOH

HCOH

HOCH

HOCH

HCOH

CH2OH

d-Xylose

CH2OH

/-Xylose

SPECIFIC ROTATION.

199

COH

COH

HCOH

HOCH

HCOH

HOCH

HCOH

HOCH

CH2OH

CH2OH

d-Ribose

Z-Ribose

COH
HCOH
HOCH
HOCH
HCOH
CH2OH

d-Galactoae

COH
HOCH
HCOH
HCOH
HOCH
CH2OH

J-Galactose

COH

COH

HCOH

HOCH

HOCH

HCOH

HCOH

HOCH

HCOH

HOCH

CH2OH

d-Glucose

CH2OH

Z-Glucose

CH2OH

CH2OH

CO

CO

HOCH

HCOH

HCOH

HOCH

HCOH

HOCH

CH2OH

CH2OH

d-Fructose

J-Fructose

We designate the optical activity of the carbohydrates with the
letter I- for levogyrate, d- for dextrogyrate, and r- for the racemic.
These are only partly indicative. Thus dextrorotatory glucose is
designated d-glucose, levorotatory Z-glucose, but EMIL FISCHER has
used these signs in another sense. He designates by these signs the
mutual relationship of the various kinds of sugars instead of their
optical activity. For example, he does not designate the levorotatory
fructose Z-fructose, but d-fructose, showing its close relation in stereometric
structure to dextrorotatory d-glucose. This designation is generally
accepted, and the above-mentioned signs only show the optical proper-
ties in certain cases. v

Specific rotation means the rotation in degrees produced by 1 gm. substance
dissolved in 1 cc. liquid placed in a tube 1 dcm. long. The reading is ordinarily
made at 20° C. and with the monochromatic sodium light. The specific rotation
with this light is represented by («)D, and is expressed by the following formula:

(a)D = ±~T, in which a represents the reading of degrees, 1 the length of the

tube in decimeters, and p the weight of substance in 1 cc. of the liquid. Inversely
the per cent P of substance can be calculated, when the specific rotation is known,
lOOa

by the formula P

-, in which s represents the known specific rotation.

In the determination of the change in specific rotation with various concen-
trations we must know also the amount of substance in grams in 1 gram of the
solution (p) and the specific gravity of the solution (d) at 20°. The rotation

is calculated according to the formula (a)D =

A freshly prepared solution of a substance often shows a different rotation
from one that has been allowed, to stand for some time (multirotation). The
correct values which are found on allowing the solution to stand for a sufficiently
long time can be obtained immediately by boiling or on the addition of very
little ammonia.

200 THE CARBOHYDRATES.

In order to explain multirotation HUDSON l believes that the aldoses exist in
two isomeric forms having different rotation, which on being dissolved pass
into each other by means of a reversible reaction. The two forms can be derived
because a lactone-like binding exists between the end carbon atom in the alde-
hyde group and the 7-carbon atom according to the formula

CHoOHCHOH.CH.CHOH.CHOH.C/' . In this way the end carbon becomes

-o- |X°H

asymmetric and according to whether the position of the atoms that are combined
with this carbon atom are:

C H
C or
0 OH

C OH

V

0 H

we obtain the two forms. TANRET 2 has obtained two isomeric forms of glucose,
galactose, arabinose and lactose which pass from one form to the other. The
two forms of glucose correspond according to E. H. ARMSTRONG s to the syntheti-
cally prepared a- and /3-methyl glucosides prepared by E. FISCHER (see pages
61-62).

Like the ordinary aldehydes and ketones, the sugars may be made to
take up hydrocyanic acid. Cyanhydrins are thus formed. These addi-
tion products are of special interest in that they make possible the arti-
ficial preparation of sugars rich in carbon from sugars poor in carbon.

As an example, if we start from glucose we obtain glucocyanhydrin
on the addition of hydrocyanic acid:

CH2.(OH).[CH(OH)]4.COH+HCN = CH2(OH).[CH(OH)]4.CH(OH).CN.

On the saponification of glucocyanhydrin the corresponding oxyacid is
formed.

CH2(OH).[CH(OH)]4.CH(OH).CN+2H20

= CH2(OH).[CH(OH)]4.CH(OH).COOH+NH3.

By the action of nascent hydrogen on the lactone of this acid we obtain
glucoheptose, CyHuOy and according to this principle the construction
of sugars up to nine carbon atoms has been accomplished.

The monosaccharides give the corresponding oximes with hydroxyl-
amine; thus glucose yields glucosoxime, CH2(OH).[CH(OH)14.CH:N.OH.
These compounds are of importance on account of the fact, as found by
WoHL,4 that they are the starting-point in the formation of one class

Uourn. Amer. Chem. Soc., 31,. 61, 955 (1909).

2 Bull. soc. chim., 13, 728 (1895),' 15, 195, 349 (1896).

3 Journ. Chem. Soc., 83, 1305 (1903).

4 Ber. d. d. chem. Gesellsch., 26, p. 730.

DERIVATIVES. 201

of sugars from another class, namely, the preparation of sugars poor in
carbon from those rich in carbon, for example, pentoses from hexoses
(see WOHL).

According to RUFF, by the action of hydrogen peroxide and the cata-
lytic action of ferric salts upon the carbohydrate monocarboxylic acids
the carbon chain can be shortened by the splitting off of the elements
of formic acid, and with the formation of the next lower aldose. NEU-
BERG 1 has accomplished the same result by electrolysis, and by this
method has split glucose step by step into formaldehyde.

By the action of alkalies, even in small amounts, as also of carbonates
and lead hydroxide, a reciprocal transformation of the sugars, such as
d-glucose, d-fructose, and d-mannose, may take place (LOBBY DE BRUYN
and ALBERDA VAN EKENSTEiN2), and from each of these three varieties
of sugar the two others are produced so that after a certain time the
solution contains all three sugars.

The transformation of the different varieties of sugar into each other
also occurs in the animal body. NEUBERG and MAYER 3 have shown by
experiments on rabbits the direct partial transformation of various
mannoses into the corresponding glucoses. Another example is, it
seems, the formation of galactose (or milk sugar) from glucose in the
mammary gland.

By the action of strong alkali the sugars are decomposed with the
formation of lactic acid and many other products.

With ammonia the glucoses may form compounds which have been
considered as osamines by LOBRY DE BRUYN, but to differentiate them
from the true osamines have been called osimines by E. FISCHER.* The
corresponding osaminic acid can be obtained from such an osimine by
the action of ammonia and hydrocyanic acid, and from the hydrochloric-
acid lactone of this acid the osamine is obtained by reduction with sodium
amalgam. In this manner E. FISCHER and LEUCHS artificially prepared
first d-arabinosimine from d-arabinose, then d-glucosaminic acid and
finally from its lactone d-glucosamine, which occurs in the animal
body. In a similar manner they 5 obtained Z-glueosamine from
Z-arabinose.

KNOOP and WIND AT: s 6 have obtained large amounts of methylimida-

1Ruff, Ber. d. d. chem. Gesellsch., 31 and 32; Neuberg, Biochem. Zeitschr., 7.
2Ber. d. d. chem. Gesellsch., 28, 3078; Bull. soc. chim. de Paris (3), 15; Chem.
Centralbl., 1896, 2, and 1897, 2.

3 Zeitschr. f. physiol. Chem., 37.

4 Lobry de Bruyn, Ber. d. d. chem. Gesellsch., 28; E. Fischer, ibid., 35.

5 Ibid., 35, p. 3787, and 36, 24 (1903).

6 Ibid., 38, and Hofmeister's Beitrage, 6.

202 THE CARBOHYDRATES.

CH3

zol, C — NHk , from glucose by the action of ammonium-zinc hydroxide
II >CH

CH— N^

at ordinary temperatures. This formation can be conceived as follows:
First methyl glyoxal is formed from the sugar, and then from this, or
from the sugar, formaldehyde is produced, which reacts with the methyl
glyoxal with the formation of methylimidazole according to the following
equation :

CH3CO NH3 Hx H3C.C— NHX

| ' + + >CH= || >CH+3H2O

COH NH3 W CH— N^

Methylglyoxal Formaldehyde Methylimidazole

A genetic relationship of the carbohydrates to histidine and the purine
bodies is thus made probable by the imidazole formation.

As the sugars are derivatives of polyhydric alcohols, they also form
esters, among which the benzoyl ester is of special interest because it is
used in the detection and isolation of the sugars and also of other car-
bohydrates. The nucleic acids probably also belong to the acid esters
of the sugars, and thus may be considered as complex phosphoric acid
esters, and perhaps the chondroitin sulphuric acid and the glucothionic
acid are sulphuric acid esters. The nature of these two groups of
sulphuric acid esters is not yet thoroughly understood.

The sugars can also combine with other bodies and with each other,
forming ether-like combinations. By the action of hydrochloric acid
as catalyst, as shown by FISCHER and collaborators, the sugars split off
water and unite with other bodies, producing lactone-like combinations,
which have bein called glucosides (see pages 61 and 200). These glucosides,
which are generally compounds with aromatic groups, occur widely dis-
tributed in the vegetable kingdom. The more complex carbohydrates
may be considered, according to FISCHER, as glucosides of the sugars.
Thus maltose, for example, is the glucOside and lactose the galactoside
of glucose. The glucosides can be split into their components by chem-
ical agents, boiling with dilute mineral acids, as well as by the action
of enzymes. The complex sugars hereby yield simple sugars and the other
glucosides yield compounds which belong either to the aromatic or the
aliphatic series besides the sugar. A long-known example of a decom-
position of this kind is the splitting of amygdalin by the enzyme emulsin
(see page 60).

With phenylhydrazine or substituted phenylhydrazines, the sugars
first yield -hydrazones with the elimination of water, and then on the fur-
ther action of hydrazine on warming in an acetic-acid solution we obtain
osazones.

HYDRAZONES AND OSAZONES. 203

The reaction takes place with the aldoses as follows:

(a) CH2(OH).[CH(OH)]3.CH(OH).CH04-H2N.NH.C6H5 =

CH2(OH).[CH(OH)]3.CH(OH)CH:N.NH.C6H6+H20.

Phenylglucosehydrazone

(6) CH2(OH)[CH(OH)]3.CH(OH).CH:N.NH.C6H5+H2N.NH.C6H5 =

CH2(OH).[CH(OH)r3.C.CH:N.NH.C6H6

N.NH.C6H6+HoO+H2.

Phenylglucosazone

and with the keteoses :

CH2(OH)[CH(OH)]3CO.CH2(OH)-fH2N.NH.C6H5 =

CH2(OH)[CH(OH)]3C.CH2OH

N.NH.C6H6+H20,
and CH2(OH)[CH(OH)]3.C.CH2(OH)

CH2(OH)[CH(OH)]3.C.CH:N.NH.C6H6 +H2O+H2.

N.NH.C6H6

The hydrogen is not evolved, but acts on a second molecule of phenylhy-
drazine and splits it into aniline and ammonia:

H2N.H.C6H6+ H2 =H2N.C6H6+NH3.

As seen from the above equations the aldoses and ketoses yield the
same osazones, while the hydrazones are different.

The osazones, which are more important than the hydrazones, are
generally yellow crystalline compounds which differ from each other in
melting-point, solubility, and optical properties, and hence have been of
great importance in the characterization of certain sugars. They have
also become of extraordinarily great interest in the study of the carbo-
hydrates for other reasons. Thus they are a very good means of pre-
cipitating sugars from solution in which they occur mixed with other
bodies, and they are of the greatest importance in the artificial prepara-
tion of sugars. On cleavage, by the brief action of gentle heat and fum-
ing hydrochloric acid (for disaccharides still better with benzaldehyde),1
the osazones yield so-called osones, which on reduction yield aldoses or
more often ketoses. The hydrazones can be much more readily retrans-
formed into the corresponding sugar, especially by decomposition with
benzaldehyde (HERZFELD) or with formaldehyde (RUFF and OLLEN-
DORFF 2), whereby the sugar is replaced by the aldehyde used.

An important property, although not applicable to all sugars, is their
ability to undergo fermentation, especially their ability to undergo
alcoholic fermentation with alcohol-yeast. We must state, however,
that the power of fermentation with pure 3reast has been shown only for

1 E. Fischer and Armstrong, Ber. d. d. chem. Gesellsch., 35.

2 Herzfeld, ibid., 28; Ruff and Ollendorff, ibid., 32.

204 THE CARBOHYDRATES.

the hexose group, and in fact all the hexoses do not ferment, and they
do not all ferment with the same readiness. d-Glucose and d-mannose
ferment readily, but d-galactose only with difficulty. The /-forms of
the above-mentioned sugars do not ferment, and from the racemic forms
of these sugars the optical Z-antipode can be prepared by the fermenta-
tion of the d-sugar. Among the ketoses the d-fructose ferments while
the sorbose does not. Among the sugars containing nine atoms of car-
bon, the nonoses, the mannonose ferments while the glucononose does
not. In regard to the fermentability of the triose, dioxy acetone, see
page 205. The different behavior of the various sugars with yeast stands
in fixed relation to their configuration, and is not only of great importance
for the behavior of the sugar in lower organisms, but also for their behavior
in higher developed organisms. Thus the investigations of NEUBERG
WOHLGEMUTH l upon arabinose and of NEUBERG and MAYER 2 on man-
noses have shown that in rabbits the Z-arabinose and the d-mannose are
much better utilized than d- and r-arabinose or I- and r-mannose. See
also pages 61-62.

In the alcoholic fermentation the sugar is decomposed according
to the general equation C6Hi206 = 2C2H6O+2C02. The exact process
is not clear, and seems to be rather complicated. On page 52 it has
been mentioned that for the action of the fermentation enzymes the
presence of a dialyzable substance present .in the press-juice is neces-
sary (HARDEN and YOUNG 3). On the other hand the fermentation
power of the press-juice can also be considerably increased by the addi-
tion of secondary sodium phosphate. The phosphoric acid in the press-
juice after fermentation is only partly precipitable with magnesia mixture
(HARDEN and YOUNG). The most favorable action of boiled press-
juice is inhibited by lipase (BUCHNER and KLATTE). According to
HARDEN and YOUNG we must consider the action of boiled press-juice
and of phosphate in that first a hexose-phosphoric acid ester is produced
with the simultaneous formation of carbon dioxide and alcohol, according
to the following formula:

The hexose phosphate can then be split into hexose and phosphate
by a special enzyme. The hexose phosphoric acid has been isolated
as a lead salt by YOUNG. Glucose, fructose and mannose produce in
their fermentation the same hexose phosphoric acid. According to

1 Zeitschr. f. physiol. Chem., 35.'
*Ibid., 37.

3 Literature in Harden and Young, Bioch. Zeitschr., 32, 173 (1911) as well as
Buchner and Klatte, ibid., 8, 520 (1908).

ALCOHOLIC FERMENTATION. 205

IWANOFF l the phosphoric acid combination is a triose phosphate which
is fermented, with the formation of carbon dioxide, alcohol and phos-
phoric acid, by the dead and not by the living yeast. On the contrary
LEBEDEW finds the same formula as YOUNG 2 for the phosphoric acid
ester. IWANOFF as well as EULER and their collaborators admit that
the formation of phosphoric acid esters is brought about by a special
enzyme.3 According to IWANOFF and to LEBEDEW the sugar is first
fermented after it has combined with the phosphoric acid. It seems,
according to all evidence, that phosphoric acid esters of carbohydrates
are formed and that these are in some way of importance for the accom-
plishment of the fermentation. It is not probable that in the fermenta-
tion the hexose does not directly break into alcohol and C02. It is
generally admitted that the process takes place through intermediary
steps. Lactic acid is considered as one of these, although in fact, this
acid is not fermented with the formation of alcohol. Recently BUCHNER
and MEISSENHEIMER 4 have proposed dioxyacetone (HOCEb.CO.CH^OH)
as a probable intermediary step. They found that dioxyacetone was
very readily fermented by press-juice in the presence of common salt
and indeed with the formation of alcohol and carbon dioxide. This has
been substantiated by LEBEDEW.S HARDEN and YOUNG disputed the
possibility that dioxyacetone is an intermediary step in the alcoholic
fermentation of sugar because it is more slowly fermented than the
sugars.6

Besides ethyl alcohol and carbon dioxide there are formed in the fer-
mentation of sugar, although in small amounts, several higher alcohols
which form the so-called fusel oil. The most important constituents
of fusel oil are isoamylalcohol, d-amylalcohol, isobutylalcohol and normal
propylalcohol in varying proportions. The formation of fusel oil was
ascribed for a long time to the action of bacteria until F. EHRLICH 7
found that the higher alcohols could be produced from certain amino-
acids by the living activity of yeast. From an amino-acid probably
the corresponding oxyacid is formed first by the splitting off of ammonia,

1 Centralbl. f. Bakfc. 24, 1 (1909).

2Bioch. Zeitschr. 36, 248 (1911).

3Euler and Kullberg, Zeitschr. f. physiol. Chem., 74, 15 (1911); 80, 175 (1912);
Bioch. Zeitschr., 37, 133 (1911).

4 In reference to the intermediary products in alcoholic fermentation see Buchner
and Meissenheimer, Jter. d. d. chem. Gesellsch., 43, 1773 (1910) which also contains
the literature.

5Compt. Rend., 153, 136 (1911).

s Bioch. Zeitschr., 40, 458 (1912).

7 Zeitschr. f. Ver. d. d. Zuckerind, 55, 539 (1905) also Ber. d. d. chem. Gesellsch,
40, 1027, 2538 (1907); Bioch. Zeitschr. 1, 8 (1906); 8, 438 (1908); 18, 391 (1909).

206 THE CARBOHYDRATES.

and then from this by loss of C02 the alcohol is derived. The ammonia
is assimilated by the yeast. If the amino-acid is in the racemic form
then only the one component occurring naturally is transformed into
alcohol while the other remains in great part unchanged. In this manner
leucine is converted into isoamylalcohol according to the following equa-
tion:

/CH3
HOCO.CH(NH2).CH2.CH< +H20 =

Leucine XCH3

/CH3
NH3+C02+HOCH2.CH2.CH<

Isoamylalcohol \QH3

Other examples of the same kind is the formation of d-amylalcohol
from d-isoleucine and of isobutylalcohol from a-amino-valeric acid.
The formation of higher alcohols takes place with yeast poor in nitrogen
and in the presence of large amounts of sugar. In an analogous man-
ner, under the influence of yeast in the presence of sugar and inor-
ganic nutritive salts, from tyrosine tyrosol (p-oxyphenylethyl alcohol)
HO.C6H4.CH2.CH2.OH is derived and from tryptophane we get tryptophol
(/3-indoxy lethyl alcohol) 1 .

C.CH2.CH2OH

NH

Other fermentation processes which are brought about by yeast but without
the presence of sugar have been studied by NEUBERG 2 and his collaborators.
Among these we will mention the decomposition of pyroracemic acid (pyruvic
acid) into carbon dioxide and acetaldehyde :

HO.CO.CO.CH3 =C02+HOC.CH3.

The enzyme active in this fermentation is called carboxylase. If the pyro-
racemic acid exists in the form of an alkali salt then the cleavage takes place ac-
cording to the formula,

2KO.CO.CO.CH3+H20=C02+2HOC.CH3H-K2C03

and alkali carbonate is formed from a neutral salt. In this case the aldehyde
is condensed by the alkali to aldol, the first polymerization product of acetaldehyde.

The previously mentioned (page 41) lactic acid fermentation of
various sugars is caused by the action of different varieties of bacteria.
The equation represents a cleavage of one hexose molecule into two
lactic acid molecules C6H] 2O6 = 2HOCO.CHCOH) .CH3. Nothing positive

1 Ber. d. d. chem. Gesellsch., 44, 139 (1910); 45, 883 (1912).

2 Bioch. Zeitschr., 31, 170 (1910); 32, 323; 36, 60, 68, 76 (1911); 47, 405, 413 (1912).

PENTOSES. 207

is known as to how this cleavage occurs. According to BUCHNER and
MEISSENHEIMER l the fermentation with the enzymes contained in the
bacteria produces chiefly the racemic, inactive form of the acid. This
also occurs as a rule by the action of living bacteria. In reference to the
formation of lactic acid within the organism see Chapter X.

The monosaccharides are colorless and odorless bodies, neutral in
reaction, with a sweet taste, readily soluble in water, generally soluble
with difficulty in absolute alcohol, and insoluble in ether. Some of them
crystallize well in the pure state. They are strong reducing substances.
They reduce metallic silver from ammoniacal silver solutions and they
also reduce other metallic oxides such as copper, bismuth and mercury
oxides, on heating in alkaline solution. This behavior is of great
importance in the detection and quantitative estimation of the sugars.

The simple varieties of sugar occur in part in nature as such, already
formed, which is the case with both of the very important sugars, glucose
and fructose. They also occur in great abundance in nature as more
complex carbohydrates (di- and polysaccharides) ; also as ester-like
combinations with different substances, as so-called glucosides.

Among the groups of monosaccharides known at the present time,
those containing less than five and more than six carbon atoms in the
molecule have no great importance in biochemistry, although they are
of high scientific interest. Of the two groups the hexoses are the more
abundant and are of special interest. The pentoses are becoming of
greater importance, not only for the chemistry of plants, but also for
the chemical processes in the animal body.

Pentoses (C5Hi005).

As a rule the pentoses do not occur as such in nature. They are
obtained from animal tissues, organs and fluids as cleavage products
of the nucleic acids, or nucleoproteins. The pentoses are chiefly obtained
from the plant kingdom, where nucleic acids also occur, by the hydro-
lytic cleavage with dilute mineral acids, of more complex carbohydrates,
the so-called pentosans. The pentosans exist very widely distributed
in the plant kingdom, and are of especially great importance in the build-
ing up of certain plant constituents. Methyl pentosans and methyl
pentoses also occur in the plants, and of these, the methyl pentose, rham-
nose, which occurs in several glucosides, must be specially mentioned.

The pentoses were first found in the animal kingdom by SALKOWSKI
and JASTROWITZ in the urine of a person addicted to the morphine habit,

1 Ann. d. Chem. u. Pharm., 349, 125 (1906).

208 THE CARBOHYDRATES.

and later by SALKOWSKI and others in human urine. Small quantities
of pentoses have been detected by KULZ and VOGEL 1 in the urine of
diabetics, as also in dogs with pancreas diabetes or phlorhizin diabetes.
Pentose has also been found by HAMMARSTEN among the cleavage
products of a nucleoprotein obtained from the pancreas, or from the
corresponding guanylic acid, and seems also, according to the observa-
tions of BLUMENTHAL, to be a constituent of nucleoproteins of various
organs, such as the thymus, thyroid, brain, spleen, and liver. Their
occurrence in the nucleic acids has been previously discussed. In regard
to the quantity of pentoses found in the various organs, we must refer
to the works of GRUND and of BENDIX and EBSTEIN and MANCiNi.2

The pentosans (STONE, SLOWTZOFF) as well as the pentoses are of the
greatest importance as foods for herbivorous animals. In regard to the
value of the pentoses, the researches of SALKOWSKI, CREMER, NEUBERG,
and WoHLGEMUTH3 upon rabbits and hens show that these animals
can utilize the pentoses. The question whether the pentoses are active
as glycogen-formers is still an open one (see Chapter VII). The pen-
toses seem to be absorbed by human beings and in part utilized, but
they pass in part into the urine even when small quantities are taken.4

The natural pentoses are reducing aldoses, and as a rule do not belong
to the sugars fermentable by yeast. Still, the observations of SAL-
KOWSKI, BENDIX, SCHONE and TOLLENS seem to indicate that the pen-
toses are fermentable.5 They are readily decomposed by putrefaction
bacteria. With phenylhydrazine and acetic acid they give crystalline
osazones which are soluble in hot water, and whose melting-points and
optical behavior are important for the detection of the pentoses. On
heating with hydrochloric acid they yield furfurol, but no levulinic acid.
In this reaction furfuran is formed from the pentose molecule, and then

1 Salkowski and Jastrowitz, Centralbl. f. d. med. Wissensch., 1892, 337 and 593;
Salkowski, Berl. klin. Wochenschr., 1895; Bial, Zeitschr. f. klin. Med., 39; Bial and
Blumenthal, Deutsch. med. Wochenschr., 1901, No. 2; Kiilz and Vogel, Zeitschr. f.
Biologie, 32.

2 Hammarsten Zeitschr. f. physiol. Chem., 19; also Salkowski, Berl. klin. Wochen-
schr., 1895; Blumenthal, Zeitschr. f. klin. Med., 34; Grund, Zeitschr. f. physiol. Chem.
35; Bendix and Ebstein, Zeitschr. f. allgemein. Physiol., 2; Mancini, Chem. Centralbl.,
1906.

3 Stone, Amer. Chem. Journ., 14; Slowtzoff, Zeitschr. f. physiol. Chem., 34; Sal-
kowski, ibid., 32; Cremer, Zeitschr. f. Biologie, 29 and 42; Neuberg and Wohlgemuth,
Zeitschr. f. physiol. Chem., 35.

4 See Ebstein, Virchow's Arch., 129; Tollens, Ber. d. deutsch. chem. Gesellsch., 29,
1208; Cremer, 1. c.; Lindemann and May, Deutsch. Arch. f. klin. Med., 56; Salkowski,
Zeitschr. f. physiol. Chem., 30.

5 Salkowski, Zeitschr. f. physiol. Chem., 30; Bendix, see Chem. Centralbl., 1900, 1;
Schone and Tollens, ibid., 1901, 1.

PENTOSE REACTIONS. 209

HC— CH

I! II

from this its aldehyde, the furfurol HC C.CHO. The furfurol pass-

O

ing over, on distilling with hydrochloric acid, can be detected by the aid
of aniline-acetate or xylidine acetate paper, which is colored a beautiful
red by furfurol. In the quantitative estimation we can use the method
suggested by TOLLENS, which consists in converting the furfurol in the
distillate into phloroglucide by means of phloroglucin and weighing (see
TOLLENS and KROBER, GRUND, BENDIX and EBSTEIN), or according to
JOLLES 1 by bisulphite and retitrating with iodine solution. In using
these methods it must be borne in mind that glucuronic-acid compounds
also yield furfurol under the same conditions. The two following pentose
reactions, as suggested by TOLLENS, are especially applicable.

The orcin-hydrochloric acid test. Mix with the solution or the substance
introduced into water an equal volume of concentrated hydrochloric acid, add
some orcin in substance, and heat. In the presence of pentoses the solution
becomes reddish-blue, then bluish-green, and on spectroscopic examination an
absorption-band is observed between C and D. If it is cooled and shaken with
amyl alcohol, a bluish-green solution which shows the same band is obtained.

The phloroglucin-hydrochloric acid test. This test is performed in the
same manner as the above, using phloroglucin instead of orcin. The solution
becomes cherry-red on heating and then becomes cloudy and hence a shaking out
with amyl alcohol is very necessary. The red amyl-alcohol solution shows an
absorption-band between D and E. The orcin test is better for several reasons
than the phloroglucin test (SALKOWSKI and NEUBERG 2). In regard to the use
of these tests in urine examination see Chapter XIV.

Many modifications of these tests have been suggested. BRAT 3 performs
the orcin reaction by the addition of NaCl and heating to only 90-95°. BIAL 4
uses a hydrochloric acid containing ferric chloride for the orcin test and claims
to get a greater delicacy. On too strong and too long heating (l|-2 minutes),
when using this modification, a confusion with sugars of the six carbon series may
occur (BiAL, VAN LEERSUM).S According to R. ADLER and 0. ABLER the phlo-
roglucin and orcin tests can be made with glacial acetic acid and a few drops
hydrochloric acid instead of with the hydrochloric acid alone. These investigators
also use a mixture of equal volumes of aniline and glacial acetic acid as a reagent
for pentoses. On the addition of a little pentose to the boiling mixture a beautiful
red color of furfurol-anilme acetate is obtained. A. NEUMANN 6 performs the
orcin test with glacial acetic acid and adds concentrated sulphuric acid drop
by drop. On following the exact instructions not only do the pentoses give
this reaction, but also glucuronic acid, glucose, and fructose give characteristic

1 Bendix and Ebstein, 1. c., which contains the literature; Jolles, Ber. d. d. chem.
Gesellsch., 39 and Zeitschr. f. anal. Chem., 46.

2 Salkowski, Zeitschr. f. physiol. Chem., 27; Neuberg, ibid., 31.

3 Zeitschr. f. klin. Med., 47.

4 Deutsch. med. Wochenschr., 1902 and 1903, and Zeitschr. f. klin. Med., 50.

5 Bial, Zeitschr. f. klin. Med., 50; van Leersum, Hofmeister's Beitrage, 5.

« 6R. and O. Adler, Pfliiger's Arch., 106; A. Neumann, Berl. klin. Wochenschr.,
1904.

210 THE CARBOHYDRATES.

colored solutions with special absorption-bands which can be made use of in
identifying the various sugars. FR. SACHS has tested BIAL'S test and has given
special precautions to prevent confusion with glucuronic acid. JOLLES l pre-
cipitates (from urine) the pentoses as osazones, distills the precipitate with
hydrochloric acid, and tests the distillate with BIAL'S reagent.

In performing the above two tests for pentose it must be borne in mind that
glucuronic acid gives the same reactions and also that the colors alone are not
sufficient. The spectroscopic examination must therefore never be omitted.
Both tests are to be considered as tests of detection rather than definite pentose
reactions, and therefore for a positive detection of pentoses we must prepare also
the osazones or other compounds.

Arabinoses. The pentose isolated by NEUBERG from human urine
is r-arabinose. It can be isolated from the urine as the diphenylhydra-
zone, from which the arabinose can be separated by splitting with for-
maldehyde. The inactive r-arabinose seems to be the pentose regularly
occurring in pentosuria and thus far, in only one case, has Z-arabinose
been found. /-Arabinose is said to pass into the urine after partaking of
certain fruits, such as plums, in large amounts (C. BARSZCZEWSKI 2) .

The r-arabinose is crystalline, has a sweetish taste, and melts at
163-164° C. Its diphenylhydrazone, which, according to NEUBERG
and WoHLGEMUTH,3 can be used in its quantitative estimation, melts
at 206° C., is insoluble in cold water and alcohol, but readily soluble
in pyridine. The osazone melts at 166-168° C.

The dextrorotatory Z-arabinose is obtained by boiling gum arabic or
cherry gum with dilute sulphuric acid. The d-arabinose has been pre-
pared synthetically. The phenylosazone of Z-arabinose melts at 160°.
The Z-arabinose which crystallizes in plates or prisms melts at about
164°. The specific rotation is (a)D = +104.5°.

Xyloses. The Z-xylose occurs extensively in the plant kingdom and
is prepared from wood-gum by the action of dilute acid. Xylose is
crystalline, melts at 150-153° C., dissolves very readily in water but
with difficulty in alcohol, is faintly dextrorotatory, (a)D= +18.1°, and
gives a phenylosazone which melts at 155-158° C., and according to
TOLLENS and MUTHER a diphenylhydrazone which melts at 107-108°.
According to BERTRAND xylose can be transformed into xylonic acid,
CH2(OH)[CH(OH)]3COOH, by bromine-water and the brom-cadmium
compound or the brucine salt (NEUBERG) of this acid is well suited for
the detection and isolation of Z-xylose. On oxidation with nitric acid
the optically inactive trioxyglutaric acid, with a melting-point of 152° C.
is obtained.

1 Fr. Sachs, Biochem. Zeitschr., 1 and 2; Jolles, ibid., 2, Centralbl. f. inn. Med.,
1907, and Zeitschr. f. anal. Chem., 46.

2 Neuberg, Ber. d. d. chem. Gesellsch., 33; Barszczewski, Maly's Jahrsb., 27, 733.

3 Zeitschr. f. physiol. Chen?., 35.

HEXOSES. 211

According to NEUBERG and to REWALDJ the pentose obtained from a
pancreas nucleoprotein and the pentose isolated by NEUBERG and BRAHN
from inosinic acid is identical with Z-xylose.

Ribose. This pentose has been prepared synthetically by E. FISCHER.
The phenylhydrazone melts at 154-155° C, the p-bromphenylhydrazone
at 164-165° C. The osazone is identical with arabinosazone. On oxida-
tion it yields an optically inactive trioxyglutaric acid, which melts at 170-
171° C. d-ribose is, according to LEVENE and JACOBS, the pentose of
inosinic acid, guanylic acid and yeast nucleic acid. According to these
workers the pentose exists in these nucleic acids in a glucoside-like com-
bination with the purine bases, as so-called nucleosides. It must be
remarked that NEUBERG adheres to his claim that Z-xylose exists at
least in the pancreas.2

Hexoses (C6Hi2O6).

The most important and best-known simple sugars belong to this
group, and most of the other bodies which have been considered as car-
bohydrates in the past are anhydrides of this group. Certain hexoses,
such as glucose and fructose, either occur in nature already formed
or are produced by the hydrolytic splitting of other more complicated
carbohydrates or glucosides. Others, such as mannose or galactose,
are formed by the hydrolytic cleavage of other natural products, while
some, on the contrary, such as gulose, talose, and others, are obtained
only by artificial means.

All hexoses, as also their anhydrides, yield levulinic acid, CsHsOs,
besides formic acid and humus substances on boiling with dilute min-
eral acids. Oxymethyl furfurol, CeHeOs, occurs here as an intermediary
step and this then quantitatively decomposes into levulinic acid and
formic acid.3 Some of the hexoses, as above stated, are fermentable
with yeast.

Some hexoses are aldoses, while others are ketoses. Belonging to the
first group we have MANNOSE, GLUCOSE, and GALACTOSE, and to the other
FRUCTOSE, and also SORBINOSE.

The most important syntheses of the carbohydrates have been made
by E. FISCHER and his pupils, chiefly within the members of the hexose
group. " A short summary of the syntheses of hexoses will be given.

' 1Tollens and Miither, Ber. d. d. chem. Gesellsch., 37; Bertrand, Bull. soc. chim.
(3), 5; Neuberg, Ber. d. d. chem. Gesellsch., 35; Neuberg and Brahn, Biochem. Zeitchr.,
5; Rewald, Ber. d. d. chem. Gesellsch., 42 (1909).

2 E. Fischer, Ber. d. d. chem. Gesellsch, 24, Levene and Jacobs, ibid, 42, 2102, 2469
2474, 3247 (1909); 43, 3147 (1910); Neuberg, ibid., 42, 2806 (1909); 43, 3501 (1910).

3Kiermeyer, Chem. Zeitung., 1895; v. Ekenstein and Blanksma, Ber. d. d. chem.
Gesellsch., 43, 2355 (1910).

212 THE CARBOHYDRATES.

The first artificial preparation of a sugar was made by BUTLEROW. On
treating trioxymethylene, a polymer of formaldehyde, with lime-water he obtained
a faintly sweetish syrup called methylenitan. LOEW l later obtained a mixture
of serveral sugars, from which he isolated a fermentable sugar, called methose,
by condensation of formaldehyde in the presence of bases. The most important
and comprehensive syntheses of sugar have been performed by E. FISCHER 2

The starting-point of these syntheses is ot-acrose, which occurs as a condensa-
tion product of formaldehyde. The name a-acrose has been given to this body
because it is obtained from acrolein bromide by the action of bases (FISCHER).
It is also obtained admixed with (3-acrose on the oxidation of glycerin with
bromine in the presence of sodium carbonate and treating the resulting mixture
with alkali. On the oxidation with bromine a mixture of glvceric aldehyde,
CH2OH.CH(OH).CHO, and dioxyacetone, CH2(OH).CO.CH2OH, is obtained.
These two bodies may be considered as true sugars, namely, glyceroses or trioses.
It seems as if a condensation to hexoses takes place on treatment with alkalies.

a-acrose may be isolated from the above mixture and obtained pure by first
converting it into osazone and then retransforming this into the sugar, a-acrose
seems to be identical with r-fructose. With yeast one-half, the levogyrate
d-fructose ferments, while the dextrogyrate /-fructose remains. The r- and
/-fructose may be prepared in this way.

On the reduction of a-acrose we obtain a-acrite, which is identical with r-
mannite. On oxidation of r-mannite we obtain r-mannose, from which only
/-mannose remains on fermentation. On further oxidation of r-mannose it
yields r-mannonic acid. The two active mannonic acids may be separated from
each other by the fractional crystallization of their strychnine or morphine salts.
The two corresponding mannoses may be obtained from these two acids, d- and
/-mannonic acids, by reduction.

rf-fructose can be obtained from d-mannose with the osazone as an inter-
mediary step and it remains now to speak of the formation of glucoses. The
d- and /-mannonic acids are partly converted into d- and /-gluconic acids on heat-
ing with quinoline, and d- or /-glucose is obtained on the reduction of these acids ;
/-glucose is best prepared from /-arabinose by means of the cyanhydrin reaction,
using /-gluconic acid as the intermediate step. The combination of /- and d-
gluconic acids, forming r-gluconic acid, yields r-glucose on reduction.

The artificial preparation of sugars by means of the condensation of formalde-
hyde has received special interest because, according to BAEYER'S assimilation
hypothesis, in plants formaldehyde is first formed by the reduction of carbon
dioxide, and the sugars are produced by the condensation of this formaldehyde.
BOKORNY 3 has shown, by special experiments on algae Spirogyra, that formalde-
hyde sodium sulphite was split by the living algae cells. The formaldehyde set
free is immediately condensed to carbohydrate and precipitated as starch.4

Among the hexoses known at the present time only glucose, fructose,
and galactose are really of physiological-chemical interest; therefore of
the other hexoses only mannose will be incidentally mentioned.

d-Glucose (grape sugar) also called dextrose and diabetic sugar —
occurs abundantly in the grape, and also, often accompanied with fructose

1 Butlerow, Ann. d. Chem. u. Pharm., 102; Compt. rend., 53; O. Loew, Journ. f.
prakt. Chem. (N. F.), 33, and Ber. d. deutsch. chem. Gesellsch., 20, 21, 22.

2 Ber. d. d. chem. Gesellsch., 21, and 1. c., p. 197.

3 Biolog. Centralbl. 12, pp. 321 and 481.

4 In regard to the syntheses of sugar see also W. Lob and Pulvermacher Bioch.
Zeitschr, 23, 10 (1909), 26, 231 (1910).

GLUCOSE. 213

(levulose), in honey, sweet fruits, seeds, roots, etc. It occurs in the
human and animal intestinal tract during digestion, also in small quan-
tities in the blood and lymph, and as traces in other animal fluids and
tissues. It occurs only as traces in urine under normal conditions,
while in diabetes the quantity is very large. It is formed in the hydro-
lytic cleavage of starch, dextrin, and other compound carbohydrates,
as also in the splitting of glucosides. The question whether glucose
can be formed in the body from proteins or from fats is disputed and will
be discussed in a following chapter (VII).

Properties of Glucose. Glucose crystallizes sometimes with 1 mole-
cule of water of crystallization in warty masses consisting of small leaves
or plates, and sometimes when free from water in fine needles or prisms.
The sugar containing water of crystallization melts even below 100° C.
and loses its water of crystallization at 110° C. The anhydrous sugar
melts at 146° C., and is converted into glucosan, CeHioOs, at 170° C.
with the elimination of water. On strongly heating it is converted into
caramel and then decomposes.

Glucose is readily soluble in water. This solution, which is not as
sweet as a cane-sugar solution of the same strength, is dextrogyrate and
shows strong birotation. The specific rotation is dependent upon the
concentration of the solution, as it increases with an increase in the con-
centration. A 10 per cent solution of anhydrous glucose can be taken as
-J-52.50 at 20° C.1 Glucose dissolves sparingly in cold, but more freely
in boiling alcohol. One hundred parts alcohol of sp. gr. 0.837 dissolves
1.95 parts anhydrous glucose at 17.5° C. and 27.7 parts at the boiling
temperature (ANTHON2). Glucose is insoluble in ether.

If an alcoholic caustic-potash solution is added to an alcoholic solu-
tion of glucose, an amorphous precipitate of insoluble sugar-potash
compound is formed. On warming this compound it decomposes easily
with the formation of a yellow or brownish color, which is the basis of
MOORE'S test. Glucose also forms compounds with lime and baryta.

MOORE'S Test. If a glucose solution is treated with about one
quarter of its volume of caustic potash or soda and warmed, the solution
becomes first yellow, then orange, yellowish-brown, and lastly dark
brown. It has at the same time a faint odor of caramel, and this odor
is more pronounced on acidifiying.3

Glucose forms several crystallizable combinations with NaCl of
which the easiest to obtain is (CeH^Oe^.NaCl+H^O, which forms

1 For further information see Tollens' Handbuch der Kohlehydrate, 2. Aufl., 44.

2 Cited from Tollens' Handbuch.

3 In regard to the products formed in this reaction, see Framm, Pfliiger's Arch., 64;
Neff, Annal. d. Chem. u. Pharm., 357; Buchner and Meisenheimer, Ber. d. d. chem.
Gesellsch., 39; Meisenheimer, ibid., 41.

214 THE CARBOHYDRATES.

large colorless six-sided double pyramids or rhomboids with 13.52 per
cent NaCl.

Glucose in neutral or very faintly acid (organic acid) solution under-
goes alcoholic fermentation with beer-yeast : CeH^Oe = 2C2H5.OH+2CO2.
In the presence of acid milk or cheese the glucose undergoes lactic-acid
fermentation, especially in the presence of a base such as ZnO or CaCOs.
The lactic acid may then further undergo butyric-acid fermentation:

Glucose reduces several metallic oxides, such as copper, bismuth,
and mercuric oxide, in alkaline solutions, and the most important
reactions for sugar are based on this fact.1

TROMMER'S test is based on the property that glucose possesses of
reducing cupric hydroxide in alkaline solution into cuprous oxide. Treat
the glucose solution with about -J- 1 vol. caustic soda and then carefully
add a dilute copper-sulphate solution. The cupric hydroxide is thereby
dissolved, forming a beautiful blue solution, and the addition of copper
sulphate is continued until a very small amount of hydroxide remains
undissolved in the liquid. This is now warmed, and a yellow hydrated
suboxide or red suboxide separates even below the boiling temperature.
If too little copper salt has been added, the test will be yellowish-brown
in color, as in MOORE'S test; but if an excess of copper salt has been added,
the excess of hydroxide is converted on boiling into a dark-brown hydrate
which interferes with the test. To prevent these difficulties the so-
called FEHLING'S solution may be employed. This solution is obtained
by mixing just before use equal volumes of an alkaline solution of Rochelle
salt and a copper-sulphate solution (173 grams Rochelle salt and about
50-60 grams NaOH per liter and 34.65 grams crystalline copper sulphate
per liter) . This solution is not reduced or noticeably changed by boiling.
The tartrate holds the excess of cupric hydroxide in solution, and an excess
of the reagent does not interfere in the performance of the test. In
the presence of sugar this solution is reduced.

According to BENEDICT 2 this test is more delicate if sodium carbonate is
used instead of sodium hydroxide in the preparation of FEHLING'S solution.

BOTTGER-ALMEN'S test is based on the property glucose possesses
of reducing bismuth oxide in alkaline solution. The reagent best adapted
for this purpose is obtained, according to NYLANDER'SS modification of
ALMEN'S original test, by dissolving 4 grams of Rochelle salt in 100 parts
of 10 per cent caustic-soda solution and adding 2 grams of bismuth
subnitrate and digesting on the water-bath until as much of the bismuth

1 In regard to the products produced see Neff, Annal. d. Chem. u. Pharm., 357.

2 Journ. of biol. Chem., 3.

3 Zeitschr. f. physiol. Chem., 8.

GLUCOSE. 215

salt is dissolved as possible. If a glucose solution is treated with about
xV vol., or with a larger quantity of the solution when large quantities
of sugar are present, and boiled for a few minutes, the solution becomes
first yellow, then yellowish-brown, and finally nearly black, and after a
time a black deposit of bismuth (?) settles.

The property that glucose has of reducing an alkaline solution of
mercury on boiling is the basis of KNAPP'S reaction with alkaline mercuric
cyanide, and of SACHSSE'S reaction with an alkaline potassium-mercuric
iodide solution.

On heating with PHENYLHYDRAZINE ACETATE a glucose solution
gives a precipitate consisting of fine yellow crystalline needles which are
almost insoluble in water, but soluble in boiling alcohol, and which separate
again, on treating the alcoholic solution with water. The crystalline
precipitate consists of phenylglucosazone (see page 203). This com-
pound melts when pure at 205° C. It must be borne in mind that
the melting-point of this and other osazones is somewhat variable, depend-
ing upon the rapidity of the heating, the diameter of the tube and the
thickness of the sides of the tube.1 The osazone dissolves readily in
pyridine (0.25 gram in 1 gram), and precipitates again from this solu-
tion as crystals on the addition of benzene, ligroin, or ether. According
to NEUBERG 2 this behavior can be used in the purification of the osazone.
The diphenylhydrazone and the methyl phenylhydrazone are also of
interest.

Glucose is not precipitated by a lead-acetate solution, but is almost
completely precipitated by a solution of ammoniacal basic lead acetate.
On warming, the precipitate becomes flesh-color or rose-red (RUBNER'S
reaction 3) .

If a watery solution of glucose is treated with benzoylchloride and
an excess of caustic soda, and shaken until the odor of benzoylchloride
has disappeared, a precipitate of benzoic-acid ester of glucose will be
produced which is insoluble in water or alkali (BAUMANN4).

If J-l cc. of a dilute watery solution of glucose is treated with a few
drops of a 10 per cent alcoholic solution (free from acetone) of a-naphthol,
on the slow addition of 1-2 cc. of concentrated sulphuric acid a beautiful
reddish-violet ring forms at the juncture of the liquids, or on shaking, the
entire mixture becomes a beautiful reddish-violet color (MOLISCH 5) .

1 See E. Fischer, Ber. d. d. chem. Gesellsch., 41.

2 Ber. d. d. chim. Gesellsch., 32, 3384.

3 Zeitschr. f . Biologic, 20.

4 Ber. d. deutsch. chem. Gesellsch., 19; also Kueny, Zeitschr. f. physiol. Chem., 14,
and Skraup, Wien. Sitzungsber., 98 (1888).

6 Molisch, Monatshefte f. Chem., 7, and Centralbl. f. d. med. Wissensch., 1887,
pp. 34 and 49.

216 THE CARBOHYDRATES.

This reaction depends, according to VILLE and DERRIEN, as well as to
v. EKENSTEIN and BLANKSMA 1 upon the formation of oxymethylfurfurol
which reacts with the a-naphthol. As oxymethylfurfurol is formed from
all hexoses, hence MOLISCH'S reaction is a general reaction for hexoses.

DIAZOBENZENESULPHONIC ACID gives with a glucose solution made alkaline
with a fixed alkali a red color, which after 10-15 minutes gradually changes to
violet. ORTHONITROPHENYLPROPIOLIC ACID yields indigo when boiled with a
small quantity of glucose and sodium carbonate, and this is converted into
indigo-white by an excess of sugar. An alkaline solution of glucose is colored
deep red on being warmed with a dilute solution of PICRIC ACID. The behavior
of glucose toward certain pentose reactions has been given on page 209.

A more complete description as to the performance of these several
tests will be given in detail in a subsequent chapter (on the urine).

Glucose is prepared, pure, by inverting cane-sugar by the follow-
ing simple method of SOXHLET and TOLLENS, which is a modification of
SCHWARZ'S 2 method :

Treat 12 liters 90 per cent alcohol with 480 cc. fuming hydrochloric
acid and warm to 45-50° C.; gradually add 4 kilos of powdered cane-
sugar, and allow to cool after two hours, when all the sugar will have
dissolved and been inverted. To incite crystallization, some crystals of
anhydrous glucose are added, and after several days the crystals are
sucked dry by the air-pump, washed with dilute alcohol to remove
hydrochloric acid, and crystallized from alcohol or methyl alcohol.
According to TOLLENS it is best to dissolve the sugar in one-half its
weight of water on the water-bath and then add double this volume of
90-95 per cent alcohol.

In detecting glucose in animal fluids or extracts of tissues we may
make use of the above-mentioned reduction tests, the optical deter-
mination, fermentation, and phenylhydrazine tests. For the quantitative
estimation the reader is referred to the chapter on the urine. Those
liquids containing proteins must first have these removed by coagulation
with heat and addition of acetic acid, or by precipitation with alcohol
or metallic salts, before testing for glucose. In regard to the difficulties
of operating with blood and serous fluids we refer the student to larger
works.

Mannoses. d-Mannose, also called seminose, is obtained with d-fructose on
the careful oxidation of rf-mannite. It is also obtained on the hydrolysis of
natural carbohydrates, such as salep slime and reserve cellulose (especially
from the shavings of the ivory-nut). It is dextrorotatory, readily ferments
with beer-yeast, gives a hydrazone not readily soluble in water, and an osazone
which is identical with that from d-glucose.

d-Galactose (not to be mistaken for lactose or milk-sugar) is obtained on
the hydrolytic cleavage of milk-sugar, and by the hydrolysis of many other

1 Bull. soc. chim. (4), 5, 895 (1909); Ber. d. d. chem. Gesellsch., 43, 2358 (1910).

2 Tollens, Handbuch der Kohlehydrate, 2. Aufl. I, 39.

FRUCTOSE. 217

carbohydrates, especially varieties of gums and mucilaginous bodies.
It is also obtained on heating cerebrin, a nitrogenized glucoside prepared
from the brain, with dilute mineral acids.

It crystallizes in needles or leaves which melt at 168° C. It is some-
what less soluble in water than glucose. It is dextrogjo-ate, and according
to NEUBERG1 has a rotation (a)D=+81°. With ordinary yeast galac-
tose is slowly, but nevertheless completely, fermented. It is fermented
by a great variety of yeasts (E. FISCHER and THIERFELDER), but not by
Saccharomyces apiculatus,2 which is of importance in physiological-
chemical investigations. Galactose reduces FEHLING'S solution to a
less extent than glucose, and 10 cc. of this solution are reduced, accord-
ing to SOXHLET, by 0.0511 gram galactose in 1 per cent solution. Its
phenylosazone melts according to NEUBERG at 196-197° C., and is soluble
with difficulty in hot water, but with relative ease in hot alcohol. Its
solution in glacial acetic acid is optically inactive. In the test with
hydrochloric acid and phloroglucin galactose gives a color similar to that
of the pentoses, but the solution does not give the absorption spectrum.
On oxidation it first yields galactonic acid and then mucic acid, and
these serve in the detection of galactose.

d-Fructose (levulose) also fruit-sugar, occurs, as above stated, mixed
with glucose, extensively distributed in the vegetable kingdom and
also irr honey. It is formed in the hydrolytic cleavage of cane-sugar
and several other carbohydrates, but it is very readily obtained by the
hydrolytic splitting of inulin. In extraordinary cases of diabetes mellitus
we find fructose in the urine. NEUBERG and STRAUSS 3 have detected
fructose with positiveness in human blood-serum, and exudates in cer-
tain cases.

Fructose crystallizes with comparative difficulty in coarse crusts
or warts or in fine needles. C. MORNER* has obtained crystals 2-3 mm.
long which belonged to the rhombic system, and neither melted nor lost
in weight on heating to 100° C. The melting-point is 110° C. Fructose
is readily soluble in water, but almost insoluble in cold absolute alcohol,
though rather readily in boiling alcohol. Its aqueous solution is levogy-
rate. C. MORNER found the rotation for a 10 and 20 per cent solution
was (a)D=— 93° and —94.1° respectively. Fructose ferments with
yeast, and gives the same reduction tests as glucose, and also the same
osazone. It gives a compound with lime which is less soluble than the
corresponding glucose compound. Fructose is not precipitated by
sugar of lead or basic lead acetate.

1 See C. Oppenheimer, Handb. d. Biochem. 1, p. 197.

2 See F. Voit, Zeitschr. f. Biol., 28 and 29.

3 Zeitschr. f. physiol. Chem., 36, which also contains the older literature.

4 Svensk. Farmac. Tidskr, No. 6, 1907. See also Maly's Jahresb., 37, p. 95.

218 THE CARBOHYDRATES.

Fructose does not reduce copper to the same extent as glucose.
Under similar conditions the reduction relationship is 100 : 92.08.

In detecting fructose and those varieties of sugar which yield fructose on
cleavage we make use of the following reaction, suggested by SELIWANOFF which
consists in heating with hydrochloric acid and resorcinol. This depends upon
the formation of oxymethylfurfurol and is therefore obtained by all hexoses.
As the ketoses give about 20 per cent oxymethylfurfurol and the aldoses only
1 per cent the reaction is more readily obtained with the ketohexoses than with
the aldohexoses (y. EKENSTEIN and BLANKSMA, page 216). To a few cubic
centimeters of fuming hydrochloric acid add an equal volume of water and a small
quantity of the sugar solution or of the solid substance and a few crystals of
resorcinol, and apply heat. The liquid becomes a beautiful red, and gradually
& substance precipitates which is red in color and soluble in alcohol. According
to OFNER x the mixture must not contain more than 12 per cent HC1, and the
boiling must not be continued longer than twenty seconds, if it is boiled for
a longer time and with more hydrochloric acid this reaction is also given with
the aldoses. R. and 0. ADLER 2 perform the test with glacial acetic acid and a
drop of hydrochloric acid and some resorcinol, in which case a reaction with
aldoses is not obtained. SELIWANOFF'S reaction, according to ROSIN, 3 may
be made more delicate by a combination with the spectroscopic examination.
In regard to its use in urine examinations see Chapter XIV.

The naphtho-resorcinol reaction as suggested by B. TOLLENS and RORIVE 4
can be carried out as follows: A few particles of the sugar and about the same
quantity of naphthoresorcinol are treated with about 10 cc. of a mixture of equal
volumes of water and concentrated hydrochloric acid of sp. gr. 1.19. This is
slowly heated to boiling over a low flame, and is continued for 1-3 minutes.
The fluid becomes more purple or violet than with SELIWANOFF'S resorcin test.
The spectroscopic examination shows a faint band in the green.

According to NEUBERG,6 methylphenlhydrazine is an excellent substance
to use for the separation and detection of fructose, as it gives a characteristic
fructose methylphenylosazone. This osazone when recrystallized from alcohol
melts at 153°. It shows a dextrorotation of 1° 40' when 0.2 gram of the osazone
is dissolved in 4 cc. pyridine and 6 cc. absolute alcohol.

OFNER has made objections to the use of methylphenjdhydrazine in the detec-
tion of fructose. He has obtained the osazone from glucose and methylphenylhy-
drazine, although the osazone is formed much more quickly with fructose ^than
with glucose. Only when the separation of the osazone crystals with methyl-
phenylhydrazine after the addition of acetic acid takes place within five hours
at ordinary temperatures is the presence of fructose positively proven (OFNER 6).

The use of secondary asymmetric hydrazines as a general reagent for ketoses
and as a means of separation from aldoses is objected to by OFNER.

d-Sorbinose (sorbin) is a ketose obtained from the juice of the berry of the
mountain ash under certain conditions. It is crystalline and levogyrate, and
is converted into d-sorbite by reduction.

1 Monatshefte f . Chem., 25.

2 See footnote 6, p. 209.

3 Ber. d. d. chem. Gesellsch., 38.

4 Ibid., 41, p. 1783 and Tollens, ibid., 41, p. 1788. See also Mandel and Neuberg,
Biochem. Zeitschr., 13.

5 Ibid., 35; also Neuberg and Strauss, ibid., 36.

6 Ibid., 37, and Zeitschr. f. physiol. Chem., 45.

GLUCOSAMINE. 219

Appendix to the M on osaccha rides.
a. Ammo-sugars.

The most important amino-sugar is the already mentioned glucosamine.

CH2OH

d-Glucosamine (chitosamine), CeHiaNOo, = A^' TTT , whose synthet-

. JN ±±2

COH

ical preparation has been given on page 201 was first prepared by
LEDDERHOSE 1 from chitin by the action of concentrated hydrochloric
acid. Recently it has been obtained as a cleavage product of several
mucin substances and proteins (see pages 84 and 168). Glucosamine is,
as E. FISCHER and LEUcns2 have shown, a derivative of glucose or of
d-mannose (probably glucose), and is an a-amino-sugar.

The free base, which can crystallize in needles, is readily soluble in
water giving an alkaline reaction, and quickly decomposes. The charac-
teristic hydrochloride forms colorless crystals which are stable in the air
and readily soluble in water, soluble with difficulty in alcohol, and insoluble
in ether. The solution is dextrorotatory, (<*)D = +70.15° to 74.64°, at vari-
ous concentrations.3 Glucosamine has a reducing action similar to that of
glucose, and gives the same osazone, but is not fermentable. With
benzoyl-chloride and caustic soda it gives a crystalline ester. In alkaline
solution it gives with phenylisocyanate a compound which can be con-
verted into its anhydride by acetic acid, and is used in the separation and
detection of glucosamine (STEUDEL).4 On oxidation with nitric acid it
yields norisosaccharic acid, whose lead salt can be separated, and whose
salts with cinchonine or quinine are soluble with difficulty in water and can
also be used very successfully in the detection of glucosamine (NEUBERG and
WOLFF 5). On oxidation with bromine, chitaminic acid (d-glucosaminic
acid) is produced, and this is converted into chitaric acid, CeHioOe,
by nitrous acid. On treatment with nitrous acid glucosamine yields
a non-fermentable sugar called chitose.

EHRLICH 6 has suggested a test which does not respond with the free glucos-
amine, but with the mucins and other protein bodies containing an acetylated
glucosamine. It consists in warming the substance, which has been previously

1 Zeitschr. f. physiol. Chem., 2 and 4.

2 Ber. d. d. chem. Gesellsch., 36.

3 See Hoppe-Seyler-Thierfelder's Handbuch, 8, Aufl.; Sundvik, Zeitschr. f. physiol
Chem., 34.

4 Zeitschr. f . physiol. Chem., 34.

* Ber. d. d. chem. Gesellsch., 34.

* Mediz. Woche, 1901, No. 15; see Langstein, Ergebnisse der Physiol., I, Abt. 1, 88.

220 THE CARBOHYDRATES.

treated with alkali, with a hydrochloric-acid solution of dimethylaminobenzalde-
hyde, when a beautiful red color is obtained.

Glucosamine is best prepared from decalcified lobster-shells by treat-
ing with hot concentrated hydrochloric acid.1 In regard to its prepara-
tion from protein substances we must refer to the works cited on page
84, footnote 5.

Albamine (diglucosamine), (CeHnCXN^+HaO, is the name given by S. FRAN-
KEL 2 to a body which he isolated from the products of the hydrolysis of ovalbumin
with baryta, as well as in its digestion. Albamine is amorphous, dextrogyrate,
and reduces after boiling with acids. As hydrolytic cleavage product it yields
d-glucosamine.

Galactosamine is claimed to have been found by SCHULZ and DITTHORN in
a glycoprotein of the spawn of the frog. This claim is not generally accepted.
v. EKENSTEIN and BLANKSMA 3 obtained galactose on the hydrolysis of the slimy
envelope of frog eggs.

According to OFFER,4 pentosamine occurs in the liver of the horse. Accord-
ing to OFFER, the pentose derivative, which he calls dipentosamine (C5H703.NH2)2+
H20 and a second, perhaps a diacetyl-pentosamine 2(CH3CO)CioHi8N207 (?),
also occur in the liver. The first gives pentose reactions and reduces FEHLING'S
solution after boiling with acid. The only amino-sugar positively detected in
the annual organs is glucosamine.

The amino-sugars, as intermediary bodies between the carbohydrates
and oxyamino-acids, are of great physiological interest, and this interest
has become still more important since NEUBERG was first able to pre-
pare the corresponding amino-aldehyde from glycocoll and then also
from other amino-acids. From the ethyl ester of glycocoll in acid solu-
tion NEUBERG 5 obtained the amino-acetaldehyde. NH2.CH2.CHO, by
treatment with sodium amalgam. This aldehyde is very unstable and
has a tendency to condensation with ring formation, and NEUBERG
obtained therefrom by oxidation with corrosive sublimate and caustic
soda, pyrazine according to the equation:

.LI

i

NH2

N
H2+CHO ^\

| HC CH

CHO CH2+0= || +3H20

| HC CH

NH2 V/

N

1 See Hoppe-Seyler-Thierfelder's Handbuch, 8. Aufl.

2 Monatsh. f. Chem., 19.

3 Schulz and Ditthorn, Zeitschr. f. physiol. Chem., 29; v. Ekeastein and Blanksma,
Chem. Centralbl., 1907, 2, p. 1001.

4 Hofmeister's Beitrage, 8.

6 Ber. d. d. chem. Gesellsch., 41.

.GLUCURONIC ACIDS. 221

On account of this tendency to ring-formation the amino-acetalde-
hyde as well as the amino-aldehydes as a group, stand, according to NEU-
BERG, in close relationship to many ring systems, such as imidazole,
piperazine, pyrazine, pyridine and others, and also to the alkaloids.

The amino-sugars, like the amino-aldehydes, can also unite, form-
ing ring compounds, and this seems to be the case on the decomposi-
tion of free glucosamine in aqueous solution, which occurs with access
of air (LOBRY DE BRUYN). As found by STOLTE 1 2,5-ditetraoxybutyl
pyrazine (= fructosazine) is hereby produced according to the following
equation :

NH2 N

O4H9C4.CH+CHO O4H9C4.C CH

I +0= | || +3H20

HO CH.C4H904 HC C.C4H904

.

I
C

NH2 N

Fructosazine

The 2,5-ditetraoxybutyl pyrazine, which STOLTE obtained by LOBRY
DE BRUYN's2 method from fructose in methyl alcohol solution and
ammonia, and which he calls fructosazine, can be oxidized outside of the
body into 2,5-pyrazine dicarboxylic acid.

The same acid can be formed in the animal body (rabbits), although not
constantly, after introducing fructosazine. It also passes into the urine of
rabbits after intravenous injection of d-fructose and glycocoll (SPIRO), a behavior
which SPIRO claims indicates that carbohydrates in metabolism react with the
cleavage products of proteins. STOLTE'S experiments to decide the question
whether in the animal body the glucosamine in its decomposition passes into
fructosazine did not at first yield conclusive results. His more recent investiga-
tions 3 show on the contrary that in rabbits 2-oxymethylpyrazine-5-carboxylic
acid is formed as an oxidation product, and this can be oxidized outside of the
body into pyrazine-2, 5-dicarboxylic acid.

b. Glucuronic Acids.

The glucuronic acids occurring in the animal body either physiolog-
ically or pathologically, are conjugated acids which will be described in
detail in a subsequent chapter (XIV). We will here describe only the
d-glucuronic acid in connection with the carbohydrates.

CHO
d-Glucuronic acid (glycuronic acid), CeHioO? = (CH.OH)4, is a deriva-

COOH
tive of glucose, and has been synthetically prepared by E. FISCHER and

1 Hofmeister's Beitrage, 11.

2 Cited by Stolte, Hofmeister's Beitrage, 11.

3Spiro, Hofmeister's Beitrage, 10, p. 283; Stolte, Biochem. Zeitschr., 12.

222 THE CARBOHYDRATES.

PILOTY 1 by the reduction of the lactone of saccharic acid. On oxidation
with bromine it forms saccharic acid, and on reduction it yields gulonic-
acid lactone. SALKOWSKI and NEUBERG2 have obtained Z-xylose from
glucuronic acid by splitting oft0 CCb by means of putrefaction bacteria.

Glucuronic acid has not been found in the free state in the animal
body. It occurs to a slight extent in normal urine as a conjugated acid
(MAYER and NEUBERG). It occurs to a much greater extent in urine as
conjugated acid after the ingestion of certain aromatic and also aliphatic
substances, especially camphor and chloral hydrate. It was obtained
first by SCHMIEDEBERG and MEYER from camphoglucuronic acid, and
then by v. MERING 3 from urochloralic acid by cleavage with dilute acids.
According to P. MAYER,4 on the oxidation of glucose a partial forma-
tion of glucuronic acid and oxalic acid takes place, and therefore, according
to him, an increased elimination of conjugated glucuronic acids shows
in certain cases an incomplete oxidation of glucose. Conjugated glucu-
ronic acids may also occur in the blood (P. MAYER, LEPINE and BOULUD 5),
in the feces, and in the bile.6 NEUBERG and NEIMANN 7 have prepared
certain conjugated glucuronic acids (see Chapter XIV) synthetically,
among them being euxanthic acid. The most abundant source of glucu-
ronic acid is the artist's pigment " Jaune indien," which contains the
magnesium salt of euxanthic acid (euxanthon-glucuronic acid).

Glucuronic acid is not crystalline, but is only obtainable as a syrup.
It dissolves in alcohol and is readily soluble in water. If the aqueous
solution is boiled for an hour the acid is partly (20 per cent) converted
into the crystalline lactone, glucurone, CoHsOe, which is soluble in water
and insoluble in alcohol, and which has a melting-point of 175-178° C.
The alkali salts of the acid are crystalline. If a concentrated solution
of the acid is saturated with barium hydroxide the basic barium salt is
obtained as a precipitate. The neutral lead salt is soluble in water,
while the basic salt is insoluble. The readily crystallizable cinchonine
salt can be used in isolating glucuronic acid (NEUBERG 8) . Glucuronic
acid is dextrorotatory, while the conjugated acids are levorotatory;
they behave like glucose with the reduction tests, and do not ferment

1 Ber. d. d. chem. Gesellsch., 24.

2 Zeitschr. f. physiol. Chem., 36.

8 Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Schmiedeberg u. Meyer,
ibid.,&; v. Mering, ibid, 6.

4 Zeitschr. f. klin. Med., 47. See Chapter XIV.

6 Mayer, Zeitschr. f. physiol. Chem., 32; L4pine and Boulud, Compt. rend.., 133,
134, 138.

• See Bial, Hofmeister's Beitrage, 2, and v. Leersum, ibid., 3.

7 Zeitschr. f. physiol. Chem., 44.

8 Ber. d. d. chem. Gesellsch., 33.

DISACCHARIDES. 223

with yeast. With the phenylhydrazine test it gives crystalline com-
pounds which are not sufficiently characteristic (THIERFELDER, P.
MAYER1). By the action of 3 mol. phenylhydrazine and the necessary
amount of acetic acid upon 1 mol. glucuronic acid at 40° for a few days,
NEUBERG and NEIMANN obtained the glucuronic-acid osazone, which
was very similar to glucosazone and melted at 200-205°. With p-brom-
phenylhydrazine hydrochloride and sodium acetate, glucuronic acid gives
p-bromphenylhydrazine glucuronate, which is characterized by its insolu-
bility in absolute alcohol and by a very prominent levorotatory action.
This compound is very well suited for the detection of glucuronic acid.2
Dissolved in a mixture of alcohol and pyridine (0.2 gram substance in 4 cc.
pyridine and 6 cc. alcohol) the rotation is 7° 25', which corresponds to
(«)§= — 369°. On distillation with hydrochloric acid, glucuronic acid
yields furfurol and also carbon dioxide, and on this behavior TOLLENS
and LEFEVRE3 have based their quantitative method for the estimation
of glucuronic acid.

They give the pentose reactions with phloroglucin or orcin and hydrochloric
acid, and also a good reaction with naphthoresorcinol and hydrochloric acid
(see page 218). The product produced herewith is soluble in ether with a blue,
bluish-violet or reddish-violet color, and the solution shows an absorption band
somewhat to the right and on the D-line. According to MANDEL and NEUBERG 4
this reaction is not characteristic of glucuronic acid, as many aldehyde and ketone
acids give the same reaction; still, it is important in the differentiation of the
pentoses.

Glucuronic acid is best prepared from euxanthic acid, which decom-
poses on heating it with water to 120° C. for several hours. The nitrate
from the euxanthon is concentrated at 40° C., when the anhydride
gradually crystallizes out. On boiling the mother-liquor for some time
and evaporating further, the crystals of the lactone are obtained. In
regard to the quantitative estimation of glucuronic acid we must refer
the reader to the works of TOLLENS and his collaborators and of NEUBERG
and NEIMANN.5

2. Disaccharides.

Some of the varieties of sugar belonging to this group occur ready
formed in nature. Thus we have saccharose and lactose. Some, on the
contrary, such as maltose and isomaltose, are produced by the partial

1 Thierfelder, Zeitschr. f. physiol. Chem., 11, 13, 15; P. Mayer, ibid,, -29.

2 See Netiberg, Ber d. d. chem. Gesellsch., 32; and Mayer and Neuberg, Zeitschr.
f. physiol. Chem., 29.

3 Ber. d. d. chem. Gesellsch., 40.

4 Bioch. Zeitschr. 13.

5 Tollens, Zeitschr. f. physiol. Chem., 44, which cites also the older work; Neuberg
and Neimann, ibid., 44; Neuberg, ibid., 45.

224 THE CARBOHYDRATES.

hydrolytic cleavage of complex carbohydrates. Isomaltose is also obtained
from glucose by reversion (see page 225).

The disaccharides or hexobioses are to be considered as glucosides,
each of which is derived from two monosaccharides with the exit of 1
molecule of water. Corresponding to this, their general formula is
Ci2H220n. On hydrolytic cleavage and the addition of water they
yield 2 molecules of hexoses, either 2 molecules of the same hexose or
one each of two different hexoses. Thus

Saccharose +H20 = glucose + fructose;
Maltose + H2O = glucose -f glucose ;
Lactose -f-H^O = glucose +galactose.

The configuration of the disaccharides has not been positively determined.

The fructose turns the polarized ray more to the left than the glucose
does to the right; hence the mixture of hexoses obtained on the cleavage
of saccharose has an opposite rotation to the saccharose itself. On
this account the mixture is called INVERT-SUGAR, and the hydrolytic
splitting is designated as inversion. This term, "inversion," is not only
used for the splitting of saccharose, but is also used for the hydrolytic
cleavage of compound sugars into monosaccharides. The reverse reaction,
whereby monosaccharides are condensed into complex carbohydrates, is
called reversion.

We subdivide the disaccharides into two groups, first, the group to
which saccharose belongs, where the members do not have the property
of reducing certain metallic oxides; and the second group, to which the
two maltoses and lactose belong, the members acting like monosaccharides
in regard to the ordinary reduction tests. The members of the latter
group have the character of aldehyde alcohols, and in milk-sugar the
aldehyde characteristics are connected with the glucose fraction.

Saccharose, or CANE-SUGAR, occurs extensively distributed in the
plant kingdom. It occurs to the greatest extent in the stalk of the sugar-
millet and sugar-cane, the roots of the sugar-beet, the trunks of certain
varieties of palms and maples, in carrots, etc. Cane-sugar is of extraor-
dinary great importance as a food and condiment.

Saccharose forms large, colorless monoclinic crystals. On heating it
melts in the neighborhood of 160° C., and on heating more strongly it
turns brown, forming so-called caramel. It dissolves very readily in
water, and- according to HERZFELD,1 100 parts of saturated saccharose
solution contain 67 parts of sugar at 20° C. It dissolves with difficulty
in strong alcohol. Cane-sugar is strongly dextrorotatory. The specific
rotation is only slightly modified by concentration, but is markedly

1 See Tollens' Handbuch der Kohlehydrate, 2. Aufl. 1, 154.

MALTOSE. 225

changed by the presence of other inactive substances. The specific
rotation is (a)D= +66.5°.

Saccharose acts indifferently toward MOORE'S test and to the ordinary
reduction tests. On continuous boiling it may reduce an alkaline copper
solution, perhaps on account of a partial inversion. It does not ferment
directly, but only after inversion, which can be brought about by an
enzyme (invertin) contained in the yeast. An inversion of cane-sugar also
takes place in the intestinal canal. Cane-sugar does not combine with
hydrazines. Concentrated sulphuric acid blackens cane-sugar very
quickly even at the ordinary temperature, and anhydrous oxalic acid
does the same on warming on the water-bath. Various products are
obtained on the oxidation of cane-sugar, dependent upon the variety of
oxidizing agent and also upon the intensity of the action. Saccharic acid
and oxalic acid are the most important products.

The reader is referred to complete text-books on chemistry for the
preparation and quantitative estimation of cane-sugar.

Maltose (MALT-SUGAR) is formed in the hydrolytic cleavage of starch
by malt diastase, saliva, or pancreatic juice. It is obtained from gly co-
gen under the same conditions (see Chapter VII). Maltose is also pro-
duced transitorily in the action of sulphuric acid on starch. Maltose
forms the fermentable sugar of the potato or grain mash, and also of the
beerwort.

Maltose crystallizes with one molecule water of crystallization in fine
white needles. It is readily soluble in water, rather easily in alcohol,
but insoluble in ether. Its solutions are dextrorotatory; and the specific
rotation is variable, depending upon the concentration and temperature,
but is considerably stronger than glucose,1 and is generally given as
(a)D=+137 to 138°. Maltose ferments readily and completely with
yeast, and acts like glucose in regard to the reduction tests. It yields
phenylmaltosazone on warming with phenylhydrazine for 1J hours.
This phenylmaltosazone melts at 205° C., and is more soluble in hot
water than the glucosazone. Maltose differs from glucose chiefly in the
following: It does not dissolve as readily in alcohol, has a stronger dex-
trorotatory power, and has a feebler reducing action on FEHLING'S solu-
tion; 10 cc. FEHLING'S solution are, according to SoxHLET,2 reduced
by 77.8 milligrams anhydrous maltose in approximately 1 per cent solution.

Isomaltose. This variety of sugar, as has been shown by FiscHER,3
is produced, as are dextrin-like products, by reversion, and by the action
of fuming hydrochloric acid on glucose. A re-formation of isomaltose

1 See Hoppe-Seyler-Thierfelder's Handbuch, 8. Aufl.

2 Cited from Tollens' Handbuch. der Kohlehydrate, 2. Aufl. 1, 154.

3 Ber. d. deutsch. chem. Gesellsch., 23 and 28.

226 THE CARBOHYDRATES.

and other sugars from glucose can also be brought about by means of
yeast maltase (HiLL and EMMERLING, see page 58). It is also formed,
besides ordinary maltose, in the action of diastase on starch paste, and
occurs in beer and in commercial starch-sugar. It is produced, with
maltose, by the action of saliva or pancreatic juice (KULZ and VOGEL)
or blood-serum (ROHMANN x) on starch. The formation of isomaltose
in the hydrolysis of starch has been denied by many investigators because
they considered isomaltose only as contaminated maltose.2

Isomaltose dissolves very readily in water, has a pronounced sweetish
taste, and does not ferment, or, according to some, only very slowly.
It is dextrorotatory, and has very nearly the same power of rotation as
maltose. Isomaltose is characterized by its osazone. This forms fine
yellow needles, which begin to form drops at 140° C. and melt at 150-
153° C. These are rather easily soluble in hot water and dissolve in hot
absolute alcohol much more readily than the maltosazone. Isomaltose
reduces copper as well as bismuth solutions.

Lactose (MILK-SUGAR). As this sugar occurs exclusively in the animal
world, in the milk of human beings and animals, it will be treated in a
following chapter (on milk).

3. Colloid Polysaccharides.

If we exclude the not well known trisaccharides and the tetrasaccharide
stachyose this group includes a great number of very complex carbo-
hydrates which occur only in the amorphous condition, or at least not
as crystals in the ordinary sense. Unlike the bodies belonging to the
other groups, these have no sweet taste. Some are soluble in water,
while others swell up therein, especially in warm water, and finally
some are neither dissolved nor visibly changed. Polysaccharides are
ultimately converted into monosaccharides by hydrolytic cleavage.

The polysaccharides are ordinarily divided into the following groups:
starches with the dextrins, plant gums and mucilages, and the celluloses.

Starch Group.

Starch, AMYLTJM (CeHioC^x. This substance occurs in the plant
kingdom very extensively distributed in the different parts of the plant,
especially as reserve food in the seed, roots, tubers, and trunks.

Starch is a white, odorless, and tasteless powder, consisting of small

1 Kiilz and Vogel, Zeitschr. f. Biologie, 31; Rohmann, Centralbl. f. d. med. Wis-
sensch., 1893, 849.

2 Brown and Morris, Journ. of Chem. Soc., 1895; Chem. News, 72. See also Ost
Ulrich, and Jalowetz, Ref. in Ber. d. deutsch. chem. Gesellsch., 28; Ling and Baker,
Joum. of Chem. Soc., 1895; Pottevin, Chem. Centralbl., 1899, II, 1023.

STARCH. 227

granules which have a stratified structure and different shape and size
in different plants. Starch is considered insoluble in cold water. The
grains swell up in warm water and burst, yielding a paste.

According to the ordinary opinion the starch granules consist of two
different substances, STARCH GRANULOSE and STARCH CELLULOSE (v.
NAGELI), the first of which turns blue with iodine and forms the chief
part of the granule. According to MAQUENNE and Roux x this is
not the fact. According to them the starch granule consists of two
constituents, of which the first, amylose, forms the chief mass (80-85
per cent) and the other, amylopeclin, forms only 15-20 per cent of the
granule. Amylopectin is not identical with v. NAGELI'S starch cellulose,
and the above investigators consider starch cellulose as only an insoluble
form of amylose. The amylose can occur in two forms; one, which is
soluble, is colored blue by iodine and is immediately transformed into
sugar by malt, the other is a solid substance, which is not colored
vdth iodine and resists the action of malt infusion. One modification
can be transformed into the other.

In the paste, besides amylopectin, we also have soluble amylose, and
this can, by a process called retrogradation by MAQUENNE and Roux, be
transformed into the solid modification, " artificial starch." This
solid form occurs in the starch granule, and is identical with v. NAGELI'S
starch cellulose. As the starch granules are directly colored blue by
iodine they must, besides this, also contain soluble amylose. If the author
understands the above investigators correctly the starch granules con-
tain three constituents, namely, soluble amylose, which is colored blue
by iodine ( = starch granulose), insoluble amylose, which is not colored
by iodine ( = starch cellulose), and amylopectin.

In the formation of paste the amount of amylose is not the essential
but rather the quantity of amylopectin. The amylopectin is a slime-
like substance, insoluble in boiling water and dilute alkalies, only becom-
ing pasty therein, and not colored blue by iodine. Accordingly the
paste is a solution of amylose made thick by amylopectin. The amylo-
pectin, unlike the amylose, is only slowly transformed into sugar with
dextrin formation. Starch is insoluble in alcohol and ether. On heat-
ing starch with water alone, or heating with glycerin to 190° C., or on
treating the starch grains with 6 parts dilute hydrochloric acid of sp. gr.
1.06 at ordinary temperature for six to eight weeks,2 it is converted into
soluble starch (AMYLODEXTRIN, AMIDULIN). Soluble starch is also

1v. Nageli, Botan. Mittei]., 1863; Maquenne and Roux, Compt. rend., 138, 140,
142, 146, and Bull. Soc. chim. de Paris (3), 33 and 35.

2 See Tollens' Handb., 191. In regard to other methods, see Wr6blewsky, Ber. d.
deutsch. chem., Gesellsch., 30; Syniewski, ibid.

228 THE CARBOHYDRATES.

formed as an intermediate step in the conversion of starch into sugar
by dilute acids or diastatic enzymes. Soluble starch may be precipitated
from very dilute solutions by baryta-water.1

Starch granules swell up and form a pasty mass in caustic potash or
soda. This mass gives neither MOORE'S nor TROMMER'S test. Starch
paste does not ferment with yeast. The most characteristic test for starch
is the blue coloration produced by iodine in the presence of hydriodic
acid or alkali iodides.2 This blue coloration disappears on the addition of
alcohol or alkalies, and also on warming, but reappears again on cooling.

On boiling with dilute acids starch is converted into glucose. In
the conversion by means of diastatic enzymes we have, as a rule, besides
dextrin, maltose, and isomaltose, only very little glucose. We are
considerably in the dark as to the kind and number of intermediate
products produced in this process (see Dextrins) .

Starch may be detected by means of the microscope and by the
iodine reaction. Starch is quantitatively estimated, according to SACHSSE'S
method,3 by converting it into glucose by hydrochloric acid and then
determining the glucose by the ordinary methods.

Inulin (C6HioO5)x+H2O, occurs in the underground parts of many
Composite, especially in the roots of the Inula helenium, the tubers
of the Dahlia, the varieties of Helianthus, etc. It is ordinarily obtained
from the tubers of the Dahlia.

[Inulin forms a white powder similar to starch, consisting of spheroid
crystals which are readily soluble in warm water without forming a paste.
It separates slowly on cooling, but more rapidly on freezing. Its solu-
tions are levogyrate and are precipitated by alcohol, and are colored
only yellow with iodine. Inulin is converted into the levogyrate mono-
saccharide d-fructose on boiling with dilute sulphuric acid. Diastatic
enzymes of higher animals have no, or only a very slight, action on inulin.4

According to DEAN 5 inulin occurs in combination with other substances,
levulins, which are more soluble and have less rotation. He suggests that we
limit the name inulin to that carbohydrate (or mixture of carbohydrates), which
is readily precipitable by 60 per cent alcohol and shows a specific rotation of
(a)D=-3Sto40°.

Lichenin (MOSS-STARCH) occurs in many lichens, especially in Iceland moss.
It is not soluble in cold water, but swells up into a jelly. It is soluble in hot
water, forming a jelly on allowing the concentrated solution to cool. It is colored
yellow by iodine and yields glucose on boiling with dilute acids. Lichenin is
not changed by diastatic enzymes such as ptyalin or amylopsin (NILSON 6).

1 In regard to the compounds of soluble starch and dextrins with barium hydroxide,
see Biilow, Pflviger's Arch., 62.

2 See Mylius, Ber. d. deutsch. chem. Gesellsch., 20, and Zeitsch. f. physiol. Chem., 11.

3 Tollens' Handb., 2. Aufl., 1, 187.

4 Tollens' Handbuch, 208.

5 Amer. Chem. Journ., 32.

6 Upsala Lakaref. Forh., 28.

DEXTRINS. 229

Glycogen. This carbohydrate, which stands to a certain extent
between starch and dextrin, is principally found in the animal kingdom,
hence it will be considered in a subsequent chapter (on the liver).

Dextrins and Gums.

The dextrins stand in close relation to the starches, and are formed
therefrom as intermediate products by the action of acids or diastatic
enzymes. They yield as final products only hexoses, indeed only glu-
cose, on complete hydrolysis. The vegetable gums, the vegetable
mucilages and the pectin bodies, which all stand close to the hemicellu-
loses, yield, on the contrary, abundance of pentose and, among the hex-
oses, galactose is very often found.

Dextrin (starch-gum, British gum), is produced on heating starch to
200-210° C., or by heating starch, which has previously been moistened
with water containing a little nitric acid, to 100-110° C. Dextrins are
also produced by the action of dilute acids and diastatic enzymes on
starch. There have been numerous investigations as to the steps
involved in the last-mentioned process, but they have led to conflicting
views. One of these, which used to be generally accepted, is as follows:
The first product, which gives a blue color with iodine, is soluble starch
or amylodextrin, which on further hydrolytic cleavage yields sugar and
erythrodextrin, which is colored red by iodine. On further cleavage of
this erythrodextrin more sugar and a dextrin, achroodextrin, which is
not colored by iodine, is formed. From this achroodextrin after suc-
cessive splittings we have sugar and dextrins of lower molecular weights
formed, until finally we have sugar and a dextrin, maltodextrin, which
refuses to split further, as final products. The views are rather contra-
dictory in regard to the number of dextrins which occur as intermediate
steps. The sugar formed is maltose (or in first place isomaltose), and
only very little glucose is produced. Another view is that first several
dextrins are formed consecutively in the successive splittings, by hydra-
tion, and then finally the sugar is formed by the splitting of the last
dextrin. According to MOREAU, in the first stages of saccharification
amylodextrin, erythrodextrin, achroodextrin and sugar are formed sim-
ultaneously. Other investigators, especially SYNIEWSKI, have recently
suggested other views on the subject.1

This question has taken another direction by the investigations of

1 In regard to the various views on the theories of the saccharification of starch;
see Musculus and Gruber, Zeitschr. f. physiol. Chem., 2; Lintner and Dull, Ber. d. d.
chem. Gesellsch., 26 and 28; Brown and Heron, Journ. of Chem. Soc., 1879; Brown
and Morris, ibid., 1885 and 1889; Moreau, Biochem. Centralbl., 3, 648; Syniewski,
Annal. d. Chem. u. Pharm., 309, and Chem. Centralbl., 1902, 2.

230 THE CARBOHYDRATES.

MAQUENNE, mentioned above. According to him the amylose passes
directly into maltose without the formation of dextrin by the action of
malt infusion. The dextrins produced are only formed from the amylo-
pectin, which does not undergo saccharification with freshly prepared
malt infusions, but only with older or especially active infusions. This
also explains why in the older investigations the saccharification was
only about 80 per cent while MAQUENNE has been able to completely
convert the starch into sugar by enzymotic action.

The various dextrins are very hard to isolate as chemical individuals
and to separate from each other. YOUNG 1 has tried their separation
by means of neutral salts, especially ammonium sulphate, and MOREAU
by the aid of a baryta-alcohol method. We cannot enter into the dif-
ferences as to the dextrins so separated, and only the characteristic
properties and reactions will be given for the dextrins in general.

The dextrins appear as amorphous, white or yellowish-white powders
which are/ readily soluble in water. Their concentrated solutions are
viscid and sticky, like gum solutions. The dextrins are dextrogyrate.
They are insoluble or nearly so in alcohol, and insoluble in ether. Watery
solutions of dextrins are not precipitated by basic lead acetate. Dex-
trins dissolve cupric hydroxide in alkaline liquids, forming a beautiful
blue solution, which, as is generally admitted, is reduced by pure dex-
trins. According to MOREAU pure dextrin has no reducing action. The
dextrins are not directly fermentable.

SCHARDINGER has discovered a bacillus which forms acetone from
starch and which is especially useful for the perparation of crystalline
cleavage products from starch. He obtained two crystalline substances,
dextrin a and 0, which are not fermentable by yeast and on hydrolysis
with acid yield glucose. For the a-dextrin PRINGSHEIM and LANGHANS
have determined the formula (CeHioC^ while BILTZ and TRUTHE 2
found the formula (CoHioCs^ for the /3-dextrin.

The vegetable gums are soluble in water, forming solutions which are viscid
but may be filtered. We designate, on the contrary, as vegetable mucilages
those varieties of gum which do not or only partly dissolve in water, and which
swell up therein to a greater or less extent. The natural varieties of gum and
mucilage, to which belong several generally known and important substances,
such as gum arabic, wood-gum, cherry-gum, salep, and quince mucilage, and
probably also the little-studied pectin substances, will not be treated in detail,
because of their unimportance from a physiological standpoint.

1 Journ. of Physiol., 22, which contains the older researches of Nasse, Kriiger
Neumeister, Pohl, and Halliburton. Moreau, 1. c.

2 Schardinger, Centralbl. f. Bak. u. Parasitenkunde, II, 22, 98 (1909); 29, 118 (1911);
Pringsheim and Langhans, Ber. d. d. Chem. Gesellsch., 45, 2533 (1912); Biltz and
Truthe, ibid., 46, 1377 (1913).

CELLULOSE. 231

The Cellulose Group (C6H]oO5)x.

Cellulose is that carbohydrate, or perhaps more correctly, mixture
of carbohydrates, which forms the chief constituent of the walls of the
plant-cells. This is true for at least the walls of the young cells, while
in the walls of the older cells the cellulose is extensively incrusted with a
substance called LIGNIN, and with many other cellulose derivatives and
compounds.

The true celluloses are characterized by their great insolubility. They
are insoluble in cold or hot water, alcohol, ether, dilute acids, and alkalies.
We have only one specific solvent for cellulose, and that is an ammo-
niacal solution of copper oxide called SCHWEITZER'S reagent. The
cellulose may be precipitated from this solvent by the addition of acids,
and obtained as an amorphous powder after washing with water.

Cellulose is converted into a substance, so-called AMYLOID, which
gives a blue coloration with iodine, by the action of concentrated sul-
phuric acid. With oxidizing agents (nitric acid, etc.) oxycelluloses are
produced. By the action of strong nitric acid or a mixture of nitric
acid and concentrated sulphuric acid, celluloses are converted into nitric-
acid esters or nitrocelluloses, which are highly explosive and have found
great practical use.

The ordinary celluloses when treated at the ordinary temperature
with strong sulphuric acid and then boiled for some time after diluting
with water are coverted into glucose. In this case it must be observed,
according to MAQUENNE, that it is not maltose that is produced as an
intermediate step, but another disaccharide, called cellose or cellobiose.

The cellulose, at least in part, undergoes decomposition in the intestinal
tract of man and animals. A closer discussion of the nutritive value
of cellulose will be given in a future chapter (on digestion). The great
importance of the carbohydrates in the animal economy and to animal
metabolism will also be given in the following chapters.

Hemicelluloses are, according to E. SCHULZE/ those 'constituents of the cell-
wall related to cellulose which differ from the ordinary cellulose by dissolving
on heating with strongly diluted mineral acids, such as 1.25 per cent sulphuric
acid, and of yielding arabinose, xylose, galactose, and mannose instead of glucose.
Those hemicelluloses which serve partly as reserve food and partly as support-
substance, are very widely distributed in the plant kingdom. It must be recalled
that according to BIERRY and GIAJA 2 the digestive organs of different inverte-
brates (Helix, Astacus, Maja. Hommarus) contain enzymes which have an
energetic splitting action upon such polysaccharides as well as on the natural
celluloses.

1 E. Schulze, Zeitschr. f. physiol. Chem., 16 and 19, with Castro, ibid., 36.

2 Bioch. Zeitschr., 40, 370 (1912).

CHAPTER IV.
ANIMAL FATS AND PHOSPHATIDES.

1. Neutral Fats and Fatty Acids.

THE fats form the third chief group of the organic food of man and
animals. They occur very widely distributed in the animal and plant
kingdoms. Fat occurs in all organs and tissues of the animal organism,
though the quantity may be so variable that a tabular exhibit of the
amount of fat in different organs is of little interest. The marrow con-
tains the largest quantity, having over 96 per cent. The three most
important deposits of fat in the animal organism are the intermuscular
connective tissue, the fatty tissue in the abdominal cavity, and the
subcutaneous connective tissues. In plants, the seeds and fruit and in
certain instances also the roots, are rich in fat. Fat also occurs deposited,
during the winter's rest, in the trunks of trees.

The fats consist almost entirely of so-called neutral fats, with only
very small quantities of fatty acids. The neutral fats are esters of the
triatomic alcohol, glycerin, with monobasic fatty acids. These esters
are triglycerides; that is, the hydrogen atoms of the three hydroxyl
groups of the glycerin are replaced by the fatty-acid radicals, and their
general formula is therefore, CsH5.03.R3. The animal fats consist
chiefly of esters of the three fatty acids, stearic, palmitic, and oleic acids.
In certain fats, especially in milk-fat, glycerides of fatty acids such as
butyric, caproic, caprylic, and capric acids also occur in considerable
amounts. Besides the above-mentioned ordinary fatty acids, stearic,
palmitic, and oleic acids, we also find in human and animal fat, exclusive
of certain fatty acids only little studied, the following non-volatile fatty
acids, as glycerides, namely, lauric acid, Ci2H2402, myristic acid, Ci4H2g02,
and arachidic acid, C2oH4o02- Of the unsaturated fatty acids, besides
oleic acid, we probably also have in small quantities glycerides of acids
of the linolic acid series CBH2o-402 and of the linolenic acid series,
CBH2n-6-O2. In this case the question can be raised whether or not
these acids are not derived from the phosphatides mixed with the fats.
In the plant kingdom triglycerides of other fatty acids, such as lauric
acid, myristic acid, linoleic acid, erucic acid, etc., sometimes occur

232

NEUTRAL FATS. 233

abundantly. Besides these, oxyacids and high molecular alcohols have
been found in many plant fats. The extent to which traces of these
oxyacids occur in the animal kingdom has not been thoroughly inves-
tigated, but the occurrence of monoxystearic acid seems to have been
proved.1 The occurrence of high molecular alcoh9ls, although ordinarily
only in small amounts, has on the contrary been positively shown in
animal fat.

The animal fats are of the greatest interest and consist of a mixture of
varying quantities of TKISTEARIN, TRIPALMITIN, and TRIOLEIN, having
an average elementary composition of C 76.5, H 12.0, and O 11.5 per
cent. It must be remarked that in animal fat (mutton and beef tallow)
as well as in plant fat (olive-oil) mixed triglycerides, such as dipalmityl-
olein, distearyl-palmitin and distearyl-olein, occur, and that these mixed
glycerides may also be prepared synthetically.2

Fats from different species of animals, and even from different parts
of the same animal, have an essentially different consistency, depending
upon the relative amounts of the different individual fats present. In
solid fats — as tallow — tristearin and tripalmitin are in excess, while
the less solid fats are characterized by a greater abundance of triolein.
This last-mentioned fat is found in greater quantities proportionally
in cold-blooded animals, and this accounts for the fact that the fat of
these animals remains fluid at temperatures at which the fat of warm-
blooded animals solidifies. Human fat from different organs and tissues
contains, in full numbers, 67-85 per cent triolein.3 The melting-point
of different fats depends upon the composition of the mixtures, and it
not only varies for fat from different tissues of the same animal, but also
for the fat from the same tissues in various kinds of animals.4

Neutral fats are colorless or yellowish, and, when perfectly pure,
ordorless and tasteless. They are lighter than water, on which they
float when in a molten condition. They are insoluble in water, dissolve
in boiling alcohol, but separate on cooling — often in crystals. They are
easily soluble in ether, benzene, chloroform, carbon disulphide and petro-
leum ether. The fluid neutral fats give an emulsion when shaken with
a solution of gum or albumin. With water alone they give an emulsion

1 Erben, Zeitschr. f. physiol. Chem., 30; Bernert, Arch. f. exp. Path. u. Pharm., 40.
2Guth, Zeitschr. f. Biologie, 44; W. Hansen, Arch. f. Hygiene, 42; Holde and
Stange, Ber. d. d. chem. Gesellsch., 34; Kreis and Hafner, ibid., 36.

3 See Knopfelmacher, "Untersuch. liber das Fett im Sauglingsalter," etc., Jarhbuch
f. Kinderheilkunde (N. F.), 45, which also contains the older literature; Jaeckle,
Zeitschr. f. physiol. Chem., 36.

4 According to Gilkin (Ber. d. d. chem. Gesellsch., 41) the fat from bone-marrow
and also other fats of animal and plant origin contain iron,^which cannot be removed
by water containing hydrochloric acid.

234 ANIMAL FATS AND PHOSPHATIDES.

only after vigorous and prolonged shaking, but the emulsion is not pet-
sistent. The presence of some soap causes a very fine and permanent
emulsion to form easily. Fat produces spots on paper which do not
disappear; it is not volatile; it boils at about 300° C. with partial decom-
position, and burns with a luminous and smoky flame. The fatty acids
have most of the above-mentioned properties in common with the neutral
fats, but differ from them in being soluble in alcohol-ether, in having
an acid reaction, and by not giving the acrolein test. The neutral fats
generate a strong irritating vapor of acrolein, due to the decomposition
of glycerin, C3H5(OH)3— 2H2O = C2H3.CHO, when heated alone, or
more easily when heated with potassium bisulphate or with other dehy-
drating substances.

The neutral fats may be split by the addition of the constituents of
water according to the following equation:

This splitting may be produced by the pancreatic enzyme and other
enzymes occurring in the animal and vegetable kingdoms, for example,
the castor lipase. The reverse action, namely, the synthesis of fatty acid
esters, can be brought about by enzymes, such as pancreatic lipase (see
page 60). The cleavage of the neutral fats can also be accomplished
by superheated steam or by dilute acids. We most frequently decompose
the neutral fats by boiling them with not too concentrated caustic alkali,
or, still better (in biochemical researches), with an alcoholic potash solu-
tion or with sodium alcoholate. By this procedure, which is called sapon-
ification, the alkali salts of the fatty acids (soaps) are formed. If the
saponification is made with lead oxide, then lead plaster, the lead salt of
the fatty acids is produced. By saponification is to be understood not
only the cleavage of neutral fats by alkalies, but also the splitting of neutral
fats into fatty acids and glycerin in general.

On keeping fats for. a long time in contact with air they undergo a
change, becoming yellow in color and acid in reaction, and they develop
an unpleasant odor and taste, becoming rancid. In this change a part
of the fat is split into fatty acids and glycerin, and then an oxidation
of the free fatty acids takes place, producing volatile bodies of an
unpleasant odor.

The three most important fats of the animal kingdom are stearin,
palmitin, and olein.

CH2.O.Ci8H350

Stearin, or tristearin, CsTHnoOe^CH.O.CisHasO, occurs especially in

CH2.O.Ci8H35O

the solid varieties of tallow but also in the vegetable fats. Stearic acid,
CisH36O2, is found in the free state in decomposed pus, in the expectora-

PALMITIN. 235

tions in gangrene of the lungs, and in cheesy tuberculous masses. It
occurs as lime soap in excrement and adipocere, and in this last product
also as an ammonium soap. It also exists as alkali soap in the blood,
bile, transudations and pus, and in the urine to a slight extent.

Stearin is the hardest and most insoluble of the three ordinary neutral
fats. It is nearly insoluble in cold alcohol, and soluble with great dif-
ficulty in cold ether (225 parts). It separates from warm alcohol on
cooling as rectangular, and less frequently as rhombic plates. The opinions
regarding the melting-point are somewhat varied. Pure stearin, ac-
cording to HEINTZ/ melts transitorily at 55° and permanently at 71.5°.
The stearin from the fatty tissues (not pure) melts at 63° C.
CH3

Stearic acid, (CH^ie, crystallizes (on cooling from boiling alcohol) in

COOH

large, shining, long rhombic scales or plates. It is less soluble than the
other fatty acids and melts at 68.2° C.2 Its barium salt contains 19.49
per cent barium, and its silver salt contains 27.59 per cent silver.

CH2.O.Ci6H310

Palmitin, or tripalmitin, C5iH9806, = CH.O.Ci6H3iO. Of the two solid

CH2.O.Ci6H310

varieties of fats, palmitin is the one which occurs in predominant quan-
tities in human fat (LANGER3). Palmitin is present in all animal fats
and in several kinds of vegetable fat. A mixture of stearin and palmitin
was formerly called MARGARIN. As to the occurrence of palmitic acid,
CieH32O2, about the same remarks apply as to stearic acid. The mixture
of these two acids has been called margaric acid, and this mixture occurs
—often as very long, thin, crystalline plates — in old pus, in expectora-
tions from gangrene of the lungs, etc.

Palmitin crystallizes, on cooling from a warm saturated solution in
ether or alcohol, in starry rosettes of fine needles. The mixture of pal-
mitin and stearin, called margarin, crystallizes, on cooling from a solu-
tion, as balls or round masses which consist of short or long, thin plates
or needles which often appear like blades of grass. Palmitin, like stearin,
has a variable melting- and solidifying-point, depending upon the way it
has been previously treated. The melting-point is often given as 62° C.,
but some investigators 4 claim that it melts at 50.5° C., solidifies on
further heating, and melts again at 66.5° C.

1 Annal. d. Chem. u. Pharm., 92.

2 According to Carlinfanti and Levi-Malvano, Chem. Centralbl., 1910.

3 Monatshefte f. Chem., 2; see also Jaeckle, Zeitschr. f. physiol. Chem., 36.

4 R. Benedikt, Analyse der Fette, 3. Aufl., 1897, p. 44.

236 ANIMAL FATS AND PHOSPHATIDES.

CH3
Palmitic acid, (CH2)i4, crystallizes from an alcoholic solution in tufts

COOH

of fine needles. It melts at 61° C.;1 still the admixture with stearic acid,
essentially changes them elting- and solidifying-points according to th
relative amounts of the two acids. Palmitic acid is somewhat more
soluble in cold alcohol than stearic acid; but they have about the same
solubility in boiling alcohol, ether, chloroform, and benzene. Its barium
salt contains 21.17 per cent barium, and silver salt contains 29.72 per
cent silver.

CH2.O.Ci8H33O
I Olein, or triolein, C57Hio406, = CH.O.Ci8H330, is present in all animal

CH2.O.Ci8H330

fats, and in greater quantities in vegetable fats. It is a solvent for
stearin and palmitin. The oleic acid (elaic acid), CigH^Cb, as soaps,
probably has about the same occurrence as the other fatty acids.

Olein is, at ordinary temperatures, a nearly colorless oil of a specific
gravity of 0.914, without odor or marked taste, and solidifies in crystalline
needles at -6° C. It becomes rancid quickly if exposed to the air. It
dissolves with difficulty in cold alcohol, but more easily in warm alcohol
or in ether. It is converted into its isomer, ELAIDIN, by nitrous acid.
CH3
(CH2)7

C*TT

Oleic acid, /v^, is an unsaturated acid of the series C»Hn-202. and
U-ti

(CH2)7
COOH

correspondingly takes up two halogen atoms, i.e., iodine, at the double
bondage, a factor which is the basis of v. HUBL'S method for determining
the iodine equivalent. On taking up hydrogen, which can be accomplished
by heating with hydroiodic acid and amorphous phosphorus, it is trans-
fortned into the corresponding saturated acid, namely, stearic acid. On
oxidation the double bonds are satisfied by 2HO groups, and dioxystearic
acid, CH3(CH2)7CHOH.CHOH(CH2)7COOH, is formed. Oleic acid
readily undergoes oxidation in the air with the formation of acid products,
and the occurrence of monoxystearic acid, found in animal fats in certain
instances, can be explained by this oxidation. Oleic acid on heating
yields, besides volatile fatty acids, sebacic add, CioHigO^ which melts
at 127°C; and with nitrous acid it is transformed into its isomer, solid
elaidic acid, which melts at 45° C.

Oleic acid forms at ordinary temperature a colorless, tasteless, and

1 Carlinfanti and Levi-Malvano, Chem. Centralbl. 1910.

OLEIC ACID. 237

odorless oily liquid which solidifies in crystals at about 4° C., which
latter melt at 14° C. Oleic acid is insoluble in water, but dissolves
in alcohol, ether, chloroform and petroleum ether. With concentrated
sulphuric acid and some cane-sugar it gives a beautiful red or reddish-
violet liquid whose color is similar to that produced in PETTENKOFER'S
test for bile-acids. If a solution of oleic acid in glacial acetic acid is treated
with a little chromic acid (in glacial acetic acid) and then with concen-
trated sulphuric acid, the green solution gradually becomes violet or
cherry-red, and shows two characteristic absorption bands in the green,
one a broad band near the blue and a second but fainter band near the
yellow (LIFSCHUTZ) .l The barium salt of oleic acid contains 19.65 per
cent barium and the silver salt 27.73 per cent silver.

If the watery solution of the alkali compounds of oleic acid is pre-
cipitated with ^ lead acetate, a white, tough, sticky mass of lead oleate is
obtained, which is not soluble in water and only slightly in alcohol, but is
soluble in ether. This salt is more easily soluble in benzene than the lead
salts of stearic and palmitic acids, and this behavior of the lead salts
toward ether and benzene is made use of in separating oleic acid from
the other fatty acids.

An acid related to oleic acid, DOEGLIC ACID, which is solid at 4° C., liquid at
16° C., and soluble in alcohol, is found in the blubber of the Balcena rostrata.
According to BULL this acid is probably only a mixture of oleic acid and another
acid — gadoleic acid, C^H^C^, having a melting-point of +24.5° C., and occurring
in cod-liver oil, herring oil and in whale blubber. In addition to this acid BULL
found in cod-liver oil, besides myristic, palmitic, oleic and erucic acids, another
acid, having the formula C16H3o02. According to ELLMER 2 the most abundant
acid (80-90 per cent) in cod-liver oil is therapinic acid, CigHiaC^ which is changed
into stearic acid by reduction and jecoleic acid, which seems to be identical with
BULL'S gadoleic acid. KURBATOFF has demonstrated the presence of linoleic
acid in the fat of the silurus, sturgeon, seal, and certain other animals. Drying
fats have also been found by AMTHOR and ZiNK3 in hares, wild rabbits, wild
boar, and mountain-cock.

To detect the presence of fat in an animal fluid or tissue the fat must
first be shaken out or extracted with ether. After the evaporation of
the ether the residue is tested for fat and fatty acids. The neutral
fats are differentiated from the fatty acids by the acrolein test, and the
fatty acids by the fact that their solution in a mixture of alcohol and
ether has an acid reaction. In separating the fats from cholesterin
and other non-saponifiable substances, as well as for the determination
of the kind of the various fatty bodies, they are saponified with caustic
alkali, alcoholic potash, or with sodium alcoholate. In regard to these
operations, as well as the further investigation and the separation of the

1 Zeitschr. f. physiol. Chem., 56.

2 Bull, Ber. d. d. chem., Gesellsch., 39; Ellmer, Bioch. Zeitschr., 9.

* Kurbatoff, Maly's Jahresb., 22; Amthor and Zink, Zeitschr. f. anal. Chem., 36.

238 ANIMAL FATS AND PHOSPHATIDES.

various fatty acids from each other, we must refer to more complete
hand-books.

In addition to the methods already suggested there are other chemical methods
which are important in investigating fats. Besides ascertaining the melting-
and congealing-point we also determine the following: 1. The acid equivalent,
which is a measure of the amount of fatty acids in a fat, is determined by titrat-
ing the fat dissolved in alcohol-ether with N/10 alcoholic caustic potash, using
phenolphthalein as indicator. 2. The saponification equivalent, which gives
the milligrams of caustic potash uniting with the fatty acids in the saponification
of 1 gram fat with N/2 alcoholic caustic potash. 3. REICHERT-MEISSL'S equivalent,
which gives the quantity of volatile fatty acids contained in a given amount
of neutral fat (5 grams). The fat is saponified, then acidified with mineral acid,
and distilled, whereby the volatile fatty acids pass over; the distillate is then
titrated with alkali. 4. Iodine equivalent is the quantity of iodine absorbed by
a certain amount of the fat by addition. It is chiefly a measure of the quantity
of unsaturated fatty acids, principally oelic acid or olein, in the fat. Other bodies,
such as cholesterin, may also absorb iodine or halogens. The iodine equiva-
lent is generally determined according to the method suggested by v. HUBL.
5. The acetyl equivalent measures the quantity of those constituents of fats which
contain OH groups, and is found by converting these bodies (oxyfatty acids,
alcohols and others) into the corresponding acetyl ester by boiling them with
acetic acid anhydride.

In the quantitative estimation of fats, the finely divided dried tissues
or the finely divided residue from an evaporated fluid is extracted with
ether, alcohol-ether, benzene, or any other proper extraction medium.
The lecithin (phosphatides) and other bodies are dissolved by the various
extraction media, hence the results for fats are too high. The most
exact method for the estimation of fat seems to be the method sug-
gested by KTJMAGAWA and SUTO/ who give a complete review of the
literature of the subject.

The fats are poor in oxygen, but rich in carbon and hydrogen. They
therefore represent a large amount of chemical energy, and yield correspond-
ingly large quantities of heat on combustion. They take first rank
among the foods in this regard, and are therefore of very great impor-
tance in animal life. We will speak more in detail of this significance,
also of fat formation and of the behavior of the fats in the body, in the
following chapters.

Cholesterin and isocholesterin ester, which will be discussed in a sub-
sequent chapter, as well as the following bodies, are closely related to
the fats.

Spermaceti. In the living spermaceti or white whale there is found, in a large
cavity in the skull, an oily liquid called spermaceti, which on cooling, after death,
separates into a solid crystalline part ordinarily called SPERMACETI, and into a
liquid, SPERMACETI-OIL. This last is separated by pressure. Spermaceti is also
found in other whales and in certain species of dolphin.

The purified, solid spermaceti, which is called CETIN, is a mixture of esters of
fatty acids. The chief constituent is the cetyl-palmitic ester mixed with small

1 Biochem. Zeitschr., 8. See also y. Schimidzu, ibid., 28.

PHOSPHATIDES. 239

quantities of compound esters of lauric, myristic, and stearic acids with radicals
of the alcohols, LETHAL, Ci2H25.OH, METHAL, Ci4H29.OH, and STETHAL, Ci8H37.OH.

Cetin is a snow-white mass shining like mother-of-pearl, crystallizing in plates,
brittle, fatty to the touch, and which has a varying melting-point of 30 to 50° C.,
depending upon its purity. Cetin is insoluble in water, but dissolves easily
in cold ether or volatile and fatty oils. It dissolves in boiling alcohol, but crys-
tallizes on cooling. It is saponified with difficulty by a solution of caustic potash
in water, but with an alcoholic solution it saponifies readily, and the above-men-
tioned alcohols are set free.

CH3

Ethal or cetyl alcohol, CieH^O. = (CH2)i4, which occurs in smaller quantities

CH2.OH

in beeswax, and was found by LUDWIG and v. ZEYNEK in the fat from dermoid
cysts — though this is denied by AMESEDER,1 — forms white, transparent, odorless,
and tasteless crystals which are insoluble in water but dissolve easily in alcohol
and ether. Ethal melts at 49.5° C.

SPERMACETI-OIL yields on saponification valeric acid, small amounts of solid
fatty acids, and PHYSETOLEIC ACID. This acid, which has, like hypogaeic acid,
the composition Ci6H3002, occurs also, as found by LjuBARSKY,2 in considerable
amounts in the fat of the seal. It forms colorless and odorless needle-shaped
crystals which easily dissolve in alcohol and ether and melt at 34° C.

BEESWAX may be treated here as concluding the subject of fats. It con-
tains three chief constituents: (1) CEROTIC ACID, C26H8202,3 which occurs as cetyl
ether in Chinese wax and as free acid in ordinary wax. It dissolves in boiling
alcohol and separates as crystals on cooling. The cooled alcoholic extract of
wax contains (2) CEROLEIN, which is probably a mixture of several bodies, and
(3) MYRICIN, which forms the chief constituent of that part of wax which is
insoluble in warm or cold alcohol. Myricin consists chiefly of palmitic-acid
ester of melissyl (myricyl) alcohol, CsoHei.OH. This alcohol is a silky, shining,
crystalline body melting at 85° C. DUNHAM 4 has found carnaubic acid, C^H^O-.
in a phosphatide from the ox kidney.

2. Phosphatides.

In close relation to the fats stands a group of esters containing
nitrogen, phosphoric acid and fatty__acid radicals. The representative
of this group longest known is lecithin. This latter is an ester combina-
tion of a nitrogenous base, choline, with a fatty acid-glycerophosphoric
acid, and THUDICHUM 5 has shown that a large number of more or less
analogous bodies occur in the animal body, especially in the brain.
All of these bodies have received the name phosphatides.

Those phosphatides which contain only one phosphoric acid radical
in the molecule are called monophosphatides; those with two such radicals
diphosphatides. The monophosphatides may contain one, two or more

1 Ludwig and v. Zeynek, Zeitschr. f. physiol. Chem. 23; Ameseder, <ibid., 52.

2 Journ. f. prakt. Chem. (N. F.), 57.

3 See Henriques, Ber. d. deutsch. chem. Gesellsch., 30, 1415.

4 Journ. of biol. Chem., 4.

6J. L. W. Thudichum, Die chemische Konstitution des Gehirns des Menschen,
etc., Tubingen, 1901.

240 ANIMAL FATS AND PHOSPHATIDES.

atoms of nitrogen in the molecule, and hence we differentiate between
monamido- (P : N = 1 : 1) , diamido- (P : N = 1 : 2) , triamido- (P : N = 1 : 3)
monophosphatides, etc.

So also may the diphosphatides contain 1, 2 or 3 atoms of nitrogen
for every 2 atoms of phosphorus (mono- di- or triaminodiphosphatides) .
Phosphatides with 4 or more atoms of nitrogen for every atom of phos-
phorus are also claimed to occur, but these statements seem to be uncer-
tain. On the other hand, according to THUDICHUM, non-nitrogenous
phosphatides occur in the brain; but if such be true these bodies must
not, for the present at least, be classified as phosphatides.

The phosphatides thus far investigated seem to be chiefly ester com-
binations between nitrogenous bases and fatty acid-glycerophosphoric
acid. According to THUDICHUM phosphatides exist which contain no
glycerin group and the CARNAUBON obtained by DUNHAM l from beef
kidneys seems to be such a phosphatide. The fatty acids occurring in
the phosphatides may be of different kinds. It seems that at least
one oleic acid radical, or another still less saturated fatty acid, occurs
in most of the phosphatides; still we know of phosphatides that con-
tain only saturated fatty acids. On this account the phosphatides may
be divided into saturated and unsaturated phosphatides. The unsaturated
add iodine, take up oxygen from the air and are auto-oxidizable and
are changed readily. They also give a beautiful reaction with PETTEN-
KOFER'S bile-acid test.

Choline has generally been obtained as a basic constituent of the
phosphatides. Still other not sufficiently studied bases, have been found
in the plant as well as animal phosphatides and according to TRIER2
aminoethyl alcohol is a probable generally distributed component of the
lecithins (phosphatides) .

The phosphatides are very widely distributed in the plant as well as
the animal kingdom and they must undoubtedly exist as primary cell
constituents. We differentiate between such cell constituents which seem
to be absolutely necessary for the life of the cells, and those which are
stored up as reserve material, or are products of metabolism. The first,
which seem to occur in all developing cells, have been called primary
by KossEL3 wrhile he calls the others secondary. The question as to
the division of the known cell constituents into the primary or secondary
groups in the above sense, cannot be answered positively in many cases.
In the primary group besides water and mineral bodies we include pro-
teins of various kinds, nucleic acids and the so-called lipoids (see below)
to which the phosphatides belong.

1 Zeitschr. f. physiol. Chem., 64, 303 (1910).

2 Ibid., 73, 76 and 80.

3 Verhandl. d. physiol. Gesellsch. zu Berlin, 1890-91.

PHOSPHATIDES. 241

Attention has been called in Chapter I to the importance of the phos-
phatides (lipoids) for the limiting layer of the cells as well as for the
osmotic processes 'and for the metabolism of the cells. The unsat-
urated, readily oxidizable phosphatides also play a possible role as oxygen
carriers and the phosphatides are undoubtedly of great importance as
constituents of the food-stuffs. There is also no doubt that they are
very important for development and growth. It has been found that
the amount of phosphatides is especially abundant in the new-born,
and that these latter, to a certain extent, bring into the world a store of
phosphatides and this store diminishes during growth.1

The phosphatides seem to be closely related to 'one another; they
influence the solubility and precipitation properties of one another, and
are generally precipitated as mixtures which are extremely difficult
to separate into individual constituents. They are also amorphous,
and readily oxidized, and it is easy to understand* why their preparation
in a pure state is so extremely difficult. Under these circumstances
we have no sufficient guarantee as to their chemical individuality, and
the description of their properties and composition must be accepted
with some reservation.

The phosphatides are generally amorphous, colorless or yellowish;
they melt, on warming, and burn. As a rule they are insoluble in water
and swell up therein forming colloidal solutions, which are precipitated by
certain salts. The phosphatides as above stated, belong to the lipoids
and it is for this reason that each phosphatide is dissolved by at least one
of the solvents for fats (alcohol, ether, benzene, petroleum ether, etc.).
The lipoid group cannot be otherwise characterized. Originally we
included in this group, bodies similar to fat or in certain respects related
to the fats such as phosphatides, cholesterin and cerebrosides, but later
the conception has been developed and now we consider as lipoids
those bodies that are soluble in ether or equivalent. Under these
circumstances, as the diverse known and unknown bodies, such as lactic
acid, phenols, alkaloids and extractive bodies of various kinds may
belong to the lipoid group there does not seem to be any sense in speaking
of a special lipoid group, and especially from a chemical standpoint it
would be better to drop the name entirely.

The various phosphatides shoiT a different behavior toward the
solvents for lipoids, namely some are soluble in ether while others
are insoluble therein, etc., and these differences are important for their

1 In regard to the quantity and importance of the phosphatides (the lecithins)
see Siwertzow, Bioch. CentralbL, 2, 310; Glikin, Bioch. Zeitschr., 4 and 7; Nerking,
ibid,, 10; Stoklasa, Ber. d. d. chem. Gesellsch., 29, Wien. Sitz. Ber., 104; Zeitschr.
f. physiol. Chem. 25; Danilewsky, Compt. Rend., 121 and 123 and W. Koch., Zeitschr.
f. physiol. Chem., 37; Kyes, ibid., 41 and Berl. klin. Wochenschr., 1904.

242 ANIMAL FATS AND PHOSPHATIDES.

preparation. They are generally all precipitated from their solution
by acetone although not completely, and this behavior is also of especial
importance in their preparation. The phosphatides 'are also nearly all
precipitated by metallic salts, especially by platinum chloride and cad-
mium chloride, and this method is also often used in their preparation.
The usefulness - of this method has been questioned at least for certain
phosphatides, since ERLANDSEN l showed that a decomposition occurs.

ERLANDSEN has also found that when finely divided heart-muscle, dried in
the air, is completely extracted with ether and then with alcohol, the first extract
contains the monophosphatides, and the alcohol extract contains the diamino
phosphatides which w.ere not free in the tissues, but existed in the combined state.
Whether this observation is of general importance in the preparation of pure
phosphatides remains to be seen.

As the phosphatides among themselves are rather difficult to char-
acterize and as there is^a question whether a pure phosphatide has thus far
been prepared it seems of little interest to give a review here of the division
of the isolated phosphatides among the different groups. In this chapter
we will only discuss the three most studied phosphatides, namely, lecithin,
cephalin and cuorin; the others will be treated of in the respective chapters.
Lecithins. In correspondence wdth the generally accepted view
lecithin is a monoaminomonophosphatide, which forms an ester com-
pound of glycerophosphoric acid substituted by two fatty-acid radicals
with a base called choline,2 hence there must exist several groups of
lecithins. According to the kind of fatty acid contained in the lecithin
molecule it is possible to have various lecithins, such as stearyl-, palmityl-,
and oleyl-lecithins. According to THUDICHUM 3 every true lecithin always
contains at least one oleic-acid radical. According to the investigations
of HENRIQTJES and HANSEN, COUSIN and ERLANDSEN,4 there is no ques-
tion that the so-called lecithin of the egg-yolk and muscles must contain
a fatty acid, still less saturated than oleic acid. All lecithins are mon-
aminophosphatides, according to the following type:

CH2 — 0 — fatty-acid radical.
CH — 0 — fatty-acid radical.
CH2— ON

HO-^PO

XC2H4-(X o

Nf(CH3)3
N

1 Zeitschr. f. physiol. Chem., 51.

2 Strecker, Annal. d. Chem. u. Pharm., 148; Hundeshagen, Journ. f. prakt. Chem.
(N. F.), 28; Gilson, Zeitschr. f. physiol. Chem., 12. A different view is held by
Malengreau and Pridgent, ibid., 77.

1 Thudichum, Die chemische Konstitution des Gehirns des Menschen, etc., Tubingen,
1901.

4 Henriques and Hansen, Skand. Arch. f. Physiol., 14 (1903); Cousin, Compt.
Rend., 137; Erlandsen, Zeitschr. f. physiol. Chem., 51.

LECITHINS. 243

The various lecithins stand close to each other in regard to constitu-
tion. The amount of phosphorus varies between 3.7-3.97 per cent and
the amount of nitrogen between 1.7-1.9 per cent. The so-called
di-stearyl-lecithin studied by HOPFE-SEYLER and DIACONOW/ which
probably has a different structure, has the formula C44HgoNP09. ERLAND-
SEN gives the formula C43HsoNPO9 for the lecithin isolated by him from
the heart muscles.

On saponification with alkalies or baryta-water, lecithin yields fatty
acids, glycerophosphoric acid, and choline. It is remarkable that in
the cleavage of lecithins a smaller amount of nitrogen than corresponds
to the choline is obtained. MAC LEAN 2 who has especially investigated
this could not re-obtain the total nitrogen in the lecithins as choline but
only a part thereof — from heart muscle lecithin, 42 per cent, and from egg-
yolk lecithin, 65 per cent. He is therefore of the opinion that the choline
group is not the only nitrogenous group in the lecithins and that there-
fore the generally accepted formula for lecithin is incorrect. TRIER3
has indeed obtained aminoethyl alcohol as a cleavage product from sev-
eral phosphatides, which he calls lecithins, but because of the difficulty
in preparing phosphatides in a pure condition we are not sure that he was
working with pure substances. Lecithin is slowly decomposed by
dilute acids. Besides small quantities of glycerophosphoric acid we
have large quantities of free phosphoric acid split off. The lecithins
are also decomposed by enzymes (lipase) with the splitting off of fatty
acids.

Lecithin is optically active, and as the glycerophosphoric acid which
can be split off is also active, WILLSTATTER and LUDECKE 4 claim that the
phosphoric acid is not bound on the middle unsymmetric CH group, but
rather at the end CEb group of glycerin.

Lecithin, according to HOPPE-SEYLER,S is found in nearly all animal
and vegetable cells thus far studied, and also in nearly all animal fluids.
It is especially abundant in the brain; nerves, fish eggs, yolk of the egg,
electrical organs of the Torpedo electricus, semen, and pus, and also in
the muscles and blood-corpuscles, blood-plasma, lymph, milk, especially
woman's milk, and bile. Lecithin is also found in different pathological
tissues or liquids. As the presence of lecithin is only indirectly deter-
mined by the detection of phosphorus in organic combinations, it must
be borne in mind that the above assertions relate chiefly to the occur-
rence of phosphatides.

1 Hoppe-Seyler, Med. chem. Unters., Heft 2 and 3.

2 Zeitschr. f . physiol. Chem., 59 and Bioch. Centralbl., 9.
3 1. c., Zeitschr. f. physiol. Chem., 73, 76 and 80.

4 Ber. d. d. chem. Gesellsch., 37.

6 Physiol. Chem. Berlin, 1877-81, p. 57.

244 ANIMAL FATS AND PHOSPHATIDES.

The same also applies to the claims as to the quantity of lecithin,
in various organs and tissues as well as in different ages. In these cases
the lecithin has not been prepared in a pure state, and the determina-
tions represent only the approximate quantity of phosphatides. These
determinations of SIWERTZOW, GLIKIN and NERKiNG,1 show that lecithins
(phosphatides) occur abundantly in the bone marrow, suprarenal capsule,
heart and lungs, besides in the spinal marrow, brain, and egg, and also
that the quantity varies strikingly in different varieties of animals.
NERKING found 41.7 per cent lecithin in the bone marrow and 21.33
per cent in the suprarenal capsule of the sea-urchin when calculated on
the living organs, while the corresponding results in the rabbit were 2.71
and 2.39 per cent, respectively.

The statements as to the properties of the lecithins apply chiefly to
the lecithin of the hen's egg, which since HOPPE-SEYLER and DIACONOW'S
time has been considered as distearyl-lecithin without any positive founda-
tion. Other lecithin preparations correspond essentially with this, and
certain differences between the various lecithins may be possibly due
to decomposition products or to admixture with other phosphatides.
It is still questioned whether the so-called distearyl-lecithin is a unit
body or not.

Lecithin may be obtained in grains or warty masses composed of small
crystalline plates by thoroughly cooling its solution in strong alcohol. In
the dry state it has a waxy appearance, is plastic, but forms pulverizable
masses when dried in vacuum, and is soluble in alcohol, especially on
heating (to 40-50° C.) ; it is less soluble in ether. It is dissolved also by
chloroform, carbon disulphide, benzene, and fatty oils. The solution
of lecithin from egg-yolk is dextrorotatory (ULPiANi2). P. MAYERS
claims to have prepared racemic lecithin from ordinary lecithin, and
Z-lecithin from the r-lecithin by cleavage with lipase. As he did not
make use of pure lecithin it is difficult to judge his- results. The solu-
tion of lecithin in alcohol-ether or chloroform is precipitated by acetone,
although not completely. It swells in water to a pasty mass which shows
under the microscope slimy, oily drops and threads, so-called myelin
forms (see Chapter XI). On warming this swollen mass or the concen-
trated alcoholic solution, decomposition takes place with the produc-
tion of a brown color. On allowing the solution or the swollen mass to
stand, decomposition takes place and the reaction becomes acid. Accord-
ing to the investigations of LONG4 the lecithins seem to be much more

1 Siwertzow, see Biochem. Zeitschr., 2, p. 310; Glikin, Biochem. Zeitschr., 4 and
7; Nerking, ibid., 10.

2 Chem. Centralbl, 1901, 2, 30 and 193.

3 Biochem. Zeitschr., 1.

4 Journ. of Amer. chem. Soc., 30, 1908.

LECITHINS, 245

resistant than was generally believed , and further investigations with
pure lecithin are desirable.

With considerable water the lecithin gives an emulsion or colloidal
solution which is not only precipitated by salts with divalent cations,
Ca, Mg, and others as claimed by W. KOCH, but is also precipitated accord-
ing to LONG and F. GEPHART1 by salts with monovalent cations, although
slowly. In putrefaction, lecithins yield glycerophosphoric acid and choline;
the latter further decomposes with the formation of methylamine, ammonia,
carbon dioxide, and marsh gas (HASEBROEK 2) . If dry lecithin be heated
it decomposes, takes fire, and burns, leaving a phosphorized ash. On
fusing with caustic alkali and saltpetre it yields alkali phosphates.

Lecithins combine with acids and bases. The compound with hydro-
chloric acid gives with platinum chloride a double salt which is insoluble
in alcohol, soluble in ether, and which contains 10.2 per cent platinum
(for distearyl-lecithin) . The cadmium-chloride compound, whose com-
position has been found somewhat variable by different investigators
is soluble with difficulty in alcohol, but dissolves in a mixture of carbon
disulphide and ether or alcohol. A solution of lecithin in alcohol is not
precipitated by lead acetate and ammonia.

Lecithins (and the same applies to the phosphatides in general) are
easily carried down during the precipitation of other compounds, such
as the protein bodies, and may therefore very greatly change the solubil-
ilities of other bodies. It is not known whether we are here dealing
with an adsorption or a chemical combination, and the conditions are
not the same in all cases. The combination with protein, the vitellines
and lecithalbumins have been discussed in a previous chapter, and
attention is there called to the necessity for more thorough investigation
of this subject. Further investigations of the so-called lecithin-sugar
(BING) is also desirable, as we know nothing definite as to its nature.
According to the investigations of WINTERSTEIN, HIESTAND and E.
SCHULZE, lecithins (phosphatides) containing carbohydrates occur in
the plant kingdom, and contain about 20 per cent carbohydrate. We
are still not decided whether we are here dealing with combinations
or admixtures.3 The same is true for the iron content of the lecithins or
phosphatides as observed by

1 W. Koch., Zeitschr. f. physiol. Chem., 37; Long and Gephart, Journ. of Amer.
Chem. Soc., 30; see also Forges and Neubauer, Biochem. Zeitschr., 7.

2 Zeitschr. f. physiol. Chem., 12.

3 Winterstein and Hiestand, Zeitschr. f. physiol. Chem., 47 and 54; Schulze, ibid.,
52 and 55; V. Njegovan, ibid., 76.

4 Ber. d. d. chem. Gesellsch., 41.

246 ANIMAL FATS AND PHOSPHATIDES.

Various methods have been suggested by STRECKER, HOPPE-SEYLER
and DIACONOW, THUDICHUM, GILSON, ZUELZER and BERGELL 1 for the
preparation of the lecithins. As none of these yield a positively pure
product we will here only mention them. According to ERLANDSEN'S
experience all methods which are based upon the precipitation of the
lecithin as a metallic compound should be avoided. The best method
depends upon the solubility of the lecithin in alcohol and in ether in
the cold and its precipitation by acetone (ERLANDSEN, H. E. ROAF and
E. EDIE 2) . The work of ERLANDSEN is especially referred to in the
preparation of lecithins.

For the present we have no quantitative method for estimating
lecithin. The methods used in the past, when the amount of lecithin
was calculated from the amount of phosphorus contained in the alcohol-
ether extract is useless, as in this case the phosphorous content of all
the phosphatides is determined and not alone of the lecithins. Even
the detection of choline is not evidence, as this base probably occurs also
in other phosphatides. In the detection of choline the double platinum
compound is ordinarily prepared, and this can be done as described
below. In special determinations of lecithin and cephalin KOCH used
to heat with hydroiodic acid, and determined the methyl groups split
off below 240° and those at about 300°. Instead of this he recommends
with WOODS 3 to separate the two by precipitation in alcoholic solution.
while boiling, with alcoholic lead acetate solution and a little ammonia,
which precipitates only the cephalin.

Of the cleavage products of the lecithins choline is of especially
great interest.

Choline (trimethyloxyethyl ammonium hydroxide),

/CH2.CH2.(OH)
C5H15N02, = HO.N<

\CH3)3,

stands in close "relation to jthe poisonous base neurine (trimethylvinyl

/(CHs)s

ammonium hydroxide), HO.N<T , which according to BRIEGER

XCH:CH2

can be formed from choline by the action of bacteria, and also to mus-

XCH3)3
carine, HO.N\ , which is the aldehyde of choline and occurs in

XCH2CHO

/ ° \
the fly agaric, and also to betaine, trimethyl glycocoll, (CH3)3N<( /CO,

X

1 Strecker, Annal. d. Chem. u. Pharm., 148; Hoppe-Seyler and Diaconow, 1. c.;
Thudichum, 1. c.; Gilson, Zeitschr. f. physiol. Chem., 12; Zuelzer, ibid., 27; Bergell,
Ber. d. d. chem. Gesellsch., 33.

2Erlandsen. 1. c.; Roaf and Edie, Thompson Yates Laboratory Reports, Vol. 6
part I, 1905.

3 Koch and Woods, Journ. of biol. Chem., 1.

CHOLINE. 247

which may be considered as the anhydride of the acid corresponding to
choline. Muscarine and betaine can be obtained from choline on oxida-
tion. Choline yields trimethylamine as a decomposition product, and this
seems to be formed in the transformation of choline in the animal body.

Choline occurs in the plant kingdom as well as in the animal kingdom.
MOTT and HALLIBURTON have repeatedly found choline in the blood in
degenerative diseases of the nervous system. It was first shown also in
normal blood by MARINO Zuco, 1 and this investigator first found it in
the suprarenal capsule, but designated it neurine. LOHMANN found it
later in this organ, and recently it has been found in various organs by
other investigators, especially by C. SCHWARZ and v. FURTH. The fact
that choline is a cleavage product of lecithin in the animal, and that it is
antagonistic to adrenalin (of the suprarenal capsule) by its depressing
action upon the blood pressure, and that it has an exciting action upon
certain secretions (LOHMANN, . THEISSIER and THEVENOT, v. FURTH
and SCHWARZ 2), gives choline great physiological importance. The
physiological action of choline is still very much disputed.

Choline is a syrupy fluid, readily miscible with absolute alcohol.
Hydrochloric acid gives with it a compound which is very soluble in water
and alcohol, but insoluble in ether, chloroform, and benzene. This com-
pound forms a double combination with platinum chloride, is soluble in
water, insoluble in absolute alcohol and ether, and crystallizing from
water in monoclinic system and this form is strongly double-refractive.
From a mixture of water and alcohol it crystallizes in the regular form
(octahedral). Both forms can be changed from one to the other
and are used according to KAUFFMANN and VORLANDER 3 in the detec-
tion of choline. Choline also forms a crystalline double compound
with mercuric chloride and with gold chloride. Choline is precipitated
by potassium iodide and iodine (GULEWITSCH), and potassium triiodide
can be used for the quantitative estimation of this base (STANEK4).
On heating the free base it decomposes into trimethylamine, ethylene
oxide, and water.

In preparing choline from lecithins, and also for the detection of
lecithin in an alcohol-ether extract, proceed as follows: The residue from

1 Mott and Halliburton, Philos. Trans., Ser. B, 191 (1899) and 194 (1901); Marino
Zuco, see Maly's Jahresber., 24, pp. 181 and 698.

2 Lohmann, Pfliiger's Arch., 118 and 122; v. Fiirth and Schwarz, ibid., 124, which
also contains the literature.

3 See Gulewitsch, Zeitschr. f. physiol. Chem., 24; Kauffmann and Vorlander, Ber.
d. d. chem. Gesellsch., 43.

4 Gulewitsch, Zeitschr. f. physiol. Chem., 24; Stanek, ibid., 56. In regard to the
quantitative estimation see also Kiesel, ibid., 53; Stanek, ibid., 54; Moruzzi, ibid.,
55; and MacLean, ibid., 55.

248 ANIMAL FATS AND PHOSPHATIDES.

the above, or the solid lecithin is boiled one hour with baryta-water,
filtered, and the excess of baryta precipitated by CC>2; filter while hot,
concentrate to a syrup, and extract with absolute alcohol, when the
insoluble barium glycerophosphate remains; then precipitate the filtrate
with an alcoholic platinum chloride solution.

€H2.OH

Glycerophosphoric acid, C3H9P06 =CH.OH , is a bibasic acid which prob-

CH2— 0\
OH-)PO
OH/

ably occurs in the animal fluids and tissues only as a cleavage product of lecithins.
According to WILLSTATTER and LUDECKE x the glycerophosphoric acid split off
from lecithins is optically active. Its barium and potassium salts are levorotatory,
and behave in certain respects differently from the corresponding salts of syn-
thetically prepared glycerophosphoric acid. The Ba and Ca salts of glycero-
phosphoric acid are crystalline and are more soluble in cold than in warm water.
The acid itself is a syrupy fluid.

Cephalin is also a monoaminophosphatide whose formula, based upon
the investigations of THUDICHUM, KOCH, THIERFELDER and STERNA is
probably C42Hs2NPOi3. The views of these investigators as to the con-
stitution of this body, which is difficult to purify, differ very considerably.
According to THUDICHUM, on cleavage it yields neurine, glycerophosphoric
acid, stearic acid, and a specific fatty acid, cephalic add. According to
KOCH it contains, on the contrary, only one methyl group attached to
nitrogen, and is therefore probably dioxystearylmonomethyl lecithin.
FRANKEL and DIMITZ found no choline, while according to COUSIN it
yields, like lecithin, stearic acid, an unsaturated fatty acid, glycero-
phosphoric acid and choline as decomposition products. The glycero-
phosphoric acid from brain cephalin gives, according to FRANKEL and
DIMITZ, a dextrorotatory Ba salt and is therefore not identical with the
glycerophosphoric acid from lecithin. According to these investigators
the cephalin of the human brain is a mixture of palmityl and stearyl-
cephalin. Besides these two fatty acids cephalin also contains an unsat-
urated fatty acid, cephalinic add, which according to PARNASS is related
to leinoleic acid or perhaps identical therewith.

From the investigations carried on thus far we can conclude that
cephalin differs from lecithin in that it contains cephalinic acid, another
glycerophosphoric acid and probably no choline but a monomethyl
base. Cephalin has probably never been obtained in a pure form.

1 Ber d. d. chem. Gesellsch., 37.

* Thudichum, 1. c.; Koch, Zeitschr. f. physiol. Chem., 36; Thierf elder and Stern,
ibid. 53.

3Frankel and Dimitz, Bioch. Zeitschr., 21; Parnas, ibid., 22; Cousin, Compt.
Rend. soc. biol., 62.

CEPHALIN AND CUOR1N. 249

The cephalin from the brain has, according to FALK/ a different composi-
tion than that of the nerves and certain observations indicate that there
are several cephalins.

Cephalin occurs quite abundantly in the brain and also in nerves
and in the egg-yolk. The statements as to its further occurrence in the
animal kingdom require substantiation.

Cephalin is amorphous, not very plastic, and more easily triturated
than lecithin. It is readily soluble in cold ether, in chloroform and
benzene but differs from lecithin by being insoluble or soluble with difficulty
in alcohol. As unsaturated phosphatide it gives, like lecithin, a positive
reaction with PETTENKOFER'S bile-acid test. The cadmium- and plat-
inum chloride combinations are soluble in ether. Cephalin is obtained
from the brain, after dehydration with acetone, by extracting with ether
and precipitating the concentrated ethereal extract with alcohol. In
regard to the preparation and detection of cephalin we must refer to more
extensive hand-books.

The purest phosphatide prepared thus far seems to be cuorin, dis-
covered by ERLANDSEN.

Cuorin, C7iHi25NP202i, is a monaminodiphosphatide prepared by
ERLANDSEN 2 from the heart muscle of the ox, and which has an iodine
equivalent of 101. It yields as cleavage products 3 molecules fatty acids
of unknown nature, partly or entirely belonging to the series CnH2n-402
and CnH2n-6O2; also glycerin, phosphoric acid and a base which is
not well known, but it is not choline. Cuorin is autooxidizable, and gives
PETTENKOFER'S bile-acid test.

Cuorin is amorphous, yellowish-brown and similar to rosin. It
gives a neutral solution with water which is like an emulsion. Cuorin
does not reduce FEHLING'S solution, even after boiling with acids. It is
soluble in ether, chloroform, petroleum ether and carbon disulphide.
It dissolves with difficulty in benzene; it is insoluble in ethyl and methyl
alcohol and in acetone. Cuorin is precipitated from its alcohol-ether
solution by cadmium or platinum chloride.

1 Bioch. Zeitschr., 13 and 16.

2 Zeitschr. f. physiol. Chem., 51, where the method of preparation is described.

CHAPTER V.

THE BLOOD

THE blood is. ; to be considered from a certain standpoint as a fluid
tissue; it consists of a transparent liquid, the blood-plasma, in which
a vast number of solid particles, the red and white blood-corpuscles (and
the blood-plates), are suspended.

Outside of the organism the blood, as is well known, coagulates more
or less quickly; but this coagulation is accomplished generally in a
few minutes after leaving the body. All varieties of blood do not coagulate
with the same degree of rapidity. Some coagulate more quickly, others
more slowly. In vertebrates with nucleated blood-corpuscles (birds,
reptiles, batrachia, and fishes) DELEZENNE has shown that the blood
coagulates very slowly if it is collected under such precautions that it
does not come in contact with the tissues. On contact with the tissues or
with their extracts it coagulates in a few minutes. The blood with
non-nucleated blood-corpuscles (mammals), on the contrary, coagulates
very rapidly. The coagulation of the blood in these cases may also be
somewhat retarded by preventing the blood from coming in contact
with the tissues (SPANGARO, ARTHtis1). Among the varieties of blood
of mammals thus far investigated the blood of the horse coagulates most
slowly. The coagulation may be more or less retarded by quickly cool-
ing; and if we allow equine blood to flow directly from the vein into a
glass cylinder which is not too wide and which has been cooled, and let it
stand at 0° C., the blood may be kept fluid for several days. An upper
amber-yellow layer of plasma gradually separates from a lower red layer
composed of blood-corpuscles with only a little plasma. Between these
is observed a whitish-gray layer which consists of white blood-corpuscles*

The plasma thus obtained and filtered is a clear amber-yellow alkaline
(toward litmus) liquid which remains fluid for some time when kept
at 0° C., but soon coagulates at the ordinary temperature.

The coagulation of the blood may be prevented in other ways. After
the injection of peptone, or, more correctly, proteose solutions into

1 Delezenne, Compt. rend. soc. de biol., 49; Spangaro, Arch. ital. de Biol., 32;
Arthus, Journ. de Physiol. et Pathol., 4.

250

PREVENTION OF COAGULATION. 251

the blood (in the living dog), it does not coagulate on leaving the veins
(FANO, ScHMiDT-MtiLHEiM1). The plasma obtained from such blood
by means of centrifugal force is called peptone-plasma. According to
ARTHUS and HTJBER 2 the caseoses and gelatoses act like fibrin proteosc
in dogs. Eel serum and certain lymph-forming extracts of organs
(see Chapter VI) have an analogous action. The coagulation of the
blood of warm-blooded animals is prevented by the injection of an
effusion of the mouth of the officinal leech or a solution of the active
substance of such an infusion, hirudin (FRANZ), into the blood current
(HAYCRAFT3). If the blood is allowed to flow directly, while stirring
it, into a neutral salt solution — best a saturated magnesium-sulphate
solution (1 vol. salt solution and 3 vols. blood) — we obtain a mixture
of blood and salt which remains uncoagulated for several days. The
blood-corpuscles, which, because of their adhesiveness and elasticity,
would otherwise easily pass through the pores of the filter-paper, are
made solid and stiff by the salt, so that they may be easily filtered
off. The plasma thus obtained, which does not coagulate spontaneously,
is called salt-plasma.

An especially good method of preventing coagulation of blood con-
sists in drawing the blood into a dilute solution of potassium oxalate,
so that the mixture contains 0.1 per cent oxalate (ARTHUS and PAGES4).
The soluble calcium salts of the blood are precipitated by the oxalate,
and hence the blood loses its coagulability. On the other hand, HORNE 5
found that chlorides of calcium, barium, and strontium, when present
in large amounts (2-3 per cent), may prevent coagulation for several
days. According to ARTHUS 6 a non-coagulable blood-plasma may be
obtained by drawing the blood into a sodium-fluoride solution until it
contains 0.3 per cent NaFl.

On coagulation there separates in the previously fluid blood an insoluble
or a very difficultly soluble protein substance, fibrin. When this separa-
tion takes place without stirring, the blood coagulates in a solid mass,
which, when carefully severed from the sides of the vessel, contracts,
and a clear, generally yellow-colored liquid, the blood-serum, exudes.
The solid coagulum which encloses the blood-corpuscles is called the
blood-clot (placenta sanguinis). If the blood is beaten during coagula-
tion, the fibrin separates in elastic threads or fibrous masses, and the

JFano, Arch f.. (anat. u.) Physiol., 1881; Schmidt-Mulheim, ibid., 1880.

2 Arch, de Physiol. (5), 8.

3 Haycraft, Proc. Physiol. Soc., 1884, 13, and Arch. f. exp. Path. u. Pharm., 18;
Franz, Arch. f. exp. Path. u. Pharm., 49.

4 Archives de Physiol. (5), 2, and Compt. Rend., 112.
6 Journ. of Physiol., 19.

6 Journ. de Physiol. et Path., 3 and 4.

252 THE BLOOD.

defibrinated blood which separates is sometimes called cruor l and con-
sists of blood-corpuscles and blood-serum, while uncoagulated blood
consists of blood-corpuscles and blood-plasma. The essential chemical
difference between blood-serum and blood-plasma is that the blood-
serum does not contain even traces of the mother-substance of fibrin,
the fibrinogen, which exists in the blood-plasma, while the serum is pro-
portionally richer in another body, the fibrin ferment (see below).

I. BLOOD-PLASMA AND BLOOD-SERUM.
The Blood-plasma.

In the coagulation of the blood a chemical transformation takes
place in the plasma. A part of the proteins separate as insoluble fibrin.
The albuminous bodies of the plasma must therefore be first described.
They are, as far as we know at present, fibrinogen, nucleoprotein, ser-
globulins, and seralbumins.

Fibrinogen occurs in blood-plasma, chyle, lymph, certain transudates
and exudates, in bone-marrow (P. MULLER), and perhaps also in other
lymphoid organs. The seats of formation of fibrinogen are, according
to MATHEWS, the leucocytes, especially of the intestine, according to
MULLER, the bone-marrow and probably other lymphoid organs such
as the spleen and lymph glands, and according to DOYON and NOLF,
the liver. The statement that the intestinal wall is a seat of formation
of fibrinogen, a view that had been held by DASTRE, is substan-
tiated not only by the direct researches of MATHEWS, but also by the
older and substantiated opinion that the blood from the mesentery vein
is richer in fibrinogen than the arterial blood. This origin of fibrinogen
has been shown to be improbable by the recent researches of DOYON, CL.
GAUTIER and MOREL. The occurrence of fibrinogen in the bone-marrow
and other lymphoid organs as shown by MULLER, and an increase of
fibrinogen in the blood as well as in the bone-marrow of animals immunized
with certain bacteria, especially pus-staphylococci, indicates the forma-
tion of fibrinogen in this tissue. The relation between the quantity of
fibrin and leucocytosis as shown by many investigators such as LANG-
STEIN and MAYER, MORAWITZ and REHN, also indicate such a formation
of fibrinogen. The observations of DOYON, GAUTIER and MA WAS that
a rapid re-formation of fibrinogen takes place in splenectornized animals

1 The name cruor is used in different senses. We sometimes mean thereby only
the blood when coagulated in a red solid mass, in other cases the blood-clot after the
separation of the serum, and again the sediment consisting of red blood-corpuscles
which is obtained from defibrinated blood by means of centrifugal force or by letting
it stand.

FIBRINOGEN. 253

without any changes in the bone-marrow speak against the especially
great importance of the spleen and bone-marrow for the formation of
fibrinogen. That the liver takes part in the formation of fibrinogen
is implied by the fact that the quantity of fibrinogen in the blood
strongly diminishes after the extirpation of the liver (NOLF), and that
fibrinogen may indeed be entirely absent in the blood in phosphorus
poisoning (CoRiN and ANSIAUX, JACOB Y, DOYON, MOREL, and KAREFF x),
and that the blood of the hepatic vein, according to DOYON, MOREL
and KAREFF, is richer in fibrinogen than the blood from other vessels,
and finally according to WHIFFLE and HuRWiTZ2 in chloroform poison-
ing the fibrinogen content of the blood diminishes with the injury to
the liver and rises again with restitution of the organ.

Fibrinogen has the general properties of the globulins, but differs
from other globulins as follows: In a moist condition it forms white
flakes which are soluble in dilute common salt solutions, and which
easily conglomerate into tough, elastic masses or lumps. The solution
in 5-10 per cent NaCl coagulates on heating at 52-55° C., and the faintly
alkaline or nearly neutral weak salt solution coagulates at 56° C., or at
exactly the same temperature at which the blood-plasma coagulates.
Fibrinogen solutions are precipitated by an equal volume of a saturated
common salt solution, and are completely precipitated by adding an excess
of NaCl in substance (thus differing from serglobulin). A salt-free
solution of fibrinogen in as little alkali as possible gives with CaCb
a precipitate which contains calcium and soon becomes insoluble. In
the presence of NaCl or by the addition of an excess of CaCk the precipitate
does not appear.3 A neutral solution of fibrinogen is precipitated by
a concentrated solution of sodium fluoride when added in a sufficient quan-
tity. Fibrinogens from different kinds of blood behave somewhat dif-
ferently in this regard. According to HUISKAMP 4 fibrinogen from horse-
blood hardly dissolves in NaCl of 3-5 per cent at ordinary temperatures,
while it does dissolve at 40-45°. It also dissolves in ammonia of 0.05

1 P. Miiller, Hofmeister's Beitrage, 6; Mathews, Amer. Journ. of Physiol., 3; Nolf,
Bull. Acad., Roy. Belg., 1905, and Arch, intern, de Physiol., 3, 1905; Langstein, and
Mayer, Hofmeister's Beitrage, 5; Morawitz and Rehn, Arch. f. exp. Path. u. Pharm.,
58; Corin and Ansiaux, Maly's Jahresber., 24; Jacoby, Zeitschr. f. physiol. Chem.,
30; Doyon, Morel and Kareff, Compt. Rend., 140; Doyon, Morel, and Peju, Comp.
rend. soc. biolog., 58; Doyon, Cl. Gautier, and Morel ibid., 62; Doyon, Gautier
and Mawas, ibid., 64.

2 Doyon, Morel and Kareff, Journ. de Physiol., 8 (1906); Whipple and Hurwitz,
Journ. of exp. Med. 13. See also Meek, Amer. Journ. of Physiol., 30.

3 See Hammarsten, Zeitschr. f. physiol. Chem., 22; Cramer, ibid., 23.

4 Huiskamp, ibid., 44 and 46. In regard to fibrinogen the reader is referred to
the author's investigations. Pfliiger's Archiv., 19 and 22, and Zeitschr. f. physiol.
Chem., 28.

254 THE BLOOD.

per cent, and on the addition of 3-5 per cent NaCl this solution can be
neutralized. The fibrinogen prepared by HUISKAMP in this way retained
its typical properties. Fibrinogen differs from the myosin of the muscles,
which coagulates at about the same temperature, and from other pro-
tein bodies, in the property of being converted into fibrin under certain
conditions. Fibrinogen has a strong decomposing action on hydrogen
peroxide. It is quickly made insoluble by precipitation with water or
with dilute acids. Its specific rotation is (a)D=— 52.5° according to

MlTTELBACH.1

Fibrinogen may be easily separated from the salt-plasma or oxalate-
plasma by precipitation with an equal volume of a saturated NaCl solu-
tion. It must be observed that the oxalate-plasma can only be employed
after the precipitate, containing proenzymes, and produced by exposure
to cold, has settled and been filtered off. If this is not done then the
fibrinogen is always impure. For further purification the precipitate
is pressed, redissolved in an 8-per cent salt solution, the filtrate pre-
cipitated by a saturated salt solution as above, and after being treated
in this way three times, the precipitate at last obtained is pressed between
filter-paper and finely divided in water. The fibrinogen dissolves with
the aid of the small amount of NaCl contained in itself, and the solution
may be made salt-free by dialysing with very faintly alkaline water. The
fibrinogen can be almost freed from fibrin-globulin, which will be spoken
of later, by precipitating with double the volume of saturated sodium-
fluoride solution, redissolving in water with 0.05-per cent ammonia,
and then neutralizing this solution, treated with NaCl, and repeating
this several times. Fibrinogen may also, according to RE YE,2 be prepared
by fractionally precipitating the plasma with a saturated solution of
ammonium sulphate. We have no knowledge as to the purity of the
fibrinogen so prepared. The methods for the detection and quantitative
estimation of fibrinogen in a liquid were formerly based on its property
of yielding fibrin on the addition of a little blood, of serum, or of fibrin
ferment. REYE has suggested the fractional precipitation with ammonium
sulphate as a quantitative method. The value of this method has not
been sufficiently tested.

Fibrinogen stands in close relation to its transformation product,
fibrin.

Fibrin is the name of that protein body which separates on the so-
called spontaneous coagulation of blood, lymph, and transudates as well
as in the coagulation of a fibrinogen solution after the addition of serum
or fibrin ferment (see below).

If the blood is beaten during coagulation, the fibrin separates in
elastic, fibrous masses. The fibrin of the blood-clot may be beaten to

1 Zcitschr. f. physiol. Chem., 19.

2 W. Reye, Ueber Nachweis und Bestimmung des Fibrinogens, Inaug-Diss. Strass-
burg, 1898.

FIBRIN. 255

small, less elastic, and not particularly fibrous, lumps. The typical
fibrous and elastic white fibrin, after washing, stands, in regard to its
solubility, close to the coagulated proteins. It is insoluble in water,
alcohol, or ether. It expands in hydrochloric acid of 1 p. m., as, also in
caustic potash or soda of 1 p. m., to a gelatinous mass, which dissolves at
the ordinary temperature only after several days; but at the temperature
of the body it dissolves more readily, although still slowly. Fibrin may
be dissolved by dilute salt solutions, after a long time, at the ordinary
temperature, or much more readily at 40° C.; and this solution takes place,
according to ARTHUS and HUBERT and also DASTRE/ without the aid
of micro-organisms. This action is due to proteolytic enzymes car-
ried down by the fibrin or enclosed within the leucocytes (RuLOT2).
According to GREEN and DASTRE 3 two globulins are formed in the solu-
tion of fibrin in neutral salt solution, and according to RULOT also pro-
teoses (and peptones) on the solution of fibrin containing leucocytes.
Fibrin, like fibrinogen, decomposes hydrogen peroxide, due to a con-
tamination with catalases, but this property is destroyed by heating or
by the action of alcohol.

What has been said of the solubility of fibrin relates only to the typical
fibrin obtained from the arterial blood of oxen or man by whipping
and washing first with water and with common salt solution, and then
with water again. The blood of various kinds of animals yields fibrin
with somewhat different properties, and according to FERMI 4 pig-fibrin
dissolves much more readily than ox-fibrin in hydrochloric acid of 5 p. m.
Fibrins of varying purity or originating from blood from different parts
of the body have unlike solubilities.

The fibrin obtained by beating the blood, and purified as above
described, is always contaminated by secluded blood-corpuscles or
remains thereof, and also by lymphoid cells. It can be obtained pure
only from filtered plasma or filtered transudates. For the preparation
of pure fibrin, as well as for the quantitative estimation of it, the spon-
taneously coagulating liquid is at once, or the non-spontaneously coagu-
lating liquid only after the addition of blood-serum or fibrin ferment,
thoroughly beaten with a whale-bone, and the separated coagulum is
washed first in water and then with a 5-per cent common salt solution,
and again with water, and finally extracted with alcohol and ether. If
the fibrin is allowed to stand for some time in contact with the Hood
from which it was formed, it partly dissolves (fibrinolysis — DASTRE 5).
This fibrinolysis must be prevented in the exact quantitative estimation

1 Arthus and Hubert, Arch, de Physiol. (5) 5; Dastre, ibid., (5) 7.

2 Arch, intern, de Physiol., 1.

3 Green, Journ. of Physiol., 8; Dastre, 1. c.

4 Zeit^chr. f. Biologic, 28.

6 Archives, de Physiol. (5), 5 and 6.

256 THE BLOOD.

of fibrin (DASTRE). The blood constituents that are active in fibrinolysis
are still unknown, but they are without doubt of enzymotic nature.
It must be mentioned that a strong fibrinolysis takes place in blood
after acute phosphorus poisoning ( JACOB Y and others), after extirpation
of the liver (NOLF), and also when the coagulability of the blood has been
reduced by the injection of proteoses (NoLF, RULOT 1).

A pure fibrinogen solution may be kept at the ordinary temperature
until putrefaction begins without showing a trace of fibrin coagula-
tion. But if to this solution is added a water-washed fibrin-clot or a
little blood-serum, it immediately coagulates, and may yield a perfect
typical fibrin. The transformation of the fibrinogen into fibrin requires
the presence of another body contained in the blood-clot and in the serum.
This body, whose importance in the coagulation of fibrin was first observed
by BucHANAN2, was later rediscovered by ALEXANDER SCHMIDT^ and
designated as fibrin ferment or thrombin. The nature of this enzymotic
body has not been ascertained with certainty. Even after careful
purification it gives very faint protein reactions and it is a much disputed
question whether it is a globulin or a nucleoprotein. It is a fad that
powerfully active solutions of thrombin can be obtained that do
not give either the reactions for globulins or nucleoproteins. Fibrin fer-
ment is produced, according to PEKELHARiNG,4 by the influence of soluble
calcium salts on a preformed zymogen existing in the non-coagulated
plasma. SCHMIDT admits the presence of such a mother-substance
of the fibrin ferment in the blood, and calls it prolhrombin. The con-
version of this mother-substance into thrombin is a very complicated
process, which will be discussed under the coagulation of the blood.
Thrombin behaves like other enzymes in that the very smallest amount of
it produces an action, and its solution becomes inactive on heating. The
velocity of coagulation is dependent upon the quantity of thrombin,
and indeed a time law has been proposed for the action of thrombin.
According to FULD the action of thrombin, at least within certain limits,
follows SCHUTZ'S law, and according to STROMBERG the thrombin follows
in its action a time law, which at least in the beginning, corresponds to

1 Jacoby, Zeitschr. f. physiol. Chem., 30; Nolf, Arch, intern, de Physiol., 3, 1905;
Rulot, 1. c.

2 London Med. Gazette, 1845, 617. Cit. by Gamgee, Journal of Physiol., 1879.

3 Pfltiger's Arch., 6; see also Zur Blutlehre, 1892, and Weitere Beitrage zur Blut-
lehre, 1895.

4 Pekelharing, Verhandl. d. Kon. Akad. d. Wetensch. te Amsterdam, 1892, Deel 1;
ibid., 1895, and Centralbl. f. Physiol., 9; Wright, Proc. Roy. Irish Acad. (3), 2; The
Lancet, 1892, and On Wooldridge's Method, etc., British Med. Journal, 1891; Lilien-
feld, Hamatol. Untersuch. Arch. f. (Anat. u.) Physiol., 1892; Ueber Leukocyten und
Blutgerinnung, ibid.', Halliburton and Brodie, Journal of Physiol., 17 and 18; Huis-
kamp, Zeitschr. f. physiol. Chem., 32; Pekelharing and Huiskamp, ibid., 39.

FIBRIN FORMATION. 257

SCHUTZ'S law while on increasing dilution deviates more and more
and finally shows a proportionally slow, and more irregular procedure.
MARTIN 1 has found another law from experiments with plasma and snake-
poisons containing thrombin. According to him the behavior is as
follows : As in the casein coagulation with rennin, the celerity of coagula-
tion is inversely proportional to the quantity of ferment; and LOEB
has observed a similar conduct with invertebrates. The optimum of the
thrombin action lies at about 40° C.; at 70-75° C., in neutral solution,
the enzyme is destroyed. According to HOWELL and RETTGEE 2 throm-
bin, under proper conditions, can withstand boiling for a short time. The
question as to whether the thrombin found in different animals is the
same substance or whether we have several thrombins, has not been
decided. The latter is not improbable; nevertheless a definite specificity
of different thrombins has not been observed with certainty.

The isolation of thrombin has been tried in several ways. Ordinarily,
it may be prepared by the following method, proposed by ALEX. SCHMIDT:
Precipitate the serum or defibrinated blood with 15-20 vols. of alcohol
and allow it to stand a few months. The precipitate is then filtered
off and dried over sulphuric acid. The ferment may be extracted from
the dried powder by means of water. Other methods have been suggested
by HAMMARSTEN, PEKELHARING, and HOWELL.S According to a method
suggested by HAMMARSTEN a solution of thrombin so poor in lime salts
that it contains only 0.3-0.4 p. m. solids and about 0.0007 p. m. CaO
can be prepared.

If a fibrinogen solution containing salt, as above prepared, is treated
with a solution of thrombin, it coagulates at the ordinary temperature
more or less quickly and yields a typical fibrin. Besides the thrombin,
the' presence of neutral salts is necessary, for ALEX. SCHMIDT has shown
that fibrin coagulation does not take place without them. The presence
of soluble calcium salts is not, as is generally assumed, a positive con-
dition for the formation of fibrin, because, thrombin can transform
fibrinogen into typical fibrin in the absence of lime salts precipitable by
oxalate.4 The fibrin is not richer in lime than the fibrinogen used in its
preparation if the fibrinogen and thrombin solutions are employed as lime-
free as possible, and the view that the fibrin formation is connected with
a taking up of lime has been shown to be untenable (HAMMARSTEN).
The quantity of fibrin obtained on coagulation is always smaller than

1 Martin, Journ. of PhysioL, 32; Fuld, Hofmeister's Beitrage, 2; Loeb, ibid., 9;
Stromberg, Biochem. Zeitschr., 37.

2 Howell, Amer. Journ. of Physiol., 26; Rettger, iUd., 24.

3 Hammarsten, ibid., 18; Pekelharing, 1. c.; Howell, 1. c.

4 See Hammarsten, Zeitschr. f . physiol. Chem., 22, which also cites the works of
Schmidt and Pekelharing, and ibid., 28.

258 THE BLOOD.

the amount of fibrinogen from which the fibrin is derived, and we always
find a small amount of protein substance in the solution. It is therefore
not improbable that the fibrin coagulation, in accordance with the views
first proposed by DENIS, is a cleavage process in which the soluble fibrinogen
is split into an insoluble protein, the fibrin, which forms the chief mass,
and a soluble protein substance which is produced only in small amounts.
We find a globulin-like substance which coagulates at about 64° C. in
blood-serum as well as in the serum from coagulated fibrinogen solutions.
This substance is called fibrin-globulin by HAMMARSTEN. The investiga-
tions of HUISKAMP have shown that this substance is not formed as a
cleavage product from pure fibrinogen, but occurs in plasma or in fibrinogen
solutions not purified from sodium fluoride or perhaps in loose com-
bination with fibrinogen. The view that a cleavage takes place in the
coagulation of the fibrinogen has not been supported by these investi-
gations.1

Opinions are not unanimous in regard to the enzyme nature of throm-
bin and the enzymotic formation of fibrin, and there are, indeed, investiga-
tors who consider the coagulation as another process. A more thorough
discussion of this subject can take place only in connection with the
coagulation of the blood.

Nucleoprotein. This substance, which, as above-mentioned, is considered
by PEKELHARING and HUISKAMP as identical with the prothrombin or thrombin,
occurs in the blood-plasma as well as in the serum, and is precipitated from the
latter with the globulin. It is similar to the globulin in that it is readily soluble
in neutral salt solution, and can be completely salted out on saturation with
magnesium sulphate, and separates only incompletely on dialysis. It is much
less soluble than serglobulin in an excess of dilute acetic acid, and coagulates
at 65-69° C. C. G. LIEBERMEISTER 2 found only 0.08-0.09 per cent phosphorus
in the nucleoprotein, which indicates that the nucleoprotein was contaminated
with other proteins. He also found that the substance was soluble in acetic acid
with difficulty, a property which is used by PEKELHARING as an important means
of separating the compound proteins from the globulins.

Serglobulins, also called paraglobulin (KUHNE), fibrinoplastic substance
(ALEX. SCHMIDT), serum-casein (PANUM3), occur in the plasma, serum,
lymph, transudates and exudates, in the white and red corpuscles, and
probably in many animal tissues and form-elements, though in small
quantities. They are also found in the urine in many diseases.

The so-called serglobulin is without doubt not an individual sub-
stance, but consists of a mixture of two or more protein bodies which

^ee Hammarsten, Zeitschr. f. physiol. Chem., 28; Heubner, Arch. f. exp. Path.
u. Pharm., 49, and Zeitschr. f. physiol. Chem., 45; Huiskamp, ibid., 44 and 46.

2 Hofmeister's Beitrage, 8; Pekelharing and Huiskamp, 1. c. footnote 1, page 256.

'Kiihne, Lehrbuch d. physiol. Chem., Leipzig., 1866-68; Alex. Schmidt, Arch. f.
(Anat. u.) Physiol., 1861-62; Panum, Virchow's Arch., 3 and 4.

SERGLOBULINS. 259

cannot be completely and positively separated from each other. The
mixture of globulins obtained from blood-plasma or blood-serum by
saturation with magnesium sulphate or half-saturation with ammonium
sulphate consists of nucleoprotein, fibrin-globulin, and the true serglobulin
or mixture of globulins.

The nucleoprotein has been previously discussed. The fibrin-globulin,
which occurs in the serum only in small amounts, can be completely pre-
cipitated by NaCl. It has the general properties of the globulins, but
differs from the serglobulins by a lower coagulation temperature, 64-
66° C., and also in that it is precipitated by (NH^SCU even in 28 per
cent solution.

Serglobulins. If the globulin obtained by saturation with magnesium
sulphate is dialyzed, then, as has been known for a long time and further
substantiated by MARCUS, only a part of the globulin separates out,
while a portion remains in solution and cannot be precipitated by the
addition of acid. For this reason MARCUS l also differentiates between
a water-soluble globulin and one insoluble in water. According to the
later investigations of HOFMEISTER and PiCK2 the part insoluble in
water corresponds chiefly to a globulin fraction readily precipitated by
(NH4)2SO4 (by 28-36 vols. per cent saturated solution), and the part
soluble in water corresponds to a fraction difficult to precipitate (by
36-44 vols. per cent saturated solution). The first fraction is called
euglobulin and the second pseudoglobulin. According to FORGES and
SpiRO3 the serglobulins can be separated by (NH^SCU into three
fractions whose precipitation limits are 28-36, 33-42, and 40-46 vols.
per cent saturated solution. All three fractions contain globulin insoluble
in water. FREUND and JOACHIM 4 have found that the euglobulin as
well as the pseudoglobulin fraction is a mixture of globulin soluble in
water and globulin insoluble in water, and consequently the number
of different globulins in the serum may be still greater.

It follows from all these investigations that either the difference between
the globulin soluble in water and that insoluble is not sufficient or that the frac-
tional precipitation with ammonium sulphate is not suited for the separation
of the various globulins. This latter seems to be the case, as shown by MELLANBY
HASLAM and recently by WIENER. 5 It must not be forgotten that the globulin
fractions are always contaminated with other serum constituents, and that these
may influence the solubility and precipitability. As HAMMARSTEN has shown,
a water-soluble globulin, can be transformed into a globulin insoluble in water

1 Zeitschr. f. physiol. Chem., 28.

2 Hofmeister's Beitrage, 1.

3 Hofmeister's Beitrage, 3.

4 Zeitschr. f. physiol. Chem., 36.

5 Mellanby, Journ. of Physiol., 36; Haslam, ibid., 32; Wiener, Zeitschr. f. physiol
Chem., 74.

2CO THE BLOOD.

by careful purification, and also the reverse, namely, a globulin insoluble in
water can sometimes be converted into one soluble in water by allowing it to lie
in the air. An insoluble protein like casein can also, according to HAMMARSTEN, *
have the solubilities of a globulin due to contamination with constituents of the
serum, and K. MORNER 2 has also shown that a contamination of the serum-
globulins with soap can essentially modify the precipitation of these globulins.
Under these circumstances the above assumptions in regard to the different
globulin fractions must be accepted with great caution.

The investigations made thus far upon the so-called serglobulin,
have not led to any positive results. That this globulin, with the
exception of the enzymes, antienzymes, immune bodies, and other
unknown substances which are carried down by the various fractions, is
a mixture of globulins there seems to be no doubt. The serglobulin or
the globulin mixture which is obtained from the serum by the methods
to be described has the following properties:

In a moist condition it forms snow-white flaky masses, neither tough
nor elastic, which always contain thrombin and hence can bring about
coagulation in a fibrinogen solution. The neutral solution is only incom-
pletely precipitated by NaCl added to saturation, and is not precipitated
by an equal volume of a saturated salt solution. It is only partly
precipitated by dialysis or by the addition of acid. On saturation with
magnesium sulphate, or one-half saturation with ammonium sulphate
a complete precipitation is obtained. The coagulation temperature
is, with 5-10 per cent NaCl in solution, 69-76°, but more often 75° C.
The specific rotation of the solution containing salt is (a)j>=— 47.8°
for the serglobulin from ox-blood (FREDERICQ 3) . The various globulin
fractions do not differ essentially from each other in their coagulation
temperatures, specific rotation, refraction coefficient (REiss4), and their
elementary composition. The average composition is, according to
HAMMARSTEN, C. 52.71, H 7.01, N 15.85, S 1.11 per cent. K. MORNER 5
found 1.02 per cent sulphur and 0.67 per cent lead-blackening sulphur.
All the sulphur seems to exist as cystine.

Serglobulin contains, as K. MORNER first showed, a carbohydrate
group which can be split off. LANGSTEIN 6 has obtained several car-
bohydrates from the blood-globulin, namely, glucose, glucosamine,

1 See Hammarsten, Ergebnisse, d. PhysioL, 1, Abt. 1.

2 Zeitschr. f. physiol. Chem., 34.

3 Bull. Acad. Roy. de Belg. (2), 50. In regard to paraglobulin, see Hammarsten,
Pfliiger's Arch., 17 and 18, and Ergebnisse d. Physiol., 1, Abt. 1.

4 Hofmeister's Beitrage, 4.

6 Zeitschr. f. physiol. Chem., 34.

6M6rner, Centralbl. f. Physiol., 7; Langstein, Munch, med. Wochenschr., 1902,
1876, and Wien. Sitzungsber., 112, Abt. 116, 1903; Monatsheft f. Chem., 25; Hof-
meister's Beitrage, 6; see also footnote 5, p. 84.

SERALBUMINS. 261

and carbohydrate acids of unknown kinds. It has not been shown
whether these small amounts of carbohydrate are derived from the globulin
or from other contaminating bodies. According to ZANETTI and also
BYWATERS, the blood-serum contains a glucoproteid, seromucoid, and
the investigations of EICHHOLZ l seem to show that the globulins are
contaminated by a glucoproteid. According to LANGSTEIN the sugar
is not only mixed with the globulin, but it exists in a combined form,
probably in loose combination.

Serglobulin (the euglobulin) may be easily separated as a fine floc-
culent precipitate from blood-serum by neutralizing or making faintly
acid with acetic acid and then diluting with 10-20 vols of water. For
further purification this precipitate is dissolved in dilute common salt
solution, or in water with the aid of the smallest possible amount of
alkali, and then reprecipitated by diluting with water or by the addition
of a little acetic acid. All the serglobulin may also be separated from
the serum by means of magnesium or ammonium sulphate; in these
cases it is difficult to completely remove the salt by dialysis. As long
as we are not agreed as to the number of globulins in the serum, it is
not necessary to give a method of separating the various globulins in this
mixture. Thus far the fractional precipitation with (NH^SO* has
chiefly been used. The serglobulin from blood-serum is always contam-
inated with lecithin and thrombin. A serglobulin free from thrombin
may be prepared from ferment-free transudates, as sometimes from
hydrocele fluids, and this shows that serglobulin and thrombin are dif-
ferent bodies. For the detection and the quantitative estimation of
serglobulin we may use the precipitation by magnesium sulphate added
to saturation (HAMMARSTEN) , or by an equal volume of a saturated
neutral ammonium-sulphate solution (HOFMEISTER and KAUDER and
POHL 2) . In the quantitative estimation the precipitate is collected on a
weighed filter, washed with the salt solution employed, dried with the
filter at about 115° C., then washed with boiling-hot water, so as to
completely remove the salt, extracted with alcohol and ether, dried,
weighed, and incinerated to determine the ash. This method, according
to the investigations of WIENER,S can only be used when the serum is
sufficiently diluted with water.

Seralbumins are found in large quantities in blood-serum, blood-
plasma, lymph, transudates, and exudates. Probably they also occur
in other animal fluids and tissues. The proteins which pass into the
urine under pathological conditions consist largely of seralbumin.

The seralbumin, like the serglobulin, seems also to be a mixture of
at least two protein bodies. The preparation of crystalline seralbumin

1 Zanetti, Chem. Centralbl., 1898, I, p. 624; Bywaters, Journ. of Physiol., 35, and
Biochem. Zeitschr., 15; Eichholz, Journ. of Physiol., 23.

2 Hammarsten, 1. c.; Hofmeister, Kauder and Pohl, Arch. f. exp. Path. u. Pharm., 20.

3 Zeitschr. f. physiol. Chem., 74.

262 THE BLOOD.

(from horse-serum) was first performed by GURBER. It crystallizes
with difficulty from other blood-sera (GRUZEWSKA). Even from horse-
serum only a portion, according to ROBERTSON 1 not more than 40 per
cent, of the albumin can be obtained as crystals, and it is also pos-
sible that the amorphous albumin, which is precipitated by ammo-
nium sulphate with difficulty, represents two seralbumins (MAXIMO-
WITSCH). According to GURBER and MICHEL it would seem that the
crystalline seralbumin is also a mixture, but this is disproved by the obser-
tions of SCHULZ, WICHMANN, and KRiEGER2. We know nothing as
to the behavior of the amorphous fraction of the seralbumin in this respect.
Because of the different coagulation temperatures, HALLIBURTON claims
the existence of three different albumins in the blood-serum, a view
which has been disputed by several experimenters, and recently by
HOUGARDY. On the other hand, the earlier investigations of KAUDER,
as well as the more recent work of OPPENHEIMER,S seem to indicate a
non-unit nature of the seralbumins, but this question is still an open
one.

The crystalline seralbumin may perhaps be a combination with
sulphuric acid (K. MORNER, INAGAKI). The coagulated albumin obtained
from the aqueous solution of the crystals with the aid of alcohol has
almost the same elementary composition (MICHEL) as the amorphous
mixture of albumin prepared from horse-serum (HAMMARSTEN and
K. STARKE4). The average composition was C 53.06, H 6.98. N 15.99,
S 1.84 per cent. K. MORNER, after the removal of the sulphuric acid
from crystalline albumin, found 1.73 per cent total sulphur, which prob-
ably exists only as cystine. LANGSTEIN 5 has been able to split off a nitrog-
enous carbohydrate (glucosamine) from crystalline seralbumin. The
quantity was so small that the question is still undecided whether or
not the carbohydrate was a contamination. The fact that ABDER-
HALDEN, BERGELL, and DORPINGHAUS 6 were able to prepare a seral-
bumin entirely free from carbohydrate and which did not respond to
MOLISCH'S very delicate reaction, seems to, be decisive on this point.
The specific rotation of crystalline seralbumins from horse-serum was
found by MICHEL to be (a)D=— 61 to 61.2°, and by MAXIMOWITSCH on
the contrary (a)D= -47.47°.

1 Journ. of biol. Chem., 13.

2 In regard to the literature on the crystalline seralbumins, see Schulz, Die Kristal-
lisation von Eiweissstoffen, Jena, 1901; Maximowitsch, Maly's Jahresber., 31, 35.

3 Halliburton, Journ. of PhysioL, 5 and 7; Hougardy, Centralbl. f. Physiol., 15,
665; Oppenheimer, Verhandl. d. physiol. Gesellsch., Berlin, 1902.

4 Michel, Verhandl. d. phys-med. Gesellsch. zu Wurzburg, 29, No. 3; K. Starke,
Maly's Jahresber., 11; K. Morner, 1. c.; Inagaki, Biochem, Centralbl., 4, p. 515.

6 K. Morner, 1. c.; Langstein, Hofmeister's Beitrage, 1.
6 Zeitschr. f. physiol. Chem., 41.

SERUM PROTEINS. 263

The crystalline and amorphous seralbumin in aqueous solution give
the ordinary albumin reactions. The coagulation* temperature of a
1-per cent solution, poor in salts is about 50° C., but rises with the quan-
tity of salt. The coagulation of the mixture of albumins from serum
generally takes place at 70-85° C., but is essentially dependent upon
the reaction and the amount of salt present. Up to the present time no
seralbumin solution has been prepared free from mineral bodies. A
solution as free from salts as possible does not coagulate either on boil-
ing or on the addition of alcohol. On the addition of a little common
salt it coagulates in both cases.1

Seralbumin differs from the albumin of the white of the hen's egg in
the following particulars: It is more levogyrate; the precipitate formed
by hydrochloric acid easily dissolves in an excess of the acid; it is rendered
less insoluble by alcohol.

In preparing the seralbumin mixture, first remove the globulins,
according to JOHANSSON, by saturating with magnesium sulphate at
about 30° C. and filtering at the same temperature. The cooled filtrate
is separated from the crystallized salt and is treated with acetic acid so
that it containes about 1 per cent. The precipitate formed is filtered
off, pressed, dissolved in water with the addition of alkali to neutral
reaction, and the solution freed from salt by dialysis. The mixture of
albumins may be obtained in a solid form from the dialyzed solution,
either by evaporating the solution at a gentle temperature or by pre-
cipitating with alcohol, which must be quickly removed. STARKE2
has suggested another method, which is also to be recommended. The
crystalline seralbumin may be prepared from serum freed from globulin
by half saturating with ammonium sulphate, by the addition of more
salt until a cloudiness appears, and then proceeding according to the
suggestion of GURBER and MICHEL. On acidifying with acetic acid
or sulphuric acid the crystallization may be considerably accelerated.3
The quantity of seralbumin is best calculated as the difference between
the total proteins and the globulins. A method for the quantitative
estimation of globulins and albumins in blood serum by refractometric
means has been suggested by ROBERTSON/*

Summary of the elementary composition of the above-mentioned and described
proteins (from horse-blood) :

Fibrinogen

C

... 52 93

H

6 90

N
16 66

s
1.25

O
22.26

(H AMMARSTE N)

Fibrin
Fibrin-globulin . . . .

. . . 52.68
.. . 52.70

6.83
6.98

16.91
16.06

1.10

22.48

«
«

Serglobulin.

52 71

7 01

15 85

1 11

23 32

1 1

Seralbumin.

... 53 08

7 10

15 93

1 90

21 86

(MICHEL)

1 In regard to the relationship of neutral salts to heat coagulation,, see J. Starke,
Sitzungsber. d Gesellsch. f. Morph. u. Physiol. in Miinchen, 1897.

2 Johansson, Zeitschr. f. physiol. Chem., 9; K. Starke, Maly's Jahresber., 11.

3 See Hopkins and Pinkus, Journ. of Physiol., 23; Krieger, Ueber die Darstellung
krystallinscher tierischer Eiweissstoffe, Inaug.-Dissert. Strassburg, 1899.

4 Journ. of biol. Chem., 11.

264 THE BLOOD.

Proteose-like substances have been found in blood-serum by several
investigators, and NOLF l has shown that after the abundant introduc-
tion of proteoses into the intestine, they pass into the blood. BOR-
CHARDT 2 has also been able to show that not only after the introduction
of elastin-proteose per os, but also after feeding dogs with not over-
abundant quantities of elastin, a proteose, hemielastin, passes into the
blood and can indeed be eliminated in the urine. The question whether
the proteoses are normal constituents of the blood under ordinary con-
ditions is still much disputed. The difficulty in deciding this ques-
tion lies in the fact that in the removal of the proteins a small amount
of proteose-like substance is formed from other proteins (namely from the
globin of the blood pigment), and on the other hand the proteoses can be
precipitated with the other bodies. The question as to the physiological
occurrence of proteoses in the blood or plasma must be considered as
still undecided.3

In close relation to the proteoses stands perhaps the above-men-
tioned seromucoid, which was discovered by ZANETTI and especially
studied by BYWATERS. It is a glycoprotein which is soluble in water,
and precipitated by alcohol. Seromucoid contains, according to BY-
WATERS,4 11.6 per cent N, 1.8 per cent S, and yields approximately 25
per cent glucosamine. The quantity in the blood is 0.2-0.9 p. m.

The Blood-serum.

As above stated, the blood-serum is the clear liquid which is pressed
out by the contraction of the blood-clot. It differs chiefly from the
plasma in the absence of fibrinogen and in containing an abundance
of fibrin ferment. Otherwise considered qualitatively, the blood-serum
contains the same chief constituents as the blood-plasma.

Blood-serum is a sticky liquid which is more alkaline toward litmus
than the plasma. The specific gravity in man is 1.027 to 1.032, average
1.028. The color is more or less yellow; in human blood-serum it is
pale yellow with a shade toward green, and in horses it is often amber-
yellow. The serum is ordinarily clear; after a meal it may be opales-
cent, cloudy, or milky white, according to the amount of fat contained
in the food.

Besides the above-mentioned bodies, the following constituents are
found in the blood-plasma or blood-serum:

1 Bull. Acad. Roy. Belg., 1903 and 1904.

2 Zeitschr. f. physiol. Chem., 51 and 57.

3 See especially Abderhalden, Zeitschr. f. physiol. Chem., 51, and Biochem. Zeitschr.,
8 and 10, and E. Freund, ibid., 7 and 9, which also contains the literature.

4 Biochem. Zeitschr., 15.

BLOOD SERUM. 265

Fat occurs from 1-7 p. m. in fasting animals. After partaking of
food the amount is increased to a great extent. Fatty acids, or soaps,
glycerin (NiCLOUx, FR. TANGL, and ST. WEISER l) phosphatides and
cholesterin are also found. Cholesterin occurs, according to HURTHLE 2,
at least in part, as fatty-acid esters (serolin according to BOUDET) . Accord-
ing to LETSCHE 3 free cholesterin probably also occurs in the serum.

Sugar seems to be a physiological constituent of the plasma and
serum. According to the investigations of many workers4 the sugar
found is glucose. STRAUSS 5 has also detected fructose in blood-serum
and in transudates and exudates. The question as to the occurrence
of other varieties of sugar, such as isomaltose (PAVY and SIAU) and pen-
tose (LEPINE and BOULUD 6) , in blood serum is still undecided. ASHER
and ROSENFELD and MICHAELIS and RON A in a more conclusive manner,
have shown that at least a considerable part of the sugar can be removed
from the blood by dialysis, hence it must exist in solution in the free
state. These observations do not exclude the possibility of the existence
of another part of the sugar which is in combination with protein.
LEPINE and BOULUD 7 could only obtain a diffusion of the sugar by a
short dialysis from serum 12 hours old, but not from perfectly fresh
serum, an observation which somewhat diminishes the conclusiveness
of MICHAELIS and RONA'S experiment with 24-hour dialysis. A fur-
ther testing of this question is therefore very desirable.

The quantity of sugar in the serum or plasma is for man 0.6-1 p. m.
calculating the total reduction as glucose, and in animals about the same
but in rabbits considerably higher or 2.2 p. m.8 Besides the sugar, the
blood contains, as first shown by J. OTTO, also another or perhaps several
reducing substances, a part existing in the serum and another part in the
blood-corpuscles. We will discuss the nature of these bodies as well
as the so-called virtual sugar and glycolysis in speaking of the division
of the sugar in the blood-corpuscles and plasma in connection with

1 Nicloux, Compt. Rend. soc. biol., 55; Tangl and St. Weiser, Pfliiger's Arch., 115.

2 Hiirthle, Zeitschr. f. physiol. Chem., 21, where Boudet is also cited. In regard
to the quantity of these esters in bird-serum, see Brown, Amer. Journ. of Physiol., 2.

3 Zeitschr. f. physiol. Chem., 53.

4 See v. Mering, Arch. f. (Anat. u.) Physiol., 1877 (this article contains numerous
references); Seegen, Pfliiger's Arch., 40; Miura, Zeitschr. f. Biologic, 32.

6 Fortschritte d. Mediz., 1902.

6 Pavy and Siau, Journ. of Physiol., 26; Lepine and Boulud, Compt. Rend., 133,
135, and 136.

7 Rosenfeld, Centralbl. f. Physiol., 19, p. 449; Le"pine and Boulud, Compt. Rend.,
143; Asher, Biochem. Zeitschr., 3; Michaelis and Rona, ibid., 14.

8 See E. Frank, Zeitschr. f. physiol. Chem., 70; Lyttkens and Sandgren, Bioch.
Zeitschr., 21 and 26.

266 THE BLOOD.

the total blood. The same applies to the conjugated glucuronic acids,
which it seems, originate from the form-elements.

The blood-plasma and the serum, as well as the lymph also contain
enzymes of various kinds. According to ROHMANN, BIAL, HAMBURGER,1
and others, diastases, which convert starch and glycogen into maltose or
isomaltose, as well as a maltase, are found in the blood. The diastase,
whose quantity is very variable in the blood of different animals, seems
at least in great part, to originate in the pancreas but can also come
from other organs and according to HABERLANDT also from the leucocytes.2
HANRIOT and others have detected, in the serum, Upases or esterases which
decompose butyrin and neutral fats and other esters. The occurrence
of butyrinases which split mono- as well as tributyrin has been recently
substantiated by RONA and MICHAELIS, while the property of this lipase
of splitting olein and other neutral fats is not generally acknowledged
(ARTHTJS, DOYON and MOREL 3) .

This lipolytic property, if it exists to the extent that HANRIOT ascribes to
it, must not be confounded with the transformation of fat into unknown sub-
stances soluble in water, a phenomenon first observed by CONNSTEIN and MICHAE-
LIS and further studied by WEIGERT. The occurrence of such a body is positively
denied by G. MANSFELD.*

Besides the above-mentioned enzymes and thrombin and the gly-
colytic enzymes that will be discussed later, several other enzymes have
been found in the blood-serum, namely, oxidases, catalases, proteolytic
enzymes, among which we must mention the polypeptide-splitting
enzymes studied by ABDERHALDEN and collaborators,5 also rennin and
several antienzymes. We cannot enter into the discussion of these, nor
of the many not chemically characterized bodies which have been called
toxines and antitoxines, immune bodies, alexines, ha^molysines, cytotoxines,
etc., and which have been discussed in Chapter I. The various enzymes
and antienzymes, and the above mentioned bodies are as a rule pre-
cipitated with the globulin, but differ among each other in that some are

1R6hmann; Rohmann and Hamburger, Ber. d. deutsch. chem. Gesellsch., 25
and 27; Pfluger's Arch., 52 and 60; Bial, Ueber das diast. Ferm., etc., Inaug.-Diss.
Breslau, 1892 (older literature). See also Pfluger's Arch., 52, 54, and 55; Wohlgemuth,
Bioch. Zeitschr., 21.

2 Wohlgemuth, 1, c.; Moeckel and Rost, Zeitschr. f. physiol. Chem., 67; Clerk
and Loeper, Compt. Rend. soc. biol., 66; Haberlandt, Pfluger's Arch., 132.

3 Hanriot, Compt. Rend. soc. biol., 48 and 54, Compt. Rend. 123 and 132; Rona,
and Michaelis, Bioch. Zeitschr., 31; Rona, ibid., 33; Arthus, Journ. de Physiol. et
de Pathol., 4; Doyon and Morel, Compt. Rend. soc. biol., 54; Achard and Clerc.
(Lipase in Disease), Compt. Rend., 129, and Arch. d. med. exper., 14.

4 Connstein and Michaelis, Pfluger's Arch., 65 and 69; Weigert, ibid., 82; Mansfeld,
Centralbl. f. Physiol., 21.

5 Zeitschr. f. physiol. Chem., 51, 53, 55.

BLOOD SERUM. 267

carried down by the euglobulin, while the others are carried down by
the pseudoglobulin fraction.

The non-protein organic constituents of the serum have been given
especial and careful study by E. LETSCHE l and he has found, besides the
previously known bodies, that the serum contains several acids, among
which there are two nitrogeneous acids whose nature has not been studied.
These, including other nitrogenous substances found by him, represent
a part of the so-called rest nitrogen, i.e., that nitrogen which remains in
the serum after the complete removal of the coagulable proteins. As
representatives of the bodies occurring as rest nitrogen in the serum we
must in the first place mention urea, also creatine, carbamic add, ammonia >
hippuric acid, phosphocarnic add (PANELLA), traces of indol (HERVIEUX),.
perhaps also uric add found by ABELES 2 in human blood, while LETSCHB
could not find any in horse-blood.

According to BROWINSKI proteic acids (see Chapter XIV) occur in
the serum and CZERNECKIS has investigated the quantity of proteic
acid nitrogen in serum and transudates under different conditions. The
occurrence of proteoses is, as above mentioned, somewhat disputed.
We have several investigations on the occurrence of amino-acids (v.
BERGMANN, HOWELL, LETSCHE, ABDERHALDEN and others) which make
the occurrence of these very probable, and recently BINGEL has been able
to show the presence of glycocoll in normal ox-blood. Otherwise the
amino-acids have often been sought for in normal blood but in vain;
still recently certain investigators like VAN SLYKE and MEYER4 have
shown the presence of amino-acids in the blood under normal con-
ditions. In dog blood after 24 hours' starvation they found 3-5 milli-
grams of amino-acid nitrogen in 100 parts blood. Under pathological
conditions lysine (NEUBERG and RICHTER 5) , leudne and tyrosine have
been found. Also purine bases and bile adds have been found in the
serum under pathological conditions. That the quantity of rest nitro-
gen is larger during digestion than in starvation requires further con-
firmation.6

1 Zeitschr. f . physiol. Chem., 53.

2Panella, cited in Virchow's Jahresb., 1902, 150, Hervieux Compt. Rend. soc.
biol., 56; Abeles, Wien. med. Jahrb., 1887.

3 Browinski, Zeitschr. f. physiol. Chem., 54 and 58; Czernecki, Maly's Jahresb.,
39 and 40.

4v. Bergmann, Hofmeister's Beitrage, 6; Howell, Amer. Journ. of Physiol., 17;
Letsche, 1. c.; Abderhalden, Zeitschr. f. physiol. Chem., 72; Bingel, ibid., 57; D.
v. Slyke and Meyer, Journ. of biol. Chem., 12.

5 Deutsch. med. Wochenschr., 1904.

6v. Bergmann and Langstein, Hofmeister's Beitrage, 6; Hohlweg and Meyer,
ibid., 11.

268 THE BLOOD.

As rest-carbon MANCINI l designates that carbon which is not precipitated
by phosphotungstic acid. It originates in great part from the urea and sugar
and amounts to 0.076-0.089 gram in 100 cc.

The pigments of the blood-serum are very little known. Besides
other pigments horse-serum, contains, as first shown by HAMMARSTEN,
bilirubin, which, according to RANG, is the only pigment of the serum of
this animal. This pigment occurs, although in small amounts, sometimes
in the serum of other animals and, according to BIFFI and GALLIC is
especially abundant in the blood of new-born.

Urobilin is not, according to ATJCHE, ROTH and HERZFELD, a physi-
ological serum-pigment. Urobilinogen may occur in extraordinary
cases according to HILDEBRANDT/ and on allowing the blood in such
cases to stand urobilin may be formed therefrom. The yellow coloring-
matter of the serum seems to belong to the group of luteins, which
are often called lipochromes or fat-coloring matters. From ox-serum
KRTJKENBERG 4 was able to isolate with amyl alcohol a so-called lipo-
chrome whose solution shows two absorption-bands, of which one encloses
the line F and the other lies between F and G.

The mineral bodies in serum and plasma are qualitatively, but not
quantitatively, the same. A part of the calcium, magnesium, and
phosphoric acid is removed on the coagulation of the fibrin. By means
of dialysis, the presence of sodium chloride, which forms the chief mass
or 60-70 per cent of the total mineral bodies, lime-salts, sodium car-
bonate, and traces of sulphuric and phosphoric acids and of potassium,
may be directly shown in the serum.5 Traces of silicic acid, fluorine,
copper, iron, and manganese, are claimed to have been found in the serum.
As in most animal fluids, the chlorine and sodium are in the blood-
serum in excess of the phosphoric acid and potassium (the occurrence
of which in the serum is even doubted). The acids present in the ash
are not sufficient to saturate the bases found, a condition which shows
that a part of the bases is combined with organic substances, perhaps
proteins. This also coincides with the fact that the great part of
the alkalies does not exist in the serum as diffusible alkali compounds,
carbonate and phosphate, but as non-diffusible compounds, protein
combinations. According to HAMBURGER 37 per cent of the alkali of
the serum from horse-blood was diffusible and 63 per cent non-diffusible.

1 Bioch. Zeitschr., 26 and 32.

2 Hammarsten, see Maly's Jahresb., 8 (1878); Ranc, Compt. Rend. soc. biol., 62;
Biffi and Galli, Journ. de Physiol. et Path., 9 (1907).

8 Auche, Compt. Rend. soc. biol., 67; Roth and Herzfeld, Deutsch. med. Wochenschr.,
37; Hildebrandt, Munch. Med. Wochenschr., 57.
4 Sitz.-Ber. d. Jen. Gesellsch. f. Med., 1885.
6 See Giirber, Verhandl. d. phys.-med. Gesellsch. zu Wurzburg, 23.

BLOOD SERUM. 269

According to RONA and TAKAHASHI 1 25-30 per cent of the calcium is
non-diffusible, probably combined with proteins.

Iodine, which seems to be habitually found, is also considered as
a mineral constituent of the plasma or serum (GLEY and BOURCET),
while arsenic, although not found in all blood, occurs in human blood
(GAUTIER, BOURCET 2). Iodine occurs to a greater extent in menstrual
blood than in other blood and does not exist as a salt, but as an organic
compound (BOURCET).

The gases of the blood-serum, which consists chiefly of carbon dioxide
with only a little nitrogen and oxygen, will be described when treating
of the gases of the blood.

We have only a few analyses of blood-plasma. As an example the
results of the analyses of the blood-plasma of the horse will be given
below. The analysis No. 1 was made by HoppE-SEYLER.3 No. 2 is the
average of the results of three analyses made by HAMMARSTEN. The
figures are given for 1000 parts of the plasma.

No. 1. No. 2.

Water 908.4 917.6

Solids 91.6 82.4

Total proteins • 77 . 6 69 . 5

Fibrin 10.1 6.5

Globulin 38.4

Seralbumin 24 . 6

Fat 1.21

Extractive substances 4.01 ion

Soluble salts 6.4 [ 1J'y

Insoluble salts 1.7 J

LEWINSKY4 has determined the total proteins and the individual
proteins in the blood-plasma of man and animals with the following
results :

Total Protein. Albumin. Globulin. Fibrinogen.

Man 72.6 40.1 28.3 4.2

Dog 60.3 31.7 22.6 6.0

Sheep 72.9 38.3 30.0 4.6

Horse 80.4 28.0 47.9 4.5

Pig 80.5 44.2 29.8 6.5

ABDERHALDEN has made complete analyses of the blood-serum of
several domestic animals. From these analyses, as well as from those
made by HAMMARSTEN of the serum from human, horse, and ox-blood,
it follows that the amount of solids ordinarily varies between 70-97
p. m. The chief mass of the solids consists of proteins, about 55-84
p. m. In hens HAMMARSTEN found much lower values, namely, 54

1 Hamburger, Arch. f. (Anat. u.) Physiol., 1898; Rona and Takahashi, Bioch.
2eitschr., 31.

2 Gley and Bourcet, Compt. Rend., 130; Bourcet, ibid., 131; Gautier, ibid., 131.

3 Cit. from v. Gorup-Besanez's Lehrbuch der physiol. Chem., 4. Aufl., 346.

4 Pfluger's Arch., 100.

270 THE BLOOD.

p. m. solids, with only 39.5 p. m. protein, and HALLIBURTON found only
25.4 p. m. protein in frog's blood. The relation between globulin and
seralbumin is, as shown by the analyses of HAMMARSTEN, HALLIBUR-
TON, and RuBBRECHT,1 very different for various animals, but may also
vary considerably in the same species of animal. In human blood-
serum HAMMARSTEN found more seralbumin than globulin, and the
relation of serglobulin to seralbumin was as 1:1.5. LEWINSKY found the
relationship in man greater than 1, indeed 1:1.39-2.13. In regard to the
quantity of the remaining organic constituents of the serum we refer
the reader to ABDERHALDEN'S complete analyses.

In starvation it seems, as first found by BURCKHARDT and then sub-
stantiated by other investigators, that the quantity of globulins relative
to that of albumin in dogs and also in rats (ROBERTSON 2) , is increased.
According to ROBERTSON, in the horse, ox and rabbit the reverse exists,
namely, the amount of albumin relative to the globulin increases in
starvation. A change in the relation with a decrease in the albumin
and an increase in the globulin may also occur in animals which have
been made sick or in part immune by inoculation with pathogenic
micro-organisms (LANGSTEIN and MAYER 3) . The total protein content
is raised in nearly all cases. The amount of fibrinogen in the plasma
is especially increased by pneumococci, streptococci, and pus-staphy-

loCOCCi (P. MtJLLER4).

The quantity of mineral bodies in the serum has been determined by
many investigators. The conclusion drawn from the analyses is that
there exists a rather close correspondence between human and animal
blood-serum, and it is therefore sufficient to here give the analysis of C.
SCHMIDT 5 of (1) human blood, and BUNGE and ABDERHALDEN'S analyses
(2) of serum of ox, bull, sheep, goat, pig, rabbit, dog, and cat. The results
correspond to 1000 parts by weight of the serum.

i 2

K20 0.387-0.401 0.226-0.270

Na20 4.290-4.290 4.251-4.442

Cl 3.565-3.659 3.627-4.170

CaO. 0. 155-0 . 155 0. 119-0. 131

MgO 0.101 0.040-0.046

PA, (inorg.) 0.052-0.085

1 Abderhalden, Zeitschr. f. physiol. Chem., 25; Hammarsten, Pflliger's Arch., 17;
Halliburton, Journ. of Physiol., 7; Rubbrecht, Travaux du laboratoire de 1'institut
de physiologie de Liege, 5, 1896.

2 Burckhardt, Arch. f. exp. Path. u. Pharm., 16; Githens, Hofmeister's Beitrage,
5; see also Morawitz, ibid., 7, and Inagaki, Zeitschr. f. Biol., 49; Robertson, Journ.
of bioL Chem., 13.

3 Hofmeister's Beitrage, 5.

4 Ibid., 6.

6 Cit. from Hoppe-Seyler, Physiol. Chem., 1881, p. 439.

BLOOD SERUM. 271

A MACALLUM l has determined the quantity of mineral bodies in
the serum of certain cold-blooded animals (fishes, shark, lobster and
others). The amount of sodium and chlorine in the serum of these animals
living in sea-water was much greater than in warm-blooded animals.

Even if we bear in mind that certain bodies, such as carbon dioxide,
are driven off during incineration, and that other bodies, such as sul-
phuric acid and phosphoric acid, are formed from sulphurized and
phosphorized organic substances, still quantitative analyses like the
above are not sufficient for the scientific demands of to-day. They
do not show the true composition, and especially do not give an explana-
tion of the number of different ions present in the serum or in other fluids,
a question which is of the greatest physiological importance. An
answer to these questions is obtainable only by physico-chemical investiga-
tions, which have thus far been used chiefly in determining the molecular
concentration, the amount of electrolytes and non-electrolytes, and the
degree of dissociation.

The average depression of the freezing-point of mammalian blood
corresponds, as given in Chapter I, closely to a 9 p. m. (A = about
— 0.56°) solution of common salt, and at the present time such a solution
is considered as a physiological salt solution for man and other mammalia.
In lower animals and fish the conditions are otherwise, as shown in
the above-mentioned chapter.

There are recorded a great number of investigations on the changes
in the osmotic pressure or the molecular concentration of the blood-
serum under various physiological conditions as well as in disease, but
still it is no doubt too early to draw any definite conclusions from these
observations.

The degree of dissociation (see Chapter I) of sera has been determined
by several investigators, and according to HAMBURGER2 it lies between
0.65 and 0.82. The molecular concentration, which represents the
total number of molecules and ions per liter, is according to BURGARSKY
and TANGL, on an average about 0.320 mol. per liter. They also
found that about three-fourths of the total number of dissolved mole-
cules in blood-serum were electrolytes, although the serum contained
about 70-80 p. m. protein and 10 p. m. inorganic bodies, and also that
three-fourths of the quantity of electrolytes consisted of NaCl.

In the determination of the alkalinity of blood and blood-serum,
up to the present time, we have estimated the amount of alkali by titra-
tion with an acid. We cannot dispense with such determinations, although

1 Proc. Roy. Soc., ser. B., 82.

2 Osmotisher Druck und lonenlehre, Wiesbaden, 1902-1904, where the literature
on the physical chemistry of the blood can be found.

272 THE BLOOD.

they do not yield any information as to the true alkalinity, apart from
the fact that the results are dependent upon the indicator used, because
we understand as true alkalinity the concentration of the hydroxyl
ions. The Na2CO3 is in aqueous solution more or less dissociated into
2Na+ and COa", depending upon the dilution. The C0s= ions com-
bine partly with the H+ ions of the dissociated water, forming HCOa"",
and the corresponding HO~ ions produce the alkaline reaction. If now
by the addition of a little acid, a few of the HO~ ions are removed,
the equilibrium is then disturbed, a new quantity of Na2COs is dissociated,
and this process is repeated every time a new quantity of acid is added
until all the carbonate is dissociated. The dissociation of the carbonate
existing in the original concentration, upon which the number of HO~~
ions is dependent, cannot therefore be determined by titration.

For these reasons we generally determine the quantity of HO
and H ions in the serum and blood by methods based upon NERNST'S
theory for the electromotive force of gas-chains. According to these
investigations it has been found that the concentration of the hydroxyl
ions in blood-serum and blood is only a little higher than in distilled
water (see Chapter I page 76, and the reaction of the blood below).

H. THE FORM-ELEMENTS OF THE BLOOD.
The Red Blood-corpuscles.

The blood-corpuscles are round, biconcave disks without membrane
and nucleus, in man and mammalia (with the exception of the llama,
the camel, and their congeners). In the latter animals, as also in birds,
amphibia, and fish (with the exception of the Cyclostoma) the cor-
puscles have in general a nucleus, are biconvex and more or less ellip-
tical. The size varies in different animals. In man they have an average
diameter of 7 to 8 /i (M= 0.001 mm.) and a maximum thickness of 1.9 /z.
They are heavier than the blood-plasma or serum, and therefore sink
in these liquids. In the discharged blood they may sometimes lie with
their flat surfaces together, forming a cylinder like a roll of coin (rouleaux) .
The reason for this phenomenon, which is considered as an agglutination,
has not been sufficiently studied, but as it may be observed in defibrinated
blood it seems probable that the formation of fibrin has nothing to
do with it.

The number of red blood-corpuscles is different in the blood of various
animals. In the blood of man there are generally 5 million red cor-
puscles in 1 c.mm., and in woman 4 to 4.5 million.

The blood-corpuscles consist principally of two chief constituents,
the stroma, which forms the real protoplasm, and the intraglobular
contents, whose chief constituent is haemoglobin. We cannot state

RED BLOOD-CORPUSCLES. 273

anything positive for the present in regard to a more detailed arrange-
ment, and the views on this subject are somewhat divergent. The two
following views are more or less related to each other. According to
one view the blood-corpuscles consist of a membrane which encloses a
haemoglobin solution, while the other view considers the stroma as a proto-
plasmic structure soaked with haemoglobin. This latter view is in accord
with the assumption as to an outside boundary-layer. Thus accord-
ing to HAMBURGER the stroma forms a protoplasmic net in whose meshes
there exists a red fluid or semi-fluid mass which consists in great meas-
ure of haemoglobin. This mass represents the water-attracting force
of the blood-corpuscles, and besides this it is also considered that the
outer protoplasmic boundary is semi-permeable, i. e., permeable to water
but not permeable to certain crystalloids. The researches of KOPPE,
ALBRECHT, PASCUCCI, RYWOSCH/ and others indicate the presence of a
special envelope or boundary-layer, and there is no doubt that the outer
layer contains so-called lipoids, such as cholesterin, lecithin, and similar
bodies.

The red blood-corpuscles retain their volume in a salt solution which
has the same osmotic pressure as the serum of the same blood, although
they may change their form in such solutions, becoming more spherical,
and may also undergo a chemical change. Such a salt solution is iso-
tonic with the blood-serum, and its concentration for a NaCl solution is
approximately 9 p. m. for human and mammalian blood. A solution
of greater concentration, a hyperisotonic solution, abstracts water from
the blood-corpuscles until osmotic equilibrium is established, hence the
corpuscles shrink and their volumes become smaller. In solutions of
less concentration, hypisotonic solutions, the corpuscles swell, due to
the taking up of water, and this swelling may be so great, on diluting
the blood with water, that the haemoglobin is separated from the stroma
and passes into the watery solution. This process is called haemolysis
(see Chapter I).

A haemolysis may also be brought about by alternately freezing and
thawing the blood, as well as by the action of various chemical substances,
which act as protoplasmic poisons. These bodies are ether, chloroform
alkalies, bile-acids, solanin, saponin, and also the saponin substances,
which have a very strong haemoloytic action, also metabolic products of
bacteria, higher plants and animals (snakes, toads, bees, spiders and
others) and also bodies occurring in blood serum of normal or immunized
animals.

1See Hamburger, Osmotischer Druck und lonenlehre, 1902; Koppe, Pfliiger's
Arch., 99 and 107; Albrecht, Centralbl. f. Physiol., 19; Pascucci, Hofmeister's
Beitrage, 6; Rywosch, Centralbl. f. Physiol., 19.

274 THE BLOOD.

When the haemoglobin is separated from the so-called stroma by a
sufficiently strong dilution with water the stroma is found in the solution
in a swollen condition. By the action of carbon dioxide, by the careful
addition of acids, acid salts, tincture of iodine, or certain other bodies,
this residue, rich in proteins, condenses, and in many cases the form of
the blood-corpuscles may be again obtained. This residue, the so-
called ghosts or stromata of the blood-corpuscles, can also be directly
colored in dilute blood by methyl violet and in this way detected, and
attempts have been made to isolate it for chemical investigation. In
the following pages we mean by the name stroma only that residue which
remains after the removal of haemoglobin and other bodies soluble in
water.

To isolate the stromata from the blood-corpuscles, they are washed
first by diluting the blood with 10-20 vols. of a 1-2 per cent common
salt solution and then separating the mixture by centrifugal force or
by allowing it to stand at a low temperature. This is repeated a few
times until the blood-corpuscles are freed from serum. These purified
blood-corpuscles are, according to WOOLDRIDGE, mixed with 5-6 vols.
of water and then a little ether is added until complete solution is obtained.
The leucocytes gradually settle to the bottom, a movement which may
be accelerated by centrifugal force, and the liquid which separates there-
from is very carefully treated with a 1-per cent solution of KHS04 until
it is about as dense as the original blood. The separated stromata are
collected on a filter and quickly washed. PASCUcci,1 on the contrary,
treats the mass of corpuscles with 15-20 vols. of a one-fifth saturated
ammonium-sulphate solution, allows the corpuscles to settle, siphons
off the fluid, repeatedly centrifuges, allows the residue to dry quickly
(on porcelain plates) at the ordinary temperature, and then washes
with water until the blood-pigments and the other soluble bodies are
dissolved out.

WOOLDRIDGE found as constituents of the stromata lecithin, choles-
terin, nucleoalbumin, and a globulin which, according to HALLIBURTON,
is pr&bably a nucleoproteid which he calls cell-globulin. No nuclein
substances or seralbumin or proteoses could be detected by HALLIBUR-
TON and FRIEND. According to PASCUCCI, the stromata (from horse-
blood) consists of one-third cholesterin and lecithin (besides a little
cerebroside), and two-thirds protein substances and mineral bodies.
The nucleated red blood-corpuscles of the bird contain, according to
PL6sz and HoppE-SEYLER,2 a protein (nucleoprotein) which swells to a
slimy mass in a 10-per cent common salt solution, and which seems to

1 Hofmeister's Beitrage, 6.

2 Wooldridge, Arch. f. (Anat. u.) Physiol., 1881, 387; Halliburton and Friend,
Journal of Physiol., 10; Halliburton, ibid., 18; P16sz, Hoppe-Seyler's Med. chem.
Untereuch., 510.

RED BLOOD-CORPUSCLES. 275

be closely related to the hyaline substance (hyaline substance of ROVIDA),
occurring in the lymph-cells. In the mass extracted by alcohol from
the blood-corpuscles of the hen, ACKERMANN found 3.93 per cent phos-
phorus and 17.2 per cent nitrogen, which on calculation gave 42.10 per
cent nucleic acid and 57.82 per cent histone. PIETTRE and VILA l found,
in the -stromata, 0.3 per cent phosphorus in the horse and 2.3-2.6 per
cent in birds (ducks and hens) , calculated on the ash-free substance. They
found the quantity of nitrogen to be 11.7 and 13.21 per cent for the horse
and dog respectively. The non-nucleated red blood-corpuscles are,
as a rule, very poor in protein, but rich in haemoglobin; the nucleated
corpuscles are richer in protein and poorer in haemoglobin than the
non-nucleated. The reducing substances, and in certain animal sugars,
probably also conjugated glucuronic acids and several enzymes, among
which occurs the proteolytic enzyme studied by ABDERHALD'EN and col-
laborators,2 belong to the stromata. It is difficult to decide in many
cases whether the enzymes found in the blood belong to the fluid or to
the various kinds of form-elements.

A gelatinous, fibrin-like protein body may be obtained from the red
blood-corpuscles under certain circumstances. This fibrin-like mass
has been observed on freezing and then thawing the sediment of the
blood-corpuscles, or on discharging the spark from a large Leyden jar
through the blood, or on dissolving the blood-corpuscles of one kind of
animal in the serum of another (LANDOIS, stroma-fibriri) ; i.e., in the so-
called hcemagglutination, a clumping of the red blood-corpuscles into
clusters takes place. This agglutination can be brought about by bodies
similar to the hsemolysines and also by serum constituents produced
normally or by immunization. It has not been shown that a fibrin for-
mation from the stroma takes place, nor is it probable. Fibrinogen
has been detected only in the red corpuscles of frog's blood (ALEX. SCHMIDT
and SEMMERS).

Closely related to the anatomical and chemical structure of the ei«ythro-
cytes is the question, which is important for the metabolism in the blood,
as to the permeability of the erythrocytes, that is, their power of tak-
ing up substances of different kinds. This question as well as the per-
meability of the blood-corpuscles for anions under the influence of carbon
dioxide has been discussed in Chapter I, pages 7 and 8.

The mineral bodies of the red corpuscles will be treated in connection
with their quantitative constitution.

1 Ackermann, Zeitschr. f. physiol. Chem., 43; Piettre and Vila, Compt. Rend., 143.

2 Zeitschr. f. physiol. Chem., 51, 53 and 55.

3Landois, Centralbl. f. d. med. Wissensch., 1874, 421; Schmidt, Pfliiger's Arch.,
11, 550-559.

276 THE BLOOD.

The constituent of the blood-corpuscles existing in greatest quantity
is the red pigment haemoglobin.

Blood-pigments.

According to HOPPE-SEYLER the coloring-matter of the red blood-
corpuscles is not in a free state, but combined with some other sub-
stance. The crystalline coloring-matter, the haemoglobin or oxyhsemo-
globin, which may be isolated from the blood, is considered, according
to HOPPE-SEYLER, as a cleavage product of this compound, but it acts
in many ways unlike the questionable compound itself. This compound
is insoluble in water and uncrystallizable. It strongly decomposes
hydrogen peroxide without being oxidized itself; it shows a greater resist-
ance to certain chemical reagents (as potassium ferricyanide) than the
free coloring-matter; and, lastly, it gives off its loosely combined oxygen
much more easily in vacuum than the free pigment. To distinguish
between the cleavage products, the haemoglobin, and the oxyhaemoglobin,
HOPPE-SEYLER calls the compound of the blood-coloring matter of the
venous blood-corpuscles phlebin, and that of the arterial arterin. Other
investigators, such as H. U. KOBERT and BoHR,2 the latter calling the
pigment of the blood-corpuscles hcemochrom, are of a similar opinion.
Since the above-mentioned combinations of the blood-coloring matters
with other bodies, for example (if they really do exist) with lecithin, have
not been closely studied, the following statements will apply only to the
free pigment, the haemoglobin.

The color of the blood depends in part on kcemoglobin and in part
on a molecular combination of this substance with oxygen, the oxy-
hamoglobin. We find in blood after asphyxiation almost exclusively
haemoglobin, in arterial blood disproportionately large amounts of
oxy haemoglobin, and in venous blood a mixture of both. Blood-color-
ing matters are also found in striated as well as in certain smooth muscles,
and lastly in solution in different invertebrates, although this pigment
is not quite identical with that from higher animals. The quantity of
haemoglobin in human blood may indeed be somewhat variable under
different circumstances, but amounts to about 14 per cent on an average,
or 8.5 grams for each kilo of the weight of the body.

Haemoglobin belongs to the group of compound proteins, and yields
as cleavage products, besides very small amounts of volatile fatty acids
and other bodies, chiefly a protein globin, and a coloring-matter, hwmo-

2 Hoppe-Seyler, Zeitschr. f/physiol. Chem., 13, 479; H. U. Robert, Das. Wirbeltier-
blut in mikro-kristallogr. Hinsicht, Stuttgart, 1901; Bohr, Centralbl. f. Physiol., 17,
p. 688.

BLOOD-PIGMENTS.

277

chromogen (about 4 per cent), containing iron, which in the presence of
oxygen is easily oxidized into hcematin.

As first shown by SCHUNCK and MARCHLEWSKI, and especially by the
work of the latter, a close relation exists between chlorophyll and the
blood-pigment, because a derivative of the first, phylloporphyrin,
stands very close in certain respects to a derivative of the blood-pigment
hsematoporphyrin. By the investigations of NENCKI in conjunction
with MARCHLEWSKI and ZALESKI, x it was shown that haemopyrrol could
be prepared from the derivatives of both the leaf-pigment and the blood-
pigments by reduction, and also the investigations of PILOTY and WILL-
STATTER on chlorophyll and blood pigments have further developed
the interesting biological fact that the chlorophyll and blood pigments
are closely related bodies.

The haemoglobin prepared from different kinds of blood has not
exactly the same composition, which seems to indicate the presence
of different haemoglobins. The analyses by different investigators of
the haemoglobin from the same kind of blood do not always agree with
one another, which probably depends upon the somewhat varying methods,
of preparation. The following analyses are given as examples of the
constitution of different haemoglobins:

Haemoglobin from the C

Dog 53.85

" 54.57

Horse 54.87

" 51.15

Ox 54.66

Pig 54.17

" 54.71

Guinea-pig 54 . 12

Squirrel 54 . 09

Goose 54.26

Hen 52.47

That the repeatedly observed quantity of phosphorus in the haemo-
globin of birds (Inoko and others) is due to a contamination has been
proved by ABDERHALDEN and MEDIGRECEANU. In the haemoglobin
from the horse (ZINOFFSKY), the pig, and the ox (HUFNER), we have
1 atom of iron to 2 atoms of sulphur, while in the haemoglobin from the
dog (JAQTJET) the relation is 1 to 3. From the data of the elementary
analysis, as also from the amount of loosely combined oxygen, HUFNER 1
has calculated the molecular weight of dog-haemoglobin as 14,129, and

7
7
6

6

7
7

7
7
7
7
7

H
.32
.22
.79
.76
.25
.38
.38
.36
.39
.10
.19

N
16.17
16.38
17.31
17.94
17.70
16.23
17.43
16.78
16.09
16.21
16.45

0.
0.
0
0
0
0.
0
0
0
0
0

s

390
568
.650
.390
447
660
479
.580
400
540
,857

0
0.
0
0
0
0
0
0
0
0
0

Fe
.430
336
.470
.335
.400
.430
.399
.480
.590
.430
.335

0

21.84
20.93
19.73
23.43
19.543
21.360
19.602
20.680
21 . 440
20.690
22.500

(HOPPE-SEYLEB)

(JAQUET)

(KOSSEL)

(ZINOFFSKY)

(HUFNER)

(OTTO)
(HUFNER)

(HOPPE-SEYLER)

t 1

( (
(JAQUET)

'Schunck and Marchlewski, Annal. d. Chem. u. Pharm., 278, 284, 288, 290;
Nencki, Ber. d. deutsch. chem. Gesellsch., 29; Marchlewski, and Nencki, Ber. d. d.
chem., Gesellsch., 34; Nencki and Zaleski, ibid., Marchlewski, Chem. Centralbl.,
1902, I, 1016; Zaleski, Zeitschr. f. physiol. Chem., 37. The literature and the works
of Willstatter and Piloty will be given under haemopyrrol, page 297.

278 THE BLOOD.

the formula CeseHK^sN^FeSaOisi. According to the more recent
determinations of HUFNER and JAQUET, ox-hsemoglobin contains an
average of 0.336 per cent iron, and the human haemoglobin, according
to BUTTERFIELD 1 contains 0.334 per cent iron. From the iron a molec-
ular weight of 16,669 may be calculated. BARCROFT and HILL have
arrived at exactly the same value by using an entirely different method
and HUFNER and GANSSER 2 have attempted to learn the size of the molec-
ular weight of haemoglobin by means of osmotic pressure determinations,
and they found the following approximate results: for horse-haemoglobin
15,115 and for ox-haemoglobin 16,321. The haemoglobin from various
kinds of blood not only shows a diverse constitution, but also a different
solubility and crystalline form, and a varying quantity of water of crys-
tallization; hence we infer that there are several kinds of haemoglobin.
BOHR is a very zealous advocate of this supposition. He has been able
to obtain haemoglobins from dog- and horse-blood, by fractional crystalliza-
tion, which had different powers of combining with oxygen and contained
different quantities of iron. HOPPE-SEYLER had already prepared two
different forms of haemoglobin crystals from horse-blood, and BOHR
concludes from all these observations that the ordinary haemoglobin
consists of a mixture of different haemoglobins. In opposition to this
statement, HUFNER 3 has shown that only one haemoglobin exists in ox-
blood, and that this is probably true for the blood of many other animals.
Oxyhaemoglobin, which has also been called H^EMATOGLOBULIN or
H^EMATOCRYSTALLIN, is a molecular combination of haemoglobin and
oxygen. For each molecule of haemoglobin 1 molecule of oxygen is
present, as shown by the investigations of HUFNER as well as HUFNER
and GANSSER, and the amount of loosely combined oxygen which is united
to 1 gram of haemoglobin (of the ox) has been determined by HUFNER 4
as 1.34 cc. (calculated at 0° C. and 760 mm. mercury).

According to BOHR, the facts are different. He differentiates between four
oxyhsemoglobins, according to the quantity of oxygen which they absorb, namely

1 Hoppe-Seyler, Med. chem. Untersuch., 370; Jaquet, Zeitschr. f. physiol. Chem.,
14, 296; Kossel, ibid,. 2, 150; Zinoffsky, ibid., 10; Hiifner, Beitr. z. Physiol., Festschr.
f. C. Ludwig, 1887, 74-81, Journ. f. prakt. Chem. (N. F.), 22; Otto, Zeitschr. f. physiol.
Chem., 7; Inoko, ibid., 18; Abderhalden and Medigreceanu, ibid., 59; Hiifner and
Jaquet, Arch. f. (Anat. u.) Physiol., 1894; E. Butterfield, Zeitschr. f. physiol. Chem.,
62.

2 Barcroft and Hill, Journ. of Physiol. 39; Hiifner and Gansser, Arch. f. (Anat. u.)
Physiol., 1907.

3 Bohr, "Sur les combinaisons de 1'hernoglobine avec 1'oxygene," Extrait du
Bulletin de l'Acade*mie Royale Danoise des sciences, 1890; also Centralbl. f. Physiol.
1890, 249. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 2; Hiifner, Arch. f. (Anat. u.)
Physiol., 1894.

4 Arch. f. (Anat. u.) physiol., 1901, Suppl.

OXYHAEMOGLOBIN. 279

tt.? /3-, 7- and S-oxyhsemoglobin, all having the same absorption-spectrum, and 1
gram combining with respectively 0.4, 0.8, 1.7, and 2.7 cc. oxygen at the tem-
perature of the room and with an oxygen pressure of 150 mm. mercury. The
7-oxyha3moglobin is the ordinary one obtained by the customary method of
preparation. BOHR designates as a-oxyhsemoglobin the crystallin powder
obtained by drying 7-oxyhaemoglobin in the air. On dissolving a-oxyhsemo-
globin in water it is converted into jS-oxyhsemoglobin without decomposition,
and the quantity of iron is increased. On keeping a solution of 7-oxyha3moglobin
in a sealed tube it is transformed into S-oxhyaamoglobin, although the exact
conditions under which this change takes place are not known. According to
HUFNER J these are nothing but mixtures of genuine and partly decomposed
haemoglobins.

The ability of haemoglobin to take up oxygen seems to be a function
of the iron it contains, and when this is calculated as about 0.33-0.40
per cent, then 1 atom of iron in the haemoglobin corresponds to about
2 atoms or 1 molecule of oxygen. By increasing the partial pressure as
well as by increasing the quantities of oxygen, the haemoglobin in solu-
tion takes up more oxygen, until it is completely saturated, when 1 mole-
cule of haemoglobin is combined with 1 molecule of oxygen. With reduced
oxygen pressure a dissociation must naturally take place and oxygen
is given off, and a re-formation of haemoglobin takes place, and this makes
it possible to expel completely the oxygen from an oxyhaemoglobin solu-
tion or blood by means of vacuum, or by passing an indifferent gas
through the solution. The equilibrium between oxyhaemoglobin, haemo-
globin, and oxygen depends, therefore, according to HUFNER, upon
a mass action, corresponding to the formula Hb-f-C^^HbCV BoHR2
has arrived at the conclusion that not only a double dissociation takes
place, in which a dissociation of the oxygen-iron combination in the
oxyhaemoglobin occurs, but also a dissociation of the haemoglobin
into a ferruginous as well as into a non-ferruginous part. Correspond-
ingly he has suggested another formula and hence the dissociation curves
for oxyhaemoglobin given by HUFNER and BOHR are different.

Important investigations have recently been carried out on this
question by BARCROFT and his co-workers CAMIS and ROBERTS from
which it follows that a generally valid dissociation curve cannot be given,
as the curve direction is dependent upon the nature and concentration
of the salts present in the solution. A haemoglobin solution with the salts
of the blood-corpuscles of the dog gives a dissociation curve of dog-blood
while with the salts from human blood-corpuscles it gives a curve like
human blood. In the presence of salts the dissociation follows BOHR'S
formula, and on the contrary while a salt-free haemoglobin solution follows
the oxygen combination according to the mass-action law of HUFNER.

1 Arch. f. (Anat. u.) physiol., 1894.

2 Bohr, Centralbl. f. physiol., 17, pp. 682 and 688.

280 THE BLOOD.

Thus far there does not seem to be any necessity for considering the
gas combination in the blood and in haemoglobin solutions to be adsorp-
tion processes as suggested by W. OsTWALD.1

That the gas combining ability of an isolated pure haemoglobin cannot
be compared with the gas combining ability of the so-called native haemo-
globin of the blood has been suggested by many experimenters. In this
connection we must mention the observations of MANCHOT 2 who found
that the combining ability of the blood for gases such as 02, CO,NO,C2H4
could be increased at least to a certain limit by increasing the dilution
so that at 8-10 times the dilution the combining power was close to the
limit value of 2 mol. gas for each atom of iron.

The elucidation of these mentioned conditions is of the greatest
importance, as the knowledge of the various conditions which influence
the taking up and the giving up of oxygen by the haemoglobin is of the
greatest importance for our knowledge of the taking up of oxygen in the
lungs and the giving up of the same to the tissues.

Oxyhaemoglobin which is generally considered as a weak acid, is
according to GAMGEE,S dextrorotatory. The specific rotation for light
of medium wave-length of C is (a) C = about +10°, which corresponds
also for carbon-monoxide haemoglobin. The haemoglobin is also, like
carbon-monoxide haemoglobin (COHb) and methaemoglobin (MHb),
diamagnetic, while the haematin, which is richer in iron, is strongly mag-
netic (GAMGEE4). On passing an electric current through an oxyhaemo-
globin solution, the pigment first separates unchanged at the anode in a
colloidal but still soluble form, and is then gradually transferred to the
cathode in the colloidal state (GAMGEE 5) . According to GAMGEE, the
haemoglobin probably exists in such a colloidal condition in the blood-
corpuscles.

Oxyhaemoglobin has been obtained in crystals from several varieties
of blood. These crystals are blood-red, transparent, silky, and may
be 2-3 mm. long. The Oxyhaemoglobin from squirrel's blood crystallizes
in six-sided plates of the hexagonal system; the other varieties of blood
yield needles, prisms, tetrahedra, or plates which belong to the rhombic
system.6 The quantity of water of crystallization varies between

1 Barcroft with Camis, Journ. of Physiol., 39; with Roberts, ibid.; W. Ostwald,
Kolloid-Zeitschr., 2, cited in Maly's Jahresb., 38, 187.

2 Annal. d. Chem. u. Pharm.; 370 and Zeitschr. f. physiol. Chem., 70.

3 Hofmeister's Beitrage, 4.

4 Proceedings of Roy. Society, 68.

5 Ibid., 70.

6 The observation of Uhlik (Pfliiger's Arch., 104) that the haemoglobin from horse-
blood can also crystallize in hexagonal plates seems to be due to the fact that he had
haemoglobin and not Oxyhaemoglobin.

OXYH^EMOGLOBIN. 281

3-10 per cent for the different oxyhsemoglobins. When completely
dried at a low temperature over sulphuric acid the crystals may be
heated to 110-115° C. without decomposition. At higher temperatures,
somewhat above 160° C., they decompose, giving an odor of burned horn,
and leave, after complete combustion, an ash consisting of oxide of
iron. The oxyhsemoglobin crystals from difficultly crystallizable blood,
for example from such as ox's, human, and pig's blood, are easily
soluble in water. The oxyhsemoglobins from easily crystallizable
blood, as from that of the horse, dog, squirrel, and guinea-pig, are soluble
with difficulty in the order above given. The oxyhsemoglobin dissolves
more easily in a very dilute solution of alkali carbonate than in pure water,
and this solution may be kept. The presence of a little too much alkali
causes the oxyhaemoglobin to decompose quickly. The crystals are
insoluble in absolute alcohol without decolorization. According to
NENCKI 1 it is converted into an isomeric or polymeric modification,
called by him parahoemoglobin. Oxyhsemoglobin is insoluble in ether,
chloroform, benzene, and carbon disulphide.

A solution of oxyhsemoglobin in water is precipitated by many metallic
salts, but is not precipitated by sugar of lead or basic lead acetate. On
heating the watery solution it decomposes at about 70° C., and splits
off protein and hsematin when sufficiently heated. It is also readily
decomposed by acids, alkalies, and many metallic salts. It gives the
ordinary reactions for proteins with those protein reagents which first
decompose the oxyhsemoglobin with the splitting off of protein. Oxy-
hsemoglobin, like the other blood-pigments, has a direct oxidizing action
upon tincture of guaiacum. It has, on the other hand, like all blood-
pigments containing iron, the property of an " ozone transmitter " in
that it turns tincture of guaiacum blue in the presence of reagents con-
taining peroxide, such as old turpentine.

A sufficiently dilute solution of oxyhsemoglobin or arterial blood
shows a spectrum with two absorption-bands between the FRAUN-
HOFER lines D and E (spectrum Plate 1). The one band, a, which is nar-
rower but darker and sharper, lies on the line D; the other, broader,
less defined and less dark band, /3, lies at E. The middle of the first
band corresponds to a wave-length X = 579 and the second X = 542. On
dilution the band |8 first disappears. By increased concentration of the
solution the two bands become broader, the space between them smaller
or entirely obliterated, and at the same time the blue and violet part
of the spectrum is darkened. Besides these two bands we can also observe

1 Nencki and Sieber, Ber. d. d. chem. Gesellsch., 18. According to Kriiger (see
Biochem. Centralbl., I, 40, 463) haemoglobin is somewhat changed by alcohol as well
as by chloroform.

282 THE BLOOD.

by the aid of special appliances (L. LEWIN, MIETHE, and STENGER) the
band first described by SORET and then by GAMGEE in the ultra-violet
portion. This violet band, X = 415, is of importance in the detection
of very small quantities of blood. While the two oxyhsemoglobin bands
are still detectable in a dilution of 1 :14,700 the violet band may be seen,
according to LEWIN, MIETHE and STENGER l in a dilution of 1 :40,000.

The observation of PIETTRE and VILA that so-called laky blood and oxyhaemo-
globin solutions in thick layers also show a third band in the red (X = 634) depends
in all probability, as also claimed by VILLE and DERRIEN, upon a partial forma-
tion of methsemoglobin which according to ARON 2 exists preformed in all blood.

A great many methods have been proposed for the preparation of
oxyhsemoglobin crystals, but in their chief features they all agree with
the following one suggested by HOPPE-SEYLER: The washed blood-
corpuscles (best those from the dog or the horse) are stirred with 2
vols. of water and then shaken with ether. After decanting the ether and
allowing the ether which is retained by the blood solution to evaporate
in an open dish in the air, cool the filtered blood solution to 0° C., add
while stirring one-fourth vol. of alcohol also cooled, and allow to stand
a few days at —5° to —10° C. The crystals which separate may be
repeatedly recrystallized by dissolving in water of about 35° C., cooling,
and adding cooled alcohol as above. Lastly, they are washed with
cooled water containing alcohol (one-quarter vol. alcohol) and dried
hi vacuum at 6° C. or a lower temperature.3

For the preparation of oxyhsemoglobin crystals in small quantities
from easily crystallizable blood, it is often sufficient to stir a drop of
blood with a little water on a microscope slide and allow the mixture
to evaporate so that the drop is surrounded by a dried ring. After
covering with a cover-glass, the crystals gradually appear radiating from
the ring. These crystals are formed more surely if the blood is first
mixed with some water in a test-tube and shaken with ether, and a drop
of the lower deep-colored liquid treated as above on the slide.

Haemoglobin, also called REDUCED HAEMOGLOBIN or PURPLE CRUORIN
(STOKES4), occurs only in very small quantities in arterial blood, in
larger quantities in venous blood, and is almost the only blood-coloring
matter after asphyxiation.

Hsemoglobin is much more soluble than the oxyhsemoglobin, and
it can therefore be obtained as crystals only with difficulty. These

, cited in Maly's Jaresb., 8; Gamgee, Zeitschr. f. Biol., 34; Lewin, Miethe
and Stenger, Pfliiger's Arch., 118; Lewin and Miethe, ibid., 121.

2 Piettre and Vila, Compt. Rend., 140; Ville and Derrien, ibid., 140; Aron, Biochem.
Zeitschr., 3.

3 In regard to the preparation of oxyhsemoglobin, see also Hoppe-Seyler-Thier-
felder's Handbuch, 8. Aufl.; also the works cited in footnote 1, p. 278; also Schuur-
manns-Stekhoven, Zeitschr. f. physiol. Chem., 33, 296; see also Bohr, Skand. Arch.
f. Physiol., 3; J. Offringa, Bioch. Zeitschr., 28.

4 Philosophical Magazine, 28, No. 190, Nov., 1864.

METH^EMOGLOBIN. 283

crystals are as a rule isomorphous with the corresponding oxyhsemo-
globin crystals, but are darker, having a shade toward blue or purple,
and are decidedly more pleochromatic. The haemoglobin from horse-
blood has also been obtained by UHLIK 1 in hexagonal plates. Its
solutions in water are darker and more violet or purplish than solu-
tions of oxyhsemoglobin of the same concentration. They absorb the
blue and the violet rays of the spectrum in a less marked degree, but
strongly absorb the rays lying between C and D. In proper dilution
the solution shows a spectrum with one broad, not sharply denned band
between D and E, whose darkest part corresponds to the wave-length
X = 559 (spectrum Plate, 2). This band does not lie in the middle
between D and E, but is toward the red end of the spectrum, a little
over the line D. This pigment also gives a band in the ultra-violet,
X = 429. A haemoglobin solution actively absorbs oxygen from the air
and is converted into an oxyhsemoglobin solution.

A solution of oxyhaemoglobin may be easily converted into a solution
having the spectrum of haemoglobin by means of a vacuum, by passing
an indifferent gas through it, or by the addition of a reducing substance,
as, for example, an ammoniacal ferrous-tartrate solution (STOKES' reduc-
tion liquid). If an oxyhaemoglobin solution or arterial blood is kept in
a sealed tube, we observe a gradual consumption of oxygen and a reduc-
tion of the oxyhaemoglobin into haemoglobin. If the solution has a
proper concentration, a crystallization of haemoglobin may occur in the
tube at lower temperatures (HUFNER 2) .

Methaemoglobin. This name has been given to a coloring-matter
which is easily obtained from oxyhaemoglobin as a transformation prod-
uct and which has been correspondingly found in transudates and cystic
fluids containing blood, in urine in haematuria or haemoglobinuria, and
also m urine and blood on poisoning with potassium chlorate, amyl
nitrite or alkali nitrite, and many other bodies.

Methaemoglobin does not contain any oxygen in molecular or dis-
sociable combination, but still the oxygen seems to be of importance in
the formation of methaemoglobin, because it is formed from oxyhsemo-
globin and not from haemoglobin in the absence of oxygen or oxidizing
agents. If arterial blood be sealed up in a tube, it gradually consumes
its oxygen and becomes venous, and by this absorption of oxygen a little
methaemoglobin is formed. The same occurs on the addition of a small
quantity of acid to the blood. By the spontaneous decomposition
of blood some methaemoglobin is formed, and by the action of ozone,
potassium permanganate, potassium ferricyanide, chlorates, nitrites,

1 Pfliiger's Arch., 104.

2 Zeitschr. f. physiol. Chem., 4; see also Uhlik, 1. c.

284 THE BLOOD.

nitrobenzene, pyrogallol, pyrocatechin, acetanilide, and certain other
bodies on the blood an abundant formation of methsemoglobin takes place.
By the action of light, HASSELBACH,1 especially by the use of rays hav-
ing a wave-light below 310 M M, obtained methsemoglobin from oxyhsemo-
globin, but not from haemoglobin in the absence of oxygen, and by this
behavior pure methsemoglobin can be prepared.

According to the investigations of HUFNER, KULZ, and OTTO 2
methsemoglobin contains just as much oxygen as oxy haemoglobin, but
it is more strongly combined, a view which is accepted by most investiga-
tors. According to HUFNER and v. ZEYNEK we can admit in the methsemo-
globin formation of an expulsion of oxygen and a combination of two

/OR
hydroxyl groups; methsemoglobin would then be Hb<^ . Accord-

[b^ .

NOH

ing to others, HOPPE-SEYLER, KUSTER, LETSCH the methsemoglobin
contains less oxygen than the oxyhsemoglobin and is HbO or HbOH.
A methsemoglobin solution is converted into a hsemoglobin solution
by reducing substances. The reaction taking place in the forma-
tion of methsemoglobin from oxyhsemoglobin by the action of potassium
ferricyanide has been quantitatively followed by v. REiNBOLD.3 He
found that one molecule of KsFe(CN)6 was necessary to transform 1
molecule oxyhsemoglobin or to drive off 1 molecule of oxygen from the
oxyhsemoglobin. The reaction takes place according to the equation:

/O

Hb< | ^K3Fe(CN)6+H20 = Hb.OH+K3HFe(CN)6+02
X)

and from his investigations he gives the formula Hb.OH to methsemo-
globin, in correspondence to the views of KUSTER.

According to HUFNER and REINBOLD 4 1 gram methsemoglobin can
take up 2.685 cc. nitric oxide.

Methsemoglobin crystallizes, as first shown by HUFNER and OTTO,
in brownish-red needles, prisms, or six-sided plates. It dissolves easily
in water; the solution has a brown color and becomes a beautiful red
on the addition of alkali. The solution of the pure substance is not
precipitated by basic lead acetate alone, but by basic lead acetate and

. Zeitschr., 19.

2 See Otto, Zeitschr. f. physiol. Chem., 7; v. Zeynek, Arch. f. (Anat. u.) Physiol.,
1899; Hiifner, ibid.

3 Kiister, Zeitschr. f. physiol. Chem., 66; Letsche, ibid., 80; v. Reinbold, Zeitschr.
f. physiol. Chem., 85.

4 Arch, f . (Anat. u.) Physiol., 1904, Suppl.

METH^MOGOBIN. CYANMETH^EMOGLOBIN. 285

ammonia. The absorption-spectrum of a watery or acidified solution
of methaemoglobin is, according to JADERHOLM and BERTIN-SANS, very
similar to that of hsematin in acid solution, but is easily distinguished
from the latter since, on the addition of a little alkali and a reducing
substance, the former passes over to the spectrum of reduced haemoglobin,
while a hsematin solution under the same conditions gives the spectrum
of an alkaline h^mochromogen solution (see below). According to
ARAKI and DITTRICH, a neutral or faintly acid methaemoglobin solution
shows only one characteristic band, a, between C and D, whose middle
corresponds to about X = 634. The two bands between D and E are
only due to contamination with oxyhaemoglobin (MENZIES, LEWIN,
MIETHE and STENGER. According to HASSELBACH'S 1 experience a
pure neutral solution of methaemoglobin gives four absorption bands cor-
responding to a maxima X = 630, 580, 540 and 500. Methsemoglobin
in alkaline solution shows two absorption-bands which are like the
two oxyhaemoglobin bands, but they differ from these in that the band
0 is stronger than a. By the side of the band a and united with it by a
shadow lies a third fainter band between C and D, near to D. (Spec-
trum Plate, 4.)

The claims as to the action of sodium fluoride upon haemoglobin and methaemo-
globin are somewhat contradictory.2

Crystallized methaemoglobin may be easily obtained by treating a
concentrated solution of oxyhaemoglobin with a sufficient quantity of
concentrated potassium-ferricyanide solution to give the mixture a porter-
brown color. After cooling to 0° C. add one-fourth vol. cooled alcohol
and allow the mixture to stand a few days in the cold. The crystals
may be easily purified by recrystallizing from water by the addition
of alcohol. According to HASSELBACH this method ordinarily gives
impure products while a pure preparation can be obtained by the action
of l^ht (see above).

Cyanmethaemoglobin (cyanhsemoglobin) is, according to HALDANE, identical
with photomethaemoglobin (Bocx), which is produced by the influence of sun-
light upon a methaemoglobin solution containing potassium ferricyanide. It
was first carefully described by R. ROBERT and obtained in a crystalline form
by v. ZEYNEK.S It is immediately formed in the cold by the action of a hydro-
cyanic-acid solution upon methaemoglobin, but is formed by its action upon oxy-

1 Jaderholm, Zeitschr. f. Biol. 16; Bertin-Sans, Comp. Rend., 106; Araki, Zeitschr. f.
physiol. Chem., 14; Dittrich. Arch. f. exp. Path. u. Pharm., 29; Menzies, Journ. of
PhysioL, 17; Lewin and collaborators, footnote 1, page 282; Hasselbach, Bioch.
Zeitschr., 19, and Proceedings of the 7th Internat. Congr. of Appl. Chem., London,
1909. Important references on methsemoglobin are given by Otto, Pfliiger's Arch., 31.

2 Piettre and Vila, Compt. Rend., 140; Ville and Derrien, ibid., 140.

3 Haldane, Journ. of PhysioL, 25; Bock, Skand. Arch. f. Physiol., 6; Robert,
Pfliiger's Arch., 82; v. Zeynek, Zeitschr. f. physiol. Chem., 33. See also Leers,
Biochem. Zeitschr., 12.

286 THE BLOOD.

haemoglobin only at the body temperature. The neutral or faintly alkaline
solutions show a spectrum which is very similar to the hemoglobin spectrum.
The question as to a special cyanmethsemoglobin is still disputed.

Acid haemoglobin is a coloring-matter produced by the action of very weak
acids upon oxyhsemoglobin, which according to HARNACK 1 is not, as used to be
admitted, identical with methffimoglobin.

Carbon-monoxide Haemoglobin 2 is the molecular combination between

1 molecule of haemoglobin and 1 molecule of CO, according to HtJFNER,3
which contains 1.34 cc. of carbon monoxide (at 0° and 760 mm. Hg)
for 1 gram haemoglobin. This combination is stronger than the oxygen
combination of haemoglobin. The oxygen is for this reason easily driven
out of oxyhsemoglobin by carbon monoxide, and this explains the poison-
ous action of this gas, which kills by the expulsion of the oxygen of the
blood. In regard to the division of the blood-pigments between the car^
bon monoxide and oxygen under different partial pressures of both gases
in the air, we must refer to the investigations of HUFNER, DOUGLAS and

HALDANE.4

The carbon monoxide can be driven out by a vacuum as well as by
passing an indifferent gas, or oxygen, or nitric oxide, through the solu-
tion for a long time, and in these cases haemoglobin, oxyhaemoglobin,
or nitric-oxide haemoglobin are formed. The carbon-monoxide is also
expelled by potassium ferricyanide and methaemoglobin is formed
(HALDANE 5) . The above-mentioned behavior found by MANCHOT for
the absorption of oxygen, namely, that the amount of gas taken up
increases with the dilution of the blood so that for every atom of iron

2 mol. of gas are absorbed applies also for the carbon-monoxide haemo-
globin as well as for the nitric-oxide haemoglobin, which will be discussed
further on.

Carbon-monoxide haemoglobin is formed by saturating blood or
a haemoglobin solution with carbon monoxide, and may be obtained
as crystals by the same means as oxyhaemoglobin. These crystals are
isomorphous with the oxyhaemoglobin crystals, but are less soluble and
more stable, and their bluish-red color is more marked. For the detec-

1 Zeitschr. f. physiol. Chem., 26.

2 In reference to carbon-monoxide haemoglobin, see especially Hoppe-Seyler, Med.-
chem. Untersuch., 201; Centralbl. f. d. med. Wissensch., 1864 and 1865; Zeitschr.
f. physiol. Chem., 1 and 13.

3 Arch. f. (Anat. u.) Physiol., 1894. On the dissociation constant of carbon-
monoxide haemoglobin, see ibid., 1895. In regard to the contradictory statements
of Saint-Martin and others and their disapproval, see Hiifner, Arch. f. (Anat. u.)
Physiol., 1903.

4 Hiifner, Arch. f. exp. Path. u. Pharm., 48; Douglas and Haldane, Journ. of
Physiol., 44.

5 Journ. of Physiol., 22.

CARBON-MONOXIDE HAEMOGLOBIN. 287

tion of carbon-monoxide haemoglobin, its absorption-spectrum is of the
greatest importance. This spectrum shows two bands which are very
similar to those of oxyha3moglobin, but they occur more toward the violet
part of the spectrum. The middle of the first band corresponds to
X = 570, and the second to X = 542 (LEWIN, MIETHE and STENGER).
These bands do not change noticeably on the addition of reducing
substances; this constitutes an important difference between carbon-
monoxide haemoglobin and oxyhaemoglobin. If the blood contains oxy-
haBmoglobin and carbon-monoxide haemoglobin at the same time, we
obtain on the addition of a reducing substance (ammoniacal ferro-tar-
trate solution) a mixed spectrum originating from the haemoglobin and
carbon-monoxide haemoglobin. Carbon-monoxide haemoglobin also gives
a band in the violet A = 416.

A great many reactions have been suggested for the detection of
carbon-monoxide haemoglobin in medico-legal cases. A simple and at
the same time a good one is HOPPE-SEYLER'S alkali test. The blood is
treated with double its volume of caustic-soda solution of 1.3 sp. gr.,
by which ordinary blood is converted into a dingy brownish mass, which
when spread out on porcelain is brown with a shade of green. Carbon-
monoxide blood gives under the same conditions a red mass, which if
spread out on porcelain shows a beautiful red color. Several modifica-
tions of this test have been proposed. Another very good reagent is tan-
nic acid, which gives with dilute normal blood a brownish-green precip-
itate and with carbon-monoxide blood a pale crimson-red precipitate.1

As according to BOHR there are several oxyhaemoglobins, so also accord-
ing to BOHR and BocK,2 there are several carbon-monoxide hemoglobins, with
different amounts of carbon monoxide. As haemoglobin can unite with oxygen
and carbon dioxide simultaneously, as shown by BOHR and TROUP, so also can it
unite with carbon monoxide and carbon dioxide simultaneously and independently
of each other.

Carbon-monoxide methaemoglobin has been prepared by WEIL and v. ANREP
by the action of potassium permanganate on carbon-monoxide hemoglobin,
but this is contradicted by BERTIN-SANS and MOITESSIER.S Sulphur methaemo-
globin is the name given by HOPPE-SEYLER to that coloring-matter which is
formed by the action of sulphureted hydrogen upon oxyhaemoglobin and which
is generally designated sulphcemoglobin. The solution has a greenish-red, dirty
color, and shows two absorption-bands between C and D. This coloring-matter
is claimed to be the greenish color seen on the surface of putrefying flesh. Accord-
ing to HARNACK the conditions are different when H2S is passed through an
oxygen-free solution of haemoglobin (or carbon-monoxide haemoglobin). The

1 In regard to this test (as suggested by Kunkel) and others we refer to Kostin,
Pfliiger's Arch., 84, which contains a very excellent summary of the literature on the
subject. See also de Domenicis, Chem. Centralbl., 1908, 2, p. 66.

2 Centralbl. f. Physiol., 8, and Maly's Jahresber., 25.

3 v. Anrep, Arch. f. (Anat. u.) Physiol., 1880; Sans and Moitessier, Compt. Rend.,
113.

288 THE BLOOD.

sulphsemoglobin thus formed shows one band in the red between C and D.
According to CLARKE and HURTLEY l the formation of sulphsemoglobin takes
place after the reduction to haemoglobin.

Carbon-dioxide Haemoglobin, Carbohoemoglobin. Haemoglobin, accord-
ing to BOHR and ToRUP,2 also forms a molecular combination with
carbon dioxide whose spectrum is similar to that of haemoglobin. Accord-
ing to BOHR there are three different carbohaemoglobins, namely, a-,
P-, and 7-carbohaemoglobin, in which 1 gram combines with respectively
1.5, 3; and 6 cc. CO2 (measured at 0° C. and 760 mm.) at 18° C. and a
pressure of 60 mm. mercury. If a haemoglobin solution is shaken with a
mixture of oxygen and carbon dioxide, the haemoglobin combines loosely
with the oxygen as well as with the carbon dioxide, independently of
each other, just as if each gas existed alone (BOHR). He considers
that the two gases are combined with different parts of the haemoglobin,
that is, the oxygen with the pigment nucleus and the carbon dioxide
with the protein component. Attention must be called to the fact that,
as observed by TORUP, haemoglobin is in part readily decomposed by
the carbon dioxide with the splitting off of some protein.

Nitric-oxide Haemoglobin is also a crystalline molecular combina-
tion which is even stronger than the carbon-monoxide haemoglobin.
Its solution shows two absorption-bands, which are paler and less sharp
than the carbon-monoxide haemoglobin bands, and they do not dis-
appear on the addition of reducing bodies. Haemoglobin also forms a
molecular combination with acetylene and ethylene.

Haemorrhodin is the name given by LEHMANN to a beautiful red pigment
soluble in alcohol and ether, which is extracted from meat and meat products
by boiling alcohol and which seems to be produced by the action of small amounts
of nitrites. Another pigment isolated by LEWIN 3 from the blood of animals .
poisoned by phenylhydrazine, has been called hcemoverdin. By heating a solu-
tion of blood-pigment treated with caustic potash and mixed with alcohol to
60° C. we obtain, according to v. KLAVEREN, a pigment which he calls kathcemo-
globin, but called by ARNOLD,4 who first obtained it, neutral hcpmatin, which is
produced by the splitting off of a ferruginous complex. This pigment still con-
tains protein, but is poorer in iron than the hemoglobin or methsemoglobin and
probably forms an intermediary product in the conversion of the above into
hsematin.

Decomposition products of the blood-pigments. By its decomposi-
tion, haemoglobin yields, as previously stated, a protein, which has been

1 Hoppe-Seyler, Med.-chem. Untersuch., 151. See Araki, Zeitschr. f. physiol.
Chem., 14; Harnack, 1. c.; Clarke and Hurtley, Journ. of Physiol., 36.

2 Bohr, Extrait, du Bull, de 1'Acad. Danoise, 1890; Centralbl. f. Physiol.. 4 and
17; Torup, Maly's Jahresber., 17.

3 K. B. Lehmann, Sitzungsber. d. phys.-med. Gesellsch. Wiirzburg, 1899; Lewin,
Compt. Rend., 133.

4 v. Klaveren, Zeitschr. f. physiol. Chem., 33; Arnold, ibid, 29.

HSEMOCHROMOGEN. 289

called globin (PREYER, SCHULZ), and a ferruginous pigment as chief prod-
ucts. According to LAWROW 94.09 per cent protein, 4.47 per cent
hsematin, and 1.44 per cent other bodies are produced in this decom-
position. The globin, which was isolated and studied by SCHULZ/
differs from most other proteins by containing a high amount of carbon,
54.97 per cent., with 16.98 per cent of nitrogen. It is insoluble in water,
but very easily soluble in acids or alkalies. It is not dissolved by ammonia
in the presence of ammonium chloride. Nitric acid precipitates it in
the cold, but not when warm. It may be coagulated by heat, but the
coagulum is readily soluble in acids. Because of these reactions it is
considered as a histone by SCHULZ.

On hydrolytic cleavage globin (from horse-blood) yields, accord-
ing to AEDERHALDEN,2 the ordinary cleavage products of the proteins
and especially leucine, 29 per cent. It is also important to call attention
to. the large amount of histidine, 10.96 per cent, while the quantities of
arginine and lysine were only 5.42 and 4.28 per cent respectively.

The pigment split off is different, depending upon the conditions
under which the cleavage' takes place. If the decomposition takes place
in the absence of oxygen, a coloring-matter is obtained which is called
by HOPPE-SEYLER hcemochromogen, by other investigators (STOKES)
reduced hcematin. . In the presence of oxygen, hsemochromogen is quickly
oxidized to hsematin, and there is therefore obtained in this case hcematin
as a colored decomposition product. As hsemochromogen is easily
converted by oxygen into hsematin, so this latter may be reconverted
into hsemochromogen by reducing substances.

Haemochromogen was discovered by HOPPE-SEYLER.S It is, accord-
ing to HOPPE-SEYLER, the colored atomic group of haemoglobin and of
its combinations with gases, and this atomic group is combined with
proteins in the pigment. The characteristic absorption of light depends
on the hsemochromogen, and it is also this atomic group which binds, in
the oxyhsemoglobin, 1 molecule of oxygen and, in the carbon-monoxide
hsemoglobin, 1 molecule of carbon monoxide with 1 atom of iron. Hsemo-
chromogen is produced in an alkaline solution of hsematin by the action
of reducing bodies. By the reduction of hsematin in alcoholic ammoniacal
solution by means of hydrazine v. ZEYNEK4 was able to obtain the solid
brownish-red ammonia combination. A crystalline combination between
pyridine and hsemochromogen can be obtained according to KALMUS

1 Lawrow, ibid., 26; Schulz, ibid., 24; Preyer, Die Blutkristalle, Jena, 1871.

2 Zeitschr. f. physiol. Chem., 37; with Baumann, ibid., 51.

3 Ibid., 13.

4 Zeitschr. f. physiol., Chem., 25.

290 THE BLOOD.

and v. ZEYNEK1 from haemoglobin and pyridine by boiling, or from
hsematin and hsemin and pyridine after the addition of hydrazin-hydrate.

Haemochromogen also combines, as HOPPE-SEYLER first showed, with
carbon monoxide. This compound, which in aqueous solution gives
a spectrum similar to oxyhaemoglobin, has been obtained by PREGL2
in the solid condition as a deep-violet powder which is insoluble in
absolute alcohol. In opposition to haemoglobin the hsemochromogen
combines with oxygen more firmly than with carbon monoxide. The
assumption of HOPPE-SEYLER, that this compound is a combination of 1
molecule hsemochromogen and therefore contains 1 molecule carbon
monoxide for 1 molecule of iron has been experimentally substantiated
by HUFNER and KUSTER and by PREGL. 3

An alkaline haemochromogen solution has a beautiful cherry-red
color. It shows two absorption-bands, first described by STOKES (spec-
trum Plate, 6), one of which is dark and whose center corresponds to
X = 556.4 between D and E, and a second broader band, less dark, which
covers the FRAUNHOFER lines E and b. The middle of this band cor-
responds to X = 526 to 530 according to LEWIN, MIETHE and STENGER.
In acid solution haemochromogen shows four bands, which, according
to JA.DERHOLM,4 depend on a mixture of hsemochromogen and haemato-
porphyrin (see below), this last formed by a partial decomposition
resulting from the action of the acid.

MiLROY,5 from an alcoholic solution of haematin containing oxalic
acid, after driving out the air by means of hydrogen gas, gradually obtained
an acid solution of reduced haematin (hsemochromogen) by means of
zinc dust. This solution showed one absorption-band between D and E.

Haemochromogen may be obtained as crystals by the action of caustic
soda on haemoglobin at 100° C. in the absence of oxygen (HOPPE-SEYLER).
By the decomposition of haemoglobin by acids (of course in the absence
of air) we obtain hsemochromogen contaminated with a little haemato-
porphyrin. An alkaline haemochromogen solution is easily obtained by
the action of a reducing substance (STOKES' reduction liquid) on an
alkaline haematin solution. An ammoniacal solution of haematin on
reduction with hydrazine yields haemochromogen very easily. An alco-
holic, alkaline hydrazine solution is also recommended by RIEGLER 6
as a reagent for blood-pigments, converting them into hsemochromogen.

Haematin, also called OXYH^EMATIN, is sometimes found in old transu-
dates. It is formed by the action of the gastric or pancreatic juices on

1 E. Kalmus, Zeitschr. f. Chem., 70; v. Zeynek, ibid., 70.

2 Ibid., 44.

3 Hufner and Kuster, Arch. f. (Anat. u.) PhysioL, 1904, Suppl. Pregl, 1. c.

4 Nord. Med. Arkiv., 16.
6 Journ. of Physiol., 32.

6 Zeitschr. f. anal. Chem., 43.

HAEMATIN. 291

oxyhsemoglobin, and is, therefore, found in the feces after hemorrhage
in the intestinal canal, and also after a meat diet and food rich in blood.
It is stated that hsematin may occur in urine after poisoning with arseniu-
reted hydrogen. As shown above, the hsematin is formed by the decom-
position of oxyhsemoglobin, or at least of haemoglobin, in the presence
of oxygen.

The views in regard to the composition of hsematin are rather con-
tradictory, which seems to be due to the fact that the substance hsemin
(see below), from which the formula of hsematin is derived, has a some-
what different composition, dependent upon various conditions. Accord-
ing to HOPPE-SEYLER hsematin has the formula C34H34N4FeOs, and
from the recent investigations upon hsemin, which will be mentioned
below, this formula seems to be now generally accepted. According
to this formula 1 atom of iron occurs with every 4 atoms of nitrogen.
According to CLOETTA, and also ROSENFELD/ hsematin has the formula
, with 1 atom of iron for every 3 atoms of nitrogen.

v. ZEYNEK has prepared a haematin by the digestion of an oxyhsemoglobin
solution with pepsin-hydrochloric acid, from which he then prepared hsemin.
As this hsematin of v. ZEYNEK was readily convertible into hsemin, and while the
ordinarily prepared haematin from hsemin cannot be retransformed into hsemin,
KUSTER considers that these two forms of hsematin are not identical. The
first he calls a-hsematin and the ordinary which is a polymeric 'body, he calls
^-hsematin. That a retransformation of hsemin is possible from ordinary haematin
is still admitted by PILOTY and ELLiNGER.2

Hsematin contains at least three hydroxyl groups, one of which acts
as hydroxyl ion and seems to be united with the iron, and is replaced
in the hsemin formation (see below) by the chlorine. By means of the
two others, salts with metals as well as alkyl derivatives may be formed,
which latter (as hsemin derivatives) have been especially studied by NENCKI
and ZALESKI and KusTER.3 Hsematin dissolves in concentrated sulphuric
acid and is converted into hsematoporphyrin, with the splitting off of
iron. On heating dry hsematin it yields an abundance of pyrrol. The
products produced on the oxidation and reduction of hsematin and the
question as to the constitution of hsematin will be discussed in connec-
tion with hsematoporphyrin.

Hsematin is amorphous, dark brown or bluish-black. It may be
heated to 180° C. without decomposition; on burning it leaves a residue

1 Hoppe-Seyler, Med.-chem. Untersuch., p. 525; Cloetta, Arch. f. exp. Path. u.
Pharm., 36; Rosenfeld, ibid., 40.

2v. Zeynek, Zeitschr. f. physiol. Chem., 30 and 49; Kuster, ibid., 66 and Ber.
d. d. chem. Gesellsch., 43; Piloty, Annal. d. Chem. u. Pharm., 377; Eppinger, Unters.
iiber den Blutfarbstoff. Dissert. Munchen, 1907.

3 Nencki and Zaleski, Zeitschr. f. physiol. Chem., 30; Kuster, Ber. d. d. Chem.
Gesellsch. -13 and 45, and Zeitschr. f. physiol. Chem., 82.

292 THE BLOOD.

consisting of iron oxide. It is insoluble in water, dilute acids, alcohol,
ether, and chloroform, but it dissolves slightly in warm glacial acetic acid.
Hsematin dissolves in acidified alcohol or ether. It easily dissolves in
alkalies, even when very dilute. The alkaline solutions are dichroic;
in thick layers they appear red by transmitted light and in thin layers
greenish. The alkaline solutions are precipitated by lime- and baryta-
water, as also by solutions of neutral salts of the alkaline earths. The
acid solutions are always brown.

An acid haematin solution (spectrum Plate, 4), absorbs the red part
of the spectrum only slightly and the violet parts strongly. The solu-
tion shows a rather sharply defined band between C and D, whose posi-
tion may change with the variety of acid used as a solvent. Between
D and F a second, much broader, less sharply defined band occurs, which
by proper dilution of the liquid is converted into two bands. The one
between b and F, lying near F, is darker and broader; the other, between
D and E, lying near E, is lighter and narrower. Also by proper dilution
a fourth very faint band is observed between D and E, lying near D.
Haematin may thus in acid solution show four absorption-bands; ordi-
narily one sees, distinctly, only the bands between C and D and the broad,
dark band — or the two bands— between D and F. In alkaline solution,
haematin (spectrum Plate, 5), ishows a broad absorption-band, which
lies in greatest part between C and D, but reaches a little over the line
D toward the right in the space between D and E. As the position of
the hsematin bands in the spectrum is quite variable, the exact wave-
lengths corresponding thereto cannot be given exactly.

Haemin, H^MIN CRYSTALS, or TEICHMANN'S CRYSTALS. Haemin is
formed, as generally admitted, by the replacement of an HO group by
chlorine in the haematin, and is the starting point in the preparation of
the latter.

The statements as to the composition of haemin differ quite considerably,
and various haemins have been accepted, which is partly due to the fact,
as first shown by NENCKI and ZALESKI, that haemin combines with acid
and alkyl radicals and can also give addition products with other
bodies. Thus for example the methylhaemins, carefully studied by
KUSTER, especially monomethylhaemin, is produced in the preparation
of haemin according to MORNER'S method (see below) by means of methyl
alcohol. These behaviors have been further explained by the work
of numerous investigators, especially by KUSTER, and most investigators
generally admit that only one haemin exists whose general formula is
C34H3304N4FeCl. According to PILOTY the formula is C34H3204N4FeCl
while PIETTRE and VILA 1 deny this formula and claim to have

1 Nencki and Zaleski, Zeitschr. f. physiol. Chem., 30; Nencki and Sieber, Arch,
f. exp. Path. u. Pharm., 18 and 20, and Ber. d. d. chem. Gesellsch., 18; Schalfejeff

ELEMIN. 293

prepared a hsemin free from chlorine, from pure crystalline oxy-
hsemoglobin.

Hsemin crystals form, in large masses, a bluish-black powder, but are
so small that they can be seen only by aid of the microscope. They
consist of dark-brown or nearly brownish-black long, rhombic, or spool-
like crystals, isolated or grouped as crosses, rosettes, or stellar forms.
Cubical crystals may also occur, according to CLOETTA. They are
insoluble in water, dilute acids at the normal temperature, alcohol, ether,
and chloroform. They are slightly soluble in glacial acetic acid with heat.
They dissolve in acidified alcohol, as also in dilute caustic alkalies or
carbonates; and in the last case they form, besides alkali chlorides,
soluble hsematin alkali, from which the hsematin may be precipitated by
an acid. As shown by PILOTY and EPPINGER and then also by v. SIE-
WERT,1 crystalline hsemin can be reobtained from the hsematin.

On shaking with cold aniline and treating first with acetic acid and then
with ether, KUSTER obtained a product, dehydrochloride hsemin, which was
poor in the elements of hydrochloric acid, and which again took up HC1 and was
converted into hsemin. By the action of boiling aniline, hydrogen is driven out
and a combination with aniline, without loss of iron, takes place.

The principle of the preparation of hsemin crystals in large quan-
tities is as follows: The washed sediment from the blood-corpuscles
is coagulated with alcohol or by boiling after dilution with water and
the careful addition of acid. The strongly pressed but not dry mass
is rubbed with 90-95 per cent alcohol which has been previously treated
with oxalic acid or J-l per cent concentrated sulphuric acid, and this
is allowed to stand several hours at the temperature of the room. The
filtrate is warmed to about 70° C., treated with hydrochloric acid (for
each liter of filtrate add 10 cc. 25 per cent hydrochloric acid diluted
with alcohol — MORNER), and allowed to stand in the cold. The crystals,
which separate in one or two days, are first washed with alcohol and then
with water. On dissolving the hsemin in chloroform containing quinine
and treating the filtrate with alcoholic hydrochloric or acetic acid we can
recrystallize the hsemin according to SCHALFEJEFF. By adding glacial
acetic acid saturated with salt to a solution of hsematin in chloroform
containing quinine PILOTY and EPPINGER obtained crystalline hsemin.
For particulars as to the various methods of preparation and purification
we refer the reader to the above-cited works of NENCKI and SIEBER,
MORNER, NENCKI and ZALESKI (ScHALFEjEFF),and especially to KtJSTER.2

Hsematin is obtained on dissolving the hsemin crystals in very dilute
caustic alkali and precipitating with an acid.

with Nencki and Zaleski, 1. c.; Bialobrzeski, Arch, des scienc. biol. de St. Pe'tersbourg-
5; K. Morner, Nord. Med. Arkiv. Festband, 1897, Nos. 1 and 26, and Zeitschr. f.
physiol. Chem., 41; Zaleski, ibid., 37; Helper and Marchlewski, ibid., 41 and 42;
Kiister, ibid., 40 and 82 and footnote 1, page 292; Piettre and Vila, Compt. Rend., 141,
p. 734; Piloty, 1. c.

1 Piloty and Eppinger, 1. c.; v. Siewert, Arch. f. exp. Path. u. Pharm., 58,

2Kiister, Zeitschr., f, pbysiol. Chem., 40.

294 THE BLOOD.

In preparing hsemin crystals in small quantities proceed in the fol-
lowing manner: The blood is dried after the addition of a small quantity
of common salt, or the dried blood may be rubbed with a trace of the
same. The dry powder is placed on a microscope slide, moistened
with glacial acetic acid, and then covered with the cover-glass. Add,
by means of a glass rod, more glacial acetic acid by applying the drop
at the edge of the cover-glass until the space between the slide and the
cover-glass is full. Now warm over a very small flame, with the pre-
caution that the acetic acid does not boil and pass with the powder from
under the cover-glass. If no crystals appear after the first warming
and cooling, warm again, and if necessary add some more acetic acid.
After cooling, if the experiment has been properly performed, a number
of dark-brown or nearly black hsemin crystals of varying forms will
be seen.

In regard to the preparation and properties of the iodine-, bromine-,
and acetone-hsemin we refer to the work of STRZYZOWSKI, MERUNO
wicz and ZALESKi.1

By the action of acids upon hsemochromogen, hsematin, or haemin, a
new iron-free pigment, which was first closely studied by HOPPE-SEYLER
and called hcematoporphyrin, is produced. According to the method of
preparation, hsematoporphyrins having different solubilities, and whose
relation to each other is not perfectly clear, are produced, but all show
the same characteristic absorption-spectrum. The best-studied hsema-
toporphyrin is the one obtained according to NENCKI and SIEBER'S
method, by the action of glacial acetic acid saturated with hydrobromic
acid upon hsemin crystals, best at the temperature of the body (NENCKI
and ZALESKI). Another porphyrin is the mesoporphyrin obtained by
NENCKI and ZALESKI 2 by the reduction of hsemin in glacial acetic acid
by hydriodic acid and iodophosphonium.

Haematoporphyrin, Cs^ssN^e, which, according to recent molec-
ular weight determinations must perhaps be doubled (PILOTY) occurs
according to MAC MUNN 3 as a physiological pigment in certain animals.
A porphyrin occurs, as shown by GARROD and SAILLET, as a normal con-
stituent in human urine, although only as traces and it has also been
observed several times in large amounts in the urine after the use of
sulphonal (see Chapter XIV). This urine porphyrin is generally con-
sidered as hsematoporphyrin.

In the production of hsBmatoporphyrin from haemin or hsematin the iron is
split off. Opinions are not unanimous in regard to this process. According

1 Strzyzowski, Therap. Monatsh., 1901 and 1902; Merunowicz and Zaleski, Bull,
de 1'Acad. d. Scienc. de Cracovie, 1907.

2 Hoppe-Seyler, Med.-chem. Untersuch., 528; Nencki and Sieber, Monatshefte f.
Chem., 9, and Arch. f. exp. Path. u. Pharm., 18, 20, and 24; Nencki and Zaleski,
Zeitschr. f. physiol. Chem., 30.

3 Piloty, Annal. d. Chem. u. Pharm., 388; MacMunn, Journ. of Physiol., 7.

H^EMATOPORPHYRIN. 295

to PILOTY two carboxyl groups are formed with the taking up of water and
these occur to a certain extent latent in lactam combination in the hsBmin.
According to KtisTER,1 who admits of two already formed carboxyls in the hsemin,
two hydrox3rls are produced secondarily in the hsematoporphyrin, in that (by the
action of the glacial acetic acid and hydrobromic acid) primarily an attachment
of hydrobromic acid takes place and then from this as intermediary product,
by the action of water, bromine is split off and is replaced by hydroxyl. In the
formation of mesoporphyrin the procedure is still different because among others,
mesoporphyrin contains 2 oxygen atoms less than the hsematoporphyrin.

On the gentle reduction of hsemin with glacial acetic acid, hydriodic acid
and red phosphorus, PILOTY and FINK obtained besides mesoporphyrin a second
body, phonoporphyrin, which differs from the mesoporphyrin by containing
more oxygen, a brown color and almost complete insolubility in dilute hydro-
chloric acid. It is not reduced to mesoporphyrin by hydriodic acid but yields
hsematinic acid and methyl-ethyl maleic imide on oxidation with chromic acid.
They obtained no other cleavage products from hsemin under the above men-
tioned experimental conditions. The two porphyrins were produced in about
equal quantities and they formed about 90 per cent of the calculated cleavage
products. They each represent one-half of the hsemin, whose formula corresponds
to CesHeiNsOsFeoClo which must be doubled. As these two porphyrins yield
methyl ethylmaleic imide, while this is not the case with either the hsemin or the
hsematoporphyrin, it is believed that both are combined together in the hsemin
or hsematoporphyrin with that part of their molecules which allow of the maleic
imide formation.

By the action of glacial acetic acid, and hydriodic acid upon hsemin in the
cold (room temperature) in the presence of iodophosphonium, H. FISCHER and
BARTHOLOMAUS have obtained a beautiful crystalline, colorless product, por-
phyrinogen, whose formation and behavior have been further studied by RosE.2
Porphyrinogen, CsJ^aN^ is formed from hsemin, the meso- and the hsemato-
porphyrin in acid, and from the meso- or hsematoporphyrin also in alkaline
reduction. Porphyrinogen can be transformed into mesoporphyrin by oxidative
action of various kinds. Like the latter it yields ha3matinic acid as well as methyl
ethyl maleic imide as oxidation products.

Hsematoporphyrin is closely related to the bile pigment bilirubin
(see Chapter VII) and also stands in close relation with the urinary
pigment, urobilin. By action of reducing substances several investiga-
tors (HOPPE-SEYLER, NENCKI and SIEBER, LE NOBEL, MACMUNN and
others) have obtained pigments similar to urobilin, and by experiments
with rabbits, NENCKI and RoTSCHY3 have proved that hsematoporphyrin
introduced into the animal body may in part be transformed into a
urobilin substance.

In connection with the question of the behavior of hasmatoporphyrin in the
animal body, the poisonous action of this body, as discovered by HAUSMANN,
and which manifests itself as a photobiological sensibiliaation, is of interest.
HAUSMANN has found that white mice that have had hsematoporphyrin injected

1 Piloty, 1. c.; Kiister, Ber. d. d. chem. Gesellsch., 45.

2 Piloty and Fink, Ber. d. d. chem. Gesellsch., 46, 2021; H. Fischer and Bartholo-
maus, ibid., 46, 511; Rose, Zeitschr. f. physiol. Chem., 84.

3 Hoppe-Seyler, Med. Chem. Unters. p. 533; Le Nobel, Pfliiger's Arch., 40;
Nencki and Sieber, 1. c.; MacMunn, Proc. Roy. Soc., 30, and Journ. of Physiol., 10;
Nencki and Rotschy, Monatsh. f. chem., 10.

296 THE BLOOD.

subcutaneously and then exposed to bright light die very quickly with character-
istic symptoms, while control animals kept in the dark show no symptoms of disease.
H. FISCHER and MEYER-BETZ l have also shown that in this regard a certain
difference exists between the hsematoporphyrin and the mesoporphyrin. The
perfectly pure crystalline mesoporphyrin does not show the photobiological
action which occurs with crystalline hsematoporphyrin.

Of especial interest is the close relationship of the hsematoporphyrin
to certain chlorophyll derivatives, especially to phylloporphyrin,.
Cs2H36N402. Phylloporphyrin is similar to the above-mentioned meso-
porphyrin, C34H3gN4O4, and the absorption spectrum of the brom-
porphyrins, bromphylloporphyrin and brommesoporphyrin as prepared
by SCHUNCK and MARCHLEWSKI, seems to be almost identical. Just as
from mesoporphyrin, with sodium chloride, glacial acetic acid and an
iron salt we can regenerate a product very similar to hsemin (ZALESKI)
so MARCHLEWSKI 2 has been able under similar conditions to prepare from
phylloporphyrin a pigment, phyllohcemin, which contained iron and was
similar to hsemin. A comparison of the cleavage products gives still
more conclusive and important proofs of the close relationship of the
blood and leaf pigments.

We have important investigations of KUSTER,S PILOTY, WILLSTATTER,
H. FISCHER 4 and their collaborators upon the constitution of hsBmin and
hsematoporphyrin. The constitution of chlorophyll has been explained
by the pioneering researches of WILLSTATTER.

On the oxidation of hsematin in glacial acetic acid by potassium
dichromate or chromium trioxide KUSTER obtained the imide of the

/CO— C.CH2.CH2.COOH
tribasic hcematinic add, HN< , which is a deriva-

XCO— C.CH3
tive of maleic acid, and from which methylethylmaleic acid anhydride,

xCO— C.CH2CH3

O\ , can be readily obtained. The same hsematinic

XCO— C.CH3

1 Hausmann, Bioch. Zeitschr., 30; Hans Fischer and Meyer-Betz. Zeitschr. f,
physiol. Chem., 82.

2 The pertinent literature will be found in L. Marchlewski, Die Chemie der Chlor-
ophylle und ihre Beziehung zur Chemie des Blutfarbstoffes, 1909 and Ber. d. d. chem.
Gesellsch., 45.

3 Beitrage zur Kenntnis. des Hamatins, Tubingen, 1896; Ber. d. d. chem. Gesellsch. r
27, 30, 32, 35, 43, and 45, Annal. d. Chem. u. Pharm., 315, and Zeitschr. f. physiol.
Chem., 28, 40, 44, 54, 55, 61, 62, and 82.

4 The work of Piloty and collaborators may be found in Annal. d. Chem. u. Pharm. r
366, 37?, 388, 390, and 392 and Ber. d. d. chem. Gesellsch., 42, 43, and 45. In regard
to the work of Willstatter and the literature on chlorophyll (to 1911) see Willstatter
in Abderhalden's Bioch. Handlexicon, Bd. VI and Annal. d. Chem. u. Pharm., 378,
380, 382, and 385. Hans Fischer and collaborators, Zeitschr. f. physiol. Chem., 82,
and footnote 2, p. 297, and the literature on bilirubin Chapter VII.

CONSTITUTION OF THE BLOOD-PIGMENTS. 297

acid imide, which also hsemato- and mesoporphyrin give, were obtained by
MARCHLEWSKI on the oxidation of phylloporphyrin and by WILLSTATTER,

/CO— C.C2H5
besides methylethylmaleic imide, HN<; , on the oxidation of

XCO— C.CH3

certain chlorophyll derivatives. The same two products were obtained
by KUSTER l on the oxidation of mesoporphyrin while hsemin and hsema-
to porphyrin gave no methylethylmaleic imide.

It has been known for a long time that hsemin and hsematoporphyrin
gave an abundance of pyrrol on heating, and that phylloporphyrin has a
similar behavior was first shown by SCHUNCK and MARCHLEWSKI.
That at least one pyrrol, of the pyrrol mixture, the so-called hsemopyrrol
is common to both the blood and leaf pigments has been shown by the
investigations of MARCHLEWSKI and his collaborators, and from hsemo-
pyrrol KUSTER was the first to obtain methylethylmaleic imide on oxida-
tion, showing that hsemopyrrol was probably a dimethylethylpyrrol.
This behavior has been further developed by the investigations of
PILOTY and WILLSTATTER on the reduction products of the blood and
leaf pigments and by H. FISCHER and BARTHOLOMAUS 2 on the sub-
stituted pyrrols.

WILLSTATTER obtained a pyrrol mixture from hsemin and hsemato-
porphyrin, as well as from chlorophyll derivatives, by reduction, from
which he isolated three different pyrrols. The first, which he calls hoemo-
pyrrol, was perhaps not perfectly pure, consisted at least in great part
of the cryptopyrrol (FISCHER and BARTHOLOMAUS. c-hsemopyrrol of
PILOTY and STOCK) which is identical with the 2, 4-dimethyl-3-ethyl

H3C.C- -C.CoH5
pyrrol = || prepared synthetically by KNORR and HESS.S

HC— NH— C.CH3

The second hsemopyrrol, which he calls isohcemopyrrol ( = hsemopyrrol
of FISCHER and BARTHOLOMAUS, B-hsemopyrrol of PILOTY and STOCK)
is also a trisubstituted pyrrol, namely 2, 3-dimethyl-4-ethylpyrfol

C2H5.C- C.CH3

II II

HC— NH— C.CH3
These two dimethylethylpyrrols give with nitrous acid the correspond-

1 Ber. d. d. chem. Gesellsch., 45.

2Piloty, Annal. d. Chem. u. Pharm., 377, 388, 390, and 392; Willstatter and
Asahina, ibid., 385. In this article will be found on pages 189 and 190 the references
to the literature on the investigations of Marchlewski and others on hsemopyrrol.
H.k Fischer and Bartholomaus, Zeitschr. f. physiol. Chem., 77 and 80 and Ber. d. d.
chem. Gesellsch., 45.

3 Piloty and Stock, Annal. d. Chem. u. Pharm., 392; Knorr and Hess, Ber. d.
d. chem. Gesellsch., 44 and 45.

298 THE BLOOD.

ing oxime of methylethylmaleic imide. The third pyrrol found by
WILLSTATTER, which he calls phyllopyrrol, is a tetra-substituted pyrrol,

H3C.C- -C,C2H5
namely, 2, 3, 5 trimethyl-4-ethylpyrrol =

H3C.C-NH-C.CH3

The statement that the hsemopyrrol of WILLSTATTER is in part derived
from cryptopyrrol is not correct, and must be changed because of the
investigations of PILOTY and STOCK, who find that the hsemopyrrol of
WILLSTATTER (and ASAHINA) undoubtedly contains cryptopyrrol, but
consists chiefly of the B-hsemopyrrol, consequently isohsemopyrrol. Ac-
cording to more recent investigations of PILOTY and STOCK 1 the hsemo-
pyrrol question is even more complicated than was expected.

In the crude pyrrol obtained by the reduction of the blood pigments
several other pyrrol bodies have been found, for example the phonopyrrol
of PILOTY which has not been sufficiently explained. According to
GRABOWSKI and MARCHLEWSKi2 as well as to PILOTY and STOCK, the
crude hsemopyrrol contains also disubstituted pyrrol, namely, £1, /3-methyl-
ethylpyrrol. On fusing haematoporphyrin or hsematopyrrolidinic acid
(see below) with caustic alkali we obtain, according to PILOTY, a mixture
of pyrrols among which we will mention 2, 3-dimethylpyrrol
HC— — C.CH3

, which has been studied by PILOTY and WILKE.S
HC-NH-C.CHs

By the reduction of hsematoporphyrin and hsemin by various methods,
PILOTY and co-workers have obtained, besides hsemopyrrol, several
acids namely hsematopyrrolidinic acid, phonopyrrolcarboxylic acid
(isophonopyrrolcarboxylic acid) and xanthopyrrolcarboxylic acid. The
hsematopyrrolidinic acid seems from the most recent investigations
not to be a unit substance. PILOTY obtained from it phonopyrrolcar-

H3C.C— -C.CH2.CH2.COOH
boxylic add, CgHi3NO2 = which on

H3C.C— NH— CH
treatment with nitrous acid lost a methyl group and was converted into

H3C.C==C.CH2CH2.COOH.
the oxime of hsematinic acid, The acid

HONG— NH— CO

received this name because, according to PILOTY, it yields a special
dimethylethylpyrrol, called phonopyrrol by him. The question as to the
nature of xanthopyrrolcarboxylic acid and its occurrence has not been

1 Piloty and Stock, Annal. d. Chem. u. Pharm., 392 and Ber. d. d. chem. Gesellsch.,
46, 1008.

2 Grabowski and Marchlewski, Zeitschr. f. physiol. Chem., 81.

3 Piloty and Wilke, Ber. d. d. chern. Gesellsch., 45.

PYRROLE DERIVATIVES. 299

answered; still there does not seem to be any doubt that there exists
an isophonopyrrolcarboxylic acid, which can be obtained from the blood
as from the bile pigments. From the mixture of acid cleavage products
obtained by the reduction of hsemin with hydriodic acid, and glacial acetic
acid PILOTY and DORMANN 1 have obtained as well characterized prod-
ucts, phonopyrrolcarboxylic acid and isophonopyrrolcarboxylic acid and
also xanthopyrrolcarboxylic acid, CioHisNCb and they consider the
existence of this acid as positively proved. The melting-point of the
crystalline acid was 108°, the picrate 143°, and the oxime 208°. The
corresponding values for isophonopyrrolcarboxylic acid was 122°, 146°
and 210° respectively. An isomeric xanthopyrrolcarboxylic acid, called
D-phonopyrrolcarboxylic acid, seems also to occur.

It is extremely difficult to correlate the somewhat contradictory
statements of the various authors in this subject and to draw quite
positive conclusions from these statements. It is nevertheless positive
that from the hsemopyrrol mixture the three pyrrols, cryptopyrrol,
isohaemopyrrol and phyllopyrrol can be obtained and also that there
are two hsemopyrrolcarboxylic acids (phonopyrrol- and isophonopyrrol-
carboxylic acids), of which one possibly is related to the crypto-
pyrrol and the other to the isohaemopyrrol. On account of the uncer-
tainty of the experimental foundation it is difficult to enter into a
discussion of the variously proposed hypothetical constitutional formulae
for the derivatives of the blood pigments. The same is true for the
disputed question as to the form of binding of the iron in hsematin and
in haemin. It is generally admitted that the iron here is trivalent. The
views are different in regard to the valence of the iron in haemoglobin,
namely, MANCHOT considers that haemoglobin is a ferric combination
while KUSTER 2 on the contrary considers it a ferrous combination.

Haematoporphyrin gives with hydrochloric acid a compound which
crystallizes in long brownish-red needles. If the solution in hydrochloric
acid is nearly neutralized with caustic soda and then treated with sodium
acetate, the pigment separates out as amorphous, brown flakes not
readily soluble in amyl alcohol, ether, or chloroform, but readily soluble
in ethyl alcohol, alkalies, and dilute mineral acids. The compound
with sodium crystallizes as small tufts of brown crystals and several
other salts of haematoporphyrin are known such as the methyl and ethyl
esters. The acid alcoholic solutions have a beautiful purple color,
which become violet-blue on the addition of large quantities of acid.
The alkaline solution has a beautiful red color, especially when too much
alkali is not present.

1 Piloty and Dormann, Ber. d. d. chem. Gesellsch., 46.

2 Manchot, Zeitschr. f. physiol. Chem., 70; Kiister, ibid., 71.

300 THE BLOOD.

An alcoholic solution of hsematoporphyrin, acidulated with hydro-
chloric or sulphuric acid, shows two absorption-bands (spectrum Plate,
7), one of which is fainter and narrower and lies between C and D, near
D. The other is much darker, sharper, and broader, and lies midway
between D and E. An absorption extends from these bands toward
the red, terminating with a dark edge, which may be considered as a
third band between the other two.

'> A dilute alkaline solution shows four bands, namely, a band between
C and D; a second, broader band surrounding D and with the greater
part between D and E', a third, between D and E, nearly at E; and
lastly, a fourth, broad and dark band between b and F. On the addi-
tion of an alkaline zinc-chloride solution the spectrum changes more
or less rapidly,1 and finally a spectrum is obtained with only two bands,
one of which surrounds D and the other lies between D and E. If an
acid hsematoporphyrin solution is shaken with chloroform, a part of the
pigment is taken up by the chloroform, and this solution often shows a
five-banded spectrum with two bands between C and D. The position
of the hsematoporphyrin bands in the spectrum differ with the various
methods of preparation and other conditions, so that they do not cor-
respond to the same wave length. These facts coincide well with the
recent investigations of A. ScnuLz;2 according to which the appearance
of the spectrum is not only dependent upon the reaction but also upon
the character of the solvent and the method of preparation.

In regard to the preparation of hsematoporphyrin, see HOPPE-SEYLER-
THIERFELDER'S Handbuch, 8. Aufl., and the works cited on page 294.

Mesoporphyrin, CsJIssN^, has the same spectrum as haematoporphyrin.
It has two oxygen atoms less, and further differs from it in that on oxidation it yields
hsematinic acid as well as methylethylmaleic imide, and does not show the
above-mentioned biological action of hsematoporphyrin.

Hsematinogen is a ferruginous pigment so named by FREUND,S which he
obtained by carefully extracting blood with alcohol containing hydrochloric
acid. It is closely related to hsematin, but is not sufficiently characteristic and is
not considered as a cleavage product.

A question of great interest is whether it is possible to produce the
blood-pigment from its cleavage products. In this respect certain recent
investigations are interesting. ZALESKI obtained from mesoporphyrin
hydrocloride dissolved in 80 per cent acetic acid saturated with NaCl
and heated to 50°-70°, a hsemin-like pigment by the addition of a solu-
tion of iron in acetic acid, and this pigment had a spectrum in acid
solution very similar to that of hsematin, although not identical with it.

1 See Hammarsten, Skand. Arch. f. Physiol., 3, and Garrod, Journ. of Physiol., 13.

2 Arch. f. (Anat. u.) Physiol., 1904, Suppl.

3 Wien. klin. Wochenschr., 1903.

HAEMATOIDIN. 301

ZALESKI considers this pigment as a hydrogenized haemin. A regenera-
tion of haematin from haematoporphyrin has been performed by LAID-
LAW. If haematoporphyrin is dissolved in dilute ammonia and warmed
with STOKES' solution and hydrazine hydrate, iron is taken up again
and haemochromogen is produced, which is changed into hsematin by
shaking with air. According to HAM and BALEAN/ it is possible to pro-
duce haemoglobin from haemochromogen and globin, and it is indeed
possible that other proteins can replace globin in this formation.

Haematoidin, thus called by VIRCHOW, is a pigment which crystallizes
in orange-colored rhombic plates, and which occurs in old blood extrav-
asations, and whose origin from the blood-coloring matters seems to
be established (LANGHANS, CORDUA, QUINCKE, and others2). A solu-
tion of haematoidin shows no absorption-bands, but only a strong absorp-
tion from the violet to the green (EWALD 3) . According to most observers,
haematoidin is identical with the bile-pigment bilirubin. It is not
identical with the crystallizable lutein from the corpora lutea of the ovaries
of the cow (PICCOLO and LiEBEN,4 KUHNE and EWALD).

In the detection of the above-described blood-coloring matters the
spectroscope is the only entirely trustworthy means of investigation.
If it is only necessary to test for blood in general and not to determine
definitely whether the coloring-matter is haemoglobin, methaemoglobin
or haematin, then the preparation of haemin crystals is an absolutely
positive test. In regard to the detection of blood in urine, see Chapter
XIV, and for the detection of blood in intestinal contents, in pathological
fluids and in chemi co-legal cases we must refer the reader to more extended
text-books.

The methods proposed for the quantitative estimation of the blood-
•coloring matters are partly chemical and partly physical.

Among the chemical methods to be mentioned is the incineration of the
.blood and the determination of the amount of iron contained in the ash from
which the amount of hemoglobin may be calculated. We must refer to works
on chemical methods of investigation in regard to these methods.

The physical methods consist either of colorimetric or of spectroscopic
investigations.

The principle of HOPPE-SEYLER'S colorimetric method is that a measured
quantity of blood is diluted with an exactly measured quantity of water
until the diluted blood solution has the same color as a pure oxyhaemo-
globin solution of a known strength. The amount of coloring-matter
present in the undiluted blood may be easily calculated from the degree
of dilution. In the colorimetric testing we use a glass vessel with parallel

1 Zaleski, Zeitschr. f. physiol. Chem., 43; Laidlaw, Journ. of Physiol., 31; Ham
and Balean, ibid., 32.

2 A comprehensive review of the literature pertaining to haematoidin may be found
in Stadelmann, Der Icterus, etc., Stuttgart, 1891, pp. 3 and 45.

3 Zeitschr. f. Biologic, 22, 475.

4 Cit. from Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 1878.

302 THE BLOOD.

sides containing a layer of liquid 1 cm. thick (HOPPE-SEYLER'S hsematinom-
eter). The use of HOPPE-SEYLER'S colorimetric double pipette is more
advantageous. Other good forms of apparatus have been constructed
by GIACOSA and ZAN GERMEISTER.1 Instead of an oxyhaemoglobin solu-
tion we now generally use a carbon-monoxide haemoglobin solution as
a standard liquid because it may be kept for a long time. The blood
solution in this case is saturated with carbon monoxide.2

The quantitative estimation of the blood-coloring matters by means
of the spectroscope may be done in different ways, but at the present
time the spectrophotometric method is chiefly used, and this seems to be
the most reliable. This method is based on the fact that the extinction
coefficient of a colored liquid for a certain region of the spectrum is directly
proportional to the concentration, so that C : E — C\ : E\, when (7 and
Ci represent the different concentrations and E and E\ the corresponding

C C

coefficients of extinction. From the equation —=-Fr-: it follows that for

hi JUii

one and the same pigment this relation, which is called the absorption
ratio, must be constant. If the absorption ratio is represented by A,
the determined extinction coefficient by E, and the concentration (the
amount of coloring-matter in grams in 1 cc.) by C, then C = AE.

Different forms of apparatus have been constructed (VIERORDT and
HUFNER 3) for the determination of the extinction coefficient, which is
equal to the negative logarithm of those rays of light which remain
after the passage of the light through a layer 1 cm. thick of an absorbing
liquid. In regard to this apparatus the reader is referred to other text-
books.

For purposes of control the extinction coefficients are determined in two dif-
ferent regions of the spectrum. HUFNER has selected (a) the region between the
two absorption-bands of oxyhaemobglobin, especially between the wave-lengths
554 MM and 565 MM and (6) the region of the second band, especially the inter-
val between the wave-lengths 531.5 MM and 542.5 MM- The constants or the
absorption ratio for these two regions of the spectrum are designated by HUFNER
by A and A'. Before the determination the blood must be diluted with water,
and if the proportion of dilution of the blood be represented by V, then the con-
centration or the amount of coloring-matter in 100 parts of the undiluted blood

C = 100. V. A. tfand
C = 100. V.A'.E'.

The absorption ratio or the constants in the two above-mentioned regions
of the spectrum have been determined for oxy haemoglobin, haemoglobin, carbon-
monoxide haemoglobin, and methsemoglobin, as follows:

Oxyhsemoglobin A0 =0.002070 and A'0 =0.001312

Haemoglobin AT =0.001354 and A'r =0.001778

Carbon-monoxide haemoglobin . .'A« =0.001383 and A 'c =0.001263

Methsemoglobin Am =0.002077 and A'm = 0.001754

JF. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 16; G. Hoppe-Seyler, ibid., 21;
Winternitz, ibid.; Giacosa, Maly's Jahresber., 26; Zangermeister, Zeitschr. f. Biol-
ogie, 33.

2 See Haldane, Journ. of Physiol., 26.

3 See Vierordt. Die Anwendung des Spektralapparates zu Photometric, etc. (Tubin-
gen, 1873), and Hiifner. Arch. f. (Anat. u.) Physiol., 1894, and Zeitschr. f. physiol
Chem., 3, v. Noorden, ibid., 4; Otto, Pfliiger's Arch, 31 and 36.

QUANTITATIVE ESTIMATION OF BLOOD-PIGMENTS. 303

From what has been said above about the absorption behavior,
the concentration and the extinction coefficient it follows that the quo-

77"

tient of the extinction coefficient — measured at two different parts of

E

the spectrum, independently of the concentration, is a characteristic
constant for the respective pigments. According to HUFNER'S figures
this quotient for oxyhaemoglobin is 1.58, for haemoglobin 0.76, for
carbon-monoxide haemoglobin 1.10 and for methsemoglobin 1.19. BUT-
TERFIELD l who has made a thorough investigation on this, finds the
figure 1.58 for normal and pathological human blood as well as for
crystalline human, horse and ox oxyhaemoglobin.

The quantity of each coloring-matter may be determined in a mixture
of two blood-coloring matters by this method; this is of special impor-
tance in the determination of the quantity of oxyhsemoglobin and
haemoglobin present in blood at the same time.

In order to facilitate these determinations, HtJFNER2 has worked
out tables which give the relation between the two pigments existing
in a solution containing oxyhaemoglobin and another pigment (haemo-
globin, methaemoglobin, or carbon-monoxide haemoglobin), and thus
allowing of the calculation of the absolute quantity of each pigment.

Among the many apparati constructed for clinical purposes for the
quantitative estimation of haemoglobin, FLEISCHL'S hcemometer, which has
undergone numerous modifications, HENOCQUE'S hcematoscope, and
SAHLI'S hcemometer, are to be specially mentioned. In regard to these
apparati we must refer to larger hand-books and text-books on clinical
methods.

Many other pigments are found besides the often-occurring haemoglobin
in the blood of invertebrates. In a few Arachnidse, Crustacea, Gasteropodae
and Cephalopoda? a body analogous to haemoglobin, containing copper, hcemo-
cyanin, has been found by FREDERICQ. By the taking up of loosely bound oxygen
this body is converted into blue oxyhcemocyanin, and by the escape of the oxygen
becomes colorless again. According to' HENZE 1 gram hsemocyanin combines
with about 0.4 cc. oxygen. It is crystalline and has the following composition:
C 53.66; H 7.33; N 16.09; S 0.86; Cu 0.38; 0 21.67 per cent. On hydrolytic
cleavage with hydrochloric acid HENZE found the following division of the nitro-
gen in hsemocyanin: Of the total nitrogen 5.78 per cent was split off as ammonia,
2.67 per cent as humus nitrogen, 27.65 per cent as diamino nitrogen, and 63.39
per cent as monamino nitrogen. He found no arginine in the cleavage products,
but could detect histidine, lysine, tyrosine, and glutamic acid. A coloring-
matter called chlorocruorin by LANKESTER is found in certain Chaatopodse.
HcBmerythrin, so called by KRUKENBERG but first observed by SCHWALBE. is 'a
red coloring-matter from certain Gephyrea. Besides hasmocyanin we find in the
blood of certain Crustacea the red coloring-matter tetronerythrin (HALLIBURTON),
which is also widely spread in the animal kingdom. Echinochrom, so named

1 Zetischr. f. physiol. Chem./62.

2 Arch. f. (Anat. u.) Physiol., 1900.

304 THE BLOOD.

by MAcMuNN,1 is a brown coloring-matter occurring in the peri visceral fluid of
a variety of echinoderms. According to HENZE 2 the Ascidia contain a brown
pigment which contains vanadium but does not hold any oxygen in a dissociable
form.

The quantitative constitution of the red blood-corpuscles. The amount
of water varies in different varieties of blood-corpuscles between 570-
644 p. m., with a corresponding amount, 430-356 p. m., of solids. The
chief mass, about TQ--fc, of the dried substance consists of haemoglobin
(in human and mammalian blood).

According to the analyses of HOPPE-SEYLER 3 and his pupils, the red
corpuscles contain in 1000 parts of the dried substance:

Haemoglobin. Protein. Lecithin. Cholesterin.

Human blood 868-944 122-51 7.2-3.5 2.5

Dog's " 865 126 5.9 3.6

Goose's " 627 364 4.6 4.8

Snake's " 467 525

ABDERHALDEN found the following composition for the blood-
corpuscles from the domestic animals investigated by him: Water,
591.9-644.3 p. m.; solids 408.1-335.7 p. m.; haemoglobin, 303.3-331.9
p. m.; protein, 5.32 (dog)-7.85 p. m. (sheep); cholesterin, 0.388 (horse)
-3.593 p. m. (sheep) ; and lecithin, 2.296 (dog)-4.855 p. m.

Of special interest is the varying proportion of the haemoglobin to the
protein in the nucleated and in the non-nucleated blood-corpuscles. These
last are much richer in haemoglobin and poorer in protein than the
former.

The amount of mineral bodies in various species of animals is different.
According to BUNGE and ABDERHALDEN the red corpuslces from the pig,
horse, and rabbit contain no soda, while those from man, the ox, sheep,
goat, dog, and cat are relatively rich in soda. In the five last-mentioned
species the amount of soda was 2.135-2.856 p. m. The quantity of potash
was 0.257 (dog)-0.744 p. m. (sheep). In the horse, pig, and rabbit
the quantity of potash was 3.326 (horse)-5.229 p. m. (rabbit). Human
blood-corpuscles contain, according to WANACH, about five times as
much potash as soda, on an average 3.99 p. m. potash and 0.75 p. m.
soda. The nucleated erythrocytes of the frog, toad, and turtle also

. iFredericq, Extrait des Bulletins de 1'Acad. Roy. de Belgique (2), 46, 1878; Lan-
kester, Journ. of Anat. and Physiol., 2 and 4; Henze, Zeitschr. f. physiol. Chem.,
33 and 43; Krukenberg, see Vergl. physiol. Studien Reihe 1, Abt. 3, Heidelberg,
1880; Halliburton, Journal of Physiol., 6; MacMunn. Quart. Journ. Microsc. Science,
1885.

2 Zeitschr. f. physiol. Chem., 72 and 79.

3 Med.-chem. Untersuch., 390 and 393.

WHITE BLOOD-CORPUSCLES. 305

contain, according to BOTTAZZI and CAPPELLI/ considerably more
potassium than sodium. Lime is claimed to be absent in the blood-
corpuscles, but according to HAMBURGER2 this is not true for at least
ox-blood, and magnesia occurs only in small amounts: 0.016 (sheep)
-0.150 p. m. (pig). The blood-corpuscles of all animals investigated
contain chlorine, 0.460-1.949 p. m. (both in horse), generally 1 to 2
p. m., and also phosphoric acid. The amount of inorganic phosphoric
acid shows great variation: 0.275 (sheep)-1.916 p. m. (horse). All
of the above figures are calculated on the fresh, moist blood-corpuscles.

By quantitative determinations of the swelling and shrinking of the cells
under the influence of NaCl solutions of various concentration, or of serum of
various dilutions, HAMBURGER has attempted to determine for the erythrocytes,
as well as the leucocytes, the percentage relationship between the two chief con-
stituents of the cells (the frame and the intracellular fluid). He found that the
volume of the frame-substance for both varieties of blood-corpuscles of the horse
was equal to 53-56.1 per cent. The volume for the red blood-corpuscles was
for the rabbit 48.7-51; hen, 52.4-57.7, and for the frog, 72-76.4 per cent.
KOEPPE has raised objections to these determinations.3

The White Blood- corpuscles and the Blood-plates.

The White Blood-corpuscles, also called LEUCOCYTES or Lymphoid
Cells, are of different kinds, and ordinarily we differentiate between
the small forms poor in protoplasm, called lymphocytes, and the larger,
granular, often more nucleated forms, called leucocytes. The poly-
nuclear leucocytes occur in greater abundance in the blood than the
lymphocytes. In human and mammalian blood, most of the white
blood-corpuscles are larger than the red blood-corpuscles. They also
have a lower specific gravity than the red corpuscles, move in the circulat-
ing blood nearer to the walls of the blood-vessels, and also have a slower
motion.

The number of white blood-corpuscles varies not only in the different
blood-vessels, but also under different physiological conditions. On
an average there is only 1 white corpuscle for 350-500 red corpuscles.
According to the investigations of ALEX. SCHMIDT 4 and his pupils,
the leucocytes are destroyed in great part on the discharge of the blood
before and during coagulation, so that discharged blood is much poorer
in leucocytes than the circulating blood. The correctness of this state-
ment has been denied by other investigators.

1 Bunge, Zeitschr. f. Biologie, 12, and Abderhalden, Zeitschr. f. physiol. Chem.,
23 and 25; Wanach, Maly's Jahresber., 18, 88; Bottazzi and Cappelli, Arch. Ital.
de Biologie, 32.

2 Zeitschr. f. physik. Chem. 69.

3 Hamburger, Arch. f. (Anat. u ) Physiol., 1898; Koeppe, ibid., 1899 and 1900.

4 Pfliiger's Arch., 11 and Kruger, Arch. f. exp. Path. u. Pharm., 51.

306 THE BLOOD.

From a histological standpoint we generally, as above indicated,
discriminate between the different kinds of colorless blood-corpuscles.
Chemically considered, however, there is no known essential difference
between them, and what little we do know chemically is chiefly in con-
nection with the leucocytes. With regard to their importance in the
coagulation of fibrin, ALEX. SCHMIDT and his pupils distinguish between
the leucocytes which are destroyed in the coagulation and those which
are not. The last mentioned give with alkalies or common-salt solutions
a slimy mass; the first do not show such behavior.

The protoplasm of the leucocytes has, during life, amoeboid move-
ments which serve partly to make possible the wandering of the cells,
and partly to aid in the absorption of smaller grains or foreign bodies
and make the phagocytosis possible. The action of various agents
such as hyper- and hypotonic salt solutions, of foreign ions, such as
iodine, bromine, and salts of the alkaline earths upon the chemotaxis
and the phagocytic activity of the leucocytes has been thoroughly
studied by HAMBURGER and DE HAAN/ and among other things they have
shown that the Ca causes an accellerating influence upon phagocytosis
which is peculiar for Ca and does not depend upon its properties as a
divalent ion. Because of the contractibility of the leucocytes, the
occurrence of myosin in them has been admitted even without any
special proof therefore. We know nothing positively whether in the
leucocytes, or in the cells, in general, globulins occur with traces
of albumins, because cell constituents which used to be called globulins
have on more careful investigation been found to be nucleoalbumins
or nucleoproteins. The substance observed by HALLIBURTON,2 and
occurring in all cells, which coagulates at 47 to 50° C., is considered as
a true globulin. ALEX. SCHMIDT claims to have found serglobulin in
equine-blood leucocytes which have been washed with ice-cold water.

The proteins of the leucocytes as well as the cells in general are prin-
cipally compound proteins. For the present it is impossible to state to
what extent the nucleoalbumins occur in leucocytes or cells, because in the
past no careful differentiation was made between the nucleoalbumins
and nucleoproteins. The nucleoproteins are without any doubt the
principal constituents of the protoplasm of the white blood-corpuscles,
and one of these it seems is identical with the so-called hyaline substance
of ROVIDA, which yields a slimy mass when treated with alkalies or
NaCl solutions and which occur in pus-cells.

On digesting the leucocytes with water, a solution of a protein body

1 Bioch. Zeitschr., 24 and 26.

2 See Halliburton, On the chem. Physiol. of the animal cell. King's College, London,
Physiol. Labor. Collected papers, 1893.

LEUCOCYTES. 307

is obtained which can be precipitated by acetic acid and which forms the
chief mass of the leucocytes. This substance, which is undoubtedly
concerned in the coagulation of the blood, has been described under
different names, such as tissue fibrinogen (WOOLDRIDGE) cytoglobin and
prdglobulin (ALEX. SCHMIDT) or nucleohistone (KOSSEL and LILIENFELD J)
and consists, chiefly at least, of nucleoprotein. The ordinary view that
this is nucleohistone does not seem to be correct, according to the invest-
igations of BANG,2 and further proof is necessary.

Besides these constituents of the protoplasm of the leucocytes we
must also include lecithin and especially phosphatides, cholesterin, glu-
cothionic add (in pus-corpuscles, MANDEL and LEVENES), purine bodies
derived from the nuclein substances and glycogen. According to HOPPE-
SEYLER glycogen is a constant constituent of all cells having amoeboid
movement, and he found it in the colorless blood-corpuscles but not in
the non-mobile pus-cells. Nevertheless glycogen has also been found
in pus-cells by SALOMON4 and by others. The glycogen found by
HUPPERT, CZERNY, DASTRE,5 and others in blood and lymph probably
originated from the leucocytes. Enzymes also occur in the leucocytes
and the proteolytic enzymes are of special importance. According to
OPIE and BARKER two proteolytic enzymes occur in the leucocytes,
one of which is active in alkaline solution and occurs in the polynuclear
cells while the other is active in acid solution and occurs in the large
mononuclear cells. According to FIESSINGER and MARIE, the leucocytes
contain a proteolytic enzyme which forms peptone, leucine and tyrosine
from protein and which is probably identical with the proteolytic enzyme
discovered earlier by ACHALME in pus. It acts best in faintly alkaline
solution, but also in weak acid reaction, and is destroyed at 75-80° C.
It occurs in the polynuclear leucocytes but principally in those which have
a medullary origin, while it is absent in the leucocytes of the lymph series.
The lipase occurring in pus and in blood seems, according to the above
experimenters, to originate in the lymphocytes. TSCHERNORUZKI 6 has

1 See Wooldridge, Die Gerinnung des Blutes (published by M. v. Frey, Leipzig, 1891);
A. Schmidt, Zur Blutlehre, Leipzig, 1892; Lilienfeld, Zeitschr. f. physiol. Chem., 18.
2 1. Bang, Studier over Nukleoproteider, Kristiania, 1902.

3 Biochem. Zeitschr., 4.

4 In regard to the literature on Glycogen, see Chapter VII.

5 Huppert, Centralbl. f. Physiol., 6, 394; Czerny, Arch. f. exp. Path. u. Pharm.,
31; Dastre, Compt. Rend., 120, and Arch, de Physiol. (5), 7. See also Hirschberg,
Zeitschr. f. klin. Med., 54.

6 In regard to the enzymes see Erben, Jochmann and E. Miiller, Jochmann and
Lockemann, Hofmeister's Beitrage, 11, which contains the literature. Opie, Journ.
of exper. Medicine, 8; with Barker, ibid., 9; Fiessinger, and Marie, Journ. de physiol.
et de pathol. generate, 11, which also contains the literature and Compt. rend. soc.
biol, 66, 67; Tschernoruzki, Zeitschr. f. physiol. Chem., 75.

308 THE BLOOD.

also shown the presence of amylase (diastase), catalase, nudease and per-
oxidase in the poly nuclear leucocytes.

The blood-plates (BrzzozEKo), hsematoblasts (HAYEM), whose nature,
preformed occurrence, and physiological importance have been much
questioned, are pale, colorless, gummy disks, round or somewhat oval
in shape, generally with a diameter one-half or one-third that of the
blood-corpuscles. In mammalia their number, according to AYNAUD,
is on an average 500,000 in 1 c.mm. They change their shape readily,
attack foreign bodies and agglutinate under conditions which AYNAUD
has carefully studied. Human blood-plates consist, according to DEETJEN/
of a nucleus and a hyaline protoplasm. They are very sensitive toward
alkalies and much more so than the plates from other mammalia. They
are destroyed in a concentration of hydroxyl ions, Con = lXlO"5 and
in a concentration of H ions, CH = 2X10~4.

According to the researches of KOSSEL and of LILIENFELD 2 the blood-
plates consist of a chemical combination between protein and nuclein,
and hence they are also called nuclein-plates by LILIENFELD, and are
considered as derivatives of the cell nucleus. It seems certain that the
blood-plates have some connection with the coagulation of blood. The
views on this question, especially in regard to the manner in which these
plates act in coagulation, are unfortunately very divergent.

HI. THE BLOOD AS A MIXTURE OF PLASMA AND BLOOD-CORPUSCLES.

The blood in itself is a thick, sticky, light or dark red liquid, opaque
even in thin layers, having a salty taste and a faint odor differing in
different kinds of animals. On the addition of sulphuric acid to the
blood the odor is more pronounced. In adult human beings the specific
gravity ranges between 1.045 and 1.075. It has an average of 1.058
for grown men and a little less for women. LLOYD JONES found that the
specific gravity is highest at birth and lowest in children until about
two years old, and in pregnant women. The determinations of LLOYD
JONES, HAMMERSCHLAG,3 and others show that the variation of the specific
gravity, dependent upon age and sex, corresponds to the variation in
the quantity of haemoglobin.

The determination of the specific gravity is accurately obtained

1 Aynaud, Maly's Jahresb., 39; Deetjen, Zeitschr. f. physiol. Chem., 63.

2 In regard to the literature of the blood-plates, see Lilienfeld, Arch. f. (Anat. u.)
Physiol., 1892, and "Leukocyten und Blutgerrinnung," Verhandl. d. physiol. Gesellsch.
zu Berlin, 1892; and also Mosen, Arch. f. (Anat. u.) Physiol., 1893, and Maly's Jahres-
ber., 30 and 31.

3 Lloyd Jones, Journ. of Physiol., 8; Hammerschlag, Wien. klin. Wochenschrift,
1890, and Zeitschr. f. klin. Med., 20.

ALKALINITY OF THE BLOOD. 309

by means of the pyknometer. For clinical purposes, where only small
amounts are available, it is best to proceed by the method as suggested
by HAMMERSCHLAG. Prepare a mixture of chloroform and benzene of
about 1.050 sp. gr. and add a drop of the blood to this mixture. If the
drop rises to the surface then add benzene, and if it sinks add chloroform.
Continue this until the drop of blood suspends itself midway and then
determine the specific gravity of the mixture by means of an areometer.
This method is not strictly accurate and must be performed quickly.
In regard to the necessary details refer to ZUNTZ and A. LEVY.1

The reaction of the blood is alkaline toward litmus, and various bodies
such as alkali carbonates, the phosphates, alkali-protein combinations,
the amino-acids and carbon dioxide all take part in bringing about the
normal reaction. According to HENDERSON 2 the normal reaction is
also partly brought about by ammonia formation and partly by the
phosphates, in that the kidneys secrete acid salts (phosphates) and return
alkali to the blood and regulate the reaction of the blood.

In considering the alkalinity of the blood we must, as previously
remarked, differentiate between the amount of titratable alkali in the blood
and the true alkalinity, i.e., the amount of hydroxyl or hydrogen ions
in the blood.

We have a large number of determinations of the quantity of titratable
alkali, calculated as Na2CO3, in fresh as well as defibrinated blood of
animals and man, and in the latter case under healthy and diseased con-
ditions. As these determinations have been carried out with dif-
ferent methods which were not without error they cannot be given any
great importance. The results found generally vary between 3 and 6
p. m. Na2COa and for man the figures below 3.3 p. m. and above 5.3 p. m.
are considered as pathological. The alkaline reaction diminishes out-
side of the body, and indeed the more quickly the greater the original
alkalinity of the blood. This depends on the formation of acid in the
blood, in which the red-blood corpuscles seem to take part in some way or
another. After excessive muscular activity the alkalinity is diminished
(PEIPER, COHNSTEIN), and it is also decreased after the continuous
ingestion of acids (LASSAR, FREUDBERG,3) and others.

1 Zuntz, Pfliiger's Arch., 66; Levy, Proceed. Roy. Soc., 71.

2Amer. Journ. of Physiol., 21, and Journ. of biol. Chem., 9; see also Robertson,
ibid., 6 and 7.

3Peiper, Virchow's Arch., 116; Cohnstein, ibid., 130, which also cites the works
of Minkowski, Zuntz, and Geppert; Freudberg, ibid., 125 (literature); in regard to the
methods for the estimation of the alkalinity see, besides the above-mentioned authors,
v. Jaksch, Klin. Diagnostik; v. Limbeck, Wien. med. Blatter, 18; Wright, The Lancet.
1897; Biernacki, Beitrage zur Pneumatologie, etc., Zeitschr. f. klin. Med., 31 and
32; Hamburger, Eine Methode zur Trennung, etc., Arch., f. (Anat. u.) Physiol. >

310 THE BLOOD.

The methods for the determination of the true reaction of animal
fluids, also the blood, have been given in Chapter I. For the true
alkalinity of the blood, as first shown by HOBER and especially by
HASSELBALCH and LUNDSGAARD, the carbon dioxide is of the greatest
importance in that with an increasing carbon-dioxide tension the con-
centration of the H ions increase. Thus HASSELBALCH and LUNDS-
GAARD 1 found that a rise in the carbon-dioxide tension of 30-50 mm.,
that is a rise of 20 mm., increased the concentration of the H ions about
36 per cent.

For the determination of the true reaction the temperature at which
the measurement is made is of the greatest importance. As the dis-
sociation constant of water strongly rises with the temperature, the
HO ion concentration of the blood must rise with the temperature, and
we can believe that the alkalinity of the blood at body temperature
must be 2-3 times greater than when measured at 18° and that this
alkalinity increases 15-20 per cent when the normal temperature of the
body (38°) rises to that of a high fever (42°).

The true alkalinity of the blood is somewrhat variable under different
conditions. In this connection it must be remarked that also age and
other conditions have an action upon the alkalinity. As the determin-
ations are made with different, and not always exact methods, and some-
times without consideration of the action of carbon dioxide and tem-
perature, it is extremely difficult to give satisfactory average results.
Under these circumstances it is perhaps sufficient to refer to the figures
given in Chapter I (page 76).

The alkali of the blood as above mentioned exists in part as alkaline
salts, carbonate and phosphate, and partly in combination with protein
or haemoglobin. The first are often spoken of as readily diffusible alkalies,
while the others are not or are only diffusible with difficulty (see page
268) . The quantity of the first, in human blood, is about one-fifth of the
total alkali (BRANDENBURG). The readily as well as the difficultly
diffusible alkali is divided between the blood-corpuscles and plasma, and
the blood-corpuscles seem to be richer in difficultly diffusible alkali than
the plasma or serum. This division may be changed by the influence of
even very small amounts of acid, even of carbonic acid, and also, as shown
by ZUNTZ, LOEWY and ZUNTZ, HAMBURGER, LIMBECK, and GtJRBER,2

1898. See also Maly's Jahresber., 29, 30, and 31; Salaskin and Pupkin, Zeitschr. f.
physiol. Chem., 42, and O. Folin, ibid., 43; Laitinen, Hammarsten's Festschr., 1905 ;
Westenrijk, Arch. f. exp. Path. u. Pharm. Suppl., 1908, Schmiedeberg-Festschrift.

1 The literature may be found in Sorenssen, Messung und Bedeutung der Wasser-
stoff-ionkonzentrationen, Ergbn. d. Physiol., 12; Hasselbalch and Lundsgaard, Bioch.
Zeitschr., 38, and Skand. Arch. f. Physiol., 27; Hasselbalch, Bioch. Zeitschr., 30; Lunds-
gaard, ibid., 41.

2 Zuntz, in Hermann's Handbuch der Physiol., 4, Abt. 2; Loewy and Zuntz,

BLOOD-CORPUSCLES AND GASES. VISCOSITY. 311

by the influence of the respiratory exchange of gas. The blood-corpuscles
give up a part of the alkali united with protein to the serum by the action
of carbon dioxide, hence the serum becomes more alkaline. The equilib-
rium of the osmotic tension in the blood-corpuscles and in the serum
is thus disturbed; the blood-corpuscles swell up because they take up
water from the serum, and this then becomes more concentrated and richer
in alkali, protein, and sugar. Under the influence of oxygen, the cor-
puscles take their original form again and the above changes are reversed.
The blood-corpuscles for this reason are less biconcave in their small
diameter in venous than in arterial blood (HAMBURGER).

These conditions have been further studied by v. KORAN YI and
BENCE/ and they have investigated the relation between the changes of
the volume of the blood-corpuscles and the electrical conductivity, the
refractivity of the serum and the viscosity of the blood. The refrac-
tion coefficient of the serum is highest with an increase in the amount of
carbon dioxide, while it is lowest when the blood is rich in oxygen and
poor in carbon dioxide. They consider this as an action of acid, as a
similar rise is observed after the addition of acid, while after the addition
of alkali a fall in the refraction coefficient of the serum takes place, and
these same changes can be brought about by C(>2 or by a current of oxygen.
With an increase in the amount of carbon dioxide, the conductivity
of the blood diminishes; the viscosity is, on the other hand, highest
when the blood is richest in carbon dioxide. If the CO2 is driven off
by O the viscosity diminishes to a minimum, and on leading in more
oxygen it rises again. The changes in viscosity of the blood runs parallel
with .the volume changes of the blood-corpuscles, and changes in the
viscosity, which can be brought about by the removal of carbon dioxide,
cause a change in the electric charge of the blood-corpuscles (v. KORANYI
and BENCE). The viscosity of the blood is a variable quantity which,
besides the gas content of the blood, is also dependent upon many other
circumstances (ADAM 2) and which is different at various ages and under
unequal physiological and pathological conditions.

The color of the blood is red — light scarlet-red in the arteries and dark
bluish-red in the veins. Blood free from oxygen is dichroic, dark red
by reflected light and green by transmitted light. The blood-coloring
matters occur in the blood-corpuscles. For this reason blood is opaque

Pfliiger's Arch., 58; Hamburger, Arch. f. (Anat. u.) Physiol., 1894 and 1898, and
Zeitschr. f. Biologie, 28 and 35; v. Limbeck, Arch. f. exp. Path. u. Pharm., 35; Giirber,
Sitzungsber. d. phys. med. Gesellsch. zu Wiirzburg, 1895.

1 Pfluger's Arch., 110.

2 In regard to the viscosity of the blood and the literature of the subject, see R.
Hober in Oppenheimer's Handb. der Bioch., 2, p. 12-18. See also Adam, Zeitschr.
f. klin. Med., 68.

312 THE BLOOD.

in thin layers. If the haemoglobin is removed from the stroma and
dissolved by the blood liquid by any of the above-mentioned means
(see page 273), the blood becomes transparent and has then a " lake
color." l Less light is now reflected from its interior, and this laky
blood is therefore darker in thicker layers. On the addition of salt
solutions to the blood-corpuscles they shrink, more light is reflected,
and the color appears lighter. A great abundance of red corpuscles
makes the blood darker, while by diluting with serum or by a greater
abundance of white corpuscles the blood becomes lighter in appearance.
The different colors of arterial and of venous blood depend on the vary-
ing quantities of gas contained in these two varieties of blood, or, bet-
ter, on the different amounts of oxyhsemoglobin and haemoglobin they
contain.

The most striking property of blood consists in its coagulating within
a shorter or longer time, but as a rule very shortly after leaving the veins.
Different kinds of blood coagulate with varying rapidity; in human
blood the first marked sign of coagulation is seen in two to three minutes,
and within seven to eight minutes the blood is thoroughly converted into
a gelatinous mass. If the blood is allowed to coagulate slowly, the red
corpuscles have time to settle more or less before the coagulation, and
the blood-clot then shows an upper yellowish-gray or reddish-gray layer
consisting of fibrin enclosing chiefly colorless corpuscles. This layer
has been called crusta inflammatoria or phlogistica, because it has been
especially observed in inflammatory processes and is considered one
of the characteristics of them. This crusta, or " buffy coat," is not
characteristic of any special disease, and it occurs chiefly when the blood
coagulates slowly or when the blood-corpuscles settle more quickly
than usual. A buffy coat is often observed in the slowly coagulating
equine blood. The blood from the capillaries is not supposed to have the
power of coagulating.

Coagulation is retarded by cooling, by diminishing the oxygen, and by
increasing the amount of carbon dioxide, which is the reason that venous
blood and to a much higher degree blood after asphyxiation coagulates
more slowly than arterial blood. The coagulation may be retarded or
prevented by the addition of acids, alkalies, or ammonia, even in small
quantities; by concentrated solutions of neutral alkali salts and alkaline
earths, alkali oxalates and fluorides; also by egg-albumin, solutions of
sugar or gum, glycerin, or much water; also by receiving the blood in
oil. Coagulation may be prevented by the injection of a proteose solu-
tion or of an infusion of the leech into the circulating blood, but this

1 R. Du Bois-Reymond presents objections to the general use of the above terms
in Centralbl. f. Physiol., 19, p. 65.

COAGULATION OF THE BLOOD. 313

infusion also acts in the same way on blood just drawn. Coagulation
is also hindered by snake poison (cobra-poison), and bacterial toxines.
The coagulation may be facilitated by raising the temperature; by con-
tact with foreign bodies, to which the blood adheres; by stirring or beat-
ing it; by admission of air; by diluting with very small amounts of
water; by the addition of platinum-black or finely powdered carbon;
by the addition of laky blood, which does not act by the presence of
dissolved blood-coloring matters, but by the stromata of the blood-corpus-
cles; and also by the addition of the leucocytes from the lymphatic
glands, or of a watery saline extract of the lymphatic glands, testicles, or
thymus and various other organs (DELEZENNE, WRIGHT, ARTHUS/ and
others).

An important question to answer is why the blood remains fluid
in the circulation, while it quickly coagulates when it leaves the circula-
tion. The reason why blood coagulates on leaving the body is therefore
to be sought for in the influence which the walls of the living and unin-
jured blood-vessels exert upon it. These views are derived from the
observations of many investigators. From the observations of HEWSON,
LISTER, and FREDERICQ it is known that when a vein full of blood is
ligatured at the two ends and removed from the body, the blood may remain
fluid for a long time. BRUCKE 2 allowed the heart removed from a tortoise
to beat at 0° C., and found that the blood remained uncoagulated for
some days. The blood from another heart quickly coagulated when
collected over mercury. In a dead heart, as also in a dead blood-vessel,
the blood soon coagulates, and also when the walls of the vessel are changed
by pathological processes.

What then is the influence which the walls of the vessels exert on
the liquidity of the circulating blood? FREUND found that the blood
remains fluid when collected by means of a greased canula under oil or
in a vessel smeared with vaseline. If the blood collected in a greased
vessel be beaten with a glass rod previously oiled, it does not coagulate,
but it quickly coagulates on beating it with an unoiled glass rod or when
it is poured into a vessel not greased. The non-coagulability of blood
collected under oil was confirmed later by HAYCRAFT and CARLIER.
FREUND found on further investigation that the evaporation of the
upper layers of blood or their contamination with small quantities of dust
causes a coagulation even in a vessel treated with vaseline. According

1Delezenne, Arch, de Physiol. (5), 8; Wright, Journ. of Physiol., 28; Arthus,
Journ. de Physiol. et Pathol., 4.

2Hewson's works, edited by Gulliver, London, 1876, cited from Gamgee, Text-
book of Physiol. Chem., 1, 1880; Lister, cited from Gamgee, ibid.- Fredericq, Recher-
ches sur la constitution du plasma sanguin, Gand, 1878; Briicke, Virchow's Arch. 12.

314 THE BLOOD.

to FREUND 1 it is this adhesion between the blood and a foreign substance
— and the diseased walls of the vessel also act as as such — that gives the
impulse toward coagulation, while the lack of adhesion prevents the
blood from coagulating. BORDET and GENGOU 2 have also shown that
the plasma obtained by centrifuging blood collected in a paraffined
vessel, and perfectly free from form-elements, can be kept without coagulat-
ing in a paraffined vessel, and that it does coagulate on being transferred
to an unparaffined vessel. The adhesion of the plasma to a foreign
body may also, in the absence of form-elements, give the impulse to
coagulation. That this adhesion of the form-elements is of great impor-
tance cannot be denied and is also generally accepted. By this adhesion
the form-elements undergo certain changes which seem to stand in a
certain relation to the coagulation of the blood.

The views in regard to these changes are, unfortunately, very diver-
gent. According to ALEX. SCHMIDT 3 and the Dorpat school an abun-
dant destruction of the leucocytes, especially polynuclear leucocytes,
takes place in coagulation, and important constituents for the coagula-
tion of the fibrin pass into the plasma. A direct relation between the
destruction of leucocytes and coagulation is denied by many investigators,
while according to other experimenters the essential factor is not a
destruction of the leucocytes, but an elimination of constituents from
the cells into the plasma. This process is called plasmoschisis by LowiT.4
The passage of cell constituents into the plasma before coagulation must
not necessarily be considered as a phenomenon of death, as it may just
as well be a secretory process (ARTHUS, MORAWITZ, DASTRE 5).

Great importance has also been ascribed to the blood-plates in coagula-
tion as certain investigators (BIZZERO, LILIENFELD, SCHWALBE, MORA-
WITZ, BURKER, DEETJEN, LE SOURD and PAGNIEZ) found that they
induce, accelerate or make coagulation possible. According to VINCI and
CHISTONI they are not necessary as they are absent in the blood of
birds, which coagulates rapidly, and also in the lymph, of the dog, rabbit
and cat. They may nevertheless accelerate coagulation and they are
necessary for the contraction of the clot. According to AYNAUD they

1 Freund, Wien. med. Jahrb., 1886; Haycraft and Carlier, Journ. of Anat. and
Physiol., 22.

2 Annal. de 1' Institute Pasteur, 17.

3 Pfluger's Arch./ 11. The works of Alex. Schmidt are found in Arch. f. Anat.
und Physiol., 1861, 1862; Pfluger's Arch., 6, 9, 11, 13. See especially Alex. Schmidt,
Zur Blutlehre (Leipzig, 1892), which also gives the work of his pupils, and Weitere
Beitrage zur Blutlehre, 1895.

4 Wien. Sitzungsber., 89 and 90, and Prager med. Wochenschr., 1889, referred
to in Centralbl. f. d. med. Wissensch., 28, 265.

6 Morawitz, Hofmeister's Beitrage, 5; Arthus, Compt. rend. soc. biolog., 55;
Dastre, ibid., 55.

FORM-ELEMENTS AND COAGULATION. 315

are not necessary for the contraction of the clot nor for the coagulation
as a whole, and they are absent in the lymph and serous fluids. Accord-
ing to PETRONE 1 they indeed have a function in retarding coagulation.

WOOLDRIDGE 2 takes a very peculiar position in regard to this question: he
considers the form-elements as only of secondary importance in coagulation.
As he has found, a peptone-plasma which has been freed from all form-con-
stituents by means of centrifugal force yields abundant fibrin when it is not
separated from a substance which precipitates on' cooling. This substance,
which WOOLDRIDGE has called A-fibrinogen, seems to all appearances to be a
nucleoproteid, which, according to the unanimous view of several investigators,
originates from the form-elements of the blood, either the blood-plates or the
leucocytes and the generally accepted view as to the great importance of the
form-elements in the coagulation of the blood is not really contrary to WOOL-
DRIDGE'S experiments.

There is great diversity of opinion in regard to those bodies which
are eliminated from the form-elements of the blood before and during
coagulation.

According to ALEX. SCHMIDT the leucocytes, like all cells, contain
two chief groups of constituents, one of which accelerates coagulation,
wrhile the other retards or hinders it. The first may be extracted from the
cells by alcohol, while the other cannot be extracted. Blood-plasma
contains only traces of thrombin, according to SCHMIDT, but does con-
tain its antecedent, prothrombin. The bodies which accelerate coagu-
lation are neither thrombin nor prothrombin, but they act in this wise
in that they split off thrombin from the prothrombin. On this account
they are called zymoplastic substances by ALEX. SCHMIDT. The nature
of these bodies is unknown, and SCHMIDT has given no opinion as to
their relation to the lime salts, which have been found to have zymoplastic
activity by other investigators.

The constituents of the cells which hinder coagulation and which are insoluble
in alcohol-ether are compound proteins, and have been called cytoglobin and
preglobulin by SCHMIDT. The retarding action of these bodies may be sup-
pressed by the addition of zymoplastic substances, and the yield of fibrin on coagula-
tion in this case is much greater than in the absence of the compound protein
retarding coagulation. This last supplies the material from which the fibrin is
produced. The process is, according to SCHMIDT, as follows: The preglobulin
first splits, yielding serglobulin, then from this the fibrinogen is derived, and from
this latter the fibrin is produced. The object of the thrombin is two-fold. The
thrombin first splits the fibrinogen from the paraglobulin, and then converts the

1See footnote 2, p. 308. Also Schwalbe, liters, z. Blutgerinnung, etc., Braun-
schweig, 1900; Morawitz, Deutsch. Arch. f. klin. Med., 79, and Hofmeister's Beitrage,
4 and 5; Biirker, Pfliiger's Arch., 102, and Centralbl. f. Physiol., 21; Deetjen, 1. c.;
Le Sourd and Pagniez, Journ. de Physiol., 11; Vinci and Chistoni, Chem. Centralbl.,
1909, 2, 838, and Maly's Jahresb., 39; Aynaud, Maly's Jahresb., 39; 165; Petrone,
Maly's Jahresber., 31, p. 170.

2 Die Gerinnung des Blutes (published by M. v. Frey, Leipzig, 1891).

316 THE BLOOD.

fibrinogen into fibrin. The assumption that fibrinogcn can be split from para-
globulin has not sufficient foundation and is even improbable.

According to SCHMIDT the retarding action of the cells is prominent
during life, while the accelerating action is especially pronounced out-
side of the body or by coming in contact with foreign bodies. The paren-
chymous masses of the organs and tissues, through which the blood flows
in the capillaries, are those cell-masses which serve to keep the blood
fluid during life.

LILIENFELD has given further proof as to the occurrence, in the form-
elements of the blood, of bodies which accelerate or retard the coagula-
tion. According to this author the nature of these bodies is very markedly
different from SCHMIDT'S idea. While, according to SCHMIDT, the coagula-
tion accelerators are bodies soluble in alcohol, and the compound proteins
exhausted with alcohol act only retardingly on coagulation, LILIENFELD
states that the substance which acts acceleratingly and retardingly
on coagulation are contained in a nucleoprotein, namely, nucleohistone.
Nucleohistone readily splits into leuconuclein and histone, the first
of which acts as a coagulation-excitant, while the other, introduced
into the blood-vascular system, either intravascular or extravascular, robs
the blood of its property of coagulating. Introduced into the circulatory
system the nucleohistone splits into its two components. It therefore
causes extensive coagulation on one side and makes the remainder of
the blood uncoagulable on the other. This theory as well as that of
SCHMIDT is not based upon sufficiently demonstrated facts.

BRUCKE showed long ago that fibrin left an ash containing calcium
phosphate. The fact that calcium salts may facilitate or even cause a
coagulation, in liquids poor in ferment, has been known for several years,
through the researches of HAMMARSTEN, GREEN, RINGER and SAINS-
BURY. The necessity of the lime salts for the coagulation of blood and
plasma was first shown positively by the important investigations of
ARTHUS and PAGES. Recent investigations of SABBATANI 1 have also
shown the importance of calcium salts or the free calcium ions for
coagulation without explaining the mode of their action.

According to the generally accepted view of ARTHUS and PAGES the soluble
lime salts precipitable by oxalate are necessary requisites for the fermentive
transformation of fibrinogen, because thrombin remains inactive in the absence
of soluble lime salts. This view is untenable, as shown by the researches of
ALEX. SCHMIDT, PEKELHARING, and HAMMARSTEN. 2 Thrombin acts as well in
the absence as in the presence of precipitable lime salts.

1 Hammarsten, Nova Acta, reg. Soc. Scient. Upsala, (3), 10, 1879; Green, Journ.
of PhysioL, 8; Ringer and Sainsbury, ibid., 11 and 12; Arthus et Pages and Arthus,
see footnote 4, p. 251; Hammarsten, Zeitschr. f. physiol. Chem., 22; Sabbatani,
cited, Centralbl. f. PhysioL, 16, 665.

2 Hammarsten, Zeitschr. f. physiol. Chem., 22, where the other investigators are
cited.

COAGULATION OF THE BI^rOD. 317

According to PEKELHARING l thrombin is the lime compound of
prothrombin, and the process of coagulation consists, according to him,
in the thrombin transferring the lime to the fibrinogen, which is thereby
converted into an insoluble lime compound, fibrin. Among the objec-
tions to this theory can be mentioned, the fact that fibrin has not
been obtained absolutely free from lime, but still so poor in lime
(HAMMARSTEN 2) that if the lime belongs to the fibrin, its molecule
must be more than ten times greater than the haemoglobin molecule,
which is not probable. These as well as many other observations
indicate that the lime is carried down by the fibrinogen only as a
contamination.

If, as it seems, the lime is not of importance in the transformation
of fibrinogen into fibrin in the presence of thrombin, still this does not
contradict the above-mentioned observations of ARTHUS and PAGES that
the lime salts are necessary for coagulation of blood and plasma. It
is very probable that the lime salts, as admitted by PEKELHARING, are
a requisite for the transformation of prothrombin into thrombin.

If we attempt to summarize the more or less contradictory investi-
gations and views as given in the preceding pages, we can consider the
following facts as conclusive: In the first place, two bodies, the fibrin-
ogen and the thrombin, are necessary for the coagulation. The fibrinogen
exists preformed in the plasma. The thrombin, on the contrary, does
not occur in living blood, at least not in appreciable amounts as such,
but is formed from another substance, the prothrombin. The presence
of calcium salts is necessary for the formation of this thrombin, while
the calcium salts are not necessary for the enzymotic transformation
of fibrinogen into fibrin. Besides the calcium salts also other substances,
the zymoplastic active substances, are active in the formation of thrombin
from its mother-substance, and these zymoplastic substances stand in
some relation to the form-elements of the blood.

The formation of thrombin and the relation of the form-elements
therewith are still unexplained and disputed questions.

It is a question whether the mother-substance of thrombin exists in
the plasma of the circulating blood or whether it is a body eliminated
from the form-elements before coagulation. We have two opposing
views on this question, namely, those of ALEX. SCHMIDT and of PEKEL-
HARING. According to SCHMIDT prothrombin occurs preformed in the
circulating plasma, and it is transformed into thrombin by the zymo-
plastic substances which pass out from the form-elements. PEKEL-
HARING, on the contrary, holds the view that the plasma does not contain

1 See footnote 4, p. 256, and especially Virchow's Festschrift, 1, 1891.
2Zeitschr. f. physiol Chem., 28.

318 THE BLOOD.

appreciable amounts of prothrombin. This body, according to him,
passes before coagulation from the form-elements into the plasma, and
is there converted into thrombin by the calcium salts. The observa-
tion that uncoagulated leech-plasma does not coagulate on the addition
of calcium salts, while it does coagulate on the addition of prothrombin
solutions, seems to support this view; still it is not quite conclusive.
Leech-extract contains a body, hirudin, which, seems to be an anti-
body toward thrombin and quantitatively neutralizes it. On the addi-
tion of prothrombin, new thrombin may be formed, which may act
if the hirudin is not present in too great an excess.

Other observations which dispute the occurrence of prothrombin in
the circulating plasma can be explained in various ways and it is the
general view at present that the prothrombin is a preformed constituent
of the plasma. l

Although the opinions are rather united as to the occurrence of at
least three bodies, fibrinogen, prothrombin (thrombogen) and lime salts
in the plasma, still the question arises how the thrombin is formed from
the thrombogen. The zymoplastic substances must be here considered,
and the starting-point in these new investigations is the accelerat-
ing action upon coagulation, of different tissue extracts, an action which
has been known for a long time and was especially studied by DELE-
ZENNE on the plasma from bird's blood. Unfortunately we are not in
accord as to the nature and manner of action of the active constituents
of these extracts. According to MORAWITZ the active body is not
thrombin, but another substance called thrombokinase, besides lime-
salts, which are necessary for the transformation of prothrombin (throm-
bogen according to MORAWITZ). The production of thrombokinase is,
according to MORAWITZ, a general property of the protoplasm, and also
occurs in the leucocytes (and blood-plates). Three substances are nec-
essary, according to his view, for the formation of thrombin, namely:
thrombogen, thrombokinase and lime salts. Thrombogen is, he claims,
not quite identical with the prothrombin (other investigators), which
he calls a-prothrombin, but is a mother-substance of it. The process
of thrombin formation can be given as follows: The kinase first trans-
forms the thrombogen into a-prothrombin, which latter then is converted
into thrombin (a) by the lime salts.

1 Arthus, Journ. de Physiol. et Pathol., 3 and 4, and Compt. rend. soc. biol., 56.
The works of Morawitz may be found in Hofmeister's Beitrage, 4 and 5, Deutsch.
Arch. f. klin. Med., 79 and 80, and in Oppenheimer's Handb. der Bioch., 2; Fuld,
Centralbl. f. Physiol., 17, p. 529; with Spiro, Hofmeister's Beitrage, 5; Schittenhelm
and Bodong, Arch. f. exp. Path. u. Pharm., 54; Bordet and Gengou, Annak Institut
Pasteur, 18. For more recent literature see Loeb, Biochem. Centralbl., 6, p. 907.
P. Nolf, Arch, internat. de Physiol., 6, 1908.

COAGULATION OF THE BLOOD. 319

The thrombokinase does not occur to any appreciable extent in
the circulating blood, but is supplied by the form-elements. The
accelerating action upon coagulation of tissues or parts of tissues depends,
as above stated, upon their content of kinase; but it also in part depends
upon the fact that the tissue fluids excite the secretory activity of the form-
elements.

FULD 1 has arrived at about the same results independently of MORA-
WITZ, but he has selected other names. The three substances, throm-
bogen, kinase, and thrombin are called by him plasmozym, cytozym,
and holozym. The principal reason why circulating blood remains fluid is,
according to FULD, because the cytozym is only slowly formed therein
and the ferment (holozym) produced thereby is quickly changed into an
inactive form. Another reason is that the blood contains an antibody
for the fibrin ferment. The assumption of ALEXANDER SCHMIDT that
the blood contains substances retarding coagulation (anti-thrombins)
has recently also received support by the observations of FULD and
SPIRO, MORAWITZ, LOEB, NOLF, PUGLIESE, HOWELL 2 and others. Accord-
ing to HOWELL the non-coagulability of circulating blood depends on
the fact that the antithrombin prevents the activation of the prothrombin
into thrombin.

According to the theory of MORAWITZ, FULD and SPIRO, which is the
most accepted, of those substances necessary for coagulation, only
the thrombokinase (the cytozym) is absent in the circulating blood,
and this is the reason why the circulating blood remains fluid. The
reason why the plasma does not contain any thrombokinase lies in the
fact that the healthy endothelium of the vessels does not have any irritat-
ing action upon the form-elements, and therefore no mentionable quan-
tity of kinase is given off under these circumstances. Such an elimina-
tion occurs first outside of the blood vessels, and indeed very quickly
in contact with foreign bodies. The formation of thrombin from the
thrombogen takes place in an unknown manner by the action of the
kinase only in the presence of lime salts (in the plasma), and this throm-
bin then transforms the fibrinogen into fibrin.

A serum poor in ferment and having a weak action can be reactivated by the
addition of acid or alkali (ALEX. SCHMIDT, MORAWITZ), and in this action, accord-
ing to MORAWITZ, a thrombin ()8) is produced which is somewhat different from
a-thrombin. The /3-thrombin is produced from a special /3-prothrombin which
never occurs in the plasma, but only in the serum. FULD explains this by
affirming that the a-thrombin is changed in the serum into metazym (/3-pro-

1 Centralbl. f. Physiol., 17. See also Fuld and Spiro, Hofmeister's Beitrage, 5.

2Fuld and Spiro, 1. c.; Morawitz, 1. c.; Loeb, Hofmeister's Beitrage, o; Nolf, Arch,
internat. de Physiol., 6; Pugliese, Biochem. Centralbl., 5, p. 930; Howell, Amer.
Journ. of Physiol., 29.

320 THE BLOOD.

thrombin), which is then transformed by the alkali or acid into neozym (=/3-
thrombin). Nevertheless it is a fact that the quantity of thrombin in the serum
diminishes after coagulation, and that the thrombin action is considerably increased
by the addition of alkali or acid as well as by zymoplastic substances. The above
view as to the occurrence of different thrombins has not sufficient basis, and
PEKELHARISG l has also raised objections thereto.

The theories of MORAWITZ, FULD and SPIRO at least stand in accord
with several known facts but do not take sufficient account of the action
of the zymoplastic substances of ALEX. SCHMIDT. Thrombokinase is
precipitated, by alcohol and is not thermostabile, while the zymoplastic
substances, of SCHMIDT are thermostabile and soluble in alcohol. The
thrombokinase cannot therefore be identical with these zymoplastic
substances, and hence this theory does not explain the action of these
latter. Further, the mode of action of tissue extracts is unexplained,
and is a much disputed subject. It can be said that these two views are
in the main opposed to each other. According to one (ALEX. SCHMIDT,
ARTHUS, MORAWITZ and others) they do not act like fibrin ferment,
but have an indirect action. According to the other (PEKELHARING,
HUISKAMP, DELEZENNE and LOEB 2) they are thrombin, or at least bodies
having an analogous action.

CRAMER and PRINGLES have made the important observation that a
carefully prepared oxalate plasma when filtered through a Berkefeld
filter does not coagulate on adding calcium chloride, while the unfiltered
but centrifuged plasma does coagulate. The reason for this lies in the
fact that centrifuged plasma contains blood-plates, which are absent
in the filtered plasma. By means of these blood-plates, which yield
thrombokinase, the coagulation is produced on the addition of calcium
chloride. The points in NOLF'S theory of coagulation that are difficult
to understand as well as the observations of FREUND (page 313) and of
BORDET and GENGOU (page 314) are explained by this observation.

L. LoEB,4 who has carried out complete investigations on the coagulation of
blood, especially of Crustacea, has arrived at the following view: The coagula-
tion in the Crustacea can, according to him, be of two kinds. It may in part
be an agglutination of the amrebocytes and in part a fibrin formation from a fibrino-
gen of the plasma. This latter coagulation is essentially the same as occurs in
vertebrates. The substance acting here as the excitant for the coagulation is
also active in the absence of lime salts, and behaves therefore like a thrombin.
The tissues contain constituents which accelerate coagulation, which LOEB calls
coagulins, which are not identical with the coagulins of the clot or the blood serum,

1 Bioch. Zeitschr., 11.

2Huiskamp. Zeitschr. f. physiol. Chem., 34, 39; Delezenne, Arch. de. physiol.,
1897; Loeb, Biochem. Centralbl, 6, pages 829 and 889.

3 Quarterly Journ. of exp. Physiol., 6.

4 Medical News, New York, 1903, and Virchow's Arch., 176; Hofmeister's Beitrage,
5, 6, 8, 9, and Biochem. Centralbl., 6, pages 829 and 889.

COAGULATION OF THK BLOOD. C21

and these have also, although only in the presence of lime salts (if the author
understands LOEB), a direct coagulating action upon fibrinogen. According to
LOEB the tissue coagulins do not act as kinases in the invertebrates, and he also
finds it improbable that they would act as kinases in the vertebrates. Under
favorable conditions the combined blood and tissue coagulins are more active
than the sum of the individual action. That this is due to an activation by a
kinase, which is a possible explanation, has, in LOEB'S opinion, not been proved.

The coagulins of the blood are, as above stated, according to LOEB, different
from the tissue coagulins. The latter are for different classes of animals so
adapted that they bring about a quicker coagulation in the blood of certain classes
of animals than do the bther class. The erythrocytes of mammalia (cat, dog, rabbit)
contain, on the contrary, according to LOEB and FLEISHER x coagulins of such a
specific adaptability that it is possible to differentiate between the blood corpuscles
of different kinds of mammalia or, if the erythrocytes are known, to detect an
unknown plasma.

Opinions are strikingly at variance in regard to the mode of action
of the tissue constituents which accelerate coagulation, and their nature
also is entirely unknown, hence great confusion exists on the whole in
this subject.

If we accept the fact that thrombokinase does not occur in the plasma,
but is produced under the influence of a foreign body acting as an excitant,
it is rather difficult to understand why the plasma obtained from blood
collected in a paraffined vessel and quickly and strongly centrifuged,
and which is perfectly free from form-elements, should remain fluid for
a long time in a paraffined vessel while it coagulates in an ordinary glass
vessel. NOLF has tried by his theory to explain this difficulty, as well
as the action of the alcohol-soluble zymoplastic substances (ALEX.
SCHMIDT).

According to NoLF2 the following bodies take direct part in the
coagulation of the blood, namely: Fibrinogen, thrombogen (formerly
called hepatothrombin by him) thrombozym ( = thrombokinase of MORA-
WITZ) and lime salts. The coagulation of the blood, according to him,
is a different process from the coagulation of a fibrinogen solution by
thrombin. While in this last case the thrombin is the substance exciting
coagulation, in the other case the thrombin is a product of the coagu-
lation, as suggested by WOOLDRIDGE. In the coagulation of the plasma,
according to NOLF we have a mutual precipitation of the three above-
mentioned colloids — fibrinogen, thrombogen and thrombozym, all three
of which are contained in the fibrin clot. This latter has correspondingly
no constant composition, but varies according to the relative propor-
tions of these three colloids. In the presence of only a little fibrinogen
thrombin is produced from the three colloids (in the presence of lime
salts) ; in the presence of abundance of fibrinogen, on the contrary, fibrin

1 Loeb and Fleisher, Bioch. Zeitchr. 28.

2 Arch, internat. de Physiol., 6, Fasc., 1, 2, and 3 and 7 and 9.

322 THE BLOOD.

is formed. Thrombin is a fibrin incompletely saturated with fibrinogen,
and in the coagulation of fibrinogen with thrombin the still unsatisfied
affinities of the latter are saturated. ("La thrombine d'A. Schmidt n'est-
pas autre chose que de la fibrine insuffisamment pourvue de fibrinogene.
Dans la coagulation du fibrinogene par la thrombine les affinites restees
libres de celle-ci peuvent s'assouvir; le compose moins sature se trans-
forme en un compose plus sature.") The formation of fibrin from
fibrinogen is not, according to NOLF, an enzymotic process, and ,the
thrombin is only a residue of the fibrin remaining in solution.

In NOLF'S opinion the thrombogen is probably formed in the liver
and found to a large extent in all plasma. The thrombozym is secreted
by the leucocytes and the endothelial cells, and in opposition to MORA-
WITZ is not secreted by other cells. It is also a normal constituent of the
blood-plasma circulating in the living body. Most tissues, on the con-
trary, contain no thrombozym. The tissue extracts, NOLF believes,
also contain no substances absolutely necessary for the coagulation,
but only bodies which can have a powerful accelerating action, the
thromboplastic substances which are mixed with the thrombokinase of
MORAWITZ. The circulating blood-plasma contains all the bodies directly
necessary in the coagulation, namely, fibrinogen, thrombogen, throm-
bozym and lime salts. Besides these it also contains a substance that
inhibits coagulation, antithrombin, which is formed in the liver. There
exists, if the author understands the work of NOLF, a labile equilibrium
between the various constituents of the plasma, and this equilibrium is
destroyed in coagulation. The first impulse to coagulation is given by
the thromboplastic substances.

NOLF considers as thromboplastic active any influence of a physical
or chemical nature which, be it produced by the walls of the vessel, a
suspended body, a solvent or a dissolved body, a colloid or crystalloid,
a molecule or an ion, makes the combination of the three above colloids
possible. To the thromboplastic agents belong the walls of a glass
vessel, finely powdered glass, the precipitates of calcium oxalate or
calcium fluoride, also living protoplasm, aqueous tissue extracts, the
alcohol soluble zymoplastic substances of ALEX. SCHMIDT, and other
substances. All these agents in some way or other may serve as points
of precipitation. That a plasma free from form-elements coagulates
for example on contact with the walls of a glass vessel depends upon the
fact that the inhibitory action of the antithrombin is retarded by the
thromboplastic action of the foreign surface. Unfortunately we are
not certain as to how this thromboplastic action is brought about.

An important side of NOLF'S theory of coagulation is also the fibrinol-
ysis which is brought about by the thrombin. The proteolytic action
of the thrombin is due only to the thrombozym contained therein, and

COAGULATION OF THE BLOOD. 323

it has a proteolytic action only upon fibrin and not upon fibrinogen.
According to NOLF, coagulation is merely a preparation for the prote-
olysis, and is a nutrition phenomenon, and in addition is of special
importance, in arresting hemorrhage'. In order to prevent a rapid
fibrinolysis, the plasma also contains one or more antifibrinolytic sub-
stances, which are secreted by the liver.

What has been given contains the chief points in NOLF'S theory
of coagulation, and it is impossible in a text-book to enter more into
detail in regard to his remarkable investigations or the foundations
on which he bases his theory and the objections which can be raised
against it.

Recently other investigators as RETTGER and HOWELL have raised
objections to the view that the coagulation of the blood is an enzymotic
process. STROMBERG l also leans toward such a conception and they
all raise the objection that the quantity of fibrin increases with the quan-
tity of thrombin. This behavior, which has been known for a long time,
is of such a complicated nature, that no positive conclusions can be drawn
therefrom.

The belief of MELLANBY 2 that the plasma originally only contains one
globulin, fibrinogen, from which by enzymotic cleavage the fibrin and serglobulin
are formed, is untenable and is based upon the imperfect methods of preparing
fibrinogen that he used.

From the above description of the various theories of coagulation
it at least follows that in the study of the coagulation of the blood there
are many contradictory statements and observations, and so many obscure
points, that for the present it is impossible to give a clear, comprehensive
summary of the different views and to deduce a theory of the process
of coagulation which would embrace all the factors. In spite of this
confusion and all contradictions, still we are sure that certain bodies such
as fibrinogen and thrombin, even though this latter be an enzyme or a
colloid combination, are directly concerned in the formation of fibrin,
while other bodies act indirectly as accelerators or inhibitors of coagulation.

The bodies accelerating coagulation, with the exception of gelatin,
whose action in this regard has not been positively proved, have been
mentioned several times above. The mode of action of the bodies retard-
ing coagulation is not clear and is much disputed. Their action may,
it seems, also be more of a direct or indirect kind. Thus, for example,
the oxalate and fluoride may prevent the formation of thrombin by
precipitation of the lime. The cobra-poison seems to prevent the forma-

1Rettger, Amer. Journ. of Physiol., 24; Howell, ibid., 26; Stromberg, Bioch.
Zeitschr., 37.

2 Journ. of Physiol., 38.

324 THE BLOOD.

tion of thrombin by the action upon the thrombokinase; the hirudin 1
may, it is generally believed, as antithrombin make the thrombin inactive,
and the normal constituents of the plasma retarding coagulation perhaps
act in a similar manner. In other cases the retarding bodies act indirectly,
for they may, like the proteoses and others, cause the body to produce
special bodies which stand in close relation to intravascular coagulation.

Intravascular Coagulation. It has been shown by ALEX. SCHMIDT
and his students, as also by WOOLDRIDGE, WRIGHT,2 and others, that an
intravascular coagulation may be brought about by the intravenous
injection into the circulating blood of a large quantity of a thrombin
solution, as also by the injection of leucocytes or tissue fibrinogen (impure
nucleoprotein) . Intravascular coagulation may also be brought about
under other conditions, such as after the injection of snake-poison
(MARTIN3 and others) or certain of the protein-like colloid substances,
synthetically prepared according to GRIMAUX'S method (HALLIBURTON
and PICKERING 4). If too little of the above-mentioned bodies be injected,
then we observe only a marked retarding tendency in the coagulation
of the blood. According to WOOLDRIDGE it can generally be maintained
that after a short stage of accelerated coagulability, which may lead to a
total or partial intravascular coagulation, a second stage of a diminished
or even arrested coagulability of the blood follows. The first stage is
designated (WOOLDRIDGE) as the positive and the other as the negative
phase of coagulation. These statements have been confirmed by several
investigators.

There is no doubt that the positive phase is brought about by an
abundant introduction of thrombin, or by a rapid and abundant for-
mation of the same. The explanation of the production of the negative
phase, which can easily be brought about by pepsin proteoses, by various
bodies such as extracts of crabs' muscles and other organs, eel-serum,
enzymes, bacterial toxines, certain snake-poisons, etc., has been attempted
in different ways. The best studied is the action of proteoses, but no
conclusive results have been obtained thus far. The assertion of PICK
and SPIRO that the action of the proteoses does not depend upon the
proteoses themselves, but upon a contaminating substance, the protozym,
is claimed to be incorrect by UNDERBILL, while the recent investigations
of POPIELSKI indicate that this is correct. The bodies retarding coagu-

1 The action of hirudin is somewhat doubtful. See Schittenhelm and Bodong, 1. c.

2 A study of the Intravascular Coagulation, etc., Proceed, of the Roy. Irish Acad.
(3), 2. See also Wright, Lecture on Tissue or Cell Fibrinogen, The Lancet, 1892;
and Wooldridge's Method of Producing Immunity, etc., Brit. Med. Journ., Sept., 1891.

3 Journ. of Physiol., 15.

4 Ibid., 18.

RETARDATION OF COAGULATION. 325

lation, obtained by CONRADI 1 in autolysis, which are probably antithrom-
bins, seem to act in a different way from the proteoses, and cannot for
the present be made use of in explaining this question.

There are a large number of researches on the action of proteoses
and of other similar retarding substances by a great number of different
investigators, especially by GLEY AND PACHON, SPIRO, MORAWITZ, NOLF,
DELEZENNE, DOYON and collaborators.2 We can say with certainty
that the action is indirect, and that the liver is important for the process.
The non-coagulability of " peptone-blood " seems to be due to several
reasons, but it has not been thoroughly explained. On the one hand
such blood contains an antithrombin, and on the other it seems as if the
formation of thrombin is not sufficient, although the plasma contains
the necessary conditions for the thrombin formation, as it coagulates as
a rule on dilution with water or the addition of a little acid. This last
behavior speaks, according to MELLANBY,S for the assumption that the
liver, because of the proteose injection, gives up an excess of alkali to the
blood thus preventing the coagulation of the peptone-blood. Opinions
in regard to the occurrence of an antithrombin in the peptone-
plasma seem to be unanimous, and we have gained considerable
experience in regard to the formation of this antithrombin. According
to NOLF, the peptones (more correctly the proteoses) cause an altera-
tion in the leucocytes and the walls of the vessels, and a substance is
secreted which brings about, in the liver, the formation of antithrombin.
According to DELEZENNE the proteoses bring about a destruction of
leucocytes, and thereby a substance accelerating coagulation and another
having a retarding action is set free. The first is destroyed by the liver,
and hence the action of the retarding substance (the antithrombin) is
obtained. DOYON and co-workers have also shown that the isolated
washed liver on transfusing normal arterial blood, gives off a thermo-
stable antithrombin, which behaves like a nucleoprotein. That the liver
takes part in the retardation of coagulation is positively known.

xPick and Spiro, Zeitschr. f. physiol. Chem., 31; Underbill, Amer. Journ. of
Physiol., 9; Popielski, Arch. f. expt. Path. u. Pharm., Suppl. 1908, Schmiedeberg's
Festschrift; Conradi, Hofmeister's Beitrage, 1.

2Grosjean, Travaux du laboratoire de L. Fredericq, 4, Liege, 1892; Ledoux, ibid.,
5, 1896; Nolf, Bull. 1'Acad. roy. de Belgique, 1902 and 1905, and Biochem. Centralbl.,
3; and footnote 1, p. 318; Spiro and Ellinger, Zeitschr. f. physiol. Chem., 23; Fuld
and Spiro, 1. c.; Morawitz, 1. c. The works of the above-mentioned French investi-
gators can be found in Compt. rend. soc. biol., 46, 47, 48, 50, and 51, and Arch. d.
Physiol. (5), 7, 8, 9, and 10; see also especially Delezenne, Arch. d. Physiol. (5), 10;
Compt. rend. soc. biol., 51, and Compt. Rend., 130; Doyon, Compt. rend. soc. biol.,
68, with Morel and Policard, ibid, 70.

3 Journ. of Physiol., 38.

326 THE BLOOD.

The reason of the slow coagulation of the blood in haemophilia is not
well known. Recent investigations of MORAWITZ and LOSSEN, SAHLI,
NOLF and HENRY 1 make it very probable that the thrombokinase plays
an important part. According to SAHLI the quantity of kinase is dimin-
ished, while according to NOLF and HENRY, it is qualitatively changed
so that it is less active. Both cases explain the repeatedly observed
relation of the vessel-walls to haemophilia ' as, according to NOLF, the
thrombokinase (his thrombozym) is also secreted by the endothelial cells.

The non-coagulability of cadaver blood depends usually, according to MORA-
WITZ, 2 'upon tne fact that it contains no fibrinogen, due to a fibrinolysis.

The gases of the blood will be treated in Chapter XVI (on respiration)

IV. THE QUANTITATIVE COMPOSITION OF THE BLOOD.

The quantitative analyses of the blood are of little value. We must
ascertain on one side the relation of the plasma and blood-corpuscles to
each other, and on the other the constitution of each of these two chief
constituents. The difficulties which stand in the way of such a task,
especially in regard to the living, non-coagulated blood, have not been
removed. Since the constitution of the blood may differ not only in
different vascular regions, but also in the same region under different
circumstances, which renders a number of blood analyses necessary, it
can hardly appear remarkable that our knowledge of the constitution of
the blood is still relatively limited.

The relative volume of blood-corpuscles and serum in blood has been
determined by various methods. Of these methods that of L. and M.
BLEiBTREU,3 against which important objections have been raised by
several investigators, such as EYKMAN, BIERNACKI and HEDiN,4 must
be especially mentioned. In regard to this as well as to the method
of St. BUGARSKY and TANGL, which is based upon a difference in the
electrical conductivity of the blood and the plasma, and STEWART^
colorimetric method, we must refer to the original publications.

For clinical purposes the relative volume of corpuscles in the blood
may be determined by the use of a small centrifuge called a hcematocrit,
constructed by BLIX and described and tested by HEDIN. A measured
quantity of blood is mixed with a known volume (best an equal volume)

1 Morawitz and Lessen, Deutsch. Arch. f. klin. Med., 94; Sahli, ibid., 99; Nolf
and Henry, Revue de medicine, 29, 1909.

2 Hofmeister's Beitrage, 8.

3 Pfliiger's Arch., 51, 55, and 60.

4 Biernacki, Zeitschr. f. physiol. Chem., 19; Eykman, Pfluger's Arch., 60; Hedin,
ibid., and Skand. Arch. f. Physiol., 5.

6 Bugarsky and Tangl, Centralbl. f. Physiol., 11; Stewart, Journ. of Physiol., 24.

QUANTITATIVE COMPOSITION OF THE BLOOD. 327

of a fluid which prevents coagulation. This mixture is introduced into
a tube and then centrifuged. According to HEDIN it is best to treat the
blood, which is kept fluid by 1 p. m. oxalate, with an equal volume of
a 9 p. m. NaCl solution. After complete centrifugalization, the layer of
blood-corpuscles is read off on the graduated tube and the volume of
blood-corpuscles (or more correctly the layer of blood-corpuscles) in 100
vols. of the blood calculated therefrom. By means of comparative counts,
HEDIN and DALAND have found that an approximately constant relation
exists between the volume of the layer of blood-corpuscles and the number
of red corpuscles under physiological conditions, so that the number of
corpuscles may be calculated from the volume. DALAND 1 has shown
that such a calculation gives approximate results also in disease, when
the size of the blood-corpuscles does not essentially deviate from the
normal. In certain diseases, such as pernicious anaemia, this method
gives such inaccurate results that it cannot be used.

KOPPE 2 has shown that in centrifuging blood very rapidly, more
than 5000 turnes per minute, the blood-corpuscles may be so completely
separated that all intermediate fluid is removed. Because of the absence
of this intermediate fluid the refraction is changed; the outer layers of
the erythrocytes containing fat become transparent, and the column
of blood-corpuscles becomes transparent and laky. If the volume of
the separated column of blood-corpuscles is determined and the number
of red blood-corpuscles counted, the absolute volume of these latter
can be determined by this method.

In determining the relation between the weight of blood-corpuscles and
the weight of blood-fluid, we generally proceed in the following manner:

If any substance is found in the blood which belongs exclusively to
the plasma and does not occur in the blood-corpuscles, then the amount of
plasma contained in the blood may be calculated if we determine the
amount of this substance in 100 parts of the plasma or serum respectively
on the one side, and in 100 parts of the blood on the other. If we repre-
sent the amount of this substance in the plasma by p and that in the
blood by b, then the amount of x in the plasma from 100 parts of blood is

100.6

x= .

P

Such a substance, which occurs only in the plasma, is fibrin according
to HOPPE-SEYLER, sodium according to BUNGE (in certain kinds of blood) .
The experimenters just named have tried to determine the amount of the
plasma and blood-corpuscles, respectively, in different kinds of blood,
starting from the above-mentioned substances.

Another method suggested by HOPPE-SEYLER is to determine the
total amount of haemoglobin and proteins in a portion of blood, and on
the other hand the amount of hemoglobin and proteins in the blood-
corpuscles (from an equal portion of the same blood) which have been
sufficiently washed with common-salt solution by centrifugal force. The
figure obtained, as a difference between these two determinations, corre-
sponds to the amount of proteins which was contained in the serum of

1 Hedin, Skand. Arch. f. Physiol., 2, 134 and 361, and 5; Pfliiger's Arch., 60;
Daland, Fortschritte, d. Med., 9.

2 Pfluger's Arch., 107.

328

THE BLOOD.

Water

Solids

Haemoglobin. . .

Protein

Sugar

Chqlesterin. . . .
Lecithin ......

Fat

Fatty acids. , . .

Phosphoric ac^d

as nuclein

Soda

Potash

Iron oxide

Lime

Magnesia. . . .

Chlorine

Phosphoric acid
Inorganic P2O5. .

Pig-blood.

272.20 518.36

162.89

142.20

8.35

0.213
i.504

0.027

46.54

38.26
0.684
0.231
0.805
1.104
0.448

0 0455 0.0123

2.401
0.152

0.0689
0.0656 0.0233
0.642
0.8956 0

2.048
.1114

0.7194 0.0296 0.1140:0.0571

Ox-b'oxl.

192.65 616.25 243.86 551.14
51.15

1.100
1.220

132.85 58.249

103.10
20.89 48.901
0.708
0.835
1.129
0.625

0.0178 0.0089

0.7266 2.9084
0.2351 0.1719
0.544

0.0805
0.0056 0.0300
0.5901 2.4889
0.23920.1646

Horse-blood. Dog-blood. Bull-blood.

88

153.84
125.8
20.05

0.26
1.93

0.02
0.05

1.32
0.59

0.04
0.18
0.98
0.76

.co

II

42.65
0.90
0.31
1.05
0.50
0.36
0.01
2.62
0.15

0.07
0.03
2.20
0.15
0.05

0.03
0.60
0.67
0.54

0.073
0.027
2.453
0.156
0.041

Sheep-blood.

its

O WCC

ll

200.03 624.16
118.82; 56.63
102.80 —
2.80 46.56

— iO.708
1.147 0.891
1.329 11.088

— 0.859

— jO.4908

0.02350.0109

0.760 '2.917

0.236 '0.172

0.545 ! —

— 0.089
0.006 0.027
0.575 ;2.516
0.228 0.163
0.088 0.057

Water

Solids

Haemoglobin. . . .

Protein

Sugar

Chqlesterin

Lecithin

Fat

Fatty acids

Phosphoric acid \
as nuclein f

Soda

Potash

Iron oxide

Lime

Magnesia

Chlorine

Phosphoric acid. .
Inorganic PjOs. . .

Goat-blood.

211.35

135.86

112.50

18.76

0.601
1.339

0.028

0.755
0.236
0.547

0.014
0.514
0.243
0.097

592.54
60.25

50.96
0.822
0.698
1.127
0.0407
0.398

0.0117

2.824
0.160

0.078
0.026
2.409
0.154
0.045

Cat-blood.

270.90
163.11
143.2
11.62

0.556
1.354

0.063

1.174
0.112
0.694

0.035
0.455
0.697
0.515

524.17
41.35

33.16
0.860
0.339
0.971
0.446
0.282

0.009

2.512
0.148

0.062
0.024
2.360
0.133
0.040

Rabbit-blood.

Human Blood,
Man.

235.74

136.37

123.50

4.55

0.268
1.722

0.040

1.946
0.615

0.029
0.460
0.835
0.645

518.18
46.71

33.63
1.036
0.343
1.105
0.749
0.507

0.015

2.789
0.162

0.072
0.028
2.438
0.151
0.040

349.69
163.33

| Organic
!• bodies
| 159.59

Inorg.
3.74

0.24
1.59

0.90

439.02
47.96

43.82

4.14

1.66
0.15

1.72

Human Blood,
Woman.

272.56
123.68

120.13

3.55

0.65
1.41

0.36

551.99
51.77

46.70

5.07

1.92
0.20

0.14

the first portion of blood. If we now determine the proteins in a special
portion of serum of the same blood, then the amount of serum in the
blood is easily determined. The usefulness of this method has been
confirmed by BUNGE by the control experiments with sodium determina-
tions. If the amount of serum and blood-corpuscles in the blood is known,
and we then determine the amount of the different blood-constituents in
the blood-serum on one side and of the total blood on the other, the dis-
tribution of these different blood-constituents in the two chief components

SUGAR IN THE BLOOD. 329

of the blood, plasma and blood-corpuscles may be ascertained. In the
table on page 328 are given analyses of the blood of various animals by
ABDERHALDEN * according to HOPPE-SEYLER'S and BUNGE'S methods.
The analyses of human blood by C. SCHMIDT 2 are older and were made
according to another method, hence the results for the weights of the
corpuscles are perhaps a little too high. All the results are in parts per
1000 parts of blood.

The relation between blood-corpuscles and plasma may vary con-
siderably under different circumstances even in the same species of animal.
In animals, in most cases considerably more plasma is found, some-
times two-thirds of the weight of the blood.3 For human blood ARRONET
has found 478.8 p. m. blood-corpuscles and 521.2 p.m. serum (in defibrinated
blood) as an average of nine determinations. SCHNEIDER 4 found 349.6
and 650.4 p. m. respectively in women.

The sugar was considered as occurring only in the serum and not with
the blood-corpuscles. According to the investigations of RONA and
MICHAELIS the blood-corpuscles of the dog contain considerable amounts
of sugar; and the quantity of sugar in the blood, in the blood-corpuscles
as well as in the plasma, is increased in man with diabetes mellitus.
HoLLiNGER5 also found that in man, with normal quantity of sugar
in the blood, the sugar was distributed almost equally between the
blood-corpuscles and the plasma.

The amount of sugar in the blood-corpuscles, which was shown by
LEPINE and BOULUD before MICHAELIS and RONA, has been the sub-
ject of numerous investigations by BANG and his pupils, LYTTKENS and
SANDGREN on the one hand and by RONA, MICHAELIS, TAKAHASHI,
FRANK and others on the other hand 6. The results of these investiga-
tions are so contradictory that it is hardly possible for the present to
draw any positive conclusions. It seems to follow from them, nevertheless,
that the dog blood-corpuscles always contain sugar, while for the corpuscles
of the rabbit and man the conditions are somewhat doubtful and may
be variable (FRANK and BRETSCHNEIDER) . According to LYTTKENS
and SANDGREN the blood-corpuscles of man contain as maximum 0.06

1 Zeitschr. f. physiol. Chem., 23 and 25.

2 Cited and in part recalculated from v. Gorup-Besanez, Lehrb. d. physiol Chem.,
4. Aufl., 345.

8 See Sacharjin in Hoppe-Seyler's Physiol. Chem., 447; Otto, Pfliiger's Arch.,
35; Bunge, Zeitschr. f. Biol., 12; L. and M. Bleibtreu, Pfliiger's Arch., 61.
4Arronet, Maly's Jahresber., 17; Schneider, Centralbl, f. Physiol., 5, 362.

5 Rona and Michaelis, Bioch. Zeitschr. 16 and 18; Hollinger, ibid., 17.

6 Lepine and Boulud, Bioch. Zeitschr., 32; Lyttkens and Sandgren, ibid., 26, 31,
36; Rona with Doblin, ibid., 31, with Michaelis, ibid., 37, with Takahashi, ibid., 30;
Takahashi, ibid., 37; E. Frank, Zeitschr. f. physiol. Chem., 70, with Bretschneider,
ibid., 71 and 76; see also Oppler, ibid., 64 and 75.

330 THE BLOOD.

p. m. sugar. The blood-corpuscles of the ox, sheep, horse, pig, cat and
guinea-pig do not contain any sugar according to these last-mentioned
investigators. On the contrary the blood-plasma as well as the blood-
corpuscles contain a non-fermentable reducing substance. The quan-
tity of this in the human blood-corpuscles is 0.6 p. m. according to
LYTTKENS and SANDGREN and in the blood-corpuscles of different
animals an average of 0.44-0.8 p. m. calculated as glucose. The quan-
tity of the non-fermentable bodies in the blood-plasma of the animals
investigated by them was 0.3 to 0.5 p. m.

The quantity of glucose in the blood cannot be exactly determined.
As the blood also contains other reducing substances besides glucose the
total reduction naturally cannot be used as an exact value for the glucose
content; and it must also be added that the different methods do not
give uniform results. Thus on using the methods of KNAPP and BANG,
which give the total reduction, higher values are obtained than with
ALLIHN'S or BERTRAND'S methods, in which the quantity of precipitated
cuprous oxide is determined. The polarization method cannot give
exact results because of the presence of other optically active substances
and objections can also be raised against the fermentation method.1
On using this last method Oiro2 first observed, and was substantiated
later by others, namely BANG and his co-workers, that the blood contained
non-fermentable bodies which reduced KNAPP'S (and also BANG'S) solu-
tion. The remaining reduction "rest reduction" after the fermenta-
tion cannot be detected according to BERTRAND'S titration method.

The nature of this reducing but not fermentable substance occurring
in the plasma as well as in the blood-corpuscles is not known. The
assumption of JACOBSEN, BING, and HENRIQUESS that this question-
able substance is jecorin or lecithin sugar does not have sufficient founda-
tion, and the question of the identity with jecorin is doubtful and is con-
nected with the question as to the existence of jecorin at all. The
conjugated glucuronic acids have also been considered and according
to the investigations of MAYER, LEPINE and BOULUD 4 they occur in
blood and originate in the form-elements. For these assumptions we
do not have sufficient support, and especially we have no explanation

1 In regard to methods see Bang, Der Blutzucker, Wiesbaden, 1913 which also
describes a new method suggested by him for the determination of sugar in very small
amounts of blood.

2 Pfluger's Arch., 35.

3 Jacobson, Centralbl. f. physiol. 6; Bing, Skand. Arch. f. physiol., 9; Henriques,
Zeitschr. f. physiol Chem., 23. See also P. Mayer, Bioch. Zeitschr., 1 and 4.

4 Mayer, Zeitschr. f. physiol. Chem. ,'32; Lepine and Boulud, Compt. Rend., 133,
135, 136, 138, 141 and Journ. de Physiol., 7 (cited from Bioch. Centralbl., 4, page 421).

SUGAR IN THE BLOOD. 331

for the total rest reduction. FRANK and BRETSCHNEIDER 1 have, never-
theless, shown that the reducing substance or mixture that occurs in the
blood-corpuscles, and which does not reduce BERTRAND'S solution, but
does reduce BANG'S solution, yields a reduceable sugar on boiling with acid
which now reduces BERTRAND'S solution. The corresponding substance
in the blood-plasma has a similar behavior. If, as in the experiments
of .FRANK and BRETSCHNEIDER, the extent of reduction after acid hydrol-
ysis is about the same as the original substance (titrated according to
BANG) we cannot here be dealing with dextrins and the nature of this
body in question (or mixture) is quite unknown.

In close relation to what has been given above is the question of
" sucre immediat " and the " sucre virtuel " of LEPINE and BouLUD.2
They designate as " sucre immediat " the reduction, calculated as
sugar, of the blood immediately after leaving the blood vessels and as
" sucre virtuel " the increase in the reducing power brought on in part
by allowing the blood to stand after leaving the body, in part by the
action of invertase or emulsin at 39° C. and in part by boiling with hydro-
fluoric acid. The quantity of " sucre virtuel " in dogs amounts to an average
of 70 per cent of the "sucre immediat." The nature of the " sucre virtuel"
is not well known; from what was said above we are probably dealing here
to all appearances with very different bodies.

From what has been presented above it can be understood why the
exact sugar content of the blood is not known. In consideration of the
above mentioned difficulties and sources of error attempts have been made
to determine the sugar content of the blood and we will give the results
of some of these.

The quantity of actual sugar in the blood, amounts according to LYTT-
KENS and SANDGREN, in man to 0.63, in sheep 0.64, pig 0.82, ox 0.86,
horse 0.98, rabbit 2.22, guinea-pig 2.48 and in the cat 2.91 p. m. Small
animals with an active metabolism contain more sugar in the blood
than larger animals. According to FRANK the amount of sugar in the
blood-plasma of man lies between 0.8 and 1.1 p. m. and according to
FRANK and COBLINERS it is 1.19-1.26 p. m. in new-born.

The amount of blood sugar seems to be almost independent of the
character of the food. After feeding with large amounts of sugar or dex-
trin, BLEILE, nevertheless, has observed a considerable increase in the
sugar. The amount of sugar is not only somewhat different with
various animals but it also varies with the same animal under different

1 Zeitschr. f. physiol. Chem., 71 and 76.

2 Compt. Rend., 137, 144, 147, and Journ de Physiol. et d. Path., 11 and 13.

3 Lyttkens and Sandgren, Bioch. Zeitschr., 36; Frank and Cobliner, Zetischr. f.
physiol. Chem., 70.

332 THE BLOOD.

external conditions. When it amounts to more than 3 p. m., according
to a statement of Cl. BERNARD,1 sugar appears in the urine and a gly-
cosuria occurs, a view that has not been substantiated. On the one
hand a glycosuria may occur at a lower sugar content in the blood and
on the other hand a glycosuria may be absent for a time with a higher
sugar content. An increase in the sugar content occurs, as first shown
by BERNARD and subsequently proved by others, after drawing blood.
In this case not alone is the quantity of sugar increased but also the other
reducing substances. According to certain investigators the quantity
of these latter is especially increased (HENRIQUES, N. ANDERSON, LYTTKENS
andSANDGREN, LEPINE and BouLUD2).

BERNARD3 has shown that the quantity of sugar in the blood
diminishes more or less rapidly on leaving the veins. LEPINE, associated
with BARRAL, has specially studied this decrease in the quantity of
sugar, and calls it glycolysis. LEPINE and BARRAL, as well as ARTHUS,
have shown that this glycolysis takes place in the complete absence of
micro-organisms. It seems to be due to a soluble glycolytic enzyme whose
activity is destroyed by heating to 54° C. This enzyme is derived,
according to the above investigators, from the leucocytes and, accord-
ing to ARTHUS as well as to Do YON and MOREL 4 it occurs only in the
serum but not in the plasma. According to LEPINE,5 it has some con-
nection with the pancreas. The glycolysis is, according to ROHMANN
and SPITZER and SIEBER, an oxidation which is produced, according
to the two last-mentioned investigators, by an oxidation ferment. Accord-
ing to RONA and DOBLIN it takes place in an atmosphere of hydrogen,
which does not speak for the above view. The recent investigations of
SLOSSE, of EMBDEN and collaborators KRASKE, KONDO and K. v. NOORDEN 6

1 Bleile, Arch. f. (Anat. u.) Physiol., 1879; Bernard, Lecons sur le diabete.

2 Henriques, Zeitschr. f. physiol. Chem., 23, N. Anderson, Bioch. Zeitschr., 12;
Lyttkens and Sandgren, ibid., 26; Lepine and Boulud, Journ. de Physiol., 13.

3 Legons sur le diabete, Paris, 1877.

4 Arthus, Arch, de Physiol. (5), 3; Doyon and Morel, Compt. rend soc. biol., 55.

6 In regard to the numerous memoirs of Lepine and L6pine and Barral, see Lyon
medical., 62 and 63 ; Compt Rend. 110, 112, 113, 120 and 139; Lepine, Le ferment
glycolytique et la pathogenic du diabete (Paris, 1891), and Revue analytique et
critique des travaux, etc., in Arch, de me"d. exp6r. (Paris, 1892); Revue de medecine
1895; Etat actuel de la question de la glycolyse, Semaine medicale, 1911; Arthus,
Arch, de Physiol (5), 3, 4; Nasse and Framm, Pfliiger's Arch., 63, Paderi, Maly's
Jahresber., 26; see also Cremer, Physiologic des Glykogens in Ergebnisse d. Physiol.,
1, Abt. 1.

6 Rohmann and Spitzer, Ber. d. d. chem. Gesellsch., 28; Spitzer, Pfliiger's Arch.,
60 and 67; Sieber, Zeitschr. f. physiol Chem., 39 and 44; Rona and Doblin, Bioch.
Zeitschr., 32; Slosse, Arch, internat. de Physiol., 11; Kraske, Kondo, and v. Noorden
Bioch. Zeitschr., 45.

i LACTIC ACID FORMATION FROM SUGAR. 333

speak positively for the statement that in glycolysis a formation of lactic
acid from the sugar occurs.

That a formation of lactic acid from glucose, and indeed by means of
the leucocytes, takes place in glycolysis was shown by LEVENE and MEYER
before EMBDEN and collaborators. On continuing these investigations
LEVENE and MEYER found that fructose as well as mannose and galactose
under the same conditions with leucocytes, yield d-lactic acid while with
the investigated pentoses, arabinose and xylose, this is not the case. Accord-
ing to EMBDEN and co-workers, this formation of lactic acid takes place
probably with glyceric aldehyde, and perhaps also with small amounts
of dioxyacetone, as intermediary steps, and a formation of lactic acid from
glyceric aldehyde (and dioxyacetone) can in fact, as A. LOEB and GRIES-
BACH l have shown, be brought about by enzymotic means by the form-
elements of the blood. It seems as if several enzymes were active in the
formation of lactic acid from glucose. According to LOEB those varieties
of blood which show no glycolysis with the formation of lactic acid, or
none worth mentioning, can form lactic acid from glyceric aldehyde
and according to GRIESBACH in this last -mentioned process an enzyme
is active which is soluble in water and resistant toward the haemolysis
of the blood with water, while the action of the blood upon glucose is
destroyed in the destruction of the form-elements by haemolysis. In
regard to the formation of lactic acid from methyl glyoxal see page 584.
According to LEPINE and BOULUD a double process takes place in the
glycolysis. On one side the sugar is destroyed and on the other side
a re-formation of sugar from the "sucre virtuel" takes place. Hereby the
actual glycolysis may be greater than the visible, and the mentioned
investigators have therefore suggested a method for determining the
extent of the actual glycolysis.2

The quantity of urea, which, according to SCHONDORFF, is equally
divided between the blood-corpuscles and the plasma, is greater on tak-
ing food than in starvation (GREHANT and QUINQUAUD, SCHONDORFF)
and varies between 0.2 and 1.5 p. m. In dogs SCHONDORFF found in
starvation a minimum of 0.348 p. m. and a maximum of 1.529 p. m. at
the point of highest urea formation. GOTTLIEB obtained much lower
results by another direct method, namely, in starvation 0.1-0.2, and
after meat feeding 0.28-0.56 p. m., FOLIN and DENIS found 0.3-0.77
p. m. in the blood of the cat. In man v. JAKSCHS found 0.5-0.6 p. m.

1 Levene and Meyer, Journ. of biol. Chem., 11 and 14; A. Loeb, Bioch. Zeitschr.
49 and 50; Griesbach, ibid., 50.

2 Lepine and Boulud, Journ. de Physiol., et de Path, generate, 13.

3 Grehant et Quinquaud, Journ.de 1'anatomie et de la physiol., 20, and Compt.
Rend., 98; Schondorff, Pfliiger's Arch., 54 and 63; Gottlieb, Arch. f. exp. Path. u.
Pharm., 42; Fob'n and Denis, Journ. of biol. Chem., 11 and 12; v. Jaksch, Leyden-
Fetschr., I, 1901.

334 THE BLOOD.

urea in normal blood. The quantity of urea is somewhat increased in
fever, and in general in augmented protein metabolism the increased
urea formation is dependent upon this. A more important increase in the
quantity of urea in the blood occurs in a retarded elimination of urea,
as in cholera, also in cholera infantum, and in infections of the kidneys
and urinary passages. After ligaturing the ureters or after extirpation
of the kidneys of animals, an accumulation of urea takes place in the
blood.

v. SCHRODER first showed that the blood of the shark was very rich
in urea, and the quantity indeed amounted to 26 p. m. BAGLIONI 1
has recently shown that this large quantity of urea is of the greatest
importance, as the presence of urea in these animals is a necessary life-
condition for the heart and very probably for all organs and tissues.

The blood also contains traces of ammonia. According to HORODYN-
SKI, SALASKIN, and ZALESKi,2 the quantity in arterial dog-blood was
0.41 milligram in 100 grams of blood. According to WINTERBERG,S the
blood from healthy persons contains on an average 0.90 milligram per
100 cc.3 The .quantity of uric acid may be 0.1 p. m. in bird's
blood (v. SCHRODER4). Uric acid has only recently been positively
detected under normal conditions, while it has been found, earlier,
in the blood in gout, croupous pneumonia, and certain other diseased
conditions. FOLIN and DENIS 5 have determined the uric acid in the
blood of certain animals as well as in man by a colorimetric method
suggested by FOLIN. Normal human blood contains not less than 1
to 2-2.5 milligrams uric acid per 100 grm.; in gout they found 5.5
milligrams as maximum. They also determined the quantity of total
non-protein nitrogen and urea nitrogen in human blood. In normal
blood the first was equal to 22-26 milligrams and the last equal to 11-13
( = 24-28 urea) milligrams in 100 grams of blood.. In disease great varia-
tions were found. Lactic acid was first found in human blood by
SOLOMON and then by GAGLIO, BERLINERBLAU, and IRISAWA. The
quantity of lactic acid may vary considerably. BERLINERBLAU found
0.71 p. m. as maximum, in dog's blood. SAITO and KATSUYAMA 6 found
on an average 0.269 p. m. in hen's blood, and after carbon-monoxide
poisoning the quantity increased to 1.227 p. m. Fat and fatty acids occur

1 v. Schroder, Zeitschr. f. physiol. Chem., 14; Baglioni, Centralbl. f. Physiol., 19.

2 Zeitschr. f. physiol. Chem., 35, which also gives the older literature. t

3 Wien. klin. Wochenschr., 1897, and Zeitschr. f. klin. Med., 35.

4 Ludwig's Festschrift, 1887.

6 Journ. of biol. Chem., 13 and 14.

6Irisawa, Zeitschr. f. physiol. Chem., 17, which also gives the older literature;
Saito and Katsuyama, ibid., 32.

BLOOD IN DIFFERENT VASCULAR REGIONS. 335

perhaps only in the serum. The small traces of bile acids occurring in
normal blood, according to CROFTAN,1 are contained in the leucocytes.

The calcium occurs, with the exception perhaps of the blood cor-
puscles of the ox, only in the plasma and the same applies at least for
the principal part of the magnesium. The division of the alkali between
the blood-corpuscles and the plasma is very different, namely, the blood-
corpuscles of the pig, horse and rabbit contain no sodium, the human
corpuscles are richer in potassium and those of the ox, sheep, goat, dog
and cat are much richer in sodium than potassium. Chlorine occurs in
greater abundance in the serum of all animals than in the blood-corpuscles.
The iodine only occurs in serum, while iron regularly, almost without ex-
ception occurs in the form-elements, especially in the erythrocytes. As the
nucleoproteins contain iron, some iron occurs in the leucocytes and traces
of iron also occur in the serum. This quantity is very small under normal
conditions while in disease the relationship between the haemoglobin-
iron and the other blood-iron may, it seems, changes very distinctly.
Manganese has also been found in the blood, as well as traces of lithium
copper, lead, silver, and also arsenic in menstrual blood. The entire
blood contains in ordinary cases 770-820 p. m. water with 180-230
p. m. solids, among these 173-220 p. m. are organic and 6-10 p. m.,
inorganic. The organic consist, after substracting 6-12 p. m. extractives,
of protein and haemoglobin. The quantity of the latter in man is 130-
150 p. m. In the dog, cat, pig and horse the hemoglobin content is
about the same; in ox, bull, sheep, goat and rabbit blood it is lower
(ABDERHALDEN).

The Composition of the Blood in Different Vascular Regions and under

Different Conditions.

Arterial and Venous Blood. The most striking difference between
these two kinds of blood is the variation in color caused by their con-
taining different amounts of gas and different amounts of oxy haemoglobin
and haemoglobin. The arterial blood is light red; the venous blood is
dark red, dichroic, greenish by transmitted light through thin layers.
The arterial coagulates more quickly than the venous blood. The latter,
on account of the transudation which takes place in the capillaries, was
formerly said to be somewhat poorer in water but richer in blood-cor-
puscles and haemoglobin than the arterial blood; but this is denied by
modern investigators. According to KRVGER 2 and his pupils the quan-

1 Pfliiger's Arch., 90.

2 Zeitschr. f . Biologie, 26. This also gives the literature on the composition of
the blood in different vascular regions.

336 THE BLOOD.

tity of dry residue and haemoglobin in blood from the carotid artery and
from the jugular vein (in cats) is the same. ROHMANN and MUHSAM
could not detect any difference in the quantity of fat in arterial and venous
blood. The serum from dog's blood has, according to WIENER/ a rela-
tively higher globulin content relative to the albumin in the venous blood
as compared with the arterial blood.

Blood from the Portal Vein and the Hepatic Vein. In consequence of
the small quantities of bile and lymph formed relatively to the large
quantity of blood circulating through the liver in a given time, we can
hardly expect to detect by chemical analysis a positive difference in the
composition between the blood of the portal and hepatic veins. The
statements in regard to such a difference are in fact contradictory. For
example, DROSDOFF found more haemoglobin in the hepatic than in
the portal vein, while OTTO found less. KriioER finds that the quantities
of haemoglobin, as well as of the solids, in the blood from the vessels
passing to and from the liver are different, but a constant relation can-
not be determined. The hepatic vein, according to Do YON and collab-
orators,2 is richer in fibrinogen than the blood from the portal vein.
The disputed question as to the varying quantities of sugar in the por-
tal and hepatic veins will be discussed in a following chapter (see Chap-
ter VII, on the formation of sugar in the liver). After a meal rich in
carbohydrates, the blood of the portal vein not only becomes richer
in glucose, but may also contain dextrin and other carbohydrates (v.
MERINO, OiTO3). The amount of urea in the blood from the hepatic
vein is greater than in other blood (GREHANT and QUINQUAUD). In
portal blood FOLIN and DENIS found about the same amount of urea
as in the carotid blood. Like HORODJNSKI, SALASKIN and ZALESKi,4
they found that the portal blood was richer in ammonia than the carotid
blood. The largest amount of ammonia was always found in the blood
from the mesentery vessels of the large intestine.

Blood of the Splenic Vein is decidedly richer in leucocytes than the
blood from the splenic artery. The red blood-corpuscles of the blood
from the splenic vein are smaller than the ordinary, are less flattened, and
show a greater resistance to water. The blood from the splenic vein is
also claimed to be richer in water, fibrin, and protein than the ordinary
venous blood. According to v. MIDDENDORFF, it is richer in haemoglobin

1 Rohmann and Miihsam, Pfliiger's Arch., 46; Wiener, Zeitschr. f. physiol. Chem.,
82.

2 See footnote 2, page 253.

3 Drosdoff, Zeitschr. f. physiol. Chem., 1; Otto, Maly's Jahresber, 17; v. Mering,
Arch. f. (Anat. u.) Physiol., 1877, 214.

4 Grehant et Quinquaud, 1. c. footnote 3, page 333; Folin and Denis, Journ. of biol.
Chem., 11 and 12; Horodjnski, Salaskin and Zaleski, Zeitschr. f. physiol. Chem., 35.

BLOOD OF THE TWO SEXES. 337

than arterial blood. KRUGER l and his pupils found that the blood
from the vana lienalis is generally richer in haemoglobin and solids than
arterial blood; still the contrary is often found. The blood from the
splenic vein coagulates slowly.

The Blood from the Veins of the Glands. The blood circulates with
greater rapidity through a gland during activity (secretion) than when
at rest, and the outflowing venous blood has therefore during activity a
lighter red color and a greater amount of oxygen. Because of the secre-
tion, the venous blood also becomes somewhat poorer in water and richer
in solids.

The blood from the Muscular Veins shows an opposite behavior, for
during activity it is darker and more venous in its properties because
of the increased absorption of oxygen by the muscles and still greater
production of carbon dioxide than when at rest.

Menstrual Blood, according to an old belief, has not the power of
coagulating. This statement, is nevertheless, false, and the apparent
uncoagulability depends in part on the retarding action of the mucous
membrane of the uterus upon coagulation (CRISTEA and DENK 2) and in
part on a contamination with vaginal mucus, which disturbs the coagula-
tion. Menstrual blood, according to GAUTIER and BOURCET, contains
arsenic and is also richer in iodine than other blood (see Blood-serum,
page 269).

The Blood of the Two Sexes. Women's blood coagulates somewhat
more quickly, has a lower specific gravity, a greater amount of water,
and a smaller quantity of solids than the blood of man. The amount
of blood-corpuscles and haemoglobin is somewhat smaller in woman's
blood. The amount of haemoglobin is 146 p. m. for man's blood and 133
p. m. for woman's.

During pregnancy NASSE has observed a decrease in the specific gravity,
with an increase in the amount of water, until the end of the eighth month.
From then the specific gravity increases, and at delivery it is again
normal. The amount of fibrin is somewhat increased (BECQUEREL and
RODIER, NASSE). The number of blood-corpuscles seems to decrease.
In regard to the amount of haemoglobin the statements are somewhat
contradictory. COHNSTEIN found the number of red corpuscles diminished
in the blood of pregnant sheep as compared with non-pregnant, but
the red corpuscles were larger and the quantity of haemoglobin in the blood
was greater in the first case. MOLLENBERG found in most cases an
increase in the amount of haemoglobin in pregnancy in the last months,

1 v. Middendorff, Centralbl. f. Physiol, 2, 753; Kruger, 1. c.

2 Cristea and Denk, Maly's Jahresb., 40, 181.

338 THE BLOOD.

and according to HERMANN and NAUMANN 1 an increase in the cholesterin
ester and the neutral fats occurs in the blood during pregnancy.

The Blood at Different Periods of Life. Fetal and infant blood is
richer in erythrocytes and haemoglobin than the blood of the mother.
In animals this is true at least for the haemoglobin while the number
of erythrocytes in growing or adult animals may be greater than in new-
born animals. The highest percentage of haemoglobin in the blood has
been observed by several investigators, such as COHNSTEIN and ZUNTZ,
OTTO, WINTERNITZ, ABDERHALDEN, SCHWINGE, and others, immediately
or very soon after birth or at least within the first few days. In man
two or three days after birth the haemoglobin reaches a maximum (200-
210 p. m.) which is greater than at any other period of life. This is the
cause of the great abundance of solids in the blood of new-born infants,
as observed by several investigators. The quantity of haemoglobin and
blood-corpuscles sinks gradually from this first maximum to a minimum
of about 110 p. m. haemoglobin, which minimum appears in human beings
between the fourth and eighth years. The quantity of haemoglobin then
increases again until about the twentieth year, when a second maximum
of 137-150 p. m. is reached. The haemoglobin remains at this point
only to about the forty-fifth year, and then gradually and slowly
decreases (LEICHTENSTERN, OTTO2). According to earlier reports, the
blood at old age is poorer in blood-corpuscles and protein bodies, but
richer in water and salts.

The Influence of Food on the Blood. In complete starvation no
decrease in the amount of solid blood-constituents is found to take place
(PANUM and others). The amount of haemoglobin is increased a little,
at least in the early period (SUBBOTIN, OTTO, HERMANN and GROLL,
LUCIANI and BUFALINI), and also the number of red blood-corpuscles
increases (WORM MULLER, BUNTZENS), which probably depends partly
on the fact that the blood-corpuscles are not so quickly transformed as
the serum and partly on a greater concentration due to loss of water.

1 Nasse, Maly's Jahresber., 7; Becquerel and Rodier, Traite de chim. pathol.,
Paris, 1854; Cohnstein, Pfliiger' Arch., 34, 233; Mollenberg, Maly's Jahresber., 31,
185. See also Payer, Arch. f. Gynak., 71; Herrmann and Naumann, Bioch. Zeitschr.,
43.

2 Cohnstein and Zuntz, Pfliiger's Arch., 34; Winternitz, Zeitschr. f. physiol. Chem.,
22; Leichtenstern, Untersuch. iiber. den Hamoglobingehalt des Blutes, etc., Leipzig,
1878; Otto, Maly's Jahresber., 15 and 17; Abderhalden, Zeitschr. f. physiol. Chem.,
34; Schwinge, Pfliiger's Arch., 73 (literature). See also Fehrsen, Journ. of Physiol.,
3<K

3 Panum, Virchow's Arch., 29; Subbotin, Zeitschr. f. Biologic, 7; Otto. 1. c.,
Worm Miiller, Transfusion und Plethora, Christiania, 1875; Buntzen, see Maly's
Jahresber., 9; Hermann and Groll, Pfliiger's Arch., 43; Luciani and Bufalini, Maly's
Jahresber., 12.

INFLUENCE OF FOOD ON THE BLOOD. 339

In rabbits and to a less extent in dogs, POPEL found that complete absti-
nence had a tendency to increase the specific gravity of the blood. The
amount of fat in the blood may be somewhat increased in starvation
because the fat is taken up from the fat deposits and carried to the various
organs by the blood (N. SCHULZ, DADDI 1).

After a rich meal, or after secretion of digestive juices or absorption
of nutritive liquids, the relative number of blood-corpuscles may be
increased or diminished (BUNTZEN, LEICHTENSTERN) . The number of
white blood-corpuscles may be considerably increased after a diet rich
in proteins. After a diet rich in fat the plasma becomes, even after a
short time, more or less milky-white, like an emulsion. According to
JUST, in rabbits, on the contrary, the various food-stuffs such as carbohy-
drate, fat and protein or peptone has no influence on the number of red
and white corpuscles, which he considers as a proof for the difference
between the digestive processes in carnivora and herbivora (rabbits).
The composition of the food acts essentially on the amount of haemo-
globin in the blood. SUBBOTIN has observed in dogs after. a one-sided
feeding with food rich in carbohydrates that the amount of haemoglobin
sank, from the physiological average of 137.5 p. m. to 103.2-93.7 p. m.
TSUBOI 2 has also shown in experiments on rabbits and dogs that with
an insufficient diet of bread and potatoes, where the body gave up pro-
tein and contained relatively considerable carbohydrate, the amount
of haemoglobin decreased and the blood became richer in water. Accord-
ing to LEICHTENSTERN, a gradual increase in the amount of haemoglobin
is found to take place in the blood of human beings on enriching the food,
and according to the same investigator the blood of lean persons is gen-
erally somewhat richer in haemoglobin than blood from fat ones of the
same age. The addition of iron salts to the food greatly influences
the number of blood-corpuscles and especially the amount of haemoglobin
they contain. The action of the iron salts is obscure.3 There does not
seem to be any doubt that the iron contained in the food in the form
of organic compounds is active, but also iron salts and therapeutic iron.
According to BUNGE and his pupils the iron preparations act indirectly
only. They may combine with the sulphureted hydrogen of the intes-
tinal canal and thereby prevent the iron associated in the food as assim-

1 Popel, Arch, des scienc. biol. de St. Petersbourg, 4, 354; Schulz, Pfliiger's Arch.,
65; Daddi, Maly's Jahresber., 30.

2 Just, Centralbl. f. Physiol., 23; Subbotin, 1. c.; Tsuboi, Zeitschr. f. Biologie, 44.

3 See Bunge, Zeitschr. f. physiol. Chem., 9; Hausermann, ibid., 23, where the
works of Weltering, Gaule, Hall, Hochhaus, and Quincke are cited (the same work
contains a table of the quantity of iron in various foods); Kunkel, Pfliiger's Arch.,
61; Macallum, Journal of Physiol., 16; Abderhalden, Zeitschr. f. Biologie, 39.

340 THE BLOOD.

liable protein compounds from being eliminated as iron sulphide (BUNGE),
a view which is now generally discarded.

An increase in the number of red corpuscles, takes place after trans-
fusion of blood of the same species of animal. According to the observa-
tions of PANUM and WORM MULLER/ the blood-liquid is quickly eliminated
and transformed in this case — the water being eliminated principally
by the kidneys and the protein burned into urea, etc. — while the blood-
corpuscles are preserved longer and cause a " polycythcemia." A relative
increase in the number of red corpuscles is found after abundant transuda-
tion from the blood, as in cholera and heart-failure with considerable
congestion. An increase in the number of red blood-corpuscles has
also been observed under the influence of diminished pressure or in high
altitudes. VIAULT first called attention to the fact that the number of
red corpuscles was very great in the blood of man and animals living
in high regions. According to him the llama has about 16 million blood-
corpuscles per cubic millimeter. By observations on himself and others,
as well as on animals, VIAULT found the first effect of sojourning in high
altitudes was a very considerable increase in the number of red corpuscles,
in his own case 5-8 millions. In a young man residing for four weeks
in 2900 meters altitude, LACQUEUR observed an increase in the erythro-
cytes as well as the haemoglobin in the unit volume of the blood. The
maximum, which appeared first after 15 days, was 15 per cent increase
for the erythrocytes and 16 per cent for the haemoglobin. He also
found that dogs from whom about one-half of the blood was drawn required
at 2900 meters altitude, on an average of 16 days to replace the same,
while at the normal level this requires on an average of 27 days or in
round numbers an increase of 70 per cent. Both observations show
a re-formation of blood under the influence of high altitude. COHNHEIM
and WEBER 2 found in 23 men engaged on the Jungfrau railroad, who had
lived for a long time in high altitudes that the number of erythrocytes
(5.2-6.3 million) as well as the haemoglobin (generally 87-94 as com-
pared to 80 per cent as normal) was higher than under normal conditions,
and they consider this as a proof for the actual formation of blood-
corpuscles in high altitudes. A similar increase of the red blood-cor-
puscles, as also an increase in the quantity of haemoglobin under the
influence of diminished pressure, has been observed by many other inves-
tigators, in human beings as well as in animals. Investigators are not
united as to how this increase is brought about. The increase in the
blood-corpuscles is not absolute, but is only relative, and it is considered
by several observers that there is neither a new formation nor a dimin-

1 Panum, Virchow's Arch., 29; Worm Miiller, 1. c.

2 Lacqueur, Deutsch. Arch. f. klin. Med., 110; Cohnheim and Weber, ibid., 110.

VARIATIONS IN THE NUMBER OF RED-CORPUSCLES. 341

ished destruction of the blood-corpuscles. A relative increase may be
brought about in different ways. For example, another division of the
blood-corpuscles in the vascular system has been supposed, whereby
the blood-corpuscles accumulate in the capillaries, from which region
the blood has been examined most often (ZUNTZ). It is also claimed
that a concentration of the blood takes place by increased evaporation
(GRAWITZ), and finally an increase in the blood-corpuscles has also been
explained by assuming a contraction of the vascular system with the
pressing out of plasma (BUNGE, ABDERHALDEN *). In connection with
these experiments, it must be remarked that several trustworthy observa-
tions show that under the influence of diminished blood-pressure an
actual increase in the red blood-corpuscles takes place. These and
especially those of ZUNTZ and his co-workers have shown that under
these conditions an increased activity occurs in the red bone-marrow.
This question is still not clear. COHNHEIM and collaborators2 have
observed in man and dogs, that no essential increase in the blood-corpuscles
and haemoglobin occurs in high altitudes after 12 days. They do not
dispute the action of a continued residence in high altitudes, and they also
do not dispute such an action upon rabbits and mice. They explain
this in these animals by a concentration of the blood due to a loss of water
which is not replaced. In man and dogs on the contrary the loss of
water brought about by perspiration is immediately replaced and the
concentration of the blood prevented and the increase in the number
of blood-corpuscles and of haemoglobin is not observed.

A decrease in the number of red corpuscles occurs in anaemia from differ-
ent causes. Every excessive hemorrhage causes an acute anaemia, or, more
correctly, oligaemia. Even during the hemorrhage, the remaining blood
becomes by diminished secretion and excretion, as also by an abundant
absorption of parenchymous fluid, richer in water, somewhat poorer in
proteins, and strikingly poorer in red blood-corpuscles. The oligaemia
soon passes into an hydraemia. The amount of protein then gradually
increases again; but the re-formation of the red blood-corpuscles is slower,
and after the hydraemia follows also an oligocythaemia. After a little
time the number of blood-corpuscles rises to normal. INAGAKIS has
made thorough investigations on the changes which the number, volume
and haemoglobin content of the erythrocytes undergo after drawing blood
as well as during regeneration. It is impossible here to enter more in

1 The literature on this subject may be found in Abderhalden, Zeitschr. f. Biologic,
43; van Voornveld, Pfliiger's Arch., 92.

2 Hohenklima und Bergwanderungen, by N. Zuntz, A. Loewy, Franz Miiller, and
W. Caspari, Berlin, 1906; Otto Cohnheim, G. Kreglinger, L. Tobler, O. H. Weber,
Zeitschr. f. physiol. Chem., 78 and Cohnheim, Ergebn. d. Physiologic, 1912, 12.

3 Zeitschr. f. Biol., 49.

342 THE BLOOD.

detail as to the results, but simply to state that they substantiate the
previously known observation that, during regeneration, irregularties
may occur in the relation between the quantity of haemoglobin and the
number of erythrocytes. A considerable decrease in the number of red
corpuscles also occurs in chronic anaemia and chlorosis; still in such cases
an essential decrease in the amount of haemoglobin occurs without an
essential decrease in the number of blood-corpuscles. The decrease in the
amount of haemoglobin is more characteristic of chlorosis than a decrease
in the number of red corpuscles. The opinions on the changes in the
blood in anaemia and chlorosis differ very considerably.1

A very considerable decrease in the number of red corpuscles (300,000-
400,000 in 1 c.mm.) and diminution in the amount of haemoglobin
(J-TV) occurs in pernicious anaemia (HAYEM, LAACHE, and others2).
On the contrary, the individual red corpuscles are larger and richer in
haemoglobin than they ordinarily are, and the number stands in an inverse
relation to the amount of haemoglobin (HAYEM). Besides this the red
corpuscles often, but not always, show in pernicious anaemia remarkable
and extraordinary irregularities of form and size, which has been termed
poikilocytosis.

The number of leucocytes may, as stated above, be increased under
physiological conditions as well as after a meal rich in protein (physiological
leucocytosis) . Under pathological conditions a high leucocytosis may
occur, and this is especially found in leucaemia, which is characterized
by a very great abundance of leucocytes in the blood. The number of
leucocytes is markedly increased in this disease, and indeed, not only
absolutely, but also in relation to the number of red blood-corpuscles,
which are diminished to a considerable extent in leucaemia. Leucaemic
blood has a lower specific gravity than the ordinary blood (1035-1040),
and a paler color, as if it were mixed with pus. The reaction is alkaline,
but after death it is frequently acid, probably due to a decomposition
of lecithin, which is often considerably increased in leucaemia. Volatile
fatty acids, lactic acid, glycero-phosphoric acid, large amounts of purine
bases, and so-called CHARCOT'S crystals (see Semen, Chapter XII) have
also been found in leucaemic blood. The peptone (proteose) which is
found in the leucaemic blood after death, and which does not exist in
the fresh blood, is, according to ERBEN, 3 a digestive product which is

1 Complete analyses of chlorotic blood may be found in Erben, Zeitschr. f. klin.
Med., 47.

2 Laache, Die Anamie (Christiania, 1883), which also contains the older liter-
ature. A complete chemical analysis of the blood has been made by Erben, Zeitschr.
f. klin. Med., 40.

3 Erben, Zeitschr. f. Heilkunde, 24, and Hofmeister's Beitrage, 5. See also Schumm,
ibid., 4 and 5. See also footnote 3, page 342.

QUANTITY OF BLOOD. 343

produced by a tryptic enzyme which originates from the leucocytes
as well as by traces of a peptic enzyme. A chemical analysis of leucaemic
blood has been made by ERBEN.1

A great number of investigations have been made on the chemical
composition of blood in disease. But as we have only a few analyses
of the blood of healthy individuals, and as the possible variations under
physiological conditions are little known, it is difficult to draw any pos-
itive conclusions from the analyses of pathological blood. Unfortunately,
on account of the large number of contradictory deductions concern-
ing the composition of the blood of diseased human beings, it is impossible
to give a brief summary of the results, still the changes in the blood in
disease must be of the greatest importance.

The quantity of blood is indeed somewhat variable in different species
of animals and in different conditions of the body; in general we consider
the entire quantity of blood in adults as about ArA of the weight of the
body, and in new-born infants about yV- HALDANE and LORRAIN SMITH,2
who have determined the quantity of blood by a new method, find in
fourteen persons that it varies between -^ and ^ of the weight of the body.
According to the same method OERUMS has determined the quantity
of blood in men as about iV and in woman -fa of the weight of the body.
Fat individuals are relatively poorer in blood than lean ones. During
inanition the quantity of blood decreases less quickly than the weight
of the body (PANUM 4) , and it may therefore be also proportionally greater
in starving individuals than in well-fed ones.

By careful bleeding, the quantity of blood may be considerably dimin-
ished without any dangerous symptoms. A loss of blood amounting
to one-fourth of the normal quantity has as a sequence no lasting sink-
ing of the blood-pressure in the arteries, because the smaller arteries
accommodate themselves to the small quantities of blood by contract-
ing (WORM MULLER 5) . A loss of blood amounting to one-third of
the quantity reduces the blood-pressure considerably, and a loss of
one-half of the blood in adults is dangerous to life. The more rapid the
bleeding the more dangerous it is. New-born infants are very sensitive
to loss of blood, and likewise fat, old, and weak persons cannot stand
much loss of blood. Women can stand loss of blood better than men.

The quantity of blood may be considerably increased by the injection
of blood from the same species of animal (PANUM, LANDOIS, WORM

iZeitschr. f. klin. Med., 66 (1908).

2 Journ. of Physiol., 25.

3 Deutsch. Arch. f. klin. Med., 93 (1908).

4 Virchow's Arch., 29.

6 Transfusion und Plethora, Christiania, 1875.

344 THE BLOOD.

MULLER, PONFICK). According to WORM MULLER the normal quantity
of blood may indeed be increased as much as 83 per cent without pro-
ducing any abnormal conditions or lasting high blood-pressure. An
increase of 150 per cent in the quantity of blood may, with a considerable
variation in the blood-pressure, be directly dangerous to life (WORM
MULLER). If the quantity of blood of an animal is increased by trans-
fusion with blood of the same kind of animal, an abundant formation
of lymph takes place. The water in excess is eliminated by the urine;
and as the protein of the blood-serum is quickly decomposed, while the
red blood-corpuscles are destroyed much more slowly (TSCHIRJEW, FORS-
TER, PANUM, WORM MULLER x), a polycythsemia is gradually produced.
The quantity of blood in the different organs depends essentially on
their activity. During work the exchange of material in an organ is
more pronounced than during rest, and the increased metabolism is
connected with a more abundant flow of blood. Although the total
quantity of blood in the body remains constant, the distribution of the
blood in the various organs may be different at different times. As a
rule the quantity of blood in an organ is an approximate measure of the
more or less Active metabolism going on in it, and from this point of
view the distribution of the blood in the different organs is of interest.
According to RANKED to whom we are especially indebted for our knowl-
edge of the relation of the activity of the organs to the quantity of blood
contained therein, of the total quantity of blood (in the rabbit) about
one-fourth comes to the muscles in rest, one-fourth to the heart and the
large blood-vessels, one-fourth to the liver, and one-fourth to the other
organs.

1Panum, Nord. med. Ark., 7; Virchow's Arch., 63; Landois, Centralbl. f. d,
med. Wissensch., 1875, and Die Transfusion des Blutes, Leipzig, 1875; Worm M tiller,
Transfusion und Plethora; Ponfick, Virchow's Arch., 62; Tschirjew, Arbeiten aus
der physiol. Anstalt zu Leipzig, 1874, 292; Forster, Zeitschr. f. Biologic, 11; Panum,
Virchow's Arch., 29.

2 Die Blutvertheilung und der Thatigkeitswechsel des Organe, Leipzig, 1871.

CHAPTER VI.
CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

I. CHYLE AND LYMPH.

THE lymph is at least in part the mediator in the exchange of con-
stituents between the blood and the tissues. The bodies necessary for
the nutrition of the tissues pass from the blood into the lymph, and the
tissues deliver water, salts, and products of metabolism to the lymph.
The lymph, therefore, originates partly from the blood and partly from
the tissues. From a purely theoretical standpoint one can, according
to HEIDEHAIN, differentiate between blood-lymph and tissue-lymph
according to origin. It is impossible at the present time to separate
completely that which comes from the one or the other source.

The lymph formed in th6 different organs and tissues has a different
composition, and as the lymph is not obtained directly but only from the
large lymph vessels, hence the lymph that we use for investiga-
tions is generally a mixture, whose composition may vary under certain
conditions. The most easily obtained and best studied is the lymph
from the thoracic duct. In starving individuals this lymph, which is
called starvation lymph, does not essentially differ from other lymphs.
After fatty food the lymph, which is called digestion lymph or chyle,
differs from other lymphs by its great richness in very finely divided fat,
which gives it a milky appearance, and which has led to the old name
" lacteal fluid."

Chemically the lymph is the same as plasma, and contains, at least
to a great extent, the same bodies. The observation of ASHER and BAR-
BERA,1 that the lymph contains poisonous metabolic products, does
not contradict such an assumption, as no doubt these products are trans-
ferred to the blood with the lymph. Although the blood does not show
the same poisonous action as the lymph, still this can be explained by the
great dilution these bodies undergo in the blood, and the difference
between blood-plasma and lymph is no doubt of a quantitative nature.
This difference consists chiefly in that the lymph is poorer in proteins.

1 Zeitschr. f . Biologie 36.

345

346 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

Lymph, like the plasma, contains seralbumin, ser globulins, fibrinogen,
and fibrin ferment. The two last-mentioned bodies occur only in very
small amounts; therefore the lymph coagulates slowly (but spontaneously)
and yields but little fibrin. Like other liquids poor in fibrin ferment,
lymph does not at once coagulate completely, but repeated coagula-
tions take place.

i The extractive bodies seem to be the same as in plasma. Sugar (or
at least a reducing substance) is found in about the same quantity as in
the blood-serum, namely, about 1 p. m. The glycogen detected by DASTRE*
in the lymph occurs only in the leucocytes. According to ROHMANN and
BIAL, lymph contains a diastatic enzyme similar to that in blood-plasma,
and LUPINE2 found that the chyle of a dog during digestion has great gly-
colytic activity. Lipases may also occur in lymph. The amount of urea
has been determined by WURTZ 3 as 0.12-0.28 p. m. The mineral bodies
appear to be the same as in plasma.

fi As form-elements, leucocytes and in certain cases red blood-corpuscles
are common to both chyle and lymph. Chyle in fasting animals has the
appearance of lymph. After fatty food it is, on the contrary, milky,
due partly to small fat-globules, as in milk, and partly, indeed, mostly
to finely divided fat. The nature of the fat occurring in chyle depends
upon the kind of fat in the food. By far the greater part consists of
neutral fat, and even after feeding with large quantities of free fatty
acids, MUNK 4 found that the chyle contained chiefly neutral fat with
only small amounts of fatty acids or soaps.

The gases of the entirely normal human lymph have not thus far been
investigated. The gases from dog-lymph contain, according to HAMMAR-
STEN, only traces of oxygen, and consist of 37.4-53.1 per cent C02 and
1.6 per cent N, calculated at 0° C., and 760 mm. mercury. The chief mass
of the carbon dioxide of the lymph seems to be in firm chemical com-
bination. Comparative analyses of blood and lymph have shown that the
lymph contains more carbon dioxide than arterial, but less than venous
blood. The tension of the carbon dioxide of lymph is, according to
PFLUGER and STRASSBURG,S smaller than in venous, but greater than in
arterial, blood.

The quantitative composition of the chyle must evidently be very
variable. The specific gravity varies between 1.007 and 1.043. As an

1Compt. rend, de soc. biol., 47, and Compt. Rend., 120; Arch, de Physiol. (5), 7.

2 Rohmann and Bial, Pfliiger's Arch., 52, 53, and 55; Lepine, Compt. Rend., 110.

3 Compt. Rend., 49.

4 Virchow's Arch., 80 and 123. In regard to the analysis of the fat of chyle, see
Erben, Zeitschr. f. physiol. Chem., 30.

5 Hammarsten, Die Gase der Hundelymphe, Arbeiten aus d. physiol. Anstalt zu
Leipzig, 1871 ; Strassburg, Pfliiger's Archiv, 6.

CHYLE AND LYMPH. 347

example of the composition of human chyle two analyses will be given.
The first is by OWEN-REES, of the chyle of an executed person, and the
second by HoppE-SEYLER,1 of^the chyle in a case of rupture of the thoracic
duct. In the latter case the fibrin had previously separated. The results
are in parts per 1000.

No. 1. No. 2.

Water 904.8 940. 72 water

Solids 95.2 59.28 solids

Fibrin Traces

Albumin 70.8 36.67 albumin

Fat 9.2 7. 23 fat

2.35 soaps

Remaining organic bodies ... 10.8

0.83 lecithin

1 . 32 cholesterin

3 . 63 alcohol extractives

0 . 58 water extractives

oQl, A A j 6.80 soluble salts

10. 35 insoluble salts

The quantity of fat is very variable and may be considerably increased
by partaking of food rich in fats. I. MUNK and A. ROSENSTEIN 2 have
investigated the lymph or chyle obtained from a lymph fistula at the
end of the upper third of the leg of a girl eighteen years old and weigh-
ing 60 kg., and the highest quantity of fat in the chylous lymph was 47
p. m. after partaking of fat. In the starvation lymph from the same
patient they found only 0.6-2.6 p. m. fat. The quantity of soaps was
always small, and on partaking of 41 grams of fat the quantity of soaps was
only about gV °f the neutral fats. SCHUMM 3 found in the creamy
contents of a chylous cyst of the mesentery, 357.8 p. m. fat and compara-
tively large amounts of calcium soaps.

A great many analyses of chyle from animals have been made, and
they chiefly show the fact that the chyle is a liquid with a very changeable
composition which stands closely related to blood-plasma, but with the
principal difference that it contains more fat and less solids. The reader
is referred to special works for these analyses, as, for example, to v. GORUP-
BESANEZ'S " Lehrbuch der physiologischen Chemie," 4th edition.

The composition of the lymph is also very changeable, and its specific
gravity shows about the same variation as the chyle. In the following
analyses, 1 and 2, made by GUBLER and QUEVENNE, are the results
obtained from lymph of the upper part of the thigh of a woman aged
thirty-nine; and 3, made by v. SCHERER, is an analysis of lymph from

^wen-Rees, cited from Hoppe-Seyler's Physiol. Chem., 595; Hoppe-Seyler,
ibid., 597. See also Carlier, Brit. Med. Journ., 1902, 175, and T. Sollmann, Amer.
Journ. of Physiol., 17.

2 Virchow's Arch., 123.

3 Zeitschr. f. physiol. Chem., 49.

348 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

the sac-like dilated lymphatic vessels of the spermatic cord. No. 4
was made by C. SCHMIDT 1 the data being obtained from lymph from the
neck of a colt. The results are expressed in parts per 1000.

Water.. . 939.9 934.8 957.6 955.4

Solids 60.1 65.2 42.4 44.6

Fibrin 0.5 0.6 0.4 2.2

Albumin 42.7 42.8 34.7)

Fat, cholesterin, lecithin 3.8 9.2 [ 35.0

Extractive bodies 5.7 4.4 J

Salts 7.3 8.2 7.2 7.5

The salts found by C. SCHMIDT in the lymph of the horse have the
following composition, calculated in parts per 1000 parts of the lymph:

Sodium chloride 5 . 67

Soda 1.27

Potash.. 0.16

Sulphuric acid 0 . 09

Phosphoric acid united with alkalies 0 . 02

Earthy phosphates 0.26

In the cases investigated by MUNK and ROSENSTEIN the quantity of
solids in the fasting condition varied between 35.7 and 57.2 p. m. This
variation was essentially dependent upon the extent of secretion, so that
the low amount coincides with a more active secretion, and the reverse
in the other case. The chief portion of the solids consisted of proteins,
and the relation between globulin and albumin was as 1:2.4 to 4. The
mineral bodies in 1000 parts lymph (chylous) were: NaCl 5.83; Na2C03
2.17; K2HPO40.28; Ca3(PO4)2 0.28; Mg3(PO4)2 0.09; and Fe(PO4) 0.025.
The quantity of titratable alkali in the lymph is much smaller than in
the blood. CARLSON, GREEK and LucKHARDT2 have recently made com-
parative estimations of NaCl in blood-serum and lymph of the same
individual (horse and dog) and find that the lymph is regularly richer in
chlorides, a condition which, according to them, is difficult to reconcile
with the view of the filtration and transudation processes in the forma-
tion of lymph.

In this connection it must be recalled that according to many inves-
tigators the lymph has a somewhat higher osmotic pressure and therefore
a somewhat greater molecular concentration than the serum. CARLSON,
GREER and BECHTS found, nevertheless, that the osmotic pressure of
neck-lymph in the dog is often lower than the serum.

1 Gubler and Quevenne, cited from Hoppe-Seyler's Physiol. Chem., 591; v. Scherer,
ibid., 591; C. Schmidt, ibid., 592. See also Zaribnicky, Zeitschr. f. physiol. Chem.,
78.

2 Amer. Journ. of Physiol., 22 (1908).
« Ibid., 19(1907).

CHYLE AND LYMPH. 349

Under special conditions the lymph may be so rich in finely divided fat that
it appears like chyle. Such lymph has been investigated by HENSEN in a case of
lymph fistula in a ten-year-old boy, and by LANG 1 in a case of lymph fistula in
the upper part of the left thigh of a girl of seventeen. The lymph investigated
by HENSEN varied in the quantity of fat, as an average of nineteen analyses,
between 2.8 and 36.9 p. m.; while that investigated by LANG contained 24.85
p. m. of fat.

The quantity of lymph secreted must naturally change considerably
under various conditions, and there are no means of measuring it. The
size of the flow of lymph is, as HEIDENHAIN suggests, no measure of the
abundance of supply of nutritive material to the organs, and the lymph-
tubes act according to him as " drain-tubes/' removing the excess of
fluid from the lymph fissures as soon as the pressure therein rises to a
certain height. Attempts have been made to determine the quantity
of lymph flowing in 24 hours through the thoracic duct of animals. Ac-
cording to HEIDENHAIN the quantity averages 640 cc. for a dog weighing
10 kilos.

Determinations of the quantity of lymph in man have also been
attempted. NOEL-PATON 2 obtained 1 cc. of lymph per minute from the
severed thoracic duct of a patient weighing 60 kilos. The quantity in
the 24 hours cannot be calculated from this amount. In the case of
MUNK and ROSENSTEIN, 1134-1372 grams of chyle were collected within
12-13 hours after partaking of food. In the fasting condition or after
starving for 18 hours they found 50 to 70 grams per hour, sometimes 120
grams and above, especially in the first few hours after powerful muscular
exercise.

Several circumstances have a marked influence on the extent of lymph
secretion. During starvation less lymph is secreted than after partak-
ing of food. NASSE 3 has observed that the formation of lymph in dogs
is increased 36 per cent more after feeding with meat than after feeding
with potatoes, and about 54 per cent more than after 24 hours' depriva-
tion of food. In this connection mention must be made of the important
observations of ASHER and BARBEEA4 that with pure protein diet the
lymph current is increased in the thoracic cavity, and also that the increase
in the lymph secretion runs parallel with the elimination of nitrogen in
the urine, i.e., with the absorption of the protein from the digestive tract.

An increase in the total quantity of blood, as by transfusion of blood,
also especially in preventing the flow of blood by means of ligatures,

1 Hensen, Pfliiger's Arch., 110; Lang, see Maly's Jahresber., 4.

2 Journ. of Physiol., 11.

3 Cited from Hoppe-Seyler, Physiol. Chem., 593.

4 The works of Asher and collaborators, Barbera, Gies, and Busch, upon lymph
forrration may be found in Zeitschr. f. Biologie, 36, 37, 40.

350 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

causes an increase in the quantity of lymph. According to HEIDENHAIN,
on the contrary, a very considerable change in the pressure in the aorta
causes only a little change in the abundance of the lymph flow. The
quantity of lymph may be raised by powerfully active and passive move-
ments of the limbs (LESSER). Under the influence of curare, an increase
of the lymph secretion is observed (PASCHUTIN, LESSER 1), and the quan-
tity of solids in the lymph is also increased.

The bodies inciting lymph flow, the so-called lymphagogues, are of
especially great interest, and they may, according to HEIDENHAIN,2 be
divided into two different chief groups. The lymphagogues of the first
series — extracts of crab-muscles, blood-leech, anodons, liver and intestine
of dogs, as well as peptone and egg albumin, strawberry extracts, meta-
bolic products of bacteria and others — cause a greatly increased secre-
tion of lymph without raising the blood-pressure, and in this way the
blood-plasma becomes poorer in proteins and the lymph richer than
before. For the formation of this lymph, which HEIDENHAIN designates
blood-lymph, we must admit with him that a special secretory activity
of the capillary-wall endothelium exists. The lymphagogues of the second
series, such as sugar, urea, sodium chloride, and other salts, also cause
an abundant lymph formation. The blood, as well as the lymph, thereby
becomes richer in water. This increased amount of water depends,
according to HEIDENHAIN, upon an increased delivery of water by the
tissue-elements, and this lymph is chiefly tissue-lymph, in his opinion.
Diffusion is no doubt of great importance in the formation of this lymph ,
but the secretory activity of the endothelium is also of importance,
at least for certain bodies, such as sugar.

In the past, the formation of lymph was explained in a purely physical
way by the united action of filtration from the blood and the osmosis
between the blood and tissue-fluid. Later HEIDENHAIN and also HAM-
BURGER ascribed a special activity to the capillary endothelium, assum-
ing that they take part in the formation of lymph in a secretory manner.
The above-mentioned observations on the greater NaCl content in the
lymph as compared to the plasma as well as the regularly found higher
osmotic pressure of the lymph speak for such a view.

According to ASHER and his collaborators (BARBERA, GIES and
BUSCH) the lymph is a product of the work of the organs. Its amount
is dependent upon an increased or diminished activity of the organs,

1 Lesser, Arbeiten ars der physiol. Anstalt zu Leipzig, Jahrgang, 6; Paschutin,
ibid., 7.

2 Heidenhain, Pfliiger's Arch., 49; Hamburger, Zeitschr. f. Biologic, 27 and 30.
See especially Ziegler's Beitr. zur Path. u. zur allg. Pathol., 14, 443; also Arch. f.
(Anat. u.) Physiol., 1895 and 1896.

CHYLE AND LYMPH. 351

and the lymph is therefore a measure of the work in these. The close
relation between lymph formation and the work of organs has also been
shown for several of them, especially for the liver. STARLING has
shown that after the introduction of lymphagogues of the first series,
chiefly liver lymph is secreted, which he claims is a proof against HEIDEN-
HAIN'S view, and he explains the increased permeability of the vessel
wall by the fact that these bodies have an irritating, poisonous action.
On the contrary, ASHER explains this increased lymph flow by the state-
ment that the substance in question — as well as those influences which
incite the activity of the liver — produces an increased formation of lymph
in these organs. This view is supported by experiments upon the action
of lymphagogues on blood coagulation and liver activity (DELEZENNB
and others), for, according to GLEY, these bodies have at the same time
a lymphagogue action and an action upon the secretion of the glands.
We have no direct evidence of the action of the lymphagogues of the
first series upon the organs, but we know from KUSMINE'S work that
peptone, leech extract, and the extractives of the crab-muscles act directly
upon the liver-cells and bring about morphological changes. The con-
nection between organ activity and lymph formation has also been
shown upon muscles and glands by others besides the above-mentioned
investigators (HAMBURGER, BAINBRIDGE 1).

The extent of organ work essentially influences the quantity and
properties of the lymph. Still from this we cannot draw any posi-
tive conclusions as to whether the lymph formation is brought about
by physico-chemical processes alone or whether in this process a specific,
not closely definable secretory force is at work at the same time. In
regard to this much-disputed question, attention must be calied in the
first place to the fact that the important works of HEIDENHAIN, HAM-
BURGER, LAZARUS-BARLOW, and others, as well as the investigations of
ASHER and GIES and of MENDEL and HOOKER 2 upon the lengthy post-
mortem lymph flow, have shown that the older filtration hypothesis is
untenable.

That osmotic processes play an important role in the lymph formation
is generally admitted and that the work of the glands and tissue cells
must cause a difference in the osmotic pressure on both sides of the capillary
walls, has been shown by the researches of many investigators (KoRANYi,
STARLING, ROTH, ASHER and others). That this is so follows from
several circumstances, and especially from the fact that, in disassimila-

1 In regard to the works cited, as well as the literature upon lymph formation, see
Ellinger, "Die Bildung der Lymphe," Ergebnisse der Physiol., I, Abt. 1, 1902, and
Asher, Biochem. Centralbl., 4.

2 Amer. Journ. of Physiol., 7. See also footnote 1.

352 CHYLE, LYMPH, TKANSUDATES AND EXUDATES.

tion in the cells, bodies of high molecular weight are split into a number
of smaller molecules, which latter, either directly, if they leave the cells
and pass into the tissue-fluid, or indirectly, when they remain in the cells,
produce an increase in the osmotic tension within the cells, and in this
way cause a taking up of water from the fluid, and must therefore increase
the osmotic pressure of the tissue-fluids. As the cells can by synthesis
build up highly complex constituents from simple molecules, and as the
chief products of catabolism are carbon dioxide and water, it is difficult
to explain these intricate conditions. Still, irrespective of whatever
view, a change in one or the other direction in the osmotic pressure
upon both sides of the capillary wall must be produced thereby. Whether
this and other physico-chemical processes are alone sufficient to explain
the lymph formation (COHNSTEIN, ELLINGER) remains an open and
disputed question.1

H. TRANSUDATES AND EXUDATES.

The serous membranes are normally kept moistened by liquids whose
quantity is sufficient only in a few instances, as in the pericardial cavity
and the subarachnoidal space, for a complete chemical analysis to be
made of them. Under diseased conditions an abundant transudation
may take place from the blood into the serous cavities, into the sub-
cutaneous tissues, or under the epidermis; and in this way pathological
transudates are formed. Such true transudates, which are similar to lymph,
are generally poor in form-elements and leucocytes, and yield only very
little or almost no fibrin, while the inflammatory transudates, the so-called
exudates, are generally rich in leucocytes and yield proportionally more
fibrin. As a rule, the richer a transudate is in leucocytes the closer it
stands to pus, while a diminished quantity of leucocytes renders it more
nearly like a real transudate or lymph.

It is ordinarily accepted that filtration is cf the greatest importance
in the formation of transudates and exudates. The facts coincide with
this view that all these fluids contain the salts and extractive bodies
occurring in the blood-plasma in about the same quantity as the blood-
plasma, while the amount of proteins is habitually smaller. While the
different fluids belonging to this group have about the same quantities
of salts and extractive bodies, they differ from one another chiefly in
containing differing quantities of protein and form-elements, as well as
varying quantities of transformation and decomposition products of
these latter — changed blood-coloring matters, cholesterin, etc. The

^n this question see Ellinger, "Die Bildung der Lymphe," Ergebnisse der Phys-
iologic, I, Abt. 1, 355, and Asher, Biochem. Centralbl., 4, pp. 1 and 45.

TRANSUDATES AND EXUDATES. 353

correspondence in the amount of salts and extractive bodies present in
the blood and in transudates supplies just as little proof for a filtration
as it does for the formation of lymph; but still it cannot be doubted for
other reasons that filtration is often of great importance in the forma-
tion of a transudate. To what extent filtration is active in the perfectly
normal vascular wall cannot be answered.

The altered permeability of the capillary walls in disease is a second
important factor in the formation of transudates. The circumstance
that the greatest quantity of protein occurs in transudates in inflammatory
processes, to which is also due the abundant quantity of form-elements
in such transudates, has been explained by this hypothesis. The greater
quantity of protein in the transudates in formative irritation is in great
part explained by the large amount of destroyed form-elements. The
interesting observation made by PAUKULL,1 that in those cases in which
an inflammatory irritation has taken place the fluid contains nucleoal-
bumin (or nucleoprotein?), while this substance does not occur in
transudates in the absence of inflammatory processes, can be explained
by the presence of form-elements. Still, such a phosphorized protein
substance does not occur in all inflammatory exudates.

As the secretory importance of the capillary endothelium has been
made probable by the investigations of HEIDENHAIN, it is a priori to be
expected that an abnormally increased secretory activity of the endothe-
lium is a cause of transudates. Those observations which substantiate
such an assumption can also be explained just as well by assuming a
changed permeability of the capillary walls.

The varying quantities of protein observed by C. SCHMIDT 2 in the
tissue-fluids in different vascular regions can perhaps be explained by the
different condition of the capillary endothelium. For example, the
amount of protein in the PERICARDIAL, PLEURAL, and PERITONEAL FLUIDS
is considerably greater than in those fluids which are found in the SUB-
ARACHNOIDAL SPACE, in the SUBCUTANEOUS TISSUES, or in the AQUEOUS
HUMOR, which are poor in protein. The condition of the blood also
greatly affects the transudates, for in hydrsemia the amount of protein in
the transudate is very small. With the increase in the age of a transudate,
of a hydrocele fluid for instance, the quantity of protein is increased,
probably by resorption of water, and indeed exceptional cases may occur
in which the amount of protein, without any previous hemorrhage, is
even greater than in the blood-serum.

The proteins of transudates are chiefly seralbumin, serglobulin, and
a little fibrinogen. Proteoses and peptones do not occur, excepting

1 See Maly's Jahresber., 22.

2 Cited from Hoppe-Seyler, Physiol. Chem., 607.

354 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

perhaps in the cerebrospinal fluid, and in those cases where an autolysis
has taken place in the liquid.1 The non-inflammatory transudates do
not as a rule undergo spontaneous coagulation or do so only very slow-
ly. On the addition of blood or blood-serum they coagulate. Inflam-
matory exudates coagulate spontaneously, and PAIJKULL has shown
that these often contain nucleoprotein (or nucleoalbumin) . In inflam-
matory exudates a protein substance has been habitually observed which
is precipitated by acetic acid, but which does not occur in transudates,
or only in very small quantities. This substance, which has been observed
and studied by MOBITZ, STAEHELIN, UMBER, and RIVALTA, is claimed
by the first three observers to be free from phosphorus, while RIVALTA
considers it to be a phosphorized pseudoglobulin. UMBER calls it sero-
samucin, although it yields only very little reducing carbohydrate.
According to JOACHIM 2 it is only a part of the globulin, a view which can-
not be correct for all cases, v. HOLST 3 has so far substantiated UMBER'S
observation in that he has isolated a mucin substance from an ascitic
fluid in carcinoma of the stomach and the peritoneum, which seemed to
be identical with UMBER'S serosamucin, as well as with the synovial
mucin. There does not seem to be any doubt that in transudates and
exudates, different protein substances may occur under different cir-
cumstances, although the globulins form besides seralbumin the principal
mass of the protein bodies. Mucoid substances, which were first
observed by HAMMARSTEN in certain cases of ascites without complica-
tions with ovarial tumors, and which are cleavage products of a more
complicated substance, seem according to PAIJKULL 4 to be regular
constituents of transudates and are closely related to . the above-men-
tioned serosamucin. The occurrence of the above-mentioned substances
precipitable by acetic acid, the globulins (RIVALTA) and the nucleo-
proteins, in puncture fluids, has been recognized as of very great impor-
tance in the differential diagnosis between transudates and exudates.
There are numerous investigations on the relation between glob-
ulin and seralbumin, and JOACHIM has determined the relation between
euglobulin and the total globulin. No conclusive results can l>e drawn
from these determinations. The relation between globulin and seral-
bumin varies very much in different cases, but, as HOFFMANN and

^Umber, Munch, med. Wochenschr., 1902, and Berlin, klin. Wochenschr., 1903.
In regard to the autolysis in transudates, see also Galdi, Biochem. Centralbl., 3;
Eppinger, Zeitschr. f. Heilkunde, 25, and Zak, Wien. klin. Wochenschr., 1905.

2Paijkull, 1. c.; Moritz, Munch, med. Wochenschr., 1903; Staehelin, ibid., 1902,
Umber, Zeitschr. f. klin. Med., 48; Rivalta, Biochem. Centralbl., 2 and 5; Joachim;
Pfliiger's Arch., 93.

3 Zeitschr. f. physiol. Chem., 43.

4 Hammarsten, ibid., 15; Paijkull, 1. c.

TRANSUDATES AND EXUDATES. 355

PIGEAND l have shown, the variation is in each case the same as in the
blood-serum of the individual.

The specific gravity runs almost parallel with the quantity of protein.
The varying specific gravity has been suggested as a means of differentia-
tion between transudates and exudates by REUSS,2 as the first often show
a specific gravity below 1015-1010, while the others have a specific gravity
of 1018 or above. This rule holds good in many, but not in all cases.

The gases of the transudates consist of carbon dioxide besides small
amounts of nitrogen and traces of oxygen. The tension of the carbon
dioxide is greater in the transudates than in the blood. When mixed
with pus, the amount of carbon dioxide is decreased.

The extractives are, as above stated, the same as in the blood-plasma.
Urea seems to occur in very variable amounts. Sugar also occurs in
transudates, but it is not known to what extent the reducing power is
due to other bodies, as in blood-serum. A reducing, non-fermentable
substance has been found by PICKARDT in transudates. The sugar is
generally glucose, but fructose seems to have been found3 in several
cases. Sarcolactic add has been found by C. KULZ in the pericardial
fluid from oxen. Sucdnic add has been found in a few cases in hydrocele
fluids, while in other cases it is entirely absent. Leudne and tyrosine
have been found in transudates from diseased livers and pus-like trans-
udates which have undergone decomposition, and after autolysis. Among
other extractives found in transudates must be mentioned allantoin
(MoscATELLi4), uric add, purine bases, creatine, inosite, and pyrocate-
chin (?).

The division of the nitrogenous substances in human transudates
and exudates has so far been little studied. OTORI found that no
essential difference exists between serous exudates and transudates in
regard to the quantity of urea and amino-acids. The amount of total
nitrogen and proteins runs parallel with the specific gravity, and the
same is generally true for the absolute values for ammonia nitrogen and
purine nitrogen. According to the investigations of CZERNECKI,S in
pathological puncture fluids, also oxyproteic adds (see Chapter XIV
on the urine) occur and which represent 13.3 — 25.9 per cent of the total
nitrogen of the protein free filtrate. The question as to the amount of

1 Joachim, 1. c.; Hoffmann, Arch. f. exp. Path. u. Pharm., 16; Pigeand, see Maly's
Jahresber., 16.

2 Reuss, Deutsch. Arch. f. klin. Med., 28. See also Otto, Zeitschr. f. Heilkimde, 17.

3 Pickardt, Berl. klin. Wochenschr., 1897. See also Rotmann, Munch, med. Woch-
enschr., 1898; Neuberg and Strauss, Zeitschr. f. physiol. Chem., 36; Sittig, Bioch.
Zeitschr. 21.

4C. Kiilz, Zeitschr. f. Biologic, 32; Moscatelli, Zeitschr. f. physiol. Chem. 13.
BOtori, Zeitschr. f. Heilk. 25; Czernecki, Maly's Jahresb., 39.

356 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

urea nitrogen and amino-acid nitrogen in such fluids must, under these
circumstances, require further study.

The investigations upon the molecular concentration have shown
that no essential and constant difference exists between exudates and
transudates. The osmotic concentration and the concentration of the
electrolytes are as a rule the same as in blood-serum, although some-
times rather divergent results have been found. The concentration of
the electrolytes shows/according to BoDON,1 like the blood-serum, much
less variation than the total concentration. The alkalinity determined
by titration is about the same in transudates and exudates, and is equal
to that of the blood-serum. The determination of the HO ion concen-
tration has shown that the transudates and exudates in this regard are
about as neutral as the blood-serum (BODON).

As above stated, irrespective of the varying number of form-elements
contained in the different transudates, the quantity of protein is the most
characteristic chemical distinction in the composition of the various
transudates; therefore a quantitative analysis is of importance only
in so far as it considers .the quantity of protein. On this account, in the
following, relative to the quantitative composition, stress will be put on
the quantity of protein.

Pericardial Fluid. The quantity of this fluid is, even under physio-
logical conditions, so large that a sufficient quantity for chemical inves-
tigation has been obtained (from persons who had been executed). This
fluid is lemon-yellow in color, somewhat sticky, and yields more fibrin
than other transudates. The amount of solids, according to the analyses
performed by v. GORUP-BESANEZ, WACHSMUTH, and HoppE-SEYLER,2
is 37.5-44.9 p. m., and the amount of protein is 22.8-24.7 p. m. The
analysis made by HAMMARSTEN of a fresh pericardial fluid from a young
man who had been executed yielded the following results, calculated in
1000 parts by weight.

Water.. . 960.85
Solids 39.15

(Fibrin 0.31

Proteins 28 . 60 ] Globulin .... 5 .95

I Albumin 22.34

Soluble salts 8.60 NaCl 7.28

Insoluble salts 0. 15.

Extractive bodies 2.00

FRIEND 3 found almost the same composition for a pericardial fluid
from a horse, with the exception that this liquid was relatively richer

1 Pfliiger's Arch., 104, where literature on this subject may be found.
2v. Gorup-Besanez, Lehrbuch d. physiol Chem., 4. Aufl., 401; Wachsmuth, Vir-
chow's Arch., 7; Hoppe-Seyler, Physiol. Chem., 605.

3 Halliburton, Text-book of Chem. Physiol., etc., London, 1891.

PLEURAL FLUID. 357

in globulin. The ordinary statement that pericardial fluids are richer
in fibrinogen than other transudates is hardly based on sufficient proof.
In a case of chylopericardium, which was probably due to the rupture
of a chylous vessel, or caused by a capillary exudation of chyle because
of stoppage, HASEBROEK l found in 1000 parts of the fluid 103.61 parts
solids, 73.79 parts proteins, 10.77 parts fat, 3.34 parts cholesterin, 1.77
parts lecithin, and 9.34 parts salts.

The pleural fluid occurs under physiological conditions in such small
quantities that a chemical analysis of it cannot be made. Under patho-
logical conditions this fluid may show very variable properties. In
certain cases it is nearly serous, in others again sero-fibrinous, and in others
similar to pus. There is a corresponding variation in the specific gravity
and the properties in general. If a pus-like exudate is kept enclosed for
a long time in the pleural cavity, a more or less complete maceration
and solution of the pus-corpuscles is found to take place. The ejected
yellowish-brown or greenish fluid may then be as rich in solids as the
blood-serum; and an abundant flocculent precipitate of a nucleoalbumin
or nuceloprotein (the pyrin.of early writers) may be obtained on the
addition of acetic acid. This precipitate is soluble with difficulty in
an excess of acetic acid.

Numerous analyses, by many investigators,2 of the quantitative
composition of pleural fluids under pathological conditions have been
published. From these analyses we learn that in hydrothorax the
specific gravity is lower and the quantity of protein less than in pleuritis.
In the first case the specific gravity is generally less than 1.015, and the
quantity of protein 10-30 p. m. In acute pleuritis the specific gravity
is generally higher than 1.020, and the quantity of protein 30-65 p. m.
The quantity of fibrinogen, which in hydrothorax is about 0.1 p. m.,
may amount to more than 1 p. m. in pleuritis. In pleurisy with an
abundant accumulation of pus, the specific gravity may rise even to 1.030
according to the observations of HAMMARSTEN. The quantity of solids
is often 60-70 p. m., and may be even more than 90-100 p. m. (HAM-
MARSTEN). Mucoid substances have also been detected in pleural fluids
by PAIJKULL. Cases of chylous pleurisy are also known; in such a
case MEHU 3 found 17.93 p. m. fat and cholesterin in the fluid.

The quantity of peritoneal fluid is very small under physiological
conditions. The investigations refer only to the fluid under diseased

1 Zeitschr. f. physiol. Chem., 12.

2 See the works of Me"hu, Runeberg, F. Hoffmann, Reuss, all of which are cited in
Bernheim's paper in Virchow's Arch., 131, 274. See also Paijkull, 1. c., and Halli-
burton's Text-book, 346; Joachim, 1. c.

3 Arch. ge"n. de med., 1886, 2, cited from Maly's Jahresber., 16.

358 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

conditions (ascitic fluid). The color, transparency, and consistency of
these may vary greatly.

In cachectic conditions or a hydrsemic condition of the blood the fluid
has little color, is milky, opalescent, watery, does not coagulate spon-
taneously, has a very low specific gravity, 1.006-1.010-1.015, and is
almost free from form-elements. The ascitic fluid in portal stagnation,
or in general venous congestion, has a low specific gravity and contains or-
dinarily less than 20 p. m. protein, although in certain cases the quantity
of protein may rise to 35 p. m. In carcinomatous peritonitis it may have a
cloudy, dirty-gray appearance, due to its richness in form-elements of
various kinds. The specific gravity is then higher, the quantity of solids
greater, and it often coagulates spontaneously. In inflammatory proc-
esses it is straw- or lemon-yellow in color, somewhat cloudy or reddish,
due to leucocytes and red blood-corpuscles, and from great richness in
leucocytes it may appear more like pus. It coagulates spontaneously
and may be relatively richer in solids. It contains regularly 30 p. m.
or more protein (although exceptions with less protein occur), and may
have a specific gravity of 1.030 or above. On account of the rupture
of a chylous vessel, the ascitic fluid may be rich in very finely emulsified
fat (CHYLOUS ASCITES). In such cases 3.86-10.30 p. m. fat has been
found in the ascitic fluid (GUINOCHET, HAY x), and even 17-43 p. m.
has been found by MINKOWSKI.

As first shown by GROSS, an ascitic fluid may have a chylous appearance
without the presence of fat, i.e., pseudochylous. The cause of the chylous
properties of a transudate is not known, although numerous investigators,
such as GROSS, BERNERT, MOSSE, and STRAUSS, have studied the sub-
ject; several observations, however, seem to show that it is connected
with the amount of lecithin contained therein. In a case investigated
by H. WOLFF 2 the oleic-acid ester of cholesterin was combined either
chemically or molecularly with the euglobulin.

By admixture of ascitic fluid with that from an ovarian cyst the
former may sometimes contain pseudomucin (see Chapter XII). There
are also cases in which the ascitic fluid contains mucoids which may be
precipitated by alcohol after removal of the proteins by coagulation at
boiling temperature. Such mucoids, which yield a reducing substance
on boiling with acids, have been found by HAMMARSTEN in tuberculous
peritonitis and in cirrhosis hepatis syphilitica in men. According to the
investigations of PAIJKULL, these substances seem to occur often and
perhaps habitually in the ascitic fluids.

1 Guinochet, see Strauss, Arch, de Physiol., 18. See Maly's Jahresber., 16, 475.

2 Gross, Arch. f. exp. Path. u. Pharm., 44; Bernert, ibid., 49; Mosse, Leyden's
Festschrift, 1901; Strauss, cited in Biochem. CentralbL, 1, 437; Wolff, Hofmeister's
Beitrage, 5.

HYDKOCELE AND SPERMATOCELE FLUIDS. 359

As the quantity of protein in ascitic fluids is dependent upon the same
factors as in other transudates and exudates, it is sufficient to give the
following example of the composition, taken from BERNHEIM'S l treatise.
The results are expressed in 1000 parts of the fluid:

Max. Min. Mean. *

Cirrhosis of the liver 34.5 5.6 9.69—21.06

Bright's disease 16.11 10.10 5.6 —10.36

Tuberculous and idiopathic peritonitis ... 55 . 8 18 . 72 30 . 7 —37 . 95

Carcinomatous peritonitis 54.20 27.00 35. 1 —58.96

JOACHIM found the highest relative globulin amounts and lowest albumin
percentages in cirrhosis; in carcinoma, on the contrary, the lowest globulin and
the highest albumin. The values in cardiac stagnation stand between the cirrhosis
and carcinoma percentages.

Urea has also been found in ascitic fluids, sometimes only as traces, some-
times in larger quantities (4 p. m. in albuminuria), also uric acid, allantoin in
cirrhosis of the liver (MOSCATELLI), xanthine, creatine, cholesterin, sugar, diastatic
and proteolytic enzymes, and according to HAMBURGER 2 also a lipase.

Hydrocele and Spermatocele Fluids. These fluids differ essentially
from each other in various ways. The hydrocele fluids are generally
colored light or dark yellow, sometimes brownish with a shade of green.
They have a relatively higher specific gravity, 1.016-1.026, with a variable
but generally higher amount of solids, an average of 60 p. m. They
sometimes coagulate spontaneously, sometimes only after the addition of
fibrin ferment or blood. They contain leucocytes as chief form-elements.
Sometimes they contain smaller or larger amounts of cholesterin crystals.

The spermatocele fluids, on the contrary, are as a rule colorless,
thin, and cloudy like water mixed with milk. They sometimes have an
acid reaction. They have a lower specific gravity, 1.006-1.010, a lower
amount of solids — an average of about 13 p. m. — and do not coagulate
either spontaneously or after the addition of blood. They are, as a rule,
poor in protein and contain spermatozoa, cell-detritus, and fat-globules as
form constituents. To show the unequal composition of these two kinds
of fluids we will give the average results (calculated in parts per 1000
parts of the fluid) of seventeen analyses of hydrocele fluids and four
of spermatocele fluids made by HAMMARSTEN.S

Hydrocele. Spermatocele.

Water 938.85 986.83

Solids 61 . 15 12. 17

Fibrin 0.59

Globulin 13.25 0.59

Seralbumin 35.94 1 .82

Ether extractive bodies 4 . 02 1

Soluble salts 8.60 10.76

Insoluble salts 0 . 66 J

1 1. c. As it was impossible to derive mean figures from those given by "Bernheim,
the author has given the maximum and minimum of the averages given by him.

2 Arch. f. (Anat. u.) Physiol., 1900, 433.

3 Upsala Lakaref. Forh., 14, and Maly's Jahresber., 8, 347.

360 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

In the hydrocele fluid traces of urea and a reducing substance have been
found, and in a few cases also succinic acid and inosite. A hydrocele fluid may,
according to DEViLLARD,1 sometimes contain paralbumin or metalbumin (?).
Cases of chylous hydrocele are also known.

Cerebrospinal Fluid. The cerebrospinal fluid is thin, water-clear,
of low specific gravity, 1.007-1.008. The spina bifida fluid is very poor
in solids, 8-10 p. m. with only 0.19-1.6 p. m. protein. The fluid of
chronic hydrocephalus is somewhat richer in solids (13-19 p. m.) and
proteins. The amount of protein in the cerebrospinal fluid seems to be
rather variable under diseased conditions and FRENKEL-HEIDEN 2 found
0.875-3 p. m. protein in the lumbar fluid in progressive paralysis and
0.7-2.8 p. m. protein in tuberculous meningitis. In the perfectly fresh
fluid from healthy calves NAWRATZKI found an average of 0.22 p. m.
protein.

According to HALLIBURTON the protein of the cerebrospinal fluid
is a mixture of globulin and protease; occasionally some peptone occurs,
and more rarely, in special cases, seralbumin appears. The conclusions
of HALLIBURTON on the occurrence of protease do not coincide with the
observations of other investigators (PANZER, SALKOWSKi3). In general
paralysis, HALLIBURTON and MOTT obtained a nucleoprotein in the
cerebrospinal fluid. Choline occurs in several diseases, as in general
paralysis, brain-tumors, tabes dorsalis, and epilepsy (HALLIBURTON
and MOTT, DONATH, ROSENHEIM). According to KAUFMANN4 we
are not here dealing with choline but with another base. Glucose, or at
least a fermentable sugar, occurs habitually in the cerebrospinal fluid,
while the claims of HALLIBURTON as to the occurrence of a substance
similar to pyrocatechin could not be substantiated in calves and men
by NAWRATZKI,5 and hence this substance does not exist in all cerebro-
spinal fluids. Urea occurs in cerebrospinal fluids, but not always. In
the cases investigated by FRENKEL-HEIDEN indeed all the rest-nitrogen
occurred as urea and the urea-nitrogen varied in different pathological
cases between 0.196-1.12 p. m. Lactic Acid has been found by LEHN-
DORFF and BAUMGARTEN6 in many pathological cases. The quantity
of NaCl is regularly much greater than the KC1, 6-7 p. m. NaCl against

1 Bull. Soc. chim., 42, 617.

2 Bioch. Zeitschr., 2.

3 Halliburton's Text-book; Panzer, Wein. klin. Wochenschr., 1899; Salkowski,
Jaffa* Festschrift, 265.

4 Halliburton and Mott, Phil. Transact. Roy. Soc. London, Series B, 191; Donath,
Zeitschr. f. physiol Chem., 39 and 42; see also Mansfield, ibid., 42; Rosenheim, Journ.
of Physiol., 35; Kaufmann, Zeitschr. f. physiol. Chem., 66.

6 Zeitschr. f. physiol. Chem., 23. See also Rossi, ibid., 39 (literature).
6 Zeitschr. f. exp. Path. u. Therap., 4 (literature).

AQUEOUS HUMOR AND BLISTER-FLUID. 361

about 0.4 p. m. KC1, and the variable relation between potassium and
sodium is probably due, according to SALKOWSKI/ to the absence or pres-
ence of fever during the formation of the exudate; the amount of potas-
sium is high in the acute cases and low in the chronic ones. According
to LANDAU and HALPERN 2 a certain antagonism seems to exist between
nitrogen and sodium chloride, as the highest results of the first correspond
to the lowest results of the other. According to CAVAZZANI,S who has
especially studied the cerebrospinal fluids, the alkalinity of these fluids
is considerably less than that of the blood and independent of this last
fluid. For this and several other reasons CAVAZZANI draws the con-
clusion that the cerebrospinal fluid is formed by a true secretory process.

A large number of investigations on the cerebrospinal fluid have been made
on the fluid obtained from cadavers and in consideration of this it must be remarked
that this fluid quickly changes after death and that the results obtained therefore
are not comparable with the fluid during life.

Aqueous Humor. This fluid is clear, alkaline toward litmus, and has
a specific gravity of 1.003-1.009. The amount of solids is on an average
13 p. m., and the amount of proteins only 0.8-1.2 p, m. The protein
consists of seralbumin and globulin and very little fibrinogen and mucin.
According to GRUENHAGEN it contains paralactic acid, another dextro-
gyrate substance, and a reducing body which is unlike sugar or dextrin.
PAUTZ 4 found urea and sugar in the aqueous humor of oxen.

Blister-fluid. The content of blisters caused by burns, and of vesi-
catory blisters and the blisters of the pemphigus chronicus, is generally a
fluid rich in solids and proteins (40-65 p. m.). This is especially true
of the contents of vesicatory blisters. In a burn-blister K. MORNERS
found 50.31 p. m. proteins, among which were 13.59 p. m. globulin and
0.11 p. m. fibrin. The fluid contains a substance which reduces copper
oxide, but no pyrocatechin. The fluid of the pemphigus is alkaline in
reaction. A wound secretion collected by LIEBLEIN 6 under aseptic
conditions was alkaline in reaction, and contained less protein than the
blood-serum. It formed a slight fibrin clot, and contained proteoses
only at first or at the beginning of the abscess formation. As the wound
healed, the relation between the globulin and albumin changed, and on

1 See Salkowski, 1. c. New quantitative analyses of cerebrospinal and hydrocephalus
fluids may be found in the cited works of Nawratzki, Panzer, and Salkowski.
2Bioch. Zeitsehr., 9.

3 See Maly's Jahresber., 22, 346, and Centralbl. f. Physiol., 15, 216.

4 Gruenhagen, Pflliger's Arch., 43; Pautz. Zeitsehr. f. Biologic, 31.
6 Skand. Arch. f. Physiol., 5.

6 Habilitationsschrift Prag. 1902, printed by H. Laupp, Tubingen.

362 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

the third day of the healing the quantity of albumin was at least nine-
tenths of the total protein.

The fluid of subcutaneous oedema. This is, as a rule, very poor in
solids, purely serous, does not contain fibrinogen, and has a specific
gravity of 1.005-1.013. The quantity of proteins is in most cases lower
than 10 p. m. — according to HOFFMANN 1-8 p. m. — and in serious
affections of the kidneys, generally with amyloid degeneration, less than
1 p. m. has been shown (HOFFMANN 1). The cedematous fluid also habit-
ually contains urea, 1-2 p. m., and sugar.

The FLUID OF THE ECHiNOCOCCUs cyst is related to the transudates, and is
poor in proteins. It is thin and colorless, and has a specific gravity of 1.005-
1.015. The quantity of solids is 14-20 p. m. The chemical constituents are
sugar (2.5 p. m.), inosite, traces of urea, creatine, succinic acid, and salts (8.3-9.7
p. m.). Proteins are found only in traces, and then only after an inflammatory
irritation. In the last-mentioned case 7 p. m. proteins have been found in the fluid.

The Synovial Fluid and Fluid in Synovial Cavities around Joints,
etc." The synovia is hardly a transudate, but it is often discussed in an
appendix to the transudates.

The synovia is an alkaline, sticky, fibrous, yellowish fluid which
is cloudy, from the presence of cell-nuclei and the remains of destroyed
cells, but is also sometimes clear. Besides proteins and salts, it also
contains a mucin substance, synoviamucin (v. HOLST 2) . In pathological
synovia, HAMMARSTEN found a mucin-like substance which is not mucin.
It behaves like a nucleoalbumin or a nucleoprotein, and gives no reducing
substance on boiling with acids. SALKOWSKI 3 also found a mucin-like
substance in a pathological synovial fluid, which was neither mucin nor
nucleoalbumin. He called the substance synovin.

The composition of synovia is not constant, but is different in rest
and in motion. In the last-mentioned case the quantity of fluid is less,
but the amount of the mucin-like body, of proteins, and of .the extractive
bodies is greater, while the quantity of salts is diminished. This may
be seen from the following analyses by FRERicns.4 The figures repre-
sent parts per 1000.

I. Synovia from II. Synovia from

a Stall-fed Ox. a Field-fed Ox.

Water 969.9 948.5

Solids 30.1 51.5

Mucin-like body 2.4 5.6

Albumin and extractives 15. 7 35 . 1

Fat 0.6 0.7

Salts 11.3 9.9

1 Deutsch. Arch. f. klin. Med., 44.

2Zeitschr. f. physiol. Chem., 43.

9 Hammarsten, Maly's Jahresber., 12; Salkowski, Virchow's Arch., 131.

4 Wagner's Handworterbuch, 3, Abt. 1, 463.

PUS. 363

The synovia of new-born babes corresponds to that of resting animals.
The fluid of the bursae mucosae, as also the fluid in the synovial cavities
around joints, etc., is similar to synovia from a qualitative standpoint.

III. PUS.

Pus is a yellowish-gray or yellowish-green, creamy mass of a faint
odor and an unsavory, sweetish taste. It consists of a fluid, the pus-
serum, in which solid particles, the pus-cells, swim. The number of these
cells varies so considerably that the pus may at one time be thin and at
another time so thick that it scarcely contains a drop of serum. The
specific gravity, therefore, may also greatly vary, namely, between
1.020 and 1.040, but ordinarily it is 1.031-1.033. The reaction of fresh
pus is generally alkaline, but it may become neutral or acid from a decom-
position in which fatty acids, glycerophosphoric acid, and also lactic
acid are formed.

In the chemical investigation of pus, the pus-serum and the pus-
corpuscles must be studied separately.

Pus-serum. Pus does not coagulate spontaneously nor after the
addition of defibrinated blood. The fluid in which the pus-corpuscles
are suspended is not to be compared with the blood-plasma, but rather
with the serum. The pus-serum is pale yellow, yellowish-green, or brownish-
yellow, and has an alkaline reaction toward litmus. It contains, for the
most part, the same constituents as the blood-serum; but sometimes
besides these — when, for instance, the pus has remained in the body
for a long time — it contains a nucleoalbumin or a nucleoprotein which
is precipitated by acetic acid and is soluble with great difficulty in an
excess of the acid (pyin of the earlier, authors). This nucleoalbumin
seems to be formed from the hyaline substance of the pus-cells by macera-
tion. The pus-serum contains, moreover, at least in many cases, no
fibrin ferment. According to the analyses of HOPPE-SEYLER l the pus-
serum contains in 1000 parts:

Water.. . 913'.70 905.65

Solids 86.30 94.35

Proteins 63.23 77.21

Lecithin 1 . 50 0 . 56

Fat 0.26 0.29

Cholesterin 0.53 0.87

Alcohol extractives 1 . 52 0.73

Water extractives 11 . 53 6 .92

Inorganic salts .' , 7 . 73 7 . 77

Med.-Chem. Untersuch., 490.

364 CHYLE, LYMPH, TRANSUDATES _AND EXUDATES.

The ash of pus-serum has the following composition, calculated to
1000 parts of the serum:

i. IT.

NaCl . . 5.22 5.39

Na2SO4 0.40 0.31

Na2HPO4 . 0.98 0.46

Na2CO3 0.49 1 . 13

Ca3(PO4)2 0.49 0.31

Mg3(PO4)2 0.19 0.12

PO4 (in excess) 0.05

The pus-corpuscles are generally thought to consist chiefly of emi-
grated white blood-corpuscles, and their chemical properties have there-
fore been given in discussing these. The molecular granules, fat-
globules, and red blood-corpuscles are considered rather as casual form-
elements.

The pus-cells may be separated from the serum by centrifugal force,
or by decantation directly or after dilution with a solution of sodium
sulphate in water (1 vol. saturated sodium-sulphate solution and 9 vols.
water) and then washed by this same solution in the same manner as
the blood-corpuscles.

The chief constituents of the pus-corpuscles are proteins, of which
the largest portion seems to be a nucleoprotein which is insoluble in
water and which expands into a tough, slimy mass when treated with a
10-per cent common-salt solution. This protein substance, which is
soluble in alkali but is quickly changed thereby, is called ROVIDA'S hyaline
substance, and the property of the pus of being converted into a slime-
like mass by a solution of common salt depends on this substance. Besides
this substance, to which the nucleoprotein of the pus-cells investigated
by STRADA 1 seems to stand in close relation, we also have a globulin
which coagulates at 48-49° C., as well as serglobulin (?), seralbumin,
a substance similar to coagulated protein (MIESCHER), and lastly peptone
or proteose (HOFMEISTER 2) . It is very remarkable that no nucleo-
histone-.or histone has been detected in the pus-cells, although histone
occurs in the cells of the lymph glands.

There are also found in the protoplasm of the pus-cells, besides the
proteins, lecithin, cholesterin, glucothionic acid,3 purine bodies, fat, and soaps.
HOPPE-SEYLER has found cerebrin, a decomposition product of a pro-
tagon-like substance, in pus (see Chapter XI). KOSSEL and FREYTAG4
have isolated from pus two substances, pyosin and pyogenin, which

1 Bioch. Zeitschr., 16.

2 Miescher in Hoppe-Seyler's Med.-Chem. Untersuch., 441; Ch. Pons. Maly's Jahresb.,
39; Hofmeister, Zeitschr. f. physiol. Chem., 4.

3 Mandel and Levene, Bioch. Zeitschr., 4.
* Zeitschr. f. physiol. Chem., 17, 452.

PUS. 365

belong to the cerebrin group (see Chapter XI). HOPPE-SEYLER 1
claims that glycogen appears only in the living, contractile white blood-
cells and not in the dead pus-corpuscles. Several other investigators
have, nevertheless, found glycogen in pus. The cell-nucleus contains
nuclein and nudeoproteins.

In regard to the occurrence of enzymes in the pus-cells it must be
remarked that neither thrombin nor prothrombin is found therein,
although these bodies are generally considered as being derived from
the leucocytes, and also obtainable from the thymus leucocytes. The
occurrence in the pus-cells, besides catalases and oxidases, of a proteolytic
enzyme, is of great interest. It is not only important for the intracellular
digestion and for the amount of proteoses in the pus-cell, but also for
the solution of the fibrin clot and pneumonic infiltrations (FR. MULLER,
O. SIMON 2). Alipase, which splits neutral fats, also occurs, according to
FIESSINGER and MARIE, in pus.

The mineral constituents of the pus-corpuscles are potassium, sodium,
calcium, magnesium, and iron. A part of the alkalies exists as chlorides,
and the remainder, as well as the chief part of the other bases, exists
as phosphates.

The quantitative composition of the pus-cells from the analyses of
HOPPE-SEYLER is as follows, in parts per 1000 of the dried substance:

i. n.

Proteins 137 . 62 ]

Nuclein 342 . 57 f 685 . 85 673.69

Insoluble bodies 205. 66 J

Lecithin \ , .o oo 75 . 64

Fat / 143'83 75.00

Cholesterin 74.00 72.83

Cerebrin 51 .99 \ mo CA

Extractive bodies 44.33 /

MINERAL SUBSTANCES IN 1000 PARTS OF THE DRIED SUBSTANCE.

NaCl 4.34

Ca3(PO4)2 2.05

Mg3(P04)2 1.13

FePO4 1.06

PO4 9 . 16

Na 0.68

K Traces (?)

MIESCHER obtained other results for the alkali compounds, namely, potas-
sium phosphate 12, sodium phosphate 6.1, earthy phosphate and iron phos-
phate 4.2, sodium chloride 1.4, and phosphoric acid combined with organic sub-
. stances 3.14-2.03 p. m.

In pus from congested abscesses which has stagnated for some time
there occur peptone (proteose), leucine and tyrosine, free fatty acids and

1 Hoppe-Seyler, Physiol. Chem., 790.

2Fr. Miiller, Verhandl. Nat. Gesellsch. zu. Basel, 1901; O. Simon, Deutsch. Arch,
.f. klin. Med., 70.

366 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

volatile fatty adds, such as formic acid, butyric acid and valeric acid.
There are also found urea, glucose (in diabetes), bile-pigments, and bile-
adds (in catarrhal icterus).

As more specific but not constant constituents of the pus must be
mentioned the following: pyin, which seems to be a nucleoprotein pre-
cipitable by acetic acid, and also pyinic add and chlorrhodinic add, which
have been so little studied that they cannot be more fully treated here.

In many cases a blue, more rarely a green, color, has been observed
in the pus. This depends on the presence of micro-organisms (Badllus
pyocyaneus). From such pus FORDOS and LticKE 1 have isolated a crys-
talline blue pigment, pyocyanin, and a yellow pigment, pyoxanthose,
which is produced from the first by oxidation.

Appendix.
Lymphatic Glands, Spleen and Endocrinic Glands.

The Lymphatic Glands. The cells of the lymphatic glands are
found to contain the protein substances generally occurring in cells
(Chapter V) . According to BANG 2 they also contain histone nucleates
(nucleohistone) , but in smaller amounts and of a different variety from
the better-studied nucleohistone from the thymus gland. Proteoses
occur as products of autolysis. By a lengthy autolysis of lymph glands
REHS found ammonia, tyrosine, leucine (somewhat scanty), thymme,
and uracil among the cleavage products. Besides the other ordinary
tissue constituents, such as collagen, reticulin, elastin, and nuclein, there
occur in the lymphatic glands also cholesterin, fat, glycogen, sarcolactic
add, purine bases, and leudne. In the inguinal glands of an old woman
OIDTMANN found 714.32 p. m. water, 284.5 p. m. organic and 1.16 p. m.
inorganic substances. In the cells of the mesenteric lymphatic glands
of oxen, BANG4 found 804.1 p. m. water, 195.9 p. m. solids, 137.9 total
proteins, 6.9 p. m. histone nucleate, 10.6 p. m. nucleoprotein, 47.6 p. m.
bodies soluble in alcohol, and 10.5 p. m. mineral constituents.

The Thymus. The cells of this gland are very rich in nuclein bodies
and relatively poor in the ordinary proteins, but their nature has not been
closely studied. The chief interest is attached to the nuclein substances.
KOSSEL and LILIENFELD first prepared from the watery extract of the
gland, by precipitating with acetic acid and then further purifying, a

1 Fordos, Compt. Rend., 51 and 56; Liicke, Arch. f. klin. Chirurg., 3; Boland, Cen-
tralbl. f. Bakt. u. Parasit., I., 25.

2 Studier over Nucleoproteider, Kristiania, 1902, and Hofmeister's Beitrage, 4.

3 Hofmeister's Beitrage, 3.
«Lc.

THYMUS. 367

protein substance which has been generally called nudeohistone. By the
action of dilute hydrochloric acid upon nudeohistone it splits, according
to these investigators, into histone and leuconudein. The leuconuclein
is a true nuclein; hence it is a nucleic-acid compound with protein which
is relatively poor in protein and rich in phosphorus. The more recent
investigations of BANG, MALENGREAU, HUISKAMP and GOUBAN l upon
nudeohistone all show that this nucleoprotein is not a unit substance, but
a mixture of at least two bodies. The views of the investigators men-
tioned differ quite essentially from one another as to the nature of these
bodies, but this is partly due to the different methods used by them and
partly to the ready changeability of the substances in question.

Besides the real nudeohistone, B-nucleoalbumin of MALENGREAU,
LILIENFELD'S histone contains a second nucleoprotein which BANG and
HUISKAMP call simple nucleoprotein, while MALENGREAU designates
it A-nucleoalbumin. This protein, which contains only about 1 per cent
phosphorus and which is possibly identical with the nucleoprotein found
by LILIENFELD in the thymus, yields a nuclein, but no free nucleic acid,
on cleavage. As a second cleavage product it yields, according to MAL-
ENGREAU, the A-histone, which can be readily precipitated by magnesium
and ammonium sulphates from the ordinary B-histone of the thymus
gland. The occurrence of A-histone in the gland has been verified by
BANG, and according to BANG and HUISKAMP the A-histone is not derived
from the nucleoprotein, as these investigators claim that it yields no his-
tone. According to BANG the nucleoprotein yields only an albuminate,
besides the nuclein, as cleavage products. According to GOUBAN we
have been dealing with three substances, namely a nucleoprotein which
does not yield any histone, and two nucleohistones, which correspond
to the nucleoalbumins A and B of MALENGREAU and form the mixture
of lime-nucleohistone of HUISKAMP. They occur in this last mentioned
mixture in a somewhat modified form due to the method of preparation.

The true nudeohistone, which is much richer in phosphorus (the
calcium salt containing, according to BANG, on an average 5.23 per cent
P), yields ordinary histone (or 2 histones) as one cleavage product and
free nucleic acid as the other. According to BANG, whose statements
on this point have been substantiated by MALENGREAU, it splits on saturat-
ing with NaCl into nucleic acid and histone without yielding any other
protein. On this account BANG does not consider this body as nudeo-
histone in the ordinary sense, i.e., not as a nucleoprotein, but as a histone

1 Lilienfeld, Zeitschr. f. physiol. Chem., 18; Kossel, ibid., 30 and 31; Bang., ibid.
30 and 31. See also Arch. f. Math, og Naturvidenskab, 25, Kristiania, 1902, and
Hofmeister's Beitrage, 1 and 4; Malengreau, La Cellule, 17 and 19; Huiskamp, Zeit-
schr. f. physiol. Chem., 32, 34 and 39; Gouban, Bioch. Centralbl., 9, 803.

368 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

nucleate. We cannot say whether this applies to the two nucleohistones
(if there are two). The nucleohistone or mixture of nucleohistones
behave like an acid, whose salts, especially the calcium salt, have been
closely studied by HUISKAMP. On the electrolysis of a solution of alkali
nucleohistone in water HUISKAMP also found that the nucleohistone
collected in traces at the anode, and that the sodium compound is there-
fore ionized in the solution. The nucleic acid-calcium histone-com-
pound has been prepared, it seems, in a pure state by BANG, and he found
the following average composition: C 43.69; H 5.60; N 16.87; S 0.47;
P 5.23; Ca 1.71 per cent.

The nucleohistone prepared by HUISKAMP'S method of precipitating with
CaCU is, according to him, a mixture of two nucleohistones, of which one, the
a-nucleohistone, contains 4.5 per cent phosphorus, and the other, /3-nucleohistone
contains, on the contrary, only in round numbers 3 per cent phosphorus.1 As
the two nucleohistones are poorer in phosphorus than the nucleic acid-histone
compound analyzed by BANG, and as HUISKAMP on cleavage of his preparation
did not, like BANG and MALENGREAU, obtain pure nucleic acid, it is still a ques-
tion whether HUISKAMP was working with sufficiently pure substances.

In regard to the methods used by the above investigators in the
isolation of the bodies in question we must refer to the original publications.

In connection with the so-called nucleohistone, attention must be called to
tissue fibrinogen and cell fibrinogen, which are compound proteins, and are claimed
by certain investigators to stand in close relation to the coagulation of the blood.
These may be in part nucleoproteins and in part also nucleohistones. To this same
group belong also the important cell constituents described by ALEX. SCHMIDT 2
and called cytoglobin and preglobulin. The cytoglobin, which is soluble in water,
may be considered as the alkali compound of preglobulin. 'The residue of the
cells left after complete extraction with alcohol, water, and salt solution has
been called cytin by ALEX. SCHMIDT.

Besides the above-mentioned and the ordinary bodies belonging to
the connective-tissue group, small quantities of fat, leudne, sucdnic
add, lactic add, sugar, and traces of iodothyrin are present. According
to GAUTIER 3 arsenic also occurs in very small amounts, and no doubt
here as well as in other organs it is related to the nuclein substances. The
richness in nuclein bodies explains the occurrence of large quantities
of purine bases, chiefly adenine, whose quantity, according to KOSSEL
and ScHiNDLER,4 is 1.79 p. m. in the fresh organ and 19.19 p. m. in the
dry substance, and guanine. The bodies thymine and (uradlf) obtained,
besides lysine and ammonia, by KUTSCHER, as products of autodiges-
tion of the gland, probably have a similar origin. Among the enzymes,

1 Zeitschr. f. physiol. Chem., 39.

2 See footnote 1, p. 307.

3 Compt. Rend., 129.

4 Zeitschr. f. physiol. Chem., 13; Kutscher, Zeitschr. f. physiol. Chem., 34.

THYMUS AND SPLEEN. 369

besides arginase, guanase, adenase, and proteolytic enzyme we must
especially mention the enzyme studied by JONES, 1 which acts like a nu-
clease, splitting off phosphoric acid and purine bases, from the nucleo-
proteins. This enzyme, contrary to trypsin, acts best in acid liquids, and
is readily destroyed by alkalies at body temperature. The quantitative
composition of the lymphocytes from the thymus of a calf is, according to
LILIENFELD'S analysis, as follows. The results are given in 1000 parts
of the dried substance:

Proteids 17.7

Leuconuclein 687 . 9

Histone 86.7

Lecithin 75. 1

Fat 40.2

Cholesterin 44.0

Glycogen 8.0

The dried substance of the leucocytes amounted to an average of
114.9 p. m. Potassium and phosphoric acid are prominent mineral
constituents. LILIENFELD found KH^PCU among the bodies soluble in
alcohol.

Attention must be called to the analyses of BANG,2 which show that
the thymus contains about the same quantity of nucleoprotein, but about
five times as much histone nucleate as the lymphatic glands — calculated
in both cases upon the same amount of dry substance. OIDTMAN 3 found
807.06 p. m. water, 192.74 p. m. organic and 0.2 p. m. inorganic sub-
stances in the gland of a child two weeks old.

In regard to the functions of the thymus it seems to be the general
view that this gland takes part in the recruiting of the blood lympho-
cytes and correspondingly belong to the lymphoid organs. On the other
hand also certain other observations indicate that it may belong to the
endocrinic organs. It is generally admitted that the extirpation of the
thymus leads to a reduction and change in the formation of bone. A
certain relation also exists with the organs of generation and perhaps
a reciprocal action also exists between it and other organs with internal
secretion.

The Spleen. The pulp of the spleen cannot be freed from blood.
The mass which is separated from the spleen capsule and the structural
tissue by pressure, and which ordinarily serves as material for chemical
investigations is, therefore a mixture of blood and spleen constituents.
For this reason the proteins of the spleen are little known. The nucleo-
protein isolated by LEVENE and MANDEL 4 is to be considered as a true

1 Zeitschr. f. physiol. Chem., 41.
2 1. c., Arch. f. Math., etc.

3 Cited from v. Gorup-Besanez, Lehrb. d. physiol. Chem., 4. Aufl., p. 732.

4 Bioch. Zeitschr., 5.

370 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

spleen constituent, and this nucleoprotein yields 25 per cent glutamic
acid on hydrolysis. Histone has not been directly detected in the spleen;
but its presence is to be admitted because KRASNOSSELSKY 1 was able
to isolate a histone-peptone as sulphate from the spleen. The ferruginous
albuminate has been considered as a spleen constituent for a long time,
and especially also a protein substance which does not coagulate on boil-
ing and which is precipitated by acetic acid and yields an ash contain-
ing much phosphoric acid and iron oxide. This substance is probably
identical with the nucleoproteins which later investigators such as SATO
and CAPEZZUOLI 2 have prepared from the spleen. These nucleoproteins,
which are modified products, contain iron in variable amounts and more
or less firmly combined.

The pulp of the spleen, when fresh, has an alkaline reaction, but
quickly turns acid, due partly to the formation of free paralactic add
and partly perhaps to glycerophosphoric add. Besides these two acids
there are found in the spleen also volatile fatty adds, as formic, acetic,
and butyric acids, as well as sucdnic add, neutral fats, cholesterin, traces
of leudne, inosite (in ox-spleen), scyllite, a body related to inosite (in the
spleen of Plagiostoma), glycogen (in dog-spleen), uric add, purine bases,
and jecorin. LEVENE found a glucothionic add in the spleen, i.e., an
acid which is related to chondroitin-sulphuric acid but not identical
therewith, and which gives a beautiful violet coloration with orcin and
hydrochloric acid. The question whether this glucothionic acid originates
from the above-mentioned nucleoprotein or from the mucoid substance
has not been decided (LEVENE and MANDEL). In regard to the question
whether this acid is a unit body or not we refer to the wrork of MANDEL
and NEUBERG and LEVENE and JACOBS.3

In the human and ox-spleen BuROW4 has found three phosphatides
which all contain iron in organic combination. Among these one is a
saturated diaminomonophosphatide and the other two are unsaturated
phosphatides.

Many enzymes are found in the spleen also, and certain of these
are of special interest. To these belong the uric-acid-forming enzyme,
the xanthine oxidase (BURIAN), which occurs in the spleen of many
animals, but not in man, and which transforms the oxypurines,
hypoxanthine, and xanthine into uric acid; also the deamidizing enzymes

1 Zeitschr. f. physiol. Chem., 49.

2 Sato, Bioch. Zeitschr., 22; Capezzuoli; Zeitschr. f. physiol. Chem., 60.

3 Levene, Zeitschr. f. physiol. Chem., 37; Levene and Mandel, ibid., 45 and 47;
Mandel and Neuberg, Bioch. Zeitschr., 13; Levene, ibid., 16; Neuberg, ibid., 16; Levene
and Jacobs, Journ. of experim. Medic., 10.

4 Bioch. Zeitschr., 25.

SPLEEN. 371

guanase and adenase (LEVENE, SCHITTENHELM, JONES and PARTRIDGE,
JONES and WINTERNITZ), by the first of which the guanine is transformed
into xanthine, and by the latter the adenine into hypoxanthine. The
guanase also occurs in the spleen of the ox and horse, but not ( JONES),
or only in small amounts (SCHITTENHELM), in the pig-spleen.1 The
spleen also contains two enzymes, lienases, as shown by HEDIN (and
ROWLAND), one of which, the a-lienase, acts chiefly in alkaline solution,
while the other, 6-lienase, is active only in acid reaction. These enzymes,
which without doubt stand in close relation to the leucocytes, not only
act autolytically upon the proteins of the spleen, but they also dissolve
fibrin and coagulated blood-serum. The spleen also contains nucleases
and besides, as TANAKA 2 has found for the pig-spleen, diastase, invertin,
lipase, urease, trypsin and an erepsin like enzyme.

Among the constituents of the spleen the deposit rich in iron, which
consists of ferruginous granules or conglomerate masses of them, and
which is derived from a transformation of the red blood-corpuscles, is of
special interest. It was closely studied by NASSE. This deposit does
not occur to the same extent in the spleen of all animals. It is found
especially abundant in the spleen of the horse. NASSE 3 on analyzing
the grains (from the spleen of a horse) obtained 840-630 p. m. organic
and 160-370 p. m. inorganic substances. These last consisted of 566-
726 p. m. Fe203, 205-388 p. m. P2O5, and 57 p. m. earths. The organic
substances consisted chiefly of proteins (660-800 p. m.), nuclein (52 p. m.
maximum), a yellow coloring-matter, extractive bodies, fat, cholesterin,
and lecithin.

In regard to the mineral constituents, it is to be observed that the
amount of iron in new-born and young animals is small (LAPICQUE,
KRUGER, and PERNOU), in adults more appreciable, and in old animals
sometimes very considerable. NASSE found nearly 50 p. m. iron in the
dried pulp of the spleen of an old horse. GUILLEMONAT and LAPICQUE 4
have determined the iron in man. They find no regular increase with
growth, but in most cases 0.17-0.39 p. m. (after subtracting the blood-
iron) calculated on the fresh substance. A remarkably high amount of
iron is not dependent upon old age, but is a residue from chronic diseases.
MAGNUS-LEVY found 0.72 p. m. iron in the fresh human spleen.

xSee Chapter XIV for the literature.

2 Hedin and Rowland, Zeitschr. f. physiol. Chem., 32, and Hedin, Journ. of Physiol.,
30, and Hammarsten's Festschr., 1906; Tanaka, Bioch. Zeitschr, 37.

3 Maly's Jahresber., 19, p. 315.

4 Lapicque, ibid., 20; Lapicque and Guillemonat, Compt, rend, de soc. biol., 48.
and Arch de Physiol. (5) 8; Kriiger and Pernou, Zeitschr. f. Biologic, 27; Nasse, cited
from Hoppe-Seyler, Physiol. Chem., 720.

372 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

On the analysis of the human spleen MAGNUS-LEVY found 784.7
parts water, 215.3 parts solids, 27.7 parts fat and 27.9 parts nitrogen in
1000 parts of the fresh organ. In the dog spleen, CORPER 1 found 750
to 770 p. m. water, and 120-150 p. m. ether soluble substances, of which
one-fourth consisted of cholesterin and three-fourths of lecithin. As
purine bases he found 1.1 p. m. guanine, 0.6 p. m. adenine, 0.15 p. m.
hypoxanthine and 0.04 p. m. xanthine.

In regard to the pathological processes going on in the spleen we must
specially recall the abundant re-formation of leucocytes in leucaemia and
the appearance of amyloid substance (see page 172).

The physiological functions of the spleen are little known, with the
exception of its importance in the formation of leucocytes. Some
consider the spleen as an organ for the dissolution of the red blood-
corpuscles, and the occurrence of the above-mentioned deposit rich in
iron seems to confirm this view, but this iron could in part have another
origin. ASHER and his collaborators GROSSENBACHER, ZIMMERMANN and
H. VOGEL have found that the spleen is an organ for the iron metabolism,
as they found in a splenectomized dog that the iron elimination was much
greater than in a dog with its spleen. R. BAYER2 has made a similar
observation on a splenectomized human being, and the spleen it seems
has the purpose of retaining for the organism the iron set free in the
metabolism and also in starvation metabolism.

The spleen has also been claimed to play a certain part in digestion
especially in pancreatic digestion. This organ is said by SCHIFF, HERZEN,
and others to be of importance in the production of trypsin in the pan-
creas. The investigations of HERZEN seem to confirm this relation, but
the recent work of PRYM 3 has made the assumption doubtful.

Splenectomized dogs require according to RICHET 4 for their mainte-
nance more food, about one-third more, than normal dogs. The spleen
makes a complete utilization of the food possible or diminishes its con-
sumption..

An increase in the quantity of uric acid eliminated in splenic leucaemia
has been observed by many investigators (see Chapter XIV), while the
reverse has been observed under the influence of quinine in large doses,
which produces an enlargement of the spleen. These facts give a rather
positive proof that there is a close relation between the spleen and the

1 Magnus-Levy, Bioch. Zeitschr., 24; H. J. Corper, Journ. of biol. Chem., 11.
2Asher and Grossenbacher, Centralbl. f. Physiol.. 22, 375, and Bioch. Zeitschr, 17 ;
Zimmermann, Bioch. Zeitschr., 17; R. Bayer, Bioch. Centralbl., 9, 815.

3 Schiff, cited by Herzen, Pfliiger's Arch., 30, 295, 308, and 84, and Maly's Jahr-
esber., 18; Prym, Pfltiger's Arch., 104 and 107; see also Chapter VIII.

4 Journ. de Physiol. et de Pathol. gen., 14 and 15.

THYROID GLAND. 373

formation of uric acid. This relation has been studied by HORBAO
ZEWSKI. He has shown that when the spleen-pulp and blood of calves
are allowed to act on each other, under certain conditions and certain tem-
perature, in the presence of air, large quantities of uric acid are formed,
and he has also shown that the uric acid originates from the nucleins of
the spleen.1 This behavior is explained by the above-mentioned inves-
tigations of BURIAN, SCHITTENHELM, JONES, and others on the enzymotic
formation of uric acid, and the deamidization of the purine bodies, and a
relation between the spleen and uric-acid formation is indisputable.
Still we cannot say that the spleen shows a special relation to the uric-acid
formation as compared with other organs (see Chapter XIV).

The spleen has the same property as the liver of retaining foreign
bodies, metals and metalloids.

The Thyroid Gland. The nature of the different protein substances
occurring in the thyroid gland has not been sufficiently studied, but at
present, through the researches of OSWALD, there are known at least two
bodies which are constituents of the so-called secretion of the glands,
the colloids. One of these, iodothyreoglobulin, behaves like a globulin, while
the other is a nucleoprotein (see also GouRLAY2). The iodine present
in the gland occurs chiefly in the firct body, while the arsenic, which has
been shown to be a normal constituent by GAUTIER and BERTRAND,;*
seems to be related to the nuclein substances.

According to OSWALD the iodothyreoglobulin occurs only in those
glands which contain colloid, while the colloid-free glands, the parenchyma-
tous goitre, and the glands of the new-born contain thyreoglobulin free
from iodine. The thyreoglobulin first becomes iodized into iodothyreo-
globulin on passing from the follicle-cells. Besides these mentioned
bodies leucine, xanthine, hypoxanthine, choline, iodothyrine, lactic and
sucdnic acids occur in the thyreoidea. Like certain other organs, sub-
stances also occur in the thyroid which act upon the blood pressure and
indeed partly as vasodilator and partly depressing but whose chemical
nature has not been positively established. Among the enzymes we
find lipases and catalases which, according to JuscHTSCHENKO,4 are
related to the corresponding enzymes of the blood. MAGNUS-LEVY 5
found 757 parts water, 243 parts solids, 43.8 parts fat, 26.8 parts nitrogen,
and 0.058 parts iron in 1000 parts of the human thyroid gland.

1 Monatshefte f. Chem., 10, and Wein. Sitzungsber. Math. Nat. Klasse, 100, Abt. 3.

2 Gourlay, Journ. of Physiol., 16; Oswald, Zeitschr. f. physiol. Chem., 32, and
Biochem. Centralbl., 1, 249.

3 Gautier, Compt. Rend., 129. See also ibid., 130, 131, 134, 135; Bertrand, ibid.,
134, 135.

4 Juschtschenko, Bioch. Zeitschr., 25 and Arch, scienc. biol. de St. Petersbourg, 15.

5 Bioch. Zeitschr., 24.

374 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

In " STRUMACYSTICA " HoppE-SEYLER found hardly any protein in the smaller
glandular vessels, but an excess of mucin, while in the larger he found a great
deal of protein, 70-80 p. m.1 Cholestehn is regularly found in such cysts, some-
times in such large quantities that the entire contents form a thick mass of cho-
lesterin plates. Crystals of calcium oxalate also occur frequently. The contents
of the struma cysts are sometimes of a brown color, due to decomposed coloring-
matter, methcemoglobin (and hsematin?). Bile-coloring matters have also been
found in such cysts. (In regard to the paralbumins and colloids which have been
found in struma cysts and colloid degeneration, see Chapter XII.)

Those substances which bear a close relation to the functions of the
gland seem to be of special interest.

The complete extirpation, as also the pathological destruction, of the
thyroid gland causes great disturbances, ending finally in death. In
dogs, after the total extirpation, a disturbance of the nervous and muscular
systems occurs, such as trembling and convulsions, and death generally
supervenes shortly after, most often during such an attack. The researches
of GLEY, VASSALE and GENERAL: 2 upon various animals have shown
that for the success of the operation it is of the greatest importance
whether the parathyroids, discovered by SANDSTROM,3 are removed at
the same time or not. In herbivora (rabbits) because of the anatomical
relations, the parathyroids are seldom extirpated in the operation of
the removal of the thyroid, the tetany does not regularly occur and
the disturbance in metabolism is most striking. If these glands are
not extirpated in dogs, the tetany also does not appear, and the dis-
turbances in metabolism occur. In human beings, after the removal of
the gland by operation, different disturbances appear, such as nervous
symptoms, diminished intelligence, dryness of the skin, falling out of
the hair, and, on the whole, those symptoms which are included under
the name cachexia thyreopriva, death coming gradually. Among these
symptoms must be mentioned the peculiar slimy infiltration and exuber-
ance of the connective tissue called myxcedema.

All these conditions indicate that the thyroids belong to those glands
with internal secretion, so called endocrinic glands. The most con-
vincing proof of this is the fact that the ordinary symptoms do not occur
if a small piece of the gland is allowed to remain in the body, or even
when a piece of the gland is transplanted in any part of the body. The
observations of ASHER and FLACK 4 that the irritation of the nerves of the
thyroid causes an internal secretion from the thyroid gland into the
blood, is of great interest in this connection. A further proof of practical

iPhysiol. Chem., p. 721.

2Gley, Compt. rend. soc. biol., 1891, and Arch, de Physiol (5), 4; Vassale and
General!, Arch. Ital. d. Biol., 25 and 26.
•Upsala Lakaref. Forh., 15 (1880).
4 Asher and Flack, Zeitschr. f. Biol., 55.

THYROID GLAND. 375

importance is that the injurious results from removal of the thyroids
can be counteracted by the introduction of artificial extracts of the
thyroid gland into the body or by feeding with thyroid glands.

Of the disturbances in metabolism which occur on the extirpation
or reduction of the thyroid function (athyreoidismus or hypothyreoid-
ismus) we must especially mention the reduction in the protein catabolism
which in a starving dog without thyroids may fall to about one-half of
the starvation protein metabolism in a normal dog of the same size
(FALTA and collaborators J). The reverse is observed when large quan-
tities of the thyroid gland substance is fed, namely, a strong increase in
the protein metabolism, besides certain other symptoms. BASEDOW'S
disease is also considered as a form of hyperthyreoidismus which, by an.
increased activity of the glands, brings about an overproduction of the
specific secretion. There does not seem to be any doubt that the thyroid
glands stand in close relation to other endocrinic glands although for the
present we are unable to survey this very complicated condition. One
side of this reciprocal action with other organs, which is of special impor-
tance, is the relation of the thyroids to glycosuria, which will be discussed
in a following chapter.

The glands with internal secretion, the so-called endocrinic glands,
to which the adrenals belong, which will be discussed below, and the
hypophysis, are of especially great interest because of the reciprpcal
action which they exert among each other and with other organs. A
chemical correlation exists between different organs, of a kind, that
bodies which are formed in one organ can awaken or regulate the func-
tions of another organ or other organs. These chemically active sub-
stances, which awaken or regulate the activity of other organs have
been given the group name hormone (6p/xaw = I awaken or excite) by STAR-
LING and to this group belong the specifically active constituents of the
endocrinic glands.

It is impossible for the present to state anything about the kind of
bodies having a specific action in the thyroid gland or anything about the
importance of the bases found by certain investigators, such as S. FRANK-
EL, DRECHSEL, and KocHER,2 as these bodies have not been characterized
sufficiently. It seems proved that the specifically active substance is, as
first shown by NOTKINS and OSWALD ,4 a protein substance: NOTKIN'S
thyreoproteid, OSWALD'S thyreoglobulin. This does not conflict with the views

1 Eppinger, Falta and Rudinger, Zeitschr. f. klin. Med., 66.

2Frankel, Wein. med. Blatter, 1895 and 1896; Drechsel and Kocher, Centralbl.
f . Physiol., 9, 705.

8 Wien. med. Wochenschr., 1895, and Virchow's Arch., 144, Suppl., 224.
4 Zeitschr. f. physioi. Chem., 32, and Bioch. Centralbl., 1, 249.

376 CHYLE, LYMPH, TKANSUDATES AND EXUDATES.

of BAUMANN and Roos that the active substance is iodoihyrin, as this
can be produced as a cleavage product from the iodothyreoglobulin. In
fact OSWALD has found in the tryptic digestion of iodothyreoglobulin
that a substance similar to iodothyrin is produced; important inves-
tigations 1 nevertheless make it probable that the thyreoglobulin is
the active substance and not the iodothyrin. There are several reasons
why the action of the thyroid gland substance is not due to one substance,
but to several.

Iodothyrin is considered by BAUMANN, who first showed that the thyroid con-
tained iodine and who with Roos 2 proved the importance of this substance for the
physiological activity of the gland, as the only active substance. By boiling the
finely divided gland with dilute sulphuric acid BAUMANN obtained iodothyrin
as an amorphous, brown mass, nearly insoluble in water but readily soluble in
alkali and precipitated again by the addition of acid. The iodothyrin, which
is not a unit body, has a variable content of iodine and is not a protein substance.
According to v. FURTH and SCHWARZ it is probably a melanoid-like transforma-
tion product of the iodized protein of the gland produced by the action of the
acid.

Thyreoglobulin or iodothyreoglobulin was obtained by OSWALD from
the watery extract of the gland by half saturating with ammonium sul-
phate. It has the properties of the globulins and with the exception of
the iodine content it has about the same composition as the proteins.
The amount of iodine varies: 0.46 per cent in pigs, 0.86 per cent in oxen,
and 0.34 per cent in man. In the iodothyreoglobulin of the ox, NUREM-
BERG 3 found 0.59-0.86 per cent iodine and 1.83-2.0 per cent sulphur.
In young animals, whose glands contain no iodine, the thyreoglobulin
is iodine-free. Thyreoglobulin on taking up iodine is converted into
iodothyreoglobulin. By introducing iodine salts the iodine content of the
iodothyreoglobulin can be raised in living animals and thus the physiological
activity increased (OSWALD). The amount of iodine in the gland is
markedly dependent upon the food.

JOLIN has examined a large number of thyroid glands from healthy and
diseased persons (in SWEDEN), for their iodine content. In 28 children, ages

1 See Oswald. Arch. f. exp. Path. u. Pharm., 60; Pick and Pineles, Zeitschr. f .
exp. Path. u. Therap., 7.

2 In regard to this subject, see Baumann and Roos, Zeitschr. f. physiol. Chem., 21
and 22; also Baumann, Munch, med. Wochenschr., 1896; Baumann and Goldmann,
ibid.] Roos., ibid.', v. Fiirth and Schwarz, Pfliiger's Arch., 124. An extensive review
of the literature on the action of iodothyrin and the thyroid preparations can be found
in Roos, Zeitschr. f. physiol. Chem., 22, 18. In regard to their action in protein catabo-
lism and in metabolism, see F. Voit, Zeitschr. f. Biologie, 35; Schondorff, Pliiger's Arch.,
67, and Andersson and Bergman, Skand. Arch. f. Physiol., 8; Magnus-Levy, Zeitschr.
f. klin. Med., 32. In regard to the function of the thyroid gland see also Sw. Vincent,
Innere Sekretion etc. Ergebnisse d. Physiol., 11, 218-302.

3 Bioch. Zeitschr., 16.

ADRENAL BODIES. 377

varying between 1 and 10 years, he found an average of 0.28 p. m. iodine in the
glands. In 108 normal glands above 10 years old or adults the iodine content
varied with an average of 1.56 p. in. iodine. In glands from persons after using
iodine preparations (34 cases) the iodine content was 2.56 p. m. The amount
of silicic acid in normal thyroid glands was found by H. SCHULZ 1 to be on an
average 0.084 p. m., calculated on the dry substance. In goitres from GREIFSWALD
and ZURICH he found 0.175 and 0.434 p. m., respectively. There does not seem
to be any connection between the silicic acid content of the drinking water and
the occurrence of goitre.

We cannot enter into a discussion as to the various hypotheses and
theories in regard to the mode of action of the constituents of the thyroids.
In the tetany appearing after parathyroidectomy many investigators
find an increased elimination of calcium, nitrogen and ammonia and the
hypothesis has been suggested that the tetany depends upon an increased
irritability of the nervous system due to lack of calcium. The fact as
found by several experimenters that a diminished calcium content of the
organs in question does not occur, speaks against this theory. On the
contrary, it seems to be generally admitted that lime salts reduce or
prevent the tetany and, according to FiiouiN,2 this depends upon the lime
combining with the carbonic acid produced, which is the cause of the
tetany. The tetany is produced at least from a poison which is formed
only on the removal of the parathyroids or if it is regularly produced
it is made harmless by these organs.

G. MANSFIELD and FR. MtJLLER3 have made investigations in regard
to the action of the thyroids upon protein metabolism which indicate
that lack of oxygen acts as an excitant upon the thyroids and that the
increased protein catabolism, which occurs to a mean degree with lack
of oxygen, depends upon a hyperfunction of the thyroid glands brought
on by this condition. With greater lack of oxygen besides this a general
damage to the protoplasm of the body cells may occur.4

The Adrenal Bodies. Besides proteins, substances of the connect-
ive tissue, and salts, there occur in the suprarenal capsule inosite, purine
bases, especially xanthine (OKER-BLOM), phosphatides and glycerophos-
phoric add, which is probably a decomposition product of the latter.
The earlier . accounts of the occurrence of benzole acid, hippuric acid,
and bile-acids are, on the contrary, doubtful, and are not substantiated
by recent investigations (STADELMANN 5) . The medullary substance

1 Jolin, Hammarsten's Festschr., 1906; H. Schulz, Bioch. Zeitschr., 46.
2Compt. Rend., 148.

3 Pfluger's Arch., 143.

4 A very complete discussion of the physiology of the thyroid gland and the pertinent
literature may be found in Sw. Vincent, Ergebnisse"der Physiologic, 11, 218-302.

5Oker-Blom, Zeitschr. f. physiol. Chem., 28; Stadelmann, ibid., 18, which also
contains the literature on this subject.

378 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

contains the so-called chromaffine tissue, i.e., cells, whose substance is
colored brown by chromic acid or chromates.

Earlier investigators, like VULPIAN and ARNOLD, have found, in the
medulla, a chromogen which has been considered as connected with the
abnormal pigmentation of the skin in ADDISON'S disease. This chromogen,
which is transformed by air, light, alkalies, iodine, and other bodies into
a red pigment, seems, on the contrary, to be related to the substance
adrenalin, of the gland which produces an increase in the blood-pressure.
Choline has been shown to have a reverse effect upon this blood-pressure
raising action, and LOHMANN has shown that it is formed in the cortical
substance of the adrenals. In the cortical this last-mentioned exper-
imenter l has found besides neurin, another not known base. That
the watery extract of the adrenals has a blood-pressure raising action
was shown by OLIVER and SCHAFER, CYBULSKI and SzYMONOWicz.2
The substance which is here active was formerly called sphygmogenin
and has also other actions besides bringing about a marked increase
in blood-pressure by the strong contraction of the muscles of the periphery
vessels; for instance, it can bring about glycosuria and mydriasis, espe-
cially in the frog's eye, has been chemically investigated by numerous
experimenters.3 v. FURTH calls it suprarenin, ABEL epinephrin. and
'TAKAMINE adrenalin. This last name seems to be the most generally
accepted one.

Adrenalin (suprarenin epinephrin) (methylaminoethanolpyrocatechin)

CH

(HO)C C.CH(OH).CH2.NHCH3

(HO)C CH

V
CH

The constitution of adrenalin has been essentially proved by FRIEDMANN,*
and he has shown the correctness of the above formula, which was given by
PAULY. The synthesis of adrenalin, which was first performed by STOLZ,S is
also in accordance with this formula. By the action of methylamine upon
chloracetopyrocatechin we obtain methylaminoaceto-pyrocatechin :

C6H3(OH)2.COCH2C1+NH.CH3=C6H3(OH)2.COCH2.NHCH;.HC1,

which yields adrenalin on reduction.

1 Centralbl. f. Physiol., 21, and Pfliiger's Arch., 118 and Zeitschr. f. Biol., 56.

2 Oliver and Schafer, Proceed, of Physiol. Soc., London, 1895. Further literature
on the function of the adrenals may be found in Sw. Vincent, Innere Sekretion, etc.
Ergebnisse d. Physiol., 9, 505-585.

3 The literature on this subject may be found in Abderhalden's Bioch. Handlexikon
Bd. 5, s. 454-495.

4 Hofmeister's Beitrage, 8.

6 Ber. d. d. chem. Gesellsch., 37.

ADRENALIN. 379

The synthetically prepared adrenalin is optically inactive d-Z-adrenalin,
while that from the adrenals is optically active Z-adrenalin. FLACHER has
divided the racemic adrenalin into the two optically active components,
and the identity of the so-obtained synthetical adrenalin with the natural
has been shown by ABDERHALDEN and FR. MuLLER.1 These last inves-
tigators also found that the /-adrenalin had at least 15 times as strong
an action upon the blood-pressure as the d-adrenalin, and later ABDER-
HALDEN with THIES and SLAVU found that the /-adrenalin had also in
other respects a much stronger action than d-adrenalin.

Adrenalin crystallizes in masses of needles or rhombic leaves. It is
soluble in water, and can be precipitated from its solution by ammonia
as a crystalline substance. Its aqueous solution containing hydrochloric
acid is levorotatory: (a)D= —50.72° (ABDERHALDEN and GUGGENHEIM 2).
On heating adrenalin it turns yellowish-brown at about 205° and decom-
poses at about 218° C. Its solution turns emerald green with ferric chlor-
ide in acid solution and carmine red in alkaline solution. Adrenalin
reduces FEHLING'S solution and ammoniacal silver solution.

Among the reactions for adrenalin in solution we must especially
mention the red coloration which is obtained on the addition of an oxidizing
medium such as iodine or bi-iodate and dilute phosphoric acid and warm-
ing (FRANKEL and ALLERS), or of mercuric chloride in the presence of a
catalyst such as the lime salts in tap-water (COMESATTI). These reactions
are extremely delicate, 1 : 1000000-2000000. A still more delicate reaction
(1:5000000) is the one suggested by Ewms,3 namely a characteristic
red coloration is obtained on adding a 0.1 per cent solution of potassium
persulphate and warming gently in a boiling water-bath.

As above stated, it has been considered for some time that the color
of the skin in ADDISON'S disease was connected with the adrenals or their
chromogen. We know nothing positive in regard to this relation,
but it is nevertheless of interest that pigments, and finally melanins or
at least dark-brown substances, can be produced from adrenalin by the
action of enzymes. NEUBERG has brought about such melanin forma-
tion by the extract from the metastases of a melanoma of the adrenals
and also with the extract of the ink-sac of the sepia, and ABDERHALDEN
and GUGGENHEIM 4 with tyrosinase. This would indicate a close relation

' 1 Flacher, Zeitschr. f. physiol. Chem., 58; Abderhalden and Franz Miiller, ibid.
58; with Thies, ibid., 59; with Slavu, ibid., 59; with Kautsch and Miiller, ibid., 61 and
62; see also Frohlich, Centralbl. f. physiol., 23 and Waterman, Zeitschr. f. physiol.
Chem., 63.

2 Zeitschr. f. physiol. Chem., 57.

3 Frankel and Allers, Bioch. Zeitschr., 18; Comesatti, Munch, med. Wochenschr.
1908 and Physiol. Centralbl., 23; Ewins, Journ. of Physiol., 40.

4 Neuberg, Bioch. Zeitschr., 8; Abderhalden and Guggenheim, Zeitschr. f. physiol.
Chem., 57.

380 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

between adrenalin and tyrosine, which also gives melanin with the sepia
enzyme, and indeed tyrosine has been considered as the probable mother-
substance of adrenalin (HALLE). The investigations of E WIN'S and
LAIDAW l to prove this last-mentioned possibility have not given any
support thereto.

Besides the action of producing a rise in the blood-pressure, adrenalin
is also of special interest because, as first shown by BmM,2 it also has a
glycosuric action. We will discuss the question of adrenalin glycosuria
and the relation which seems to exist between the internal secretions
of the thyroids, the adrenals and the pancreas, when we treat of the
formation of sugar and pancreas diabetes. We cannot here enter into
the question of the reciprocal action between the adrenals and the other
organs.

The hypophysis or pituitary gland has been little studied from a chemical
standpoint. An extract of the gland shows, by its action, a certain similarity to an
extract of the adrenals in that it causes a rise in blood pressure and by causing a
dilation of the pupils of the frog's eye, Still no adrenalin could be detected in the
gland. Also no iodine occurs in the glands (WELLS, DENIS 3).

The gland consists essentially of two parts, one an outside formation of vascular-
glandular epithelium and a lower nervous part the infundibular part. The out-
side part seems to have a relation to the growth of the tissues and skeleton and
acromegalie and gigantism are claimed by many investigators to be related to this
part. The infundibular part, on the contrary, contains the specific bodies which
raises the blood -pressure and stimulates the smooth muscles of the uterus and
upon the kidney secretion. The relation of the hypophysis to other endocrinic glands
is still very much disputed.

1 Halle, Hofmeister's Beitrage 8; Ewins and Laidaw, Journ. of Physiol., 40.

2 Deutsch. Arch, f . klin. Med., 91 and Pfluger's Arch., 90.

3 H. G. Wells, Journ. of biol. Chem., 7; W. Denis, ibid., 9.

CHAPTER VII.
THE LIVER.

THE liver, which is the largest gland of the body, stands in close
relation to the glands mentioned in Chapter VI. The importance
of this organ for the assimilation of the food-stuffs and for the phys-
iological composition of the blood is evident from the fact that the
blood coming from the digestive tract, laden with absorbed bodies, must
circulate through the liver before it is driven by the heart through the
different organs and tissues. An assimilation of food-stuffs in the liver
has been positively shown in the first place for carbohydrates in that the
liver constructs a polysaccharide glycogen from hexoses, which according to
the needs is then again retransformed into glucose. The liver is a storage
organ for fats and takes up food fat as well as fat from depots (in
starvation) and as it seems, at least in part, prepares them so that they can
be further used in the animal body.

We are not clear as to what extent an assimilation of products of pro-
tein digestion takes place in the liver. The subject will be discussed in detail
under absorption in Chapter VIII. It is claimed that the liver can serve
as a storage organ for proteins,1 and it is at least certain that it retains
alien protein which is brought to it by the blood.2 The retention
of alien protein stands probably in close relationship to the ability of
the liver to take up and retain foreign substances as a group from the blood.
This is not only true for different metals but also, as shown by several
investigators,3 alkaloids which perhaps are also partly decomposed in the
liver. Toxins are also withheld by the liver and hence this organ has
a protective action against poisons.

The formation of glycogen from glucose is one of the numerous syn-
theses occurring in the liver and this is no doubt the one which takes
place to the greatest extent. Other syntheses in the liver are, for example,

1 See Seitz, Pfluger's Arch., Ill and Asher and Boehm, Zeitschr. f. Biol. 51.

2 See Reach, Bioch. Zeitschr., 16 and Pacchioni and Carlini, Maly's Jahresb., 39.

3 Roger, Action du foie sur les poisons (Paris, 1887), which quotes the works of
Schiff, Heger and others; also W. N. Woronzow, Maly's Jahresb., 40 and Z. Vamossy,
ibid., 40.

381

382 THE LIVER.

the formation of urea or uric acid (in birds) from ammonium salts, the
formation of etheral sulphuric acids and conjugated glucuronic acids
from the phenols produced in intestinal putrefaction and the recently
shown syntheses of amino-acids in the liver. On the other hand a
deamidation of amino-acids and purine bodies, hydrolyses, oxidations,
reductions and fermentative processes of various kinds occur in the liver.
Because of these diverse processes, the results of which we must espe-
cially mention the formation of bile as well as the fact that the liver is
introduced between the intestine and the general circulation, makes the
liver a central organ for metabolism.

Among the numerous chemical processes which take place in the liver
there are especially two which give special interest to this organ, namely,
the formation of glycogen or the carbohydrate metabolism in the liver,
and the formation of bile. For this reason only these two processes will
be discussed in this chapter while the others will be discussed in other
chapters and in other connection. Before we begin to discuss these two
processes a short review of the constituents and the chemical com-
position of the liver seems to be appropriate.

The reaction of the liver-cells is alkaline toward litmus during life,
but becomes acid after death, due to a formation of lactic acid, chiefly
fermentation lactic acid and other organic acids (MORISHIMA, MAGNUS-
LEVY 1). A coagulation of the protoplasmic proteins in the cells probably
takes place. A positive difference between the proteins of the dead
and the living, non-coagulated protoplasm has not been observed.

The proteins of the liver were first carefully investigated by PL6sz.
He found in the watery extract of the liver an albuminous substance
which coagulates at 45° C. (globulin, HALLIBURTON), also a globulin
which coagulates at 75° C., a nuckoalbumin which coagulates at 70° C.,
and lastly a protein body which is closely related to the coagulated albumins
and which is insoluble in dilute acids or alkalies at the ordinary tem-
perature, but dissolves on the application of heat, being converted into
an albuminate. HALLIBURTON 2 found two globulins in the liver-cells,
one of which coagulates at 68-70° C., and the other at 45-50° C. He
also found, besides traces of albumin, a nucleoprotein which possessed
1.45 per cent phosphorus and a coagulation-point of 60° C. POHL has
obtained an " organ plasma " by extracting the finely divided liver
which had previously been entirely freed from blood by washing with
8 p. m. NaCl solution, in which he was able to detect a globulin having
a low coagulation temperature. The very variable phosphorus content

1 Morishima, Arch. f. exp. Path. u. Pharm., 43; Magnus-Levy, Hofmeister's Bei-
trage, 2.

2 P16sz, Pfliiger's Arch., 7; Halliburton, Journ. of Physiol., 13, Suppl. 1892.

PROTEINS OF THE LIVER. 383

(0.28-1.3 per cent) of this globulin as well as the insolubility of the pre-
cipitates produced by little acid, in an excess of acid, and in neutral salts
seem to indicate that we here have a mixture which consists chiefly of
nucleoproteins and not of globulins. The almost complete digestibility
with pepsin-hydrochloric acid does not controvert this assumption,
because, as is known, nucleoproteins may on digestion yield no residue
(see Chapter II). Nor can we be positive concerning the nature of
the liver-globulin found by DASTRE,' having a coagulation temperature
of 56° C. The proteins extractable from the liver without modification
must be thoroughly investigated.

Besides the above-mentioned proteins, which are very soluble, the
liver-cells contain large quantities of difficultly soluble protein bodies
(see PL6sz). The liver also contains, as first shown by ST. ZALESKI
and later substantiated by several other investigators, ferruginous
proteins of different kinds.2. The chief portion of the protein substances
in the liver seems in fact to consist of ferruginous nucleoproteins. On
boiling the liver with water, such a. nucleoprotein or perhaps several
are split, and a solution is obtained containing a nucleic-acid-rich nucleo-
protein or a mixture of these which are precipitable by acids. This
protein or protein mixture, which has been called ferratin by SCHMIEDE-
BERG,3 has been studied by WoHLGEMUTH.4 The quantity of phos-
phorus was 3.06 per cent. As cleavage products on hydrolysis he found
/-xylose, or at least a pentose, the four nuclein bases, and also arginine,
lysine (and histidine?), tyrosine, leucine, glycocoll, alanine, a-prc4ine,
glutamic acid, aspartic acid, phenylalanine, oxyaminosuberic acid, and
oxydiaminosebacic acid (see Chapter II). The Z-xylose depends, no
doubt, at least in part, upon the guanylic acid isolated from the liver,
by LEVENE and MANDELA and the finding of adenine among the cleavage
products also indicates the presence of a thymonucleic acid. There does
not seem to be any doubt that the ferratin, as above stated, is a mixture,
and the correctness of this assumption is shown by the recent investiga-
tions of SCAFFIDI and SALKOWSKI.G

1 Pohl, Hofmeister's Beitrage, 7; Dastre, Compt. rend. soc. biolog., 58.

2 St. Zaleski, Zeitschr. f. physiol. Chem., 10, 486; Weltering, ibid., 21; Spitzer,
Pfliiger's Arch., 67.

3 Arch. f. exp. Path. u. Pharm., 33; see also Vay, Zeitschr. f. physiol. Chem., 20.
4Wohlgemuth, Zeitschr. f. physiol. Chem., 37, 42, and 44, and Ber. d. d. chem.

Gesellsch., 37. See on liver nucleoproteins also Salkowski, Berl. klin. Wochenschr.,
1895; Hammarsten, Zeitschr. f. physiol. Chem., 19; Blumenthal, Zeitschr. f. klin. Med.,
34.

5 Bioch. Zeitschr., 10.

6 Scaffidi, Zeitschr. f. physiol. Chem., 58; Salkowski, ibid., 58.

384 THE LIVER.

The yellow or brown pigment of the liver has been little studied. DASTRE
and FLORESCO 1 differentiate, in vertebrates and certain invertebrates, between a
ferruginous pigment soluble in water, ferrine, and a pigment soluble in chloroform
and insoluble in water, chlorochrome. They have not isolated these pigments in a
pure condition. In certain invertebrates chlorophyll originating from the food also
occurs in the liver.

The fat of the liver occurs partly as very small globules and partly
(especially in nursing children and suckling animals, as also after food
rich in fat) as rather large fat-drops. The occurrence of a fatty infiltra-
tion, i.e., a transportation of fat to the liver, may not only be produced
by an excess of fat in the food (NOEL-PATON), but also by a migration
from other parts of the body under abnormal conditions, such as poison-
ing with phosphorus, phlorhizin, and certain other bodies (LEO, LEBEDEFF,
ROSENFELD, and others 2) . The fatty infiltration occurring in poisoning,
and which is accompanied with degenerative changes in the cells, may
cause a diminution in the amount of protein and a rise in the water con-
tent. If the amount of fat in the liver is increased by an infiltration, the
water decreases correspondingly, while the quantity of the other solids
remains little changed. Changes of a kind may occur, so that, because
of the antipathy (ROSENFELD, BoTTAZZi3) existing between glycogen
and fat, a liver rich in fat is habitually poor in glycogen. The reverse
occurs after feeding with carbohydrate-rich food, namely, the liver is
rich in glycogen and poor in fat.

The composition of the liver-fat seems to vary not only in different
animals, but is variable with differing conditions. Thus NOEL-PATON
found that the liver-fat in man and several animals was poorer in oleic
acid and had a correspondingly higher melting-point than the fat from
the subcutaneous connective tissue, while ROSENFELD 4 observed the
opposite condition on feeding dogs with mutton-fat.

Several investigators, HARTLEY, LEATHES and MOTTRAM suggested
as a difference between the fat of the liver and the connective tissues,
the great amount in the first of unsaturated, higher fatty acids. Accord-
ing to HARTLEY 5 the fat of the pig liver contains palmitic acid, stearic

1 Arch, de Physiol. (5), 10.

2 Noel-Paton, Journ. of Physiol., 19; Leo, Zeitschr. f. physiol. Chem., 9; Lebedeff,
Pfliiger's Arch., 31; Athanasiu, Pfliiger's Arch., 74; Taylor, Journ. of Exp. Med., 4;
Kraus u. Sommer, Hofmeister's Beitrage, 2; Rosenfeld, Zeitschr. f. klin. Med., 36.
See also Rosenfeld, Ergebnisse der Physiologic, 1, Abt. 1, and Berl. klin. Wochenschr.
1904; Schwalbe, Centralbl. f. Physiol., 18, p. 319; Shibata, Bioch. Zeitschr., 37.

3 Arch. Ital. d. Biol, 48 (1908), cited in Bioch. Centralbl., 7, p. 833.

4 Cited by Lummert, Pfluger's Arch., 71. In regard to the liver-fat of children,
see Thiemich, Zeitschr. f. physiol. Chem., 26.

6 Hartley, Journ. of Physiol., 38; Leathes and Meyer-Wedell, ibid., 38; Mottram,
ibid., 38.

PHOSPHATIDES OF THE LIVER. 383

acid, and oleic acid which is not identical with the ordinary oleic acid, also
linoleic acid and an acid having the formula C2oH3202. A part of
these unsaturated fatty acids are contained in the phosphatides but as
the unsaturated acids are about one-half of the fatty acids they must
also occur in the fats. The abundant occurrence of unsaturated fatty
acids is considered by the above-mentioned investigators as the first
step in the cleavage of the transportable fat from the fat tissues to the
liver and destined for use in the body. There is no doubt that the phos-
phatides are of great importance for this transformation of the fat.

Phosphatides, which were formerly designated lecithin, and whose
quantity is generally calculated as such, also belong to the normal con-
stituents of the liver. The quantity (as lecithin) amounts to over 23.5
p. m. according to NoEL-PATON.1 In starvation the lecithin, according
to NOEL-PATON, forms the greater part of the ethereal extract, while
with food rich in fat, on the contrary, it forms the smaller part. In
the liver of a healthy dog BASKOFF2 found 84 p. m. phosphatides (cal-
culated as lecithin) in the dry substance. The phosphatides are undoubt-
edly of various kinds, but they have not been closely studied. Among
others, we have lecithin and the so-called jecorin. Cholesterin is also a
constituent of the liver, although only in small quantities, and KoNDO3
finds that cholesterin ester occurs in the liver.

Jecorin was first found by DRECHSEL in the liver of horses, and also in the
liver of a dolphin, and later by BALDI in the liver and spleen of other animals, in
the muscles and blood of the horse, and in the human brain. It contains sul-
phur and phosphorus, but its constitution is not positively known. Jecorin dis-
solves in ether, but is precipitated from this solution by alcohol. It reduces
copper oxide, and gives a wine-red coloration with an ammoniacal silver-solution.
On boiling with alkali and then cooling it solidifies to a gelatinous mass. MANASSE
has detected glucose as osazone in the carbohydrate complex of jecorin.

The statement by BING that jecorin is a combination of lecithin and glucose
does not follow from the analyses of jecorin thus far known. Jecorin contains
sulphur, even as much as 2.75 per cent, and further the relation of P:N in lecithin is
1:1, while in jecorin it is quite different, 1: 2 to 1: 6. According to the investiga-
tions of BASKOFF the liver jecorin, prepared according to DRECHSEL'S sugges-
tion, and when it is so pure that it is completely soluble in ether, and quantitatively
precipitated by alcohol from this solution, is a rather constant compound at
least in regard to the N, P and glucose content. BASKOFF found as average 2.55
per cent N, 2.87 per cent P, and about 14 per cent glucose. The relation P:N
was nearly 1 :2 and therefore jecorin is correspondingly a diaminomonophosphatide.

The variable composition and divergent properties of the jecorin isolated and
analyzed by various investigators 4 depends, according to BASKOFF, upon imper-

1 1. c. See also Heffter, Arch. f. exp. Path. u. Pharm., 28.

2 Zeitschr. f. physiol. Chem., 62.

3 Bioch. Zeitschr., 26.

4 Drechsel, Ber. d. sachs. Gesellsch. d. Wissensch., 1886, p. 44, and Zeitsch. f. Biol-
ogie, 33; Baldi, Arch. f. (Anat. u.) Physiol., 1887, Suppl., 100; Manasse, Zeitschr. f.
physiol. Chem., 20; Bing, Centralbl. f. Physiol., 12, and Skand. Arch. f. Physiol., 9;

386 THE LIVER.

feet purification. His investigations do not give any explanation for the quantity
of sulphur and it is very probable that jecorin is only a mixture of several bodies
among which a sulphurized and a phosphorized substance occurs. According
to BASKOFF it is very probable that the jecorin is a decomposition product of lecithin
(or other phosphatides) .

Another phosphatide, which does not reduce directly or after boiling with acid,
has been called heparphosphatide by BASKOFF. In certain respects this body is
similar to cuorin, and the relation P:N= 1.45:1, although it was not pure.

Among the extractive substances besides glycogen, which will be treated
later, rather large quantities of the purine bases occur. KOSSEL 1 found
in 1000 parts of the dried substance 1.97 p. m. guanine, 1.34 p. m. hypo-
xanthine, and 1.21 p. m. xanthine. Adenine is also contained in the
liver. In addition there are found urea and uric acid (especially in
birds), and indeed in larger quantities than in the blood, paralactic acid,
choline, leucine, taurine, and cystine. In pathological cases inosite and
amino-acids have been detected. The occurrence of bile-coloring matters
in the liver-cell under normal conditions is doubtful; but in retention of
the bile the cells may absorb the coloring-matter and become colored
thereby.

A large number of enzymes are found in the liver, such as catalases,
oxidases, aldehydases and hydrolytic enzymes of various kinds; the dias-
tase acting upon glycogen, the Upases and the different proteolytic enzymes.
Nucleases and the nucleic acid splitting enzymes of different kinds men-
tioned in Chapter II have been formed in the liver and deamidases for
amino-acids as well as purine bodies also occur in the liver. The last
group of deamidases show a different behavior in regard to their occurrence
in different animals and the same is true for the uric acid forming and
uric acid destroying enzymes (Chapter XIV). We must also mention the
arginase which splits off urea from arginine.

The proteolytic enzymes of the liver are of special interest, especially
in regard to the study of the autolysis of this organ. The processes
in the liver in phosphorus poisoning and in acute yellow atrophy of the
liver are considered as an intravitally increased autolysis. In these
cases a softening of the organ takes place, and proteoses, mono- and
diamino-acids, and other bodies are produced, which may also be found
in the urine, and although they may not all be derived from the liver
(NEUBERG and RICHTER), they are at least in part derived from this organ.
WAKEMAN has found in phosphorus poisoning that not only is the quan-
tity of nitrogen markedly diminished in the liver (of dogs), but also
that the quantity of nitrogen of the hexone bases is diminished, and

Meinertz, Zeitschr. f. physiol. Chem., 46; Siegfried and Mark, ibid.-, Paul Mayer,
Bioch. Zeitschr., 1, and Baskoff, Zeitschr. f. physiol. Chem., 57, 61, 62.
1 Zeitschr. f. physiol. Chem., 8.

IRON IN THE LIVER. 387

that the part of the protein molecule richer in nitrogen is first removed
and eliminated under these conditions. A similar condition has been
observed by WELLS in the idiophatic, acute yellow atrophy of the liver.
In consideration of the variable results for the diamino-nitrogen even
under normal conditions (GLIKIN and A. LOEWY J), it is desirable that
a greater number of observations be made on this subject. The increased
consumption of glycogen under the above-mentioned pathological con-
ditions may also be considered as an increased autolysis, while the claim
of certain observers that fat is formed in the autolysis of the liver is,
according to SAXL,2 to be considered only as a more pronounced appear-
ance of the fat previously occurring in the organ.

Besides the above-mentioned organic constituents in the liver we must
mention the glucoihionic add found by MANDEL and LEVENE,S whose
chemical individuality is doubted.

The mineral bodies of the liver consist of phosphoric acid, potassium,
S3dium, alkaline earths, and chlorine. The potassium is in excess of
the sodium. Iron is a regular constituent of the liver, but it occurs
in very variable amounts. BTJNGE found 0.01-0.355 p. m. iron in
the blood-free liver of young cats and dogs. This was calculated on the
liver substance freshly washed with a 1-per cent NaCl solution. Cal-
culated on 10 kilos bodily weight, the iron in the liver amounted to 3.4-
80.1 mg. Recent determinations of the quantity of iron in the liver of
the rabbit, dog, hedge-hog, pig, and man have been made by GUILLE-
MONAT and LAPICQUE, and in rabbits by SCAFFIDI. The variation was
great in human beings. In men the quantity of iron in the blood-free
liver (blood-pigment subtracted in the calculation) was regularly 0.23
p. m., and in women 0.09 p. m. (calculated on the fresh moist organ),
and this relation was not changed after the twentieth year. Above
0.5 p. m. is considered as pathological. According to BiELFELD,4 who
worked with another method, an even greater quantity of iron occurs in
men.

The quantity of iron in the liver can be increased by drugs contain-
ing iron. The quantity of iron may also be increased by an abundant
destruction of red blood-corpuscles, which will result from the injection

1 Neuberg and Richter, Deutsch. med. Wochenschr., 1904; Wakeman, Zeitschr. f.
physiol. Chem., 44; Wells, Journ. of Exper. Med., 9; Glikin and Loewy, Bioch. Zeitschr.,
10.

2 Hofmeister's Beitrage, 10.

3 Mandel and Levene, Zeitschr. f. physiol. Chem., 45.

4Bunge, Zeitschr. f. physiol. Chem., 17, 78; Guillemonat and Lapicque, Compt.
rend, de soc. biol., 48, with Bailie, ibid., 68; and Arch de Physiol. (5) 8; Biefeld, Hof-
meister's Beitrage, 2;"see also Schmey, Zeitschr. f. physiol. Chem., 39; Scaffidi, ibid.,
54.

388 THE LIVER.

of dissolved haemoglobin, in which process the iron combinations derived
from the blood-pigments in other organs, such as the spleen and marrow,
also seem to take part.1 A destruction of blood-pigments, with a splitting
off of compounds rich in iron, seems to take place in the liver in the for-
mation of the bile-pigments. Even in invertebrates, which have no
haemoglobin, the so-called liver is rich in iron, from which DASTRE and
FLORESCO 2 conclude that the quantity of iron in the liver of inverte-
brates is entirely independent of the decomposition of the blood-pigment,
and in vertebrates it is in part so. According to these authors the liver
has, on account of the quantity of iron, a specially important oxidizing
function, which they call the " fonction martiale " of the liver.

The richness in iron of the liver of new-born animals is of special
interest — a condition which was shown by the analyses of ST. ZALESKI,
but was especially studied by KRUGER and MEYER. In oxen and cows
they found 0.246-0.276 p. m. iron (calculated on the dry substance),
and in the cow-foetus about ten times as much. The liver-cells of a calf
a week old contain about seven times as much iron as the adult animal;
the quantity decreases in the first four weeks of life, when it reaches
about the same amount as in the adult. LAPICQUE 3 also found that in
rabbits the quantity of iron in the liver steadily diminishes from the
eighth day to three months after birth, namely, from 10 to 0.4 p. m.,
calculated on the dry substance. " The foetal liver-cells bring an abun-
dance of iron in the world to be used up, within a certain time, for a pur-
pose not well known." A part of the iron exists as phosphate, but the
greater part is in combination in the ferruginous protein bodies (ST.
ZALESKI).

The quantity of calcium oxide in the fresh, moist liver of the horse,
ox, and pig, according to TOYONAGA, amounts to 0.148-0.193 p. m., or
more than the human liver (0.101 p. m. according to MAGNUS-LEVY).
The amount of magnesium oxide was remarkably high, namely, 0.168,
O.lSSand 0.158 p. m., in the livers of the horse, ox, and pig, respectively,
but considerably less than the human liver in which MAGNUS-LEVY found
0.292 p. m. KRUGER4 found the quantity of calcium in the livers of
adult cattle and of calves to be respectively 0.71 p. m. and 1.23 p. m.
of the dried substance. In the foetus of the cow it is lower than in calves.
During pregnancy the iron and calcium in the fcetus are antagonistic;

1 See Lapicque, Compt. Rend., 124, and Schurig, Arch. f. exp. Path. u. Pharm., 41.

2 Arch. dePhysiol. (5), 10.

3 St. Zaleski, 1. c.; Kriiger and collaborators, Zeitschr. f. Biologic, 27; Lapicque,
Maly's Jahresber., 20.

4 Kriiger, Zeitschr. f. Biologic, 31; Toyonaga, Bull, of the College of Agriculture;
Tokio, 6; A. Magnus-Levy, Bioch. Zeitschr., 24.

STORAGE OF PROTEIN IN THE LIVER. 389

that is, an increase in the quantity of calcium in the liver causes a diminu-
tion in the iron, and an increase in the iron causes a decrease in the calcium.
Copper seems to be a physiological constituent, and occurs to a considerable
extent in Cephalopods (HENZE 1). Foreign metals, such as lead, zinc,
arsenic, and others (also iron), are easily taken up and combined by the
liver (SLOWTZOFF, v. ZEYNEK, and others2).

v. BIBRA 3 found in the liver of a young man who had suddenly died
762 p. m. water and 238 p. m. solids, consisting of 25 p. m. fat, 152 p. m.
protein, gelatin-forming and insoluble substances, and 61 p. m. extract-
ive substances.

MAGNUS-LEVY4 found in the liver of a healthy suicide 606 p. m.
water, 394 p. m. solids among which 212.8 p. m. fat occurred. If the
total nitrogen, 27 p. m., is calculated as protein the amount would be
approximately 169 p. m.

PROFITLICH 5 found 68.2-75.17 per cent water in the dog liver and 70.76-
72.86 per cent in the ox liver. The relation N : C in the fat and glycogen-free
dried substance was 1:3.21 in dogs and 1:3.13 in oxen or about the same as in
flesh (see Chapter XI).

The quantitative composition of the liver may show great varia-
tion, depending upon the kind and amount of the food supplied. The
amount of carbohydrate (glycogen) and fat may vary considerably,
which is due to the fact that the liver is a storage-organ for these bodies,
especially for the glycogen.

Based upon special experiments, SEITZ claims that the liver is a
storehouse for protein also. In experiments on hens and ducks which
had previously been starved, he found that the liver took up abundant
protein on feeding meat, and that its weight as compared with the weight
after starvation was doubled or quadrupled. As it is characteristic of
storage or reserve bodies that their amount in the storage-organs on
feeding with such bodies strongly increases in percentage, it is remarkable
in SEITZ'S feeding experiments that the percentage of protein in the liver
did not increase, but rather diminished slightly. In this case we did not
have a higher percentage of protein, but an increase in the weight of the
total cell mass of the organ, probably brought about by increased work
of the liver due to the protein feeding. The investigations of GRUND 6
have shown that with protein feeding in dogs, the relation P:N in the

Zeitschr. f. physiol. Chem., 33.

2Slowtzoff, Hofmeister's Beitrage, 1; v. Zeynek, see Centralbl. f. Physiol., 15.

3 See v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., p. 711.

4 Bioch. Zeitschr., 24.

5 Pfliiger's Arch., 119.

6Seitz, Pfliiger's Arch., Ill; Grund, Zeitschr. f. Biol., 54.

390 THE LIVER.

liver was not essentially changed which speaks against a simple storage
of food protein.

Glycogen and its Formation.

Glycogen was first discovered by BERNARD. It is a carbohydrate
closely related to the starches or dextrins, with the general formula
mCCoHioOs). Its molecular weight is unknown, but seems to be very
large (GATIN-GRUZEWSKA and v. KNAFFL-LENZ1). The largest quan-
tities are found in the liver of adult animals, and smaller quantities
in the muscles (BERNARD, NASSE). It is found in very small quantities
in nearly all tissues of the animal body. Its occurrence in lymphoid
cells, blood, and pus has been mentioned in a previous chapter, and it
seems to be a regular constituent of all cells capable of development.
Glycogen was first shown to exist in embryonic tissues by BERNARD
and KUHNE, and it seems on the whole to be a constituent of tissues
in which a rapid cell formation and cell development are taking place.
It is also present in rapidly forming pathological tumors (HOPPE-SEYLER) .
Some animals, as certain mussels (Bizio), Tsenia and Ascaridse (WEIN-
LAND 2), are very rich in glycogen. Glycogen also occurs in the vegetable-
kingdom, especially in many fungi.

The quantity of glycogen in the liver, as also in the muscles, depends
essentially upon the food. In starvation it disappears almost com-
pletely after a short time, but more rapidly in small than in large animals,
and it disappears earlier from the liver than from the muscles. As
shown by C. VOIT, KULZ and especially by PrLUGER,3 it never entirely
disappears in starvation, as a re-formation of glycogen always takes
place. After partaking of food, especially such as is rich in carbo-
hydrates, the liver becomes rich again in glycogen, the greatest incre-
ment occurring 14 to 16 hours after eating (KULZ). The quantity of
liver-glycogen may amount to 120-160 p. m. after partaking of large
quantities of carbohydrates, and in dogs which had been especially
fed for glycogen SCHONDORFF and GATIN-GRUZEWSKA found still higher
results, even more- than 180 p. m. Ordinarily it is considerably less,
namely, 12-30 to 40 p. m. The highest amount of glycogen in the liver
thus far observed was 201.6 p. m., found by MANGOLD 4 in the frog. The

1 Gatin-Gruzewska, Pfliiger's Arch., 103; v. Knaffl-Lenz, Zeitschr. f. physiol. Chem.,
46.

2 Zeitschr. f. Biologie, 41. The extensive literature on glycogen may be found in
E. Pfliiger, Glykogen, 2. Aufl., Bonn, 1905; and in Cremer, " Physiol. des Glykogens,"
in Ergebnisse der Physiologic, 1, Abt. 1. In the following pages we shall refer to these
works.

3 Pfliiger's Arch., 119, which contains the literature.

4 Ibid., 121.

GLYCOGEN. 391

shark, whose liver is very rich in fat, even though well nourished, only
has comparatively low values for the glycogen in the liver, 9.3-23.8
p. m. (BOTTAZZI l). According to CREMER the quantity of glycogen in
plants (yeast-cells) is, as in animals, dependent upon the food. He
finds that the yeast-cells contain glycogen, which disappears from the
cells in the auto-fermentation of the yeast, but reappears on the intro-
duction of the cells into a sugar solution.

The quantity of glycogen of the liver (and also of the muscles) is
also dependent upon rest and activity, because during rest, as in hiberna-
tion, it increases, and during work it diminishes. KULZ has shown that
by hard work the quantity of glycogen in the liver (of dogs) is reduced
to a minimum in a few hours. The muscle-glycogen does not diminish
to the same extent as the liver-glycogen. KULZ, ZUNTZ and VOGELIUS,
FRENTZEL, and others have been able to render rabbits and frogs nearly
glycogen-free by suitable strychnine poisoning. The same result is pro-
duced by starvation followed by hard work. According to GATIN-
GRUZEWSKA,2 the liver and muscles in rabbits can be made glycogen-
free after 36-40 hours by first starving one day and then injecting
adrenalin.

Glycogen forms an amorphous, white, tasteless, and non-odorous powder.
When perfectly pure, and by proper alcohol precipitation, it can be obtained
as rods or prisms which look like crystals (GATIN-GRUZEWSKA) . It
gives an opalescent solution with water which, when allowed to evaporate
on the water-bath, forms a pellicle over the surface that disappears again
on cooling. It is undecided whether we here have a true solution or
not. Like other colloids, glycogen in water under the influence of the
electric current migrates to the anode, on which it collects (GATIN-
GRUZEWSKA). According to BoiTAZZi,3 who obtained the same results,
a little acid or a little alkali modify the results so that the glycogen becomes
isoelectric. Its aqueous solution is dextrorotatory, and HUPPERT found
it to be («:)D = +196.630. GATIN-GRUZEWSKA has recently obtained
the same result by using a perfectly pure solution of glycogen. A
solution of glycogen, especially on the addition of NaCl, is colored wine-
red by iodine. It may hold cupric hydroxide in solution in alkaline
liquids, but does not reduce it. A solution of glycogen in water is not
precipitated by potassium-mercuric iodide and hydrochloric acid, but is
precipitated by alcohol (on the addition of NaCl when necessary), or

1 Arch. Ital. d. Biol., 48; cited in Bioch. Centralbl., 7, 833.

2 Compt. Rend., 142.

3 Bottazzi, Chem. Centralbl. 1909 p. 1423; Bottazzi and d'Errico (Pfliiger's Arch.,
115) have investigated the viscosity, the electrical conductivity and the freezing-point
of glycogen solutions at different concentrations.

392 THE LIVER.

ammoniacal basic lead acetate. An aqueous solution of glycogen made
alkaline with caustic potash (15 per cent KOH) is completely precipitated
by an equal volume of 96 per cent alcohol. Tannic acid also precipitates
glycogen. It gives a white granular precipitate of benzoyl-glycogen
with benzoyl chloride and caustic soda. Glycogen is completely pre-
cipitated by saturating its solution at ordinary temperatures with magne-
sium or ammonium sulphate. It is not precipitated by sodium chloride,
or by half saturation with ammonium sulphate (NASSE, NEUMEISTER,
HALLIBURTON, YouNG1). On boiling with dilute caustic potash (1-2
per cent) the ghrcogen may be more or less changed, especially if it has
been previously exposed to the action of acid or to BRUCKE'S reagent
(see below) (PFLUGER). On boiling with stronger caustic potash (even
of 36 per cent) it is not injured (PFLUGER). By diastatic enzymes
glycogen is converted into maltose or glucose, depending upon the nature
of the enzyme. It is transformed into glucose by dilute mineral acids.
According to TEBB 2 various dextrins appear as intermediary steps in
the saccharification of glycogen, depending on whether the hydrolysis
is caused by mineral acids or enzymes. The glycogen from various
animals and different organs is the same according to PFLUGER.3 Nor has
it been decided whether all the glycogen in the liver occurs as such or
whether it is in part combined with protein (PFLUGER-NERKING) . The
investigations of LoESCHCKE4 have shown that we have no positive
reasons for this assumption.

The preparation of pure glycogen (most easily from the liver) is
generally performed by the method suggested by BRUCKE, of which the
main points are the following: Immediately after the death of the animal
the liver is thrown into boiling water, then finely divided and boiled
several times with fresh water. The filtered extract is now sufficiently
concentrated, allowed to cool, and the proteins removed by alternately
adding potassium-mercuric iodide and hydrochloric acid. The glycogen
is precipitated from the filtered liquid by the addition of alcohol until
the liquid contains 60 vols. per cent. By repeating this and precipitating
the glycogen several times from its alkaline and acetic-acid solution it
is purified on the filter by washing first with 60 per cent and then with
95 per cent alcohol, then treating with ether, and drying over sulphuric
acid. It is always contaminated with mineral substances. To be able
to extract the glycogen from the liver or, especially, from muscles and
other tissues completely, which is essential in a quantitative estimation,
these parts must first be warmed for two hours with strong caustic potash
(30 per cent) on the water-bath. As the glycogen changes in this purifica-

1 Young, Journ. of Physiol., 22, citing the other investigators.

2 Journ. of Physiol., 22.
1 Pfliiger's, Arch. 129.

* Ibid., 102.

GLYCOGEN FORMERS. 393

tion, as suggested by BRUCKE, it is better, for quantitative determinations
of glycogen, to precipitate it directly from the alkaline solution by alcohol
(PFLUGER !).

The quantitative estimation is best performed according to PFLUGER' s
method, which is as follows: The finely divided organ is heated on
the water-bath for 2-3 hours in the presence of 30 per centKOH; after
diluting with water and filtering, the glycogen is precipitated with
alcohol, and the redissolved glycogen estimated in part by the polar-
iscope and in part as sugar after inversion. One part by weight of sugar
equals 0.927 part glycogen. As in the estimation the prescribed direc-
tions must be exactly followed, we must refer to the original work of
PFLUGER for the details of the method. Other methods of estimating
glycogen, such as those of BRUCKE-KULZ, PAVY, and AUSTIN, are described
in PFLUGER'S Archiv. 96. Also compare the recent works of PFLUGER.2

Numerous investigators have endeavored to determine the origin
of glycogen in the animal body. It is positively established by the
unanimous observations of many investigators3 that the varieties of
sugars and their anhydrides, dextrins and starches, have the property of
increasing the quantity of glycogen in the body. The action of inulin
seems to be somewhat uncertain.4 The statements are questioned in
regard to the action of the pentoses. CREMER found that in rabbits and
hens various pentoses, such as rhamnose, xylose, and arabinose, have a
positive influence on the glycogen formation, and SALKOWSKI obtained
the same result on feeding Z-arabinose. FRENTZEL, on the contrary,
found no glycogen formation on feeding xylose to a rabbit which had
previously been made glycogen-free by strychnine poisoning, and NEU-
BERG and WOHLGEMUTH 5 obtained similar negative results on feed-
ing rabbits with d- and r-arabinose. In general we can for the present
accept the view that the pentoses are not direct glycogen formers.

The hexoses, and the carbohydrates derived therefrom, do not all
possess the ability of forming or accumulating glycogen to the same
extent. Thus C. VOIT 6 and his pupils have shown that glucose has a
more powerful action than cane-sugar, while milk-sugar is less active
(in rabbits and hens) than glucose, fructose, cane-sugar, or maltose.

The following substances when introduced into the body also increase the
quantity of glycogen in the liver: Glycerin, gelatin, arbutin, and likewise, accord-

1 See also the method suggested by Gautier, Compt. Rend., 129.

2 Pfliiger's Arch., 103, 104, 121 and especially 129.

3 In reference to the literature on this subject, see E. Kiilz. Pfliiger's Arch., 24,
and Ludwig, Festschrift, 1891; also the cited works of Pfliiger and Cremer, foot-note
2, p. 390.

4 See Miura, Zeitschr. f. Biologic, 32, and Nakaseko, Amer. Journ. of Physiol., 4.

5 Salkowski, Zeitschr. f. physiol. Chem., 32; Neuberg and Wohlgemuth, ibid., 35.
See also Pfliiger, 1. c., and Cremer, 1. c.

6 Zeitschr. f. Biologic, 28.

394 THE LIVER.

ing to the investigations of KULZ, erythrite, quercite, dulcite, mannite, inosite,
ethylene and propylene glycol, glucuronic anhydride, saccharic acid, mucic acid,
sodium tartrate, saccharin, isosaccharin, and urea. Ammonium carbonate, gly-
cocoll, and asparagine may similarly, according to ROHMANN, cause an increase
in the amount of glycogen in the liver. NEBELTHAU finds that other ammonium
salts and some of the amides, as well as certain narcotics, hypnotics, and antipyretics,
produce an increase in the glycogen of the liver. This action of the antipyretics
(especially antipyrine) had been shown by LEPINE and PoRTERET.1

The fats, according to BOUCHARD and DESGREZ, increase the glycogen
content of the muscles but not of the liver, while COUVREUR believes that
the glycogen is increased at the expense of the fat in the silkworm larva,
but these statements have been shown to be incorrect by the recent
investigations of KOTAKE and SERA.2 In general it is believed that fat
does not increase the amount of glycogen in the liver or in the animal
body, although a carbohydrate formation from glycerin, but not a gly-
cogen formation, is probable.

The question whether the proteins have the ability to increase the
glycogen content of the liver or the animal body has been long disputed.
The feeding experiments with meat or with pure proteins by older exper-
imenters, such as NAUNYN, v. MERINO and E. KULZ seem to show an
ability. But the proof of these investigations has been strongly dis-
puted by PFLUGER and later investigations of SCHONDORFF, BLUMENTHAL
and WOHLGEMUTH, as also those of BENDIX and STOOKEY3 yield contradic-
tory results. These investigations have really only historical interest,
since now a carbohydrate formation as well as a glycogen formation from
proteins have been positively observed.

If the question is raised as to the action of the various bodies on
the accumulation of glycogen in the liver, it must be recalled that a forma-
tion of glycogen takes place in this organ, as well as a consumption of the
same. An accumulation of glycogen may be caused by an increased
formation of glycogen, but also by a diminished consumption, or by both.

It is not known how the various bodies above mentioned act in this
regard. Certain of them probably have a retarding action on the trans-
formation of glycogen in the liver, while others perhaps are more com-
bustible, and in this way protect the glycogen. Some probably excite
the liver-cells to a more active glycogen formation, while others yield
material from which the glycogen is formed, and are glycogen-formers

1 Rohmann, Pfliiger's Arch., 39; Nebelthau, Zeitschr. f. Biologie, 28; Lupine and
Porteret, Compt. Rend., 107,

2 Bouchard et Desgrez, Compt. Rend., 130; Couvreur, Compt. rend, de soc. biol.,
47; Kotake and Sera Zeitschr. f. physiol. Chem., 62.

3 See the work on glycogen by Pfliiger and also Schondorff, Pfliiger's Arch., 82 and 88;
Blumenthal and Wohlgemuth, Berl. klin. Wochenschr., 1901; Bendix, Zeitschr. f. physiol.
Chem., 32 and 34; Stookey, Amer. Journ. of Physiol., 9.

FORMATION OF GLYCOGEN. 395

in the strictest sense of the word. The knowledge of these last-mentioned
bodies is of the greatest importance in the question as to the origin of
glycogen in the animal body, and the chief interest attaches to the
question: To what extent are the two chief groups of food, the proteins
and carbohydrates^ glycogen-formers?

The great importance of the carbohydrates in the formation of gly-
cogen has given rise to the opinion that the glycogen in the liver is pro-
duced from sugar by a synthesis in which water separates with the for-
mation of an anhydride (LUCHSINGER and others). This theory (anhy-
dride theory) has found opponents because it neither explains the forma-
tion of glycogen from such bodies as proteins, carbohydrates, gelatin,
and others, nor the circumstance that the glycogen is always the same,
independent of the properties of the carbohydrate introduced, whether
it is dextrogyrate or levogyrate. This last circumstance does not now
present any special difficulty, since we know that the simple sugars can
easily be transformed into each other. It was formerly the opinion of
many investigators that all glycogen is formed from protein, and that
this splits into two parts, one containing nitrogen and the other being
free from nitrogen; the latter is the glycogen. According to these
views, the carbohydrates act only in that they spare the protein and
the glycogen produced therefrom (sparing theory of WEISS, WOLFFBERG,
and others J).

In opposition to this theory C. and E. VOIT and their pupils have
shown that the carbohydrates are " true " glycogen-formers. After par-
taking of large quantities of carbohydrates, the amount of glycogen stored
up in the body is sometimes so great that it cannot be covered by the
proteins decomposed during the same time, and in these cases a gly-
cogen formation from the carbohydrates must be admitted. According
to CREMER only the fermentable sugars of the six carbon series or their
di- and polysaccharides are true glycogen-formers. For the present, only
glucose, fructose, and to a much less degree galactose (WEINLAND 2),
and perhaps also d-mannose (CREMER) are designated as true glycogen-
formers. Other monosaccharides may indeed, according to CREMER,
influence the formation of glycogen, but they are not converted into
glycogen, and hence are called only pseudoglycogen-formers.

The poly- and disaccharides may, after a cleavage into the cor-
responding fermentable monosaccharides, serve as glycogen-formers.
This is true for at least cane-sugar and milk-sugar, which must first

1 In regard to these two theories, see especially Wolffberg, Zeitschr. f . Biologic, 16.

2E. Voit, Zeitschr. f. Biologic, 25, 543, and C. Voit, ibid., 28. See also Kausch
and Socin, Arch. f. exp. Path. u. Pharm., 31; Weinland, Zeitschr. f. Biologic, 40 and
38; Cremer, ibid., 42, and Ergebnisse der PhysioL, 1.

396 THE LIVER.

be inverted in the intestine. These two varieties of sugar, therefore,
cannot, like glucose and fructose, serve as glycogen-formers after sub-
cutaneous injection, but reappear almost entirely in the urine (DASTRE,
FR. VOIT) . Maltose, which is inverted by an enzyme present in the blood,
passes only to a slight extent into the urine (DASTRE and BOURQUELOT
and others), and it can, like the monosaccharides, even after subcuta-
neous injection, be used in the formation of glycogen (FR. Von).1 Of
the disaccharides the maltose and the cane-sugar are strong glycogen-
formers while milk-sugar has only a weak action.

The ability of the liver to form glycogen from monosaccharides has
also been shown by K. GRUBE in a very interesting and direct manner,
by perfusion experiments with solutions of various carbohydrates. In
these perfusion, experiments on tortoise livers, glucose produced an
abundant glycogen formation, while with fructose and galactose it was
less abundant. Pentoses, disaccharides, casein and amino-acids (gly-
^cocoll, alanine and leucine) were inactive while on the contrary glycerin
and also formaldehyde acted as glycogen-formers. The formation of
glycogen from formaldehyde is disputed by SCHONDORFF and GRLBE.2

After PAVYS first showed the occurrence of carbohydrate groups in
ovalbumin, other investigators were able to split off glucosamine from
this and other protein substances (see Chapter II), and the question
arose whether the amino-sugar could serve in the formation of glycogen.
The investigations carried out in this direction by FABIAN, FRANKEL
and OFFER, CATHCART and BIAL, have shown that the glucosamine
introduced into the organism is in part eliminated unchanged in the
urine and has no glycogen-forming action. No definite conclusions
can be drawn from this on the behavior of the carbohydrate groups,
which exist not as free groups but combinedjvith the protein molecules.
The investigations of FORSCHBACH on the behavior of glucosamine
chained to an acid-group in an amide-like combination, as well as the
investigations of KURT MEYER and STOLTE,4 have yielded no proofs for
the theory that glycogen is formed from glucosamine.

Whether or not, or to what extent, the glucoproteins by their glucosa-
mine component take part in the sugar or glycogen formation in the animal

1Dastre, Arch, de Physiol. (5) 3, 1891; Dastre and Bourquelot, Compt. Rend., 98;
Fritz Voit, Verhandl. d. Gesellsch. f . Morph. u. Physiol. in Miinchen, 1896, and Deutsch.
Arch. f. klin. Med., 58. In regard to the glycogen formation after intravenous injection
of sugar see FREUND and POPPER, Bioch. Zeitschr., 41.

2 Pfliiger's Arch., 138; Grube, ibid., 118, 121, 122, 126 and 139.

3 The Physiology of the Carbohydrates, London, 1894.

4 Fabian, Zeitschr. f. physiol. Chem., 27; Frankel and Offer, Centralbl. f. Physiol.,
13; Cathcart, Zeitschr. f. physiol. Chem., 39; Bial, Berl. klin. Wochenschr., 1905;
Forschbach, Hofmeister's Beitrage, 8; Meyer, ibid., 9; Stolte, ibid., 11.

FORMATION OF GLYCOGEN. 397

body is difficult to answer for the present, as but little is known of the
quantity of these substances in the body, and our knowledge of the amount
of carbohydrate which can be split off from the various protein substances
is also very meager.

If the proteins are to be counted, and this is in agreement with the
generally accepted view, among those bodies which increase the glycogen
of the body, then we must ask the question: Do the proteins act only
indirectly as pseudoglycogen-formers, or are they direct glycogen-
formers which can serve as material for the formation of glycogen or
sugar? This question stands in close relation to the sugar formation
and sugar elimination in the various forms of glycosuria, and will be best
discussed below in connection with the question of diabetes.

Glycogen is a reserve-food deposited, in the liver and which, like other
carbohydrates can be transformed into fat, and it is generally admitted
that such a fat formation from glycogen also takes place in the liver.
There is no doubt that the glycogen deposited in the liver is formed in
the liver-cells from the sugar; but where does the glycogen existing in
the other organs, such as the muscles, originate? t Is the glycogen of the
muscles formed on the spot or is it transmitted to the muscles by the blood?
These questions cannot at present be answered with certainty, and the
investigations on this subject by different experimenters have given
varying results. The experiments of KtJLZ,1 in which he studied the
glycogen formation by passing blood containing cane-sugar through
the muscle, have led to no conclusive results, while the perfusion exper-
iments of HATCHER and WOLFF with glucose seem to indicate a glycogen
formation from sugar in the muscles. The investigations of DE FILIPPI 2
on dogs with so-called Eck's fistula also show a glycogen formation from
sugar in the muscles. In the Eck fistula operation the portal vein is
ligated near the liver hilus and sewed to the inferior vena cava and an
opening established between the two veins so that the portal blood flows
directly into the vena cava without passing through the liver. In
well-nourished animals, operated upon in this manner, the livers had the
same properties as those from starving animals, while, on the contrary,
the muscles contained quantities of glycogen which corresponded to
those found in a normal over-fed dog.

If it be true that the blood and lymph contain a diastatic enzyme
which transforms glycogen into sugar, and also that the glycogen regularly
occurs in the form-elements and is not dissolved in the fluids, it seems
probable that the glycogen in solution is not transmitted by the blood to

1See Minkowski and Laves, Arch. f. exp. Path. u. Pharm., 23; Kiilz, Zeitschr. f.
Biologic, 27; Hatcher and Wolff, Journ. of Biol. Chem., 3.
2 Zeitschr. f. Biol., 49 and 50.

398 THE LIVER.

the organs, but perhaps more likely, if the leucocytes do not act as car-
riers, it is formed on the spot from the sugar.1 The glycogen formation
seems to be a general function of the cells. In adults, the liver, which
is very rich in cells, has the property, on account of its anatomical posi-
tion, of transforming large quantities of sugar into glycogen.

This glycogen, which is deposited in the liver as reserve-food, in order
that it can be useful to the body, must at least in greater part be trans-
formed into sugar and supplied to the various organs by the blood. The
question now arises whether there is any foundation for the statement
that the liver glycogen is transformed into sugar.

As first shown by BERNARD and redemonstrated by many inves-
tigators, the glycogen in a dead liver is gradually changed into sugar,
and this sugar formation is caused, as BERNARD supposed and then shown
by numerous investigators by a diastatic enzyme whose relation to the
diastatic enzyme of the blood is not quite clear.2

This post-mortem sugar formation led BERNARD to the assump-
tion of the formation of sugar from glycogen in the liver during life.
BERNARD suggested the following arguments for this theory: The liver
always contains some sugar under physiological conditions, and the
blood from the hepatic vein is always somewhat richer in sugar than the
blood from the portal vein. BERNARD'S views found in SEEGEN an active
supporter, as he tried to show by numerous experiments the physio-
logical sugar content of the liver as well as the high sugar content of the
blood of the liver veins. On the other hand the correctness of the
observations of BERNARD and SEEGEN is disputed by many investigators
such as PAVY, RITTER, SCHIFF, EULENBERG, LUSSANA, MOSSE, N. ZUNTZ
and others,3 and in regard to the sugar content in the two kinds of
blood we have come to the general conclusion that when only the stasis
and other disturbing influences of the operation are prevented, the blood
of the liver veins, if at all, is only slightly richer in sugar than the blood
of the portal vein.4

The circumstance that the blood-sugar rapidly sinks to J-J of its
original quantity, or even disappears when the liver is cut out of the
circulation, indicates a vital formation of sugar in the liver (SEEGEN,
BOCK and HOFFMANN, KAUFMANN, PAVY and others). In geese whose

1 ^ee Dastre, Compt. rend, de soc. biol., 47, 280, and Kaufmann, ibid., 316.
'Rohmann, Verb. d. Ges. deutsch. Naturf. u. Aerzte. Breslau, 1903; Borchardt,
Pfliiger's Arch., 100; Zegla, Bioch. Zeitschr., 16; E. Starkenstein, ibid., 24.

3 In regard to the literature on sugar formation in the liver see Bernard, Legons sur
le diabete, Paris, 1877; Seegen, Die Zuckerbildung im Tierkorper, 2. Aufl. Berlin,
1900; M. Bial, Pfliiger's Arch., 55, 434.

4 Seegen, Die Zuckerbildung, etc., and Centralbl. f. Physiol., 10, 497 and 822;
Zuntz, ibid., 561; Mosse, Pfliiger's Arch., 63; Bing, Skand. Arch. f. Physiol., 9.

SUGAR FORMATION FROM GLYCOGEN. 399

livers were removed from the circulation, MINKOWSKI found no sugar
in the blood after a few hours. On removing the liver from the
circulation by tying all the vessels to and from the organ, the quan-
tity of sugar in the blood is not increased (ScHENCK1). An important
proof of the possibility of a vital formation of sugar from the liver gly-
cogen lies in the fact that we shall learn below of certain poisons and
operative changes which may cause an abundant elimination of sugar,
but only when the liver contains glycogen.

A vital formation of sugar from the liver glycogen is now generally
accepted. Most investigators consider this as an enzymotic transforma-
tion of the glycogen by means of the liver diastase, while certain inves-
tigators such as DASTRE, NOEL-PATON, E. CAVAZZANI, McGuiGAN and
BROOKS 2 and others explain it by a special activity of the protoplasm.
BANG 3 has studied the formation of sugar in frogs' livers, which had not
appreciably changed in weight in a RINGER'S solution which was isotonic
with the frog blood and which correspondingly had retained their vital
properties. This sugar formation does not depend upon a protoplasmic
activity but is of an enzymotic nature. It is caused by a diastase,
which in Rana esculent a occur in great part in a latent, inactive form
due to the inhibitory action of the liver lipoids. Common salt is espe-
cially important as an activator for this enzyme. The surviving frog
liver is stimulated to a strong sugar production by adrenalin, and this
sugar formation is also of an enzymotic nature. The action of the
adrenalin consists in an activation of the liver diastase, brought about
in various ways.

The relation of the sugar eliminated in the urine under certain
conditions, such as in diabetes mellitus, certain intoxications, lesions
of the nervous system, etc., to the glycogen of the liver is also an important
question.

It does not enter into the plan and scope of this book to discuss in
detail the various views in regard to glycosuria and diabetes. The
appearance of glucose in the urine is a symptom which may have essen-
tially different causes, depending upon different circumstances. Only
a few of the most important points will be mentioned.

The blood always contains about the average of 1 p. m., while the
urine has in it at most only traces of glucose. When the quantity of

1 Seegen, Bock, and Hoffmann, see Seegen, 1. c.; Kaufmann, Arch, de Physiol. (5),
8; Tangl and Harley, Pfliiger's Arch., 61; Pavy, Journ. of Physiol., 29, Minkowski,
Arch. f. exp. Path. u. Pharm., 21; Schenck, Pfliiger's Arch., 57.

2 See Dastre, Noel-Paton, Cavazzani and their work cited in Pick, Hofmeister's
Beitrage, 3, and McGuigan and Brooks, Amer. Journ. of Physiol., 18; R. G. Pearce,
ibid., 25.

3 Bioch. Zeitschr., 49.

400 THE LIVER.

sugar in the blood rises above this average, sugar passes into the urine,
sometimes even with slight rise and in other cases with stronger rise.
The. kidneys have the property to a certain extent of preventing the
passage of blood-sugar into the urine; and it follows from this that an
elimination of sugar in the urine may be caused partly by a reduction
or suppression of this above-mentioned activity, and partly also by an
abnormal increase of the quantity of sugar in the blood.

The first seems, according to v. MERINO and MINKOWSKI, and others
to be the case in phlorhizin diabetes, v. MERING found that a strong
glycosuria appears in man and animals on the administration of the
glucoside phlorhizin. The sugar eliminated is not derived from the
glucoside alone. It is formed in the animal body, and in fact from the
carbohydrates, or as generally admitted on prolonged starvation, from
the protein substances of the body (LUSK). The quantity of sugar in
the blood is not increased, but rather diminished, in phlorhizin diabetes
(MINKOWSKI), which does not indicate increase in the sugar production
but rather an increased excretion of the sugar by the kidneys. The
fact that after extirpation of the kidney in phlorhizin diabetes no rise
in the blood-sugar is observed, and that after the injection of phlorhizin
in the renal artery of one side the urine secreted by this kidney contains
sugar sooner and more abundantly than the urine from the other kidney
(ZUNTZ), tends to favor this view. The experiments especially performed
by PAVY, BRODIE, and SIAU upon blood containing phlorhizin and sur-
viving kidneys also indicate the same, namely, that the phlorhizin acts
upon the kidneys and the researches of ERLANDSEN also lead to the same
conclusion. He found that on combining the phlorhizin action with
bleeding that the glycosuria was increased while after bleeding alone
without phlorhizin poisoning the hyperglycaBmia was absent. While
v. MERING and others believe in an increased permeability of the kidneys
for sugar, produced by the phlorhizin LEPINE l is of the view that the
phlorhizin causes a formation of glucose from the virtual sugar in the
kidneys. PAVY is, on the contrary, of the opinion that the kidneys,
under the influence of the phlorhizin, split off sugar from a substance

1 In regard to the literature on phlorhizin diabetes see v. Mering, Zeitschr. f. klin.
Med., 14 and 16; Minkowski, Arch. f. exp. Path. u. Pharm., 31; Moritz and Prausnitz,
Zeitschr. f. Biologic, 27 and 29; Kiilz and Wright, ibid., 27, 181; Cremer and Rioter,
ibid., 28 and 29; Contejean, Compt. rend, de soc. biol., 48; Lusk, Zeitschr. f. Biologic,
36 and 42; Levene, Journal of PhysioL, 17; Pavy, ibid., 20, and with Brodie and Siau,
29; Arteaga, Amer. Journ. of PhysioL, 6; O. Loewi, Arch. f. exp. Path. u. Pharm., 47;
N. Zuntz, Arch. f. (Anat. u.) PhysioL, 1895; Stiles and Lusk, Amer. Journ. of PhysioL,
10; Lusk, ibid., 22; Cremer, Ergebnisse der PhysioL, 1, Abt. 1; Erlandsen, Bioch.
Zeitschr., 23 and 24; Lupine, Compt. rend. soc. biol., 68; Lusk, Ergebnisse der PhysioL,
Bd. 12, 315-392, and the monographs upon diabetes.

GLYCOSURIAS. 401

circulating in the blood, perhaps from a protein with loosely combined
carbohydrate groups.

GRUBE from experiments upon the surviving tortoise liver has made the
suggestion that it is not the kidneys which are first attacked by the phlorhizin
action in phlorhizin glycosuria but the liver. Important experimental evidence
against this view has been raised by SCHONDORFF and SucKROW.1

Another form of glycosuria which according to certain investigators
is to be connected with a changed permeability of the kidneys (UNDER-
BILL and CLOSSON) is the glycosuria first observed by BOCK and HOFF-
MANN after the intravascular injection of large quantities of a 1-per
cent salt solution, which is also of great interest because, as shown by
MARTIN FiscHER,2 it can be again arrested by an injection of a salt solu-
tion containing CaC^. There are investigators who attempt to connect
this glycosuria with the adrenals and a hyperglyca3mia.

With the exception of these two forms of glycosuria, the phlorhizin
diabetes and the salt-glycosuria, and also the glycosuria produced by
certain kidney poisons, all other forms of glycosuria or diabetes, as far
as known at present, depend on a hyperglyccemia.

A hyperglyca3mia may be caused in various ways. It may be caused,
for example, by the introduction of more sugar than the body can destroy.

The ability of the animal body to assimilate the different varieties
of sugar has naturally a limit. If too much sugar is introduced into the
intestinal tract at one time, so that the so-called assimilation limit
(see Chapter VIII, on absorption) is overreached, then the excess of
absorbed sugar passes into the urine. This form of glycosuria is called
alimentary glycosuria,3 and is caused by the passage of more sugar into
the blood than the liver and other organs can destroy.

As the liver cannot transform into glycogen all the sugar which comes
to it in these, to a certain extent physiological, alimentary glycosurias,
it is possible that a glycosuria may also be produced under pathological
conditions, even by a moderate amount of carbohydrate (100 grams
glucose), which a healthy person could overcome. This is true, among
other cases, in various affections of the cerebral system and in certain
chronic poisonings. Certain observers include the lighter forms of

1 Grube, Pfliiger's Arch., 128; Schondorff and Suckrow, ibid., 138. See also the
opposed view of Underbill, Journ. of biol. Chem., 13.

2 Bock and Hoffmann, Arch. f. (Anat. u.) Physiol., 1871; M. Fischer, University
of California publications Physiol., 1903 and 1904, and Pfluger's Arch., 106 and 109;
Underbill and Closson, Amer. Journ. of Physiol., 15, and Journ. of Biol. Chem., 4.

3 In regard to alimentary glycosuria see Moritz, Arch. f. klin. Med., 46, which also
contains the earlier literature; B. Rosenberg, Ueber das Vorkommen der alimentaren
Glykosurie, etc. (Inaug.-Dissert. Berlin, 1897); van Oondt, Munch, med. Wochen-
schr., 1898; v. Noorden, Die Zuckerkrankheit, 3. Aufl., 1901.

402 THE LIVER.

diabetes, where the sugar disappears from the urine when the carbohy-
drates are cut off as much as possible from the food, in this class of gly-
cosuria.

A hyperglycaemia which passes into a glycosuria may also be brought
about by an excessive or sudden formation of sugar from the glycogen
and other substances within the animal body.

To this group of glycosurias belongs, it seems, the adrenalin glycosuria,
in which an increased mobilization of the carbohydrate occurs, espe-
cially the liver glycogen. Several circumstances indicate this origin
of the sugar. Thus, after adrenalin injection the glycogen disappears
from the liver and, according to MiCHAUD,1 adrenalin is without action
in dogs with Eck fistula. The activity of the adrenalin in starving
animals whose livers are very poor in glycogen speaks for the possibility
that the sugar also may in part have another origin than that from the
liver glycogen.

Adrenalin glycosuria takes, to a certain degree, a central position and
as such a glycosuria we consider also several other forms of glycosuria
caused by hyperglycaemia. This is for example the case with the gly-
cosuria after BERNARD'S sugar puncture or piqfire. That the glycosuria
produced after piqure is due to an increased transformation of the gly-
cogen, follows from the fact that no glycosuria appears, under the above-
mentioned circumstances, when the liver has been previously made free
from glycogen by starvation or other means. The close relation of
this form of hyperglycaemia and glycosuria to the adrenals follows from
the fact that the sugar puncture is without action after the extirpation
of the two adrenals. In rats, SCHWARZ found, after such a double extirpa-
tion of the adrenals, that the liver was glycogen free and he considers
this lack of glycogen as the cause for the inaction of the piqure under
these conditions. According to KAHN and STARKENSTEIN 2 the conditions
must be different, as they found in rabbits who remained alive a year
after the total extirpation of the adrenals, that the liver had a normal
amount of glycogen and that the sugar puncture nevertheless was with-
out action. Adrenalin caused glycosuria in such animals.

It is generally admitted that the stimulation which the sugar center
in the fourth ventricle exerts, through the sympathetic nerve reaches to
the adrenals and causes a secretion of adrenalin, which increases the sugar
formation. Certain circumstances, for example, that a glycosuria can be
brought about in starving animals > in which the piqure is without action,
by adrenalin, make the mechanism of this glycosuria somewhat uncer-

1 Verhandl. d. deutsch. Kongr. f. inn. Med. Wiesbaden, 1911.
2Schwarz, Pfluger's Arch., 134; Kahn and Starkenstein, ibid., 139; Kahn, ibid.,
140; Starkenstein, Arch. f. exp. Path. u. Therap., 10.

GLYCOSURIAS. 403

tain. Under all circumstances the sugar puncture glycosuria stands in
close relation to the adrenals and is generally considered as an adrenalin-
glycosuria. The same is true for the glycosuria after splanchnic stimula-
tion and probably for several other forms of glycosuria. In the gly-
cosuria produced by stimulation of the central vagus, according to
BANG, LJUNGDAHL and BOHM/ the hyperglycsemia (in rabbits) depends
upon an increased destruction of the glycogen of the muscles and not of
the liver.

Many investigators consider the glycosuria appearing after the occur-
rence of dyspncc,2 produced in various ways, and also after certain poisons
such as carbon monoxide, curare, ether, chloroform, strychnine, morphine,
piperidin and others as adrenalin glycosurias. That also in many of
such cases the glycosuria is brought about by an increased glycogen
destruction is not doubted. In certain cases, as in carbon monoxide
poisoning, a formation of sugar has been claimed from protein, because
STRAUB and ROSENSTEINS found that this glycosuria only occurred in
those animals that had a sufficient quantity of protein at their disposal.
Protein starvation and simultaneous abundant carbohydrate supply cause a
disappearance of this glycosuria.

A hyperglycsemia and glycosuria may also be caused by a decreased
ability of the animal to consume or to utilize the sugar or to transform
it into glycogen. In this case the sugar must accumulate in the blood,
and the formation of severe cases of diabetes mellitus is now generally
explained by this process.

The inability of diabetics to destroy or consume the sugar does not
seem to be connected with any decrease in the oxidative energy of the
cells. The oxidative processes are not generally diminished in diabetes
(SCHULTZEN, NENCKI and SIEBER), and this has recently been sub-
stantiated by BAUMGARTEN.4 This latter investigator made experiments
with several bodies which on account of their aldehyde nature were
closely related to sugar or were cleavage or oxidation products of it,
namely, glucuronic acid, d-gluconic acid, c?-saccharic acid, glucosamine,

1 Hofmeister's Bietrage, 10.

2 On the importance of the oxygen and the carbon dioxide content of the blood
for the non-appearance or appearance of glycosuria see Underbill, Journ. of biol. Chem.,
1; Penzoldt and Fleischer, Virchow's Arch., 87; Sauer, Pfliiger's Arch., 49, 425, 426;
Macleod, Amer. Journ. of Physiol., 19, with Briggs, Cleveland Med. Journ., 1907;
Eddie, Bioch. Journ., 1, with Moore and Roaf, ibid., 5; Henderson and Underbill,
Amer. Journ. of Physiol., 28.

3 Straub, Arch. f. exp. Path. u. Pharm., 38; Rosenstein, ibid., 40.

4Schultzen, Berl. klin. Wochenschr., 1872; Nencki and Sieber, Journ. f. prakt.
Chem. (N. F.), 26, 35; Baumgarten, " Ein Beitrag zur Zenntniss des Diabetes mel-
litus," Habilitationschrift, also Zeitschr. f. exp. Path. u. Therap., 2, 1905.

404 THE LIVER.

mucic acid, and others, and he found that diabetics destroyed or burned
these bodies to the same extent as healthy individuals. Besides this
it must be remarked that the two varieties of sugar, glucose and fructose,
which are oxidized with the same readiness, act differently in diabetics.
According to RULZ and other investigators fructose is, contrary to
glucose, utilized to a great extent in the organism, but this in man is,
not always the case or at least to a less extent than in certain animals.
In animals with pancreas diabetes (see below) fructose 1 may cause
a deposition of glycogen in the liver while with glucose this does not
occur. The combustion of protein and fat takes place as in healthy
subjects, and the fat is completely burned into carbon dioxide and water.
In this diabetes the ability of the cells to utilize the glucose suffers diminu-
tion, and the explanation of this has been sought in the fact that the glu-
cose is not previously split before combustion.

GOa

The variation in the respiratory quotient, i.e., the relation ~^~, seems

to show an insufficiency of the glucose combustion in the tissues in
diabetes. As will be thoroughly explained in a subsequent chapter, this
quotient is greater the more carbohydrates are burned in the body, and
it is correspondingly smaller when protein and fat are chiefly burned.
The investigations of LEO, HANRIOT, WEINTRAUD and LAVES,2 and
others have shown that in severe cases of diabetes, in the starving con-
dition, the low quotient is not raised after partaking of glucose, as in
healthy individuals, but that it is raised after feeding fructose, which is
also of value to diabetics.

The poverty of the organs and tissues of diabetics in glycogen indicates
that the glycogen in them is more abundantly transformed into sugar.
From what has been said above in regard to the different behavior of
fructose and glucose in the glycogen formation in diabetes, indicates that
in diabetes, also an inability of the body to transform glucose into glycogen
exists and that the lack of glycogen may come about in this way.

Indeed it has been suggested that a preliminary transformation of
glucose into glycogen is necessary before it can be burned in the animal body.
This assumption is without foundation, at least for the glycogen for-
mation in the liver, as the animal body as is shown with experiments
on dogs, can assimilate and burn considerable quantities of carbohy-
drates even after the liver is excluded ( WEHRLE, VERZAR 3) . The admitted

1Kiilz, Beitrage zur Path. u. Therap. des Diabetes mellitus (Marburg, 1874), 1;
Weintraud and Layes, Zeitschr. f. physiol. Chem., 19; Haycraft, ibid.; Minkowski,
Arch. f. exp. Path. u. Pharm., 31.

2 See v. Noorden, Die Zuckerkrankheit, 3. Aufl., 1901.

3 Wehrle, Bioch. Zeitschr., 34; Verzar, ibid., 34.

PANCREAS DIABETES. 405

ability of the liver in diabetes to use fructose and not glucose in the
formation of glycogen is, according to E. NEUBAUER,1 not characteristic
for diabetes, because it also occurs in phosphorus poisoning. Whether
the different behavior of the two kinds of sugar actually depends upon
a diminished ability of the liver in diabetes to form glycogen from glucose
or to another unknown circumstance has not been sufficiently proved.
In experiments on tortoise livers, by perfusion of RINGER'S solution con-
taining sugar, NISHI 2 found that the livers of diabetic animals formed
as much glycogen as the livers of normal animals. These results, which
cannot be applied to other animals, require at least further investiga-
tion.

The relation of the pancreas to diabetic glycosuria is of the greatest
importance for its proper understanding.

The investigations of MINKOWSKI, v. MERINO, DOMINICIS, and later
of many other investigators,3 show that a true diabetes of a severe
kind is caused by the total or almost total extirpation of the pancreas
of many animals, especially dogs. As in man in severe forms of diabetes,
so also in dogs with pancreatic diabetes, an abundant elimination of
sugar takes place even on the complete exclusion of carbohydrates from
the food.

Artificial pancreas diabetes may indeed also in other respects present
the same picture as diabetes in man, but there exist important differences
between these two.4 It is generally accepted that in pancreas diabetes
a diminished consumption exists, i.e., diminished utilization, which
does not exclude an increased sugar formation from other bodies not
carbohydrates.

Many important observations show that a close relation exists between
the liver and pancreas diabetes. PFLUGER has also especially shown
that in diabetes produced by SANDMEYER'S method (partial extirpa-
tion with subsequent destruction of the remains of the gland in the abdom-
inal cavity, when the animal remains alive for a longer time than after
total extirpation) the liver does not lose weight, although the total weight
of the animal diminishes greatly, while in starvation without diabetes

1 Arch. f. exp. Path. u. Pharm., 61.

2 Ibid., 62.

3 See Minkowski, Untersuchungen iiber Diabetes mellitus nach Exstirpation des
Pankreas (Leipzig, 1893); v. Noorden, Die Zuckerkrankheit (Berlin, 1901), which
contains a very complete index of the literature. In regard to diabetes see also Cl.
Bernard, Le§ons sur le diabete (Paris), Seegen, Die Zuckerbildung im Thierkorper
(Berlin, 1890), and Pfluger, Des Glykogen, 2. Aufl., 1905, and especially v. Noorden's
Hanb. d. Pathol. des Stoffwechsels, 2. Aufl., 1907, Bd. 2, Chapter I.

4 See Falta " Ueber den Eiweissumsatz beirn Diabetes mellitus." Berl. klin. Woch-
enschr., 1908, and Zeitschr. f. klin. Med., 66; Gigon, Deutsch. Arch. f. klin. Med., 97.

406 THE LIVER.

the liver loses weight more than the other parts of the body. PFLUGER
concludes from this that the liver in diabetes works actively, and is the
most important seat of production of diabetic sugar.

PFLUGER has found that in frogs the total extirpation of the duodenum
causes a strong and continuous glycosuria and based upon his investigations
and those of other investigators, he believes that a certain relation exists between
the duodenum and pancreas diabetes. The question as to the occurrence of a
duodenal diabetes has been the subject of numerous investigations but the works
of EHRMANN, MINKOWSKI and ROSENBERG x show that such a view is untenable.

There does not seem to be any doubt as to the existence of a certain
relationship between the pancreas to the adrenals and adrenalin gly-
cosuria. The glycosuric action of adrenalin could be prevented by
ZUELZER by the injection of pancreas extracts, and this statement is
confirmed by FRUGONI by experiments with pancreatic juice or pancreatic
extracts, v. FURTH and SCHWARZ 2 have confirmed the correctness of
ZUELZER' s statement but dispute the fact that we are here dealing with
an antagonistic hormone action as they have obtained similar results also
with other bodies, for example with turpentine.

Very stimulating views on the relationship of pancreas diabetes
to the adrenals and the thyroids have been given by FALTA, EPPINGER
and RuDiNGER.3 According to these investigators a reciprocal retarda-
tion exists between the pancreas and thyroid as between the pancreas
and the adrenals while a mutual accelerating action exists between the
thyroids and the adrenals. In depancreatized dogs the retarding action
of the pancreas upon the thyroids is removed, and in this way we explain
the strong increase in the protein, fat (MOHR) and salt-metabolism
(FALTA and WHITNEY 4) observed in pancreas diabetes. By the removal
of the retarding action of the pancreas upon the adrenals, the mobiliza-
tion of the carbohydrates by means of the adrenalin is increased, and
herein, as well as the diminished sugar utilization, lies the reason for the
strong elimination of sugar. The relations between the above three
glands is still further described by the above-mentioned authors, but we
cannot enter more into detail in regard to the interesting question,
which requires further study.

The conditions in pancreas diabetes are certainly very complicated,
and the reasons for this are still very uncertain. Most investigators are of

1 Rosenberg, Bioch. Zeitschr., 18, which contains the literature.

2 Frugoni, fieri, klin. Wochenschr., 45, 1908; v. Fiirth and Schwarz, Bioch. Zeitschr.,
31.

3 Eppinger, Falta and Rudinger, Zeitschr. f. klin. Med., 66, which also contains the
literature on adrenalin diabetes.

*Mohr, Zeitschr. f. exp. Path. u. Therap., 4; Falta and Whitney, Hofmeister'a
Beitrage, 11.

PANCREAS DIABETES. GLYCOLYSIS. 407

the opinion that we are here dealing with the abolition of one or more
bodies which are considered as products of the internal secretion of the
glands (hormones according to STARLING) and which in an unknown
manner regulate the sugar destruction or carbohydrate metabolism.

The assumption of an internal secretion is based on the investiga-
tions of MINKOWSKI, HEDON,' LANCERAUX, THIROLOIX, and others l
upon the action of the subcutaneous transplantation of the gland.
According to these investigations a subcutaneously transplanted piece
of the gland can completely perform the functions of the pancreas as
to the sugar exchange and the sugar elimination, because on the removal
of the intra-abdominal piece of gland, the animal in this case does not
become diabetic, but if the subcutaneously embedded piece of pancreas
is subsequently removed, an active elimination of sugar appears immedi-
ately. As this occurs also on completely cutting off the nerve supply,
it is explained by the assumption of a formation of a special product
in the gland, which passes into the blood; on the other hand ZUELZER,
DOHM and MARKER 2 have made preparations from the pancreas which,
in dogs as well as in man, cause a diminution in the elimination of sugar
(and acetone bodies) in diabetes and an improvement in the general con-
dition.

This internal secretion of the pancreas has in recent times been sup-
posed to be connected with the so-called islands of LANGERHANS; but no
positive results have been obtained in this connection. Nor are we
acquainted with the kind of active substance here formed.

The glycolytic property of the blood as shown by LUPINE was con-
sidered for a time to be due to a glycolytic enzyme formed in the pancreas,
and pancreas diabetes used to be explained by the fact that the action
of this enzyme was removed when the gland was extirpated. This
glycolysis is not sufficient, even if it is derived from the pancreas, to
explain the transformation of the large quantity of sugar in the body,
and for the destruction of sugar we are also obliged to accept a glycolysis
in the organs and tissues. Opinions in regard to this glycolysis differ
in certain points. According to one view (SPITZER and others) special
oxidases are active in the glycolysis, while another (STOKLASA3) con-
considers the glycolysis as analogous to alcoholic fermentation, where we
have processes brought on by special tissue zymases, in which lactic

1 See Minkowski, Arch. f. exp. Path. u. Pharm., 31; He*don, Diabete pancreatique,
Travaux de Physiologic (Laboratoire de Montpellier, 1898), and the works on diabetes.

2 Deutsch. med. Wochenschr., 1908.

3Hofmeisters Beitrage, 3, Centralbl. f. Physiol., 16, 17, 18; Ber. d. d. chem. Ge-
sellsch., 38; also with Czerny, ibid., 36; with Jelinek, Simacgk and Vitek, Pfliiger's
Arch., 101.

408 THE LIVER.

acid is an intermediary step. Many 1 objections have been advanced
against the view of STOKLASA that in animal as well as in plant tissues,
in anaerobic respiration, an alcoholic fermentation may occur as this
observed action of the tissues could only be brought about by the presence
of micro-organisms.

That lactic acid can be an intermediary step in the destruction of
sugar in the animal body cannot be denied. On the contrary it follows
from several circumstances which will be mentioned in Chapter
X. (muscle) on the origin of lactic acid that such a condition exists
and the following observations of A. R. MANDEL and LusK2 on the
relation of lactic acid to diabetes indicate the same. These exper-
imenters showed after phosphorus poisoning in dogs, that the blood and
urine contained abundance of lactic acid, and on producing phlorhizin-
diabetes it disappeared from these fluids, and also that phosphorus poison-
ing does not cause a lactic acid formation in dogs with phlorhizin-
diabetes. Although it is difficult to give a satisfactory interpretation
of these observations, it is still very probable that in the elimination
of the sugar in phlorhizin-diabetes a mother-substance of the lactic acid
is lost.

We do not agree as to the ways and means which bring about
the so-called glycolysis, and another disputed question is whether the
glycolysis can be produced by one organ or only by the combined action
of several organs. COHNHEIM 3 found that a cell-free fluid can be obtained
from a mixture of pancreas and muscle, which destroys glucose, while
the pancreas alone does not have this action, and the muscle only to a
slight extent. The pancreas does not contain, according to COHNHEIM,
a glycolytic enzyme, but a substance resistant to boiling temperatures,
which is soluble in water and alcohol, and which, like an amboceptor,
activates a glycolytic proenzyme which exists in the muscle fluid, but
which is inactive alone and which retards glycolysis when it exists in
excess.

The statements of COHNHEIM have been disputed, and recently LEVENE
and MEYER4 have shown that we are not here dealing with a disap-
pearance of glucose by glycolysis, but more likely with a disappearance

1 See the works of O. Cohnheim, Zeitschr. f. physiol. Chem., 39, 42, 43; Batelli,
Compt, rend., 137; Portier, Compt. rend. soc. biol., 57; Harden and Maclean, Journ.
of Physiol., '42 and 43.

2 Amer. Journ. of Physiol., 16.

3 Cohnheim, Zeitschr. f. physiol. Chem., 39, 42, 43, and 47.

4Stocklasa and collaborators, Centralbl. f. Physiol., 17, and Ber. d. d. chem.
Gesellsch., 36 and 38; Feinschmidt, Hofmeister's Beitrage, 4; Hirsch, ibid.', Claus and
Embden, ibid., 6; Arnheim and Rosenbaum, Zeitschr. f. physiol. Chem., 40; Braun-
stein, Zeitschr. f. klin. Med., 51; Levene and Meyer, Journ. of biol. Chem., 9.

ORIGIN OF THE SUGAR. 409

due to synthesis, where a disaccharide is formed. According to J. DE
MEYER * neither the pancreas nor the tissues as a whole contain any
glycolytic enzymes. According to him only the blood has a glycolytic
action, and this action is supported by a body acting as an amboceptor
and produced in the pancreas. Our knowledge as to the existence of the
glycolysis and the mode of action of the pancreas in the metabolism of
sugar in the animal body is very meager and incomplete.

Where does the sugar eliminated in diabetes originate? Does it
depend entirely upon the carbohydrates of the food or the store of car-
bohydrates in the body, or has the body the power of producing sugar
from other material? To LUTHJE belongs the credit for positively
deciding this question. He has made experiments on dogs with pan-
creas diabetes, in which on a protein diet free from carbohydrates so much
sugar was eliminated that it could not possibly be accounted for by
the store of glycogen or other carbohydrate-containing substances in the
body. Similar experiments were also performed later by PFLUGER,2
with the results that the power of the animal body to produce sugar from
non-carbohydrate material is now definitely proved.

Is this sugar produced from protein or fat, or from both? This ques-
tion so far has not been answered, and it is the subject of continuous
dispute. It is not possible to enter into an exhaustive and detailed
discussion of the question in a text-book, and we will only mention,
briefly, certain of the most important observations and historical points.

The largest amount of sugar which we can obtain theoretically from
protein is 8 grams of sugar from 1 gram of protein nitrogen, if we admit
that all the carbon of the protein, with the exception of that necessary
to form ammonium carbonate, is used for the formation of sugar. These
results are still somewhat too high for the average carbon and nitrogen
content of the proteins and the values D:N = 6.6 is probably more correct.3
The actual relation between glucose and nitrogen in the urine, i.e.,
the quotient D: N, has been repeatedly determined in various forms of
diabetes, and in depancreatized dogs it is generally 2.8 and in starving
dogs or dogs fed with protein and poisoned with phlorhizin it is equal to
3.65 (LUSK). It may undergo considerable variation, and in certain
cases it may indeed be lower than 1 as well as higher than 8, and high
results have been repeatedly obtained in cases of human diabetes. From
these quotients conclusions have been drawn as to the amount of sugar

1 Cited from Centralbl. f. Physiol., 20 and 23. See also Lupine, Etat actuel de la
question de la Glycolyse, La semaine medicale, 1911.

2 Lttthje, Deutsch. Arch. f. klin. Med., 79, and Pfliiger's Arch., 106; Pfliiger, Pflii-
ger's Arch., 108.

3 See Falta, Zeitschr. f. klin. Med., 65; see also Gigon, Deutsch. Arch. f. klin. Med., 97.

410 THE LIVER.

formed, as well as the origin of the sugar, but according to the views of
HAMMARSTEN such conclusions are mostly very uncertain. The sugar
eliminated by the urine represents the difference between the total sugar
production of the body and the quantity of sugar burned or utilized.
Only under the supposition that the body cannot burn or utilize any
sugar, is the sugar of the urine a measure of the quantit}' produced,
and this seems to be the case in phlorhizin diabetes ; but it is
difficult to decide how these suppositions apply to the different forms
of diabetes. Still several observations seem to show that in the different
forms of diabetes variable amounts of the sugar are burned, and only
in special cases can we draw approximately accurate conclusions.

The property of protein of increasing the elimination of sugar is
considered as an important proof of the formation of sugar from protein.
In this regard those experiments are of special interest in which the
diabetic animal is allowed to starve until the urine is poor in sugar or
indeed free from sugar, and then on feeding with protein, an abundant
elimination of sugar is produced. If we do not accept the view in this
case that the protein, but rather the fat, was the material from which
the sugar was produced, still we must admit either of a sugar-sparing
action due to protein or of a strong sugar formation from fat, incited
by the protein.

A sparing in the sense that the protein is oxidized instead of the sugar,
and in this manner protects it, is naturally possible only under the sup-
position that the body can burn at least a part of the sugar, otherwise
there would be nothing to spare and nothing to protect from burning.
The assumption of such an indirect action of proteins is difficult to recon-
cile with the common view of the inability of the body to burn sugar
in diabetes. LUTHJE l has communicated one experiment among others,
in which a dog with pancreas diabetes, \\hose weight before starvation
was 18 kilos, with nineteen days' starvation eliminated an average of
10.4 grams sugar for the last six days of starvation. By exclusive pro-
tein feeding the quantity of sugar per day could be raised to a maximum
of 123.0 grams, and as average it was 97.5 grams for the ten protein
days. The protein, therefore, had protected daily an average of 87
grams sugar from burning, which is hardly possible; and if in the diabetic
animal we admit of this considerable power of burning sugar, the quotient
D:N becomes valueless as a measure of the quantity of sugar formed.

If, on the contrary, we admit of an indirect action of proteins in
that they incite a sugar formation from fat, perhaps by a certain very
important increase in the activity of the liver, we are opposed by the
great difficulty that, according to known laws of metabolism, the pro-

1 Deutsch. Arch. f. klin. Med., 79.

SUGAR FORMATION FROM PROTEINS. 411

teins do not raise the fat metabolism, but rather diminish it. The pro-
tein displaces a corresponding quantity of fat from the metabolism,
and if the fat were the only source of sugar then in this case we would
expect a diminished elimination of sugar instead of an increased one.
Nevertheless the above action of protein upon sugar elimination is much
more easily explained by the assumption of a sugar formation from pro-
tein than from fat.

The action of monamino-acids upon the carbohydrate metabolism
has also given important ground for the assumption of a sugar forma-
tion from protein. That a deamidation occurs in the animal body was
shown by the earlier observations of BAUMANN and BLENDERMANN.
Further proofs of this were furnished by the investigations of NEUBERG
and LANGSTEIN, where in feeding experiments with alanine they found
abundance of lactic acid in the urine, and P. MAYER 1 observed glyceric
acid in the urine after the subcutaneous injection of diaminopropionic
acid. As from amino-acids by deamidation ketone acids or oxyacids
may be formed (see Chapter XIV) it would be of interest to test the action
of amino-acids upon the carbohydrate metabolism. Several investiga-
tions have been carried on with this in view, such as those of LANGSTEIN
and NEUBERG, R. COHN and F. KRAUS, which have shown a very prob-
able formation of carbohydrate under the influence of amino-acids; but
the investigations of EMBDEN and SALOMON, and of EMBDEN and ALMAGIA
have positively shown, in a dog without a pancreas, that the amino-
acids can bring about a re-formation of carbohydrate. LUSK alone
and with RINGER 2 have shown the same for several amino-acids by
experiments on dogs poisoned with phlorhizin. According to the exper-
iments and calculations of the two last mentioned investigators glycocoll
and alanine can be completely transformed into glucose. Of the four
carbon atoms of aspartic acid and of the five carbon atoms of glutamic
acid three appear as glucose.

The investigations of WEINLANDS tend to prove a sugar formation
from protein. He studied the formation of sugar in the chrysalis pulp
of the Calliphora and showed that the sugar formed thereby did not orig-
inate from the fat, but that the protein was the only material from

1Baumann, Zeitschr. f. physiol. Chem., 4; Blendermann, ibid., 6; Neuberg and
Langstein, Arch. f. (Anat. u.) Physiol., 1903, Suppl.; Mayer, Zeitschr. f. physiol. Chem.,
42.

2 Langstein and Neuberg, 1. c.; Cohn, Zeitschr. f. physiol. Chem., 28; F. Kraus,
Berl. klin. Wochenschr., 1904; Embden and Salomon, Hofmeister's Beitrage, 5 and
6, and with Almagia, ibid., 7; Lusk, Amer. Journ. of Physiol., 22; Ringer and Lusk,
Zeitschr. f. physiol. Chem., 66.

3 Zeitschr. f. Biol., 49 (N. F., 31); with Krummacher, ibid., 52.

412 THE LIVER.

which the sugar was formed. The formation of sugar from protein is
now generally considered as positively proved.

DAKIN 1 has found with experiments with phlorhizinized dogs that
serine, cysteine, proline, ornithine and arginine yield abundant sugar in
glycosuric animals. Valine, leucine, isoleucine, lysine, hisfcidine, phenyl-
alanine and tryptophane gave relatively little sugar or none at all.
The ammo-acids with straight chains (with the exception of lysine) give
sugar while those with branched chains do not. Proline is the only
cyclic amino-acid, which yields abundance of sugar. Arginine is the
only one with more than five carbon atoms which yields sugar and the
sugar comes in this case from the ornithine components.

If we assume a formation of sugar from fat, we must differentiate
between the two components of neutral fats, that is, between the glyc-
erin and the fatty acids. A formation of sugar from glycerin can
be considered as proved by the investigations of CREMEB, and especially
those of LUTHJE 2 and in the following we will discuss only the forma-
tion of sugar from the fatty acids.

The formation of sugar from fat seems to occur in the plant king-
dom, and as the chemical processes in the animal and plant life are in
principle the same, it makes the possibility of a sugar formation from
fat very probable. Such an origin of sugar in the animal body is accepted
by many investigators, especially by PFLTJGER and several French observ-
ers, among whom \ve must specially mention CHAUVEAU and KAUF-

MANN.3

When food as free from carbohydrate as possible is taken, the quo-
tient D:N is high, i.e., higher than 8, as well as when the quantity of
sugar is so large that it cannot be accounted for by the calculated
protein (and carbohydrate) metabolism, then if the observations are
otherwise free from error we can admit of a formation of sugar from fat.
Several such cases of diabetes in man have been published (RUMPF,
ROSENQVIST, MOHR, v. NOORDEN, ALLARD, FALTA and co-workers and
others), and also in animals (HARTOGH and ScnuMM4). Although these
researches are not fully conclusive, still certain of them indicate a prob-
able formation of sugar from fat. We also have several conditions which

1 Journ. of biol. Chem., 14, 321.

2 Cremer, Sitzungsber. d. Ges. f. Morph. u. Physiol. Miinchen, 1902; Luthje, Deutsch.
Arch. f. klin. Med., 80.

3 Kaufmann, Arch. f. Physiol. (5), 8, where Chauveau's work is cited.

4Rumpf, Berl. klin. Wochenschr., 1899; Rosenqvist, ibid.', Mohr, ibid., 1901; v.
Noorden, Die Zuckerkrankheit, 3. Aufl. Berlin, 1901; Allard, Arch. f. exp. Path. u.
Pharm., 57; Falta and co-workers, Zeitschr. f. klin. Med., 66; Hartogh and Schumm,
Arch. f. Path. u. Pharm., 45. See also the works of O. Loewi, ibid., 47, and Lusk,
Zeitschr. f. Biologic, 42.

SUGAR FORMATION FROM FATS. 413

indicate the same, namely, that in phlorhizin diabetes after the disap-
pearance of the liver-glycogen the fat which migrates to the liver serves
as material for the formation of sugar (PFLUGER). These observations
make the formation of sugar from fat highly probable and the same is true
for the observations of JuNKERSDORF.1 He found that in an animal made
glycogen free, by starvation and with phlorhizin poisoning, that toward
death, the nitrogen as well as the sugar elimination increased but that
the D : N ratio was higher than with the sugar formation from protein
alone. His calculations are not free from exception.

On the other hand there are many observations on animals and
also clinical observations which oppose the theory of the formation
of sugar from fat in diabetes. LUSK found in a dog with phlorhizin
diabetes that the quotient D:N = 3.65:1 was not changed on feeding fat,
and he has published further results of experiments 2 which show that
active muscular work, which strongly increases the fat decomposition,
does not change the quotient in dogs with phlorhizin diabetes. It is
difficult to draw positive conclusions from these experiments, still LUSK
seems to deny the formation of sugar from fat.

Attempts have been made to solve the question as to the material
from which sugar is formed by the determination of the respiratory
quotient and comparing this with the quotient D:N. The calculations
in this direction have not led to positive results.3 As the quotient D : N
is not an accurate measure of the quantity of sugar formed, and as we,
as yet, do not know the quantity of oxygen necessary to form sugar
from protein, HAMMARSTEN believes that it is just as impossible to con-
clude from the respiratory quotient that sugar is formed from the fats
as from the proteins.

We have no complete proofs for the formation of sugar from fat, still
we can indicate the probable proofs therefor. There is really no objec-
tion from a theoretical standpoint to the assumption that the body has
the power of producing sugar from protein as well as from fat, and such
a power does not seem improbable.

As a formation of sugar from protein is now generally considered as
proved, it follows that the protein can yield material for the formation
of glycogen and that it is a true glycogen-former. PFLUGER and JUNKERS-
DORF 4 have given direct proof for this. They fed a dog, which had
previously been made glycogen-free by starvation and phlorhizin injec-

1 Pfliiger's Arch., 137.

2 Amer. Journ. of Physiol., 22.

3 Magnus-Levy, Zeitschr. f. klin. Med., 56; Pfliiger's Arch., 108; Mohr, Zeitschr.
f. exp. Path. u. Therap., 4.

4 Pfluger's Arch., 131.

414 THE LIVER.

tions, with abundance of codfish and then found so much glycogen (6.46
per cent in the liver and 1 per cent in the muscle) that a re-formation of
glycogen must have undoubtedly occurred. By special control exper-
iments with fat feeding they also showed that the glycogen did not orig-
inate from the fat but must unquestionably have come from the protein.
Carbohydrates and proteins are without question true glycogen-formers,
while the question in regard to fats is still open.

The Bile and Its Formation.

By the establishment of a biliary fistula, an operation which was
first performed by SCHWANN in 1844 and which has been improved lately
by DASTBE and PAWLOW/ it is possible to study the secretion of the bile.
This secretion is continuous, but with varying intensity. It takes
place under a very low pressure; therefore an apparently unimportant
hindrance in the outflow of the bile, namely, a stoppage of mucus in the
exit, or the secretion of large quantities of viscous bile, may cause stagna-
tion and absorption of the bile by means of the lymphatic vessels (absorp-
tion icterus).

The quantity of bile secreted in the twenty-four hours in dogs can be
exactly determined. The quantity secreted by different animals varies,
and the limits are 2.9-36.4 grams of bile per kilo of weight in the twenty-
four hours.2

The reports as to the extent of bile secretion in man are few and
not to be depended on. NOEL-PAYTON, MAYO-ROBSON, HAMMARSTEN,
PFAFF and BALCH, and BRAND 3 found a variation between 514 and
1083 cc. per twenty-four hours. Such determinations are of doubtful
value, because in most cases it follows from the composition of the
collected bile that the fluid is not the result of a secretion of normal liver
bile.

The quantity of bile secreted is, however, as shown by STADEL-
MANN,4 subject to such great variation, even under physiological con-
ditions, that the study of 'those circumstances which influence the secre-
tion is very difficult and uncertain. The contradictory statements
by different investigators may probably be explained by this fact.

^chwann, Arch. f. (Anat. u.) Physiol., 1844; Dastre. Arch, de Physiol. (5) 2;
Pawlow, Ergebnisse der Physiol., 1, Abt. 1.

2 In regard to the quantity of bile secreted in animals see Heidenhain, Die Gallenab-
sonderung, in Hermann's Handbuch der Physiol., 5, and Stadelmann, Der Icterus und
seine verschiedenen Formen (Stuttgart, 1891).

3 Noel-Payton, Rep. Lab. Roy. Coll. Edinburgh, 3; Mayo-Robson, Proc. Roy. Soc.,
47; Hammarsten, Nova Act. Reg. Soc. Scient. Upsala (3), 16; Pfaff and Balch, Journ.
of Exp. Med., 1S97; Brand, Pfliiger's Arch., 90.

4 Stadelmann, Der Icterus, etc., Stuttgart, 1891.

BILE SECRETION. 415

In starvation the secretion diminishes. According to LUKJANOW
and ALBERTONi,1 under these conditions the absolute quantity of solids
decreases, while ' the relative quantity increases. After partaking of
food the secretion increases again. The findings are very contradictory
in regard to the time necessary, after partaking of food, before the
secretion reaches its maximum. After a careful examination and com-
pilation of all the existing reports, HEiDENHAiN2 has come to the con-
clusion that in dogs the curve of rapidity of secretion shows two maxima,
the first at the third to fifth hour and the second at the thirteenth to
fifteenth hour after .partaking of food. According to BARBERA the
time when the maximum occurs is dependent upon the kind of food.
With carbohydrate food it is two to three hours, after protein food three
to four hours, and with fat diet it is five to seven hours, after feeding.
According to LoEB3 the maximum occurs in dogs one to two hours after
feeding with meat, casein or gliadin.

According to earlier observations, the proteins of all the various
foods cause the greatest secretion of bile, while the carbohydrates dimin-
ish the secretion, or at least excite it much less than the proteins. This
coincides with the recent observations of BARBERA. The authorities
by no means agree as to the action of the fats. While many older
investigators have not observed any increase, but rather the reverse
in the secretion of bile after feeding with fats, the researches of BARBARA
show an undoubted increase in the secretion of bile on fat feeding, greater
even than after carbohydrate feeding. According to ROSENBERG olive-
oil is a strong cholagogue, a statement which, according to other inves-
tigators— MANDELSTAMM, DOYON and DUFOURT ! — has not been proved.

As BARBERA has shown, a close relation exists between the bile
secretion and the quantity cf urea formed, as an increase in the first
goes hand in hand with an increase of the latter. The bile is, therefore,
according to him, a product of disassimilation, whose quantity rises and
falls with the degree of activity of the liver.

The question whether there exists special medicinal bodies, so-called
cholagogues, which have a specific excitant action on the secretion of

1 Lukjanow, Zeitschr. f. physiol. Chem., 16; Albertoni, Recherches sur la se*cre*tion
biliaire, Turin, 1893.

2 Hermann's Handb., 5, and Stadelmann, Der Icterus, etc.

3 Barbara, Centralbl. f. Physiol., 12 and 16; A. Loeb, Zeitschr. f. Biol., 55.

4 Barbera, Bull, della scienz. med. di Bologna (7), 5, Maly's Jahresber., 24, and
Centralbl. f. Physiol., 12 and 16; Rosenberg, Pfliiger's Arch., 46; Mandelstamm, Ueber
den Einfluss einiger Arzneimittel auf Sekretion und Zusammensetzung der Galle (Dis-
sert. Dorpat, 1890); Doyon and Dufourt, Arch, de Physiol. (5), 9. In regard to the
action of various foods on the secretion of bile see also Heidenhain, 1. c.; Stadelmann,
Der Icterus; and Barbera, 1. c.

THE LIVER.

bile, has been answered in very different ways. Many, especially the
older investigators, have observed an increase in the bile secretion after
the use of certain therapeutic agents, such as calomel, rhubarb, jalap,
turpentine, olive-oil, etc.; while others, especially the more recent inves-
tigators, have arrived at quite opposite results. From all appearances
this contradiction is due to the great irregularity of the normal secretion,
which might readily cause mistakes in tests with therapeutic agents.

SCHIFF'S view, that the bile absorbed from the intestinal canal increases
the secretion of bile and hence acts as a cholagogue, seems to be a pos-
itively proved fact by the investigations of several experimenters.1
Sodium salicylate is also perhaps a cholagogue (STADELMANN, DOYON
and DUFOURT, WINOGRADOW) and according to PETROWA 2 in dogs sodium
benzoate, thymole, phenol, menthol and all such bodies which are
conjugated to ethereal sulphuric acid in the animal body, increase the
secretion of bile.

Acids, and especially, under normal conditions, hydrochloric acid,
seem to be physiological excitants for bile secretion. According to
FALLOISE and FLEIG the acids act upon the duodenum and the upper
part of the jejunum, and the action is brought about by a secretin forma-
tion similar to the action of acids upon the secretion of pancreatic juice
(see Chapter VIII). According to FALLOISE 3 chloral hydrate introduced
into the duodenum causes a secretion of bile in an analogous manner, by
the aid of a special chloral secretin.

The bile is a mixture of the secretion of the liver-cells and the so-
called mucus which is secreted by the glands of the biliary passages
and by the mucous membrane of the gall-bladder. The secretion of the
liver, which is generally poorer in solids than the bile from the gall-
bladder, is thin and clear, while the bile collected in the gall-bladder
is more ropy and viscous on account of the absorption of water and the
admixture of " mucus," and cloudy because of the presence of cells,
pigments, and the like. The specific gravity of the bile from the gall-
bladder varies considerably, being in man between 1.010 and 1.040.
Its reaction is alkaline to litmus. The color changes in different animals:
golden-yellow, yellowish-brown, olive-brown, brownish-green, grass-green
or bluish-green. Bile obtained from an executed person immediately
after death is golden-yellow or yellow with a shade of brown. Still cases

1 Schiff, Pfliiger's Arch., 3. See Stadelmann, Der Icterus, and the dissertations of
his pupils, especially Winteler, " Experimentelle Beitrage zur Frage des Kreislaufes
der Galle " (Inaug.-Diss. Dorpat, 1892), and Gartner, " Experimentelle Beitrage zur
Physiol. und Path, der Gallensekretion " (Inaug.-Dis. Jurjew, 1893); also Stadelmann,
" Ueber den Kreislauf der Galle," Zeitschr. f. Biologic, 34.

2 Zeitschr. f. physiol. Chem., 74 (literature). See also footnote 4, page 415.

3 Falloise, Bull. Acad. Roy. de Belg., 1903; Fleig, ibid., 1903.

BILE-SALTS. 417

occur in which fresh human bile from the gall-bladder has a green color.
The ordinary post-mortem bile has a variable color. The bile of cer-
tain animals has a peculiar odor; for example, ox-bile has an odor of
musk, especially on warming. - The taste of bile is also different in
different animals. Human as well as ox-bile has a bitter taste, with a
sweetish after-taste. The bile of the pig and rabbit has an intensely
persistent bitter taste. On heating bile to boiling it does not coagulate.
It contains (in the ox) only traces of true mucin, and its ropy properties
depend, it seems, chiefly on the presence of a nucleoalbumin similar to
mucin (PAIJKULL). The bile from the animals investigated by HAM-
MARSTEN showed a similar behavior. HAMMARSTEN l has, on the con-
trary, found a true mucin in human bile. To all appearances this mucin
originates from the billiary passages, as he found it in the bile flowing
from the hepatic duct, and also because the mucous membrane of the
gall-bladder, according to WAHLGREN,2 does not in man secrete any
mucin, but a mucin-like riucleoalbumin.

The specific constituents of the bile are bile-adds combined with alkalies,
bile-pigments, and, besides small quantities of lecithin and phosphatides,
cholesterin, soaps, neutral fats, urea, ethereal sulphuric acid, traces of
conjugated glucuronic adds, enzymes and mineral substances, chiefly chlorides,
besides phosphates of calcium, magnesium, and iron. Traces of copper
also occur.

Bile-salts. The bile-acids, which thus far have best been studied,
may be divided into two groups, the glycocholic and taurocholic add
groups. As found by HAMMARSTEN 3 a third group of bile-acids occurs
in the shark, which are rich in sulphur, and like the ethereal sulphuric
acids they split off sulphuric acid on boiling with hydrochloric acid.
All glycocholic acids contain nitrogen, but are free from sulphur and
can be split, with the addition of water, into glycocoll (amino-acetic acid)
and a nitrogen-free acid, a cholic acid. All taurocholic acids contain
nitrogen and sulphur and are split, with the addition of water, into
taurine and a cholic acid. The reason for the existence of different glyco-
cholic and taurocholic acids depends on the fact that there are several
cholic acids.

The conjugated bile-acid found in the shark, and called scymnol-sulphuric add
by HAMMARSTEN, yields as cleavage products sulphuric acid and a non-nitrogenous
substance, scymnol (C27H460S), which gives the characteristic color reactions of
cholic acid.

1 Paijkull, Zeitschr. f. physiol. Chem., 12; Hammarsten, 1. c., Nova Act. (3), 16,
and Ergebnisse der Physiol., Bd. 4.

2 Maly's Jahresber., 32.

3 Hammarsten, Zeitschr. f. physiol. Chem., 24.

418 THE LIVER.

The different bile-acids occur in the bile as alkali salts, generally
the sodium compounds, even in sea-fishes, although this is contrary to
the earlier observations (ZANETTi1). In the bile of certain animals we
find almost solely glycocholic acid, in others only taurocholic acid, and
in still others a mixture of both (see below).

All alkali salts of the biliary acids are soluble in water and alcohol,
but insoluble in ether. Their solution in alcohol is therefore precipitated
by ether, and this precipitate, with proper care in manipulation, gives,
for nearly all kinds of bile thus far investigated, rosettes or balls of fine
needles, or four- to six-sided prisms (PLATTNER'S crystallized bile). Fresh
human bile also crystallizes readily. The bile-acids and their salts
are optically active and dextrorotatory. The salts of the different bile-
acids act somewhat differently toward neutral salts. The alkali salts
of the ordinary and best-studied bile-acids from man, ox, and dog are,
according to TENGSTROM,2 precipitated by ammonium and magnesium
sulphates, and also, in pure form, by sodium nitrate and sodium chloride
(added to saturation). Potassium and sodium sulphates do not precip-
itate them. The alkali salts cannot be directly precipitated from the
bile by NaCl, on account of the presence of bodies retarding precipita-
tion, among which we find oil-soaps.

The bile-acids are dissolved by concentrated sulphuric acid at the
ordinary temperature, forming a reddish-yellow liquid which has a beautiful
green fluorescence. According to PREGL an oxidation with a reduction
of the sulphuric acid into sulphur dioxide takes place. The fluorescent
substance has been called dehydrocholan (see below) by PREGL.3 On
carefully warming with concentrated sulphuric acid and a little cane-
sugar, the bile-acids give a beautiful cherry-red or reddish-violet liquid.
PETTENKOFER'S reaction for bile-acids is based on this behavior.

PETTENKOFER'S test for bile-acids is performed as follows: A small
quantity of bile in substance is dissolved in a small porcelain dish in con-
centrated sulphuric acid and warmed, or some of the liquid contain-
ing the bile-acids is mixed with concentrated sulphuric acid, taking special
care in both cases that the temperature does not rise higher than 60-
70° C. Then a 10-per-cent solution of cane-sugar is added, drop by
drop, continually stirring with a glass rod. The presence of bile is indi-
cated by the production of a beautiful red liquid, whose color does not
disappear at the ordinary temperature, but becomes more bluish-violet
in the course of a day. This red liquid shows a spectrum with two absorp-
tion-bands, the one at F and the other between D and E, near E.

1 See Chem. Centralbl., 1903, 1, 180.

2 Zeitschr. f. physiol. Chem., 41.

3 Zeitschr. f. physiol. Chem., 45.

GLYCOCHOLIC ACID. 419

This extremely delicate test fails, however, when the solution is
heated too high, or if an improper quantity — generally too much — of
the sugar is added. In the last-mentioned case the sugar easily car-
bonizes and the test becomes brown or dark brown. The reaction fails
if the sulphuric acid contains sulphurous acid or the lower oxides of
nitrogen. Many other substances, such as proteins, oleic acid, amyl
alcohol, and morphine, give a similar reaction, and therefore in doubt-
ful cases the spectroscopic examination of the red solution must not be
forgotten.

PETTENKOFER'S test for the bile-acids depends essentially on the
fact that furfurol is formed from the sugar by the sulphuric acid (MYLIUS).
According to MYLIUS and v. UDRANSZKY 1 a 1 p. m. solution of furfurol
should be used. Dissolve the bile, which must first be decolorized by
animal charcoal, in alcohol. To each cubic centimeter of alcoholic
solution of bile in a test-tube add 1 drop of the furfurol solution and
1 cc. concentrated sulphuric acid, and cool when necessary, so that the
test does not become too warm. This reaction, when performed as
described, will detect ^r to j^- milligram cholic acid (v. UDRANSZKY).
Other modifications of PETTENKOFER'S test have been proposed.

The reaction with furfurol is not identical with that obtained with
cane-sugar, according to VILLE and DERRIEN, and the absorption-bands
do not occur in the same place in the two cases. The reaction with cane-
sugar does not depend, according to these investigators, upon a furfurol
formation from the sugar. The acid hydrolyzes the sugar, and from the
fructose produced, 4-methyl-2-oxyfurfurol is formed by the further action
of the acid, and this gives the color reaction with the cholic acid. Instead
of furfurol other aldehydes such as vanillin and anisaldehyde can be
used according to VILLE and DERRIEN. 2

Glycocholic Acid. The constitution of the glycocholic acid occurring
in human and ox-bile, and which has been most studied, is represented
by the formula C26H43NOe. Glycocholic acid is absent, or nearly so,
in the bile of carnivora. On boiling with acids or alkalies this acid,
which is analogous to hippuric acid, is converted into cholic acid and
glycocoll.

By the action of hydrazine hydrate upon the ethyl ester of cholic acid
BONDI and MULLER 3 prepared first cholic-acid hydrazide, and then, by
the action of nitrous acid upon this, they obtained the cholic-acid azide,
C23H3Q03CO.N3, and finally from this last in alkaline solution with glyco-

1 Mylius, Zeitschr. f. physiol. Chem., 11; v. Udranszky, ibid., 12.

2 Ville and Derrien, Chem. Centralbl. 1909, 2, 1699 and Compt. rend, soc., biol. 64
and 66.

3 Zeitschr. f. physiol. Chem., 47.

420 THE LIVER.

coll they synthetically prepared the alkali salt of glycocholic acid, at
the same time splitting off nitrogen.

Glycocholic acid crystallizes in fine, colorless needles or prisms. It
is soluble with difficulty in water (in about 300 parts cold and 120 parts
boiling water), and is easily precipitated from its alkali-salt solution
by the addition of dilute mineral acids. According to BONDI l glyco-
cholic acid is a rather strong acid, about as acid as lactic but much
stronger than acetic acid. This last-mentioned acid precipitates gly-
cocholic acid from the solution of its alkali salts in water. It is readily
soluble in strong alcohol, but with great difficulty in ether. The solu-
tions have a bitter but at the same time sweetish taste. The acid melts
between 132-152°, depending upon the method of preparation. Accord-
ing to LETSCHE, the acid containing water of crystallization (1J mol.)
deflagrates on heating rapidly at 126°, and at 130° an active frothing is
observed. The acid free from water of crystallization deflagrates at
130-132°, and decomposes at 154-155° C. with frothing. The salts of
the alkalies and alkaline earths are soluble in alcohol and water.

The solution of the alkali salt in water can be salted out by NaCl,
but not by KC1. The salts of the heavy metals are mostly insoluble or
soluble with difficulty in water. The solution of the alkali salts in water
is precipitated by sugar of lead, cupric and ferric salts, and silver nitrate.

On boiling with water glycocholic acid is probably transformed into its
physical isomer paraglycocholic acid, according to LETSCHE,2 and this
crystallizes in long leaves which, when containing water of crystalliza-
tion, show ready deflagration at 186° and decompose with frothing at
198° C. On solution in alcohol or dilute alkalies the paraglycocholic
acid passes into the ordinary glycocholic acid.

Glycocholeic Acid is a second glycocholic acid, first isolated by WAHL-
GREN 3 from ox-bile, and has the formula C^eH^sNOs or C2?H45NO5.
This acid, which on hydrolytic cleavage yields glycocoll and choleic
acid, has also been detected in human bile and the bile of the musk-ox
(HAMMARSTEN 4) .

Glycocholeic acid may, like glycocholic acid, crystallize in tufts of
fine needles, but is often obtained as short thick prisms. It is much more
insoluble in water, even on boiling, than glycocholic acid, and it melts
at 175-176° C. The alkali salts are soluble in water, have a pure bit-
ter taste, and are more readily precipitated by neutral salts (NaCl) than
the glycocholates. The solution of the alkali salts is not only precipitated

1 Zeitschr. f. physiol. Chem., 53.

2 Ibid., 60 and 73.

3 Ibid., 36.

., 43.

TAUROCHOLIC ACID. 421

by the salts of the heavy metals, but also by the salts of barium, cal-
cium and magnesium.

The principle in the preparation of the pure glycocholic acids con-
sists in treating a 2-3 per cent solution of bile free from mucus, when
rich in glycocholic acid (so-called HUFNER'S bile x), with ether, and then
with 2 per cent hydrochloric acid. If the bile is not directly precipitable
with hydrochloric acid (bile relatively poor in glycocholic acid), then
precipitate the chief mass of the glycocholic acid with ferric chloride,
or better with lead acetate, decompose the precipitate with soda and treat
the 2 per cent" solution as above stated with ether and hydrochloric acid.
The crystalline and washed mass is boiled with water, and on cooling
glycocholic acid crystallizes out, and then this is recrystallized from
water or from alcohol by the addition of water. The residue that remains
after boiling in water (paraglycocholic acid and glycocholeic acid) is
converted into their barium salts, and after a complicated method (see
WAHLGREN) the glycocholeic acid is obtained. The reader is referred to
more exhaustive works for other methods of preparation.

Hyoglycocholic Acid, C27H43N05, is the crystalline glycocholic acid obtained
from the bile of the pig. It is very insoluble in water. The alkali salts, whose
solutions have an intensely bitter taste; without any sweetish after-taste, are
precipitated by CaCl2, BaCl2, and MgCl2, and may be salted out like a soap by
Na2S04 when added in sufficient quantity. According to Piettre it can be salted
out entirely, free from sulphur, by caustic alkali which is not possible by other
methods. By precipitation with NaCl in such quantity that the precipitate re-
dissolves on warming, HAMMARSTEN 2 obtained the alkali salt, as macroscopic
crystals, on cooling. Besides this acid there occurs in the bile of the pig still
another glycocholic acid ( JOLIN 3) .

The glycocholate in the bile of rodents is also precipitated by the above
mentioned earthy salts, but cannot, like the corresponding salt in human or ox-
bile, be directly precipitated on saturating with a neutral salt (Na2S04). Guano
bile-acid possibly belongs to the glycocholic-acid group, and is found in Peruvian
guano, but has not been thoroughly studied.

Taurocholic Acid. This acid, which is found in the bile of man, car-
nivora, oxen, and a few other herbivora, such as sheep and goats, has the
constitution C26H4sNS07. On boiling with acids and alkalies it splits
into cholic acid and taurine. Taurocholic acid has also been prepared
synthetically by BONDI and MULLER, using the same method as they used
for glycocholic acid.

Taurocholic acid can be readily obtained, by the method suggested
by HAMMARSTEN,4 as groups of fine needles or as beautiful prisms on
slow crystallization. The crystals do not change in the air, but they
decompose above 100°. They are soluble in alcohol but insoluble in

1 Hiifner, Journ. f. prakt. Chem. (N. F.), 10, 19, and 25.

2 Not published. M. Piettre, Recherches sur la bile, Laval, 1910.

3 Zeitschr. f. physiol. Chem., 12 and 13.

4 Ibid., 43.

422 THE LIVER.

ether, benzene, and acetone. Taurocholic acid is very soluble in water,
and the solution has a very sweet taste, with only a slight bitter taste.
It can hold the difficultly soluble glycocholic acid in solution. This is
the reason why a mixture of glycocholate with a sufficient quantity of
taurocholate, which often occurs in ox-bile, is not precipitated by a dilute
acid. Its salts are, as a rule, readily soluble in water, and the solutions
of the alkali salts are not precipitated by copper sulphate, silver nitrate
or lead acetate. Basic lead acetate gives, on the contrary, a precipitate
which is soluble in boiling alcohol. The alkali salts are not only pre-
cipitated from their solution by the same neutral salts that precipitate
glycocholic acid, but also by potassium chloride, and by sodium and
potassium acetates.

Taurocholeic Acid is a second taurocholic acid, detected by HAMMAR-
STEN in dog-bile and isolated by GULLBRING l from ox-bile, and has the
formula C26H45NS06 or C27H4?NSO6. Thus far it has been obtained
only in the amorphous form. It is readily soluble in water, and has a
disagreeably bitter taste. It is also readily soluble in alcohol, but insoluble
in ether, acetone, chloroform, and benzene. The alkali salt, soluble in
water, can be salted out by NaCl as a pasty mass. The solutions of the
salts can be precipitated by ferric chloride. The cleavage products are
taurine and choleic acid.

The taurocholic acids are most simply prepared from bile, free from
glycocholic acid or poor therein, such as fish- or dog-bile, easiest from the
latter. The aqueous solution of the mucus-free bile is almost completely
precipitated by ferric chloride. The precipitate is worked for tauro-
choleic acid and the filtrate for taurocholic acid. The iron is first removed
from the filtrate by Na2CO3, and then the faintly alkaline filtrate satu-
rated with NaCl. The taurocholate separates out and after further
purification is decomposed by alcohol containing hydrochloric acid. The
taurocholic acid is precipitated from the alcoholic filtrate by ether and
recrystallized from alcohol containing water by the addition of ether.
The taurocholeic acid is obtained from the above iron precipitate by treat-
ing it with soda, and decomposing the alkali salt of the taurocholeic acid
with alcohol, containing HC1, and precipitating the acid from the alcoholic
solution with ether and repeating this precipitation from alcohol by ether.

Cheno-taurocholic Acid. This is the most essential acid of goose-bile and has
the formula C»H«N8Oe. This acid, but little studied, is amorphous and solu-
ble in water and alcohol.

The taurocholic acids differ from the glycocholic acids in being
readily soluble in water. In the bile of the walrus, on the contrary, a
relatively insoluble, readily crystallizable taurocholic acid occurs, which

1 Hammarsten, Zeitschr. f. physiol. Chem., 43; Gullbring, ibid., 45.

CHOLIC ACID. 423

can be precipitated from the solution of the alkali salts by the addition
of mineral acids, like glycocholic acid (HAMMARSTEN *).

As repeatedly mentioned above, the two bile-acids split on boiling
with acids or alkalies into non-nitrogenous cholic acids and into glycocoll
or taurine. Of the various cholic acids the following have been best
studied.

Cholic Acid or Cholalic Acid. The ordinary cholic acid obtained as
a decomposition product of human and ox-bile, which occurs, regularly
in the contents of the intestine, and also in the urine in icterus, has, accord-
int to STKECKER and nearly all recent investigators, the constitution

fCHOH
C24H4o05, = C2oH3i | (CH^OH^. According to MYLius,2 cholic acid is a

ICOOH

monobasic alcohol-acid with one secondary and two primary alcohol
groups. CURTIUS 3 has shown by preparing the cholamine, C23H3903.NH2,
from the above-mentioned (p. 419) cholic-acid azide, with cholic-acid
urethane as an intermediary step, that the carboxyl group is not imme-
diately connected with the CHOH group, but is combined with the chief
nucleus without the neighboring secondary alcohol group. On oxida-
tion it first yields dehydrocholic acid, C24Hs405 (HAMMERSTEN) from
which by electric reduction, SCHENCK obtained the reducto-dehydro-
cholic acid, C24H.3&O5. On further oxidation bilianic add, C24H340g
(CLEVE), is obtained, or, more correctly, according to LATSCHINOFF,
LASSAR-COHN and PREGL, a mixture of bilianic and isobilianic acids
discovered by LATSCHINOFF. On oxidation, bilianic acid yields dlianic
add (LASSAR-COHN), whose formula, according to PREGL,4 is C2oH28Og.

The products formed on a more active oxidation are of great interest.
If we discard the still somewhat problematic cholesterinic acid, we
find in these products in the first place choloidanic add which has also
been* called cholecamphoric acid and has the formula, CisH^sOg, accord-
ing to PREGL. This acid, as well as the acid obtained by LETSCHE 5
on the oxidation of cholic acid and with the formula, CigH^gOio, have
been obtained by PREGL 6 from the three most closely studied cholic acids,

1 Zeitschr. f. physiol. Chem., 61.

2 The important researches of Strecker on the bile-acids may be found in Annal. d.
Chem. u. Pharm., 65, 67, and. 70; Mylius, Ber. d. deutsch. chem. Gesellsch., 19.

3 Ibid., 39.

4 Hammarsten, Ber. d. deutsch. chem. Gesellsch., 14; Schenck, ibid., 63 and 69;
Cleve, Bull. Soc. chim., 35; Latschinoff, Ber. d. d. chem. Gesellsch., 15; Lassar-Cohn,
Ber. d. d. chem. Gesellsch., 32; Pregl, Wein. Sitzungsber., Ill, 1902.

5 Zeitschr. f. physiol. Chem., 61.
<> Ibid., 65.

424 THE LIVER

namely from cholic acid, choleic acid and desoxycholic acid, and these
three acids are identically constructed in regard to their 19 carbon atoms.

The choloidanic acid is interesting in several respects. PANZERX has obtained
from it by distillation with soda-lime, a hydrocarbon, CnHie, a homologue of
benzene, and on the oxidation of the cholic acid he has obtained an acid with the
formula, C80i206, which he considers as an oxyhexahydro-benzene-l-4-dicarboxylic
acid and from which he obtained paraoxybenzaldehyde. PREGL has obtained
from choloidanic acid, by heating, pyrocholoidanic acid, Ci6H2o04 which he con-
siders as parabenzoic acid d-methyl-n-capric acid, and is produced from the
hexahydrobenzene derivative by total dehydrogenation of a benzene derivative.

v. FURTH and collaborators have investigated the products obtained on the
dry distillation of cholic acid at ordinary pressure, and WIELAND and WEIL2 on
such distillation in vacuum. In the first case chiefly hydrocarbons with 12 to
17 carbon atoms were obtained, and in the second instance chiefly an unsaturated
acid, C24H34O2, was obtained, and in both cases these products and their double
bindings have been carefully investigated. We must wait for further developments
in these investigations before we attempt to draw any positive conclusions from
them.

From the investigations on the cholic acids carried out thus far we
are not able to draw any positive conclusions on their constitution, but
that they are derivatives of hexahydrobenzene, is very probable for sev-
eral reasons.

Cholic acid crystallizes partly in rhombic plates or prisms with one
molecule of water, and partly in larger rhombic tetrahedra or octahedra
with one molecule of alcohol of crystallization (MTLIUS). These crystals
quickly become opaque and porcelain-white in the air. They are quite
insoluble in water (in 4000 parts cold and 750 parts boiling), rather
soluble in alcohol, but soluble with difficulty in ether. The amorphous
cholic acid is less insoluble. The solutions have a bitter-sweetish taste.
The crystals lose their alcohol of crystallization only after a lengthy
heating to 100-120° C. The acid free from water and alcohol melts at
195-196° C. According to BONDI and MULLER the melting-point of the
perfectly pure acid is 198° C. It forms a characteristic blue compound
with iodine (MYLIUS). If finely powdered cholic acid is added to 25
per cent hydrochloric acid at the ordinary temperature, a beautiful violet-
blue coloration gradually appears, and this color is permanent for some
time and then becomes gradually green and yellow. The blue solution
shows an absorption band in the neighborhood of the D line (HAMMAR-

STEN 3) .

The alkali salts are readUy soluble in water, but when treated with a
concentrated caustic or carbonated alkali solution, they may then be

1 Panzer, Zeitschr. f. physiol. Chem., 48 and 60.

2 v. Fiirth with Link, Bioch. Zeitschr., 26, with Ishihara, ibid., 43; Wieland and
Weil, Zeitschr. f. physiol. Chem., 80.

3 Zeitschr. f. physiol. Chem., 61.

CHOLEIC ACID. 425

separated as an oily mass which becomes crystalline on cooling. The
alkali salts are not readily soluble in alcohol, and on the evaporation of
the alcohol they may crystallize. The specific rotatory power of the
sodium salt 1 is (a)D = +30.61° (2.29 per cent concentration) to +27.46°
(7.59 per cent concentration). The watery solution of the alkali salts,
when not too dilute, is precipitated immediately or after some time by
lead acetate or by barium chloride. The barium salt crystallizes in fine,
silky needles, and is rather insoluble in cold, but somewhat easily soluble
in warm water. The barium salt, as well as the lead salt, which is
insoluble in water, is soluble in warm alcohol.

Choleic Acid (C25H4204, LATSCHINOFF) is another cholic acid which,
according to LASSAR-CoHN,2 has the formula, C24H4o04. This acid,
which occurs in varying but always small quantities in ox-bile, and also
in gall-stones (H. FISCHER and P. MEYER 3) yields dehydrocholeic add,
C24Hs404, and then cholanic acid, C24Hs4O7, and isocholanic add on
oxidation.

Choleic acid crystallizes when free from water in hexagonal vitreous
prisms with pointed ends, melting at 185-187° C. The crystalline acid
containing water melts at 135-140° C. (LATSCHINOFF). The acid
dissolves in water with difficulty and is also relatively difficultly soluble
in alcohol. It has an intensely bitter taste and gives the MYLIUS iodine
reaction for cholic acid, and also the color reaction of cholic acid with
hydrochloric acid. The specific rotation is (a) D = +48.87° (VAHLEN).
The barium salt which crystallizes from the hot alcoholic solution as
spherical aggregations of radial needles is more difficultly soluble in
water than the corresponding cholate.

Desoxycholic Acid, C24H4oO4, is the name given by MYLIUS 4 to a
cholic acid isolated by him from putrid ox-bile, also in gall-stones (Ktis-
TER) and in faeces (FISCHER 5), and which is formed from the cholic acid
(on the putrefaction of the bile) by reduction. This last is still very
improbable, and the investigations of EKBOM do not support such an
assumption. On using perfectly pure cholic acid he was able to regain
it almost quantitatively after the action of metallic sodium on the alcoholic
solution of the acid, or of zinc and alkali. By treatment with zinc
and glacial acetic acid a reaction took place, but the product was a
mixture of mono- and diacetyl derivatives. The observation of PREGL

1 See Vahlen, Zeitschr. f. physiol. Chem., 21.

2 Latschinoff, Ber. d. deutsch. chem. Gesellsch., 18 and 20; Lassar-Cohn, ibid., 26,
and Zeitschr. f. physiol. Chem., 17. See also Vahlen, Zeitschr. f. physiol. Chem., 23.

3 Zeitschr. f. physiol. Chem., 76.

4 Ber d. d. chem. Gesellsch, 19 and 20.

6 Kiister, Zeitschr. f. physiol. Chem., 69; H. Fischer, ibid., 73.

426 THE LIVER.

that desoxycholic acid, like choleic acid, yields dehydrocholeic acid and
cholanic acid as oxidation products, makes the formation of desoxycholic
acid from cholic acid by reduction very improbable. The conclusion
of LATSCHINOFF that both choleic and desoxycholic acids are identi-
cal, is not to be accepted on account of the different properties of the
two acids, and as shown by LANGHELD and also found by HAMMARSTEN/
both acids can be detected in the same perfectly fresh ox-bile. PREGL 2
has given important proofs that we are here dealing with two different,
probably, isomeric acids. He found that the two acids yielded dehydro-
choleic acid on oxidation but that the dehydro-acid was not the same
in both cases. The choleic acid yielded a dehydro-acid with a lower
melting-point and a weaker specific rotation than the desoxycholic acid.
The desoxycholic acid crystallizes from glacial acetic acid in needles
with 1 molecule acetic acid, having a melting-point of 144-145°. The
melting-point of the acid crystallized from alcohol-ether is 153-155°,
and for the anhydrous acid or crystallized from acetone it is 172-173°.
It is soluble with difficulty in water, more readily soluble in alcohol, but
somewhat less soluble in glacial acetic acid than choleic acid. It has an
intensely bitter taste. The acid does not give a blue iodine compound,
and no color reaction with hydrochloric acid. Its barium salt is soluble
with difficulty in cold water, but dissolves in boiling alcohol and crys-
tallizes on cooling.

The cholic acids are best prepared from ox-bile, which is boiled for
24 hours with 5-10 per cent caustic soda. The crude acid is precipitated
by hydrochloric acid, dissolved in ammoniacal water and precipitated
by BaCl2. The precipitate contains essentially choleic and desoxy-
cholic acids, while the nitrate contains a part of these and the chief part
of the cholic acid. In regard to the further rather complicated method
of separating the various acids, as also in regard to the many methods
suggested for the preparation of the pure cholic acids, we must refer to
more extensive hand-books.3

Fellic Acid, CtMwO* is a cholic acid, so called. by SCHOTTEN, which he obtained
from human bile, along with the ordinary acid. This acid is crystalline, is insolu-
ble in water, and yields barium and magnesium salts, which are very insoluble.
It does not respond to PETTENKOFER'S reaction easily and gives a more reddish-
blue color. The existence of this acid is still doubtful.

The conjugate acids of human bile have not been sufficiently investi-
gated. To all appearances human bile contains under different circum-

1 Ekbom, Zeitschr. f. physiol. Chem., 50; Pregl, Wien. Sitz-Ber. Bd., Ill, Math.
Naturw. Kl., 1902; Latschinoff, Ber. d. d. Chem. Gesellsch., 20; Langheld, ibid.,
41; Hammarsten in Abderhalden's Handbuch d. bioch. Arbeitsmethoden Bd. 2, 2.

2 Zeitschr f. physiol. Chem., 65.

3 Abderhalden's Handbuch d. bioch. Arbeitsmethoden Bd. II. 2; also Pregl and
Buchtala, Zeitschr. f. physiol. Chem., 74 and Schryver, Journ. of Physiol., 44.

SPECIAL CHOLIC ACIDS. 427

stances various conjugate bile-acids. In some cases the bile-salts of
human bile are precipitated by BaCb and in others not. According to
the statements of LASSAR-COHN l three cholic acids may be prepared
from human bile, namely, ordinary CHOLIC ACID, CHOLEIC ACID, and

FELLIC ACID.

Lithofellic Acid, C2oH3604, is the acid related to cholic acid which occurs in
the oriental bezoar stones, which is insoluble in water, comparatively easily solu-
ble in alcohol, but only slightly soluble in ether.2

Lithocholic Acid, C^HUOs, is a cholic acid found by H. FISCHER 3 in gall-
stones. It melts at 184-186° and is tasteless.

The hyo-glycocholic and cheno-taurocholic acids, as well as the
glycocholic acid of the bile of rodents, yield corresponding 'cholic acids.
This also seems to be the case with the glycocholic acid of the hippopota-
mus-bile, which stands very close to the pig-bile (HAMMARSTEN 4). In the
polar bear a third cholic acid exists besides cholic and choleic acids.
It ite called ursocholeic add, CigHsoCU or CigH^gCU (HAMMARSTEN5).
Also in the bile of other animals (walrus,, seal) HAMMARSTEN 6 has found
special cholic acids, phoccecholic acids, of which one, the a-acid crystallizes
from benzene or petroleum ether in six-sided thin plates which melt at
152-154° C. Its formula seems to be C22H3e05. The other, fl-phocse-
cholic acid has the formula C24H4o05 and is isomeric with cholic acid.
The isocholic acid melts at 220-222° C.

On boiling with acids, on putrefaction in the intestine, or on heating,
cholic acids lose water and are converted into anhydrides, the so-called
dyslysins. The dyslysin, C24H3eO3, corresponding to ordinary cholic
acid, which occurs in fseces, is amorphous, insoluble in water and alkalies.
Choloidic acid, €24^304, is called the first anhydride or an intermediary
product in the formation of dyslysin. On boiling dyslysins with caustic
alkali they are reconverted into the corresponding cholic acids.

THE DETECTION OF BILE-ACIDS IN ANIMAL FLUIDS. To obtain the
bile-acids pure so that PETTENKOFER'S test can be applied to them, the
protein and fatt must first be removed. The protein is removed by
making the liquid first neutral and then adding a great excess of alcohol,
so that the mixture contains at least 85 vols. per cent of water-free alcohol.
Now filter, extract the precipitated protein with fresh alcohol, unite all
filtrates, distil the alcohol, and evaporate to dryness. The residue is

'Schotten, Zeitschr. f. physiol. Chem., 11; Lassar-Cohn, Ber. d. deutsch. chem.
Gesellsch., 27.

2 See Jiinger and Klages, Ber. d. deutsch. chem. Gesellsch. 28 (older literature).

3 Zeitschr. f. physiol. Chem., 73.

4 Ibid., 74.
* Ibid., 36.

6 Ibid., 61 and 68.

428 THE LIVER.

completely exhausted with strong alcohol, filtered, and the alcohol entirely
evaporated from the filtrate. The residue is extracted with ether and"
dissolved in water, and filtered if necessary, and the solution precipitated
by basic lead acetate and ammonia. The washed precipitate is dissolved
in boiling alcohol, filtered while warm, and a few drops of soda solution
added. Then evaporate to dry ness, extract the residue with absolute
alcohol, filter, and add an excess of ether. The precipitate now formed
may be used for PETTENKOFER'S test. It is not necessary to wait for
cystallization; but one must not consider the crystals which form in the
liquid as being positively crystallized bile. It is also possible for needles
of alkali acetate to be formed. In this connection it must be remarked
that a confusion with phosphatides, which also give PETTENKOFER'S
reaction, is not excluded, and a further testing and separation are advisable.

Bile-pigments. The bile-coloring matters known thus far are rela-
tively numerous, and in all probability there are still more of them. Most
of the known bile-pigments are not found in the normal bile, but occur
either in post-mortem bile or principally in the bile concrements. The
pigments which occur under physiological conditions in human bile are
the reddish-yellow bilirubin, the green biliverdin, and sometimes also
urobilin (and urobilinogen) or a closely related pigment. The pigments
found in gall-stones are (besides the bilirubin and biliverdin) choleprasin,
bilifuscin, biliprasin, bilihumin, bilicyanin and (choletelinf). Besides
these, others have been noticed in human and animal bile by various
observers. The two above-mentioned physiological pigments, bilirubin
and biliverdin, are those which serve to give the golden-yellow or orange-
yellow or sometimes greenish color to the bile; or when, as is most fre-
quently the case in ox-bile, the two pigments are present in the bile at
the same time, they produce the different shades between reddish-brown
and green.

Bilirubin. This pigment has the formula, CioHis^Os, or according
to ORNDORFF and TEEPLE and KUSTER/ more correctly C32H36N4O6,
and is designated by the names CHOLEPYRRHIN, BILIPH^EIN, BILIFULVIN,
and H^EMATOIDIN. It occurs chiefly in the gall-stones as calcium bilirubin.
Bilirubin is present in the liver-bile of all vertebrates, and in the bladder-
bile especially in man and carnivora; sometimes, however, the latter
may have a green bile when fasting or in a starving condition. It also
occurs in the contents of the small intestine, in the blood serum of the
horse, in old blood extravasations (as hsematoidin) , and in the urine and
the yellow-colored tissue in icterus.

On reduction with sodium amalgam MALY obtained a reduction
product, which he called hydrobilirubin, with the formula, C

1 Orndorff and Teeple, Salkowski's Festschrift, Berlin, 1904; Klister, Zeitschr. f.
Physiol. Chem., 59.

BILIRUBIN. 429

and which shows great similarity to the urinary pigment, urobilin, as well
as to stercobilin found in the contents of the intestine (MASIUS and
VANLAIR 1). The reduction products have been carefully investigated
by H. FISCHER and then by PAUL MEYER and F. MEYER-BETZ. They
have found that hydrobilirubin is a mixture of bodies, among which there
is one which forms at least one-half and therefore, called hemibilirubin,
gives colorless crystals, and according to FISCHER and MEYER-BETZ
is identical with the urobilinogen of the urine. The formula of this
body is, C32H44N4Oe or CssH^^Oe. The other body is amorphous
but in properties and composition shows great similarity to the hemi-
bilirubin. The analyses correspond closely to the formula, C32H4oN4Oe.
This body as well as the hemibilirubin yields hsematinic acid and methyl-
ethylmaleic imide on oxidation. As KUSTER 2 first showed, bilirubin
yields hsematinic acid as oxidation product. It does not on the con-
trary yield methylethyl maleic imide.

PILOTY and TnANNHAUSER3 obtained bilinic acid, CuH^e^Os
from bilirubin on reduction with hydriodic acid and iodophosphonium.
This acid corresponded to the h^matopyrrolidine carboxylic acid obtained
from ha3matoporphyrin. This bilinic acid is identical with the bilirubinic
acid described below and hence has this name.4 They also obtained an
isomeric acid to phonopyrrolic acid, the isophonopyrrol carboxylic acid
and in the potash fusion they found partly a dimethyl- and partly a
trimethylpyrrol. From bilinic acid they later obtained on mild oxida-
tion an intensely yellow colored acid, the dehydrobilinic acid.

From bilirubin and hemibilirubin, on heating with sodium methylate,
H. FISCHER and RosE5 have obtained 2, 4, 5- trimethylpyrrol-3-propionic
acid which was previously obtained by H. FISCHER and BARTHOLOMAUS
from phonopyrrolcarboxylic acid. From bilirubinic acid on the con-
trary, with the same procedure they did not obtain this acid but another,
xanthobilirubinic add, CnH^^Oa, which is probably identical with
dehydrobilinic acid, and which contains two atoms of hydrogen less than
.bilirubinic acid, and which can be retransformed into the latter by glacial
acetic acid and hydriodic acid. As bilirubin, as well as hemibilirubin,
yields xanthobilirubinic acid as a side product with sodium methylate,
these experimenters consider this as a proof that the bilirubinic acid con-

1 Maly, Wien. Sitzungsber., 57, and Annal. d. Chem., 163; Masius and Vanlair,
Centralbl. f. d. med. Wissensch., 1871, 369.

2 Hans Fischer, Zeitschr. f. physiol. Chem., 73, with Paul Meyer, ibid., 75, with
Meyer-Betz, ibid., 75; Kiister, ibid., 26 and Ber. d. d. chem. Gesellsch, 32 and 35.

3 Piloty and Thannhauser, Annal. d. chem. u. Pharm., 390 and Ber d. d. chem.
Gesellsch., 45.

4 Piloty, Ber. d. d. chem. Gesellsch., 46, 1000; H. Fischer, ibid., 46, 1574.
6 Ber. d. d. chem. Gesellsch., 46, 439.

430 THE LIVER.

figuration exists already formed in these two bodies, and that the above-
mentioned tetrasubstituted acid, which is not obtained from bilirubinic
acid, must come from a special third pyrrol nucleus in the bilirubin and
hemibilirubin. Hsematinic acid (KUSTER from bilirubin) and methyl-
ethylmaleic imide (H. FISCHER and MEYER from hemibilirubin) have
been obtained from the two other pyrrol nuclei. Haematinic acid as
well as methylethylmaleic imide have also been obtained from biliru-
binic acid.

FISCHER and ROSE 1 have earlier obtained, from hemibilirubin as
well as from the above-mentioned bodies and from bilirubin, by reduc-
tion with hydriodic acid, glacial acetic acid, a new crystalline acid,
the bilirubinic acid, CirH^^Os. This acid, to which PILOT Y and
THANNHAUSER'S bilinic acid stands in close relation, yields hsematinic
as well as methylethylmaleic imide on oxidation. By changing the
method of reduction FISCHER and ROSE 2 have obtained cryptopyrrol
and isophonopyrrolcarboxylic acid from bilirubin. The bilirubinic acid
also yielded the same products.

The close relation of the blood pigments to the bile pigments was
first shown by KUSTER when he obtained the two hsematinic acids (as
imide) as oxidation products of these. This close relation is further
shown by the investigations given above although it is perhaps too
early to draw positive conclusions in regard to the structure of the two
groups of pigments and the differences existing between them.

Bilirubin is sometimes amorphous and sometimes crystalline. The
amorphous bilirubin is a reddish-yellow or reddish-brown powder; the
crystals have a reddish-yellow, reddish-brown, or more reddish color,
and sometimes they have nearly the color of crystalline chromic acid.
The crystals, which can easily be obtained by allowing a solution of bili-
rubin in chloroform to evaporate spontaneously, are reddish-yellow,
rhombic plates, whose obtuse angles are often rounded. On crystalliz-
ing from hot dimethylaniline it forms, on cooling, broad columns with
both ends sharply cut (KUSTER 3). On dissolving in chloroform both
kinds of crystals are converted into long needles or whetstones.

Bilirubin is insoluble in water, behaves like an acid, and occurs in
animal fluids as soluble alkali bilirubin. It is very slightly soluble in
ether, benzene, carbon disulphide, amyl alcohol, fatty oils, and glyc-
erin. It is somewhat more soluble in alcohol. In cold chloroform it
dissolves with difficulty, and is much more readily soluble in warm chloro-
form. Its solubility varies, and supersaturated solutions are readily
formed (ORNDORFF and TEEPLE). The varying solubility of bilirubin

^ - - - , — _.- - _.--.--. • .-_-

1 Zeitschr. f. physiol. Chem., 82.

2 Ber. d. d. chem. Gesellsch, 45.

1 Ibid., 30 and 35, and Zeitschr. f. physiol. Chem., 47.

BILIRUBIN. 431

in chloroform depends, according to KUSTER, on the fact that in its
preparation, derivatives which are readily soluble and contain chlorine
or other transformation products are formed, or perhaps the bilirubin
goes over into polymeric modifications having different solubilities. In
cold dimethylaniline it dissolves in the proportion of 1 : 100, and in hot
dimethylaniline much more readily. Its solutions show no absorption-
bands, but only a continuous absorption from the red to the violet end
of the spectrum, and they have a decided yellow color, even on diluting
greatly (1:500000), in a layer 1.5 cm. thick. The combinations of
bilirubin with alkali are insoluble in chloroform, and the bilirubin in solu-
tion in chloroform can be removed from this solution by shaking with
dilute alkali (differing from lutein). Solutions of bilirubin-alkali in
water are precipitated by the soluble salts of the alkaline earths and also
by metallic salts. If a dilute solution of alkali bilirubin in water is
treated with an excess of ammonia and then with a zinc-chloride solution,
the liquid is first colored deep orange and then gradually olive-brown
and then green. This solution first gives a darkening of the violet and
blue part of the spectrum, and then the bands of alkaline cholecyanin
(see below), or at least the bands of this pigment in the red between
C and D, close to C. This is a good reaction for bilirubin. The fol-
lowing reaction has been suggested by AucHls l. Treat 5 cc. of an alcoholic
solution of bilirubin (1:20000) which contains 1 drop of ammonia in
100 cc., with 5 to 6 drops of an alcoholic zinc acetate solution (1:1000)
and then 1 drop alcoholic iodine solution (1:100) when a beautiful bluish-
green coloration with a beautiful garnet-red fluorescence is obtained on
shaking. The spectrum shows a dark band between B and C, and a
pale band at D. If a few drops of hydrochloric acid are added to the
solution the color becomes violet, the fluorescence disappears and the
two JAFFE'S cholecyanin bands appear. This reaction is extremely
delicate.

As EHRLICH first showed, bilirubin forms combinations with diazo
compounds, which have been closely studied by PROSCHER, ORNDORFF
and TEEPLE.2 A test suggested by EHRLICH for bilirubin is based upon
this behavior with sulphodiazobenzene.

If an alkaline solution of bilirubin be allowed to stand in contact
with the air, it gradually absorbs oxygen, and green biliverdin is formed*
This process is accelerated by warming. According to KUSTER, in this
case the alkali also has a splitting action upon the pigment, and among
the products formed we find haematinic acid. Biliverdin is formed only

1 Compt. rend. soc. biol., 64.

2Ehrlich, Zeitschr. f. anal. Chem., 23; Proscher, Zeitschr. f. phyeiol. Chem., 39;
Orndorff and Teeple, 1. c.

432 THE LIVER.

from bilirubin by oxidation under special conditions (KUSTER). A green
coloring-matter similar in appearance is formed by the action of other
reagents such as Cl, Br, and I. According to JOLLES/, biliverdin is
produced by the use of HUBL'S iodine solution, while according to others
(THUDICHUM, MALT 2) substitution products of bilirubin are formed.

GMELIN'S Reaction for Bile-pigments. If one carefully pours nitric
acid, containing some nitrous acid, under an aqueous solution of alkali
bilirubin, there is obtained a series of colored layers at the juncture of the
two liquids in the following order from above downward: Green, blue,
violet, red, and reddish-yellow. This color reaction, GMELIN'S test,
is very delicate, and serves to detect the presence of one part bilirubin
in 80,000 parts liquid. The green ring must never be absent; and also the
reddish-violet must be present at the same time, otherwise the reaction
may be confused with that for lutein, which gives a blue or greenish ring.
The nitric acid must not contain too much nitrous acid, for then the reac-
tion takes place too quickly and it does not become typical. Alcohol
must not be present in the liquid, because, as is well known, it gives a
play of colors, in green or blue, with the acid.

HAMMARSTEN'S Reaction. An acid is first prepared consisting of 1
vol. nitric acid and 19 vols. hydrochloric acid (each acid being about
25 per cent). One volume of this acid mixture, which can be kept for
at least a year, is, when it has become yellow by standing, mixed with
4 vols. alcohol. If a drop of bilirubin solution is added to a few cubic
centimeters of this colorless mixture a permanent beautiful green color
is obtained immediately. On the further addition of the acid mixture
to the green liquid all the colors of GMELIN'S scale, as far as choletelin,
can be produced consecutively.

HUPPERT'S Reaction. If a solution of alkali bilirubin is treated with
milk of lime or with calcium chloride and ammonia, a precipitate is
produced consisting of calcium bilirubin. If this moist precipitate, which
has been washed with water, is placed in a test-tube and the tube half
filled with alcohol which has been acidified with hydrochloric acid, and
heated to boiling for some time, the liquid becomes emerald-green or
bluish-green in color.

In regard to the modifications of GMELIN'S test and certain other
reactions for bile-pigments, see Chapter XIV (Urine).

That the characteristic play of colors in GMELIN'S test is the result
of an oxidation is generally admitted. The first oxidation step is the

1 Kttster, Ber. d. d. chem. Gesellsch., 35 and 59; Jolles, Journ. f .prakt. Chem. (N.F.),
59, and Pfliiger's Arch., 75.

2Thudichum, Journ. of Chem. Soc. (2)^13, and Journ. f. prakt. Chem. (N.F.),
53; Valy, Wien. Sitzungsber., 72.

BILIRUBIN, BILIVERDIN. 433

green biliverdin. Then follows a blue coloring-matter which HEINSIUS
and CAMPBELL call bilicyanin, and STOKVIS calls cholecyanin, and which
shows a characteristic absorption-spectrum. The neutral solutions of
this coloring-matter are, according to STOKVIS, bluish-green or steel-blue
writh a beautiful blue fluorescence. The alkaline solutions are green
and have no marked fluorescence, and show three absorption-bands:
one, sharp and dark, in the red between C and D, nearer to C; a second,
less well defined, covering D; and a third between E and F, near E.
The strongly acid solutions are violet-blue and show two bands, described
by JAFFE between the lines C and E, separated from each other by a
narrow space near D. A third band between b and F is seen with dif-
ficulty. The next oxidation step after these blue coloring-matters is
a red pigment, and lastly a yellowish-brown pigment, called choletelin,
by MALY, which in neutral alcoholic solutions does not give any absorp-
tion-spectrum, but in. acid solution gives a band between b and F. On
oxidizing cholecyanin with lead peroxide, STOKVIS 1 obtained a product
which he calls choletelin, which is quite similar to urinary urobilin, to
be discussed later.

Bilirubin is best prepared from gall-stones of oxen, these concretions
being very rich in calcium bilirubin. The finely powdered concrement
is first exhausted with ether and then with boiling water, so as to remove
the cholesterin ^nd bile-acids. In order to remove the mineral con-
stituents it is better to use 10 per cent acetic acid instead of hydrochloric
acid (KUSTER 2). A green pigment is now removed by extraction with
alcohol, and the choleprasin is extracted with hot glacial acetic acid.
After washing with water it is dried, and extracted repeatedly with boil-
ing chloroform. The bilirubin separates from the chloroform as crusts,
which are treated once or twice in the above manner. It is then extracted
with alcohol and precipitated from its chloroform solution by alcohol, or
crystallized from boiling dimethylaniline. Further details are given by

KtJSTER.3

The quantitative estimation of bilirubin may be made by the spectro-
photometric method, according to the steps suggested for the blood-
coloring matters.4

Biliverdin, CieHis^CU or C32H36N4Cg. This body, which is formed
by the oxidation of bilirubin, occurs in the bile of many animals, in
vomited matter, in the placenta of the bitch (?), in the shells of birds'

1 Heinsius and Campbell, Pfliiger's Arch., 4; Stokvis, Centralbl. f. med. Wis-
sensch., 1872, 785; ibid., 1873, 211 and 449; Jaffe, ibid., 1868; Maly, Wien. Sitzungs-
ber., 59.

2 Zeitschr. f. physiol. Chem., 47.
» Ibid., 59.

4 See also Herzfeld, Zeitschr. f. physiol. Chem., 77 and 78.

434 THE LIVER.

eggs, in the urine in icterus, and sometimes in gall-stones, although in
very small quantities.

Biliverdin is amorphous; at least it has not been obtained in well-
defined crystals. It is insoluble in water, ether, and chloroform (this is
true at least for the artificially prepared biliverdin) but is soluble in
alcohol or glacial acetic acid, showing a beautiful green color. It is dis-
solved by alkalies, giving a brownish-green color, and this solution is
precipitated by acids, as well as by calcium, barium, and lead salts.
Biliverdin gives HUPPERT'S, GMELIN'S, and HAMMARSTEN'S reactions,
commencing with the blue color. It is converted into hydrobilirubin
by nascent hydrogen. On allowing the green bile to stand, also by the
action of ammonium sulphide, the biliverdin may be reduced to bilirubin
(HAYCRAFT and SCOFIELD *).

Biliverdin is most simply prepared by allowing a thin layer of an
alkaline solution of bilirubin to stand exposed to the air in a dish until
the color is brownish-green. The solution is then precipitated by hydro-
chloric acid, the precipitate washed with water until no HC1 reaction
is obtained, then dissolved in alcohol and the pigment again separated by
the addition of water. Any contaminating bilirubin may be removed
by means of chloroform. KUSTER has shown that the biliverdin is only
formed by the oxygen of the air from bilirubin under certain conditions:
The presence of 2 molecules caustic alkali with the addition of water so
that the solution contains 0.2 per cent and, a temperature not above 5° C.
HUGOUNENQ and DOYON 2 prepared biliverdin from bilirubin by the
action of sodium peroxide and a little hydrochloric acid.

Choleprasin is a green pigment isolated by KUSTER 3 from gall-stones, which
is soluble in glacial acetic acid but insoluble in alcohol. It differs from the other
bile-pigments by containing sulphur. On distillation with zinc powder it gives
the pyrrol reaction, and on oxidation with chromic acid, KUSTER could not
observe any formation of haematinic acid.

Bilifuscin, so named by STADELER.4 is an amorphous brown pigment soluble
in alcohol and alkalies, almost insoluble in water and ether, and soluble with great
difficulty in chloroform (when bilirubin is not present at the same time). Pure
bilifuscin does not give GMELIN'S reaction. This is also true fof the bilifuscin
prepared by v. ZUMBUSCH,S which is more like a humin substance, and the formula
of which is, CeJIg'eNyOu. Bilifuscin has been found in gall-stones. Biliprasin
is a green pigment prepared by STADELER from gall-stones, and is generally con-
sidered as a mixture of biliverdin and bilirubin. DASTRE and FLORESCO,6 on the

1 Centralbl. f. Physiol., 3, 222, and Zeitschr. f. physiol. Chem., 14.

2 Hugounenq et Doyon, Arch, de Phyisol. (5), 8; Kiister, Zeitschr. f. physiol. Chem.,
59.

3 Zeitschr. f. physiol. Chem., 47.

4 Cited from Hoppe-Seyler, Physiol. u. Path. chem. Analyse, 6. Aufl., p. 225.
6 Zeitschr. f. physiol. Chem., 31.

6 Arch, de Physiol. (5), 9.

SPECIAL BILE PIGMENTS. 435

contrary, consider biliprasin as an intermediate step between bilirubin and bili-
verdin. According to them it occurs as a physiological pigment in the bladder-
bile of several animals, and is derived from bilirubin by oxidation. This oxida-
tion is brought about by an oxidative ferment existing in the bile. Bilihumin
is the name given by STADELER to that brownish amorphous residue which is left
after extracting gall-stones, with chloroform alcohol, and ether. It does not
give GMELIN'S test. Bilicyanin is also found in human gall-stones (HEINSIUS and
CAMPBELL). Cholohcematin, so-called by MACMUNN, is a pigment often occurring
in sheep- and ox-bile and characterized by four absorption-bands, which is
formed from hffimatin by the action of sodium amalgam. In the dried condition,
as when obtained by the evaporation of the chloroform solution, it is green, and in
alcoholic solution olive-brown. This pigment, which has also been found by
HAMMARSTEN in the bile from the musk-ox and hippopotamus, is, according' to
MARCHLEWSKI, identical with the crystalline bilipurpurin isolated by LOEBISCH
and FISCHLER from ox-bile. This latter pigment, according to MARCHLEWSKI,
is not a bile-pigment, but phylloerythrin, a transformation product of chlorophyll.
Fhylloerythrin has been detected by MARCHLEWSKI x in the excrement of cows
fed on green grass.

GMELIN'S and HUPPERT'S reactions are generally used to detect the
presence of bile-pigments in animal fluids or tissues. The first, as a rule,
can be performed directly, and the presence of proteins does not interfere
with it, but, on the contrary, it brings out the play of colors more strik-
ingly. If blood-coloring matters are present at the same time, the bile-
coloring matters are first precipitated by the addition of sodium phos-
phate and milk of lime. This precipitate containing the bile-pigments
may be used directly in HUPPERT'S reaction, or a little of the precipitate
may be dissolved in HAMMARSTEN'S reagent. Bilirubin is detected in
blood, according to HEDENIUS, by precipitating the proteins with alcohol,
filtering and acidifying the filtrate with hydrochloric or sulphuric acid,
and boiling. The liquid becomes of a greenish color. Serum and serous
fluids may be boiled directly with a little acid after the addition of alcohol.
According to OBERMEYER and POPPER 2 the alcoholic filtrate from the
protein precipitation can be tested with an alcoholic solution of iodine
or ferric chloride.

Besides the bile-acids and the bile-pigments, there occur in the bile
also cholesterin, lecithin, jecorin or other phosphatides (HAMMARSTEN),
palmitin, stearin, olein, myristic add (LASSAR-CoHN3), soaps, ethereal
sulphuric adds, conjugated glucuronates, diastatic and proteolytic enzymes,
oxidases and catalases. Choline, and glycerophosphoric add, when they
are present, may be considered as decomposition products 01 lecithin.
Urea occurs, though only in traces, as a physiological constituent of human,

1 MacMunn, Journ. of Physiol., 6; Loebisch and Fischler, Wien. Sitzungsber., 112
(1903); Marchlewski, Zeitschr. f. physiol. Chem., 41, 43, and 45; Hammarsten, ibid.,
43, and investigations not published.

2 Hedenius Upsala Lakaref. Forh., 29 and Maly's Jahresber., 24; Obermeyer and
Popper, Wien. med. Wochenschr., 60.

8 Zeitschr. f. physiol. Chem., 17; Hammarsten, ibid., 32, 36 and 43.

436 THE LIVER.

ox-, and dog-bile. Urea occurs in the bile of the shark and ray in such
large quantities that it forms one of the chief constituents of the bile.1
The mineral constituents of the bile are, besides the alkalies, to which the
bile-acids are united, sodium and potassium chloride, calcium and
magnesium phosphate, and iron — 0.04-0.115 p. m. in human bile,
chiefly combined with phosphoric acid ( YOUNG 2). Traces of copper
are habitually present, and traces of zinc are often found. Sulphates are
entirely absent, or occur only in very small amounts.

The quantity of iron in the bile varies greatly. According to Novi
it is dependent upon the kind of food, and in dogs it is lowest with a bread
diet and highest with a meat diet. According to DASTRE this is not the
case. The quantity of iron in the bile varies even though a constant
diet is maintained, and the variation is dependent upon the- forma-
tion and destruction of blood. According to BECCARIS the iron does
not disappear from the bile in inanition, and the percentage shows no
constant diminution. The question as to the extent of elimination by
the bile of the iron introduced into the body has received various answers.
There is no doubt that the liver has the property of collecting and retain-
ing iron, as well as other metals, from the blood. Certain investigators,
such as Novi and KUNKEL, are of the opinion that the iron introduced
and transitorily retained in the liver is eliminated by the bile, while
others, such as HAMBURGER, GOTTLIEB, and ANSELM,4 deny any such
elimination of iron by the bile.

Quantitative Composition of the Bile. Complete analyses of human
bile have been made by HOPPE-SEYLER and his pupils. The bile was
removed from the gall-bladder of cadavers, hence these analyses can
be of little interest. Older and less complete analyses of perfectly fresh
human bile have been made by FRERICHS and v. GoRUP-BESANEz.5
The bile analyzed by them was from perfectly healthy persons who
had been executed or accidentally killed. The two analyses of
FRERICHS are, respectively, of (I) an 18-year-old and (II) a 22-year-
old male. The analyses of v. GORUP-BESANEZ are of (I) a man of 49
and (II) a woman of 29. The results are, as usual, in parts per 1000.

1 Hammarsten, ibid., 24.

2 Journ. of Anat. and Physiol., 5, 158.

3 Novi, see Maly's Jahresber., 20; Dastre, Arch, de Physiol. (5), 3; Beccari, Arch,
ital. de Biol., 28.

4Kunkel, Pfliiger's Arch., 14; Hamburger, Zeitschr. f. physiol. Chem., 2 and 4;
Gottlieb, ibid., 15; Anselm, " Ueber die Eisenausscheidung der Galle," Inaug.-Diss.
Dorpat, 1891. See also the works cited in footnote 3, p. 339.

6 See Hoppe-Seyler Physiol. Chem., 301; Socoloff, Pfliiger's Arch., 12; Trifanow-
ski, ibid., 9; Frerichs in Hoppe-Seyler's Physiol. Chem., 299; v. Gorup-Besanez, ibid.

COMPOSITION OF THE BILE. 437

FREKICHS. v. GORUP-BESANEZ.

I. II. I. II.

Water 860.0 859.2 822.7 898.1

Solids 140.0 140.8 177.3 101.9

Biliary salts 72.2 91.4 107.9 56.5

Mucus and pigments 26.6 29.8 22.1 .14.5

Cholesterin 1.6 2.6\ ,7 Q on n

Fat 3.2 9.2/

Inorganic substances 6.5 7.7 10.8 6.2

Human liver-bile is poorer in solids than the bladder-bile. In
several cases it contained only 12-18 p. m. solids, but the bile in these
cases is hardly to be considered as normal. JACOBSEN found 22.4-22.8
p. m. solids in a specimen of bile. HAMMARSTEN, who had occasion to
analyze the liver-bile in seven cases of biliary fistula, has often
found 25-28 p. m. solids. In a case of a corpulent woman the quantity
of solids in the liver-bile varied between 30.10-38.6 p. m. in ten days.
BRAND 1 observed still higher figures, more than 40 p. m., in two
cases. This investigator suggests that the bile from an imperfect
fistula, when it is partly absorbed, is richer in solids than when it comes
from a perfect fistula.

The molecular concentration of human bile, according to BRAND,
BONANNI, and STRAUSS,2 is generally identical with that of the
blood, although the amount* of water and solids varies. The freezing-
point varies only between —0.54° and —0.58°. This constancy of the
osmotic pressure is explained by the fact that in concentrated biles with
larger amounts of organic substances (with larger molecules) the amount
of inorganic salts is lower.3

Human bile, sometimes, but not always, contains sulphur in an ethereal
sulphuric-acid-like combination (HAMMARSTEN, OERUM, BRAND). The
quantity of such sulphur may even amount to J-f of the total sulphur.
We do not know the nature of these ethereal sulphuric acids. According
to OERUM 4 they are not precipitated by lead acetate, but are precipitated
by basic lead acetate, especially with ammonia. Human bile is habitually
richer in glycocholic than in taurocholic acid. In six cases of liver-bile
analyzed by HAMMARSTEN the relation of taurocholic to glycocholic acid
varied between 1 : 2.07 and 1 : 14.36. The bile analyzed by JACOBSEN
contained no taurocholic acid.

As an example of the composition of human liver-bile the following

1 Jacobsen, Ber. d. deutsch. chem. Gesellsch., 6; Hammarsten, Nova Acta Reg.
Soc. Scient. Upsala, 16; Brand, Pfliiger's Arch., 90.

2 Brand, 1. c.; Bonanni, Biochem. Centralbl., 1; Strauss, Berl. klin. Wochenschr.,
1903.

3 See Brand, I. c.; Hammarsten, 1. c.

4 Skand. Archiv. f. Physiol., 16.

438 THE LIVER.

results of three analyses made by HAMMARSTEN are given. The results
are calculated in parts per 1000 : l

Solids . . 25 . 200 35 . 260 25 . 400

Water 974.800 964.740 974.600

Mucin and pigments 5 . 290 4 . 290 5 . 150

Bile-salts 9.310 18.240 9.040

Taurocholate 3.034 2.079 2.180

Glycocholate 6.276 16.161 6.860

Fatty acids from soaps 1 . 230 1 . 360 1 . 010

Cholesterin 0.630 1.603 1.500

Lecithin \ n 990 ° 574 °-650

Fat / U> U 0.956 0.610

Soluble salts 8.070 6.760 7.250

Insoluble salts 0.250 0.490 0.210

Among the mineral constituents the chlorine and sodium occur to
the greatest extent. The relation between potassium and sodium
varies considerably in different samples, Sulphuric acid and phosphoric
acid occur only in very small quantities.

BAGINSKY and SOMMERFELD 2 found true mucin, mixed with some
nucleoalbumin, in the bladder-bile of children. The bile contained
on an average 896.5 p. m. water; 103.5 p. m. solids; 20 p. m. mucin;
9.1 p. m. mineral substances; 25.2 p. m. bile-salts (of which 16.3 p. m.
were glycocholate and 8.9 p. m. taurochojate) ; 3.4 p. m. cholesterin;
6 p. m. lecithin; 6.7 p. m. fat, and 2.8 p. m. leucine.3

The quantity of pigment in human bile is, according to NOEL-PATON,
0.4-1.3 p. m. (in a case of biliary fistula). The method used in deter-
mining the pigments in this case was not quite trustworthy. Accurate
results for dog-bile obtained by spectrophotometric methods are on
record. According to STADELMANN"* dog-bile contains on an average
0.6-0.7 p. m. bilirubin. At the most only 7 milligrams of pigment are
secreted per kilo of body in the twenty-four hours.

In animals the relative proportion of the two acids varies, con-
siderably. It has been found, on determining the amount of sulphur,
that, so far as the experiments have gone, taurocholic acid is the pre-
vailing acid in carnivorous mammals, birds, snakes, and fishes. Among
the herbivora, sheep and goats have a predominance of taurocholic
acid in the bile. Ox-bile sometimes contains taurocholic acid in excess,
in other cases glycocholic acid predominates, and in a few cases the
latter occurs almost alone. The bile of the rabbit, hare, kangaroo,

Decent quantitative analyses may be found in Brand, 1. c.; v. Zeynek, Wien.
klin. Wochenschr., 1899; Bonanni, 1. c.

2 Verhandl. d. physiol. Gesellsch. zu Berlin, 189-95.

* Analyses of bile from children may be found in Heptner, Maly's Jahresber., 30.

*Noel-Paton, Rep. Lab. Roy. Soc. Coll. Phys. Edinburgh, 3; Stadelmann, Der
Icterus.

CONSTITUTION OF THE BILE. 439

hippopotamus, and orang-utang (HAMMARSTEN l) contains, like the
bile of the pig, almost exclusively glycocholic acid. A distinct influence
on the relative amounts of the two bile-acids exerted by differences in
diet has not been detected. HITTER 2 claims to have found a decrease
in the quantity of taurocholic acid in calves when they pass from the
milk to the vegetable diet.

In the above-mentioned calculation of the taurocholic acid from the
quantity of sulphur in the bile-salt, it must be remarked that no definite
conclusion can be drawn from such a determination, since it is known
that other kinds of bile (e. g., human and shark bile) contain sulphur in
compounds other than taurocholic acid.3

The phosphorized constituents of bile are not well known; never-
theless, there is no doubt that bile contains other phosphatides besides
lecithin (HAMMARSTEN). These phosphatides are in part precipitated in
the precipitation of the bile-salts and they in part keep the bile-salts in
solution, preventing their complete precipitation, and hence they have a
double disturbing action in the quantitative analysis of bile. Those biles
richest in phosphatides, so far as known, are the following, in the order of
their amount: Polar bear, man (in special cases), dog, black bear, orang-
utang. The bile of certain fishes contains but little .phosphatides

(HAMMARSTEN4).

The cholesterin, which, according to several investigators, originates
not only from the liver but also from the biliary passages, occurs in
larger quantities in the bladder-bile than in the liver-bile, and is present
to a greater extent in the non-filtered than in the filtered bile (DoyoN
and DUFOURT). The quantity seems to be very variable and in patients
with bile fistulas BACMEisTER5 found 0.24-0.59 p. m. The gases
of the bile consist of a large quantity of carbon dioxide, which increases
with the amount of alkalies, only traces of oxygen, and a very small
quantity of nitrogen.

Little is known in regard to the composition of the bile in disease. The quantity
of urea is found to be considerably increased in uraemia. Leucine and tyrosine are
observed in acute yellow atrophy of the liver and in typhoid. Traces of albumin
(without regard to nucleoalbumin) have several times been found in the human
bile. The so-called pigmentary acholia, or the secretion of a bile containing
bile-acids but no bile-pigments, has also been repeatedly noticed. In all such
cases observed by HITTER he found a fatty degeneration of the liver-cells, in return
for which, even in excessive fatty infiltration, a normal bile containing pigments
was secreted. The secretion of a bile nearly free from bile-acids has been

1 See Ergebnisse der Physiol., 4.

2 Cited from Maly's Jahresber., 6, 195.

8 Hammarsten, Zeitschr. f. physiol. Chem., 32, and Ergebnisse, der Physiol., 4.
4 Zeitschr. f . physiol. Chem., 36, and Ergebnisse der Physiol., 4. y

6 Doyon and Dufort, Arch, de Physiol. (5), 8; Bacmeister, Bioch. Zeitschr., 26. ;

440 THE LIVER.

observed by HOPPE-SEYLER * in amyloid degeneration of the liver. In animals,
dogs, and especially rabbits, it has been observed that the blood-pigments pass
into the bile in poisoning and in other conditions, causing a destruction of the
blood-corpuscles, as also after intravenous hemoglobin injection (WERTHEIMER
and MEYER, FILEHNE, STERN 2). Albumin can pass into the bile after the intra-
venous injection of a foreign protein (casein) (GURBER and HALLAUER), as well
as after poisoning with phosphorus or arsenic (PILZECKER), or after the irrita-
tion of the liver by the introduction of ethyl alcohol or amyl alcohol (BRAUER).
Sugar occurs in bile only in exceptional cases.3

The physiological secretion of the gall-bladder in man is, according
to WAHLGREN4 a viscous, alkaline fluid with 11.24-19.63 p. m. solids.
The mucilaginous properties are not due to mucin, but to a phosphorized
protein substance (nucleoalbumin or nucleoprotein) .

Instead of bile there is sometimes found in the gall-bladder under pathological
conditions a more or less viscous, thready, colorless fluid which contains pseudo-
mucins or other peculiar protein substances.5

Chemical Formation of the Bile. The first question to be answered
is the following: Do the specific constituents of the bile, the bile-acids
and bile-pigments originate in the liver; and if this is the case, do they
come from this organ alone, or are they also formed elsewhere?

The investigations of the blood, and especially the comparative
investigations of the blood of the portal and hepatic veins under normal
conditions, have not given any answer to this question. To decide this,
therefore, it is necessary to extirpate the liver of animals or to isolate
it from the circulation. If the bile constituents are not formed in the
liver, or at least not alone in this organ, but are eliminated only from
the blood, then, after the extirpation or removal of the liver from the
circulation, an accumulation of the bile constituents is to be expected
in the blood and tissues. If the bile constituents, on the contrary, are
formed exclusively in the liver, then the above operation naturally would
give no such result. If the ductus choledochus is tied, then the bile
constituents will be collected in the blood or tissues whether they are
formed in the liver or elsewhere.

From these principles KOBNER has tried to demonstrate by exper-
iments on frogs that the bile-acids are produced exclusively in the liver.
While he was unable to detect any bile-acids in the blood and tissues of

fitter, Compt. Rend., 74, and Journ.de 1'anat. et de la physiol. (Robin), 1872;
Hoppe-Seyler, Physiol. Chem., 317.

2 Wertheimer and Meyer, Compt. Rend., 108; Filehne, Virchow's Arch., 121; Stern,
ibid., 123.

8 Giirber, and Hallauer, Zeitschr. f. Biologic, 45; Pilzecker, Zeitschr. f. physiol.
Chem., 41; Brauer, ibid., 40.

4 See Maly's Jahresber., 32.

5 Winternitz, Zeitschr. f. physiol. Chem., 21; Solhnann, Amer. Medicine, 5 (1903),

FORMATION OF BILE PIGMENTS. 441

these animals after extirpation of the liver, he was able to discover them
on tying the ductus choledochus. The investigations of LUDWIG and
FLEISCHL 1 show that in the dog the bile-acids originate in the liver alone.
After tying the ductus choledochus, they observed that the bile constituents
were absorbed by the lymphatic vessels of the liver and passed into the
blood through the thoracic duct. Bile-acids could be detected in the
blood after such an operation, while they could not be detected in the
normal blood. But when the common bile and thoracic ducts were both
tied at the same time, then not the least trace of bile-acids could be
detected in the blood, while if they are also formed in other organs and
tissues they should have been present.

From earlier reports of CLOEZ and VULPIAN, as well as VIRCHOW, the bile-
acids also occur in the suprarenal capsule. These claims have not been confirmed
by later investigations of STADELMANN and BEiER.2 At the present time there
is no ground for supposing that the bile-acids are formed elsewhere than in
the liver.

It has been undoubtedly proved that the bile-pigments may be formed
in other organs besides the liver, for, as is generally admitted, the color-
ing-matter ha3matoidin, which occurs in old blood extravasations, is
identical with the bile-pigment bilirubin (see page 301). LATSCHEN-
BERGER3 also observed in horses, under pathological conditions, a
formation of bile-pigments from the blood-coloring matters in the tissues.
The occurrence of bile-pigments in the placenta also seems to depend
on their formation in that organ, while the occurrence of small quantities
of bile-pigments in the blood-serum of certain animals probably depends
on an absorption of these substances.

Although the bile-pigments may be formed in other organs besides
the liver, still it is of first importance to know what bearing this organ
has on the elimination and formation of bile-pigments. In this regard
it must be recalled that the liver is an excretory organ for the bile-pig-
ments circulating in the blood. TARCHANOFF observed in a dog with
biliary fistula, that intravenous injection of bilirubin causes a very
considerable increase in the bile-pigments eliminated. This statement
has been later confirmed by the investigations of Vossius.4

Numerous experiments have been made to decide the question whether
the bile-pigments are only eliminated by the liver, or whether they are
also formed therein. By experimenting on pigeons, STERN was able

1 Kobner, see Heidenhain, Physiologic der Absonderungsvorgange, in Hermann's
Handbuch, 5; Fleischl, Arbeiten aus der physiol. Anstalt zu Leipzig, Jahrgang, 9.

2 Zeitschr. f. physiol. Chem., 18, in which the older literature may be found.

3 See Maly's Jahresber., 16, and Monatshefte f . Chem., 9.

4 Tarchanoff, Pfliiger's Arch., 9; Vossius, cited from Stadelmann, Der Icterus.

442 THE LIVER.

to detect bile-pigments in the blood-serum five hours after tying the
biliary passages alone, while after tying all the vessels of the liver and also
the biliary passages, no bile-pigments could be detected either in the
blood or the tissues of the animal, which was killed 10-24 hours after
the operation. MINKOWSKI and NAUNYN 1 also found that poisoning
with arseniureted hydrogen produces a liberal formation of bile-pig-
ments, and the secretion, after a short time, of a urine rich in biliverdin
in previously healthy geese. In geese with extirpated livers this does
not occur.

With experiments on dogs, WHIPPLE and HOOPER 2 found after intra-
venous injection of blood-corpuscles of the same animal heemolyzed
with water, that a transformation of the haemoglobin into bile-pigments
occurred with the same rapidity in normal animals as with animals with
Eck fistulas, or with such a fistula and the hepatic artery ligatured. The
formation of bile-pigments also occurred on removing the liver, spleen
;and abdomen from the circulation, as well as by circulation through the
head and thorax. A transformation of haemoglobin into bile-pigments,
at least in dogs, can take place easily without the medium of the liver
and these experimenters suggest the possibility that the endothelial
cells are here active.

No such experiments can be carried out on mammalia, as they do
not live long enough after the operation; still there is no doubt that this
organ is the chief seat of the formation of bile-pigments under physiolog-
ical conditions.

In regard to the materials from which the bile-acids are produced,
it may be said with certainty that the two components, glycocoll and
taurine, which are both nitrogenized, are formed from the protein bodies.
The close relation of taurine to the cystine group of the protein mole-
cule has been especially shown by the investigations of FREIDMANN,
(see Chapter III), and recently v. BERGMANNS has shown by feeding
dogs with sodium cholate and cystine that the animal body can trans-
form cystine into taurine, and that the taurine of the bile originates
from the proteins of the food. In regard to the origin of the non-nitro-
genized cholic acid, which was formally considered as originating from
the fats, nothing is positively known; to all appearances it is from
proteins.

The blood-coloring matters are considered as the mother-substances
of the bile-pigments. If the identity of hsematoidin and bilirubin was

1 Stern, Arch. f. exp. Path. u. Pharm., 19; Minkowski and Naunyn, ibid., 21.

2 Journ. of exp. Med., 17.

3 Hofmeister's Beitrage, 4. See also Wohlgemuth, Zeitschr. f. physiol. Chem., 40.

FORMATION OF BILE PIGMENTS. 443

settled beyond a doubt, then this view might be considered as proved.
Independently, however, of this identity, which is not admitted by
all investigators, the view that the bile-pigments are derived from the
blood-coloring matters has strong arguments in its favor. It has been
shown by several experimenters that a yellow or yellowish-red pigment
can be formed from the blood-coloring matters, which gives GMELIN'S
test, and which, though it may not form a complete bile-pigment, is
at least a step in its formation (LATSCHENBERGER) . The previously
mentioned relationship between the blood and bile-pigments must be
recalled, and the formation of bilirubin from the blood-pigments is
shown, according to the unanimous observations of several investi-
gators,1 by the fact that the appearance of free haemoglobin in the plasma,
produced by the destruction of the red corpuscles by widely differing
influences (see below) or by the injection of haemoglobin solution, causes
an increased formation of bile-pigments. The amount of pigments in
the bile is not only considerably increased, but the bile-pigments may
even pass into the urine under certain circumstances (icterus). After
the injection of haemoglobin solution into a dog either subcutaneously
or in the peritoneal cavity, STADELMANN and GORODECKI 2 observed an
increase of 61 per cent in the secretion of pigments by the bile, which
lasted for more than twenty-four hours. Recently BRUSCH and YOSH-
IMOTO,3 by quantitative estimations of the bile-pigments and urobilin
in animals with bile fistulas with ligated ductus choledochus, have
shown the increased formation of bile-pigments after the injection of
known amounts of haematin, and in this manner further proved the
genetic relationship between the bile-pigments and hsematin.

If bilirubin, which contains no iron, is derived from ha3matin, which
contains iron, then iron must be split off. The question in what form or
combination the iron is split off is of special interest, and also whether
it is eliminated by the bile. This latter does not seem to be the case,
at least to any great extent. In 100 parts of bilirubin which are eliminated
by the bile there are only 1.4-1.5 parts iron, according to KUNKEL, while
100 parts haematin contain about 9 parts iron. MINKOWSKI and BASE-
RiN4 also found that the abundant formation of bile-pigments occurring
in poisoning by arseniureted hydrogen does not increase the quantity
of iron in the bile. The quantity apparently does not seem to correspond
with that in the decomposed blood-coloring matters. It follows from the

1 See Stadelmann, Der Icterus, etc., Stuttgart, 1891.

2 See Stadelmann, ibid.

3 Zeitschr. f. exp. Path. u. Therap., 8.

4 Kunkel, Pfltiger's Arch., 14; Minkowski and Baserin, Arch. f. exp. Path. u.
Pharm., 23.

444 THE LIVER.

researches of several investigators l that the iron is, at least chiefly,
retained by the liver as a ferruginous pigment or protein substance.

What relation does the formation of bile-acids bear to the forma-
tion of bile-pigments? Are these two chief constituents of the bile derived
simultaneously from the same material, and can we detect a certain
connection between the formation of bilirubin and bile-acids in the liver?
The investigations of STADELMANN teach us that this is not the case.
With increased formation of bile-pigments the amount of bile-acids
is decreased, and the introduction of hemoglobin into the liver strongly
increases the formation of bilirubin, but simultaneously strongly decreases
the production of bile-acids. According to STADELMANN the formation
of bile-pigments and bile-acids is due to a special activity of the cells.

An absorption of bile from the liver, and the passage of the bile con-
stituents into the blood and urine occurs in retarded discharge of the
bile, and usually in different forms of hepatogenic icterus. But bile-
pigments may also pass into the urine under other circumstances, espe-
cially when a solution or destruction of the red blood-corpuscles takes
place in animals through injection of water or a solution of biliary salts,
through poisoning by ether, chloroform, arseniureted hydrogen, phos-
phorus, or toluylenediamine, and in other cases. This also occurs in man
in severe infectious diseases where the red blood-corpuscles are dissolved
or destroyed. It has also been claimed many times that a transformation
of blood-pigments into bile-pigments occurs elsewhere than in the liver,
namely, in the blood. Such a belief has been made very improbable
and in some of the above-mentioned cases, as after poisoning with phos-
phorus, toluylenediamine, and arseniureted hydrogen, it has been dis-
proved by direct experiment.2 In these cases we are also dealing with
an abundant working up of the blood-pigments in the liver.

Bile Concretions.

The concrements which occur in the gall-bladder vary considerably
in size, form, and number, and are of three kinds, depending upon the
kind and nature of the bodies forming their principal mass. One group of
gall-stones contains lime-pigment as chief constituent, another cholesterin,
and the third calcium carbonate and phosphate. The concrements
of the last-mentioned group occur very seldom in man. The so-called
cholesterin-stones are those which occur most frequently in man, while

*See Naunyn and Minkowski, Arch. f. exp. Path. u. Pharm., 21; Latschenberger,
I.e.; Neumann, Virchow's Arch., Ill, and the literature in footnote 2, p% 383.

* The literature belonging to this subject is found in Stadelmann, Der Icterus, etc.,
Stuttgart, 1891.

BILE CONCRETIONS. CHOLESTERIN. 445

the lime-pigment stones are not found very often in man, but often in
oxen.

The pigment-stones are generally not large in man, but in oxen and
pigs they are sometimes found the size of a walnut or even larger. In
most eases they consist principally of calcium-bilirubin with little or no
biliverdin, and they also often contain very small amounts of cholic acids.
Sometimes also small black or greenish-black, metallic-looking stones
are found, which consist chiefly of bilifuscin along with biliverdin. Iron
and copper seem to be regular constituents of pigment-stones. Man-
ganese and zinc have also been found in a few cases. The pigment-stones
are generally heavier than water.

The cholesterin-stones, whose size, form, color, and structure may vary
greatly, are often lighter than water. The fractured surface is radiated,
crystalline, and frequently shows crystalline, concentric layers. The
cleavage fracture is waxy in appearance, and the fractured surface when
rubbed by the finger-nail also becomes like wax. By rubbing against
each other in the gall-bladder they often become faceted or take other
remarkable shapes. Their surface is sometimes nearly white and wax-
like, but generally their color is variable. They are sometimes smooth,
in other cases they are rough or uneven. The quantity of cholesterin
in the stones varies from 642 to 981 p. m. (RITTER x). The cholesterin-
stones sometimes contain variable amounts of lime-pigments, which may
give them a very changeable appearance.

Cholesterin. The formula for this body, although not positively
determined, is generally given as C2?H46O (OBERMULLER) or C2?H44O
(MAUTHNER and SUIDA).

Because of the fact that from cholesterin, hydrocarbons which
have been called cholesteriline, cholesterone and cholesterilene, can be
prepared in different wrays, it was believed that a certain analogy exists
between the cholesterin and the terpenes. The color reactions as
well as the recent investigations on the constitution of cholesterin indicate
that this body belongs to the terpenes.

The constitution of cholesterin has not been completely determined,
although we have the very laborious and thorough investigations of
many workers of whom we especially mention MAUTHNER and SUIDA,
WINDAUS, STEIN, DIELS and ABDERHALDEN.S From these investigations
we conclude that cholesterin is a monoatomic, unsaturated, secondary
alcohol whose hydroxyl group exists in a hydrogenized ring, between

1 Journ. de Tanat. et de la physiol. (Robin), 1872.

2 The literature on cholesterin can be found in Windaus, Arch. d. Pharm., 246,
Hft. 2, and in Abderhalden's Bioch. Handlexikon, Bd., 3, and also in Glikin, Bioch.
Centralbl., 7, 372-377.

446 THE LIVER.

two methyl groups, and which also contains an isopropyl group.
It is also generally admitted that cholesterin contains only one double
bond, which occurs in a vinyl group, CH:CH2, at the end. No constitu-
tional formula for cholesterin can be given; still there is no doubt but
that it is a complex terpene which stands in close relation to retene as
well as to the cholic acids.

By the reduction of cholesterin by metallic sodium and amyl alcohol, DIELS
and ABDERHALDEN as well as NEUBERG and RAUCHWERGER obtained a dihydro-
cholesterin, the a-cholestanol, C27H480. On treating cholestenon, the ketone of
cholesterin, DIELS and ABDERHALDEN obtained a second dihydrocholesterin,
the (3-cholestanol, which WILLSTATTER and E. W. MAYER obtained directly from
cholesterin in ethereal solution by reduction with hydrogen and platinum-black.
According to DIELS and LINN x 0-cholestanol is obtained from cholestenon by
the action of sodium and amyl alcohol, and a-cholestanol with sodium amylate.
The relation of these bodies to each other is still not understood. These dihydro-
cholesterins have a physiological interest in regard to the question whether they
are identical or not with koprosterin, which will be discussed below.

On heating cholesterin, when contaminated with iron, to 300-320°, according
to DIELS and LiNN,2 it in part yields cholestenon and partly an isomeric cholesterin,
the ^-cholesterin. This last body can be retransformed into cholesterin by the
saponification of the cholesteryl benzoate.

Cholesterin occurs in small amounts in nearly all animal fluids and
juices. It occurs only rarely in the urine, and then in very small quanti-
ties. It is also found in the different tissues and organs, especially
abundant in the brain and the nervous system; further, in the yolk of
the egg, in semen, in wool-fat (together with isocholesterin), and in
sebum. It also appears in the contents of the intestine, in excrements,
and in the meconium. It especially occurs pathologically in gall-stones
as well as in atheromatous cysts, in pus, in tuberculous masses, old
transudates, cystic fluids, sputum, and tumors. It does not exist free
in all cases; for example, it exists in part as fatty-acid esters in wool-
fat, blood, lymph, brain, vernix caseosa and epidermis formations. Sev-
eral kinds of cholesterin, called phytosterines, have been found in the
vegetable kingdom.

Cholesterin which has been crystallized from warm alcohol on cooling,
and also that which is present in old transudates, contains one molecule
of water of crystallization, and melts at 148.5° C. when' dried in a vacuum,
and forms colorless, transparent plates whose sides and angles frequently
appear broken, and whose acute angle is often 76° 30' or 87° 30'. In
large quantities it appears as a mass of white plates which shine like
mother-of-pearl and have a greasy touch.

Cholesterin is insoluble in water, dilute acids, and alkalies. It is
neither dissolved nor changed by boiling caustic alkali. It is easily

1 Willstatter and Mayer, Ber d. d. chem. Gesellsch., 41; Diels and Linn, ibid,, 41.

2 Ibid., 41.

CHOLESTERIN. 447

soluble in boiling alcohol, and crystallizes on cooling. It dissolves -readily
in ether, chloroform, and benzene, and also in the volatile or fatty oils.
It is dissolved to a slight extent by alkali salts of the bile-acids, better
in the presence of oleic soap (GERARD 1). The solutions in ether and
chloroform are levorotatory, (a)D= —31.12° (2 per cent ethereal solu-
tion).

Among the many compounds of cholesterin the propionic ester
C2Hs.CO.O.C27H45 is of special interest because of the behavior of the
fused compound on cooling, and it is used in the detection of choles-
terin. For the detection of cholesterin use is made of its reaction with
concentrated sulphuric acid, which gives colored products.

If a mixture of five parts sulphuric acid and one part water acts on
cholesterin crystals, they show colored rings, first a bright carmine-red
and then violet. This test is employed in the microscopic detection
of cholesterin. Another test, and one very good for the microscopical
detection of cholesterin, consists in treating the crystals first with the
above dilute acid and then with some iodine solution. The crystals
will be gradually colored violet, bluish-green, and a beautiful blue.

SALKQ^SKI'S 2 Reaction. The cholesterin is dissolved in chloroform
and then treated with an equal volume of concentrated sulphuric acid.
The cholesterin solution becomes -first bluish-red, then gradually more
violet-red, while the sulphuric acid appears dark red with a greenish
fluorescence. If the chloroform solution is poured into a porcelain dish
it becomes violet, then green, and finally yellow.

LIEBERMANN-BURCHARD'S 3 Reaction. Dissolve the cholesterin in
about 2 cc. chloroform and add firslTlO drops of acetic anhydride and then
concentrated sulphuric acid drop by drop. The color of the mixture
will first be a beautiful red, then blue, and finally, if not too much
cholesterin or sulphuric acid is present, a permanent green. In the pres-
ence of very little cholesterin the green color may appear immediately.

NETJBERG-RAUCHWERGER'S 4 Reaction. With rhamnose, or better still
with S-methylfurfurol and concentrated sulphuric acid, an alcoholic
solution of cholesterin gives a pink ring, or after mixing the liquids and
cooling, a pink solution. On proper dilution an absorption-band can
be seen just beginning before E and whose other side coincides with 6.
This reaction is of interest because it is also given by bile-acids, some
camphor derivatives, abietinic acid, and a hydride of retene.

1 Compt. rend. soc. biol., 58.

2 Pfliiger's Arch., 6.

3 C. Liebermann, Ber. d. deutsch. chem. Gesellsch., 18; 1804, H. Burchard, Bei-
trage zur Kenntnis der Cholesterine, Rostock, 1899.

4 Salkowski's Festschrift, 1904.

448 THE LIVER.

LiFSCHUTz's1 Reaction. Dissolve a few milligrams of cholesterin
in 2-3 cc. glacial acetic acid, add a little benzoylsuperoxide thereto, and
boil once or twice. On adding 4 drops concentrated sulphuric acid to
the cooled solution and shaking, a pure green coloration is obtained,
which changes immediately into blue or with violet-red as an intermediary
color. An absorption-band is formed between C and d, and a broad band
at D. In this reaction an oxidation of the cholesterin occurs, and LIF-
SCHUTZ 2 therefore uses the glacial acetic acid-sulphuric acid reaction
(color and spectrum) for the detection of oxidation products of choles-
terin in blood and tissues.

Pure, dry cholesterin when fused in a test-tube over a low flame with two or
three drops of propionic anhydride yields a mass which on cooling is first violet,
then blue, green, orange, carmine-red, and finally copper-red. It is best to re-fuse
the mass on a glass rod and then to observe the rod on cooling, holding it against
a dark background (OBERMULLER).S

SCHIIT'S Reaction. If a little cholesterin is placed in a porcelain dish with
the addition of a few drops of a mixture of 2 or 3 vols. of concentrated hydrochloric
acid or sulphuric acid and 1 vol. of a rather dilute solution of ferric chloride, and
carefully evaporated to dryness over a small flame, a reddish-violet residue is
first obtained and then a bluish-violet.

If a small quantity of cholesterin is evaporated to dryness with a drop of
concentrated nitric acid, one obtains a yellow spot which becomes deep orange-red
with ammonia or caustic soda (not a characteristic reaction).

Cholesterin combines with saponin (WINDAUS, YAGI) and when a solution
of cholesterin in boiling 95 per cent alcohol is treated with a warm 1 per cent
solution of digitonin in 90 per cent alcohol, a precipitate of digitonin-cholesteride
is obtained. If the amount of the washed and dried digitonin-cholesteride is
multiplied by 0.25 the quantity of cholesterin is obtained (WINDAUS 4). The
cholesterin esters are not precipitated by digitonin.

Koprosterin is the name given by BONDZYNSKI to the cholesterin which was
isolated by him from human feces, although it was prepared earlier by FLINT
and designated as stercorin. It dissolves in cold, absolute alcohol and very readily
in ether, chloroform, and benzene. It crystallizes in fine needles which melt at
95-96° C. (89-90° according to HAUSMANN), and is dextrorotatory («)D = +24°.
It gives the same color reactions as cholesterin, with the exception that it does
not give a reaction with propionic anhydride. According to BONDZYNSKI and
HUMNICKI it is a dihydrocholesterin, with the formula CarH^O, which is formed
in the human intestine by the reduction of ordinary cholesterin. According to
KUSUMOTO as well as DOREE and GARDNER, koprosterin also occurs in the intes-
tine of dogs. The koprosterin prepared by H. FISCHER from human feces seems
to be identical with that prepared by BONDZYNSKI. It is remarkable that BOEHM 5

1 Ber. d. d. chem. Gesellsch., 41.

2 Zeitschr. f. physiol. Chem., 50, 53, 58, and Ber. d. d. chem. Gesellsch., 41.
8 Zeitschr. f . physiol. Chem., 15.

4 Windaus, Zeitschr. f. physiol. Chem., 65; Yagi, Arch. f. exp. Path. u. Pharm., 64.

6 Bondzynski, Ber. d. deutsch. chem. Gesellsch., 29; Bondzynski and Humnicki,
Zeitschr. f. physiol. Chem., 22; Flint, ibid., 23, and Amer. Journ. Med. Sciences,
1862; Miiller, Zeitschr. f. physiol. Chem., 29; Hausmann, Hofmeister's Beitrage, 6;
Kusumoto, Bioch. Zeitschr., 14; Dore"e and Gardner, Proc. Roy. Soc. London, 80,
Ser. B.; H. Fischer, Zeitschr. f. physiol. Chem., 73; Boehm, Bioch. Zeitschr., 33.

CHOLESTERINS. 449

found a dihydrocholesterin in the contents in a part of the ileum which had been
disconnected from the other part of the intestine for 14 years. This had the
same optical rotation and the same melting-point, 142-143° C., as the dihydro-
cholesterin (j3-cholestanol) prepared by DIELS and ABDERHALDEN, WILLSTATTER
and MAYER.

Hippokoprosterin is another cholesterin richer in hydrogen, which BONDZYNSKI
and HUMNICKI found in the feces of the horse. Its formula is CmH^O. According
to DOREE and GARDNER it is not an animal cleavage product, but a constituent
of the grass used as fodder. It melts at 78.5-79.5° C.

Isocholesterin is a cholesterin, so called by SCHULZE, x with the formula
C26H440, which occurs in wool-fat, and is therefore found to a great extent in
so-called lanolin. It gives the LIEBERMANN-BURCHARD reaction, but does not
give SALKOWSKI'S reaction. It melts at 138-138.5° C. The specific rotation in
7 per cent ethereal solution is («)D = 4-59.1°.

Spongosterin, C^EUO is the^name given by HENZE2 to a cholesterin isolated by
him from a silicious sponge. It is very similar to cholesterin, but is not identical
with it or with phytocholesterins. It gives the LIEBERMANN-BURCHARD reaction as
well as SALKOWSKI'S reaction, but the last test is not quite so beautiful a red.
OBERMULLER'S reaction is negative. Melting-point 123-124°.

Bombicesterin is the name given by MENOZZI and MORESCHI 3 to a cholesterin
isolated by them from the chrysalis of the silkworm, which has a melting-point
of 148° and a specific rotation of («)D= -34°.

The cholesterin occurring in the intestine is derived in part from the
food, in part from the bile and part, as shown from the contents of a
ligatured portion of the intestine (see Chapter VIII), from the epithelium
or the secretion of the intestinal mucosa. That a part of the cholesterin
of the intestine disappears has been shown by KUSUMOTO, although
it remains undecided whether this takes place by bacterial decomposi-
tion or by absorption. LEVITES 4 on the contrary, recovered the cho-
lesterin introduced into dogs almost quantitatively. The behavior of
cholesterin in metabolism is not well known; LIFSCHUTZ believes that he
has detected by his color-reaction the oxidation products of cholesterin
in the blood and in bone-fat.

The cholesterins belong to the so-called lipoids, which have been
mentioned in previous chapters (I and VI), and are of the greatest
importance as constituents of the outer envelope of erythrocytes and
the cells in general. Cholesterin is also of great interest because it inhibits
or prevents the haemolysis produced by certain bodies, and therefore
acts as a certain protective power in the animal bod}r. This action of
the cholesterins in regard to inhibiting the hsemolytic action of saponin,

1 Ber. d. deutsch. chem. Gesellsch., 6; Journal f. prakt. Chem. (N. F.), 25; and
Zeitschr. f. physiol. Chem., 14, 522. See also E. Schulze and J. Barbieri, Journal f.
prakt. Chem. (N. F.), 25, 159. In regard to the formula for isocholesterin, see
Darmstadter and Lifschiitz, Ber. d. deutsch. chem. Gesellsch., 31, and E. Schulze,
ibid., 1200.

2 Zeitschr. f. physiol. Chem., 41 and 55.

« Cited from Chem. Centralbl., 1908, 1377 and 1910, 872.
4 Zeitschr. f. physiol. Chem., 57.

450 THE LIVER.

as first discovered by RANSOM, is destroyed, as shown by HAUSMANN, by
replacing the hydroxyl groups. These combinations between cholesterin
and saponins have been studied by MADSEN and NOGUCHI and by

WlNDAUS.1

The so-called cholesterin-stones are best employed in the preparation
of cholesterin. The powder is first boiled with water and then repeatedly
boiled with alcohol. The cholesterin which on cooling separates from the
warm filtered solution is boiled with a solution of caustic potash in alcohol
so as to saponify any fat. After the evaporation of the alcohol the choles-
terin is extracted from the residue with ether, by which the soaps are
not dissolved; filter, evaporate the ether, and purify the cholesterin by
recrystallization from alcohol-ether. The cholesterin may be extracted
with fat from tissues and fluids by first extracting with ether and then
proceeding as suggested by RiTTER.2 The assential points in his method
consist in saponifying the fat with sodium alcoholate, removing the alcohol
by evaporating to dryness with NaCl, and finally extracting the dried
pulverized mass with ether. After evaporating the ether the residue is
dissolved in as little alcohol as possible and the cholesterin precipitated
by the addition of water. It is ordinarily easily detected in transudates
and pathological formations by means of the microscope. In regard to
the methods of preparation, detection and quantitative estimation of
cholesterin we refer to the larger text-books.

1 Ransom, Deutsch. med. Wochenschr., 1901; Hausmann, Hofmeister's Beitrage,
6; Madsen and Noguchi, Kgl. Dansk. Vidensk. Selskabs. Forh., 1904; Windaus, Ber.
d. d. chem. Gesellsch., 42.

2 Zeitschr. f. physiol. Chem., 34. See also Corper, Journ. of biol. Chem., 11.

CHAPTER VIII.
DIGESTION.

THE purpose of digestion is to separate those constituents of the
food which serve as nutriment for the body from those which are use-
less, and to separate each in such a form that it may be taken up by the
blood from the alimentary canal and employed for various purposes in
the organism. This demands not only mechanical, but also chemical,
action. The first action, which is essentially dependent upon the physical
properties of the food, consists in a tearing, cutting, crushing, or grinding
of the food, while the second serves chiefly in converting the nutritive
bodies into a soluble and easily absorbable form, or in splitting them into
simpler compounds for use in the animal syntheses. The solution of the
nutritive bodies may take place in certain cases by the aid of water alone,
but in most cases a chemical metamorphosis or cleavage is necessary;
this is effected by means of the acid or alkaline fluids secreted by the
glands. The study of the processes of digestion from a chemical stand-
point must therefore begin with the digestive fluids, their qualitative
and quantitative composition, as well as their action on the nutriments
and foods.

I. THE SALIVARY GLANDS AND THE SALIVA.

The salivary glands are partly albuminous glands (as the parotid
in man and mammals, and the submaxillary in rabbits), partly mucous
glands (as some of the small glands in the buccal cavity and the subi-
lingual and submaxillary glands of many animals), and partly mixed
gland's (as the submaxillary gland in man) . The alveoli of the albuminous
glands contain cells which are rich in protein but which contain no mucin.
The alveoli of the mucin-glands contain cells rich in mucin but poor in
protein. Cells arranged in different ways, but rich in proteins, also occur
in the submaxillary and sublingual glands. According to the analyses
of MAGNUS-LEVY 1 the human salivary glands contain 274 p. m. solids,
of which 114 p. m. was fat and 154 p. m. was protein. Among the
solids we find mucin, proteins, nucleoproteins, nuclein, enzymes and their

1 Bioch. Zeitschr., 24.

451

452 DIGESTION.

zymogens, besides extractive bodies, leucine, purine bases, and mineral
substances.

The occurrence of a mucinogen has not been proved. On the complete removal
of all mucin E. HOLMGREN l found no mucinogen in the submaxillary gland of
the ox, but a mucin-like gluconucleoproteid.

The saliva is a mixture of the secretion of the above-mentioned groups
of glands; therefore it is proper that a study be made of each of the dif-
ferent secretions by itself and then of the mixed saliva.

The submaxillary saliva in man may be easily collected by intro-
ducing a canula through the papillary opening into Wharton's duct.

The submaxiliary saliva has not always the same composition or
properties; this depends essentially, as shown by experiments on animals,
upon the conditions under which the secretion takes place. That is to
say, the secretion is partly dependent on the cerebral system, through
the facial fibers in the chorda tympani, and partly on the sympathetic
nervous system, through the fibers entering the vessels in the gland. In
consequence of this dependence the two distinct varieties of submaxillary
secretion are distinguished as chorda- and sympathetic saliva. A third
kind of saliva, the so-called paralytic saliva, is secreted after poisoning
with curare or after the severing of the glandular nerves.

The difference between chorda- and sympathetic saliva (in dogs)
consists chiefly in their quantitative constitution; the less abundant
sympathetic saliva is more viscous and richer in solids, especially in
mucin, than the more abundant chorda-saliva. The specific gravity of
the chorda-saliva of the dog is 1.0039-1.0056, and contains 12-14 p. m.
solids (EcKHARD2). The sympathetic has a specific gravity of 1.0075-1.018,
with 16-28 p. m. solids. The freezing-point of the chorda-saliva obtained
from dogs on electric stimulation varies, according to NoLF,3 between
A =-0.193° and -0.396°, with a content of 3.3-6.5 p. m. salts and 4.1-
11.5 p. m. organic substances. The osmotic pressure is on an average a
little higher than one-half the osmotic pressure of the blood-serum. The
spontaneously secreted submaxillary saliva is ordinarily somewhat diluted.
On changing the 'osmotic pressure of the blood the osmotic pressure
of the saliva, according to JAPPELLI,4 changes in the same direction.
According to DEMOOR, LOCKE'S solution with some dog serum is well
suited by transfusion to keep the submaxillary gland of the dog in
activity, while ox serum is unsuited.5 The gases of the chorda-saliva

1 Upsala Lakaref. Forh. (N. F.), 2; also Maly's Jahresber., 27.

2 Cited from Kuhne's Lehrb. d. physiol. Chem., 7.

3 See Maly's Jahresber., 31, 494.

4 Jappelli, ibid., 48 and 51.

5 Arch, intern, de Physiol., 10 (1911).

SALIVA. 453

have been investigated by PFLUGER.1 He found 0.5-0.8 per cent oxygen,
0.9-1 per cent nitrogen, and 64.73-85.13 per cent carbon dioxide —
all results calculated at 0° C. and 760 mm. pressure. The greater part
of the carbon, dioxide was chemically combined. ^ OO 3

The two kinds of submaxillary secretion just named have not thus
far been separately studied in man. The secretion may be excited by an
emotion, by mastication, and by irritating the mucous membrane of the
mouth, especially with acid-tasting substances. The submaxillary saliva
in man is ordinarily clear, rather thin, a little ropy, and froths easily.
Its reaction is alkaline toward litmus. The specific gravity is 1.002-
1.003, and it contains 3.6-4.5 p. m. solids.2 As organic constituents
are found mucin, traces of protein and diastatic enzyme, which latter is
absent in several species of animals. The inorganic bodies are alkali
chlorides, sodium and magnesium phosphates, and bicarbonates of the
alkalies and calcium. Potassium sulphocyanide occurs in this saliva.

The Sublingual Saliva. The secretion of this saliva is also influenced
by the cerebral and the sympathetic nervous system. The chorda-saliva,
which is secreted only to a small extent, contains numerous salivary
corpuscles, but is otherwise transparent and very ropy. Its reaction
is alkaline, and it contains, according to HEiDENHAiN,3 27.5 p. m. solids
(in dogs).

The sublingual secretion in man is clear, mucilaginous, more alka-
line than the submaxillary saliva, and contains mucin, diastatic enzyme,
and potassium sulphocyanide.

Buccal mucus can be obtained pure, from animals only, by the method
suggested by BIDDER and SCHMIDT, which consists in tying the exit to
all the large salivary glands and cutting off their secretion from the mouth.
The quantity of liquid secreted under these circumstances (in dogs) was
so very small that the investigators named were able to collect only 2
grams of buccal mucus in the course of one hour. It is a thick, ropy,
sticky liquid containing mucin; it is rich in form-elements, above all
in flat epithelium cells, mucous cells, and salivary corpuscles. The
quantity of solids in the buccal mucus of the dog is, according to
BIDDER and SCHMIDT,* 9.98 p. m.

Parotid Saliva. The secretion of this saliva is also partly dependent
on the cerebral nervous system (n. glossopharyngeus) and partly on the
sympathetic. The secretion may be excited by emotions and by irri-

1 Pfliiger's Arch., 1.

2 See Maly's "Chemie der Verdauungssafte und der Verdauung," in Hermann's
Handb., 5, part II, 18. This article contains also the pertinent literature.

3 Studien. d. physiol. Instituts zu Breslau, Heft 4.

4 Die Verdauungssafte und der Stoffwechsel (Mitau and Leipzig, 1852), p. 5.

454 DIGESTION.

tation of the glandular nerves, either directly (in animals), or reflexly,
by mechanical or chemical irritation of the muccus membrane of the
mouth. Among the chemical irritants the acids take first place. Mas-
tication also exercises a strong influence upon the secretion of parotid
saliva, which is specially marked in certain herbivora.

Human parotid saliva may be readily collected by the introduction
of a canula into STENSON'S duct. This saliva is thin, less alkaline than
the submaxillary saliva (the first drops are sometimes neutral or acid),
without special odor or taste. It contains a little protein but no mucin,
which is to be expected from the construction of the gland. It also con-
tains a diastatic enzyme, which, however, is absent in many animals.
The quantity of solids varies between 5 and 16 p. m. The specific gravity
is 1.003-1.012. Potassium sulphocyanide seems to be present, though
it is not a constant constituent. KULZ 1 found a maximum of 1.46 per
cent oxygen, 3.8 per cent nitrogen, and in all 66.7 per cent carbon dioxide
in human parotid saliva. The quantity of firmly combined carbon dioxide
was 62 per cent.

The quantity and composition of the saliva, from the mucin glands
as well as from the albuminous glands, show differences in the various
classes of animals but these cannot be entered into here. According
to PAWLOW 2 and his pupils the quantity as well as the composition of
the saliva of the various glands and the mixed saliva in dogs is to a great
degree dependent upon the psychical stimulation, but also upon the
kind of substances introduced into the mouth, and an adaptation of
the glands for various mechanical and chemical irritants is found to occur.

POPIELSKI 3 disputes the existence of such an accommodation (in
dogs) to the kind of food and to the kind of stimulation. In man an
accommodation of the salivary glands, to the needs, has also been sug-
gested but the statements are still .not unanimous.1 See also Chapter
I (page 53).

The mixed buccal saliva in man is a colorless, faintly opalescent,
slightly ropy, easily frothing liquid without special odor or taste. It
is made turbid by epithelium cells, mucous and salivary corpuscles,
and often by food residues. Like the submaxillary and parotid saliva,
on exposure to the air it becomes covered with an incrustation consist-
ing of calcium carbonate and a small quantity of an organic substance,

1 Zeitschr. f . Biologie, 23.

2 Arch, internal, de Physiol., 1, 1904. See also Boos, Maly's Jahresber., 36. 390,
and Neilson and Terry. Amer. Journ. of Physiol., 15, as well as the work of Mendel
and Underbill, Journ. of biol. Chem., 3.

"Popielski, Pfluger's Arch., 127; Zebrowski, Pfluger's Arch., 110; Neilson and
Lewis, Journ. of biol. Chem., 4, with Scheele, ibid., 5; Carlson and Chittenden, Amer.
Journ. of Physiol., 20.

MIXED SALIVA. 455

or it gradually becomes cloudy. Its reaction is generally alkaline to
litmus. The degree of alkalinity varies considerably not only in dif-
ferent individuals but also in the same individual during different parts
of the day, so that it is difficult to state the average alkalinity. Accord-
ing to CHITTENDEN and ELY it corresponds to the alkalinity of 0.8 p. m.
Na2COs solution, or to 0.2 p. m. solution according to COHN. According
to FOA the actual alkalinity (OH-ion concentration) is always consider-
ably less than that found by titration, and the reaction determined
electrometrically is very nearly neutral. The reaction may also be acid,
as found to be the case by STICKER some time after a meal, but this is
not true, at least for all individuals. The specific gravity varies between
1.002 and 1.008, and the quantity of solids between 5 and 10 p. m.
According to CoHN,1 A= —0.20° on an average, and the amount of NaCl
is 1.6 p. m. The solids, irrespective of the form-constituents men-
tioned, consist of protein, murin, oxidases,2 two enzymes, ptyalin and
maltase, as well as a dipeptid and a tripeptid splitting enzyme3 and
mineral bodies. It is also claimed that urea is a normal constituent of
the saliva. The mineral bodies are alkali chlorides, bicarbonates of
the alkalies and calcium, phosphates, and traces of sulphates, nitrites,
ammonia, and sulphocyanides, which latter average about 0.1 p. m.
(MuNK and others). Smaller quantities, 0.03-0.04 p. m., are found in
the saliva of non-smokers (SCHNEIDER and KRUGER), while from ordin-
ary smokers the quantity of sulphocyanides may rise to 0.2 p. m.
(FLECKSEDER 4) .

Sulphocyanides, which, although not constant, occur in the saliva of
man and certain animals, may be easily detected by acidifying the saliva
with hydrochloric acid and treating with a very dilute solution of ferric
chloride. As control, especially in the presence of very small quantities,
it is best to compare the test with another test-tube containing an equal
amount of acidulated water and ferric chloride. Other methods have
been suggested by GSCHEIDLEN, SOLERA, and GANASSINI. The quantita-
tive estimation can be done according to MUNK'S 5 method.

1 Chittenden and Ely, Amer. Chem. Journ., 4, 1883; Chittenden and Richards,
Amer. Journ. of Physiol., 1; Foa, Compt. rend. soc. biol., 58; Sticker, cited from
Centralbl. f. Physiol., 3, 237; Cohn, Deutsch. med. Wochenschr., 1900.

2 Bogdanow-Beresowski, cited from Biochem. Centralbl., 2, 653; Herlitzka, Maly's
Jahresber., 40; ^panjer-Herford, Virchow's Arch., 205.

'Warfield, Johns Hopkins Hosp. Bull. 22 (1911); Koelker, Zeitschr. f. physiol.
Chem., 76, (1911).

4 Munk, Virchow's Arch., 69; Schneider, Amer. Journ. of Physiol., 5; Kriiger,
Zeitschr. T. Biologic, 37; Fleckseder, Centralbl. f. innere Med., 1905. In regard to
the variation in the amount of various constituents in saliva see Fleckseder, 1. c., and
Tezner, Arch, internat. de Physiol., 2.

5 Gscheidlen, Maly's Jahresber., 4; Solera, see ibid., 7 and 8; Munk, Virchow's
Arch., 69; Ganassini, Biochem. Centralbl., 2, p. 361.

456 DIGESTION.

Ptyalin, or salivary diastase, is the amylolytic enzyme of the saliva.
This enzyme is found in human saliva,1 but not in that of all animals,
especially not in the typical carnivora. It occurs not only in adults,
but also in new-born infants. In opposition to ZWEIFEL'S views, BER-
GER 2 claims that it is present not only in the parotid gland of children,
but also in the mucin glands.

According to H. GOLDSCHMIDT s the saliva (parotid saliva) of the horse does
not contain ptyalin, but a zymogen of the same, while in other animals and man
the ptyalin is formed from the zymogen during secretion. In horses the zymogen
is transformed into ptyalin during mastication, and bacteria seem to give the
impulse to this change. During precipitation with alcohol the zymogen is changed
into ptyalin.

Ptyalin has not been isolated in a pure form up to the present time.
It can be obtained purest by COHNHEIM'S 4 method, which consists in
carrying the enzyme down mechanically with a calcium-phosphate
precipitate, and washing the precipitate with water, which dissolves the
ptyalin, and from which it can be obtained by precipitating with alcohol.
For the study or demonstration of the action of ptyalin one employs a
watery or glycerin extract of the salivary glands, or simply the saliva
itself.

Ptyalin, like other enzymes, is characterized by its action. This
consists in converting starch into dextrins and sugar. Our knowledge
as to the process going on here is just as uncertain as our knowledge
on the formation of sugar from starch (see page 229). The nature of
the sugar thus produced is known with certainty. For a long time it
was considered that glucose was the sugar formed from starch and
glycogen, but SEEGEN and O. NASSE have shown that this is not true.
MUCULTJS and v. MERING have shown that the sugar formed by the
action of saliva, amylopsin, and diastase upon starch and glycogen is
for the most part maltose. This has been substantiated by BROWN
and HERON. E. KULZ and J. VoGEL5 have also demonstrated that
in the saccharification of starch and glycogen, isomaltose, maltose, and
some glucose are formed, the varying quantities depending upon the
amount of ferment and the length of its action. The formation of

1 In regard to the variation in the quantity of ptyalin in human saliva see Hof-
bauer, Centralbl. f . Physiol., 10, and Chittenden and Richards, Amer. Journ. of Physiol.,
1; Schiile, Maly's Jahresber., 29; Tezner, 1. c.

2 Zweifel, Untersuchungen iiber den Verdauungsapparat der Neugeborenen (Berlin,
1874); Berger, see Maly's Jahresber., 30, 399.

8 Zeitschr. f . physiol. Chem., 10.

4 Virchow's Arch., 28.

6 Seegen, Centralbl. f . d. med. Wissensch., 1876, and Pfliiger's Arch., 19; Nasse,
ibid., 14; Musculus and v. Mering, Zeitschr. f. physiol. Chem., 2; Brown and Heron,
Liebig's Annal., 199 and 204; Kiilz and Vogel, Zeitschr. f. Biologic, 31.

PTYALIN. 457

glucose is claimed by TEBB, ROHMANN, and HAMBURGER 1 to be only a
product of the inversion of the maltose by the maltase.

The action of ptyalin in various reactions has been the subject of
numerous investigations.2 Natural alkaline saliva is very active, but
it is not so active as when made neutral. It may be still more active
under certain circumstances in faintly acid reaction, and according to
CHITTENDEN and SMITH it acts better when enough hydrochloric acid
is added to saturate the proteins present than when only neutralized.
When the acid-combined protein exceeds a certain amount, then the
diastatic action is diminished. The addition of alkali to the saliva
decreases its diastatic action; on neutralizing the alkali with acid or
carbon dioxide the retarding or preventive action of the alkali is arrested.
According to SCHIERBECK, carbon dioxide has an accelerating action in
neutral liquids, while EBSTEIN claims that it has, as a rule, a retarding
action. Organic as well as inorganic acids, when added in sufficient
quantity, may stop the diastatic action entirely. The degree of acidity
necessary in this case is not always the same for a certain acid, but is
dependent upon the quantity of ferment. The same degree of acidity
in the presence of large amounts of ferment has a weaker action than in
the presence of smaller quantities. Hydrochloric acid is of special
physiological interest in this regard, for it prevents the formation of
sugar even in very small amounts (0.03 p. m.). Hydrochloric acid has
not only the property of preventing the formation of sugar, but, as
shown by LANGLEY, NYL£N, and others, may entirely destroy the
enzyme. This is important in regard to the physiological significance
of the saliva.

Foreign substances, such as metallic salts,3 have different effects.
Certain salts, even in small quantities, completely arrest the action;
for example, HgCk accomplishes this result completely in the presence
of only 0.05 p. m. Others have an accelerating action, and this seems
to apply to the salts of the saliva. According to GUYENOT the saliva
has a weaker action the more it is freed from salts by dialysis. On the

1 Tebb, Journ. of Physiol., 15; Rohmann, Ber. d. deutsch. chem. Gesellsch., 27;
Hamburger, Pfliiger's Arch., 60.

2 See Hammarsten, Maly's Jahresber., 1; Chittenden and Griswold, Amer. Chem.
Journ., 3; Langley, Journal of Physiol., 3; Nyle"n, Maly's Jahresber., 12, 241; Chit-
tenden and Ely, Amer. Chem. Journ., 4; Langley and Eves, Journal of Physiol., 4;
Chittenden and Smith, Yale College Studies, 1, 1885, 1; Schlesinger, Virchow's Arch.,
125; Schierbeck, Skand. Arch. f. Physiol., 3; Ebstein and C. Schulze, Virchow's
Arch., 134; Klibel, Pfltiger's Arch., 56.

3 See O. Nasse, Pfliiger's Arch., 11, and Chittenden and Painter, Yale College
Studies, 1, 1885, 52; Kiibel, Pfliiger's Arch., 76; Patten and Stiles, Amer. Journ. of
Physiol., 17.

458

DIGESTION.

addition of salts the dialyzed saliva becomes active again, especially
on the addition of calcium or potassium chloride (see also page 71).
ROGER1 believes that the presence of phosphates is a necessity for the
action of saliva. The amount of salts added is of special importance
for the action of the saliva, and one salt, which in small quantities has
an accelerating action, may in large quantities have a retarding action.
The presence of peptone has an accelerating action on the sugar forma-
tion (CHITTENDEN and SMITH and others).

To show the action of saliva or ptyalin on starch the three ordinary
tests for glucose may be used, namely, MOORE'S or TROMMER'S test or
the bismuth test (see Chapter III). It is also necessary, as a control,
to first test the starch-paste and the saliva for the presence of glucose.
The steps in the transformation of starch into amidulin, erythrodextrin,
and achroodextrin may be shown by testing with iodine.

Maltase occurs in saliva to only a slight extent. It converts maltose
into glucose. According to STICKER, 2 saliva also has the power of split-
ting sulphureted hydrogen from the sulphur oils of radishes, onions,
and certain other vegetables.

The quantitative composition of the mixed saliva must vary consider-
ably, not only because of individual differences, but also because under
varying conditions there may be an unequal division of the secretion
from the different glands. We give herewith a few analyses of human
saliva as examples of its composition. The results are in parts per 1000.

g

a

•

fc .

t>

w

on

S*

^;

i a

•
9

8

b

5
1

1

1

fc

fl

K W
? 0

i

§

M

HO

w

H

2*

m

s

H

B

w

Water

992 9

995 16

994 1

988 3

994 7

994 2

Solids

7.1

4 84

5 9

11 7

5 3

3 5-8 4

5 8

in

filtered

saliva

Mucus and epithelium ....

1.4

1.62

2.13

2 2

Soluble organic substances .

3.8

1.34

1.42

3 27

1 4

(Ptyalin of early investigators.)

Sulphocyanides

0 06

0 10

0 064

0 04

to

0.090

Salts

1 9

1 82

2 19

1 30

0 0

^uyenot, Compt. rend, soc, biol., 63; Roger, ibid., 65; see also Bang, Bioch.
Zeitschr., 32 (1911).

2 Munch, med. Wochenschr., 43.

» Zeitschr. f. physiol. Chem., 5. The other analyses are cited from Maly, Chemie
der Verdauungssafte, Hermann's Handbuch d. Physiol., 5, Part II, 14.

SECRETION OF SALIVA. 459

HAMMERBACHER found in 1000 parts of the ash from human saliva: potash
457.2, soda 95.9, iron oxide 50.11, magnesia 1.55, sulphuric anhydride (S03) 63.8,
phosphoric anhydride (P2(X) 188.48, and chlorine 183.52.

The quantity of saliva secreted during twenty-four hours cannot
be exactly determined, but has been calculated by BIDDER and SCHMIDT
to be 1400-1500 grams. The most abundant secretion occurs during
meal-times. According to the calculations and determinations of
TuczEK,1 in man 1 gram of gland yields 13 grams of secretion in the course
of one hour during mastication. These figures correspond fairly well
with those representing the average secretion from 1 gram of gland in
animals, namely, 14.2 grams in the horse and 8 grams in oxen. The
quantity of secretion per hour may be 8 to 14 times greater than the entire
mass of glands, and there is probably no gland in the entire body, so far
as is known at present — the kidneys not excepted — whose ability of
secretion under physiological conditions equals that of the salivary glands.
But as the secretion of saliva is so very variable under different con-
ditions no positive results can be given as to the extent. A remark-
ably abundant secretion of saliva is induced by pilocarpine, while
atropine, on the contrary, inhibits it.

That the secretion of saliva, even if we do not consider such sub-
stances as ptyalin, mucin, and the like, is not a process of filtration,
follows for many reasons, especially the following: The salivary glands
have a specific property of eliminating certain substances, such as
potassium salts (SALKOWSKI 2), iodine, and bromine compounds, but
not others, for example, iron compounds and glucose. It is also notice-
able that the saliva is richer in solids when it is eliminated quickly by
gradually increased stimulation, and in larger quantities than when the
secretion is slower and less abundant (HEIDENHAIN). The amount of
salts increases also to a certain degree by an increasing rapidity of
elimination (HEIDENHAIN, WERTHER, LANGLEY and FLETCHER, Novi3).

Like the secretion processes in general, the secretion of saliva is
closely connected with the processes in the cells. The chemical processes
going on in these cells during secretion are still unknown.

The Physiological Importance of the Saliva.— The quantity of water
in the saliva renders possible the action of certain bodies on the organs
of taste, and it also serves as a solvent for a part of the nutritive sub-
stances. The importance of the saliva in mastication is especially
marked in herbivora, and there is no question as to its importance in

1 Bidder and Schmidt, 1. c., 13; Tuczek, Zeitschr. f. Biologie, 12.

2 Virchow's Arch., 53.

3 Heidenhain, Pfliiger's Arch., 17; Werther, ibid., 38; Langley and Fletcher,
Proc. Roy. Soc., 45, and especially Phil. Trans. Ro}7. Soc. London, 180; Novi, Arch,
f. (Anat. u.) Physiol., 1888.

460 DIGESTION.

facilitating the act of swallowing. The saliva containing mucin is espe-
cially important in this regard, and PAWLOW'S school has shown that the
secretion also regulates itself in this regard. The saliva is also of import-
ance, as it serves in washing out the mouth and thereby acts as a pro-
tection against destructive substances or bodies foreign to the mouth.
The power of converting starch into sugar is not inherent in the saliva
of all animals, and even when it possesses this property the intensity
varies in different animals. In man, whose saliva forms sugar rapidly,
a production of sugar from (boiled) starch undoubtedly takes place in the
mouth, but how far this action proceeds after the morsel has entered the
stomach depends upon the rapidity with which the acid gastric juice mixes
with the swallowed food, and also upon the relative amounts of the
gastric juice and food in the stomach. The large quantity of water which
is swallowed with the saliva must be absorbed and pass into the blood,
and it must in this way go through an intermediate circulation in the
organism. Thus the organism possesses in the saliva an active medium
by which a constant stream, conveying the dissolved and finely divided
bodies, passes into the blood from the intestinal canal during digestion.
The relation of the saliva or the salivary glands to the secretion of gastric
juice will be mentioned in the next section.

Salivary Concrements. The so-called tartar is yellow, gray, yellowish-gray,
brown or black, and has a stratified structure. It may contain more than 200
p. m. organic substances, which consist of mucin, epithelium, and LEPTOTHRIX-
CHAINS. The chief part of the inorganic constituents consists of calcium car-
bonate and phosphate. The salivary calculi may vary in size from that of a
small grain to that of a pea or still larger (a salivary calculus has been found
weighing 18.6 grams), and they contain variable quantities of organic substances
(50-380 p. m.), which remain on extracting the calculus with hydrochloric acid.
The chief inorganic constituent is calcium carbonate.

H. THE GLANDS OF THE MUCOUS MEMBRANE OF THE STOMACH, AND

THE GASTRIC JUICE.

The glands of the mucous coat of the stomach have long been
divided into two distinct classes. Those which occur in the greatest
abundance and which have the greatest size in the fundus are called
fundus, rennin or pepsin glands, and the others, which occur only in
the neighborhood of the pylorus, have received the name of pyloric
glands, sometimes also, though incorrectly, called mucous glands. The
division of these two forms of glands in the mucous membrane of the
stomach is essentially different in various animals. The mucous coat-
ing of the stomach is covered throughout with a layer of columnar
epithelium , which is generally considered as consisting of goblet cells that
produce mucus by a metamorphosis of the protoplasm.

GASTRIC JUICE. 461

The fundus glands contain two kinds of cells: ADELOMORPHIC or chief
cells, and DELOMORPHIC or COVER cells, the latter formerly called RENNIN
or pepsin cells. Both kinds consist of protoplasm rich in proteins;
but their relation to coloring-matters seems to show that the protein
substances of both are not identical. The nucleus must consist princi-
pally of nuclein. Besides the above-mentioned constituents, the fundus
glands contain as more specific constituents several enzymes or their
zymogens, besides a little fat and cholesterin.

The pyloric glands contain cells which are generally considered as
related to the above-mentioned chief cells of the fundus glands. As
these glands were formerly thought to contain a larger quantity of
mucin, they were also called mucous glands. According to HEIDEN-
HAIN, independent of the columnar epithelium of the excretory ducts,
they take no part worthy of mention in the formation of mucus, which
according to his views is effected by the epithelium covering the mucous
membrane. The pyloric glands also seem to contain zymogens. Alkali
chlorides, alkali phosphates, and calcium phosphates are found in the
mucous coating of the stomach.

The Gastric Juice. The observations of HELM and BEAUMONT on
persons with gastric fistula led to the suggestion that gastric fistulas
be made on animals, and this operation was first performed by BASSOW l
in 1842 on a dog. VERNEUIL performed the same on a man in 1876 with
successful results. PAWLOW2 has recently improved the surgery of
gastric fistula and has added much to the study of gastric secretion.

As most investigations upon gastric digestion, and also upon diges-
tion as a whole, are based on observations upon dogs and then upon man,
and for this reason, when not otherwise stated, in this chapter on the
study of digestion we give the conditions in dogs and man.

The secretion of gastric juice is not continuous, at least in man and
in the mammals experimented upon. It only occurs under psychic
influence, and also by stimulation of the mucous membrane of the stomach
or the intestine. The most exhaustive researches on the secretion of
gastric juice (in dogs) have been made by PAWLOW and his pupils.

In order to obtain gastric juice free from saliva and food residues, they arranged
besides a gastric fistula also an cesophageal fistula from which the swallowed food
could be withdrawn with the saliva without entering the stomach, and in this
manner an apparent or sham feeding was possible. In this way it was possible to

1 Helm, Zwei Krankengeschichten, Wien, 1803, cited from Hermann's Handbuch,
5, part II, 39: Beaumont, "The Physiology of Digestion," 1833; Bassow, Bull, de
la floe, des natur. de Moscou, 16, cited from Maly in Hermann's Handbuch, 5, 38;
Verneuil, see Ch. Ricbet, "Du sue gastrique chez Fhomme," etc. (Paris, 1878), 158.

2 Pawlow, The Work of the Digestive Glands, (translated by Thompson, Phila-
delphia, 1910), where the works of his pupils are also mentioned. See also Ergebnisse
der Physiologic, 1, Abt. 1.

462 DIGESTION.

study the influence of psychical moments on one side and the direct action of food
on the mucous membrane on the other. After a method suggested by HEIDENHAIN
and later improved by PAWLOW and CHIGIN, they have succeeded in preparing
a blind sac by partial dissection of the fundus part of the stomach, and the secre-
tion processes could be studied in this sac while the digestion in the other parts
of the stomach was going on. In this way they were able to study the action of
different foods on the secretion.

The most essential results of the investigations of PAWLOW and his
pupils are as follows: Mechanical stimulation of the mucosa does not
produce any secretion. Mechanical irritation of the mucous membrane
of the mouth causes no reflex excitation of the secretory nerves of the
stomach. There are two moments which cause a secretion, namely,
the psychical moment — the passionate desire for food and the sensa-
tion of satisfaction and pleasure on partaking it — and the chemical
moment, the action of certain chemical substances on the mucous mem-
brane of the stomach. The first moment is the most important. The
secretion occurring under its influence by the vagus fibers appears earlier
than that produced by chemical irritants, but only after an interval of
at least 4J minutes. This secretion is more abundant but less contin-
uous than the " chemical." It yields a more acid and active juice than
the latter. As chemical excitants which cause a secretion reflexively
through the stomach mucosa we include water (slight action) and cer-
tain unknown extractive substances contained in meat and meat extracts,
in impure peptone, and also, it seems, in milk. According to HERZEN
and RADZIKOWSKI 1 and others, alcohol is also a strong agent in produc-
ing a flow of juice. The claims in regard to the action of sodium chloride
and alkali carbonates are somewhat disputed. That the alkali carbonates
retard or inhibit secretion is the opinion of many, but from more
recent determinations2 it would seem as if the concentration of the car-
bonate as well as of sodium chloride exercises a certain influence, so that
a weaker concentration is indifferent or retarding, while somewhat stronger
concentration has an accelerating action upon secretion, though inves-
tigators are not agreed as to results. Bitter substances partaken of in
small amounts a certain time before a meal increase the secretion, while
larger amounts have a retarding action (Bomssow, STBASHESKO3).
Fats have a retarding action on the appearance of secretion and diminish
the quantity of juice secreted as well as the amount of enzyme. The
substances, such as egg-albumin, which do not act as chemical stimulants,

1 Pfliiger's Arch., 84, 513.

2 See Rozenblatt, Bioch. Zeitschr, 4; Mayeda, ibid., 2; Pimenow, Bioch, Centralbl.,
6; Lonnquist, Maly's Jahresb., 36.

3 Borissow, Arch. f. exp. Path. u. Pharm., 61; Strashesko, see Biochem. Centralbl.,,
4, 148.

GASTRIC JUICE. 463

may be digested by the " psychical " secretion, and then perhaps cause
a chemical secretion by their decomposition products.

The secretion in the stomach may also be influenced by the small
intestine, and in this way, as shown by the investigations of PAWLOW
and his pupils, the fats have a retarding action upon the secretion of
juice and upon digestion by acting reflexly upon the duodenal mucosa.
In dogs on feeding fat (oil) with food containing starch, the secretion of
gastric juice remains reduced during the entire period of feeding, and
fat in connection with protein food has a similar action, with the excep-
tion that in this case the retarding action is observed only in the first
hours of digestion. According to PIONTKOWSKI l the oil-soaps differ from
the neutral fats by having a strong action on the flow of juice, and this
is the reason why about five to six hours after a meal with fat the secre-
tion of juice is stopped, as just at this time the soaps are being formed.
According to FROUIN the food in the intestine produces a secretion of
gastric juice which continues after the action of the psychic moment
has ceased. LECO^CB-2- arrived at similar results, and he ascribes a less
subordinate importance to the chemical secretion as compared with the
psychic secretion, than PAWLOW does.

The behavior of the different parts of the stomach in secretion is
also of interest. The work of PAWLOW and his pupils GROSS and
KRSHYSCHKOWSKY, has shown that meat and its extractives as well as
the digestion products and milk especially act upon the pyloric part,
although not entirely, while they are inactive upon the fundus. Alcohol
also acts upon the fundus part. PopiELSKi3 found that meat extracts
had an exciting action upon the secretion of gastric juice, even when intro-
duced subcutaneously. In close relation to what has been said above
stands the observation of EDKINS that the pylorus part of the stomach
contains a substance, a prosecretin, which by acids and certain other
substances is transformed into a secretin, which when introduced into
the blood circulation causes a secretion of gastric juice. HEMMETER,
claims that a secretin for the secretion of gastric juice is also produced
in the salivary glands. The extirpation of all the salivary glands in
dogs causes a marked diminution in the secretion of gastric juice, while
the intravenous or peritioneal injection of an extract of the salivary
glands of dogs produces a secretion of gastric juice. EMSMANN4 has
also obtained bodies having a similar action, from the mucosa of the

1 See Biochem. Centralbl., 3, 660.

2Frouin, Compt. rend. soc. biol., 53; Leconte, La Cellule, 17.

3 Gross, Bioch. Centralbl., 5, 669; Krshyschkowsky, Maly's Jahresb., 36, 403;
Popielski, ibid., 39.

4Edkins, Journ. of Physiol., 34; Hemmeter, Bioch. Zeitschr., 11; Emsmann,
Intern. Beitr. zu. Path. u. Ther. d. Ernahrungs storungen 3 (1911).

464 riGESTIOX.

duodenum, jejunum, and ileum as well as from the liver and pancreas
by hydrochloric acid.

We know very little, positively, in regard to the gastric secretion in
man. According to the earlier authorities the irritants may be mechan-
ical, thermic, and chemical. Among the chemical excitants we include
alcohol and ether, which in too great a concentration bring about no
physiological secretion, but rather the transudation of a neutral or
faintly alkaline fluid. Certain acids, such as carbonic acid, neutral
salts, meat extracts, spices, and other bodies also belong to this group.
The reports on this subject are unfortunately very uncertain and con-
tradictory.

The question as to how far the observations made by PAWLOwand
his school can be applied to man is of special interest. Many observa-
tions on this question have been collected 1 and they compare favor-
ably with the observations made upon dogs. Thus in man a psychic
secretion of gastric juice can be brought about, and it has also been
observed that it can be stopped by emotions. As in clogs, so also in man,
after sham feeding, a secretion takes place after a pause, the duration
of which varies in different cases. In some cases, as in dogs after meat
feeding, the pause was about five minutes. The chewing of indifferent
bodies did not affect the glands, while bodies acting upon the organs of
smell and taste had an exciting action. UMBER observed besides this, that
after the introduction of a nutritive enema into the rectum, a secretion
of gastric juice was produced by reflex action.

From these observations of HORNBORG and UMBER, as well as from
some earlier observations of SCHULE, TROLLER, RIEGEL, and ScHEUER,2
we conclude that in man the psychic secretion is much less than that
produced by the introduction of food or bodies having a pleasant taste.
That the preparation of the food in the mouth has an essential influence
upon the secretion is proved without doubt, but we do not agree as to
how this action takes place. Certain experimenters consider the secreted
and swallowed saliva as the most essential factor in this action, while
others believe that the act of chewing, and still others that the chemical
action and the sense of taste, are the most important.

In regard to the action of saliva, HEMMETER finds that after the
extirpation of the salivary glands, the introduction into the stomach
of chewed food soaked with dog-saliva, has no special action upon the

1Hornborg, Maly's Jahresb., 33, 547; Umber, Berl. klin. Wochenschr., 1905;
Cade and Latarjet, Compt. rend. soc. biol., 57; Kaznelson, Pfliiger's Arch., 118;
Bogen, ibid., 117; Bickel, Deutsch. med. Wochenschr., 32, and Maly's Jahresb., 36,
411. See also Maly's Jahresb. 39, 40, and Bioch, Centralbl. 12.

2 The literature may be found in Umber's work, 1. c.

COMPOSITION OF THE GASTRIC JUICE. 465

secretion of juice. On the other hand FROUIN 1 observed that the intro-
duction of saliva into the large stomach of dogs, acts favorably upon
the secretion in the small stomach (see page 462), and the acidity as well
as the digestive activity of the juice is increased. This action does not
depend, according to FROUIN, upon the alkali of the saliva.

The Qualitative and Quantitative Composition of the Gastric Juice.
The human gastric juice, which can seldom be obtained pure and free
from residues of the food or from mucus and saliva, is a clear, or only
very faintly cloudy, and nearly colorless fluid of an insipild, acid taste
and strong acid reaction. It contains, as form-elements, glandular cells
or their nuclei, and more or less changed columnar epithelium.

The acid reaction of the gastric juice depends on the presence of free
acid, whi|ch, as has been learned from the investigations of C. SCHMIDT,
RICHET, and others, consists, when the gastric juice is pure and free
from particles of food, chiefly or in large part of hydrochloric acid. CON-
TEJEAN 2 regularly found traces of lactic acid in the pure gastric juice
of fasting dogs. After partaking of food, especially after a meal rich in
carbohydrates, lactic acid occurs abundantly, and sometimes acetic
and butyric acids. In new-born dogs the acid of the stomach is lactic
acid, according to GMELIN.S The quantity of free hydrochloric acid in
the gastric juice is, according to PAWLOW and his pupils, in dogs 5-6
p. m., and in cats an average of 5.20 p. m. HC1. In man the results
obtained are variable but regularly much lower. Since it has been
possible to obtain pure human gastric juice for investigation it has been
found (UMBER, HORNBORG, BICKEL, SoMMERFELD4) that the amount
of hydrochloric acid is about 4-5 p. m. There is hardly any doubt that
at least a part of the hydrochloric acid of the gastric juice does not
exist free in the ordinary sense, but combined with organic substances.
The results obtained in testing for the acidity of gastric juice by phys-
ical methods are almost identical with those obtained by titration (P.

FRANCKEL5).

The specific gravity of gastric juice is low, 1.001-1.010. It is corre-
spondingly poor in solids. Earlier analyses of gastric juice from man,
the dog, and the sheep were made by C. SCHMIDT. 6 As these analyses

1 Compt. rend. soc. biol., 62.

2 Bidder and Schmidt, Die Verdauungssafte, etc., 44; Richet, 1. c.; Contejean, Con-
tributions a Petude de la physiol. de 1'estomac, Theses, Paris, 1892.

3 Pfliiger's Arch., 90 and 103.

4 See Richet, 1. c.; Contejean, 1. c.; Verhaegen, "La Cellule," 1896 and 1897;
Sommerfeld, Bioch, Zeitschr, 9, and also footnote 1, page 464, and the literature on
the estimation of hydrochloric acid in the gastric juice contents (p. 489) ; see also
Cohnheim and Dreyfus, Zeitschr. f. physiol. Chem. 58 (1908).

6 Zeitschr, f. exp. Path. u. Therap., 1.

6 i.e.

466 DIGESTION.

refer only to impure gastric juice they are of little value. RosEMANN,1
who has investigated the gastric juice secreted by a dog after sham
feeding, found an average of 4:22 p. m. solids, among which 1.32 p. m.
were mineral bodies and about 2.90 p. m. organic substance. The
amount of nitrogen in one case was 0.36 p. m., in another 0.54 p. m. and
the quantity of HC1 was about 5.6 p. m. The ash consisted chiefly of
potassium chloride, namely 980-990 p. m. of the inorganic part. NENCKI
and SiEBER2 found 3.06 p. m. solids in the pure gastric juice of a dog.
NENCKI 3 found 5 milligrams sulphocyanic acid per liter of gastric juice
of a dog.

In the ash of human gastric juice after sham-feeding ALBU4 found
356.2 p. m. K20; 226.5 p. m. Na2O, and 497.3 p. m. Cl. The amount
of salts insoluble in water was 23.9 p. m. In hyperacidity he found
almost the same composition.

Besides the free hydrochloric acid, pepsin, rennin, and a lipase are
the other physiologically important constituents of gastric juice.

Pepsin. This enzyme is found, with the exception of certain fishes,
in all vertebrates thus far investigated.

Pepsin occurs in adults and in new-born infants. This condition
is different in new-born animals. While in a few herbivora, such as the
rabbit, pepsin occurs in the mucous coat before birth, this enzyme is
entirely absent at the birth of those carnivora which have thus far been
examined, such as the dog and cat.

In various invertebrates enzymes have also been found which have
a proteolytic action in acid solutions. It has been shown that these
enzymes, nevertheless, are not in all animals identical with ordinary
pepsin. According to KLUG and WnoBLEWSKi5 the pepsins found
in man and various higher animals are somewhat different, an observa-
tion which according to the experience of HAMMARSTEN is very prob-
able. Enzymes also occur in various plants and animal organs, although
not identical with pepsin, but which act in acid reaction. The enzyme
obtained from the Nepenthes, which dissolves proteins only in acid
reaction, stands very close to pepsin. An enzyme more closely related
to'trypsin or erepsin (see sections III and IV) is, on the contrary,
GLAESSNER'S pseudopepsin, which according to him is the only peptic
enzyme in the pyloric end. Pseudopepsin, whose existence is disputed
by KLUG, while others (REACH, PEKELHARING) affirm its occurrence in

1 Pfliiger's Arch., 118.

2 Zeitschr. f. physiol. Chem., 32.

3 Ber. d. d. Chem. Gesellsch., 28.

4 Zeitschr. f. Path. u. Therap., 5.

6 Klug. Pfliiger's Arch. 60; Wr6blewski, Zeitschr. f. physiol. Chem., 21.

PEPSIN. 467

the mucous membrane, cannot, according to HAMMARSTEN, either be the
only or the most prominent peptic enzyme of the pyloric part. According
to GLAESSNER, it also acts in neutral and alkaline reaction and yields
tryptophane among other cleavage products. According to BERGMANN l
it is identical with erepsin (see below). Among the enzymes of the
mucosa of the stomach belongs the so-called antipepsin discovered by
WEiNLAND,2 which has a retarding action upon pepsin digestion and,
as some claim, prevents the self-digestion of the mucous membrane.

Pepsin is as difficult to isolate in a pure condition as are other
enzymes. The pepsin prepared by BRUCKE and SUNDBERG gave negative
results with most reagents for proteins, and showed nevertheless a
powerful action, which seems to indicate that it was very pure. SCHOU-
MOW-SiMANOWSKi, NENCKi and SIEBER, have designated as the true
enzyme the substance containing chlorine, which they obtain by strongly
cooling the gastric juice. That this precipitate is not a chemical indi-
vidual, and hence cannot be pepsin, follows from the investigations of
PEKELHARING. While pepsin, according to NENCKI and SIEBER, was
rich in phosphorus and contained nucleoprotein, PEKELHARING'S pepsin
was free from phosphorus and yielded no nucleoprotein. FRIEDENTHAL
and MiYAMOTA3 have also shown that the pepsin is still active after
the splitting off of the nuclein complex (and also the protein). As pepsin
is readily precipitated with the proteins and combines therewith, it is
difficult to decide whether pepsin is a protein substance or not, and the
question as to its nature is still undecided, just as is the case with other
enzymes. As ordinarily known, pepsin, at least in an impure form, is
soluble in water and glycerin. It is precipitated by alcohol, but is only
slowly destroyed thereby. In aqueous solution its action is quickly
destroyed on heating to boiling. According to BiERNACKi4 pepsin
in neutral solutions is destroyed by heating to 55° C. In the dry state
it can be heated to over 100° C. without losing its activity. In the
presence of 2 p. m. HC1 a temperature of 55° C. is not injurious, and the
compound with acid is more resistant than the free pepsin (GROBER 5) .
Pepsin in acid solution is destroyed by heating to 65° C. for five minutes.

1 Glaessner, Hofmeister's Beitrage, 1; Klug, Pfliiger's Arch., 92; Reach, Hofmeis-
ter's Beitrage, 4; Pekelharing, Arch, des scienc., biolog., St. Petersbourg 11; Pawlow-
Festband, 1904; Bergmann, Skand. Arch. f. Physiol., 18.

2 Zeitschr. f. Biologic, 44.

3 Briicke, Wien. Sitzungsber,. 43; Sundberg, Zeitschr. f. physiol. Chem., 9; Schou-
mow-Simanowski, Arch. f. exp. Path. u. Pharm., 33; Pekelharing, Zeitschr. f. physiol.
Chem., 22 and 35; Nencki and Sieber, ibid., 32; Friedenthal and Miyamota, Centrabl.
f. Physiol, 15, 785.

4 Zeithschr. f . Biologic, 28.

8 Arch. f. exp. Path. u. Pharm., 51.

468 DIGESTION.

On adding peptone or certain salts the pepsin may be heated to 70° C.
for the same time without destruction.

The behavior of pepsin on heating its acid solution is influenced not
only by the degree of acidity, but by the duration of heating and also
by the amount of other bodies in the solution. If an acid (0.2 per cent
HC1) infusion of the calf's stomach be warmed for several days to about
40 or 45° C., a part of the pepsin is destroyed, but we obtain in this
manner an infusion which still dissolves proteins but has no rennin action
(HAMMARSTEN *). The pepsin from different animals acts differently
in this regard and the pepsin of the pike stomach is very quickly destroyed
at 37-40° C.

Pepsin is extraordinarily sensitive to the action of alkalies, not only
caustic, and carbonated, but also against the hydroxides of the alka-
line earths. It is easily made inactive by these substances. If the
action of the alkali is not too strong then, as shown by PAWLOW and
TiCHOMiROW,2 the enzyme can in part be reactivated by the addition
of acid if the greater part (about four-fifths), of the alkalinity be neutral-
ized by the addition of acid and then after some hours more acid be added.
If the entire quantity of acid be added at one time the reactivation does
not take place.

The only property which is characteristic of pepsin is that it dissolves
protein bodies in acid but not in neutral or alkaline solutions, with the
formation of proteoses, peptones, and other products.

The methods for the preparation of relatively pure pepsin depend,
as a rule, upon its property of being thrown down with finely divided
precipitates of other bodies, such as calcium phosphate or cholesterin.
The rather complicated methods of BRUCKE and SUNDBERG are based
upon this property. PEKELHARING makes use of a prolonged dialysis
and precipitation with 0.2 p. m. HC1.

Very permanent pepsin solutions, from which the enzyme with con-
siderable protein can be precipitated by alcohol, may be prepared by
extraction with glycerin. Solutions having a strong action may also
be prepared by making an infusion of the gastric mucosa of an animal
in acidified water '(2-5 p. m. HC1). This is unnecessary, as we can obtain
pure gastric juice according to PAWLOW'S method, and also because very
active commercial preparations of pepsin can be bought in the market.

The Action of Pepsin on Proteins. Pepsin is inactive in neutral or
alkaline reactions, but in acid liquids it dissolves coagulated protein
bodies. The protein always swells and becomes transparant before
it dissolves. Unboiled fibrin swells up in a solution containing 1 p, m.
HC1, forming a gelatinous mass, m and does not dissolve at ordinary tem-

1 Zeitschr. f. physiol. Chem., 56. 2 Ibid., 54.

PEPSIN. 469

perature within a couple of days. Upon the addition of a little pepsin,
however, this swollen mass dissolves quickly at ordinary temperatures.
Hard-boiled egg albumin, cut in thin pieces with sharp edges, is not
perceptibly changed by dilute acid (2-4 p. m. HC1) at the temperature
of the body in the course of several hours. But the simultaneous pres-
ence of pepsin causey the edges to become clear and transparent, blunt
and swollen, and the protein gradually dissolves.

From what has been said above in regard to pepsin, it follows that
proteins may be employed as a means of detecting pepsin in liquids.
Ox-fibrin may be employed as well as coagulated egg albumin, which
latter is used in the form of slices with sharp edges. As the fibrin is
easily digested at the normal temperature, while the pepsin test with
egg albumin requires the temperature of the body, and as the test with
fibrin is somewhat more delicate, it is often preferred to that with egg
albumin. When we speak of the " pepsin test " without further explana-
tion we ordinarily understand it as the test with fibrin.

This test, nevertheless, requires care. The fibrin used should be
ox-fibrin and not pig-fibrin, which last is dissolved too readily with dilute
acid alone. The unboiled ox-fibrin may be dissolved by acid alone
without pepsin, but this generally requires more time. In testing with
unboiled fibrin at normal temperature, it is advisable to make a control
test with another portion of the same fibrin with acid alone. Since at
the temperature of the body unboiled fibrin is more easily dissolved by
acid alone, it is best always to work with boiled fibrin.

As pepsin has not thus far been prepared in a positively pure condi-
tion, it is impossible to determine the absolute quantity of pepsin in a
liquid. It is possible only to compare the relative amounts of pepsin
in two or more liquids, which may be done in several ways.

The older method, that of BRUCKE, consists in diluting the two pepsin solu-
tions to be compared with certain proportions of 1 p. m. hydrochloric acid, so
that when the amount of pepsin contained in the original solution is equal to 1,
each solution contains a degree of dilution, p, corresponding to 1, $, i, f, &,
etc. A flock of fibrin or a piece of hard-boiled egg is added to each test and the
time noted when each test begins to digest and when it ends. The relative
amount of pepsin is calculated from the rapidity of digestion as follows: The tests
P=i> !> TGJ °f °ne series is digested in the same time as tests p = l, %, \ of the
other series, hence the first solution contained four times as much pepsin.
GRUTZNER l has improved this test by using fibrin colored with carmine, and on
comparing with carmine solutions of known dilution he determines colorimetrically
the rapidity of digestion.

METT'S Method. Draw up white of egg in a glass tube 1-2 millimeters in
diameter, coagulate it by plunging it into water at 95° C., and cut the ends off
sharply; then add two tubes to each test-tube with a few cubic centimeters
of the acid pepsin solution; allow them to digest at body temperature, and after
a certain time, generally, after ten hours, measure the lineal extent of the digested

1 Griitzner, Pfliiger's Arch., 8 and 106. See also A. Korn, " Ueber Methoden Pepsin
quantitativ zu bestimmen," Inaug.-Dissert., Tubingen, 1902.

470 DIGESTION.

layer of albumin in the various tests, bearing in mind that the digested layer at
each end must not be longer than 6-7 millimeters. The quantity of pepsin in
the comparative tests is as the square of the millimeters of the albumin-column
dissolved in the same time. Thus if in one case 2 millimeters of albumin were
dissolved and in the other 3 millimeters, then the quantity of pepsin is as 4:9.
If the fluid removed from the stomach, which is rich in bodies having a disturb-
ing influence upon pepsin digestion, is to be tested, then the liquid must be first
properly diluted with hydrochloric acid (NIERENSTEIN and SCHIFF l).

Objections have been raised against these methods from several sides, and they
are in fact very uncertain. HUPPERT and E. SCHUTZ measure the relative
quantities of pepsin from the amount of secondary proteoses formed under cer-
tain conditions. The proteoses were determined by the polariscope. J. SCHUTZ
determines the total proteose-nitrogen, and SPRIGGS 2 finds that the change in
the viscosity is a measure of the amount of pepsin.

VOLHARD and LOHLEIN 3 use an acid casein solution for the pepsin determina-
tion, and determine, after precipitation with sodium sulphate, the acidity of the
filtrate of the digested test as well as of the original control solution. The casein
is precipitated as an acid compound by the sulphate, and the filtrate separated
from the precipitate contains less acid than the original solution. In propor-
tion as the digestion progresses less substance is precipitated by the sulphate,
and the acidity of the filtrate becomes correspondingly higher. The increase
in acidity in the different portions varies within certain limits as the square root
of the quantity of ferment.

JACOBY suggested a method which is based on the fact that a cloudy solution
of ricin becomes clear by the action of pepsin-hydrochloric acid, and indeed with
varying rapidity with different quantities of pepsin. This method, which requires
further testing, seems to be delicate and is of value, as is doubtless the following
method of FULD and LEVisoN.4 This is based on the property that edestan can
be precipitated from acid solution by NaCl, but not the proteoses formed therefrom.

A solution of 1 p. m. edestin in hydrochloric acid (ITT normal) is prepared
whereby the edestin is changed into edestan. The activity of a gastric juice (or
a pepsin-hydrochloric acid solution) is tested in the following manner: the solu-
tion to be tested is placed in decreasing quantities in a series of test-tubes and
allowed to act upon an equal quantity of the edestan solution, 2 cc.,and the
minimum of juice determined which is necessary to digest the solution, within
one-half an hour and at room temperature, so that on the addition of solid
NaCl and shaking no precipitate occurs. GROSS 5 suggested a similar method by
using an acid casein solution and precipitating with sodium acetate.

The rapidity of the pepsin digestion depends on several circumstances.
Thus different adds are unequal in their action; hydrochloric acid shows
in slight concentration, 0.8-1.8 p. fm., a more powerful action than any
other acid, whether inorganic or organic. In greater concentration other
acids may have a powerful action; but no constant relation has been
found between the strength of various acids and their action in pepsin
digestion, and the reports of the action of different acids are contradic-

lMett, see Pawlow, I.e.; 28; Nierenstein and Schiff, Berl. klin. Wochenschr., 40;
Jastrowitz, Bioch. Zeitschr., 2.

2 Huppert and Schiitz, Pfluger's Arch., 80; J. Schutz, Zeitschr. f . physiol. Chem.,
30; Spriggs, ibid., 35.

3 Hofmeister's Beitrage, 7.

4 Jacoby, Bioch. Zeitschr., 1; Fuld and Levison, ibid. 6.

5 Berl. klin. Wochenschr., 45.

PEPSIN DIGESTION. 471

tory.1 Sulphuric acid, it seems, has a weaker action than the other
inorganic acids. The degree of acidity is also of the greatest importance.
With hydrochloric acid the degree of acidity is not the same for differ-
ent protein bodies. For fibrin it is 0.8-1 p. m., for myosin, casein, and
vegetable proteins about 1 p. m., for coagulated egg albumin, on the
contrary, about 2.5 p. m. In regard to the dependence of the extent
of transformation upon the quantity of enzyme and the time of diges-
tion we refer to page 58. The kind of protein is of importance, for
example, for besides what was said above in regard to the fibrin, hard-
boiled egg albumin is much easier digested by an acidity of 1-2 p. m.
HC1 than liquid egg albumin, which is rather resistant to the action
of gastric juice. The accumulation of products of digestion has a retard-
ing action on digestion (page 65), although, according to CHITTENDEN
and AMERMAN,2 the removal of the digestion products by means of dialysis
does not essentially change the relation between the proteoses and true
peptones. Pepsin acts more slowly at low temperatures than it does at
higher ones. It is even active in the neighborhood of 0° C., but with
increasing temperature the rapidity of digestion also increases until
about 40° C., when the maximum is reached. If the swelling up of the
protein is prevented, as by the addition of neutral salts, such as NaCl,
in sufficient amounts, or by the addition of bile to the acid liquid,
digestion can be prevented to a greater or less extent. Foreign bodies
of different kinds produce dissimilar effects, in which naturally the
variable quantities in which they are added are of the greatest impor-
tance. Salicylic acid and carbolic acid, and especially sulphates
(PFLEIDERER), retard digestion, while arsenious acid promotes it (CHIT-
TENDEN), and hydrocyanic acid is relatively indifferent. Salts of the
alkali and alkaline earth metals have a strong retarding action in strong
concentration. By experiments with salt solutions so strongly diluted
that the action, on account of the strong dissociation, was brought about
by ions and not by the electrolytically neutral molecules (min. ^ and
max. J normal salt solutions), J. ScnuTz3 found that the anions had a
much greater retarding action upon pepsin digestion than the cations.
Of these latter the sodium cation had the strongest retarding action.
Alcohol in large quantities (10 per cent and above) disturbs the digestion,
while small quantities act indifferently. Metallic salts in very small
quantities may indeed sometimes accelerate digestion, but otherwise

1 See Wr6blewski, Zeitschr. f. physiol. Chem., 21, and especially Pfleiderer, Pfliiger's
Arch., 66, which also gives references to other works; Larin, Biochem. Centralbl., 1,
484; and A. Pick, Wein. Sitzungsber., M. N. Klasse, 112.

2 Journ. of Physiol. 14.

1 Hofmeister's Beitrage, 5.

472 DIGESTION.

they tend to retard it. The action of metallic salts in different cases
can be explained in various ways, but they often seem to form with pro-
teins insoluble or difficultly soluble combinations. The alkaloids may
also retard the pepsin digestion (CHITTENDEN and ALLEN1)- A very
large number of observations have been made in regard to the action
of foreign substances on artificial pepsin digestion, but as these observa-
tions have not given any direct result in regard to the action of the
same substances in natural digestion, as well as upon secretion and
absorption, we will not discuss them here.

The Products of the Digestion of Proteins by Means of Pepsin and Acid.
In the digestion of nucleoproteins or nucleoalbumins an insoluble residue
of nuclein or pseudonuclein always remains, although under certain
circumstances a complete solution may occur. Fibrin also yields an
insoluble residue, which consists, at least in great part, of nuclein,
derived from the form-elements inclosed in the blood-clot. This residue
which remains after the digestion of certain proteins was called dyspep-
tone by MEISSNER. This name is therefore not only unnecessary but
indeed erroneous, as this residue does not consist of bodies related to the
peptones. In the digestion of proteins, substances similar to acid albu-
minates, parapeptone (MEISSNER 2), antialbumate, and antialbumid
(KUHNE), may also be formed. On separating these bodies the filtered
liquid, neutralized at boiling-point, contains proteases and peptones in
the old sense, while the so-called KUHNE true peptone and the other
cleavage products are obtained only after a longer and more intense
digestion. The relation, between the proteoses, changes very much in
different cases and in the digestion of the proteins. For instance, a larger
quantity of primary proteoses is obtained from fibrin than from hard-
boiled egg albumin or from the proteins of meat; and the different
proteins, according to the researches of KLUG,3 yield on pepsin diges-
tion unequal quantities of the various digestive products. In the diges-
tion of unboiled fibrin an intermediate product may be obtained in the
earlier stages of the digestion — a globulin which coagulates at 55° C»
(HASEBROEK 4) . For information in regard to the different proteoses
and peptones which are formed in pepsin digestion see pages 127 to 136.

Action of Pepsin-Hydrochloric Acid on Other Bodies. The gelatin-
forming substances of the connective tissue, of the cartilage, and of the

1 Studies from the Lab. Physiol. Chem. Yale University, 1, 76. See also Chitten-
den and Stewart, ibid. 3, 60.

2 The works of Meissner on pepsin digestion are found in Zeitschr. f. rat. Med., 7,
8, 10, 12, and 14.

3 Pfliiger's Arch., 65.

4 Zeitschr. f. physiol. Chem., 11.

PEPSIN DIGESTION. 473

bones, from which last the acid dissolves only the inorganic substances,
is converted into gelatin by digesting with gastric juice. The gelatin is
further changed so that it loses its property of gelatinizing and is con-
verted into gelatoses and peptone (see page 120). True mucin (from the
submaxillary) is dissolved by the gastric juice, yielding substances similar
to peptone, and a reducing substance similar to that obtained by boil-
ing with a mineral acid. Mucoids from tendons, cartilage, and bones
dissolve, according to POSNER and GiES,1 in pepsin-hydrochloric acid,
but leave a residue which amounts to about 10 per cent of the original
material and which, as it seems, consists in great part, if not entirely,
of a combination of proteid with glucothionic acid (Chapters VI and
VII). The solution contains primary, and secondary mucoproteoses
and mucopeptones. The former contain glucothionic acid, but the latter
do not. Elastin is dissolved more slowly and yields the previously men-
tioned substances (page 117). Keratin and the epidermal formations
are insoluble. The nudeins are dissolved with difficulty, and the cell
nuclei, therefore, remain in great part undissolved in the gastric juice.
According to LONDON 2 and his collaborates the nucleic acids are not
attacked in the stomach. The animal cell-membrane is, as a rule, more
easily dissolved the nearer it stands to elastin, and it dissolves with
greater difficulty the more closely it is related to keratin. The mem-
brane of the plant-cell is not dissolved. Oxyhcemoglobin is changed into
haematin and protein, the latter undergoing further digestion. It is
for this reason that blood is changed into a dark-brown mass in the
stomach. The gastric juice does not act upon }at, but, on the contrary,
dissolves the c/ell-membrane of fatty tissue, setting the fat free. < Gastric
juice has no action on starch or the simple varieties of sugar. The
statements in regard to the ability of gastric juice to invert cane-sugar
are very contradictory. At least this action of the gastric juice is not
constant, and if it is present at all, it is probably due to the action of the
acid.

Pepsin alone, as above stated, has no action on proteins, and an acid of the
intensity of the gastric juice can only very slowly, if at all, dissolve coagulated
albumin at the temperature of the body. Pepsin and acid together not only
act more quickly, but qualitatively they act otherwise than the acid alone, at
least upon dissolved protein. This has led to the assumption of the presence of
a pepsin-hydrochloric acid whose existence and action are only hypothetical.
As pepsin digestion, it seems, yields finally the same products as the hydrolytic
cleavage with acids, we can say for the present only that this enzyme acts like
other catalysts in very powerfully accelerating a process which would also pro-
ceed without the catalvte.

1 Amer. Journ. of Physiol., 11.

8 Zeitschr. f. physiol. Chem. 70, 72.

474 DIGESTION.

Rennin or CHYMOSIN is the enzyme, which is especially character-
ized by the fact that it coagulates milk or casein solutions containing
lime in neutral or indeed faintly alkaline reaction. It must probably
be considered as a proteolytic enzyme. Rennin is habitually found in
the neutral, watery infusion of the fourth stomach of the calf and sheep,
especially in an infusion of the fundus part. In other mammals and in
birds it is seldom found, and in fishes hardly ever in the neutral infusion.
In these cases, as in man and the higher animals, a rennin-forming sub-
tance, a rennin zymogen, occurs, which is converted into rennin by the
action of an acid (HAMMARSTEN). HEDIN has obtained a retarding
solution by treating a neutral infusion of the stomachs of various animals
with dilute ammonia and then neutralizing. These solutions entirely
or partly retard the action of the rennin from the same animal and is
destroyed by acid with the setting free of rennin. HEDIN therefore
considers the rennin zymogen as a combination between rennin and an
inhibitory substance, in which combination the inhibitory body is
destroyed by treatment with acid; consequently the rennin appears in
an active form.

According to BANG the rennin of the human and pig stomachs differs
from that of the calf's stomach in being much more resistant to acids,
more easily destroyed by ^alkalies, and that its action is much more
accelerated by calcium chloride than that from the calf's stomach.1
Active rennin occurs in the human stomach under physiological condi-
tions, but may be absent under special pathological conditions.2

According to the experience of HAMMARSTEN the rennin of the pike
and of the dog differs from that of the calf, and HEDIN 3 finds in the specific
kind of inhibitory action of rennin produced by means of ammonia
treatment as well as by immune serum, a proof that the rennin enzyme
of different kinds of animals differ more or less from • each other. In
regard to this inhibition see pages 62-64.

Enzymes having a rennin action has also been found in the blood
and several organs of higher animals as well as in invertebrates. Sim-
ilar enzymes are also very widely distributed in the plant kingdom and
numerous micro-organisms have the ability to produce rennin.

1 Deutsch. med. Wochenschr., 1899, and Pfluger's Arch., 79.

2 Schumburg, Virchow's Arch., 97. A good review of the literature may be found
in Szydlowski, Beitrage zur Kenntnis des Labenzym nach Beobachtungen an Saug-
lingen, Jahrb. f. Kinderheilkunde (N. F.), 34. See also Lorcher, Pfluger's Arch., 69.
which also contains the pertinent literature. An excellent review of the literature
on rennin and its action may be found in E. Fuld, Ergebnisse der Physiol., 1, Abt. 1,
468.

3 Hammarsten, Upsala Lakaref. forh. 8, 78 (1872). Zeitschr. f. physiol. Chem.
56, 18 (1908), 68, 119 (1910); Hedin, ibid. 72, 187, 74, 242, 76, 355 (1911), 77, 229 (1912).

RENNIN. 475

The law given on page 58 in which the time of coagulation is
inversely proportional to the amount of enzyme, is true for calf rennin
(FuLD x) and for sheep rennin (HEDiN2). The other rennins investi-
gated do not follow this law at 37° C., which, according to VAN DAM, is
due in the case of the pig rennin to its less resistance toward the alkali
of the milk.3

Rennin is just as difficult to prepare in a pure state as the other
enzymes. The purest rennin enzyme thus far obtained did not give the
ordinary protein reactions. On heating its solution rennin is more or
less quickly destroyed, depending upon the length of heating and upon
the concentration. If an active and strong infusion of the gastric mucosa
of the calf's stomach in water containing 3 p. m. HC1 is heated to 40-45°
C. for 48 hours, the rennin or nearly all, is destroyed, while the pepsin
remains. A pepsin solution free from rennin can be obtained in this
way.

A much-discussed question, is, whether the digestion of protein and
the rennet action are brought about by two special enzymes, or represent
two different enzyme actions, or whether there is only one enzyme, the
pepsin, which has both actions. The supporters of this last view dispose
of the question in different ways. Some, like PAWLOW and PARAST-
SCHUK, consider the rennet action simply as the reverse of the synthetical
action of pepsin, a view which is improbable in the highest degree.
Others, such as SAWJALOW4 and GEWIN, consider, on the contrary, that
the coagulation of milk is only a pepsin action and indeed as the first
step in the beginning of proteolysis, namely, the beginning of peptic
digestion of casein. ROKOCZY 5 believes in the presence of two enzymes
in the calf's stomach, one of which, the rennin, disappears on the increas-
ing age of the animal.

The simultaneous occurrence in the animal and plant kingdoms of
enzymes having a proteolytic and rennet action and the parallelism of
the pepsin and rennet action indicates an identity of both enzymes and
enzyme actions. This parallelism in fact does not prove much, because
it has mostly been studied in acid reaction, while rennet is character-
istically active in neutral or faintly alkaline reaction.

At the same time HAMMARSTEN 6 finds that in acid reactions no

1 Hofmeister's Beitrage 2.

2 Not published investigations.

3 Zeitschr. f. physiol. Chem. 64, 316 (1910).

4 The recent literature on this question can be found in Hammarsten, Zeitschr.
f. physiol. Chem., 56, 18 (1909).

5 Ibid. 68, 421 (1910), 73, 453 (1911).

6 Zeitschr. f. physiol. Chem. 68, 119 (1910), which also contains the recent literature.

476 DIGESTION.

parallelism exists in the two enzyme actions with extracts of the dog's
and calf's, stomach, and, also on testing the two enzyme actions upon the
same casein solution no parallelism was present. The pathological cases
in man, if the observations are reliable, where only one enzyme action
occurs, seems to dispute the identity of the action of these two enzymes.
This opposition is also shown by the fact that pepsin, so far as known,
only has a digestive action in the presence of free H ions, while the
coagulation of milk occurs in the absence of these and indeed in the
presence of HO ions. Among other facts which contravene the identity
is the fact that a pepsin solution can be prepared which has a digestive
action but cannot coagulate milk, and the reverse, namely, rennet solu-
tions can be made which coagulate milk but do not have digestive action
in acid reaction (HAMMARSTEN1). The observations of DuccEScm,2
that pepsin but no rennin occurs in the stomach of the Didelphys, also
conflict with the identity of the two enzymes.

The views of NENCKI and SIEBERS take a certain reconciliary posi-
tion. According to them pepsin forms a gigantic molecule which has
various side-chains, one of which has digestive action in acid solution
while the others coagulate milk. This view coincides well with most
of the observations made thus far.

In regard to the formation of pla steins under the influence of rennin
solutions and other enzyme solutions, see Chapters I and II.

Gastric Lipase (STOMACH STEAPSIN). F. VoLHARD4 made the dis-
covery that the gastric juice has a strong fat-splitting action only when
the fat is in a fine emulsion, as in the yolk of the egg, in milk or in cream.
Considerable controversy has arisen in regard to the importance of the
splitting of fat, and the occurrence of a special gastric lipase is indeed
disputed. From numerous observations it follows without question
that in man and many animals a gastric lipase occurs and is secreted
with the gastric juice. Nevertheless the extent of fat splitting in the
stomach is generally not very grqat. In its action this lipase follows
SCHUTZ'S rule and in its other properties it seems to vary in different
animals.

The question whether the cover cells, principally, or the chief cells

1 Zeitschr. f. physiol. Chem., 56.

2 Centralbl., f. Physiol. 22, 784.

3 Zeitschr. f. physiol. Chem., 32.

4Volhard, Munch, med. Wochenschr., 1900, and Zeitschr. f. klin. Med., 42, 43.
See also Stade, Hofmeister's Beitrage, 3; A. Fromme, ibid., 7; A Zinsser, ibid.; H.
Engel., ibid.; and Inouye, Arch. f. Verdauungskrank., 9; Falloise, Arch, internat. d.
Physiol., 3 and 4; London, Zeitschr. f. physiol, Chem., 50; Levites, ibid., 49; Laqueur.
Hofmeister's Beitrage, 8, 281; Heinsheimer, Deutsch. med. Wochenschr., 32, and
Arbeiten aus d. pathol. Institute, Berlin (Hirschwald, 1906).

FORMATION OF HYDROCHLORIC ACID. 477

also, or both, take part in the formation of free acid is disputed.1 There
can be no doubt that the hydrochloric acid of the gastric juice origin-
ates in the chlorides of the blood, because, as is well known, a secretion
of perfectly typical gastric juice takes place in the stomachs of fasting
animals or those which have starved for some time. As the chlorides
of the blood are derived from the food, it is easily understood, as shown
by CAHN,2 that in dogs after a sufficiently long common-salt starva-
tion, the stomach secreted a gastric juice containing pepsin, but no free
hydrochloric acid. On the administration of soluble chlorides, a gastric
juice containing hydrochloric acid was immediately secreted. The
conditions are not so simple, because in the first case not only does the
amount of hydrochloric acid diminish but, as shown by WOHLGEMTJTH
and then by KUDO, the quantity of juice diminishes greatly, and on the
introduction of NaCl the quantity of juice secreted increases. Accord-
ing to PuGLiESE3 the gastric juice in starvation, after a certain time,
has a neutral reaction, and the introduction of NaCl does not now change
its properties. In the secretion of free acid it is assumed by PUGLIESE
that the gland cells, which decompose the chloride, have sufficient
amounts of protein at their disposal. On the introduction of alkali
iodides or bromides, KULZ, NENCKI and SCHOUMOW-SIMANOWSKI 4 have
shown that the hydrochloric acid of the gastric juice is replaced by HBr,
and to a less extent by HI. The secretion of free hydrochloric acid
from the alkaline blood has been explained in various ways, but as yet
no satisfactory theory has been suggested.5

In regard to the -secretion of pepsin we must recall that this last
is not already produced, but is formed from a preliminary step, a pep-
sinogen or propepsin. LANGLEY 6 has positively shown the existence
of such a substance in the mucous coat. This substance, propepsin,
shows a comparatively strong resistance to dilute alkalies (a soda solu-

1 See Heidenhain, Pfluger's Arch., 18 and 19, and Hermann's Handbuch, 5, part I,
" Absonderungsvorgange;" Klemensiewicz, Wien. iSitzungsber,. 71; Frankel, Pfluger's
Arch., 48 and 50; Contejean. 1. c.; Kranenburg, Archives Teyler, Ser. II, Haarlem,
1901; and Mosse, Centralbl. f. Physiol., 17, 217; Fitzgerald, Proc. Roy. Soc. B. 82,
83; L6pez-Suarez, Bioch. Zeitschr. 46, 490 (1912).

2 Zeitschr. f . physiol. Chem., 10.

3Wohlgemuth, Arbeiten aus d. pathol. Institute, Berlin, 1906; Kudo, Bioch.
Zeitschr. 16, 217 (1909), Pugliese, Maly's Jahresb., 36, 394.

4K\ilz, Zeitschr. f. Biologic, 23; Nencki and Schoumow, Arch, des sciences biol.
de St. Petersbourg, 3.

5 Koeppe, Pfluger's Arch., 62; Benrath and Sachs, ibid., 109; Maly, see v. Bunge's
Lehrbuch der physiol. u. pathol. Chem., 4. Aufl., 1898; Schwarz, Hofmeister's Bei-
trage. 5,

6 Schiff, Legons, sur la physiol. de la digestion, 1867, 2; Langley and Edkins, Journ.
of Physiol., 7.

478 DIGESTION.

tion of 5 p. m.) which easily destroy pepsin (LANGLEY). Pepsin, on the
other hand, withstands better than propepsin the action of carbon dioxide,
which quickly destroys the latter. The occurrence of a rennin zymogen
and possibly also of a steapsinogen, in the mucous coat has been men-
tioned above.

The question in what cells the two zymogens, especially the pro-
pepsin, are produced, has been extensively discussed for several years.
Formerly, it was the general opinion that the cover cells were pepsin
cells, but since the investigations of HEIDENHAIN and his pupils, LANGLEY
and others, the formation of pepsin has been attributed to the chief
cells.1

The Pyloric Secretion. That part of the pyloric end of the dog's
stomach which contains no fundus glands was dissected by KLEMENSIE-
wicz, one end being sewed together in the shape of a blind sac and the
other sewed into the stomach. From the fistula thus created he was
able to obtain the pyloric secretion of a living animal, later the secretion
from a pyloric fistula has been obtained in other ways. This secretion
is alkaline, viscous, jelly-like, rich in mucin, of a specific gravity of
1.009-1.010, containing 16.5-20.5 p. m. solids. It habitually con-
tains pepsin, which has been proved by HEIDENHAIN by observations
on a permanent pyloric fistula, and the amount may sometimes be con-
siderable. CONTEJEAN investigated the pyloric secretion in other ways,
and finds that it contains both acid and pepsin. The alkaline reaction
of the secretions investigated by HEIDENHAIN and KLEMENSIEWICZ
is due, according to CONTEJEAN, to an abnormal secretion caused by the
operation, because the stomach readily yields an alkaline juice instead
of an acid one under abnornal conditions. The reports of HEIDENHAIN
and KLEMENSIEWICZ have nevertheless been substantiated by AKER-
MANN, KRESTEFF, SCHEMLAKINE and others.2

The secretion of gastric juice under different conditions may vary
considerably. The statements concerning the quantity of gastric juice
secreted in a certain time are therefore unreliable. ROSEMANN ob-
served, on sham feeding in dogs, a secretion of 917 cc. in the course of
3J hours — a considerable quantity. KUDO 3 found more pepsin in the
secreted juice when the quantity of juice was less.

The Chyme and the Digestion in the Stomach. By means of the
chemical stimulation caused by the food, a copious secretion of gastric

1 See footnote 1, p. 477.

"Heidenhain and Klemensiewicz, 1. c.; Contejean, 1. c., Chapter II, and Skand.
Arch f. Physiol, 6; Akermann, ibid., 5; Kresteff, Maly's Jahresber., 30; Schemia-
kine Arch, des scienc. biolog. de St. Pe"tersbourg, 10.

1 Rosemann, Pfluger's Arch. 118; Kudo, Bioch. Zeitschr. 16.

CHYME AND DIGESTION IN THE STOMACH. 479

juice occurs, which gradually mixes with the swallowed food, and digests
it more or less strongly. The material in the stomach during digestion,
which has a pasty or thick consistency, and is called chyme, is not a
homogeneous mixture of the ingesta with the various digestive fluids,
gastric juice, saliva, and gastric mucus, but the conditions seem to
be more complicated.

From the investigations of several workers,1 on the movements of
the stomach, we conclude that this organ in carnivora and also in man
consists of two physiologically different parts, the pylorus and the
fundus. The greater fundus part, which serves essentially as a reservoir,
may be a rhythmic, strong contraction of the muscle, acting like a
sphincter between it and the pylorus part, be separated from the latter,
and according to some observers so completely so that during contrac-
tion scarcely anything passes from the fundus to the pylorus part.
Differing from the fundus part the pylorus is the seat of very powerful
contractions by which its contents are intimately mixed with gastric
juice and are also driven through the pyloric valve into the intestine.

The contents of the pylorus part have an acid reaction, and a strong
pepsin digestion takes place in the contents, which are thoroughly mixed
with gastric juice. The contents of the fundus, on the contrary, show
a different behavior, for here, as ELLENBERGER first showed, a special
stratification of the various solid food-stuffs takes place.

By very instructive investigations on different animals (frogs, rats,
rabbits, guinea-pigs, and dogs) GRUTZNER2 later showed that when
the aminals are fed with food having different colors, and the stomach
removed after a certain time, and the contents frozen, the frozen sec-
tions show a regular stratification of the contents. These layers are
so arranged that the food first taken is found in direct contact with the
mucosa, while the food taken later is enclosed by that partaken of first,
and this prevents contact with the walls of the stomach. The empty
stomach, whose walls touch each other, is so filled that, as a rule, the
foodstuffs taken later are in the middle of the older food.

Because of this fact only the foodstuffs which lie close to the surface
of the mucous membrane undergo the process of peptic digestion, and
it is principally these ingesta, which lie on the surface and are laden with
pepsin and mixed with gastric juice, which are shoved to the pylorus
end, here mixed and digested, and finally moved into the intestine

1 Hofmeister and Schiitz. Arch. f. exp. Path. u. Pharm., 20; Moritz. Zeitsehr. f.
Biologic, 32; Cannon, Amer. Journ. of Physiol., 1; Schemiakine, 1. c.; Cathcart,
Journ. of Physiol., 1911, 42.

2 See Eilenberger, Pfluger's Arch., 114, and Scheunert, ibid., 144; Griitzner, ibid.,
106.

480 DIGESTION.

The fundus part is therefore less a digestion-organ than a storage-organ,
and in the interior of the same, the food may remain for hours without
coming in contact with a trace of gastric juice.

What has been said above applies at least to solid food. We have
no extensive observations on the behavior of fluids or semifluid food.
According to GRUTZNER, in these cases, as well as in the above-mentioned
experiments, the swallowed foodstuffs are not irregularly mixed together.
Fluids quickly leave the stomach, which is also the case with a mixture
of solid and fluid food.

Milk is an exception because it coagulates and the clot remains in the
stomach while the whey quickly leaves the stomach.

The fact that only that part of the ingesta lying on the mucous
membrane is mixed with gastric juice, while the mass in the interior is
not acid in reaction, is of special importance for the digestion of starches
in the stomach. By this we can explain why the salivary diastase,
although sensitive toward acids, can continue its action for a long time
in the contents of the stomach. That this is true was first found by
ELLENBERGER and HOFMEISTER and then by CANNON and DAY 1 by
special experiments upon animals. The occurrence of sugar and dex-
trin in the contents of the human stomach has been repeatedly observed.
In carnivora, whose saliva shows scarcely any diastatic action, it is
a priori not expected that there should be a diastatic action in the
stomach, but the conditions are different in herbivora, where an abun-
dant digestion of starch takes place in the various stomachs, according
to the different species.

The gastric contents which have been prepared in the pylorus part
are passed through the pylorus into the intestine intermittently. This
material is generally fluid, but it is possible that pieces of solid food
may also occur, and this has often been observed. Thin or plastic
food leaves the stomach earlier than solid food, and it is obvious that the
time in which the stomach unburdens itself depends naturally upon
the coarseness or fineness of the food. This depends essentially upon
the reflex action- of the stomach or intestine, causing an opening or
closing of the pylorus, which action is dependent upon the quantity and
character of the food, the amount of fat, and the degree of acidity in
the contents of the stomach and intestine. The emptying of the food
into the small intestine causes, as shown by PAWLOW, a closing of the
pylorus by chemo-reflex in which the hydrochloric acid and the fat take
part, and we thus find in this regard an alternate action between the
stomach and duodenum.

1 Ellenberger and Hofmeister, Maly's Jahresb., 15 and 16; Cannon an 1 Da/, Amer.
Journ. of Physiol.. 9.

DIGESTION IN THE STOMACH. 481

This alternate action, according to CANNON l is due to the fact that
the acid in the pylorus which acts upon the sphincter and makes pos-
sible the passage of the fluid chyme by the contraction of the muscles
of the stomach. In the intestine the acid has a reverse stimulation
upon the sphincter and causes a contraction of the same. As soon as
the acid is neutralized the contractions of the sphincter cease and the
passage of new portions of the chyme occur. If the flow of bile and pan-
creatic juice is prevented, and the neutralization of the acid contents
of the stomach in the intestine is retarded, then the stomach does not
eject its contents so often. The duration of gastric digestion varies
according to conditions, and in consequence the reports of observers are
widely divergent. BEAUMONT 2 found in his extensive observations
on the Canadian hunter St. MARTIN that the stomach, as a rule, is
emptied 1J-5J hours after a meal, depending upon the character of the
food.

The time in which different foods leave the stomach also depends
upon their digestibility. Respecting the unequal digestibility in the
stomach we must differentiate between the rapidity with which the food-
stuffs are chemically transformed and that with which they leave the
stomach and pass into the intestine. This distinction is especially
important, and it is evident that the main factors governing speed of
digestion and the time required before the food leaves the stomach are the
kind of food and the fineness of its subdivision, and its action upon the
gastric secretion, upon the pyloric reflexes, etc.

The observations of BOLDYREFF and others3 on the action of fats
and fatty acids and not too dilute hydrochloric acid (stronger than 0.2
per cent) are conclusive concerning the manner in which the properties
of the food act upon the gastric secretion and upon the digestion in the
stomach as a whole. Irrespective of the reducing action of the fats
upon the extent and digestive power of the gastric juice BOLDYREFF
found after food very rich in fat that the bile, pancreatic juice and intes-
tinal juice migrate from the intestine into the stomach so that the diges-
tion in the stomach in these cases is essentially brought about by the
pancreatic juice.

We have numerous investigations on the rapidity with which the
food is digested in the stomach of dogs, but we must especially mention

1 Amer. Journ. of Physiol., 20.

2 The Physiology of Digestion, 1833.

3 Boldyreff, Pfluger's Arch., 121, 140; Migay, Maly's Jahresb. 39; Best and
Cohnheim, Zeitschr. f. Physiol. Chem. 69; Cathcart, Journ. of Physiol. 42. See
also Abderhalden and Medigreceanu, Zeitschr. f. physiol. Chem., 57.

482 DIGESTION.

the researches of E. ZuNZ,1 LONDON 2 and his co-workers. LONDON,
POLOWZOWA and SAGELMANN have observed that all the foodstuffs do
not leave the stomach with the same rapidity, indeed, by feeding with
bread (POLOWZOWA), the carbohydrates leave more quickly than the
protein, and with a mixture of gliadin and beef-fat (SAGELMANN) the
protein left the stomach more quickly than the fat. This is in accord
with the recent observations of LONDON and SIVRE which show that the
fats remain longest in the stomach, the starches the shortest and meat
takes a middle position.3 According to these authors the stomach has
a sort of " selective capacity," but this is strongly disputed by SCHEUN-
ERT and GRIMMER.4 Nevertheless the researches of CANNON 5 on cats,
making use of another method, have shown that this is true. After
preliminary hunger the animals received different food, such as meat,
fat, and carbohydrate mixed with bismuth subnitrate and then with
the aid of the RONTGEN rays the time was noted when the food passed
into the intestine. The carbohydrate leaves the stomach first, the
proteins next, and the fats last. If the carbohydrate is given before
the protein food, then it leaves the stomach with ordinary rapidity;
while if protein food and then carbohydrate is given the passage of the
carbohydrate is retarded. A mixture of protein food and carbohydrates
leaves the stomach more slowly than carbohydrates alone, but faster
than protein food alone. The fat, which remains in the stomach for a
long time and leaves the stomach only in amounts which are absorbed
or removed from the duodenum, retards the passage of the protein foods
as well as the carbohydrates. TANGL and ERD^LYI 6 have found in
regard to the different kinds of fat, that a fat leaves the stomach the slower
according to the height of its melting-point. According to LONDON and
SCHWARZ/ with mixed protein feeding, the digestion in the stomach
is regulated by that kind of protein, which, when alone, is removed from
the stomach the slowest.

The reason why different foodstuffs leave the stomach with unequal rapidity
is explained by CANNON by the above-mentioned action of the hydrochloric acid

1 E. Zunz, Hofmeister's Beitrage, 3; Annal de la soc. roy. des scienc. med. Bruxelles
12, 13, and Memoires publ. par 1'Acad. roy. Belg., 1906, 1907, and 1908. Intern.
Beitr. zu. Path. u. Ther. der Ernahrungsstorungen 2; Bull, de TAcad. roy. de med.
de Belgique, 24 (1910).

2 The numerous works of London and co-workers will be found in Zeitschr. f.
physiol. Chem., 45-58, 55-58, 60-74.

3 London with Polowzowa, Zeitschr. f . physiol. Chem., 49, with Sagelmann, ibid.,
52; London and Sivre', ibid. 60 (1909).

4Scheunert, Zeitschr. f. physiol. Chem., 51; Grimmer, Bioch. Zeitschr., 3.

6 Amer. Journ. of Physiol., 12 and 20; Amer. Journ. Med. Sciences, 138, 504 (1909).

•Bioch. Zeitschr. 34, 94 (1911).

'Zeitschr. f. physiol. Chem. 68, (1910).

DIGESTION IX THE STOMACH. 483

upon the pyloric sphincter. The proteins combine with the hydrochloric acid
and hence its action upon the sphincter becomes weaker, while this is not the
case with the carbohydrates. If the carbohydrates are moistened with alkali
they leave the stomach more slowly than usual, and the acid proteins, on the con-
trary, leave the stomach earlier than other proteins.

As our knowledge of the digestibility of the different foods in the
stomach is slight and uncertain, so also our knowledge of the action of
other bodies, such as alcoholic drinks, bitter principles, spices, etc., on
the natural digestion is very uncertain and imperfect. The difficulties
which stand in the way of this kind of investigation are very great, and
therefore the results obtained thus far are often ambiguous or conflict
with each other. For example, certain investigators have observed that
small quantities of alcohol or alcoholic drinks do not prevent but rather
facilitate digestion; others observed only a disturbing action, while
still others report having found that the alcohol first acts somewhat as
a disturbing agent, but afterward, when it is absorbed, produces and abun-
dant secretion of gastric juice, and thereby facilitates digestion. The
accelerating action of alcohol upon the flow of gastric juice has been
mentioned on page 464.

In regard to the importance of the stomach we used to be of the
general opinion that an abundant peptonization of protein does not
occur in the stomach, and that the food rich in protein is only
chiefly prepared in the stomach for the real digestion in the intestine.
That the stomach, at least the fundus, acts in the first place as a storage
chamber, follows from the shape of this organ, especially in certain
animals, and this function becomes especially prominent in certain riew-
born animals, as dogs and cats. In these animals the gastric secretion
contains acid but no pepsin, and the casein of the milk is precipitated
by the acid alone as solid lumps or as a solid coagulum filling the
stomach. Gradually small quantities of this coagulum pass into the
intestine and an overburdening of the intestine is thus prevented. In
other animals, as the snake and certain fishes which swallow entire animals,
the major part of the digestive work goes on in the stomach. The
importance of the stomach for digestion cannot therefore be established
in all instances. It varies in different animals and differs even in indi-
vidual animals of the same species, depending upon the fineness or coarse-
ness of the food, upon the greater or less rapidity with which pepton-
ization takes place, and also upon the rapid or slow increment in the
quantity of hydrochloric acid, etc.

In regard to the extent of chemical digestive work, i.e., in the first
place the destruction of protein in the stomach, we have numerous
researches, some carried out by the use of older methods and others
by using newer and more reliable methods. Among these latter we
must mention those of ZUNZ, LONDON and collaborators, TOBLER, LANG

484 DIGESTION.

and CoHNHEiM.1 These investigations refer to the conditions in dogs,
and as shown by ROSENFELD 2 in horses, and by LOTSCH 3 in pigs, that
the conditions are different in other animals. The following description
applies only to dogs.

In the dog ABDERHALDEN, LONDON and co-workers4 have shown
that in the stomach proteoses and peptones are formed, but no amino-
acids, or at least not in any mentionable quantity. The scanty occurrence
of amino-acids is substantiated by the observations of ZUNZ and others 5
that the amount of amino-nitrogen titratable with formol in the stomach
contents, is only small.

In like manner we must agree in the belief that a part of the protein
always leaves the stomach undigested and that the principal mass, about
80 per cent, passes into the intestine more or less digested. Besides this
it also seems as if the peptones occur in the pylorus part to a greater
extent than the proteoses, while in the fundus part the reverse is .the
case. Of the dissolved protein of the entire stomach-contents about
60 per cent exists as proteoses. Opinions are also contradictory in
regard to the absorption of the decomposition products of the proteins
in the stomach. While several investigators, like TOBLER, LANG, COHN-
HEIM, ZUNZ and others accept such an absorption, LONDON and co-workers
positively deny this.

The digestion of sundry foods is not dependent on one organ alone,
but is divided among several. For this reason it is to be expected that
the various digestive organs can act for one another to a certain extent,
and that therefore the work of the stomach could be taken up more or
less by the intestine. This in fact is the case. Thus the stomachs of
dogs and cats have been completely extirpated or nearly so (CZERNY,
CARVALLO and PACHON, LONDON and collaborators), or that part
necessary in the digestive process has also been eliminated by plugging
the pyloric opening (LUDWIG and OGATA), and in both cases it was pos-
sible to keep the animal alive, well fed, and strong for a shorter or
longer time. The extirpation of the stomach has also been repeatedly

Nobler, Zeitschr. f. physiol. Chem., 45; Lang, Bioch. Zeitschr., 2; Cohnheim,
Munch, med. Wochenschr., 1907. In regard to the works of Zunz, London, and
collaborators, see footnotes 1, 2 and 3, p. 482.

2 Rosenfeld, Ueber die Eiweissverdauung im Magen des Pferdes, Inaug.-Dissert.,
Dresden, 1908.

8 Lotsch. Zur Kenntnis der Verdauung von Fleisch im Magen und Diinndarm des
Schweines, Inaug.-Dissert. Freiburg i. Sa., 1908; see also Abderhalden, Klingemann
and Pappenhusen, Zeitschr. f. physiol. Chem. 71, 411 (1911).

4 Abderhalden and London, with Kautsch, Zeitschr. f. physiol. Chem., 48, with
L. Baumann, ibid., 51, and with v. Korosy, ibid., 51.

6 Intern. Beitr. zu Path. u. Ther. d. Ern.-Stor. 2; London and Rabinowitsch,
Zeitschr. f. physiol. Chem. 74.

DIGESTION IN THE STOMACH. 485

performed on human beings with the same results.1 In these cases it is
evident that the digestive work of the stomach was taken up by the
intestine; but all food cannot be digested in these cases to the same
extent, and the connective tissue of meat especially is sometimes found
to a considerable extent undigested in the excrements.

It is a well-known fact that the contents of the stomach may be
kept without decomposing for some time by means of hydrochloric acid,
while, on the contrary, when the acid is neutralized a fermentation
commences by which lactic acid and other organic acids are formed.
According to COHN, an amount of hydrochloric acid above 0.7 p. m.
completely arrests lactic-acid fermentation, even under otherwise favor-
able circumstances, and according to STRAUSS and BIALOCOUR the limit
of lactic-acid fermentation lies at 1.2 p. m. hydrochloric acid united
to organic bodies. The hydrochloric acid of the gastric juice has unques-
tionably an antifermentative action, and also, like all dilute mineral
acids, an antiseptic action. This action is of importance, as many path-
ogenic micro-organisms may be destroyed by the gastric juice. The
common bacillus of cholera, certain streptococci, etc., are killed by the
gastric juice, while others, especially as spores, are unacted upon. The
fact that gastric juice can diminish or retard the action of certain tox-
albumins, such as tetanotoxine and diphtheria toxine, is also of great
interest (NENCKI, SIEBER, and ScnouMOWA2).

Because of this antifermentative and antitoxic action of gastric juice
it is considered that the principal importance of this juice lies in
its antiseptic action. The fact that intestinal putrefaction is not
increased on the extirpation of the stomach, as derived from experi-
ments made on man and animal,3 does not uphold this view.

Since the hydrochloric acid of the gastric juice prevents the con-
tents of the stomach from fermenting, with the generation of gas, those
gases which occur in the stomach probably depend, at least in great
measure, upon the swallowed air and saliva, and upon those gases gen-
erated in the intestine and returned through the pyloric valve. PLANER
found in the stomach-gases of a dog 66-68 per cent N, 23-33 per cent

1 Czerny, cited from Bunge, Lehrbuch d. physiol. u. path. Chem. 4. Aufl., Theil 2,
173; Carvallo and Pachon, Arch. d. Physiol. (5), 7; Ogata, Arch. f. (Anat. u.) Physiol.
1883; Grohe, Arch. f. exp. Path. u. Pharm. 49; London and collaborators, Zeitschr.
f. physiol. Chem. 74, 328 (1911) ; in regard to the case in man, see Schlatter in
Wr6blewski, Centralbl. f. Physiol. 11, p. 665, and the surgical journals.

2 Cohn, Zeitschr. f. physiol. Chem., 14; Strauss and Bialocour, Zeitschr. f. klin.
Med., 28. See also Kiihne, Lehrb., 57; Bunge, Lehrb. d. Physiol., 4. Aufl., 148 and
159; Hirschfeld, Pfliiger's Arch., 47; Nencki, Sieber, and Schoumowa, Centralbl. f.
Bacteriol., etc., 23. In regard to the action of gastric juice upon pathogenic microbes
we must refer the reader to hand-books of bacteriology.

3 See Carvallo and Pachon, 1. c., and Schlatter in Wr6blewski, 1. c.

486 DIGESTION.

CO2, and only a small quantity, 0.8-6.1 per cent, of oxygen. SCHIER-
BECK 1 has shown that a part of the carbon dioxide is formed by the
mucous membrane of the stomach. The tension of the carbon dioxide
in the stomach corresponds, according to him, to 30-40 mm. Hg in the
fasting condition. It increases after partaking food, independently of
the kind of food, and may rise to 130-140 mm. Hg during digestion.
The curve of the carbon-dioxide tension in the stomach is the same as
the curve of acidity in the different phases of digestion, and SCHIER-
BECK also found that the carbon-dioxide tension is considerably increased
by pilocarpine, but diminished by nicotine. According to him, the
carbon dioxide of the stomach is a product of the activity of the secretory
cells.

After death, if the stomach still contains food, autodigestion goes
on not only in the stomach, but also in the neighboring organs, during
the slow cooling of the body. This leads to the question, Why does the
stomach not digest itself during life? Ever since PAVY has shown that
after tying the smaller blood-vessels of the stomach of dogs the cor-
responding part of the mucous membrane was digested, efforts have
been made to find the cause in the neutralization of the acid of the gas-
tric juice by the alkali of the blood. That the reason for the non-
digestion during life is to be sought for in the normal circulation of the
blood cannot be contradicted; but the reason is not to be found in the
direct neutralization of the acid. The investigations of FERMI and
OTTE 2 show that the blood circulation acts in an indirect manner by the
normal nourishment of the cell protoplasm, and this is the reason why
the digestive fluids, the gastric juice as well as the pancreatic juice, act
differently upon the living protoplasm as compared with the dead. We
know nothing about this resistance of the living protoplasm. Some
claim that it is closely connected with occurrence of different inhibitory
substances in the gastric mucosa. Of these the substance found by
WEINLAND is thermolabile while that of DANILEWSKY, HANSEL and
SCHWARZ is resistant toward heat.3 Without mentioning the still un-
known nature of these bodies, the neutral gastric juice, as well as an
acid infusion of the mucosa, has such a strong digestive action that the
inhibiting action of the mentioned substances can only be shown under
special conditions, and it is therefore difficult to conceive how these sub-
stances could have a protective action in life.

Planer, Wien. Sitzungsber., 42; Schierbeck, Skand. Arch. f. Physiol., 3 and 5.

2Pavy, Phil. Transactions, 153, Part I, and Guy's Hospital Reports, 13; Otte,
Travaux du laboratoire de 1'Institut de Physiol. de Liege, 5, 1896, which also contains
the literature.

3Weinland, Zeitschr. f. Biologic, 44; Hansel, Biochem. Centralbl., 1, p. 404,
arid 2, p. 326; Schwartz, Hofmeister's Beitrage, 6.

EXAMINATION OF THE GASTRIC CONTENTS. 487

Under pathological conditions irregularities in the secretion may
occur. The quantity of enzymes may be diminished and both enzymes
or, as found in certain cases, one (the rennin), may be absent. The
hydrochloric acid may also be absent or may exist in very small amounts.
A pathological high degree of acidity of the pure juice is not very prob-
able, while on the contrary a hypersecretion of gastric juice in different
forms does occur.

In testing the gastric juice or the filtered stomach contents, diluted
with digestive hydrochloric acid, for pepsin, we make use of the pepsin
tests given on pages 469, 470. In testing for rennin the liquid must be
first carefully neutralized, and 1-2 cc. of this liquid added to 10 cc.
milk. In the presence of appreciable quantities of rennin, the milk
should coagulate at room temperature within 10-20 minutes without
changing its reaction. The addition of lime salts is unnecessary, and
may readily lead to erroneous conclusions.

In many cases it is especially important to determine the degree of
acidity of the gastric juice. This may be done by the ordinary titration
methods. Phenolphthalein must not be used as an indicator, as too
high results are produced in the presence of large quantities of proteins.
Good results may be obtained, on the contrary, by using very delicate
litmus paper. Although the acid reaction of the contents of the stomach
may be caused simultaneously by several acids, still the degree of acidity
is here, as in other cases, expressed in only one acid, e.g., HC1. Gen-
erally the acidity is designated by the number of cubic centimeters of
N/10 sodium hydroxide required to neutralize the several acids in
100 cc. of the liquid of the stomach. An acidity of 43 per cent means
that 100 cc. of the liquid of the stomach required 43 cc. of N/10 sodium
hydroxide to neutralize it.

It is also important to be able to ascertain the nature of the acid or
acids occurring in the contents of the stomach. For this purpose, and
especially for the detection of free hydrochloric acid, a great number of color
reactions have been proposed which are all based upon the fact that the
coloring substance gives a characteristic color with very small quanti-
ties of hydrochloric acid, while lactic acid and the other organic acids
do not give these colorations, or only in a certain concentration, which
can -hardly exist in the contents of the stomach. These reagents are a
mixture of FERRIC-ACETATE and POTASSIUM-SULPHOCYANIDE solutions
(MOHR'S reagent has been modified by several investigators), METHYL-
ANILINE-VIOLET, TROP^JOLIN 00, CONGO RED, MALACHITE-GREEN, PHLORO-

GLUCINOL-VANILLIN, DiMETHYLAMiNOAZOBENZENE, and others. As reagents
for free lactic acid, UFFELMANN suggests a strongly diluted, amethyst-blue
solution of FERRIC CHLORIDE and CARBOLIC ACID, or a strongly diluted
nearly colorless solution of FERRIC CHLORIDE. These give a yellow
color with lactic acid, but not with hydrochloric acid or with volatile
fatty acids.

The value of these reagents in testing for free hydrochloric acid or lactic
acid is still disputed. Among the reagents for free hydrochloric acid it seems
STEENSMA'S l modification of GUNZBURG'S test with phloroglucinol-vanillm, and

1 Bioeh. Zeitschrift, 8.

488 DIGESTION.

the test with tropseolin 00, performed at a moderate temperature as suggested by
BOAS, and the test with dimethylaminoazobenzene, which is the most delicate,
seem to be the most valuable. If these tests give positive results, then the presence
of hydrochloric acid may be considered as proved. A negative result does not
eliminate the presence of hydrochloric acid, as the delicacy of these reactions
has a limit, and also the simultaneous presence of protein, peptones, and other
bodies influences the reactions more or less. The reactions for lactic acid may
also give negative results in the presence of comparatively large quantities of
hydrochloric acid in the liquid to be tested. Sugar, sulphocyanides, and other
bodies may act with these reagents like lactic acid.

In testing for lactic acid it is safest to shake the material with ether and test
the residue after the evaporation of the solvent. On the evaporation of the ether
the residue may be tested in several ways. BOAS utilizes the property possessed
by lactic acid of being converted into aldehyde and formic acid on careful oxida-
tion with sulphuric acid and manganese dioxide. The aldehyde is detected by
its forming iodoform with an alkaline iodine solution or by its forming aldehyde-
mercury with NESSLER'S reagent. CRONER and CRONHEIM * have suggested
another method.

The quantitative estimation consists in the formation of iodoform with N/10
iodine solution and caustic potash, adding an excess of hydrochloric acid and
titrating with a N/10 sodium-arsenite solution, and retitrating with iodine solu-
tion, after the addition of starch-paste, until a blue coloration is obtained. This
method presupposes the use of ether entirely free from alcohol. For details see
the original publication and the modification of this method suggested by

JERUSALEM.2

In order to be able to judge correctly of the value of the different
reagents for free hydrochloric acid, it is naturally of greatest importance
to be clear in regard to what we mean by free hydrochloric acid. It is
a well-known fact that hydrochloric acid combines with proteins, and a
considerable part of the hydrocholoric acid may therefore exist in the
contents of the stomach, after a meal rich in proteins, in combination
with them. This hydrochloric acid combined with proteins cannot
be considered as free, and it is for this reason that certain investigators
consider such methods as those of SJOQVIST, which will be described
below, as of little value. However, it must be remarked that, according
to the unanimous experience of many investigators, the hydrochloric
acid combined with proteins is physiologically active and in this regard
we must refer to the recent investigations of ALB. MULLER and J. ScnuTz.3
Those reactions (color reactions) which only respond to actually free
hydrochloric acid do not show the physiologically active hydrochloric
acid. The suggestion of determining the " physiologically active "
hydrochloric acid instead of the " free " seems to be correct in principle;
and as the conceptions of free and of physiologically active hydrochloric
acid are not the same, it must always be well denned whether one wishes
to determine the actually free or the physiologically active hydro-

1 Boas. Deutsch. med. Wochenschr., 1893, and Miinchener med. Wochenschr. 1893,
Croner and Cronheim, Berl. klin. Wochenschr., 1905. See also Thomas, Zeitschr. f.
physiol. Chem., 50.

2 Bioch. Zeitschr., 12.

8 Alb. M'iiller, Deutsch. Arch. f. klin. Med., 88, and Pfliiger's Arch., 116; J. Schiitz.
Wien, klin. Wochenschr., 20, and Wien. med. Wochenschr., 1906 (older literature).

SECRETION OF BRUNNER'S GLANDS. 489

|

chloric acid before any conclusions are drawn as to the value of a certain
reaction.

The acid reaction may be partly due to free acid, partly to acid salts (mono-
phosphates), and partly^to both. According to LEO,* one can test for acid phos-
phates by calcium carbonate, which is not neutralized therewith, while the free
acids are. If the gastric content has a neutral reaction after shaking with cal-
cium carbonate, and the carbon dioxide is driven out by a current of air, it con-
tains only free acid; if it has an acid reaction, acid phosphates are present,
and if it is less acid than before, it contains both free acid and acid phosphate.
It must not be forgotten that a faint acid reaction may, after treatment with cal-
cium carbonate, also be due to the protein. This method can likewise be applied
in the estimation of free acid.

Various titration methods have been suggested for the estimation of the
free hydrochloric acid, but these cannot yield conclusive results for the reasons
given in Chapter I. For this determination physico-chemical methods (page 74),
are necessary, but they have not been used to any great extent for clinical pur-
poses. HOLMGREN 2 has suggested a method for estimating hydrochloric acid
based upon the adsorption phenomenon.

A great number of methods have been suggested for the quantitative estima-
tion of the total acidity, among which we must mention those of K. MORNER and
SJOQVIST, which are extensively used. As the value of a Special determination
of the free and total hydrochloric acid is doubtful, or at least disputed, and also as
the question is chiefly of clinical interest we must refer to the hand-books of clinical
investigations of v. JAKSCH, EULENBURG, KOLLE, and WEINTRAUD and of SAHLI.
The same applies to the tests for volatile fatty acids.

HI. THE GLANDS OF THE MUCOUS MEMBRANE OF THE INTESTINE AND

THEIR SECRETIONS.

The Secretion of Brunner's Glands. These glands are partly con-
sidered as small pancreatic glands and partly as mucous or salivary
glands. Their importance is not the same in all animals. According
to GRUTZNER they are in dogs closely related to the pyloric glands and
contain pepsin. This also coincides with the observations of GLAESSNER
and of PONOMAREW, which differ from each other only in that PONO-
MAREW finds that the secretion is inactive in alkaline reaction and con-
tains only pepsin, while GLAESSNER claims it is active in both acid and
alkaline reaction and that it contains pseudopepsin. According to
ABDERHALDEN and RONA the pure duodenal secretion of the dog contains
a proteolytic enzyme which does not belong to the trypsin type but
rather to the pepsin variety. The statements as to the occurrence of a
diastatic enzyme in BRUNNER'S glands are disputed. SCHETJNERT and
GRIMMER 3 indeed found diastatic enzyme in the duodenal glands of the
horse, ox, pig and rabbit, but no proteolytic or rennin enzyme.

iCentralbl, f. d. med. Wissensch., 1889, p. 481; Pfliiger's Arch., 48, and Berlin,
klin. Wochenschr., 1905, p. 1491.

2 Deutsch. med. Wochenschr., 1911, p. 247.

3Griitzner, Pfliiger's Arch., 12; Glaessner, Hofmeister's Breitrage, 1; Ponomarew,
Biochem. Centralbl., 1, 351; Abderhalden and Rona, Zeitschr. f. physiol. Chern.,
47; Scheunert and Grimmer, cited in Bioch. Centralbl., 5, 673.

490 DIGESTION.

The Secretion of Lieberkuhn's Glands. The secretion of these glands
has been studied with the aid of a fistula in the intestine according to
the method of THIRY and VELLA or of PAWLOW. According to BOLDY-
EEFF,1 in dogs, with an empty stomach, a scanty secretion lasting about
15 minutes occurs at regular intervals for about two hours. According
to BOLDYREFF the intestinal juice is obtained from a THIRY- VILLA fistula
outside of the digestion period without any apparent stimulation. Accord-
ing to this experimenter, during gastric digestion the juice is periodically
but less abundantly secreted as the time interval is much longer, namely
three, four or five hours. Otherwise it is generally admitted that the
partaking of food causes the secretion, or if this is continuous, as in
lambs (PREGL), it increases the secretion. The researches of DELE-
ZENNE and FROUIN show without question that the passage of chyme
into the intestine increases the secretion of the intestinal juice. The
acid causes a formation of secretin (see below), and this produces,
according to the above investigators, a secretion of intestinal juice.
Among the chemically active substances causing a secretion we must
mention acids in general and gastric juice. Soaps, chloral, ether and
on intravenous injection, also intestinal juice or an extract of the intes-
tinal mucosa (FROUIN), are chemical excitants of intestinal juice.
Several salts, NaCl, Na2SOi, and others, may cause an abundant secre-
tion of fluid into the intestine when injected intravenously or subcu-
taneously, as well as after direct application to the peritoneal surface
of the intestine. This action can be arrested by the antagonistic,
inhibiting action of a lime salt (MACCALLUM). Pilocarpine, which has
the power of increasing the activity of secretions, does not increase the
secretion in lambs, and in dogs it does not seem to be always active

(GAMGEE2).

Mechanical irritation of the intestinal mucosa increases the secre-
tion in dogs (THIRY) as well as in man (HAMBURGER and HEKMA), but
it is still doubtful whether we here have a perfectly physiological juice.
In the cases observed by HAMBURGER and HEKMA 3 the flow of fluid was
greatest at night as well as between five and eight o'clock in the after-
noon, and was lowest between two and five o'clock in the afternoon.
The quantity of this secretion in the course of twenty-four hours has
not been exactly determined.

irThiry, Wien, Sitz.-Ber., 50; Vella, Molieschott's Untersuch., 13; Boldyreff,
Zeitschr. f. physiol. Chem., 50, Centralbl. f.'Physiol. 24, 93 (1910).

2 Delezenne and Frouin, Compt. rend. soc. biol., 56; Frouin, ibid., 56 and 58;
MacCallum, University of California Publications, 1, 1904; Gamgee, Physiol. Chem-
istry, 2, 410 (literature).

8 Journ. de Physiol. et d. path, gen., 1902 and 1904.

INTESTINAL JUICE. 491

According to DELEZENNE and FROUIN, if any mechanical irritation
is prevented, the fluid flowing spontaneously from a fistula in a dog
is ten times more abundant in the duodenum than that in the middle
or lower part of the jejunum. In the upper part of the small intestine
of the dog, on the contrary, this secretion is scanty, slimy, and gelatin-
ous; in the lower part it is more fluid, with gelatinous lumps or flakes
(ROHMANN). Intestinal juice has a strong alkaline reaction toward
litmus, generates carbon dioxide on the addition of an acid, and contains
(in dogs) nearly a constant quantity of NaCl and Na2COs, 4.8-5 and 4-5
p. m. respectively (GUMILEWSKI, ROHMANN l). The intestinal juice
of the lamb corresponded to an alkalinity of 4.54 p. m. Na2CO3. It
contains protein (THIRY found 8.01 p. m.), the quantity decreasing
with the duration of the elimination. The quantity of solids varies.
In dogs the quantity of solids is 12.2-24.1 p. m. and in lambs 29.85 p. m.
The specific gravity of the intestinal juice of the dog, according to the
observations of THIRY, is 1.010-1.0107, and in lambs 1.0143 (PREGL).
The intestinal juice from lambs contains 18.097 p. m. protein, 1.274 p.
m. proteoses and mucin, 2.29 p. m. urea, and 3.13 p. m. remaining organic
bodies.

We have the investigations of DEM ANT, TURBY and MANNING, H.
HAMBURGER and HEKMA and NAGANO 2 on the human intestinal juice.
Human intestinal juice has a low specific gravity, nearly 1.007, about
10-14 p. m. solids, and is strongly alkaline toward litmus. The con-
tent of alkali calculated as sodium carbonate is 2.2 p. m., according
to NAGANO, HAMBURGER and HEKMA, and 5.8-6.7 p. m. Na Cl. The
determination of the freezing-point was —0.62° (HAMBURGER and HEKMA).

The intestinal juice of the dog contains, according to BOLDYREFF
a lipase which acts especially upon emulsified fat (milk), and is different
from pancreas lipase, in that its action is not accelerated by bile.
JANSEN 3 found that the lipase was secreted from a THIRY-VELLA fistula
especially under the influence of bile and acid. The intestinal juice of
animals and man also contains an enzyme, erepsin, discovered by O.
COHNHEIM, which does not ordinarily have a splitting action upon native
proteins, but upon proteoses and peptones. It also possibly contains
a nuclease, and it also has a faint amylolytic action. The juice, and to
a high degree the mucous coat, contains in^ertase and maltase, which

1 Gumilewski, Pfluger's Arch., 39. Rohmann, ibid., 41.

2 Demant, Virchow's Arch., 75; Turby and Manning, Centralbl. f. d. med. Wis-
senschaft, 1892, 945; Hamburger and Hekma, 1. c.; Nagano, Mitt, aus d. Grenzgeb.
d. Med. u. Chir., 9.

3 Boldyreff, Archiv. d. sciences biolog, de St. Petersbourg, 11; Jansen, Zeitschr.
f. physiol. Chem.lW, 400 (1910).

492 DIGESTION.

fact has been substantiated by the observations of PASCHUTIN, BROWN
and HERON, BASTIANELLI, and TEBB.1 A lactose-inverting enzyme,
a lactase, also occurs, as shown by ROHMANN and LAPPE, PAUTZ and
VOGEL, WEINLAND, and ORBAN,2 in new-born infants and young ani-
mals, and also in grown mammals which were fed upon a milk diet (see
Chapter I, page 52). The lactase can be obtained more abundantly
from the mucosa than from the juice and according to some occurs only
in the cells. The claims as to the occurrence of a glucoside splitting
enzyme are disputed (FROUIN, OMI 3) .

Besides erepsin and the other enzymes mentioned, the intestinal
mucosa also contains substances which have an inhibitory action upon
pepsin and trypsin. (DANILEWSKY and WEINLAND 4), also enterokinase
or a mother-substance of the same, and finally also the so-called pro-
secretin. These two last-mentioned bodies, which are closely connected
with the secretion of pancreatic juice, will be discussed in connection
with this digestive fluid.

V.&» The various enzymes are not formed in equal quantities in all parts
of the intestine. Diastase and invertase occur, according to BOLDY-
REFF, all through the intestine, while the lipase on the contrary does not
occur in the lower parts. The kinase occurs only in the upper part of
the intestine (BOLDYREFF, BAYLISS and STARLING, DELEZENNE). Ac-
cording to HEKMA the kinase occurs in all parts of the intestine, but
most abundantly in the duodenum and the upper part of the jejunum.
The enzymes, FALLOISE claims, generally occur in greatest abundance
in the upper parts of the intestine; but the erepsin occurs to a greater
extent in the jejunum than in the duodenum. According to the investi-
gations of VERNON the behavior of erepsin is not the same in different
animals. In cats and hedge-hogs the duodenum is richer in erepsin than
the jejunum and ileum; in rabbits it is the reverse, namely, the ileum
is much richer than the duodenum. The secretin, according to BAY-
LISS and STARLING, is formed entirely in the upper part of the intestine.
The epithelium-cells of the glands or the mucous membrane are generally
considered as the • seat of formation of the enzymes, and the same is
true also for the enterokinase, according to BAYLISS and STARLING,

^aschutin, Centralbl. f. d. med. Wissensch., 1870, 561; Brown and Heron, Annal.
d. Chem. u. Pharra., 204; Bastianelli, Moleschott's Untersuch, zur Naturlehre, 14
(this contains all the older literature). See also Miura, Zeitschr. f. Biologic, 32; Wid-
dicombe, Journ. of Physiol, 28; Tebb, ibid., 15.

2 Rohmann and Lappe, Ber. d. deutsch, chem. Gessellch., 28; Pautz and Vogel,
Zeitschr. f. Biologic. 32; Weinland, ibid., 38; Orban, Maly's Jahresber., 29.

3 Frouin and Thomas, Arch, internat. de Physiol., 7; Omi, Das Verhalten- des
Salizins im tierschen Organismus, Inaug.-Dissert. Breslau, 1907.

4 See footnote 3, p. 486.

EREPSIN, 493

HEKMA, FALLOISE, and others, which, however, DELEZENNE says,1 is
formed in the leucocytes and PETER'S glands.

Erepsin. This enzyme, discovered by O. COHNHEIM, has no direct
action upon native proteins with the exception of casein, but has the
power of splitting proteoses, peptones and certain polypeptides. In
this change mono- as well as diamino-acids are produced. Erepsin
occurs in the mucous membrane and in the intestinal juice of man as
well as of dogs; the mucous membrane seems to be richer than the juice
(SALASKIN, KUTSCHER and SEEMANN2). An enzyme like erepsin also
occurs in the pancreas (BAYLISS and STARLING, VERNON), and this has
the power of acting upon casein, but not, or only faintly, upon fresh
fibrin. This erepsin is probably identical with the enzyme nitdease,
discovered by F. SACHS in the pancreas, which acts upon nucleic acids,
while NAKAYAMA claims that erepsin differs from trypsin in having a
cleavage action upon nucleic acids. Intestinal erepsin is not inhibited,
according to GLAESSNER and STAUBER, by blood-serum and differs in
this regard from trypsin. Erepsin shows a great similarity to the intra-
cellular enzymes active in autolysis, and according to VERNON and others
erepsins occur in the various tissues of invertebrates as well as verte-
brates. These tissue erepsins, which are closely related to the auto-
lytic enzymes, if they are not identical with them, behave somewhat
differently from the intestinal erepsin and are not identical therewith.
Enzymes, having an action similar to erepsin, occur, VINES believes,3
in all plants so far investigated.

Erepsin becomes inactive on heating to 59°. It works best in
alkaline solution, but has hardly any action in faint acid reaction. In
this regard, as well as by the fact that only a little ammonia is split off
by its action upon peptone substances, it differentiates itself from cer-
tain of the autolytic enzymes studied so far. The optimum of alkalinity
is, according to EuLER,4 at least in the splitting of a polypeptide, much
lower than the optimum for tryptic digestion.

The secretion of the GLANDS IN THE LARGE INTESTINE seems to con-
sist chiefly of mucus. Fistulas have also been introduced into these

1 Boldyreff, Arch. d. scienc. biolog, de St. Petersbourg, 11; Bayliss and Starling,
Journ. of Physiol., 29, 30; Hekma, 1. c.; Falloise, see Biochem. Centralbl., 4, p. 153;
Vernon, Journ. of Physiol., 33; Delezenne, Compt. rend. soc. biolog., 54 and 56.

2 Cohnheim, Zeitschr. f. Physiol. Chem., 33, 35, 36, and 47; Salaskin, ibid., 35;
Kutscher and Seemann, ibid., 35.

3 Bayliss and Starling, Journ. of Physiol., 30; Vernon, ibid., 30 and 33. See also
Cohnheim and Pletnew, Zeitschr. f. physiol. Chem. 69; F. Sachs, Zeitschr. f. physiol.
Chem., 46; Nakayama, ibid., 41; Glaessner and Stauber, Bioch. Zeitschr. 25; Vines,
Annals of Botany, 18, 19, and 23.

4 Zeitschr. f. physiol. Chem., 51,

494 DIGESTION.

parts of the intestine, which are chiefly, if not entirely, to be considered
as absorption organs. The investigations on the action of this secretion
on nutritive bodies have not as yet yielded any positive results.

IV. THE PANCREAS AND PANCREATIC JUICE.

In invertebrates, which have no pepsin digestion and which also
have no formation of bile, the pancreas, or at least an analogous organ,
seems to be the essential digestive gland. On the contrary, an anatom-
ically characteristic pancreas is absent in certain vertebrates and in
certain fishes. Those functions which should be regulated by this organ
seem to be performed in these animals by the liver, which may be
rightly called the HEPATOPANCREAS. In man and in most vertebrates
the formation of bile, and of certain secretions, containing enzymes
important for digestion, is divided between the two organs, the liver and
the pancreas.

The pancreatic gland is similar in certain respects to the parotid
gland. The secreting elements of the former consist of nucleated cells
whose basis forms a mass rich in proteins, which expands in water and
in which two distinct zones exist. The outer zone is more homogene-
ous, the inner cloudy, due to a quantity of granules. The nucleus lies
about midway between the two zones, but this position may change
with the varying relative size of the two zones. According to HEIDEN-
HAIN 1 the inner part of the cells diminishes in size during the first stages
of digestion, in which the secretion is active, while at the same time
the outer zone enlarges owing to the absorption of new material. In
the later stage, when the secretion has decreased and the absorption
of the nutritive bodies has taken place, the inner zone enlarges at the
expense of the outer, the substance of the latter having been converted
into that of the former. Under physiological conditions the glandular
cells are undergoing a constant change, at one time consuming from the
inner part and at another time growing from the outer part. The
inner granular zone is converted into the secretion, and the outer, more
homogeneous zone, which contains the repairing material, is then con-
verted into the granular substance. The so-called islands of LANGER-
HANS are related to the internal secretion or contain a substance taking
part in the transformation of the sugar of the animal body.2

The chief portion of protein substances contained in the gland con-
sists, it seems, of a protein insoluble in water or neutral salt solution and

1 Pfliiger's Arch., 10.

s See Diamare and Kuliabko, Centralbl. f. Physiol., 18 and 19; Rennie, ibid., 18;
Sauerbeck, Virchow's Arch., 177, Suppl.

PANCREAS AND PANCREATIC JUICE. 495

of nucleoproteins, while the globulin and albumin occur only to a slight
extent as compared with the nucleoproteins. Among the compound
proteins is the substance studied and isolated by UMBER but previously
discovered by HAMMARSTEN 1 and called a-proteid. This nucleopro-
tein contains, as an average, 1.67 per cent P, 1.29 per cent S, 17.12 per
cent N, and 0.13 per cent Fe. It yields, according to HAMMARSTEN,
fl-proteid on boiling, which is much richer in phosphorus than the nucleo-
protein. The native proteid (a) is the mother-substance of guanylic
acid; according to UMBER it dissolves on pepsin digestion without leaving
any residue, and yields on trypsin digestion guanylic acid on one side
and proteoses and peptones on the other. It can be extracted from the
gland by a physiological salt solution, and is precipitated by acetic acid.
Besides this compound protein the pancreas must contain at least one
other protein which is the mother-substance of the thymonucleic acid
obtainable from the pancreas.

Besides these protein substances the gland also contains several
enzymes, or more correctly zymogens, which will be discussed later.
Among the extractive bodies, which are probably in part formed by
post-mortem changes and chemical action, we must mention leucine
tyrosine, purine bases in variable quantities,2 inosite, lactic acid, volatile
fatty acids and fats. The mineral bodies vary considerably in quantity,
not only in animals and man but also in men and women (GOSSMANN).
The calcium seems, according to GOSSMANN, to exist in much greater
amount than the magnesium. According to the investigations of MAG-
NUS-LEVY the human pancreas contains 278 p. m. solids with 106 p. m.
fat and 156 p. m. protein. GOSSMANN 3 found in man 17.92 p. m. ash
and in women 13.05 p. m.

Besides the previously-mentioned (Chapter VII) relation to the trans-
formation of sugar in the aminal body, the pancreas has the property
of secreting a juice especially important in digestion.

Pancreatic Juice. This secretion may be obtained by adjusting a
fistula in the excretory duct, according to the methods suggested by
BERNARD, LUDWIG, and HEIDENHAIN, and perfected by PAWLOW,4

In herbivora, such as rabbits, whose digestion is uninterrupted, the
secretion of the pancreatic juice is continuous. In carnivora, it seems,
on the contrary, to be intermittent and dependent on the digestion.

1 Umber, Zeitschr. f. klin. Med., 40 and 43; Hammarsten, Zeitschr. f. physiol.
Chem., 19.

2 See Kossel, Zeitschr. f. physiol. Chem., 8.

3 Magnus-Levy, Bioch Zeitschr. 24; Gossmann, Maly's Jahresb. 30.

4 Bernard, Lemons de Physiol., 2, 190; Ludwig, see Bernstein, Arbeiten, ad. physiol.
Anstalt zu Leipzig, 1869; Heidenhain, Pfliiger's Arch., 10, 604; Pawlow, The Work
of the Digestive Glands, (translated by Thompson, Philadelphia, 1910), and Ergebnisse
der Physiologie, 1, Abt. 1.

496 DIGESTION.

During starvation the secretion almost stops, but commences again
after partaking of food and reaches its maximum, it is claimed by
BERNSTEIN, HEIDENHAIN, and others, within the first three hours.

PAWLOW and his pupils, especially SCHEPOWALNIKOFF, have shown
that the above-mentioned (page 492) enterokinase activates the trypsino-
gen into trypsin. These observations were later confirmed by others,
by DELEZENNE and FROTJIN, POPIELSKI, CAMUS and GLEY, BAYLISS and
STARLING, ZUNZ, and have been further studied. The pure juice con-
tains, at least as a rule, only trypsinogen, and no trypsin. By mixing
with the intestinal juice, or by contact with the intestinal mucosa, the
trypsinogen is converted into trypsin by the kinase. Enterokinase,
which itself has no action upon proteins, and therefore is not a pro-
teolytic enzyme, is not well known. It is made inactive by heating and
is therefore considered by many (including PAWLOW) as an enzyme.
Others, on the contrary, like HAMBURGER and HEKMA, DASTRE and
STASSANO, deny the enzyme nature of enterokinase because they find
that a certain quantity of intestinal juice will activate only a certain
quantity of trypsin. Enterokinase has been found in man and all
mammals investigated. According to most investigators it is formed
in the glands or the cells of the intestinal mucosa, while according to
DELEZENNE it comes from PETER'S patches and from the lymph-glands
and leucocytes, hence impure fibrin containing leucocytes acts as a kin-
ase. These deductions of DELEZENNE are disputed by BAYLISS and
STARLING, HEKMA and others.

If we accept the view that the juice secreted after partaking food
is regularly free from trypsin, still under other circumstances the juice
may contain trypsin. Thus, according to CAMUS and GLEY, the juice
secreted under the influence of secretin (see below) is not always free
from trypsin, and ZUNZ found that WITTE'S peptone or pilocarpine
causes a secretion of juice which often contained trypsin and was directly
active. According to CAMUS and GLEY not only does an exterior activa-
tion of the trypsinogen in the juice take place, but also in the interior
of the gland. An auto-activation of the juice in certain cases is also
accepted by others (SAwrrscH1).

The activation of the trypsinogen into trypsin may, in life, be brought about
— as the researches of HERZEN, which have been substantiated by GACHET and
PACHON, BELLAMY, MENDEL and RETTGER, have shown — not only in the intestine,
but also in the gland itself. This activation of the trypsinogen in the gland itself
is caused in a still undiscovered manner by a body of unknown nature formed in the
spleen, which is congested during digestion. Such a " charging " of the pancreas

1 Camus and Gley, Journ. de Physiol. et de Pathol, gen., 1907; Zunz, Recherches
sur 1'activation de sac pancreatique par les Sels., Bruxelles, 1907; Sawitsch, Zentralbl.
f. d. ges. Physiol. u. Path, des Stoffwechsels, 1909.

TRYPSINOGEN. 497

by the spleen has been repeatedly suggested by SCHIFF/ but this has recently
been denied by PRYM. According to this experimenter the extirpation of the
spleen causes no change in the properties of the pancreatic juice, and the intra-
venous injection of spleen infusion is also without action on a splenectomized
dog with permanent pancreatic fistula. The observations of HERZEN that a
spleen infusion has a strong activating action upon a weak pancreas . infusion
were substantiated by PRYM,Z but he claims that this is due essentially to micro-
organsims. Besides this the spleen itself contains proteolytic enzymes (page
371).

The conversion of the trypsinogen into trypsin in the removed gland
or in an infusion under the influence of air and water and also by other
bodies has been known for a long time. According to VERNON the tryp-
sin itself has a strong activating action upon trypsinogen, and in this
regard it is more active than enterokinase. The correctness of this
statement is still denied by BAYLISS and STARLING and by HEKMA. The
ordinary view of HEIDENHAIN, that the transformation of trypsinogen
into trypsin is also brought about by acids, has been found to be incor-
rect by HEKMA.S Besides the enterokinase and the micro-organisms,
there are other activators of the trypsinogen. As first shown by
DELEZENNE and then by ZUNZ, by further investigations the lime salts
have a special power in activating trypsinogen.4 These last do not act
immediately, but only after some time, for example, a couple of hours,
and then they activate suddenly. The lime salts are not necessary
for the digestive action of the juice, and when the activation has once
taken place, they can be removed without any harm. They probably
have a similar action as in the coagulation of the blood. According
to DELEZENNE the lime salts have the same importance in the activa-
tion of the rennin-zymogen of the juice as in the activation of the
trypsinogen. This enzyme is also activated by enterokinase. The
erepsin of the pancreatic juice (page 493) occurs as an active enzyme.

We are not quite clear whether the two other enzymes, the diastase
and lipase, are secreted as such or as zymogens. It seems, nevertheless,
that both are in part secreted as complete enzymes.

In the human embryo the trypsinogen and the erepsin (as well as also the
pepsin) appear in the fourth and fifth foetal month. The enterokinase appears
at the same time or shortly after the trypsinogen.5

1 Bellamy, Journ. of Physiol., 27; Mendel and Rettger, Amer. Journ. of Physiol., 7.
A very complete reference to the literature may be found in Menia Besbokaia Du
rapport fonctionell entre le pankreas et la rate, Lausanne, 1901.

2 Pfliiger's Arch., 104., and 107.

3 Vernon, Journ. of Physiol., 28; Hekma, Kon. Akad. v. Wetenschappen te
Amsterdam, 1903, and Arch. f. (Anat. u) Physiol., 1904; Bayliss and Starling, Journ.
of Physiol., 30

4 Delezenne, Compt. rend. soc. biol., 59, 60, 62, 63; Zunz, footnote 1, p. 496.
6 Ibrahim., Bioch. Zeitschr. 22, 24 (1909).

498 DIGESTION.

The way in which the trypsinogen is converted into trypsin is still
unknown and is the subject of dispute. According to one view, proposed
by PAWLOW and defended by BAYLISS and STARLING, the trypsinogen
is transformed into trypsin by the action of the kinase. In the opinion
of DELEZENNE, DASTRE, and STASSANO, and others,1 the trypsin, on the
contrary, is a combination of the kinase and trypsinogen, analogous to
the cytotoxines, which, according to EHRLICH'S side-chain theory, are
combinations between a complement and an amboceptor. (See page
69.)

The specific excitants for the secretion of pancreatic juice are,
according to PAWLOW and his collaborators, acids of various kinds —
hydrochloric acid as well as lactic acid — and fats, the latter acting
probably by virtue of the soaps produced therefrom. Alkalies and
alkali carbonates have, on the contrary, a retarding action. It appears
that the acids -act by irritating the mucosa of the duodenum. Accord-
ing to LONDON and SCHWARZ the secretion can also be excited from the
entire jejunum and the upper part of the ileum. The secretion becomes
weaker the further away the exciting source is from the duodenum.2
Water, which causes a secretion of acid gastric juice, likewise becomes,
indirectly, a stimulant for the pancreatic secretion, but may also be
an independent exciter. The psychical moment may, at least in the
first place, have an indirect action (secretion of acid gastric juice),
and the food seems otherwise to have an action on pancreatic secretion
by its action on the secretion of gastric juice.

The most important excitant for the secretion of juice is hydrochloric
acid, but opinions are not in unison as to the manner in which the acid
acts. PAWLOW'S school claims that the acid acts reflexly upon the
intestine, causing a secretion of juice. That a reflex action is here pro-
duced is not contradicted by the investigations of POPIELSKI, WERT-
HEIMER and LEPAGE, FLEIG,S and others. According to the researches
of BAYLISS and STARLING, which have been confirmed by CAMUS, GLEY,
FLEIG, HERZEN, DELEZENNE, and others, a second factor must also be
active here. BAYLISS and STARLING have shown that a body which
they have called secretin can be extracted from the intestinal mucosa
by a hydrochloric-acid solution of 4 p. m., and this when introduced into
the blood produces a secretion of pancreatic juice, bile, and in the
opinion of some investigators also of saliva and intestinal juice. The

1 Bayliss and Starling, Journ. of Physiol., 30 and 32, which also cities the other
investigators and also O. Cohnheim, Bioch. Centralbl. 1, 169 and S. Rosenberg, ibid.,
2, 708.

2 Zeitschr. f. physiol. Chem. 68, 346 (1910) which also contains the literature.

3 Fleig, Centralbl. f. Physiol., 16, 681, and Compt. rend. soc. biol., 55. See also
footnote 1.

SECRETION OF PANCREATIC JUICE. 499

secretin, which according to BAYLISS and STARLING/ is the same in all
vertebrates examined, is not destroyed by heat; it is therefore not
identical with etiterokinase, and is not considered an enzyme. It is
formed from another substance, prosecretin, by the action of acids.
According to DELEZENNE and POZERSKI secretin occurs as such in the
intestinal mucosa, and the acids act only by the elimination of certain
bodies having a retarding action. According to POPIELSKI secretin
action is different from acid action; and the secretin action can also be
obtained by WITTE'S peptone. He believes that the secretin is not
a specific constituent of the intestine but a body widely distributed.
GIZELT disputes the occurrence of a specific secretin and he compares
this body to peptone. GLEY has obtained a solution which had a stronger
secreting action than secretin by macerating the mucosa with proteoses.2
v. FURTH and SCHWARZS also call attention to the uncertainty of our
knowledge as to the nature of secretin. According to them secretin
is probably a mixture of bodies, among which probably the choline, found
by them in the intestinal walls, acts the role of an exciter of secretion.

A second means of causing secretion is the fat, which probably only
acts after it has been saponified. Oil-soap directly introduced into the
duodenum brings about a strong secretion of pancreatic juice (SAWITSCH,
BABKiN4), and at the same time a flow of bile, gastric juice, and the
secretion of BRUNNER'S glands occurs. The pancreatic juice secreted
under these circumstances has about the same amount of enzymes as
the juice secreted after partaking of food.

We know very little as to how the soaps act. FLEIG 5 found that by macera-
tion of the mucosa of the upper part of the duodenum with soap solution, a sub-
stance goes into solution which he calls sapoknnin, and which when introduced
into the blood brings about a strong secretion of pancreatic juice. This sapok-
rinin, which is derived from a prosapokrinin. is not an enzyme and is not identical
with secretin. After the action of chloral hydrate an abundant secretion occurs
in the duodenum (WERTHEIMER and LEPAGE), which FALLOISE considers as pro-
duced by a special secretin, chloral secretin. The secretion of pancreatic juice
can also be increased by alcohol, and FLEIG 6 claims to have obtained a secretin,
ethyl secretin, by macerating the intestinal mucosa with alcohol. Further investiga-
tions are necessary of all these so-called secretins.

1 Journ. of Physiol., 29.

2Delezenne and Pozerski, Compt. rend. soc. biol., 56; Popielski, , Centralbl. f.
Physiol., 19; Pfliiger's Arch. 128; Gizelt, Pfliiger's Arch. 123; Gley, Compt. Rend. 151,
345.

3 v. Fiirth and Schwarz, Pfliiger's Arch. 124 (literature on secretin).

4 Arch des scienc. biol. de St. Petersboiirg, 11, and Zeitschr. f. physiol. Chem., 56.
6 Compt. rend. soc. biol., 55, and Joufn. de Physiol, et de Pathol. gen., 1904.

6 Wertheimer and Lepage, Compt. rend. soc. biol., 52; Fleig, ibid., 55; Falloise,
Bull. Acad. Roy. Belg., 1903.

500 DIGESTION.

The estimation as to the quantity of pancreatic juice secreted in the
twenty-four hours differs very much. According to the determina-
tions of PAWLOW and his collaborators, KUWSCHINSKI, WASSILIEW, and
JABLONSKY,1 the average quantity (with normally acting juice) from a
permanent fistula in dogs is 21.8 cc. per kilo in the twenty-four hours.

The pancreatic juice of the dog is a clear, colorless, and odorless
alkaline fluid which when obtained from a temporary fistula is very
rich in proteins, sometimes so rich that it coagulates like the white of
the egg on heating. Besides proteins, the juice also contains the three
above-mentioned enzymes (or their zymogens), amylopsin, perhaps also
maltase, trypsin, steapsin, also an enzyme similar to erepsin, and besides
these a rennin, which was first observed by KTJHNE. Besides the above-
mentioned bodies the pancreatic juice invariably contains small quan-
tities of leucine, fat, and soaps. As mineral constituents it contains
chiefly alkali chlorides and considerable alkali carbonate, some phos-
phoric acid, lime, magnesia, and iron.

The quantity of solids in the pancreatic juice of the dog varies, as
found by MAZURKIEWICZ, BABKINE and SAWixscn,2 according to the
rapidity of secretion and the kind of excitant. As a rule the amount
of solids is in inverse proportion to the rapidity of secretion. The juice
secreted after the action of acids has the lowest amount of solids, 9-37.4
p. m. The juice after taking food is more concentrated, about 60-70
p. m. and that after vagus stimulation often contains 90 p. m. solids.
The juice analyzed by C. SCHMIDT 3 from a temporary fistula contained
99-116 p. m. solids. The quantity of mineral bodies was 8.8 p. m.

The mineral constituents consisted chiefly of NaCl, 7.4 p. m., which is remark-
able because the juice contains such a large amount of alkali carbonate. In the
juice examined by DE ZILWA 4 the quantity of alkali in the secretin juice was
5-7.9 p. m. and in the pilocarpin juice 2.9 -5.3 p. m. Na2C03.

In the pancreatic juice of rabbits 11-26 p. m. solids have been found, and
in that from sheep 14.3-36.9 p. m. In the pancreatic juice of the horse 9-15.5
p. m. solids have been found; in that of the pigeon, 12-14 p. m.

The human physiological pancreatic secretion from a fistula has been
investigated by GLAESSNER.5 The secretion was clear, foamed readily,

1 Arch, des sciences de St. Petersbourg, 2, 391. The previous claims of Bidder and
Schmidt, and others may be found in Ktihne, Lehrbuch, 114.

2 Mazurkiewicz, 1. c.; Babkin and Sawitsch, Zeitschr, f. physiol. Chem., 56.
8 Cited from Maly in Hermann's Handbuch der Physiol., 5, Theil II, 189.

4 Journ. of Physiol., 31.

6 Zeitschr. f. physiol. Chem., 40. See also Ellinger and Kohn, ibid., 45, and the
investigations upon cystic fluids from the pancreas by Schumm, ibid., 86, and Murray
and Gies, American Medicine, 4, 1902; Glaessner and Popper, Deutsch. Arch. f. klin.
Med. 94, 46; see also Wohlgemuth, Bioch. Zeitschr. 39; Bradley, Journ. of Biol.
Chem. 6.

AMYLOPSIN. STEAPSIN. 501

had a strong alkaline reaction even toward phenolphthalein, and con-
tained globulin and albumin but no proteoses and peptones. The specific
gravity was 1.0075 and the freezing-point depression was A =—0.46-
0.49°. The solids were 12.44-12.71 p. m., the total protein 1.28-1.74
p. m., and the mineral bodies 5.66-6.98 p. m. The secretion contained
trypsinogen, which was activated by the intestinal juice. Diastase and
lipase were present; inverting enzymes, on the contrary, were not. The
daily quantity of juice was 500-800 cc. The quantity of secretion, of
ferments, and of alkalinity was lowest in starvation, but soon rose with
the taking of food, and reached its maximum in about four hours.

Amylopsin, or pancreatic diastase, which, according to KOKOWIN
and ZWEIFEL, is not found in new-born infants and does not appear
until more than one month after birth, seems, although not identical
with ptyalin, to be closely related to it. Amylopsin acts very energetic-
ally upon boiled starch, and according to KUHNE also upon unboiled
starch, especially at 37 to 40° C., and according to VERNON 1 best at
35° C. It forms, similarly to the action of saliva, besides dextrin, chiefly
isomaltose and maltose, with only very little glucose (MUSCULUS and
v. MERINO, KULZ and VoGEL2). The glucose is probably formed by the
action of the invertin existing in the gland and juice. The pancreatic
juice of the dog in fact, contains, according to BIERRY and TERROINE,S
maltase, its action becomes apparent only after very faint acidification
of the juice. According to RACHFORD the action of the amylopsin is
not reduced by very small quantities of hydrochloric acid, but is dimin-
ished by larger amounts. VERNON, GRUTZNER, and WACHSMANN find
that the action is indeed accelerated by very small quantities of hydro-
chloric acid, 0.045 p. m., while alkalies in very small amounts have a
retarding action. This retarding action of alkalies and hydrochloric
acid may be stopped by bile (RACHFORD). WOHLGEMUTH as well as
Minami4 find that the action of diastase is increased to a high degree
by bile. The active constituent of the bile was soluble in water and
alcohol but was not identical with the bile salts or cholesterin. The
statements in regard to the action of lecithin are contradictory.

Steapsin, or Fat-splitting Enzyme. The action of the pancreatic
juice on fats is twofold. First, the neutral fats are split into fatty acids

1 Korowin, Maly's Jahresber., 3; Zweifel, footnote 2, p. 456, Kiihne, Lehrbuch,
117; Vernon, Journ. of Physiol., 27.

2 See footnote 5, p. 456.

3 See Tebb. Journ. of Physiol., 15; Bierry and Terroine, Compt. rend. soc. biolog.,
58; Bierry, ibid., 62.

4Rachford, Amer. Journ. of Physiol., 2; Vernon. 1. c.; Griitzner, Pfliiger's Arch.
91; Wohlgemuth, Bioch. Zeitschr. 21, 447 (1909); Minami, ibid., 39, 339 (1912).

502 DIGESTION.

and glycerin, which is an enzymotic process; and secondly, it has also
the property of emulsifying the fats.

The action of the pancreatic juice in splitting the fats may be shown
in the following way: Shake olive-oil with caustic soda and ether,
siphon off the ether and filter if necessary, then shake the ether repeatedly
with water and evaporate at a gentle heat. In this way is obtained a
residue of fat free from fatty acids, which is neutral and which dissolves
in acid-free alcohol and is not colored red by alkanet tincture. If such
fat is mixed with perfectly fresh alkaline pancreatic juice or with a freshly
prepared infusion of the fresh gland and treated with a little alkali or
with a faintly alkaline glycerin extract of the fresh gland (9 parts glyc-
erin and 1 part 1 per cent soda solution for each gram of the gland),
and some litmus tincture added and the mixture warmed to 37° C.,
the alkaline reaction will gradually disappear and an acid one take its
place. This acid reaction depends upon the conversion of the neutral
fats by the enzyme into glycerin and free fatty acids. A very much
used method consists in determining the acidity of the mixture by means
of titration before and after the action of the juice or the infusion.

The action of the pancreatic juice in splitting fats is a process analo-
gous to that of saponification, the neutral fats being decomposed, by
the addition of the elements of water into fatty acids and glycerin
according to the following equation. CaHs.Oa.Rs (neutral fat)+3H2O =
C3H5.03.H3 (glycerin) +3 (H.O.R) (fatty acid). This depends upon
a hydrolytic splitting, which was first positively proved by BERNARD
and BERTHELOT. The pancreas enzyme also decomposes other esters,
just as it does the neutral fats (NENCKI, BAAS, LOEVENHART 1 and
others). The fat-splitting action of the lipase is, according to PAW-
LOW, BRUNO and many others 2 aided in its action by the bile. ROSEN-
HEIM and SHAW-MACKENZIE found that the lipase action was accel-
erated by hsemolytic substances, as well as by normal serum; this
accelerating action was inhibited by cholesterin. The accelerating
substance of the serum was dialyzable and resistant to heat. ROSEN-
HEIM was able to. divide the lipase existing in a glycerin extract of the
pig pancreas into enzyme and co-enzyme (page 52); in diluting with
water a precipitate occurred which contained the real thermolabile
enzyme while the dialyzable, heat resisting co-enzyme remained in the

1 Bernard, Ann. de chim. et physique (3), 25; Berthelot, Jahresber, d. Chem.,
1855, 733; Nencki, Arch. f. exp. Path. u. Pharm., 20; Baas, Zeitschr. f. physiol. Chem.,
14, 416; Loevenhart, Journ. of Biol. Chem., 2; Terroine and Z. Morel, Compt. rend,
soc. biol., 65, 66.

2 Bruno, Arch, des scienc. biol. de St. Petersbourg, 7; see also Loevenhart and
Souder, Journ. of biol. Chem., 2; v. Fiirth and Schiitz, Hofmeister's Beitrage,9; Ter-
roine, Bioch. Zeitschr. 23; Compt. rend. soc. biol. 68, 439, 518, 666, 754 (1910).

TRYPSIN. 503

filtrate.1 In regard to the synthetic action of pancreatic lipase see
page 60.

The fatty acids which are split off by the action of the pancreatic
juice combine in the intestine with the alkalies, forming soaps, which
have a strong emulsifying action on the fats, and thus the pancreatic
juice becomes of great importance in the emulsification and the absorp-
tion of the fats.

Trypsin. The action of the pancreatic juice in digesting proteins
was first observed by BERNARD, but first proved by CoRViSART.2 It
depends upon a special enzyme called, by KUHNE, trypsin. This enzyme
as previously explained, does not occur in the gland as such, but as
trypsinogen. According to ALBERTONIS this zymogen is found in
the gland in the last third of the intra-uterine life. Enzymes more or
less like trypsin occur in other organs, and are very widely diffused in
the vegetable kingdom,4 in yeast and in higher plants, and are also formed
by various bacteria. The enzymes similar to trypsin occurring in the
plant kingdom are, according to VINES, a mixture of peptases, which
transform the proteins into peptone, and ereptases, which split the pep-
tones into amino-acids.

As we know of so-called antienzymes for other enzymes, so we also have anti-
trypsins, and not only in the intestinal canal but also in the blood-serum (see page
63). The results as to the possibility of producing antitrypsins by immuniza-
tion, is still disputed.

Trypsin, like other enzymes, has not been prepared in a pure con-
dition. Nothing is positively known in regard to its nature, but as
obtained thus far it shows a variable behavior (KUHNE, KLUG, LEVENE,
MAYS, and others). At least it does not seem to be a nucleoprotein, and
trypsin has also been obtained which did not give the biuret test (KLUG,
MAYS, SCHWARZSCHILD). Trypsin dissolves in water and glycerin, while
KUHNE 's trypsin was insoluble in glycerin. It is very sensitive to heat,
and even the body temperature gradually decomposes it (VERNON, MAYS).
In neutral solution it becomes inactive at 45° C. In dilute soda solu-
tion of 3-5 p. m. it is still more readily destroyed (BIERNACKI, VERNON 5).

» Journ. of Physiol. 40 (1910).

2 Gaz. hebdomadaire, 1857, Nos. 15, 16, 19, cited from Bunge, Lehrbuch, 4, Aufl.,
185.

3 See Maly's Jahresber.,'8, 254.

4 In this connection see Vines, Annals of Botany, 16, 17, 18, 19, 22, and 23, and
Oppenheimer, Die Fermente, 1910.

5Kuhne, Verb. d. naturh.-med. Vereins zu Heidelberg (N. F.), 1, 3; Klug, Math,
naturw. Ber. aus Ungarn., 18, 1902; Levene, Amer. Journ. of Physiol., 5; Mays,
Zeitschr. f. Physiol. Chem., 38; Vernon, Journ. of Physiol., 28 and 29; Biernacki,
Zeitschr. f. Biologic, 28; Schwarzschild, Hofmeister's Beitrage, 4.

504 DIGESTION.

The presence of protein or proteoses has, to a certain extent, a pro-
tective action on heating an alkaline trypsin solution, and this has
been substantiated by recent investigations of BAYLISS and VERNON.
The simpler cleavage products have a still greater protective action
(VERNON1). Trypsinogen, according to the unanimous statements
of several experimenters, is more resistant toward alkalies than trypsin.
Trypsin is gradually destroyed by gastric juice and even by digestive
hydrochloric acid alone.

The preparation of pure trypsin has been tried by various experimen-
ters. The most careful work in this direction was done by KUHNE and
MAYS. Various methods have been suggested by MAYS, but we cannot
enter into a discussion of them. A very pure preparation can be obtained
by making use of the combined salting out with NaCl and MgSCU. A
very active solution, and one that can be kept for a long time (for more
than twenty years according to HAMMARSTEN), can be obtained by extract-
ing with glycerin (HEIDENHAIN 2) . An impure but still very active
infusion can be obtained after a few days by allowing the finely divided
gland to stand with water which contains 5-10 cc. chloroform per liter
(SALKOWSKI) at the temperature of the room. Such infusions can be
obtained, nearly free from proteins, by dialyzing with running water after
the addition of toluene.

Like other enzymes, trypsin is characterized by its action, and this
action consists in dissolving protein and in splitting it into simpler prod-
ucts, mono- and diamino-acids, tryptophane, etc., in alkaline, neutral,
and indeed in very faintly acid solutions. This action has been so far
considered as characteristic for trypsin. Recent investigations seem
to indicate that this action is not due to one enzyme alone, but to the
combined action of several enzymes.

Although contrary to MAY'S statement, there is no question that
there occurs in the pancreas besides trypsin, an enzyme similar to erepsin
(BAYLISS and STARLING, VERNON 3). According to the latter this
erepsin has a strong action upon peptone, and he believes that the pep-
tone-splitting action of a pancreas infusion is in great part due to the
erepsin. The pancreas, besides these, also contains a nuclease (see page
493), whose relation to pancreas erepsin has not been determined.

The unity of trypsin has also been disputed from another point of view.
According to POLLAK the trypsin (in the ordinary sense) contains a second
enzyme, which does not act upon protein, but only upon gelatin, and he calls

1 Bayliss, Arch, des scienc. biolog. de St. Petersbourg. 11, Suppl.; Vernon, Journ,
of Physiol., 31.

2 Pfliiger's Arch., 10.

3 Bayliss and Starling, Journ. of Physiol., 30; Vernon, ibid., 30; and Zeitschr. f.
physiol. Chem., 50; Mays, ibid., 49 and 51.

ACTION OF TRYPSIN. 505

this enzyme glutinase. This glutinase is much more resistant toward acids
than trypsin, and by proper treatment with acids POLLAK was able to change a
pancreas infusion so that it acted upon gelatin and not upon certain proteins.
The correctness of these observations has, indeed, not been generally accepted,
and it is disputed by ASCOLI and NEPPi.1 According to them the action of the
trypsin is weakened by the acid, and indeed to such varying degrees for differ-
ent proteins that the action upon albumin is lost while the action upon gelatin
is noticeable. Nevertheless, we here have a warning to be careful as to the
conclusions drawn from results where impure infusions are used. For many
experiments it is undoubtedly advisable to use the natural pancreatic juice.

The following reports on the action of trypsin applies to the so-
called trypsin, with the reservation that it is perhaps not a unit enzyme.

The action of trypsin on proteins is best demonstrated by the use of
fibrin. Very considerable quantities of this protein body are dissolved
by a small amount of trypsin at 37-40° C. It is always necessary to
make a control test with fibrin alone, with or without the addition of
alkali. Fibrin is dissolved by trypsin without any putrefaction; the
liquid has a pleasant odor somewhat like bouillon. To completely
exclude putrefaction a little thymol, chloroform, or toluene should be
added to the liquid. Tryptic digestion differs essentially from peptic
digestion, irrespective of the difference in the digestive products, in that
the first takes place in neutral or alkaline reaction and not, as is neces-
sary for peptic digestion, in an acidity of 1-2 p. m. HC1, and further
by the fact that the proteins dissolve in trypsin digestion without pre-
viously swelling up.

As trypsin not only dissolves proteids, but also other protein sub-
stances such as gelatin, this latter body may be used in detecting tryp-
sin. The liquefaction of strongly disinfected gelatin is, according to
FERMI, 2 a very delicate test for trypsin or tryptic enzymes. Various
suggestions for the use of gelatin in the trypsin test have been made.
In consideration of the observations of ASCOLI and NEPPI that a trypsin
may not act upon fibrin or other proteids but still digest gelatin, it is
advisable never to make use of gelatin or proteid alone in testing for
trypsin, but always the two.

For the quantitative estimation of trypsin by measuring the rapidity of
digestion we generally make use of the method of METT, described under pepsin
digestion. Another method, suggested by WEISS, consists in determining the
nitrogen in the nitrate after coagulation with heat and acetic acid. LOHLEIN
recommends the titration method of VOLHARD as used in pepsin determinations,
and has given directions for its use. JACOB Y recommends the use of ricin, and
GROSS suggests a method based upon the precipitation of casein by acid. BAY-

1 Pollak, Hofmeister's Beitrage, 6; contradictory statements are found in Ehren-
reich, cited in Bioch. Centralbl., 4; Ascoli and Neppi, Zeitschr. f. physiol. Chem., 56.

2 Arch, f . Hyg. 12 and 55.

506 DIGESTION.

Liss follows the digestion by the electrical conductivity, and F. WEISS 1 determines
the quantity of nitrogen not precipitated by tannic acid. The formol titration
can also be used with advantage for determining the decomposition (page 166).

The reaction has a great influence upon the rapidity of the trypsin
digestion. Trypsin acts energetically in neutral, or still better in alkaline,
solutions, and according to older statements, best in an alkalinity of
3-4 p. m. Na2COs; but the nature of the protein is also of importance.
The reports in regard to the action of trypsin in various reactions are
still somewhat disputed.2 The action of the alkali depends upon the
number of hydroxyl ions (DIETZE, KANITZ), and according to KANITZS
the digestion proceeds best in those solutions which are 1/70-1/200
normal in regard to hydroxyl ions. Free mineral acids, even in very
small quantities, completely prevent the digestion. If the acid is not
actually free, but combined with protein bodies, then the digestion
may take place quickly when the acid combination is not in too great
excess (CHITTENDEN and CUMMINS). Organic acids act less disturbingly,
and in the presence of 0.2 p. m. lactic acid and the simultaneous presence
of bile and common salt, the digestion may indeed proceed more quickly
than in a faintly" alkaline liquid (LINDBERGER). The assertion of RACH-
FORD and SOUTHGATE, that the bile can prevent the injurious action
of the hydrochloric acid, and that a mixture of pancreatic juice, bile,
and hydrochloric acid digests better than a neutral pancreatic juice,
could not be substantiated by CHITTENDEN and ALBRO. That bile has
an action tending to aid the tryptic digestion has been shown by many
investigators, and recently by BRUNO, ZUNTZ and Ussow and others.4
Carbon dioxide, according to SCHIERBECK,S has a retarding action
in acid solutions, but it accelerates the tryptic digestion in faintly
alkaline liquids. Foreign bodies, such as potassium cyanide, may pro-
mote tryptic digestion, while other bodies, such as salts of mercury, iron,
and others (CHITTENDEN and CUMMINS), or salicylic acid in large quan-
tities, may have a preventive action. According to WEISS 6 the halogen

1 Weiss, Zeitschr. f. physiol. Chem., 40; Lohlein, Hofmeister's Beitrage, 7; Jacoby,
Bioch. Zeitschr., 10; Gross, Arch. f. exp. Path. u. Pharm., 58; Bayliss, Arch, des
scienc. biol. de St. Pe"tersbourg, 11, Suppl.; and Journ. of Physiol., 36; Weiss, Zeitschr.
f. physiol. Chem. 31, 78 (1900).

2 See Kudo, Bioch. Zeitschr., 15.

3 Kanitz, Zeitschr. f. physiol. Chem., 37, who also cites Dietze.

4Chittenden and Cummins, Studies from the Physiol. Chem. Laboratory of Yale
College, New Haven, 1885, 1, 100; Lindberger, Maly's Jahresber., 13; Rachford and
Southgate, Medical Record, 1895; Chittenden and Albro, Amer. Journ. of Physiol., 1,
1898; Rachford, Journ. of Physiol., 25; Bruno, 1. c.; Zuntz and Ussow, Arch. f.
(Anat. u.) Physiol., 1900.

5 Skand. Arch. f. Physiol., 3.

6 Weiss, Zeitschr. f. physiol. Chem., 40; See also Kudo, Bioch. Zeitschr. 15, 473
(1908).

PRODUCTS OF TRYPTIC DIGESTION. 507

alkali salts disturb tryptic digestion only slightly, and NaCl seems to
have the strongest action. The sulphates have a much stronger retard-
ing action than the chlorides. The nature of the proteins is also of
importance. Unboiled fibrin is, relatively to most other proteins, dis-
solved so very quickly that the digestion test with raw fibrin gives an
incorrect idea of the power of trypsin to dissolve coagulated protein
bodies in general. Boiled fibrin is digested with much greater difficulty
and also requires a higher alkalinity: 8 p. m. Na2COs (VERNON1).
The resistance of certain native protein solutions, such as blood-serum
and egg-white, against the action of trypsin is remarkable. In regard
to the inhibition of the action of trypsin see Chapter I, page 63.

The Products of the Tryptic Digestion. In the digestion of unboiled
fibrin a globulin which coagulates at 55-60° C. may be obtained as an
intermediary product (HERRMANN2). Besides this, one obtains from
fibrin, as well as from other proteins, the products previously men-
tioned in Chapter II. In trypsin digestion the cleavage may proceed
so far that the mixture fails to give the biuret reaction. This does not
indicate, as E. FISCHER and ABDERHALDEN have shown, a complete
cleavage of the protein molecule into mono- and diamino-acids, etc.
In tryptic digestion, as shown by ABDERHALDEN and REINBOLD, using
the protein edestin, and by ABDERHALDEN and VOEGTLIN 3 with casein,
a gradual cleavage of the protein takes place, and thereby certain amino-
acids, like tyrosine and tryptophane, are readily and completely split
off, while others, like leucine, alanine, aspartic acid, and glutamic acid,
are slowly and less readily split off, and others, such as a-proline, phenyl-
alanine, and glycocoll, stubbornly resist the cleavage action of the trypsin.
The polypeptide-like bodies discovered by FISCHER and ABDERHALDEN,
which are produced in digestion, and which do not give the biuret
reaction, are the atomic complexes which resist the action of trypsin.
These peptoids contain the pyrrolidine carboxylic acid and phenylal-
anine groups of the protein, but also yield other monamino-acids such
as leucine, alanine, glutamic acid, and aspartic acid. Among the above-
mentioned products we find on the autodigestion of the gland other
substances, such as oxyphenylethylamine (EMERSON), which is pro-
duced from tyrosine by fermentive CO2 cleavage, also uracil (LEVENE),
guanidine (KUTSCHER and OTORI), the purine bases, which originate
from the nuclein bodies, and choline, which latter is formed from lecithin

1 Journ. of Physiol., 28.

2 Hermann, Zeitschr. /. physiol. Chem., 11.

3 Abderhalden and Reinbold, Zeitschr. f. physiol. Chem., 44 and 46, with Voegtlin,
ibid. 53.

508 DIGESTION.

(KUTSCHER and LOHMANN 1) . If putrefaction is not completely pre-
vented, still other bodies occur which will be considered later in con-
nection with the putrefactive processes in the intestine.

The Action of Trypsin upon other Bodies. The nucleoproteins and
nucleins are so digested that the protein complex is separated from the
nucleic acid and then digested. The nucleic acids may, nevertheless,
be somewhat changed (AEAKI), which is probably brought about by
another enzyme, the nuclease (SACHS). A cleavage of nucleic acids with
the setting free of phosphoric acid and purine bases is, according to
IwANOFF,2 not brought about by trypsin. The splitting is first pro-
duced by the action of nuclease or erepsin (see page 493). Gelatin is
dissolved and digested by pancreatic juice. A cleavage with the sepa-
ration of glycocoll and leucine does not occur (KtiHNE and EWALD), or
only to a trivial extent (REICH-HERZBERGE 3) .

The gelatin-forming substance of the connective tissues is not directly
dissolved by trypsin, but only after it has been treated with acids or
soaked in water at 70° C. By the action of trypsin on hyaline cartilage
the cells dissolve, leaving the nucleus. The matrix is softened and
shows an indistinctly constructed network of collagenous substances
(KUHNE and EWALD). The elastic substance, the structureless membranes,
and the membrane of the fat-cells, are also dissolved. Parenchymatous
organs, such as the liver and the muscles, are dissolved all but the nuclei,
connective tissue, fat-corpuscles, and the remainder of the nervous
tissue. If the muscles are boiled, then the connective tissue is also
dissolved. Mudn is dissolved and split by trypsin, while chitin and horn
substance do not seem to be acted upon by the enzyme. Oxyhwmoglobin
is decomposed by trypsin with the splitting off of haematin. Trypsin
splits off large amounts of hydriodic acid from diiodotyrosine (OSWALD 4).
Trypsin has no action upon fats and carbohydrates.

The action of trypsin on simply constructed substances of known
constitution such as acid-amides, polypeptides, is of especially great
interest. In this regard we have the somewhat earlier investigations
of GULEWITSCH, GONNERMANN, and ScHWARZSCHiLD,5 but the investi-

1 Fischer and Abderhalden, Zeitschr. f. physiol. Chem., 39; Emerson, Hofmeister's
Beitrage, 1; Levene, Zeitschr. f. physiol. Chem., 37; Kutscher and Lohmann, ibid.
39; Kutscher and Otori, ibid., 43, and Centralbl. f. Physiol., 18.

2 Iwanoff, Zeitschr. f. physiol. Chem., 39, which also contains the literature; Sachs,
ibid., 46.

3 Kiihne and Ewald, Verh. d. naturh.-med. Vereins zu Heidelberg (N. F.), 1; Reich-
Herzberge, Zeitschr. f. physiol. Chem., 34.

4 Zeitschr. f. physiol. Chem. 62, 432 (1909).

5 Hofmeister's Beitrage, 4, where the other works are also cited.

PANCREATIC RENNIN. 509

gations of FISCHER and of ABDERHALDEN and their co-workers,1 are
much more complete and important. In this connection see page 62.

The behavior of the polypeptides with trypsin, or closely related
enzymes, is of the greatest interest and in many respects very im-
portant. Thus in the polypeptides we have a means of determining
the kind of enzyme, whether it belongs to the pepsin, trypsin, or erepsin
group. We know of no polypeptide which is split by pepsin; trypsin
splits only certain polypeptides, but not others, while the erepsin on the
contrary seems to split all polypeptides, occurring in nature, which
are composed of aminoacids. By the aid of the polypeptide reaction
ABDERHALDEN and co-workers have also been able to show that the
trypsin-like enzyme, occurring in the blood-plasma, is not identical
with trypsin because it does not attack glycyl-Z-tyrosine, which is split
by trypsin.

Pancreatic rennin is an enzyme found in the gland and in the juice,
which coagulates neutral or alkaline milk (KUHNE and ROBERTS and
others). This enzyme, according to PAWLOW'S school, is identical
with trypsin. The similarity of action of these two enzymes and the
fact that both are activated simultaneously from the zymogens by enter-
okinase or lime salts (DELEZENNE, WOHLGEMUTH 2) seem to point to
this identity. On the other hand the optimum of the enzyme action
for the pancreatic rennin is 60-65° C. (VERNON), which is much higher
than for the trypsin, and GLAESSNER and POPPER 3 have also observed
a case where the human pancreatic juice contained no rennin enzyme.

According to HALLIBURTON and BRODiE,4 casein is converted by the pancreatic
juice of the dog into " pancreatic casein," a substance which, in regard to solubility,
stands to a certain extent between casein and paracasein (see Chapter XIII),
and which is converted into paracasein by rennin. Further investigations on
the action of this enzyme upon milk and especially upon pure casein solutions are
very desirable.

Pancreatic Calculi. The concrement from a cystic enlargement of WIRSUNG'S
duct in a man, as analyzed by BALDONI, contained in 1000 parts as follows:
Water 34.4, ash 126.7, protein substances 34.9, free fatty acids 133, neutral fats
124, cholesterin 70.9, soaps and pigment 499.1, parts. SCHEUNERT and BERG-
HOLZ 5 have reported a pancreatic calculi in the ox.

Fischer and Bergell, Ber. d. d. chem. Gesellsch., 36 and 37; Fischer and Abder-
halden, Sitzungsber. der Kgl. Pr. Akad. d. Wissensch., Berlin, 1905. The works of
Abderhalden and co-workers cannot be specially cited, but may be found in Zeitschr.
f. physiol. Chem., 47, 48, 49, 51, 52, 53, 54, 55, and 57.

2 Kuhne and Roberts, Maly's Jahresber., 9; see also Edkins, Journ. of Physiol.,
12 (literature); Delezenne, Compt. rend. soc. biol., 62 and 63; Wohlgemuth, Bioch.
Zeitschr., 2.

* Vernon, Journ. of Physiol., 12; Glaessner and Popper, Deutsch. Arch. f. klin.
Med., 94.

4 Journ. of Physiol., 20.

5 Baldoni, Maly's Jahresb., 29, 353; Scheunert and Bergholz., Zeitschr. f. physiol.
Chem., 52.

510 DIGESTION.

Besides the enzymes which have been discussed in connection with
the pancreatic juice, the gland also contains others, among which can be
mentioned the enzyme which, according to STOKLASA and his collab-
orators, occurs principally in organs and tissues and which decomposes
sugar into alcohol and carbon dioxide, like zymase. Opinions as to the
importance of the pancreas for glycolysis are diverse, and we therefore
refer the reader to what has been previously stated on this subject in
Chapter VII, pages 407 and 408.

V. THE CHEMICAL PROCESSES IN THE INTESTINE.

The action which belongs to each digestive secretion may be essen-
tially changed under certain conditions by being mixed with other
digestive fluids for various reasons, and also by the action of the
enzymes upon each other; l and since the digestive fluids which flow
into the intestine are mixed with still another fluid, the bile, it will be
readily understood that the combined action of all these fluids in the
intestine makes the chemical processes going on therein very complicated.

As the acid of the gastric juice acts destructively on ptyalin, this
enzyme has no further diastatic action, even after the acid of the gastric
juice has been neutralized in the intestine. ROGER and SIMON 2 claim
to have observed in saliva made inactive by the gastric juice, a reac-
tivation caused by the pancreatic juice, but these investigations do
not seem to be fully conclusive. The bile has, at least in certain animals,
a slight diastatic action, which in itself can hardly be of any great
importance, but which shows that the bile has not a preventive, but
rather a beneficial influence on the energetic diastatic action of the pan-
creatic juice. Several experimenters3 have observed a beneficial action
of the bile on the diastatic action of the pancreas infusion. To this
may be added that the micro-organisms which habitually occur in the
intestine and sometimes in the food have partly a diastatic action and
partly produce a lactic-acid and butyric-acid fermentation. The
maltose which is formed from the starch seems to be converted into
glucose in the intestine. It seems conclusively that the cellulose cannot
be digested in the organism of the dog.4 LOHRISCH found that on an
average of 50 per cent of the introduced cellulose and hemicellulose was
digested in human beings and yielded the corresponding sugar. That

1 See Wr6blewski and collaborators, Hofmeister's Beitrage, 1.

2 Compt. rend. soc. biol., 62.

3 Martin arid Williams, Proceed, of Roy. Soc., 45 and 48; Bruno, footnote 2, p.
502; Buglia, Bioch. Zeitschr. 25.

4 Scheunert, cited from Bioch. Centralbl. 10, 71; see also Lorhisch, Zeitschr. f. physiol.
Chem. 69, 143 (1910) as well as Bioch. Centralbl. 8, 334.

CHEMICAL PROCESSES IN THE INTESTINE. 511

cellulose undergoes a fermentation in the intestine by the action of micro-
organisms, producing marsh-gas, acetic acid, and butyric acid, has
been especially shown by TAPPEINER; still it is not known to what extent
the cellulose is destroyed in this way.1

The bile has, as shown by MOORE and ROCKWOOD 2 and then espe-
cially by PFLUGER, the property to a high degree of dissolving fatty
acids, especially oleic acid, which itself is a solvent for other fatty acids,
and hence, as will be seen later, it is of great importance in the absorp-
tion of fat. It is also of great importance that the bile, as previously
stated, not only activates the steapsinogen, but that, as first shown by
NENCKI and RACHFORD,3 it accelerates the fat-splitting action of the
steapsin. According to v. FURTH and ScmJTZ4 the bile-salts are the
active constituents of the bile in this cleavage, and the fatty acids set
free can combine with the alkalies of the intestinal and pancreatic juices
and the bile, producing soaps which are of great importance in the
emulsification of the fats.

If to a soda solution of about 1-3 p. m. pure, perfectly neutral
olive-oil is added in not too large a quantity, a transient emulsion is
obtained after vigorous shaking. If, on the contrary, one adds to the
same quantity of soda solution an equal amount of commercial olive-
oil (which always contains free fatty acids), the vessel need only be
turned over for the two liquids to mix, and immediately there appears
a very finely divided and permanent emulsion, making the liquid appear
like milk. The free fatty acids of the commercial oil, which is always
somewhat rancid, combine with the alkali to form soaps which act to
emulsify the fats (BRUCKE, GAD, LOEWENTHAL 5) . This emulsifying
action of the fatty acids split off by the pancreatic juice is undoubtedly
assisted by the habitual occurrence of free fatty acids in the food, as
well as by the splitting off of fatty acids from the neutral fats in the
stomach (see page 476).

Bile completely prevents peptic zymolysis in artificial digestion.

1 On the digestion of cellulose see Henneberg and Stohmann, Zeitschr, f . Biologic,
21, 613; v. Knieriem, ibid., 67; Hofmeister, Arch. f. wiss. u. prakt. Thierheilkunde,
11; Weiske, Zeitsehr. f. Biologic, 22, 373; Tappeiner, ibid., 20 and 24; Mallevre,
Pfliiger's Arch., 49; Omeliansky, Arch. d. scienc. biol. de St. Petersbourg, 7; E. Miiller,
Pfliiger's Arch., 83; Lohrisch, Zeitschr. f. physiol. Chem., 47 (literature); Pringsheimr
ibid. 78, 266 (1912).

2 Proceedings of Roy. Soc., 60, and Journ. of Physiol., 21. In regard to Pfliiger's
work see Absorption.

3Nencki, Arch. f. exp. Path. u. Pharm., 20; Rachford, Journal of Physiol., 12.

4 Centralbl. f . Physiol., 20.

5 Briicke, Wien, Sitzungsber., 61, Abt. 2; Gad, Arch. f. (Anat. u.) Physiol., 1878;
Loewenthal, ibid., 1897.

512 DIGESTION.

because it retards the swelling up of the proteins. The passage of bile
into the stomach during digestion on the contrary, seems, according,
to several investigators, especially ODDI and DASTRE/ to have no dis-
turbing action on gastric digestion. According to BoLDYREFF,2 after
continuous starvation, on feeding fat and food rich in fat, as well as after
large amounts of acid, a mixture of bile, pancreatic juice, and intestinal
juice pass readily into the stomach. After food rich in fat, which
retards the secretion of gastric juice and the motility of the stomach,
a digestion due to this alkaline mixture may take place in the stomach.

Bile itself has no solvent action on proteins in neutral or alkaline
reaction, but still it may exert an influence on protein digestion in the
intestine. The acid contents of the stomach, containing an abundance
of proteins, give with the bile a precipitate of proteins and bile-acids.
This precipitate carries a part of the pepsin with it, and for this reason,
and also on account of the partial or complete neutralization of the
acid of the gastric juice by the alkali of the bile and the pancreatic
juice, the pepsin digestion cannot proceed further in the intestine.
According to BAUMSTARK and COHNHEIM 3 connective tissue is digested
on the other side of the pylorus in the intestine by the pepsin-hydrochloric
acid. On the contrary, the bile does not disturb the digestion of pro-
teins by the pancreatic juice in the intestine. The action of these diges-
tive secretions, as above stated, is not disturbed by the bile, not even
by the faintly acid reaction due to organic acids; but, on the contrary,
the action of trypsin is accelerated by the bile. In a dog killed while
digestion is going on, the faintly acid, bile-containing material of the
intestine shows regularly a strong digestive action on proteins.

The precipitate of protein and bile-salts formed on the meeting of the
acid contents of the stomach with the bile easily redissolves in an excess
of bile, and also in the NaCl formed in the neutralization of the hydro-
chloric acid of the gastric juice. This may take place even in faintly
acid reaction. Since in man the excretory ducts of the bile and the
pancreatic juice open near one another, in consequence of which the
acid contents of the stomach are probably immediately in great part
neutralized by the bile as soon as it enters, it is doubtful whether a pre-
cipitation of proteins by the bile occurs in the intestine.

Besides the previously mentioned processes caused by enzymes,
there are others of a different nature going on in the intestine, namely,
the fermentation and putrefaction processes caused by micro-organ-
isms. These are less intense in the upper parts of the intestine, but

1 Oddi, in Centralbl. f. Physiol., 1, 312; Dastre, Arch, de Physiol. (5), 2, 316.
8 Centralbl. f. Physiol., 18, 457, and Pfluger's Arch., 121.
3 Zeitschr. f. physiol. Chem., 65, 477 (1910).

PUTREFACTION IN THE INTESTINE. 513

increase in intensity toward the lower part, and decrease in the large
intestine because of the consumption of fermentable material and by
the removal of water by absorption. Fermentation processes, but only
very slight putrefaction, occur in the small intestine of man. MAC-
FAD YEN, M. NENCKI, and N. SEEBER 1 have investigated a case of
human anus prseternaturlis, in which the fistula occurred at the lower
end of the ileum, and they were able to investigate the contents of the
intestine after it had been exposed to the action of the mucous mem-
brane of the entire small intestine. The mass was yellow or yellowish-
brown, due to bilirubiD, and had an acid reaction which, on a mixed
but principally animal diet, calculated as acetic acid, amounted to 1 p. m.
The contents were nearly odorless, having an empyreumatic odor recall-
ing that of volatile fatty acids, and infrequently had a putrid odor
resembling that of indol. The essential acid present was acetic acid,
accompanied by fermentation and paralactic acid, volatile fatty acids,
succinic acid, and bile-acids. Coagulable proteins, peptone, mucin,
dextrin, sugar, and alcohol were present. Leucine and tyrosine could
not be detected.

According to the above-mentioned investigators, the proteins are
only to a very slight extent, if at all, decomposed by the microbes in
the small intestine of man. The organisms present in the small intestine
preferably decompose the carbohydrates, forming ethyl alcohol and the
above-mentioned organic acids.

Further investigations of JAKOWSKY and of AD. SCHMIDT 2 lead to
the same result, namely, that in man the putrefaction of the proteins
takes place chiefly in the large intestine, and the conditions are the
same in carnivora. In these latter it has been possible to follow the
intestinal digestion by investigating the contents of the various parts
of the intestine as well as by forming fistulas along the intestine. Again
PAWLOW and his pupils, especially LONDON 3 and his collaborators, have
essentially advanced our knowledge on this subject.

In regard to the digestion of protein, it has been found that after
feeding meat, bread, or certain protein bodies, the digestion in the
stomach and small intestine is so complete that on the passage of the
contents into the caecum all the protein is digested and absorbed.
Unboiled white of egg is an exception and is digested with difficulty.
In experiments with unboiled white of egg, LONDON and SULEIMA reob-

1 Arch. f. exp. Path. u. Pharm., 28.

2 Jakowsky, Arch, des scienc. biol. de St. Petersbourg, 1; Ad. Schmidt, Arch. f.
Verdauunskr., 4.

3 The works of London and collaborators cannot be cited in detail, but may be
found in Zeitschr. f. physiol. Chem., 46-57.

514 DIGESTION.

tained 73 per cent of the coagulable protein from a fistula in the ileum
(2-3 cm. in front of the caecum). Elastin is, according to LONDON, 1
more slowly digested in the small intestine than other proteins. KUT-
SCHEB and SEEMANN, ABDERHALDEN, LONDON and collaborators2 have
also found that non-biuret giving products and amino-acids are regularly
split off, probably by the combined action of trypsin and erepsin. These
amino-acids occur to a slight extent only, but from this no conclusion
can be drawn as to the extent of amino-acid formation, because we do
not know the extent of their absorption. The digestion of protein
in the intestine, it seems, according to ABDERHALDEN, LONDON, OPPLER
and REEMLiN,3 is similar to the artificial digestion with trypsin, namely,
that the destruction takes place step-wise, that certain amino-acids,
such as tyrosine, are split off earlier than others. ZUNZ 4 found the same
end result in the protein cleavage in the small intestine, with bread as
with meat feeding. LONDON, SCHITTENHELM and WIENER 5 found
that a cleavage of nucleic acids with the formation of nucleosides
occurred in the lower part of the jejunum and ileum.

The decomposition products of the proteins formed by the action of
gastric juice can, according to LONDON. 6 be absorbed without further
cleavage by the pancreatic juice, and a further cleavage in the intestine
seems to be more necessary for assimilation than for absorption.

The carbohydrates and the fats (LEVITES 7) may be so completely
split in the stomach and small intestine that their absorption is com-
plete before the contents pass into the caBcum. According to LONDON
and POLOWZOWA 8 a strong cleavage of starch, dextrins and disaccharides
takes place, especially in the duodenum, while the absorption is less
strong here. The carbohydrates are here prepared for the absorption
taking place in the lower parts of the intestine, though the cleavage also
goes on in the other parts, namely in the jejunum and the upper part
of the ileum.

As above remarked, ordinarily no putrefaction takes place in the
small intestine, but occurs generally only in the large intestine. This

1 London and Suleima, Zeitschr. f. physiol. Chem., 46; London, ibid., 60.
2Kutscher and Seemann. ibid., 34; Abderhalden and London, with Kautzsch,
ibid., with L. Baumann, ibid., 51, with v. Korosy, ibid., 53.

3 Zeitschr. f. physiol. Chem., 55 and 58.

4 Intern. Beitr. z. Pathol. u. Ther. d. Ernahrungsstorungen, 2, 195, 459 (1910 and
1911).

5 Zeitschr. f. physiol. Chem., 72, 459 (1911).

6 Ibid., 49.

7 Ibid., 49 and 53.

., 56.

PUTREFACTION IN THE INTESTINE. 515

putrefaction of the proteins is not the same as the pancreatic digestion.
In putrefaction the decomposition goes much further, and a mixture of
products is obtained which have become known through the labors
of numerous investigators, especially NENCKI, BAUMANN, BRIEGER, H.
and E. SALKOWSKI, and their pupils. The products which are formed
in the putrefaction of proteins are (in addition to proteases, peptones,
amino-adds, and ammonia) indol, skatol, paracresol, phenol, phenylpro-
pionic add, and phenylacetic add, also paraoxyphenylacetic add and
hydroparacoumaric add (besides paracresol, produced in the putrefaction
of tyrosin), volatile fatty adds, carbon dioxide, hydrogen, marsh-gas,
methylmercaptan, and sulphureted hydrogen. In the putrefaction of
gelatin neither tyrosine nor indol is formed, while glycocoll is produced
instead.

Among these products of decomposition a few are of special interest
because of their behavior within the organism and because after their
absorption they, pass into the urine. A few, such as the oxyacids, pass
unchanged into the urine. Others, such as phenols, are directly trans-
formed into ethereal sulphuric acids by synthesis, and are eliminated as
such by the urine; on the contrary, others, such as indol and skatol, are
converted into ethereal sulphuric acids after oxidation only (for details
see Chapter XIV). The quantity of these bodies in the urine also varies
with the extent of the putrefactive processes in the intestine; at least
this is true for the ethereal sulphuric acids. Their quantity increases
in the urine with a stronger putrefaction, and the reverse takes place,
namely, a disappearance from the urine, or a great reduction in quantity,
as BAUMANN, HARLEY and GOODBODY 1 have shown by experiments
on dogs, when the intestine is disinfected by various agents.

The gases which are produced by the decomposition processes are
mixed in the intestinal tract with the atmospheric air swallowed with
the saliva and food, and as the gas developed in the decomposition of
different foods varies, so the mixture of gases after various foods should
have a dissimilar composition. This is found to be true. Oxygen is
found only in very faint traces in the intestine; this may be accounted
for in part by the formation of reducing substances in the fermenta-
tion processes which combine with the oxygen, and partly, perhaps
chiefly, to a diffusion of the oxygen through the tissues of the walls of
the intestine. To show that these processes take place mainly in the
stomach, the reader is referred to page 486, on the composition of the
gases of the stomach. Nitrogen is invariably found in the intestine,
and it is probably due chiefly to the swallowed air. The carbon dioxide

^aumann, Zeitschr. f. physiol. Chem., 10; Harley and Goodbody, Brit. Med.
Journ., 1899.

616 DIGESTION.

originates partly from the contents of the stomach, partly from the
putrefaction of the proteins, partly from the lactic-acid and butyric-
acid fermentation of carbohydrates, and partly from the setting free of
carbon dioxide from the alkali carbonates of the pancreatic and intes-
tinal juices by their neutralization through the hydrochloric acid of
the gastric juice and by organic acids formed in the fermentation.
Hydrogen occurs in largest quantities after a milk diet, and in smallest
quantities after a purely meat diet. This gas seems to be formed
chiefly in the butyric-acid fermentation of carbohydrates, although it
may occur in large quantities in the putrefaction of proteins under certain
circumstances. There is no doubt that the methylmercaptan and sul-
phureted hydrogen which occur normally in the intestine originate from
the proteins. The marsh-gas undoubtedly originates in the putrefac-
tion of proteins. As proof of this RUGE *• found 26.45 per cent marsh-
gas in the human intestine after a meat diet. He found a still greater
quantity of this gas after a vegetable (leguminous) diet; this coincides
with the observation that marsh-gas may be produced by a fermentation
of carbohydrates, but especially of cellulose (TAPPEINER 2) . Such an
origin of marsh-gas, especially in herbivora, is to be expected. A small
part of the marsh-gas and carbon dioxide may also arise from the decom-
position of lecithin (HASEBROEK 3) .

Putrefaction in the intestine not only depends upon the composi-
tion of the food, but also upon the albuminous secretions and the bile.
Among the constituents of bile which are changed or decomposed, , there
are not only the pigments — the bilirubin yields urpbilin and a brown
pigment — but also the bile-acids, especially taurocholic acid. Glyco-
cholic acid is more. stable, and a part is found unchanged in the excre-
ment of certain animals, while taurocholic acid is so completely decom-
posed that it is entirely absent in the feces. In the fetus, on the con-
trary, in whose intestinal tract no putrefaction processes occur, undecom-
posed bilev-acids and bile-pigments are found in the contents of the
intestine. The transformation of bilirubin into urobilin does not occur,
as previously stated, in the small, but in the large intestine in man.

As under normal conditions no putrefaction, or a,t least none worth
mentioning, occurs in the small intestine, and as often nearly all the pro-
tein of the food is absorbed, it follows that ordinarily it is the secretions
and cells rich in proteins which undergo putrefaction. That the secre-
tions rich in proteins are destroyed in putrefaction in the intestine

1 Wien. Sitzungsber., 44.

2 Zeitschr. f. Biologie., 20 and 24.

3 Zeitschr. f. physiol. Chem., 12.

PUTREFACTION IN THE INTESTINE. 517

follows from the fact that putrefaction may also continue during com-
plete fasting. From the observations of MULLER l upon CETTI it was
found that the elimination of indican during starvation rapidly de-
creased and after the third day of starvation it had entirely disappeared,
while the phenol elimination, which at first decreased so that it was
nearly minimum, increased again from the fifth day of starvation, and
on the eighth or ninth day it was three to seven times as much as in man
under ordinary circumstances. In dogs, on the contrary, the elimina-
tion of indican during starvation is considerable, but the phenol elimina-
tion is slight. Among the secretions which undergo putrefaction in the
intestine, the pancreatic juice, which putrefies most readily, takes first
place.

From the foregoing facts it must be concluded that the products
formed by the putrefaction in the intestine are in part the same as
those formed in digestion. The putrefaction may be of benefit to the
organism in so far as the formation of such products as proteoses, pep-
tones, polypeptides and amino-acids is concerned. The question has
indeed been asked (PASTEUR), Is digestion possible without micro-organ-
isms? NUTTAL and THIERFELDER have shown that guinea-pigs, removed
from the uterus "of the mother by Caesarian section, could with sterile
air digest well and assimilate sterile food (milk and crackers) in the
complete absence of bacteria in the intestine, and developed normally
and increased in weight. SCHOTTELIUS 2 has arrived at other results
by experiments with hens. The chickens, hatched under sterile con-
ditions, kept in sterile rooms and fed with sterile food, had continuous
hunger and ate abundantly, but soon died, in about the same time as a
starving chicken. On mixing with the food, at the proper time, a vari-
ety of bacteria from hen feces, they gained weight again and recovered.

The bacterial action in the intestinal canal is, at least in certain cases,
as with food rich in cellulose, necessary, and it acts in the interest of the
organism. This action may, by the formation of further cleavage prod-
ucts, involve a loss of valuable material to the organism, and it is there-
fore important that putrefaction in the intestine be kept within certain
limits. If an animal is killed while digestion in the intestine is going
on, the contents of the small intestine give out a peculiar but not putres-
cent odor. Also the odor of the contents of the large intestine is far less
offensive than a putrefying pancreas infusion or a putrefying mixture
rich in protein. From this one may conclude that putrefaction in the
intestine is ordinarily not nearly so intense as outside of the organism.

1 Berlin, klin. Wochenschr., 1887.

2 Nuttal and Thierf elder, Zeitschr. f. physiol. Chem., 21 and 22; Schottelius, Arch,
f. Hygiene, 34, 42, and 67.

518 DIGESTION.

It seems thus to be provided, under physiological conditions, that
putrefaction shall not proceed too far, and the factors which here come
into consideration are probably of different kinds. Absorption is
undoubtedly one of the most important of them, and it has been proved
by actual observation that the putrefaction increases, as a rule, as the
absorption is checked and fluid masses accumulate in the intestine. The
character of the food also has an unmistakable influence, and it seems
as if a large quantity of carbohydrates in the food acts against putre-
faction (HmscHLER1). It has been shown by POHL, BIERNACKI,
ROVIGHI, WINTERNITZ, ScHMiTZ, and others 2 that milk and kephir
have a specially strong preventive action on putrefaction. This action
is not due to the casein, but chiefly to the lactose and also in part to the
lactic acid.

A specially strong preventive action on putrefaction has been
ascribed for a long time to the bile. This anti-putrid action does not
exist in neutral or faintly alkaline bile, which itself easily putrefies, but
to the free bile-acids, especially taurocholic acid (MALY and EMICH,
LiNDBERGER3). There is no question that the free bile-acids have a
strong preventive action on putrefaction outside of the organism, and
it is therefore difficult to deny such an action in the acid reacting con-
tents of the intestine. Notwithstanding this, the anti-putrid action
of the bile in the intestine is not considered by certain investigators

(VOIT, ROHMANN, HlRSCHLER and TERRAY, LANDAUER and ROSEN-

BERG 4) as of great importance.

Biliary fistulas have been established so as to study the importance
of the bile in digestion (SCHWANN, BLONDLOT, BIDDER and SCHMIDT/
and others). As a result it has been observed that with fatty foods an
imperfect absorption of fat regularly takes place and the excrement
contains, therefore, an excess of fat and has a light-gray or pale color.
The extent of deviation from the normal after the operation is essen-
tially dependent upon the character of the food. If an animal is fed
on meat and fat, then the quantity of food must be considerably increased
after the operation, otherwise the animal will become -very thin, and

1 Hirschler, Zeitschr. f. physiol. Chem., 10; Zimnitzki, ibid., 39 (literature).

2 Schmitz, ibid., 17, 401, which gives references to the older literature, and 19. See
also Salkowski, Centralbl. f. d. med. Wiss., 1893, 467, and Seelig, Virchow's Arch.,
126 (literature).

3 Maly and Emich, Monatshefte, f. Chem., 4; Lindberger, footnote 4, p. 506.
4Voit, Beitr. zur Biologie, Jubilaumschrift, Stuttgart, 1882; Rohmann, Pfluger's

Arch. 29; Hirschler and Terray, Maly's Jahresber., 26; Landauer, Math, u. Naturw.
Ber. aus Ungarn, 15; Rosenberg, Arch. f. (Anat. u.) Physiol., 1901.

6 Schwann, Miiller's Arch. f. Anat. u. Physiol., 1844; Blondlot, cited from Bidder
and Schmidt, Verdauungssafte, etc., 98.

PUTREFACTION IN THE INTESTINE. 519

indeed die with symptoms of starvation. In these cases the excrement
has the odor of carrion, and this was considered a proof of the action
of the bile in checking putrefaction. The emaciation and the increased
want of food depend, naturally, upon the imperfect absorption of the
fats, whose high calorific value is reduced and must be replaced by the
taking up of larger quantities of other nutritive bodies. If the quan-
tity of proteins and fats be increased, then the latter, which can be only
incompletely absorbed, accumulate in the intestine. This accumulation
of the fats in the intestine only renders the action of the digestive juices
on proteins more difficult, and thus increases the amount of putrefac-
tion. This explains the appearance of fetid feces, whose pale color is
not due to a lack of bile-pigments, but to a surplus of fat (ROHMANN,
VOIT). If the animal is, on the contrary, fed on meat and carbohy-
drates, it may remain quite normal, and the leading off of the bile does
not cause any increased putrefaction. The carbohydrates may be
uninterruptedly absorbed in such large quantities that they replace
the fat of the food, and this is the reason why the animal on such a diet
does not become emaciated. As with this diet the putrefaction in the
intestine is no greater than under normal conditions even though the bile
is absent, it would seem that the bile in the intestine exercises no pre-
ventive action on putrefaction.

To this conclusion the objection may be made that the carbohy-
drates, which are capable of checking putrefaction, can, so to speak,
undertake the anti-putrid action of the bile. But as there are also cases
(in dogs with biliary fistula) where the intestinal putrefaction is not
increased with exclusive meat diet,1 it is maintained that the absence
of bile in the intestine, even by exclusive carbohydrate food, does not
always cause an increased putrefaction.

Although the question as to the manner in which the putrefactive
processes in the intestine under physiological conditions are kept within
certain limits cannot be answered positively, still it may be asserted
that the faint acid reaction, and the absorption of water, and the rela-
tively rapid movement, of the contents of the small intestine and their
absorption, are important factors.

That the acid reaction in the intestine has a preventive influence on
the putrefactive processes follows from the existing relation between
the degree of acidity of the gastric juice and the putrefaction in the
intestine. Since the investigations and observations of KAST, STADEL-
MANN, WASBUTZKI, BIERNACKI and MESTER had proved that an
increased putrefaction in the intestine occurred when the quantity of
hydrochloric acid in the gastric juice was diminished or deficient,

1 See Hirschler and Terray, 1. c.

520 DIGESTION.

SCHMITZ l has shown in man that on the administration of hydro-
chloric acid, producing a hyperacidity of the gastric juice, the putrefac-
tion in the intestine may be checked. The question arises whether the
reaction in the small intestine is always acid and whether the acidity
is strong enough to prevent putrefaction. In this connection it must
be recalled that the acidity of the contents of the small intestine is not
due to hydrochloric acid, but chiefly to organic acids, acid salts, and
free carbon dioxide. There are several observations as to the reaction
of the intestinal contents, by MOORE and ROCKWOOD, MOORE and
BERGIN, MATTHES and MAKQUARDSEN, I. MUNK, NENCKI and ZALESKI,
HEMMETER,2 although they are somewhat contradictory. From these
reports one can conclude that the reaction may vary not only among
different animals, but also in the same animals under varying conditions.
There is no doubt that the acid reaction in many cases is due to the pres-
ence of organic acids. On testing with various indicators it has been
shown that sometimes the upper parts, and often the lower parts, are
acid, due to acid salts such as NaHCOs and free C02, and finally that
in certain animals the intestinal contents are alkaline throughout. The
question how, under these conditions, putrefaction is excluded, and how
the acidity of the gastric contents influences the intestinal putrefaction,
cannot be explained. It is very probable that the bacterial flora of the
intestine is of very great importance and it is possible, as BIENSTOCK
admits, that the explanation lies in an antagonistic bacterial action and
that the carbohydrates, especially lactose, which retard putrefaction,
form a good nutritive media for those bacteria which destroy the putre-
factive producers or retard their development. According to HORO-
WITZ an unequal division of the various bacteria occurs in dogs in the
different parts of the intestine and certain varieties of bacteria occur
in greater quantities than others, according to the kind of food taken.
The influence of the kind of food upon the intestinal flora has also been
studied by KENDALL. Perhaps, also, agreeing with the experience of
CONRADI and KURPJUWEIT,S the toxins produced by the intestinal bacteria
may, by their antiseptic action, keep the putrefactive processes in the
intestine within bounds.

1 Zeitschr. f. physiol. Chem., 19, 401, which includes all the pertinent literature.

2 Moore and Rockwood, Journ. of Physiol., 21; Moore and Bergin, Amer. Journ.
of Physiol., 3; Matthes and Marquardsen, Maly's Jahresber., 28; Munk, Centralbl. f.
Physiol., 16; Nencki and Zaleski, Zeitschr. f. physiol. Chem., 27; Hemmeter, Pfliiger's
Arch., 81.

3 Bienstock, Arch. f. Hygiene, 39; Horowitz. Zeitschr. f. physiol. Chem., 52; Ken-
dall, Journ. of biol. Chem. 6, 499 (1909); Conradi and Kurpjuweit, Munch, med.
Wochenschr., 1905.

FECES. 521

Feces. It is evident that the residue which remains after complete
digestion and absorption in the intestine must be different, both quali-
tatively and quantitatively, according to the variety and quantity of
the food. In man the quantity of excrement from a mixed diet is
120-150 grams, with 30-37 grams of solids, per twenty-four hours, while
the quantity from a vegetable diet, according to VoiT,1 was 333
grams, with 75 grams of solids. With a strictly meat diet the excre-
ment is scanty, pitch-like, and black. The scanty feces in starva-
tion have a similar appearance. A large quantity of coarse bread yields
a great amount of light-colored excrement. In these cases the feces
are also habitually poorer in nitrogen than after food rich hi protein.
The individuality also plays an important role in the utility of the food
and the formation of feces (ScmERBECK2). If there is a large propor-
tion of fat, it takes a lighter clay-like appearance. The decomposi-
tion products of the bile-pigments seem to play only a small part in the
normal color of the feces.

The constituents of the feces are of different kinds. In the excre-
ment are found digestible or absorbable constituents of the food, such
as muscle fibers, connective tissues, lumps of casein, grains of starch,
and fat, which have not had sufficient time to be completely digested
or absorbed in the intestinal tract. In addition the excrement con-
tains indigestible bodies, such as the remains of plants, keratin sub-
stances, and others; also form-elements originating from the mucous coat
and the glands; constituents of the different secretions, such as mucin,
cholic acid, dyslysine, and cholesterin (koprosterin or stercorin), purine
bases,3 and enzymes; mineral bodies of the food and the secretions;
and, lastly, products of putrefaction or of digestion, such as skatol, indol,
volatile fatty acids, purine bases, lime, and magnesia soaps. Occasion-
ally, also, parasites of different kinds occur; and lastly, the excrement
contains micro-organisms of various species.

That the mucous membrane of the intestine by its secretion and by
the abundant quantity of detached epithelium contributes essentially
to the formation of feces follows from the discovery first made by L.
HERMANN and substantiated by others,4 that a clean, isolated loop

1 Zeitschr. f . Biologie, 25, 264.

2 Arch. f. Hygiene, 51.

3 In regard to the purine bases in feces, see Hall, Journ. of Path, and Bacteriol.,
9; Schittenhelm, Arch. f. klin. Med., 81. Schittenhelm and Kruger, Zeitschr. f. physiol.
Chem., 45.

4 Hermann, Pfliiger's Arch., 46. .See also Ehrenthal, ibid., 48; Berenstein, ibid.,
53; Klecki, Centralbl. f. Physiol., 7; 736, and F. Voit, Zeitschr. f. Biologie, 29; v.
Moraczewski, Zeitschr. f. physiol. Chem., 25; F. Lippich; Prager med. Wochenschr.,
32.

522 DIGESTION.

of intestine collects material similar to feces. These masses are rich
in mineral substances and especially rich in bodies soluble in alcohol-
ether, among which cholesterin occurs, as previously mentioned (Chap-
ter VII). With a mixed diet with an excess of meat, the human feces
consist only in small part of food residues and consist in great part,
or after meat or milk diet, almost entirely, of intestinal secretions. Many
foods, therefore, produce a large quantity of feces chiefly by causing an
abundant secretion.1

The reaction of the feces is very variable, but in man with a mixed
diet it is neutral or faintly alkaline. It is often acid in the inner part,
while the outer layers in contact with the mucous coat have an alka-
line reaction. In nursing infants it is habitually acid. The odor is
perhaps chiefly due to skatol, which was first found in the feces by
BRIEGER, and so named by him. Indol and other substances also take
part in the production of odor. The color is ordinarily light or dark
brown, and depends above all upon the nature of the food. Medicinal
bodies may give the feces an abnormal color. The excrement is col-
ored black by bismuth, yellow by rhubarb, and green by calomel. This
last-mentioned color was formerly accounted for by the formation of a
little mercury sulphide, but now it is said that calcmel checks the putre-
faction and the decomposition of the bile-pigments, so that a part of the
bile-pigments passes into the feces as biliverdin. In the yolk-yellow or
greenish-yellow excrement of nursing infants one can detect bilirubin.
Neither bilirubin nor biliverdin seems to exist in the excrement of mature
persons under normal conditions. In adults under normal conditions
the feces contain neither bilirubin nor biliverdin. On the contrary, there
is found stercobilin (MASIUS and VANLAIR), which is identical with uro-
bilin (JAFFiD2). Bilirubin may occur in pathological cases in the feces
of mature persons. It has been observed in a crystallized state (as
hsematoidin) in the feces of children as well as of grown persons.

The absence of bile (acholic feces) causes the feces to have, as above
stated, a gray color, due to large quantities of fat; this may, however,
be partly attributed to the absence of bile-pigments. In these cases
a large quantity of crystals has been observed which consist principally
of magnesium soaps or sodium soaps. Hemorrhage in the upper parts
of the digestive tract yields, when it is not very abundant, a dark-brown
excrement, due to haematin.

1 In regard to the constitution of feces with various foods, see Hammerl, Kermauner,
Moeller, and Prausnitz, Zeitschr. f. Biologic. 35, and Poda, Micko, Prausnitz and
Miiller, ibid., 39.

2 See bile-pigments, Chapter VII, and urobilin, Chapter XIV.

MECONIUM. INTESTINAL CONCREMENTS. 523

EXCRETIN, so named by MARCET,1 is a crystalline body occurring in human
excrement, but which, according to HOPPE-SEYLER, is perhaps only impure choles-
terin (koprosterin or stercorin?). EXCRETOLIC ACID is the name given by MARCET
to an oily body with an excrementitious odor.

In consideration of the very variable composition of feces, quanti-
tative analyses are of little value and therefore will be omitted.2

Meconium is a dark brownish-green, pitchy, mostly acid mass without any
strong odor. It contains greenish-colored epithelium cells, cell-detritus, numer-
ous fat-globules, and cholesterin plates. The amount of water is 720-800,
and solids 280-200 p. m. Among the solids there are mucin, bile-pigments,
and bile-acids, cholesterin, fat, soaps, traces of enzymes, calcium and magnesium
phosphates. Sugar and lactic acid, soluble protein bodies and peptones, also
leucine and tyrosine and the other products of putrefaction occurring in the
intestine, are absent. Meconium may contain undecomposed taurocholic acid/
bilirubin and biliverdin, but it does not contain any stercobilin, which is con-
sidered as proof of the non-existence of putrefactive processes in the digestive
tract of the fetus.

The contents of the intestine under abnormal conditions are perhaps less the
subject of chemical analysis than of an inspection and microscopical investiga-
tion or bacteriological examination. On this account the question as to the
properties of the contents of the intestine in different diseases cannot be thor-
oughly treated here.3

Appendix.

INTESTINAL CONCREMENTS.

Calculi occur very seldom in the human intestine or in the intestine
of carnivora, but they are quite common in herbivora. Foreign bodies
or undigested residues of food may, when for some reason or other they
are retained in the intestine for some time, become incrusted with salts,
especially ammonium-magnesium phosphate or magnesium phosphate,
and these salts usually form the chief constituent of the concrements.
In man they are sometimes oval or round, yellow, yellowish-gray, or
brownish-gray, of variable size, consisting of concentric layers and
containing chiefly ammonium-magnesium phosphate and calcium phos-
phate, besides a small quantity of fat or pigment. The nucleus ordi-
narily consists of some foreign body, such as the stone of a fruit, a
fragment of bone, or something similar. SJOQVIST 4 has recorded an ex-
traordinary concrement consisting principally of fatty acids and a bile-acid.
In those countries where bread made from oat-bran is an important food,

1 Annal. de chim. et de phys., 59.

2 In regard to these analyses as well as to the feces under abnormal conditions
and to the pertinent literature, see Ad. Schmidt and J. Strassburger, Die Faeces des
Menschen, etc., Berlin, 1901 and 1902.

3 See Schmidt and Strassburger, 1. c.

4 Hygiea, Festband, 1908.

524 DIGESTION.

we often find in the large intestine, balls similar to the so-called hair-
balls (see below). Such calculi contain calcium and magnesium phos-
phate (about 70 per cent), oat-bran (15-18 per cent), soaps and fat (about
10 per cent). Concretions which contain very much fat (about 74 per
cent) occasionally occur, and those consisting of fibrin clots, sinews,
or pieces of meat incrusted with phosphates are also rare.

Intestinal calculi often occur in animals, especially in horses fed on bran.
These calculi, which attain a very large size, are hard and heavy (as much as 8
kilos) and consist in great part of concentric layers of ammonium-magnesium
phosphate. Another variety of concrements which occur in horses and cattle
consists of gray-colored, often very large, but relatively light stones which contain
plant residues and earthy phosphates. Stones of a third variety are sometimes
cylindrical, sometimes spherical, smooth, shining, brownish on the surface, con-
sisting of matted hairs and plant-fibers, and termed hair-balls. The so-called
" VEGAGROPIL.E," which occur in the ANTILOPE RUPICAPRA, belong to this group,
and are generally considered as nothing else than the hair-balls of cattle.

The so-called oriental bezoor-stone also belongs to the intestinal concrements,
and probably originates from the intestinal tract of the CAPRA .EGAGRUS and ANTE-
LOPE DORCAS. There may exist two varieties of bezoar-stones. One is olive-
green, faintly shining and formed of concentric layers. On heating it melts with
the development of an aromatic odor. It contains as chief constituent LITHOFELLIC
ACID, CjJEIseO^ which is related to cholic acid, and besides this a bile-acid, LITHO-
BILIC ACID. The others are nearly blackish brown or dark green, very glossy,
consisting of concentric layers, and do not melt on heating. They contain as
chief constituent ellagic acid, a derivative of gallic acid, of the formula CuHeOs,
which, according to GRABBED is the dilactone of hexaoxydiphenyldicarboxylic
acid, and which gives a deep-blue color with an alcoholic solution of ferric chlo-
ride. The last-mentioned bezoar-stone originates, to all appearances, from the
food of the animal.

Ambergris is generally considered an intestinal concrement of the sperm whale.
Its chief constituent is ambrain, which is a non-nitrogenous substance perhaps
related to cholesterin. Ambrain is insoluble in water and is not changed by boil-
ing alkalies. It dissolves in alcohol, ether, and oils.

VI. ABSORPTION.

The contents of the intestine are gradually pushed onward by the
peristalsis or rhythmical movement of the intestinal musculature, but
the mechanism is not well known.2 By these processes the intestinal
contents are intimately mixed and the constituents of the food which
are valuable to the organism are transformed, in the manner previously
mentioned, so that they are adaptable for the processes of absorption.
In discussing the absorption processes we must treat of the form into
which the different foods are changed before absorption, of the man-
ner in which this is accomplished, and lastly, of the forces which act
in these processes.
»

1 Ber. d. d. chem. Gesellsch., 36.

2 See Cannon, Amer. Journ. of Physiol., 6, 12, 29; Magnus, Pfliiger's Arch., 102,
103, 108, 111; Baumstark and Cohnheim, Zeitschr. f. physiol. Chem., 65.

ABSORPTION OF PROTEINS. 525

Before we can answer the question as to the form in which the pro-
teins are absorbed from the intestinal canal, it is of interest to learn
whether the animal body can, perhaps, also utilize such proteins as are
introduced intravenously, subcutaneously, or into a body-cavity, i.e.,
evading the intestinal canal, or, as it is called parenteral.

Since the first investigations of ZTJNTZ and v. MERING on this sub-
ject, several experimenters l have shown, without any doubt, that the
animal body can more or less completely utilize different, parenterally
introduced proteins, although different varieties of animals show a differ-
ence in this regard. Still we do not know where and how these foreign
proteins are changed and assimilated, but CRAMER ascribes great impor-
tance to the leucocytes in this regard. See ABDERHALDEN'S experiment
given on page 54.

That the animal body can also assimilate not previously digested
or split proteins introduced directly into the intestine has been shown
by BRUCKE, BAUER and VOIT, EICHHORST, CZERNY and LATSCHEN-
BERGER, VOIT and FRIEDLANDER, and others.2 In the experiments
of the two last-mentioned investigators neither casein (as milk) nor
hydrochloric-acid myosin or acid albuminate (in acid solution) was
absorbed, while, on the contrary, about 21 per cent of ovalbumin or
seralbumin and 69 per cent of alkali albuminate (dissolved in alkali)
were absorbed. MENDEL and ROCKWOOD, on the contrary, in experi-
ments with casein and edestin in the living intestinal loop, could prove
only the slightest absorption on excluding digestion as completely as
possible, while the corresponding proteoses were abundantly absorbed.

It is difficult to decide in these experiments as to how far the pro-
teins were taken up in an actually unchanged or partly modified form.
The alimentary albuminaria, observed repeatedly after the introduction
of large quantities of protein into the intestinal canal, indicates an
absorption of undigested protein under certain circumstances. To decide
this question the biological method, using the precipitine reaction, has
been made use of, and ASCOLI and ViGNO,3 using this method, claim to

1 Zuntz and v. Mering, Pfliiger's Arch., 32; Neumeister, Verb. d. phys.-med.
Gesellsch. zu Wiirzburg, 1889, and Zeitschr., f. Biologie, 27; Friedenthal and Lewan-
dowsky, Arch. f. (Anat. u.) Physiol., 1899; Munk and Lewandowsky, ibid., 1899,
Supp.; Oppenheimer, Hofmeister's Beitrage, 4; Mendel and Rockwood, Amer. Journ.
of Physiol., 12; Heilner, Zeitschr. f. Biol., 50, and Munch, med. Wochenschr., 49;
Cramer, Journ. of Physiol., 37, with Pringle, ibid.; Rona and Michaelis, Pfliiger's
Arch., 123 and 124; v. Korosy, Zeitschr. f. physiol. Chem. 62, 68 (1909), 69, 313 (1910).

2 Briicke, Wien. Sitzungsber., 59; Bauer and Voit, Zeitschr. f. Biologie, 5; Eich-
horst, Pfliiger's Arch., 4; Czerny and Latschenberger, Virchow's Arch., 59; Voit and
Friedlander, Zeitschr. f. Biologie, 33. Contradictory observations can be found in
Keller, Beitr. z. Frage d. Resorption im Dickdarm. Inaug.-Dissert. Breslau, 1909.

3 Zeitschr. f . Physiol. Chem., 39.

526 DIGESTION.

have shown the passage of non-modified protein into the blood and
lymph (page 66). Based upon many investigations on this subject
we can consider it possible that under certain circumstances, as on flood-
ing the intestinal canal with protein, with a greater permeability of the
intestinal wall, as in new-born and sucking animals, and with a dimin-
ished modification by the gastric juice, a passage of non-modified pro-
tein may take place in the blood-vessels, but that under normal con-
ditions this is not the case, or at least does not take place to any men-
tionable degree. As a rule, the absorption of protein follows a modi-
fication of it. In this connection the experiments of ORNI l are of interest
which show that the dog's intestine takes up the serum of the dog but
not that of the ox or horse. In regard to the previously split proteins
the question arises whether the proteins are chiefly absorbed as pro-
teoses or peptones or as simpler atomic complexes.

According to the earlier investigations of LUDWIG and SCHMIDT-
MULHEIM, as well as those of MUNK and RosENSTEiN,2 it is generally
agreed that the products of protein digestion do not pass into the
blood through the lymph vessels, but through the intestinal capillaries,
The question of the absorption of these products resolves itself into
the form in which they are taken up by the intestine and the form in
which they pass into the blood.

It was mentioned above that proteoses and peptones as well as non-
biuret-giving products and amino-acids have been found in the con-
tents of the intestine. The amino-acids occur to a less extent than the
proteoses and peptones. This may indicate that the amino-acids are
more abundantly formed, but also more quickly absorbed, but it may
also indicate that the amino-acids are produced to a slight extent only,
in the intestinal contents. There is no doubt that the amino-acids can
be absorbed as such, but there is still another question, namely, whether
the proteoses and peptones are absorbed as such or only after a pre-
vious cleavage into amino-acids.

NOLF and HONORE found, what was later substantiated by ZuNZ,a
that the proteos.es and peptones disappear more quickly from the
intestine than the non-biuret-giving products. This does not prove
that the proteoses are absorbed as such, but rather against such a view.
A more direct proof for the absorption of the non-split proteoses lies
in the fact, as shown by NOLF, that the proteoses when introduced in

1 Pfliiger's Arch. 126, 428 (1909).

2 Schmidt-Mulheim, Arch. f. (Anat. u.) Physiol., 1877; Munk and Rosenstein.
Virchow's Arch., 123.

3 Nolf and Honore, Arch, internal, de Physiol., 1905; Nolf, Journ. de Physiol. et
Pathol. ge"n., 1907; Zunz, MSmoires cour., etc., Acad. Roy. Med., Belg., 20, Fasc. 1.

ABSORPTION OF PROTEINS. 527

large quantities in the intestine pass in small amounts into the blood.
Another proof is the findings of BoRCHARDT,1 that after feeding dogs
with not too large amounts of elastin, the passage of a proteose, the
hemielastose, could be detected in the blood. Attention must also be
called to the fact that according to HoFMEiSTER2 the walls of the
stomach and intestine are the only parts of the body in which pro-
teoses and peptones occur during digestion.

We have reason for believing that the proteoses, as well as their
cleavage products, are taken up by the intestine, and if this is the case
the next question to be answered is, in what form do these bodies leave
the intestine and pass into the blood?

In order to decide this question the blood has been repeatedly
tested in regard to the quantity of proteoses. As seen on page 264
this has led to very contradictory results, and if we exclude those
exceptional cases where a large quantity of proteose was introduced
into the intestine at once, then we can say that the occurrence of pro-
teoses in the blood, or at least in the blood-plasma, has not been posi-
tively shown under physiological conditions.3 It can also be said that
such investigations do not prove much because of the large quantity
of blood passing through the intestine for a given time, and the quan-
tity of proteose must be so small, so that when divided in the entire blood
it can hardly be detected. It is therefore of interest that neither amino-
acids nor proteoses were found in the blood after cutting out several
organs or groups of organs so that the blood circulated only through
the intestinal canal, heart, lungs, pancreas and intercostal muscles
(KUTSCHER and SEEMANN, v. KoROSY4).

We are therefore obliged to consider that the proteoses and amino-
acids are transformed in the intestinal walls in some manner or other.
Such a belief, especially applied to the proteoses, coincides with the
observations of HOFMEISTER, that the proteoses occurring in the mucous
membrane during digestion disappear at the temperature of the room
from the removed, but still apparently living, mucous membrane after a
certain time. This also coincides well with the observations of LUDWIG
and SALViOLi.5 These investigators introduced a peptone solution into
a double-ligatured, isolated piece of the small intestine, which was kept
alive by passing defibrinated blood through it, and observed that the

1 In regard to the literature on proteoses in the blood see Chapter V, footnotes
1, 2 and 3, p. 264.

2 Zeitschr f. physiol. Chem., 6, and Arch. f. exp. Path. u. Pharm., 19, 20, 22.
8 See footnote 1.

4 Kutscher and Seemann, Zeitschr. f. physiol. Chem., 34; v. Korosy, ibid., 57.
6 Arch. f. (Anat. u.) Physiol., 1880, Supplbd. See also Cathcart and Leathes,
Journ. of Physiol., 33.

528 DIGESTION.

*

peptone disappeared from the intestine, but that the blood passing
through did not contain any peptone.

What becomes of the amino-acids in the intestinal wall? KUTSCHER
and SEEMANN have shown that the crystalline cleavage products are
so transformed in the intestinal wall that they cannot be detected.
We have here to think of two possibilities : The amino-acids are either
further split or they are used in synthesis (of proteins?).

It is a long-known fact that with the digestion and absorption an
increased elimination of nitrogen in the urine goes hand in hand. The
quantity of nitrogen eliminated in the urine after partaking of protein
corresponded, according to ASHER and HAAS/ to 65 per cent of the
nitrogen introduced. It is hardly credible that this elimination of
nitrogen depends upon an increased destruction of body protein, and
it is more probable that this represents decomposed food-protein.
But according to NENCKI and ZALESKI 2 an abundant formation of
ammonia occurs in the cells of the digestive apparatus after a rich
protein diet, so we must consider the possibility that a considerable part,
perhaps the very greatest part, of the amino-acids are deamidized in
the intestinal wall. The other part of the amino-acids may be used
in the syntheses to be mentioned below. Such a partial deamidization
of the digestive products has been shown by COHNHEIM 3 in his absorp-
tion experiments with the fish intestine.

The proteoses taken up by the intestinal mucosa, if this does take
place, can naturally undergo a further conversion into amino-acids in
the walls of the intestine. Still there are other possibilities. A direct
utilization of the proteoses in the synthesis of the proteins in the intes-
tine is not very probable, but on the contrary it is more probable that
the proteoses, in order to undergo further cleavage or further utiliza-
tion, are taken up by the leucocytes and carried off. HOFMEISTER
has advocated such a possibility for a long time. HEIDENHAIN raised
objections to this suggestion in which he called attention to the dis-
proportion between the number of leucocytes and the large quantity of
peptones (proteoses) to be absorbed, but at that time the deep cleavage
of a great part of the protein into amino-acids was not known. Recently
PRINGLE and CRAMER4 urged the theory of the importance of the
leucocytes, and the observations of iNAGAKi5 also show the possibility

1 Bioch. Zeitschr., 12.

2 Arch, des scienc. biol. de St. Pe"tersbourg, 4; Arch. f. exp. Path. u. Pharm., 37;
see also Salaskin, Zeitschr. f. Physiol. Chem., 25.

1 Zeitschr. f . physiol. Chem., 59.

4 Hofmeister, 1. c.; Heidenhain, Pfluger's Arch., 43; Pringle and Cramer, Journ.
of Physiol., 37.

6 Zeitschr. f. physiol. Chem., 50.

ABSORPTION OF PROTEINS. 529

of the leucocytes taking up the proteoses and fixing them, it seems, in
the cell substance.

It is for the present impossible to say with certainty whether or
not and to what extent the proteoses, as such, are absorbed and to give
their further fate thereafter in the intestine. The present view is prob-
ably as follows: That they do not pass as such into the blood, and
that they are transformed into amino-acids in part in the intestinal
contents and in part in the intestinal mucosa, and then from these amino-
acids the coagulable proteins are constructed by synthesis. In sup-
port of the theory of a protein synthesis from amino-acids we have a
series of experiments where deeply split or completely split proteins
were fed. In these experiments by LOEWI, HENDERSON and DEAN,
HENRIQUES and HANSEN, and especially by ABDERHALDEN and his
co-workers l on dogs, mice and rats, it was possible to keep the animals
in nitrogenous equilibrium or indeed nitrogen retention for a long time
with the cleavage products of proteins besides non-nitrogenous food-
stuffs and salts. According to the recent experiments of ABDERHALDEN
the organism can build up proteins from amino-acids when the indi-
vidual amino-acids are supplied in proportions as they exist in the cell
proteins. Certain, sometimes absent amino-acids seem to be capable
of being produced within the organism (for example, glycocoll, proline)
while other (tryptophane) cannot be produced. This explains why
gelatine which does not contain any tryptophane cannot replace protein
in the food.

The results of the experiments are generally considered as proof of
the ability of the animal body to construct proteins from amino-acids
by synthesis, and in the present state of our knowledge we can hardly
draw other conclusions from them or advance any simpler theory.

Where does the protein synthesis take place? If it were positively
sure that the amino-acids did not pass into the blood then we would
have transferred this synthesis to the intestinal walls. Otherwise we
must think in the first place of the liver; but this organ does not seem
to play an important role in this synthesis. ABDERHALDEN and LONDON 2
made an experiment on a dog with an ECK fistula (see page 397), feeding
the dog with decomposed protein, and they found that this animal
behaved exactly like a normal animal, as it was kept for eight days not
only in nitrogenous equilibrium but also in nitrogen retention. On

1 Loewi, Arch. f. exp. Path. u. Pharm., 48. See also Henderson and Dean, Amer.
Journ. of Physiol., 9; Abderhalden and Rona, Zeitschr. f . physiol. Chem., 42, 44, 47, and
52; Henriques and Hansen, ibid., 43, 49; Henriques, ibid., 54; Abderhalden with
Olinger, ibid., 57, with Messner and Windrath, ibid., 59; Abderhalden, ibid., 77, 22,
78, 1 (1912).

2 Zeitschr. f. physiol. Chem., 54.

530 DIGESTION.

the other hand it is not possible to deny the importance of the liver for
the protein syntheses. As EMBDEN and his collaborators have shown on
perfusing the liver containing a large amount of glycogen, that d-alanine
was formed and EMBDEN explains this formation by a destruction of
glucose or lactic acid and pyroracemic acid. With experiments with
blood perfusion of the liver, a-amino-acids are formed from the am-
monium salts of the corresponding a-keto-acids. The combination
NH4.O.CO.CO.R passes into HO.CO.CH(NH2).R. The cleavage
products of the carbohydrates can be converted in the liver into char-
acteristic constituents of the protein molecule.1 In this connection
we must here mention the experiments of LUTHJE 2 in which he found
a nitrogen retention after feeding only one amino-acid with abundance
of carbohydrate.

What kind of protein is formed in the synthesis? This we do not
know. ABDERHALDEN'S belief is that it is plasma protein, which, as
is well known, is the same in each animal independent of the kind of
protein introduced with the food and from which the cells of the body
then create the further protein material. Objections can be raised
against this hypothesis, but still it is worth consideration. In favor
of this we can also add that according to the investigations of FREUND
and v. KoROSY3 the blood coming from the intestine during digestion
is richer in coagulable protein than other blood, and also that this
protein, FREUND asserts, belongs to the globulin group. This globulin,
according to FREUND and TOEPFER, is not identical with the ordinary
serglobulin mixture, but is a pseudoglobulin formed in the intestine
from the food protein by synthesis, and which is more easily decom-
posed or further utilized in the liver and other organs. Further research
in this direction is necessary, as we have other investigations which
are essentially different. If a re-formation of coagulable proteins takes
place from amino-acids during digestion, it is to be expected that a
relatively greater quantity of coagulable protein should occur hi the
mucosa of the digesting intestine as compared with the non-digesting
intestine. PRINGLE and CRAMER, by a method which requires con-
firmation, claim that in the digesting animal (cat), the blood, and to a
still higher degree the intestinal mucosa, and especially the lymph nodes
of the intestine, are richer in non-coagulable protein than the starving
animal, a condition which is related to the r61e of the leucocytes in the

Zeitschr. 29, 423 (1910); 38, 393, 407, 414 (1911); 45, 1-207 (1912);
summary, 45, 201.

2 Pfliiger's Arch. 113, 547 (1906).

3v. Korosy, Zeitschr. f. physiol. Chem., 57; Freund, Zeitschr. f. exp. Path. u.
Therap., 4; G. Toepfer and Freund, and Toepfer, ibid., 3; Pringle and Cramer, Joura,
of Physiol., 37.

ABSORPTION OF PROTEINS. 531

protein assimilation. This question of the absorption of proteins in
the intestine is still unexplained in many directions.

The extent of the protein absorption is dependent essentially upon
the kind of food introduced, since as a rule the protein substances from
an animal source are much more completely absorbed than from a
vegetable source. As proof of this the following observations are
given : In his experiments on the utilization of certain foods in the intes-
tinal canal of man, RUBNER found that with an exclusively animal diet,
on partaking of an average of 738-884 grams of fried meat, or 948 grams
of eggs per day, the nitrogen deficit with the excrement was only 2.5-2.8
per cent of the total nitrogen introduced. With a strictly milk diet
the results were somewhat unfavorable, since after partaking of 4100
grams of milk the nitrogen deficit increased to 12 per cent. The con-
ditions are quite different with vegetable food, as shown by the re-
searches of MEYER, RUBNER, HULTGREN and LANDERGREN, who made
experiments with various kinds of rye bread and found that the loss of
nitrogen through the feces amounted to 22-48 per cent. Experiments
with other vegetable foods, and also the investigations of SCHUSTER,
CRAMER, MEINERT, MORI/ and others on the utilization of foods with
mixed diets, have led to similar results. With the exception of rice,
wheat bread, and certain very finely divided vegetable foods, it is found
in general that the nitrogen deficit by the feces increases with a larger
quantity of vegetable material in the food.

The reason for this is manifold. The large quantity of cellulose
frequently present in vegetable foods impedes the absorption of pro-
teins. The greater irritation produced by the vegetable food itself or
by the organic acids formed in the fermentation in the intestinal canal
causes a more violent peristalsis, which drives the contents of the intes-
tine faster than otherwise along the intestinal canal. Another and most
important reason is the fact that a part of the vegetable protein sub-
stances seems to be indigestible, and because of the difficultly digestible
vegetable food, a large quantity of digestion fluids containing nitrogen
is secreted.

In speaking of the functions of the stomach we stated that after
the removal or excision of this organ, an abundant digestion and absorp-
tion of proteins may take place. It is therefore of interest to learn how
the digestion and absorption of proteins go on after the extirpation of
the second protein-digesting organ, the pancreas. In this connection

1 Rubner, Zeitschr. f. Biologie, 15; Meyer, ibid., 7; Hultgren and Landergren,
Nord. med. Arch., 21; Schuster, in Volt's " Untersuch. d. Kost," etc., 142; Cramer,
Zeitschr. f. physiol. Chem., 6; Meinert, " Ueber Massennahrung," Berlin, 1885; Kell-
ner and Mori, Zeistchr. f. Biologie, 25.

532 DIGESTION.

there are the observations on animals after complete or partial extirpa-
tion of the gland by MINKOWSKI and ABELMANN, SANDMEYER, V. HAR-
LEY, after destroying the gland by ROSENBERG, and also in man after
closing the pancreatic duct by HARLEY and DEUCHER. In all these
cases such discrepancy of figures has resulted for the utilization of the
proteins — between 80 per cent after the apparently complete exclusion
of pancreatic juice in man (DEUCHER) and 18 per cent after extirpa-
tion of the gland in dogs (HARLEY) — that one can hardly draw any
clear conception as to the extent and importance of the trypsin diges-
tion in the intestine. That on completely preventing the entrance of
pancreatic juice only a slight diminution in the protein absorption takes
place follows from the researches of LOMBROSO and NiEMANN.1 In
order to understand, in these cases, why the digestion and absorption
took place so abundantly it would be of interest to know how other
digestion fluids act substitutingly. In this regard ZUNZ and MAYER2
found that in dogs (meat digestion) the tying of the pancreatic passages
is essentially compensated for by an increased secretion of pepsin and
other proteolytic enzymes, and that in this case the demolition of the
protein in the stomach goes further than in a normal animal.

The carbohydrates are, it seems, chiefly absorbed as monosaccharides.
Glucose, fructose, and galactose are probably absorbed as such. The
two disaccharides, saccharose and maltose, ordinarily undergo an inver-
sion in the intestinal tract and are converted into glucose and fructose.
Lactose is also, at least in certain animals, inverted in the intestine.
In other mature animals, on the contrary, if the lactase formation is not
excited by milk food, the sugar is not inverted or only to a slight
extent (VoiT and LUSK, WEINLAND, PORTIER, ROHMANN and NAGANO),
and it probably is absorbed as such in these animals if it does not under-
go fermentation, or, as ROHMANN and NAGANO 3 assumed, if it is not
transformed in the intestinal mucosa in some unknown way. An
absorption of non-inverted carbohydrates is not improbable, and accord-
ing to OTTO and v. MERING the portal blood contains, after a carbo-
hydrate diet, besides glucose, a dextrin-like carbohydrate. MoscATi4

1 Abelmann, " Ueber die Ausniitzung der Nahrungsstoffe nach Pankreasexstirpa-
tion" (Inaug.-Dissert. Dorpat, 1890), cited from Maly's Jahresber., 20; Sandmeyer,
Zeitschr. f. Biologic, 31; Rosenberg, Pfliiger's Arch., 70; Harley, Journ. of Pathol.
and Bacteriol., 1895; Deucher, Correspond. Blatt. f. Schweiz. Aerzte, 28; Lombroso,
Arch. f. exp. Path. u. Pharm., 60; Niemann, Zeitschr. f. exp. Path. u. Therap., 5;
See also Brugsch and Pletnew, Zeitschr. f. exp. Path. u. Therap. 6, 326.

2 Mem. de 1'acad. roy. de me"dic. de Belg., 18.

3 Voit and Lusk, Zeitschr. f. Biologic, 28; Rohmann and Nagano, Pfliiger's Arch.,
95, which contains the references to the literature.

4 Otto, see Maly's Jahresber., 17; v. Mering, Arch. f. (Anat., 4.) Physiol., 1877;
Moscati, Zeitschr. f. physiol. Chem., 50

ABSORPTION OF CARBOHYDRATES. 533

believes that when homogeneous starch solutions are injected intra-
venously or subcutaneously, the starch is taken up by the organs, namely
the spleen, liver and lungs, and is utilized as the starch can be changed
into glycogen. A part of the carbohydrates is destroyed by fermenta-
tion in the intestine, with the formation of lactic and acetic acids and
other bodies.

The different varieties of sugars are absorbed with varying degrees
of rapidity, but as a general thing absorption occurs very quickly. This
absorption takes place more quickly in the upper part of the intestine
than in the lower part (ROHMANN, LANNOIS and LUPINE, ROHMANN
and NAGANO 1). It is generally admitted that the simpler sugars are
more quickly absorbed than the disaccharides, while the reports as to
the absorption of the disaccharides differ somewhat (HEDON, ALBER-
TONI, WAYMOUTH REID, ROHMANN and NAGANO). There seems to be
no doubt that lactose is absorbed more slowly than the two other disac-
charides. According to the extensive experiments of ROHMANN and
NAGANO, saccharose is absorbed more quickly than maltose. NAGANO 2
contends that the pentoses are absorbed more slowly than hexoses.

On the introduction of starch even in very considerable quantities
into the intestinal tract no glucose passes into the urine, a condition
which probably depends in this case upon the absorption and assimila-
tion and the slow saccharification taking place simultaneously. If,
on the contrary, large quantities of sugar are introduced at one time,
then an elimination of sugar by the urine takes place, and this elimina-
tion of sugar is called alimentary glycosuria. In these cases the assimila-
tion of the sugar and the absorption do not take place together.

That quantity of sugar to which we must raise the ingested sub-
stance in order to produce an alimentary glycosuria gives, according
to HoFMEisTER,3 the assimilation limit for that same sugar. This limit
is different for various kinds of sugar; and it also varies for the same
sugar not only in different animals, but also in different members of the
same species, as also in the same individual under varying circum-
stances. In general it can be said that in regard to the ordinary varie-
ties of sugar, such as glucose, fructose, galactose, saccharose, maltose,
and lactose, the assimilation limit is highest for glucose and lowest for
lactose. It must be admitted that with an overabundant quantity of
sugars in the intestinal tract the disaccharides do not have sufficient
time for their complete inversion, and this has been directly shown by

1 Lannois and Lepine, Arch, de physiol. (3), 1; Rohmann, Pfliiger's Arch., 41; see
also footnote 3, p. 532.

2 In regard to the literature on the absorption of sugars, see footnote 3, p. 532.
» Arch. f. exp. Path. u. Pharm., 25 and 26.

534 DIGESTION.

ROHMANN and NAGANO. It is, therefore, not remarkable that disac-
charides, as well, have been found in the urine in cases of alimentary
glycosuria.1

The investigations of LUDWIG and v. MERING and others have
explained how the sugars enter into the blood-stream, namely, that they
as well as other bodies soluble in water do not ordinarily pass over into
the chylous vessels in measurable quantities, but are chiefly taken up by
the blood in the capillaries of the villi, and in this way pass into the mass
of the blood. These investigations have been confirmed by observa-
tions of I. MUNK and ROSENSTEIN 2 on human beings.

The reason why the sugars and other soluble bodies do not pass
over into the chylous vessels in appreciable quantity is, according to
HEiDENHAiN,3 to be found in the anatomical conditions, in the arrange-
ment of the capillaries close under the layer of epithelium. Ordinarily
these capillaries find the necessary time for the removal of the water
and the solids dissolved in it. But when a large quantity of liquid,
such as a sugar solution, is introduced into the intestine at once, this is
not possible, and in these cases a part of the dissolved bodies passes into
the chylous vessels and the thoracic duct (GINSBERG and ROHMANN 4).

The passage of sugar into the urine, when at one time large quanti-
ties of sugar are taken and the assimilation limit is exceeded, can be
best explained by the assumption that a part of the sugar escaped the
liver and passed into the large circulation, or that the liver did not have
time to retain the sugar and transform it into glycogen. According to
the observations of de FiLiPPi5 upon dogs with ECK fistula, it seems as if
the r61e of the liver in these cases is too highly estimated. An animal
with ECK fistula could take an unlimited quantity of starch without
glycosuria occurring. The assimilation limit was in these cases some-
what lower, but qualitatively they behave like normal animals and with
increasing sugar supply they could also retain increasing quantities of
sugar.

The introduction of larger quantities of sugar into the intestine at
one time can readily cause a disturbance with diarrheal evacuations
of the intestine. If the carbohydrate is introduced in the form of starch,

1 For the literature in regard to the passage of various kinds of sugars into the
urine, see C. Voit. Ueber die Glykogenbildung, Zeitschr. f. Biologic, 28, and F. Voit,
footnote 1, p. 396. See also Blumenthal, Zur Lehre von der Assimilationsgrenze
der Zuckerarten, Inaug.-Dissert. 1903, Strassburg and Brasch, Zeitschr. f. Biol., 50.

2 v. Mering. Arch. f. (Anat. u). Physiol., 1877; Munk and Rosenstein, Virchow's
Arch. 123.

3 Pfluger's Arch., 43, Suppl.

4 Ginsberg, Pfluger's Arch., 44; Rohmann, ibid., 41.

5 Zeitschr. f. Biol., 49 and 50.

ABSORPTION OF FATS. 535

then very large quantities may be absorbed without causing any dis-
turbance, and the absorption may be very complete. RUBNER found
the following: On partaking 508-670 grams of carbohydrates, as wheat
bread, per day, the part not absorbed amounted to only 0.8-2.6 per cent.
For peas, where 357-588 grams were eaten, the loss was 3.6-7 per cent,
and for potatoes (718 grams) 7.6 per cent. CONSTANTINIDI found on
partaking 367-380 grams of carbohydrates, chiefly as potatoes, a loss
of only 0.4-0.7 per cent. In the experiments of RUBNER, as also of
HULTGREN and LANDERGREN,1 with rye bread the utilization of car-
bohydrates was less complete, and the loss in a few cases rose even to
10.4-10.9 per cent. It at least follows from the experiments made thus
far that man can absorb more than 500 grams of carbohydrates per diem
without difficulty.

We generally consider the pancreas as the most important organ
in the digestion and absorption of amylaceous bodies, and it is a ques-
tion how these bodies are absorbed after the extirpation of the pan-
creas. As on the absorption of proteins, so also on the absorption of
starch, the observations have given variable results. In certain cases
the absorption was not impaired, while in others it was, on the contrary,
rather diminished, and with dogs devoid of pancreas it has been found
that the absorption was decreased to 50 per cent of the starch partaken
(ROSENBERG, CAVAZZANi2).

Em unification used to be considered as of the greatest importance
in the absorption of fats, and this emulsion occurs in the chyle on the
introduction into the intestine of not only neutral fats, but also of fatty
acids. The fatty acids do not exist as such in the emulsified fat of the
chyle. The investigations of I. MUNK, later confirmed by others, have
shown that the fatty acids undergo in great part a synthesis into neutral
fats in the walls of the intestine, and are carried as such by the stream
of chyle into the blood. This synthesis seems to take place in the
mucous membrane (MOORE and others 3) .

The assumption that the fat is absorbed chiefly as an emulsion is
partly based on the abundance of emulsified fat in the chyle after feed-
ing with fat, and partly on the fact that a fat emulsion is often found
in the intestine after such food. As an abundant cleavage of neutral

1 Rubner, Zeitschr. f. Biologie, 15 and 19; Constantinidi, ibid., 23; Hultgren and
Landergren, Nord. med. Arch. 21.

2Cavazzani, Centralbl. f. Physiol., 7. See footnote 1, p. 532; also Lombroso,
Hofmeister's Beitrage, 8.

3 Munk, Virchow's Arch., 80. See also v. Walther, Arch. f. (Anat. u.) Physiol.,
1890; Minkowski, Arch. f. exp. Path. u. Pharm., 21; Frank, Zeitschr. f. Biologie,
36; Moore, see Biochem. Centralbl., 1, 741; Frank and Hitter, Zeitschr. f. Biologie,
47; Noll, Pfliiger's Arch. 136.

536 DIGESTION.

fats occurs in the intestinal canal, and also as the fatty acids do not
occur in the chyle as such, but as emulsified fat after a synthesis with
glycerin into neutral fats, it is to be doubted whether the emulsified
fat of the chyle originates from an absorption of emulsified fat in the
intestine or from a subsequent emulsification of neutral fats formed
synthetically. This doubt has greater warrant in the observation of
FRANK l that the fatty-acid ethyl ester is extensively taken up from the
intestine, not as such, but as split-off fatty acids from which then the
neutral emulsified fats of the chyle are formed.

The assumption of an absorption of fats as an emulsion is inconsist-
ent with the fact that an emulsion produced by means of soaps is not
permanent in an acid liquid; hence we cannot consider as possible the
presence of an emulsion in the intestine so long as it is acid. This
difficulty is not too serious, as the reaction is often only due to carbonic
acid and bicarbonates, and besides as found by RUHNE and recently
shown by MOORE and KRUMBHOLZ,2 the proteins have a preserving
action upon fat emulsions.

The earlier opinions as to fat absorption were, that fat was absorbed
as soaps, soluble in water, as well as finely emulsified fat, and this last
form was considered as of the greatest importance. This view has
recently undergone essential modifications, due to the work of MOORE
and ROCKWOOD, and especially to the extensive work of PFLUGER.3

MOORE and ROCKWOOD have shown the great solvent action of the
bile for fatty acids, and on continuing these investigations further,
MOORE and PARKER have found that the bile increases the solubility
of soaps in water, and can prevent their gelatinization, a fact which is
of greater importance for the absorption of fats than the solubility of
the fatty acids in bile. The quantity of lecithin in the bile is of great
importance for the solubility therein of the fatty acids as well as the
soaps. According to the above-mentioned investigators, the absorption
of fat from the intestine is essentially dependent upon the solubility of
the soaps and free fatty acids in the bile. The neutral fats are split
and the free fatty acids are in part absorbed, dissolved as such by the
bile, and in part combined with alkalies, forming soaps. Neutral fats
are regenerated from the fatty acids, and the alkali set free from the
soaps is secreted again into the intestine and used for the re-formation

1 Zeitschr. f. Biologie, 36.

2 Kiihne, Lehr. der physiol. Chem., 122; Moore and Krumbholz, Journ. of Physiol.,
22.

3 In regard to the recent literature on fat absorption, see the works of Pfliiger,
Pfluger's Arch., 80, 81, 82, 85, 88, 89, and 90, where the work of other investigators is
cited and discussed. See also Croner, Bioch. Zeitschr. 23; Lombroso, Arch, di Fisiol. 5.

ABSORPTION OF FATS. 537

of soaps. According to CRONER the absorption of soaps occurs only
in the lower parts of the small intestine.

The importance of the bile, the soaps, and the alkali carbonates has
been closely studied, principally in the very thorough investigations of
PFLUGER. He has quantitatively determined the solvent power of
the above-mentioned bodies — each alone as well as different mixtures
of these — for the various fatty acids, and has closely studied the mode
of action of the bile. From his investigations he has arrived at the
conclusion that no unsplit fat is absorbed, that all fats, before their
absorption, must first be split into glycerin and fatty acids, and that the
bile, on account of its solvent power for soaps and fatty acids, is sufficient
for the absorption of large quantities of fat eaten. The object of the
formation of an emulsion is, according to this view, that the fat in this
condition forms such a large surface for the action of the steapsin or
the fat-splitting agents. The possibility that all the fat must be first
split and that no unsplit fat is absorbed is, according to these researches,
not to be denied.

The next question is whether all the fat or the greater part of it
passes into the blood through the lymphatics and the thoracic duct-
According to the researches of WALTHER and FRANK 1 on dogs, it seems
that only a small part of the fats, or at least of the fatty acids fed,
passes into the chylous vessels; but these observations can hardly be
applied to the absorption of neutral fats, or to the absorption in man
under normal circumstances. MUNK and RosENSTEiN,2 in their inves-
tigations on a girl with a lymph fistula, found 60 per cent of the fat
ingested in the chyle, and of the total quantity of fat i^n the chyle only
4-5 per cent existed as soaps. On feeding with a foreign fatty acid,
such as erucic acid, they found 37 per cent of the introduced body as
neutral fat in the chyle. Not all the fat introduced is found in the
chyle, and there is always a not inconsiderable part of the absorbed
fat whose fate we are not able to follow.

The completeness with which fats are absorbed depends, under nor-
mal conditions, essentially upon the kind of fat. In this regard it is
known, especially from the investigations of MUNK and ARNSCHiNK,3
that the varieties of fat with high melting-points, such as mutton-tallow,
and especially stearin, are not so completely absorbed as the fats with
low melting-points, such as hog- and goose-fat, olive-oil, etc. The kind
of fat also has an influence on the rapidity of absorption, as MUNK and
ROSENSTEIN found that solid mutton-fat was absorbed more slowly

1 Walther, Arch. f. (Anat. u.) Physiol., 1890; Frank, ibid., 1892.

2 Virchow's Arch., 123.

3 Munk, Virchow's Arch., 80 and 95; Arnschink, Zeitschr. f. Biologic.

538 DIGESTION.

than fluid lipanin. The extent of absorption in the intestinal tract is,
under physiological conditions, very considerable. In the case of a
dog investigated by VOIT it was found that out of 350 grams of fat
(butter) partaken, 346 grams were absorbed from the intestinal canal,
and according to the investigations of RUBNER 1 the human intestine
can absorb over 300 grams of fat per diem. The fats are, according
to RUBNER, much more completely absorbed when free, in the form of
butter or lard, than when inclosed in cell-membranes, as in bacon.

CLAUDE BERNARD showed long ago with experiments on rabbits in
which the ductus choledochus was made to open into the small intestine
above the pancreatic duct, that after food rich in fats the chylous vessels
of the intestine above the pancreas passages were transparent, while
below they were milk-white, and also that the bile alone cannot pro-
duce an absorption of the emulsified fat without the pancreatic juice.
DASTRE 2 has performed the reverse experiment on dogs. He tied the
ductus choledochus and adjusted a biliary fistula so that the bile flowed
into the intestine below the mouth of the pancreatic passages. On
killing the animal after a meal rich in fat the chylous vessels were first
found milk-white below the discharge of the biliary fistula. From this
DASTRE draws the conclusion that a combined action of the bile and pan-
creatic juice is important in the absorption of fats — a conclusion which
stands in accord with the experience of many others.

Through numerous observations of many investigators, such as
BIDDER and SCHMIDT, VOIT, ROHMANN, FR. MULLER, I. MUNK,S and
others, it has been shown that the exclusion of the bile from the intes-
tinal tract diminishes the absorption of fat to such an extent that only
one-seventh to about one-half of the quantity of fat ordinarily absorbed
undergoes absorption. In icterus with entire exclusion of the bile, a
considerable decrease in the absorption of fat is noticed. As under
normal conditions, so also in the absence of bile in the intestine, the lower
melting parts of the fat are more completely absorbed than those which
have a high melting-point. I. MUNK found in his experiments on dogs
with lard and mutton-tallow that the absorption of the high-melting
tallow was reduced twice as much as the lard on the exclusion of the
bile from the intestine.

We also learn from the investigations of ROHMANN and I. MUNK
that in the absence of bile the relation between fatty acids and neutral
fats is changed, namely, about 80-90 per cent of the fat existing in the

1 Voit, Zeitschr. f. Biologie, 9; Rubner, ibid., 15.

2 Arch, de Physiol. (5), 2.

3 F. Miiller, Sitzungsber. der phys.-med. Gesellsch. zu Wiirzburg, 1885; I. Mimk,
Virchow's Arch., 122. See also footnotes 4 and 5, p. 518.

ABSORPTION OF FATS. 539

feces consists of fatty acid, while under normal conditions the feces
contain 1 part neutral fat to about 2-2J parts free fatty acids. It is
not possible to state how this increased quantity of fatty acids in the fat
of the feces is produced upon the exclusion of the bile from the intestine.

There is no doubt that the bile is of great importance in the absorp-
tion of fats. Still there is also no doubt that rather considerable quan-
tities of fat may be absorbed from the intestine in the absence of bile.
What relation does the pancreatic juice bear to this fact?

Upon this point a rather large number of observations on animals
have been made by ABELMANN and MINKOWSKI, SANDMEYER, HARLEY,
ROSENBERG, H^DON and VILLE, and also on man by FR. MULLER and
DEUCHER.1 In all of these investigations a more or less diminished
absorption of fat was observed after the extirpation or destruction of
the gland, or the exclusion of the juice from the intestine. The results
are very diverse as to the extent of this diminution, as in certain cases
no absorption of fat was observed, while, in other cases, a considerable
absorption was noted in the same class of animal (dog) and even in the
same animal. According to MINKOWSKI and ABELMANN, after the total
extirpation of the pancreas, the fat of the food introduced is not absorbed
at all, with the exception of milk, of which 28-53 per cent of the fat is
absorbed. Other investigators have obtained different results, and HAR-
LEY has observed a case where in a dog an absorption of only 4 per cent
of the milk fat, or, on the complete exclusion of intestinal bacteria, even
no absorption, took place. The conditions may vary in the different
cases, and the behavior is not the same in different varieties of animals.

As shown by LOMBROSO, there exists an essential difference between
the action of the extirpation of the gland, or a prevented flow of the
secretion into the intestine. In the last case, as the experiments reported
by NIEMANN show, no essential disturbance of the absorption takes place,
while the total extirpation of the gland is followed by a marked dis-
turbance (LOMBROSO 2) . This investigator is also of the opinion that
the pancreas, independent of the external secretion in any way (by
endocrinic bodies), influences the absorption of the foodstuffs and the
activity of the pancreas enzymes in the intestine. In order to judge
this view it would be of the greatest interest to know how the exclusion
of the pancreatic juice from the intestine acts upon the other factors

1 Miiller, " Unters. iiber den Icterus," Zeitschr. f. klin. Med., 12; Hedon and
Ville, Arch, de Physiol. (5), 9; Harley, Journ. of Physiol., 18, Journ. of Pathol. and
Bacteriol., 1895, and Proceed. Roy. Soc., 61. In regard to the other authors see foot-
note 1, p. 532.

2Lombroso, see Bioch. Centralbl., 3, 67 and 566, and 4, 738; also Compt. rend,
eoc. biol., 57; Hofmeister's Beitrage, 8, 11; Pfluger'sjjArch., 112; and Arch. f. exp.
Path. u. Pharm., 56 and 60; Niemann, 1. c.

540 DIGESTION.

of the digestion, such as upon the formation of the secretions and their
activity. As to this we know at present very little, but the work of
ZUNZ and MAYER (see page 532), indicates that such a reverse action is
possible. Under these circumstances it is not possible to give LOMBROSO'S
views too great a prominence.

LOMBROSO has also found that after the extirpation of the pancreas
in the dog, sometimes more fat is eliminated than was contained in the
food; that this eliminated fat, which^ depends upon a fat secretion into
the intestinal canal, has a different composition from the introduced fat,
and that in these cases an absorption of fat also takes place. That some
fat can be absorbed in animals even in the absence of the bile as well
as pancreatic juice has been shown by the investigations of HEDON and

VlLLE and CUNNINGHAM.1

The reason for the fact that the fat absorption is diminished in the
absence of bile from the intestine must be sought for in the above-men-
tioned r61e of this fluid. It is more difficult to state why the absence
of pancreatic juice causes a reduction in the absorption of fat. The most
natural view is that the neutral fats are here less completely split, but
this does not seem to be the case, because the non-absorbed fat of the
feces consists, on the exclusion of bile and pancreatic juice (MINKOWSKI
and ABELMANN, HARLEY, HEDON and VILLE, DEUCHER), principally of
free fatty acids. A still unknown change caused by gastric or intestinal
lipase or by micro-organisms may produce a cleavage of the fat in these
cases. The imperfect fat absorption after the extirpation of the pan-
creas can possibly be explained by the removal of a considerable part
of the alkalies necessary for the formation of the emulsion and for the
solution of the fatty acids, but as SANDMEYER found in dogs deprived of
their pancreas, that the fat absorption was raised by giving chopped
pancreas with the fat, this can hardly be a sufficient explanation. The
reason for this is perhaps that after the extirpation of the pancreas the
splitting of the fat is chiefly brought about by bacteria in those parts of
the intestinal canal where the conditions for absorption are not favor-
able.

The soluble salts are also absorbed with the water. The proteins,
which can dissolve a considerable quantity of salts, such as earthy phos-
phates which are otherwise insoluble in alkaline water, are of great
importance in the absorption of such salts.

The soluble constituents of the digestive secretions can be absorbed
like the other soluble substances and toxines, and ferments may also be
absorbed, especially by a diseased change in the intestinal walls.

The occurrence of urobilin in urine attests the absorption of the bile-

1 H6don and Ville, 1. c.; Cunningham, Journ. of Physiol., 32.

ABSORPTION OF BILE. 541

constituents under physiological conditions despite the fact that the
occurrence of very small traces of bile-acids in the urine is disputed.
The absorption of bile-acids by the intestine seems to be positively proved
by other observations. TAPPEINER 1 introduced a solution of bile-
salts of a known concentration into an intestinal knot and after a time
investigated the contents. He found that in the jejunum and the ileum,
but not in the duodenum, an absorption of bile-acids took place, and
further that of the two bile-acids only the glycocholic acid was absorbed
in the jejunum. Further, SCHIFF long ago expressed the opinion that
bile undergoes an intermediate circulation, in such wise that it is
absorbed from the intestine, then carried to the liver by the blood, and
lastly eliminated from the blood by this organ. Although this view has
met with some opposition, still its correctness seems to be established by
the researches of various investigators, and more recently by PREVOST and
BINET, and specially by STADELMANN and his pupils.2 After the intro-
duction of foreign bile into the intestine of an animal, the foreign bile-
acids appear again in the secreted bile.

How does the removal of large portions of the various parts of the
intestine affect absorption? HARLEYS has been able to perform a par-
tial extirpation of the large intestine and in another instance a com-
plete extirpation. This last condition increased the feces considerably,
especially because of the large increase in the water (five-fold). Fats
and carbohydrates were absorbed just as completely as in the normal.
The absorption of the proteins, on the contrary, was reduced to only
84 per cent as compared to 93-98 per cent in normal dogs. After extir-
pation, the feces sometimes did not contain any urobilin, or only traces
thereof, while bile-pigments existed in large amounts.

ERLANGER and HEWLETT found that dogs from which 70-83 per
cent of the total length of the jejunum and ileum had been removed,
could be kept alive, like other animals, if only the food was not too rich
in fat. When the food contained large amounts of fat then 25 per
cent was evacuated by the feces as compared to 4-5 per cent in the
normal animal. Under these same conditions the amount of nitrogen
in the feces was increased to twice the normal amount. LONDON and
STASSOW* found on resection of the ileum that the eliminated diges-
tion and absorption were performed by the parts of the intestine higher

1 Wien. Sitzungsber., 77.

2 Schiff, Pfliiger's Arch., 3; Prevost and Binet, Compt. Rend., 106; Stadelmann,
see footnote 1, p. 416.

3 Proceed. Roy. Soc., 64.

4 Erlanger and Hewlett, Amer. Journ. of Physiol. 6; London and Stassow. Zeitschr.
f.physiol. Chem. 74, 349 (1911).

542 DIGESTION.

up; after resection of the jejunum the large intestine seems to have a
compensating action.

After the exclusion of the colon in rabbits, BERGMANN and HULT-
GREN 1 could find no definite action upon the availability of the cellu-
lose nor could any diminution in the utility of the other constituents
of the food be observed. ZUNTZ and USTJANZEW 2 .also found that the
removal of the caecum had no influence on the utilization of nitrogen;
but in respect of other factors they arrive at different results. They
found, namely, that the caecum of the rodent is of great importance for
the digestion of crude fiber and the pentosans. On feeding hay and
wheat to rabbits after the removal of the caecum, the digestion coefficient
for crude fiber fell from 42.8 to 23.4-18.7 per cent, and for pentosans
from 50 to 40-28.7 per cent.

The question as to the forces which are active in the intestine during
absorption has not been satisfactorily answered. Attempts have been
made to explain absorption as a filtration, due to a certain difference
in the hydrostatic pressure between the intestinal contents and the
blood. A sufficiently great difference in pressure does not seem to
exist and besides this the absorbed solution on account of its composi-
tion cannot be considered as a filtrate from the intestinal contents.
Diffusion processes without doubt play a much more important role.
These attempt to keep the same concentration of all dissolved sub-
stances on both sides of the intestinal epithelium (in intestinal contents
and in the blood). Such processes must be influenced, as mentioned
in Chapter I on the osmotic pressure, to a high degree upon the perme-
ability of the intestinal membrane for dissolved solids and for water.
Nevertheless the diffusion stream does not give sufficient explanation
for the absorption, as, according to COHNHEIM, 3 the result is different
according to whether the intestine is alive or is dead and in general a
streaming from the lumen of the intestine into the outside fluid is
noticeable in the living intestine quite independent of the differences
in concentration. How this streaming is brought about has not been
explained.

Other investigators have suggested the question whether surface-
tension forces (adsorption phenomenon) are active in absorption.4
Still it has not been possible to bring the absorbability of a substance
in simple relation to its influence on the surface-tension of the water.

1 Skand. Arch. f. Physiol., 14.

2 Verhandl. d. physiol. Gesellsch. zu Berlin, 1904-1905.

3 Zeitschr. f. physiol. Chem. 36-39.

4 J. Traube, Bioch. Zeitschr. 24, 324 (1910) which also contains literature.

THEORIES OF ABSORPTION. 543

Under these circumstances and as it is not within the scope of this
book to enter into details upon the numerous investigations as to the
theory of absorption, we must refer to larger works l and to text-books
on physiology for further information.

1 See Hober, Physikalische Chemie der Zelle, Leipzig, 1906, Koranyi and Richter,
Physikalische Chemie u. Medizin. Leipzig 1907, Bd. 1, 295. I. Munk, Ergebnisse
der Physiologic, I, Abt. 1; Hamburger, Osmotisher Druck und lonenlehre, Bd. 2,
Wiesbaden, 1904.

CHAPTER IX.
TISSUES OF THE CONNECTIVE SUBSTANCE.

I. THE CONNECTIVE TISSUES.

THE form-elements of the typical connective tissues are cells of
various kinds, of a not very well-known chemical composition, and
gelatin-yielding fibrils, which, like the cells, are imbedded in an interstitial
or intercellular substance. The fibrils consist of collagen, the interstitial
substance contains chiefly mucoid (tendon-mucoid) , besides ser globulin
and seralbumin, which occur in the parenchymatous fluid (LoEBiscn1).

The connective tissue also often contains fibers or formations con-
sisting of elastin, sometimes in such great quantities that the connective
tissue is transformed into elastic tissue. A third variety of fibers, the
reticular fibers, also occurs, and according to SIEGFRIED these consist
of reticulin.

If finely divided tendons are extracted in cold water or NaCl solu-
tions, the protein bodies soluble in the nutritive fluid in addition to a
little mucoid are dissolved. If the residue is extracted with half-
saturated lime-water, then the mucoid is dissolved and may be precipi-
tated from the filtered extract by adding an excess of acetic acid. The
extracted residue contains the fibrils of the connective tissue together
with the cells and the elastic substance.

The so-called tendon mucin is not true mucin, but a mucoid, which,
as first shown by LEVENE and then by CUTTER and GIES, contains a
part of its sulphur as an acid related to chondroitin-sulphuric acid.
These mucoids, which, according to CUTTER and GIES, are mixtures* of
several glycoproteins, contain 2.2-2.33 per cent sulphur, as shown by
the analyses of CHITTENDEN and GIES, as well as those of CUTTER and
GIES. The quantity of sulphur split off as sulphuric acid was 1.33-1.62
per cent (CUTTER and GIES) . VAN LIER 2 has prepared a substance at least
closely related to tendon mucoid from the hard skin of man and certain
animals. This mucoid yielded an ethereal sulphuric acid, a glucothionic
acid with 1.58-3.03 per cent sulphur in the barium salt, and was variable
in different animals. It gives the orcin reaction for glucuronic acid.

1 Zeitschr. f. physiol. Chem., 10.

2 Levene, ibid., 31 and 39; Cutter and Gies, Ainer. Journ. of Physiol., 6; Chitten-
den and Gies, Journ. of Exp. Med., 1; van Lier, Zeitschr. f. physiol. Chem. 61.

544

CONNECTIVE TISSUES. 545

The fibrils of the connective tissue are elastic and swell slightly in
water, somewhat more in dilute alkalies or in acetic acid. On the other
hand, they shrink by the action of certain metallic salts, such as ferrous
sulphate or mercuric chloride, and tannic acid, which form insoluble
compounds with the collagen. Among these compounds, which prevent
putrefaction of the collagen, that with tannic acid has been found of
the greatest technical importance in the preparation of leather. In
regard to the collagens, gelatins, elastins, and reticulins, see pages 116
to 121.

The tissues described under the names mucous or gelatinous tissues
are characterized more by their physical than by their chemical prop-
erties, and have been but little studied. This much, however, is
known, that the mucous or gelatinous tissues contain, at least in certain
cases, as in the Acalephse, no mucin.

The umbilical cord is the most accessible material for the investiga-
tion of the chemical constituents of the gelatinous tissues. The mucin
occurring therein yields, according to VAN LIER, an ethereal sulphuric
acid (glucothionic acid) like the tendon mucoid. C. TH. MORNER 1
has found a mucoid in the vitreous humor which contains 12.27 per
cent nitrogen and 1.19 per cent sulphur.

Young connective tissue is richer in mucoid than old. HALLIBURTON 2
found an average of 7.66 p. m. mucoid in the skin of very young children
and only 3.85 p. m. in the skin of adults. In so-called myxcedema,
in which a re-formation of the connective tissue of the skin takes place,
the quantity of mucoid is also increased.

' The connective tissue and also the elastic tissue are richer in water
and poorer in solids in young animals as compared with full-grown
animals. This may be seen from the following analyses of the Achilles
tendon (BUERGER and GIES) and of the ligamentum nuchse (VANDE-
GRIFT and GiES3):

Achilles tendon. Ligament/

Water ....

Calf.
675 . 1 p. m.

Ox.
628.7 p.
371.3 '
366.6 '
4.7
10.4
2.2
12.83
16.33
315.88
8.96

Calf.
m. 651.0p.m.
1 394.0 "
' 342.4 "
6.6 "

Ox.
575.7 p.m.
424.3
419.6
4.7
11.2
6.16
5.25
316.70
72.30
7.99

Solids

.. 324.9 "

Organic bodies

318 4 "

Inorganic bodies

6.1 "

Fat

Proteid

Elastin

Collagen

Extractives, etc. . . .

1 Zeitschr. f. physiol. Chem., 18, 250.

2 Mucin in Myxcedema: Further Analyses. King's College Collected Papers
No. 1, 1893.

'Buerger and Gies, Amer. Journ. of Physiol., 6; Vandegrift and Gies, ibid, 5.

546 TISSUES OF THE CONNECTIVE SUBSTANCE.

In regard to the mineral bodies it must be remarked that according
to the determinations of H. SCHULZ the connective tissue is rich in silicic
acid. The greatest amount was found by him, in the crystalline lens
of the ox, namely, 0.5814 gram per kilo of dried substance. In man he
found 0.0637 gram in the tendons, 0.1064 gram in the fascia, and 0.244
gram in Wharton's jelly for every kilo of dried substance. The quantity
of silicic acid is higher in the young than in the old; in man it is highest
in the embryonic connective tissue of the umbilical cord. In the last-
named substance SCHULZ also found 0.403 gram Fe2Os, 0.693 gram
MgO, 3.297 grams CaO, and 3.794 grams P2O5 for every kilo of dried
substance. The report of SCHULZ on the quantity of silicic acid does
not correspond with the investigations of FRAUENBERGER l who found,
in Wharton's jelly, only a fraction of the quantity of silicic acid that
SCHULZ gives.

II. CARTILAGE.

Cartilaginous tissues consist of cells and an original hyaline matrix,
which, however, may become changed in such wise that there appears
in it a network of elastic fibers or connective-tissue fibrils.

Those cells that offer great resistance to the action of alkalies and
acids have not been carefully studied. According to earlier opinions
the matrix was considered as consisting of a body analogous to colla-
gen, so-called chondrigen. The investigations of MOROCHOWETZ and
others, but especially those of C. MoRNER,2 have shown that the
matrix of the cartilage consists of a mixture of collagen with other
bodies.

The tracheal, thyroideal, cricoidal, and arytenoidal cartilages of
full-grown cattle contain, according to MORNER, four constituents in
the matrix, namely, chondromucoid, chondroitin-sulphuric acid, collagen,
and the albumoid.

Chondromucoid. This body, according to C. MORNER, has the com-
position C 47.30, H 6.42, N 12.58, S 2.42, O 31.28 per cent. Sulphur is
in part loosely combined and may be split off by the action of alkalies,
and a part separates as sulphuric acid when boiled with hydrochloric
acid. Chondromucoid is decomposed by dilute alkalies and yields alkali
albuminate, peptone substances, chondroitin-sulphuric acid, alkali sul-
phides, and some alkali sulphates. On boiling with acids it yields acid
albuminate, peptone substances, chondroitin-sulphuric acid, and on

1 Schulz, Pfliiger's Arch, 84 and 89, 131 and 144; Frauenberger, Zeitschr. f. physiol.
Chem., 57.

2 Morochowetz, Verhandl., d. naturh. med. Vereins zu Heidelberg, 1, Heft 5; Morner,
Skand. Arch. f. Physiol., 1.

CHONDROITIN-SULPHURIC ACID. 547

account of the further decomposition of this last body, sulphuric acid
and a reducing substance are formed.

Chondromucoid is a white, amorphous, acid-reacting powder which
is insoluble in water, but dissolves easily on the addition of a little
alkali. This solution is precipitated by acetic acid in great excess and
by small quantities of mineral acids. The precipitation may be retarded
by neutral salts or by chondroitin-sulphuric acid. The solution con-
taining NaCl and acidified with HC1 is not precipitated by potassium
ferrocyanide. Precipitants for chondromucoid are alum, ferric chloride,
sugar of lead, or basic lead acetate. Chondromucoid is not precipitated
by tannic acid, and it may by its presence prevent - the precipitation
of gelatin by this acid. It gives the usual color reactions for proteins,
namely, with nitric acid, with copper sulphate and alkali, with MIL-
LON'S and ADAMKIEWICZ-HOPKINS' reagents.

Chondroitin-sulphuric Acid, CHONDROITIC ACID. This acid, which
was first prepared pure, from cartilage, by C. MORNER and identified
by him as an ethereal sulphuric acid, occurs, according to MORNER, in
all varieties of cartilage and also in the tunica intima of the aorta and
as traces in the bone substance. K. MORNER *• has also found it in the
ox-kidney and in human urine as a regular constituent. Its occurrence
in amyloid, as mentioned on page 173, has been disputed by HANSSEN.
In the opinion of LEVENE,2 the glucothionic acid which is prepared from
tendon mucoid, and which gives the orcin reaction for glucuronic acid,
and yields furfurol on distillation with hydrochloric acid, is not identical
with the chondroitin-sulphuric acid, but is probably related thereto.

Chondroitin-sulphuric acid has the formula CisH^yNSOir, accord-
ing to ScHMiEDEBERG.3 As primary products this acid yields, on
cleavage, sulphuric acid and a nitrogenous substance, chondroitin, accord-
ing to the following equation:

Chondroitin, which is similar to gum arabic, and which is a monobasic
acid, yields acetic acid and a new nitrogenous substance, chondrosin,
as cleavage products, on decomposition with dilute mineral acids:

Chondrosin, which is also a gummy substance soluble in water, is a
monobasic acid and reduces copper oxide in alkaline solutions even

1 C. Morner, 1. c., and Zeitschr. f. physiol. Chem., 20 and 23; K. Morner, Skand.
Arch. f. Physiol., 6.

2 Zeitschr. f . physiol. Chem., 39.

3 Arch. f. exp. Path. u. Pharm., 28.

548 TISSUES OF THE CONNECTIVE SUBSTANCE.

more strongly than glucose. It is dextrogyrate, and represents the
reducing substance obtained by previous investigators in an impure
form on boiling cartilage with an acid. The products obtained on decom-
posing chondrosin with barium hydroxide tend to show, according to
SCHMIEDEBERG, that chondrosin contains the atomic groups of glucuronic
acid and glucosamine. This assumption does not seem to have sufficient
foundation. According to ORGLER and NEUBERG, chondrosin does not
give the orcin test nor does it yield furfurol. They claim that on
cleavage with baryta it yields, besides a carbohydrate complex which
has not been studied, an oxyamino-acid having the formula CeHisOeN,
a hexosamine acid or tetraoxyaminocaproic acid. In opposition to this
S. FRANKEL has found that the chondrosin gives the orcin as well as the
phloroglucin test with hydrochloric acid, and he has prepared an acid
with the formula CeHnNOe, which he calls aminoglucuronic acid,
which gives the above tests and also reduces. Among other investi-
gators, PONS and KONDO 1 have also found that chondroitin-sulphuric
acid gives the orcin test and yields furfurol, according to PONS 6.6-6.9
per cent. The chondrosin obtained after boiling with acid and distilling
off the furfurol does not, according to PONS, give furfurol, which agrees
with ORGLER and NEUBERG'S statement. From the hydrolytic products
of chondroitin-sulphuric acid with hydrochloric acid, PONS obtained
with phenylhydrazin a crystalline substance melting at 143° C.

Chondroitin-sulphuric acid appears as a white amorphous powder
which dissolves very easily in water, forming an acid solution and, when
sufficiently concentrated, a sticky liquid similar to a solution of gum
arabic. Nearly all of its salts are soluble in water. The neutralized
solution is precipitated by stannous chloride, basic lead acetate, neutral
ferric chloride, and by alcohol in the presence of a little neutral salt.
The solution, on the other hand, is not precipitated by acetic acid,
tannic acid, potassium ferrocyanide and acetic acid, sugar of lead, mer-
curic chloride, or silver nitrate. Acidified solutions of alkali chondroitin-
sulphates cause a precipitation when added to solutions of gelatin or
proteid.

. The preparation of chondromucoid, and its separation from chondroitin-
sulphuric acid can be accomplished after the method of C. MORNER, but for
details we refer to the original work.

The pre-existing chondroitin-sulphuric acid, or that formed by the
decomposition of chondromucoid, is obtained by lixiviating the cartilage
with a 5-per cent caustic-alkali solution. The alkali albuminate formed
by the decomposition of the chondromucoid can be removed from the
solution by neutralization, then the peptone precipitated by tannic acid,

1 Orgler and Neuberg, Zeitschr. f. physiol. Chem., 37; Frankel, Annal. d. Chem. u.
Pharm., 351; Pons. Arch, intern, de Physiol., 8 (1909); Kondo, Bioch. Zeitsphr., 26.

CARTILAGENEOUS TISSUE. 549

the excess of this acid removed with sugar of lead, and the lead removed
from the filtrate by H^S. If further purification is necessary, the acid is
precipitated with alcohol, the precipitate dissolved in water, this solu-
tion dialyzed and precipitated again with alcohol — this solution in water
and precipitation with alcohol being repeated a few times — and lastly
the acid is treated with alcohol and ether. Other methods for the prepara-
tion of the acid (from the septum narium of the pig) have been suggested
by SCHMIEDEBERG and KONDO.

The collagen of the cartilage gives, according to C. MORNER, a gelatin
which contains only 16.4 per cent N, and which can hardly be considered
identical with ordinary gelatin.

In the above-mentioned cartilages of full-grown animals the chon-
droitin-sulphuric acid and chondromucoid, perhaps also the collagen,
are found surrounding the cells as round balls or lumps. These balls
(MORNER'S chondrin-balls) , which give a blue color with methyl-violet,
lie in the meshes of a trabecular structure, which is colored when
brought in contact with tropseolin.

The albumoid is a nitrogenized body which contains loosely com-
bined sulphur. It is soluble with difficulty in acids and alkalies and
resembles keratin in many respects, but differs from it by being soluble
in gastric juice. In other respects it resembles elastin, but differs from
this substance in containing sulphur. This albumoid gives the color
reactions of the protein bodies.

Cartilage gelatin and the albumoid may be prepared according to
the folowing method of MORNER: First remove the chondromucoid
and chondroitin-sulphuric acid by extraction with dilute caustic potash
(0.2-0.5 per cent), remove the alkali from the remaining cartilage by
water, and then boil with water in a PAPIN'S digester. The collagen
passes into solution as gelatin, while the albumoid remains undissolved
(contaminated by the cartilage-cells). The gelatin may be purified by
precipitating with sodium sulphate, which must be added to saturation
in the faintly acidified solution, redissolving the precipitate in water,
dialyzing well, and precipitating with alcohol.

In MORNER'S experience no albumoid is found in young cartilage,
but only the three first-mentioned constituents. Nevertheless, the young
cartilage contains about the same amounts of nitrogen and mineral
substances as the old. The cartilage of the ray (Raja batis LIN.), which
has been investigated by LoNNBERG,1 contains no albumoid and only
a little chondromucoid, but a large proportion of chondroitin-sulphuric
acid and collagen.

According to PFLUGER and HANDEL,2 glycogen occurs to a slight

1 Maly's Jahresber., 19, 325.

2 Pfliiger in Pfliiger's Arch., 92; Handel, ibid.

550 TISSUES OF THE CONNECTIVE SUBSTANCE.

extent in all matrices, and of these it is richest in the cartilage. Ten-
dons, ligamentum nuchse, and cartilage of the ox contained 0.06, 0.07,
and 2.17 p. m. glycogen respectively (HANDEL).

HOPPE-SEYLER found in fresh human rib-cartilage 676.7 p. m. water,
301.3 p. m. organic, and 22 p. m. inorganic substance, and in the cartilage
of the knee-joint 735.9 p. m. water, 248.7 p. m. organic, and 15.4 p. m.
inorganic substance. PICKARDT found 402-574 p. m. water and 72.86
p. m. ash (no iron) in the laryngeal cartilage of oxen. The ash of car-
tilage contains considerable amounts (even 800 p. m.) of alkali sulphate,
which probably does not exist originally as such, but is produced in great
part by the incineration of the chondroitin-sulphuric acid and the chon-'
dromucoid. The analyses of the ash of cartilage therefore cannot
give a correct idea of the quantity of mineral bodies existing in this sub-
stance. The cartilage is richest in sodium of all the tissues of the body,
and according to BUNGE l the amount of Na and Cl is greatest in young
animals. In 1000 parts of cartilage dried at 120° C., BUNGE found 91.26
parts Na2O in the shark, 33.98 in the ox embryo, 32.45 in a fourteen-day-
old calf, and 26.4 in a ten-weeks-old calf.

Ochronose is the brown to black coloration of the cartilage which
sometimes occurs, and which has also been observed in several cases of
alcaptonuria (see Chapter XIV) or after lengthy treatment with carbolic
acid bandages (POULSEN, ADLER 2) . The nature of these melanine-
like pigments is unknown.

The Cornea. The corneal tissue, which, in a chemical sense, is con-
sidered by many investigators to be related to cartilage, contains traces
of proteid and a collagen as chief constituent, which C. MORNER 3 claims
contains 16.95 per cent N. According to him it also contains a mucoid
which has the composition C 50.16, H 6.97, N 12.79, and S 2.07 per
cent. On boiling with dilute mineral acid this mucoid yields a reducing
substance. The globulins found by other investigators in the cornea
are not derived from the matrix, according to MORNER, but from the
layer of epithelium. MORNER believes that DESCEMET'S membrane
consists of membranin (page 171), which contains 14.77 per cent N and
0.90 per cent S.

In the cornea of oxen His4 found 758.3 p. m. water, 203.8 p. m.
gelatin-forming substance, 28.4 p. m. other organic substance, besides
8.1 p. m. soluble and 1.1 p. m. insoluble salts.

1 Hoppe-Seyler, cited from Kiihne's Lehrbuch d. physiol. Chem., 387; Pickardt,
Centralbl. f. Physiol., 6, 735; Bunge, Zeitschr. f. physiol. Chem., 28.

2 See Maly's Jahresb., 40, 424, Adler, Zeitschr. f. Krebsforschung, 11.

3 Zeitschr. f. physiol. Chem., 18.

4 Cited from Gamgee, Physiol. Chem., 1880, 451.

BONE. 551

III. BONE.

The bony structure proper, when free from other formations occurring
in bones, such as marrow, nerves, and blood-vessels, consists of cells and
a matrix.

The cells have not been closely studied in regard to their chemical
constitution. On boiling with water they yield no gelatin. They
contain no keratin, which usually should not be present in the bony
structure (HERBERT SMITH 1).

The matrix of the bony structure contains two chief constituents,
namely, an organic substance, and the so-called bone-earths, lime-salts,
inclosed in or combined with it. If bones are treated with dilute hydro-
chloric acid at the ordinary temperature, the lime-salts are dissolved
and the organic substance remains as an elastic mass, preserving the
shape of the bone.

The organic matrix consists chiefly of ossein, which is generally
considered as identical with the collagen of the connective tissue. It
also contains, as HAWK and GiES2 have shown, mucoid and albuminoid.
After the removal of the lime-salts by hydrochloric acid of 2-5 p. rn.
these experimenters were able to extract the mucoid by one-half sat-
urated lime-water, and to precipitate it with 2 p. m. hydrochloric acid.
After the removal of the osseomucoid and collagen (by boiling with
water) they obtained the albuminoid as an insoluble residue.

The osseomucoid on boiling with hydrochloric acid yielded a reduc-
ing substance and sulphuric acid; 1.11 per cent sulphur appearing in
this form. The osseomucoid stands close to the chondro- and tendon
mucoid in elementary composition, as may be seen from the follow-
ing analyses:

c H N s o

Osseomucoid 47.43 6.63 12.22 2.32 31.40 (HAWK and GIES)

Chondromucoid. . .. 47.30 6.42 12.58 2.42 31.28 (C. MORNER)

Tendon mucoid 48 . 76 6 . 53 1 1 . 75 2 . 33 30 . 60 (CHITTENDEN and GIES)

Corneal mucoid.. .. 50.16 6.97 12.79 2.07 28.01 (C. MORNER)

The osseoalbuminoid is insoluble in 2 p. m. hydrochloric acid, and
in 5 p. m. Na2COs, but dissolves in 10 per cent KOH with the formation
of albuminates. The composition of chondro- and osseoalbuminoid
is as follows:

c H N s o

Osseoalbuminoid 50 . 16 7 . 03 16 . 17 1 . 18 25 . 46 \ HAWK and

Chondroalbuminoid. .. 50.46 7.05 14.95 1.86 25.68] GIES

1 Zeitschr. f. Biologic, 19.

2 Amer. Jo urn. of Physiol., 5 and 7.

552 TISSUES OF THE CONNECTIVE SUBSTANCE.

The inorganic constituents of the bony structure, the so-called
bone-earths, which, after the complete calcination of the organic sub-
stance, remain as a white brittle mass, consist chiefly of calcium and
phosphoric acid, but also contain carbonic acid and, in smaller amounts,
magnesium, chlorine, and fluorine. Iron, which has been found in bone-
ash, does not seem to belong exactly to the bony substance, but to the
nutritive fluids or to the other constituents of bones. The traces of
sulphate occurring in the bone-ash are derived, according to MORNER,
from the chondroitin-sulphuric acid. According to GABRIEL, potassium
and sodium are essential constituents of bone-earth, and this has been
substantiated by ARON 1.

The opinions of investigators differ slightly as to the manner in
which the mineral bodies of the bony structure are combined with
each other. Chlorine is present in the same form as lin apatite
3(Ca3P20s)CaCl2. If we eliminate the magnesium, the chlorine, and
the fluorine, the last, GABRIEL claims, occurring only as traces, the remain-
ing mineral bodies form the combination 3(Ca3P20s)CaCO3. In his
opinion the simplest expression for the composition of the ash of bones
and teeth is (CastPO^+CasHPaOis+Aq), in which 2-3 per cent of the
lime is replaced by magnesia, potash, and soda, and 4-6 per cent of the
phosphoric acid by carbonic acid, chlorine, and fluorine. Recently, on
the contrary, GASSMANN has given important reasons for the follow-
ing complex combination in WERNER'S 2 sense.

/OP03Ca\

Analyses of bone-earths have shown that the mineral constituents
exist in rather constant proportions, which are nearly the same in dif-
ferent animals. As an example of the composition of bone-earth we here
give the analyses of ZALESKY.S The figures represent parts per thousand:

Calcium phosphate, CasP2Og

Man.

. 838.9

Ox.
860 9

Tortoise.
859 8

Guinea-pig,
873 8

Magnesium phosphate, MgsP2O8

. 10.4

10 2

13 6

10 5

Calcium combined with CO2, Fl, and Cl. .
CO2

. 76.5
. 57.3

73.6
62 0

63.2
52 7

70.3

Chlorine

1 8

2 0

1 3

Fluorine*

2 3

3 0

2 0

1 Morner, Zeitschr. f. physiol. Chem., 23; Gabriel, ibid., 18, which also contains
the pertinent literature; Aron, Pfliiger's Arch., 106.

2 Gassmann, Zeitschr. f. physiol. Chem. 70 and 83; Werner, Ber. d. d. Chem.
Gesellsch., 40.

8 Hoppe-Seyler, Med.-chem. Untersuch., p. 19.

4 The reports as to the quantity of fluorine disagree; see Harms, Zeistchr. f.
Biologic, 38; Jodlbauer, ibid., 41.

BONE. 553

Some of the C02 is always lost on calcining, so that the bone-ash
does not contain the entire C02 of the bony substance.

GAUTIER and CLAUSMANN 1 have determined the fluorine in various
organs and tissues. In man the diaphysis end of the femur had 0.495
p. m. fluorine, and the epiphysis end 0.119 p. m. fluorine. In children
the diaphysis end of the long bones contained 0.156 p. m. fluorine
and the epiphysis end 0.037 p. m. A similar difference also occurs in
animals. Cartilage of man with 0.014 p. m. fluorine and tendons (calf)
with 0.0035 p. m. fluorine, are much poorer in fluorine than the bones.
The dentin (dog) contains 0.56 p. m. fluorine and the enamel (of a
young dog) contained 1.66. p. m. fluorine, all results obtained from the
fresh substance.

AD. CARNOT2 found the following composition for the bone-ash of
man, ox, and elephant:

Man. Oz. Elephant,

Calcium phosphate ..................... 874.5 878.7 857.2 900.3

Magnesium phosphate .................. 15.7 17.5 15.3 19 . 6

Calcium fluoride ....................... 3.5 3.7 4.5 4.7

Calcium chloride ....................... 2.3 3.0 3.0 2.0

Calcium carbonate .................... 101.8 92.3 119.6 72.7

Iron oxide ............................. 1.0 1.3 1.3 1.5

The quantity of organic substance in the bones, calculated from the
loss of weight in burning, varies between 300 and 520 p. m. This
variation may in part be explained by the difficulty in obtaining the
bony substance entirely free from water, and partly by the very variable
amount of blood-vessels, nerves, marrow, and the like in different bones.
The unequal amounts of organic substance found in the compact and
in the spongy parts of the same bone, as well as in bones at different
periods of development in the same animal, probably depend upon
the varying quantities of these above-mentioned tissues. Dentin, which
is comparatively pure bony structure, contains only 260-280 p. m.
organic substance, and HOPPE-SEYLER 3 therefore thinks it probable
that perfectly pure bony substance has a constant composition and
contains only about 250 p. m. organic substance. The question whether
these substances are chemically combined with the bone-earths or only
intimately mixed has not been decided.

The nutritive fluids which circulate through the bones have not been isolated
and we only know that they contain some protein and some NaCl and alkali
sulphate.

1 Compt. Rend., 156.

2 Ibid., 114.

* Physiol. Chem., 102-104-

554 TISSUES OF THE CONNECTIVE SUBSTANCE.

Bone Marrow. We differentiate between the red and yellow mar-
row, to which also belongs the gelatinous marrow, poor in fat, found in
fat atrophy and in old age. The difference between the first two-men-
tioned kinds of marrow lies, essentially, in the fact that the red marrow
contains a greater quantity of erythrocytes besides a higher content of
protein and less fat. The fat of the yellow marrow is, according to
NERKING/ richer in oleic acid and poorer in solid fats than the fat of the
red marrow. Besides the fat, lecithin also occurs in the bone-marrow
and this varies in amount in different animals and at various ages, as
mentioned on page 244. The protein consists of a globulin coagulating
at 47-50° C. (FORREST) and a nucleoprotein with 1.6 per cent phos-
phorus (HALLIBURTON 2) besides fibrinogen (P. MULLER 3) , traces of
albumin and proteose. In the extractives are found lactic acid, inosite,
hypoxanthine, cholesterine and bodies of an unknown kind. The quan-
titative composition of both kinds of marrow varies considerably with
the fat content, and the reports of the different investigators are corre-
spondingly discrepant (NERKING, HUTCHINSON and MACLEOD 4).

The diverse quantitative composition of the various bones of the
skeleton depends probably on the varying quantities of other tissues,
such as marrow, blood-vessels, etc., which they contain. The same
reason explains, to all appearances, the larger quantity of organic
substance in the spongy part of the bones as compared with the more
compact parts. SCHRODTS has made comparative analyses of different
parts of the skeleton of the same animal (dog) and has found an essen-
tial difference. The quantity of water in the fresh bones varies between
138 and 443 p. m. The bones of the extremities and the skull contain
138-222, the vertebra 168-443, and the ribs 324-356 p. m. water. The
quantity of fat varies between 13 and 269 p. m. The largest amount
of fat, 256-269 p. m., is found in the long tubular bones, while only
13-175 p. m. fat is found in the small short bones. The quantity of
organic substance, calculated from fresh bones, 'was 150-300 p. m., and
the quantity of mineral substances 290-563 p. m. Contrary to the
general supposition the greatest amount of bone-earths was not found
in the femur, but in the first three cervical vertebrae. In birds the
tubular bones are richer in mineral substances than the flat bones
(DURING), and the greatest quantity of mineral bodies has been found
in the humerus (HILLER, DURING 6).

1 Bioch. Zeitschr., 10.

2 Forrest, Journ. of Physiol., 17; Halliburton, ibid., 18.

3 See footnote 1, p. 253.

4 Nerking, 1. c.; Hutchinson and Macleod, Journ. of Anat. and Physiol., 36.
6 Cited from Maly's Jahresber., 6.

"Killer, cited from Maly's Jahresber., 14; Diiring, Zeitschr. f. physiol. Chem., 23.

DISEASES OF THE BONES. 555

We do not possess trustworthy information in regard to the compo-
sition of bones at different ages. The analyses by E. VOIT of bones of
dogs, and by BRUBACHER of bones of children, apparently indicate that
the skeleton becomes poorer in water and richer in ash with increase
in age. GRAFFENBERGER 1 has found in rabbits, 6|-7J years old, that
the bones contained only 140-170 p. m. water, while the bones of the
full-grown rabbit 2-4 years old contained 200-240 p. m. The bones of
old rabbits contain more carbon dioxide and less calcium phosphate.

The composition of bones of animals of different species is but little known.
The bones of birds contain, as a rule, somewhat more water than those of mam-
malia, and the bones of fishes contain the largest quantity of water. The bones
of fishes and amphibians contain a greater amount of organic substance. The
bones of pachyderms and cetaceans contain a large proportion of calcium carbo-
nate; those of granivorous birds always contain silicic acid. The bone-ash of
amphibians and fishes contains sodium sulphate. The bones of fishes seem to
contain more soluble salts than the bones of other animals.

A great many experiments have been made to determine the exchange
of material in the bones — for instance, with food rich in lime and with
food deficient in lime — but the results have always been doubtful or
contradictory. The attempts to substitute other alkaline earths or
alumina for the lime of the bones have also given conflicting results.2
On feeding sufficient calcium and phosphorus in the food ARON 3 found,
by strongly reducing the sodium and at the same time giving a large
amount of potassium, that the development of the bones was below
normal. On the administration of madder, the bones of the animal are
found to be colored red after a few days or weeks; but these experiments
have not led to any positive conclusion in regard to the growth or
metabolism in the bones.

Under pathological conditions, as in rachitis and softening of the
bones, an ossein has been found which does not give any typical gelatin
on boiling with water. This finding is still uncertain as otherwise path-
ological conditions seem to affect chiefly the quantitative composition
of the bones, and especially the relation between the organic and the inor-
ganic substance. In rachitis the bones are poorer in solids and these
are poorer in mineral substances than under normal conditions.
Attempts have been made to produce rachitis in animals by the use of
food deficient in lime. From experiments on fully developed animals
opposing results have been obtained. In young, undeveloped animals

1 Voit, Zeitschr. f. Biologie, 16; Brubacher, ibid., 27; Graff enberger in Maly's
Jahresber., 21.

2 See H. Weiske, Zeitschr. f. Biologie, 31, and W. Stoeltzner, Pfluger's Arch., 122,
and H. Stoeltzner, Bioch. Zeitechr., 12.

3 Pfluger's Arch., 106.

556 TISSUES OF THE CONNECTIVE SUBSTANCE.

ERWIN VOIT, ARON and SEBAUER and others 1 produced, by lack of
lime-salts, a change similar to rachitis. In full-grown animals the
bones were changed after a long time because of the lack of lime-salts
in the food, but did not become soft, only thinner (osteoporosis). The
attempts to remove the lime-salts from the bones by the addition of
lactic acid to the food have led to no positive results (HEITZMANN,
HEISS, BAGiNSKY2). WEISKE, on the contrary, has shown, by admin-
istering dilute sulphuric acid or monosodium phosphate with the food
(presupposing that the food gave no alkaline ash) to sheep and rab-
bits, that the quantity of mineral bodies in the bones might be dimin-
ished. On feeding continuously for a long time with a food which yielded
an acid ash (cereal grains), WEISKE observed a diminution in the min-
eral substances of the bones in full-grown herbivora.3 A few investi-
gators are of the opinion that in rachitis, as in osteomalacia, in which
disease the calcium content of the bones is also diminished, a solution
of the lime-salts by means of lactic acid takes place. This was sug-
gested by the fact that O. WEBER and C. SCHMIDT 4 found lactic acid
in the cyst-like, altered bony substance in osteomalacia.

Well-known investigators have disputed the possibility of the lime-
salts being washed from the bones in osteomalacia by means of lactic
acid. They have given special prominence to the fact that the lime-
salts held in solution by the lactic acid must be deposited on neutraliza-
tion of the acid by the alkaline blood. This objection is not very impor-
tant, as the alkaline blood-serum has the property to a high degree of
holding earthy phosphates in solution, which fact has been recently
shown by HOFMEISTER. The investigations of LEVY contradict the
claim as to the solution of the lime-salts by lactic acid in osteomalacia.
He found that the normal relation 6PO4:10Ca is retained in all parts
of the bones in osteomalacia, which would not be the case if the bone-
earths were dissolved by an acid. The decrease in phosphate occurs in
the same quantitative relation as the carbonate, and according to LEVY,
in osteomalacia the exhaustion of the bone takes place by a decalcifica-
tion in which one molecule of phosphate-carbonate after the other is
removed. This does not agree with the findings of MCCRUDDEN 5 who

1Zeitschr. f. Biologic, 16; Aron and Sebauer, Bioch. Zeitschr., 8; A. Baginsky,
Arch. f. (Anat. u.) Physiol., 1881.

2Heitzmann, Maly's Jahresber., 3, 229; Heiss, Zeitschr. f. Biologie, 12; Baginsky,
Virchow's Arch., 87.

3 See Maly's Jahresber., 22; also Weiske, Zeitschr, f. physiol. Chem., 20, and
Zeitschr. f. Biologie, 31.

4 Cited from v. Gorup-Besanez, Lehrb. de. physiol. Chem., 4. Aufl.

6 Hofmeister, Ergebn. d. Physiol. 10; Levy. Zeitschr. f. physiol. Chem. 19; McCrud-
den, Journ. of biol. Chem., 7.

TOOTH-STRUCTURE. 557

found a changed relation between the Ca and phosphoric acid in osteo-
malacia.

Rachitic bones are always poorer in mineral substances than normal bones.
The relation between Ca,P04 and C02 was found by GASSMANN to be the same
as in normal bones while he found a pathological increase in the magnesium. The
organic substance was found in rachitis to be relatively as well as absolutely
increased, at least in certain cases (GASSMANN). The statements differ in
regard to the water content. According to BRUBACHER this is larger while accord-
ing to GASSMANN it is 10 p. m. smaller than in normal bones. In opposition to
rachitis, osteomalacia is often characterized by the considerable amount of fat
in the bones, 230-290 p. m., but as a rule the composition varies so much that
the analyses are of little value. In a case of osteomalacia, CHABRIE l found a
larger quantity of magnesium than calcium in a bone. The ash contained 417
p. m. phosphoric acid, 222 p. m. lime, 269 p. m. magnesia, and 86 p. m. carbon
dioxide. MCCRUDDEN found more magnesium than calcium; other investigators
have on the contrary found more calcium than magnesium.

The tooth-structure is closely related, from a chemical standpoint,
to the bony structure.

Of the three chief constituents of the teeth — dentin, enamel, and
cement — the cement is to be considered as true bony structure, and as
such has already been discussed to some extent. Dentin has the same
composition as the bony structure, but contains somewhat less water.
The organic substance yields gelatin on boiling; but the dental tubes
are not dissolved, therefore they cannot consist of collagen. In dentin
260-280 p. m. organic substance has been found. Enamel is an epithe-
lium formation containing a large proportion of lime-salts. Correspond-
ing to its character and origin, the organic substance of the enamel
does not yield any gelatin. Completely developed enamel contains
the least water, the greatest quantity of mineral substances, and is the
hardest of all the tissues of the body. In full-grown animals it con-
tains hardly any water, and the quantity of organic substance amounts
to only 20-40-68 p. m. The relative amounts of calcium and phosphoric
acid are shown by the analyses of HOPPE-SEYLER to be about the same
as in bone-earths. The quantity of chlorine according to him is remark-
ably high, 0.3-0.5 per cent, while BERTZ 2 found that the ash of enamel
was free from chlorine and that dentin was very poor in chlorine.

CARNOT,S who has investigated the dentin from elephants, has found 4.3 p. m.
•calcium fluoride in the ash. In ivory he found only 2 p. m. Dentin from
elephants is rich in magnesium phosphate, which is still more abundant in ivory.

1 Gassmann, Zeitschr. f. physiol. Chem. 70; Brubacher, Zeitschr. f. biol. 27. See
also Cappezzuoli, Bioch. Zeitschr. 16; Chabrie, Les phe"nom6nes chim. de I'ossification,
Paris, 1895, 65.

2 See Maly's Jahresber., 30.

3 Compt. Rend., 114.

558 TISSUES OF THE CONNECTIVE SUBSTANCE.

GABRIEL found that the quantity of fluorine is very small and
amounts to 1 p. m. in ox-teeth. It is no greater in the teeth and enamel
than in the bones.1 The same investigator found that the amount of
phosphates is strikingly small in the enamel, and in the teeth consider-
able lime is replaced by magnesia. This coincides with BERTZ'S find-
ings, that dentin contains twice as much magnesia as the enamel.

According to GASSMANN,2 the teeth among themselves have dif-
ferent composition, and in man the wisdom teeth are poorer in organic
substance and richer in lime than the canine teeth. The great tend-
ency of the first to caries is probably explained by this fact. The reason
for the degeneration of the teeth is considered by C. ROSE 3 to be a lack
of earthy salts, and according to him one finds the best teeth in localities
where the drinking water has high permanent hardness.

IV. THE FATTY TISSUE.

The membranes of the fat-cells withstand the action of alcohol and
ether. They are not dissolved by acetic acid or by dilute mineral acids,
but are dissolved by artificial gastric juice. They may possibly con-
sist of a substance closely related to elastin. The fat-cells contain,
besides fat, a yellow pigment which in emaciation does not disappear
so rapidly as the fat; and this is the reason that the subcutaneous cel-
lular tissue of an emaciated corpse has a dark orange-red color. The
cells deficient in, or nearly free from fat, which remain after the complete
disappearance of the latter, seem to have an albuminous protoplasm
rich in water. : Adipose tissue is rich in a fat-splitting enzyme and in
catalases.

The less water the fatty tissue contains the richer it is in fat.
SCHULZE and REiNECKE4 found in 1000 parts:

Water. Membrane. Fat.

Fatty tissue of oxen 99 . 7 16.6 883 . 7

Fatty tissue of sheep 104.8 16.4 878.8

Fatty tissue of pigs 64.4 13.6 922.0

The fat contained in the fat-cells consists mainly of triglycerides of
stearic, palmitic, and oleic acids. Besides these, especially in the less
solid kinds of fats, there are glycerides of other fatty acids (see Chapter
IV). In all animal fats there are besides these, as FR. HOFMANN 5 has

1 See footnote 4, p. 552.

2 Zeitschr. f. physiol. Chem., 55.

3 Deutsch. Monatsh. f. Zahnheilk., 1908.

4 Annal. d. Chem. u. Pharm., 142.

6 Ludwig-Festschrift, 1874, Leipzig.

FATTY TISSUE. 559

shown, also free, non-volatile fatty acids, although in very small
amounts.

Human fat is relatively rich in olein, the quantity in the subcutaneous
fatty tissue being 70-80 per cent or more.1 In new-born infants it is
poorer in oleic acid than in adults (KNOPFELMACHER, SIEGERT, JAECKLE) ;
the quantity of olein increases until the end of the first year, when it is
about the same as in adults. The composition of the fat in man as well
as in different individuals of the same species of animals is rather variable,
a fact which is probably dependent upon the food. According to the
researches of HENRIQUES and HANSEN the fat of the subcutaneous fatty
tissue is richer in olein than that of the internal organs; this has also been
observed by LEICK and WiNKLER.2 In animals with a thick subcutaneous
fat deposit the outer layers, according to HENRIQUES and HANSEN, are
richer in olein than the inner layers. The fat of cold-blooded animals
is especially rich in olein, The fat of domestic animals has, according
to AMTHOR and ZINK, a less oily consistency and a lower iodine and
acetyl equivalent than the corresponding fat of wild animals. Under
pathological conditions the fat may have a markedly pronounced varia-
tion. The fat of lipoma seems, from JAECKLE 's experience, to be poorer
in lecithin than other fats.

The fat stored up in the organs and tissues can be changed somewhat
by the composition of the fat of the food, still, according to ABDERHALDEN
and BRAHM,S the fat actually occurring in the cells (with the exception
of the real fat cells) is not dependent in its composition upon the kind
of food fat taken.

The properties of fats in general, and the three most important varieties
of fat in particular, have been considered in a previous chapter, hence
the formation of the adipose tissue is of chief interest at this time.

The formation of fat in the organism may occur in various ways. The
fat of the animal body may consist partly of fat absorbed from the food
and deposited in the tissues, and partly of fat formed in the organism
from other bodies, such as proteins (?) or carbohydrates.

That the fat from the food which is absorbed in the intestinal canal
may be retained by the tissues has been shown in several ways. RAD-
ZIEJEWSKI, LEBEDEFF, and MUNK have fed dogs with various fats, such
as linseed-oil, mutton-tallow, and rape-seed-oil, and have afterward

JSee Jaeckle, Zeitschr. f. physiol. Chem., 36 (literature).

2 Knopfelmacher, Jahrbuch f. Kinderheilkunde (N. F.), 45 (older literature);
Siegert, Hofmeister's Beitrage, 1; Jaeckle, Zeitschr. f. physiol. Chem., 36 (literature);
Henriques and Hansen, Skand. Arch. f. Physiol., 11; Leick and Winkler, Arch. f. Path,
u. Pharm., 48.

3 Zeitschr. f. physiol. Chem., 65.

560 TISSUES OF THE CONNECTIVE SUBSTANCE.

found the administered fat in the tissues. HOFMANN starved dogs
until they appeared to have lost their fat, and then fed them upon large
quantities of fat and only little proteins. When the animals were killed
he found so large a quantity of fat that it could not have been formed
from the administered proteins alone, but the greater part must have
been derived from the fat of the food. PETTENKOFER and VOIT arrived
at similar results in regard to the action of the absorbed fats in the organ-
ism, though their experiments were of another kind. MUNK found
that on feeding with free fatty acids, these are deposited in the tissues,
not, however, as such; but they are transformed by synthesis with
glycerin into neutral fats on their passage from the intestine into the
thoracic duct. The connection between the fat of the food, and of the
body has also been shown by others, especially ROSENFELD. CORO
NEDI and MARCHETTI and in particular WINTERNITZ 1 have shown that
iodized fat is taken up in the intestinal tract and deposited in the various
organs.

Proteins and carbohydrates are considered as the mother-substances
of the fats formed in the organism.

The formation of the so-called corpse-wax, adipocere, which consists
of a mixture of fatty acids, ammonia, and lime-soaps, from parts of the
corpse rich in proteins, is sometimes given as a proof of the formation
of fats from proteins. The accuracy of this view has, however, been dis-
puted, and many other explanations of the formation of this substance
have been offered. According to the experiments of KRATTER and
K. B. LEHMANN, it seems as if it were possible by experimental means
to convert animal tissue rich in proteins (muscles) into adipocere by the
continuous action of water. Irrespective of this, SALKOWSKI has shown
that in the formation of adipocere, the fat itself takes part, in that the
olein decomposes with the formation of solid fatty acids, still it must
be considered that lower organisms undoubtedly take part in its forma-
tion. The production of adipocere as a proof of the formation of fat
from proteins is disputed by many investigators for this and other reasons.

Fatty degeneration has been considered as another proof of the
formation of fat from proteins. From the investigations of BAUER
on dogs, and LEO on frogs, it was assumed that, at least in acute poisoning
by phosphorus, a fatty degeneration, with the formation of fat from
proteins, takes place. PFLUGER has raised such strong arguments against
the older researches as well as the more recent one of POLIMANTI, who
claims to have shown the formation of fat from proteins in phosphorus

1 Coronedi and Marchetti, cited by Winternitz, Zeitschr. f. physiol. Chem., 24,
A review of the literature on fat formation may be found in Rosenfeld, Fettbildung.
in Ergebnisse der Physiologic, 1, Abt. 1.

FORMATION OF FATS. 561

poisoning, that we cannot consider the formation of fat as conclusively
proved. The investigations of LEBEDEFF, ATHANASIU, TAYLOR, SCHWALBE
and others, have shown that probably no new formation of fat from
protein took place, but rather a fat migration and that this is .actually
the case has been especially shown by ROSENFELD and recently by SHI-
BATA 1 in a conclusive manner.

Another more direct proof of the formation of fat from proteins
has been given by HOFMANN. He experimented with fly-maggots.
A number of these were killed and the quantity of fat determined. The
remainder were allowed to develop in blood whose proportion of fat
had been previously determined, and after a certain time they were killed
and analyzed. He found in them from seven to eleven times as much
fat as was contained in the maggots first analyzed and the blood taken
together. PFLUGER 2 has made the objection that a considerable number
of lower fungi develop in the blood under these conditions, in whose
cell-body fats and carbohydrates are formed from the different con-
stituents of the blood and their decomposition products, and that these
serve as food for the maggots.

WEiNLAND3 has observed the formation of higher non- volatile fatty
acids in the Calliphora larvae when they were rubbed to a homogeneous
paste after the addition of Witte's peptone. This experiment shows a
formation of fat from protein, but cannot be considered as quite con-
clusive.

As a more convincing proof of fat formation from proteins, the
investigations of PETTENKOFER and VOIT are often quoted. These
investigators fed dogs with large quantities of meat containing the least
possible proportion of fat, and found all of the nitrogen in the excreta,
but only a part of the carbon. As an explanation of these conditions
it has been assumed that the protein of the organisms splits into a
nitrogenized and a non-nitrogenized part, the former changing into the
nitrogenized final product, urea, and like products, and the other part,
on the contrary, being retained in the organism as fat (PETTENKOFER
and VOIT).

PFLUGER has arrived at the following conclusion by an exhaustive
criticism of PETTENKOFER and VOIT'S experiments and a careful recal-
culation of their balance-sheet; that these very meritorious investiga-

1 Bauer, Zeitschr. f. Biologie, 7; Leo, Zeitschr. f. physiol. Chem., 9; Polimanti,
Pfliiger's Arch., 70; Pfliiger, ibid., 51 (literature on the formation of fat from protein)
and 71; Athanasiu, ibid., 74; Taylor, Journ. Exp. Medicine, 4; see also footnote 2,
p. 384; Shibata,Bioch. Zeitschr., 37, which contains the literature; Rosenfeld, Ergebn.
d. Physiol., 1.

2 See Rosenfeld, Fettbildung, Ergebnisse der Physiologie, 1, Abt. 1.

3 Zeitschr. f. Biol., 51 and 52.

562 TISSUES OF THE CONNECTIVE SUBSTANCE.

tions, which were continued for a series of years, were subject to such
great defects that they are not conclusive as to the formation of fat
from proteins. He especially emphasizes the fact that these investigators
started from a wrong assumption as to the elementary composition of
the meat, and that the quantity of nitrogen assumed by them was too
low and the quantity of carbon too high. The relation of nitrogen to
carbon in meat poor in fat was assumed by VOIT to be as 1:3.68, while
according to PFLUGER it is 1:3.22 for fat-free meat after deducting the
glycogen, and according to RUBNER 1:3.28 without deducting the gly-
cogen. On recalculation of the figures, using these coefficients, PFLUGER
has arrived at the conclusion that the assumption as to the formation
of fat from proteins finds no support in these experiments.

In opposition to these objections, E. VOIT and M. CREMER have made
new feeding experiments, to show the formation of fat from proteins,
but the proof of these recent investigations has been disputed by PFLUGER.
On feeding a dog on meat poor in fat (containing a known quantity of
ether extractives, glycogen, nitrogen, water, and ash), KUMAGAWA 1
could not prove the formation of fat from protein. According to him
the animal body under normal conditions has not the power of forming
fat from protein.

Several French investigators, especially CHAUVEAU, GAUTIER, and
KAUFMANN,2 consider the formation of fat from proteins as positively
proved. KAUFMANN has recently substantiated this view by a method
which will be spoken of in detail in Chapter XVII, in which he studied
the nitrogen elimination and the respiratory gas exchange in conjunction
with the simultaneous formation of heat.

As we are agreed that carbohydrates and glycogen, as well as sugar,
can be formed from proteins, the fact cannot be denied that possibly
an indirect formation of fat from proteins, with a carbohydrate as an
intermediate step, can take place. The possibility of a direct fat for-
mation from proteins without the carbohydrate as intermediary must
also be generally admitted, although such a formation has not been
conclusively proved.

According to CHAUVEAU and KAUFMANN, in the direct formation of
fat from proteins, the fat is formed besides urea, carbon dioxide, and
water, as an intermediary product in the oxidation of the proteins, while
GAUTIER considers the formation of fat from proteins as a cleavage
without the taking up of oxygen. If fat is formed from protein in the
animal body, then such formation is not a splitting off of fat from the

1See Rosenfeld, Fettbildung, Ergebnisse der Physiologic, 1, Abt. 1.
2 Kaufmann, Arch, de physiol., (5) 8, where the works of Chauveau and Gautier
are cited.

FORMATION OF FATS. 563

proteins, but rather a synthesis from primarily formed cleavage products
of proteins which are poor in carbon.

The formation of fat from carbohydrates in the animal body 'was
first suggested by LIEBIG. This was opposed for some time, and until
lately it was the general opinion that a direct formation of fat from
carbohydrates not only had not been proved, but also that it was
improbable. The undoubtedly great influence of the carbohydrates on
the formation of fat as observed and proved by LIEBIG was explained
by the statement, that the carbohydrates were consumed instead of
the absorbed fat or that derived from the proteins, hence they have a
sparing action on the fat. By means of a series of nutrition experiments l
with different animals, with foods especially rich in carbohydrates it has
been apparently proved that a direct formation of fat from carbohydrates
does actually occur. The processes by which this formation takes place
are still unknown. As the carbohydrates do not contain such com-
plicated carbon chains as the fats, the formation of fat from carbohydrates
must consist of a synthesis, in which the group CHOH is converted into
CH2,' hence a reduction must occur.

After feeding with very large quantities of carbohydrates the relation between
the inspired oxygen and the expired carbon dioxide, i.e., the respiratory quotient
CO
-Q^-, was found greater than 1 in certain cases (HANRIOT and RICHET, BLEIBTREU,

KAUFMANN, LAULANI£ 2). This is explained by the assumption that the fat
is formed from the carbohydrate by a cleavage setting free carbon dioxide and water
without taking up oxygen. This increase in the respiratory quotient also depends
in part on the increased combustion of the carbohydrate.

When food contains an excess of fat, the superfluous amount is stored
up in the fatty tissue, and on partaking of food deficient in fat this
accumulation is quickly exhausted; and it is very probable that the
lipase is of importance here, as LOEVENHARTS has found that all over
the body where fat is deposited in large amounts lipase also occurs in
considerable amounts. There is perhaps not one of the various tissues
that decreases so much in starvation as the fatty tissue. The organism,
then, possesses in this tissue a depot where there is stored, during proper

1 Lawes and Gilbert, Phil. Transactions, 1859, part 2; Soxhlet, see Maly's Jahresber.,
11, 51; Tscherwinsky, Landwirthsch. Versuchsstaat, 29 (cited from Maly's Jahresber.,
13); Meissl and Stromer, Wien. Sitzungsber., 88, Abt. 3; Schultze, Maly's
Jahresber., 11, 47; Chaniewski, Zeitschr. f. Biologic, 20; Voit and Lehmann, see C.
v. Voit, Sitzungsber, d. k. bayer. Akad. d. Wissensch., 1885; I. Munk, Virchow's Arch.,
101; Rubner, Zeitschr. f. Biologic, 22; Lummert, Pfliiger's Arch., 71.

2 Hanriot and Richet, Annal. de Chim. et de Phys. (6), 22; Bleibtreu, Pfliiger's
Arch., 56 and 85; Kaufmann, Arch, de Physiol. (5), 8; Laulanie, ibid., 791.

* Amer. Journ. of Physiol., 6.

564 TISSUES OF THE CONNECTIVE SUBSTANCE.

alimentation, a nutritive substance of great importance in the develop-
ment of heat and vital force, which substance, on insufficient nutrition,
is given up as may be needed. On account of their low conducting
power, the fatty tissues become of great importance in regulating the
loss of heat from the body. They also serve to fill cavities and act as
a protection and support to certain internal organs.

CHAPTER X.

MUSCLES.

STRIATED MUSCLES.

IN the study of the muscles the chief problem for physiological chem-
istry is to isolate their different morphological elements and to investigate
each element separately. By reason of the complicated structure of
the muscles this has been thus far almost impossible, and we must be
satisfied at the present time with a few microchemical reactions in the
investigation of the chemical composition of the muscular fibers.

Each muscle-tube or each muscle-fiber consists of a sheath, the
SARCOLEMMA, which seems to be composed of a substance similar to
elastin, and containing a large proportion of protein. This last, which
in life possesses the power of contractility, has in the inactive muscle
an alkaline reaction, or, more correctly speaking, an amphoteric reac-
tion with a predominating action on red litmus paper. ROHM ANN
found that the fresh, inactive muscle shows an alkaline reaction with
red lacmoid, and an acid reaction with brown turmeric. From the effect
of various acids and salts on these coloring-matters, he concludes that the
alkalinity of the fresh muscle with lacmoid is due to sodium bicarbonate,
diphosphate, and probably also to an alkaline combination of protein
bodies, and the acid reaction with turmeric, on the contrary, to chiefly
monophosphate. The dead muscle has an acid reaction, or, more cor-
rectly, the acidity with turmeric increases on the decease of the muscle,
and the alkalinity with lacmoid decreases. The difference depends on the
presence of a larger quantity of monophosphate in the dead muscle, and
according to ROHM ANN free lactic acid is found in neither the one case
nor the other.1

If the somewhat disputed statements relative to the finer structure
of the muscles are disregarded, one can differentiate in the striated muscles
between the two chief components, the doubly refracting — anisotropous
— and the singly refracting — isotropous — substance. Both contain
abundance of protein, which form the chief part of the solids of the muscles.

1 The various reports in regard to the reaction of the muscles and the cause thereof
are conflicting. See Rohmann, Pfliiger's Arch., 50 and 55; Heffter, Arch. f. exp.
Path. u. Pharm., 31 and 38. These references contain the pertinent literature.

565

566 MUSCLES.

If the muscular fibers are treated with reagents which dissolve proteins,
such as dilute hydrochloric acid, soda solution, or gastric juice, they swell greatly
and break up into " BOWMAN'S disks." By the action of alcohol, chromic acid,
boiling water, or in general such reagents as cause a shrinking, the fibers split
longitudinally into fibrils; and this behavior shows that several chemically dif-
ferent substances of various solubilities enter into the construction of the muscular
fibers.

The protein myosin is generally considered as the principal constituent of the
diagonal disks, while the isotropous substance contains the chief mass of the
other proteins of the muscles as well as the chief portion of the extractives.
According to the observations of DANILEWSKY, confirmed by J. HOLMGREN, x
myosin may be completely extracted from the muscle without changing its struc-
ture, by means of a 5-per cent solution of ammonium chloride, which fact con-
flicts with the above view. DANILEWSKY claims that another protein-like sub-
stance, insoluble in ammonium chloride and only swelling up therein, enters essen-
tially into the structure of the muscles. The proteins, which formjbhe principal
part of the solids of the muscles, are of the greatest importance.

Proteins of the Muscles.

Like the blood which contains a fluid, the blood-plasma, which sponta-
neously coagulates, separating fibrin and yielding blood-serum, so also
the living muscle, at least of cold-blooded animals, contains, as first
shown by KUHNE, a spontaneously coagulating liquid, the muscle-plasma,
which coagulates quickly, separating a protein body, myosin, and yield-
ing also a serum. That liquid which is obtained by pressing the living
muscle is called muscle-plasma, while that obtained from the dead
muscle is called muscle-serum. These two fluids contain at least in part
different protein bodies.

Muscle-plasma was first prepared by KUHNE from frog-muscles, and later
by HALLIBURTON, according to the same method, from the muscles of warm-
blooded animals, especially rabbits. The principle of this method is as follows:
The blood is removed from the muscles immediately after the death of the animal
by passing through them a strongly cooled common-salt solution of 5-6 p. m.
Then the muscles are quickly cut and immediately frozen thoroughly so that
they can be ground in this state to a fine mass — " muscle-snow." This pulp is
strongly pressed in the cold, and the liquid which exudes is called muscle-plasma.
According to v. FURTH 2 this cooling or freezing is not necessary. It is sufficient
to extract the muscle free from blood, as above directed, with a 6 p. m. common
salt solution.

Muscle-plasma forms a yellow to brownish-colored fluid with an
alkaline reaction. It varies in different animals. Muscle-plasma from
the frog spontaneously coagulates, slowly, at a little above 0° C., but more

1 Danilewsky, Zeitschr. f. physiol. Chem., 7; J. Holmgren, Maly's Jahresber., 23.

2 See Kiihne, Untersuchungen iiber das Protoplasma, (Leipzig, 1864), 2; Hallibur-
ton, Journ. of Physiol., 8; v. Fiirth, Arch. f. exp. Path. u. Pharm., 36 and 37; Hof-
meister's Beitrage, 3, and Ergebnisse der Physiologic, 1, Abt. 1; Stewart and Soll-
mann, Journ. of Physiol., 24.

PROTEINS OF THE MUSCLES. 567

quickly at the temperature of the body. Muscle-plasma from mammals
coagulates slowly, according to vv. FURTH, even at the temperature of
the room, though only slightly, and it can hardly be considered as a
process comparable with the coagulation of the blood. Indeed the ques-
tion may be asked whether a true muscle-plasma does exist in warm-
blooded animals, or whether the fluid obtained from such muscles
exactly represents the plasma of the living muscle. According to KUHNE
and v. FURTH the reaction remains alkaline during coagulation, while
HALLIBURTON, STEWART and SOLLMANN find that it becomes acid.
Earlier investigators held that the clot consists of a globulin called
myosin, while v. FURTH claims that it consists of two coagulated pro-
teins, myosin-fibrin and myogen-fibrin.

The study of the proteins of the muscles, as well as their nomen-
clature, has changed markedly in the last few years, and it is questionable
whether an essential difference exists between the proteins of the muscle-
plasma and the muscle-serum of warm-blooded animals. Nevertheless
it is necessary to discuss separately the proteins of the dead muscle as
well as those of the muscle-plasma.

The proteins of the dead muscle are in part soluble in water or dilute
salt solutions, and in part are insoluble therein. Myosin and musculin
and also myoglobulin and myoalbumin, which exist to a very slight
extent and are perhaps only derived from the remaining lymph, belong
to the first group, and the stroma substances of the muscle-tubes belong
to the second group.

Myosin was first discovered by KUHNE, and constitutes the principal
mass of the soluble proteins of the dead muscle. It is generally considered
as the most essential coagulation product of muscle-plasma. The name
myosin, KUHNE also gives to the mother-substance of the plasma-clot,
and this mother-substance forms, according to certain investigators,
the principal mass of contractile protoplasm. The findings as to the oc-
currence of myosin in other organs besides the muscles require further
confirmation. The quantity of myosin in the muscles of different animals
varies, according to DANILEWSKY/ between 30 and 110 p. m.

Myosin, as obtained from dead muscles, is a globulin whose elementary
composition, according to CHITTENDEN and CuMMiNS,2 is, on an average,
the following: C 52.28, H 7.11, N 16.77, S 1.27, O 22.03 per cent. If
the myosin separates as fibers, or if a myosin solution with a minimum
quantity of alkali is allowed to evaporate to a gelatinous mass on a
microscope-slide, doubly refracting myosin may be obtained. Myosin
has the general properties of the globulins and is readily converted into

1 Zeitschr. f. physiol. Chem., 7.

2 Studies from the Physiol. Chem. Laboratory of Yale College, New Haven, 3, 115.

568 MUSCLES.

albuminates by dilute acids or alkalies. It is completely precipitated
upon saturation with NaCl, also by MgSO4, in a solution containing
94 per cent of the salt with its water of crystallization (HALLIBURTON).
The precipitated myosin readily becomes insoluble. Like fibrinogen it
coagulates at 56° C. in a solution containing common salt, but differs
irom it, since under no circumstances can it be converted into fibrin.
The coagulation temperature, according to CHITTENDEN and CUMMINS,
not only varies for myosins of different origin, but also for the same
myosin in different salt solutions. /

Myosin may be prepared in the following way, as suggested by HALLI-
BURTON: The muscle is first extracted by a 5-per cent magensium-
sulphate solution, and by fractional precipitation with magnesium sul-
phate the musculin and then the myosin are precipitated (see HALLI-
BURTON, 1. c.).

The older and perhaps the usual method of preparation consists,
according to DANiLEWSKY,1 in extracting the muscle with a 5-10 per cent
ammonium-chloride solution, precipitating the myosin from the filtrate
by strongly diluting with water, and redissolving the precipitate in ammo-
nium-chloride solution, and the myosin obtained from this solution is
reprecipitated either by diluting with water or by removing the salt
by dialysis.

Musculin,2 called PARAMYOSINOGEN by HALLIBURTON, and MYOSIN
by v. FURTH, is a globulin which is characterized by its low coagulation
temperature, in frogs below 40°, in mammalia 42-48°, and in birds about
51° C., and which may vary in different species of animals. It is more
easily precipitated than myosin by NaCl or MgSCU (50 per cent salt,
including water of crystallization). According to v. FURTH it is precipi-
tated by ammonium sulphate with a concentration of 12-24 per cent
salt. If the dead muscle is extracted with water a part of the musculin
goes into solution, and may be precipitated therefrom by carefully
acidifying. It separates from a dilute salt solution on dialysis. Mus-
culin readily passes into an insoluble modification which v. FURTH calls
myosin fibrin. Musculin is called myosin by v. FURTH, as he considers
it nothing but myosin. As musculin has a lower coagulation temper-,
ature and has other precipitating properties for neutral salts than the
older substance called myosin, it is difficult to accept this view.

Myoglobulin. After the separation of the musculin and the myosin from the
salt extract of the muscle by means of MgS04, the myoglobulin may be precipitated

1 Zeitschr. f. physiol. Chem., 5, 158.

2 As we have up to the present no conclusive basis for the identity of the globulins
called myosin and paramyosinogen, and also as the use of the name myosin for the
last-mentioned substance may readily cause confusion, the author does not feel
justified in dropping the old name musculin (Nasse).

PROTEINS OF THE MUSCLES. 569

by saturating the filtrate with the salt. It is .similar to serglobulin, but coagu-
lates at 63° C. (HALLIBURTON). Myoalbumin, or muscle-albumin, seems to
be identical with seralbumin (seralbumin a, according to HALLIBURTON), and
probably originates only from the blood or the lymph. Proteoses and peptones
do not seem to exist in the fresh muscles.

After the complete removal from the muscle of all protein bodies which are
soluble in water and ammonium chloride, an insoluble protein remains which
only swells in ammonium-chloride solution, and which forms with the other insoluble
constituents of the muscular fiber the " muscle-stroma" According to DANILEW-
SKY the amount of such stroma substance is connected with the muscle activity.
He maintains that the muscles contain a greater amount of this substance, com-
pared with the myosin present, when the muscles are quickly contracted and
relaxed, the correctness of which report has recently been disputed by SAXL.*

According to J. HOLMGREN, 2 this stroma substance does not belong to either
the nucleoalbumin or the nucleoprotein group. It is not a glucoproteid, as it
does not yield a reducing substance when boiled with dilute mineral acids. It is
very similar to the coagulable proteins, and dissolves in dilute alkalies, forming
an albuminate. The elementary composition of this substance is almost the same
as that of myosin. There is no doubt that the insoluble substances, myofibrin
and myosin fibrin, which are formed, according to v. FURTH, in the coagulation of
the plasma, also occur among the stroma substances. When the muscles are
previously extracted with water, the stroma substances also contain a part of the
myosin hereby made insoluble. The observations of SAXL on rabbits' muscles
agree with this view that the fresh muscle after work contains 11.5-21.6 per cent
of the total protein in an insoluble form, while the muscle after rigor mortis con-
tains on the contrary 71.5-73.2 per cent.

To the proteins insoluble in water, and neutral salts, belongs the
nucleoprotein detected by PEKELHARING, which occurs as traces and is
soluble in faintly alkaline water, and which probably originates from
the muscle nuclei. According to BOTTAZZI and DuccEScm3 the heart
muscle is richer in nucleoprotein than the skeletal muscle.

Muscle-syntonin, which may be obtained by extracting the muscles with
hydrochloric acid of 1 p.m., and which, according to K. MORNER. is less soluble
and has a greater aptitude to precipitate than other acid albumins, seems not
to occur preformed in the muscles. HEUBNER's4 mytolin is modified muscle-
proteid, chiefly myosin, which has lost a part of its sulphur by the action of alkali.

Proteins of the Muscle-plasma. As above stated, myosin was ordi-
narily considered as the coagulated modification of a soluble protein
existing in the muscle-plasma. As in blood-plasma there is present
a mother-substance of fibrin, fibrinogen, so also there exists in the
muscle-plasma a mother-substance of myosin, a soluble myosin or a
myosinogen. This body has not thus far been isolated with certainty.

1 Hofmeister's Breitage, 9.

2 See footnote 1, p. 566.

3 Pekelharing, Zeitschr. f. physiol. Chem., 22; Bottazzi and Ducceschi, Centralbl.
f. Physiol., 12.

4 Arch. f. exp. Pathol. u. Pharm., 53.

570 MUSCLES

HALLIBURTON, who has detected in the muscles an enzyme-like substance,
" myosin ferment," which is related to fibrin ferment but is not identical with it,
has also found that a solution of purified myosin, in dilute salt solution (5 per
cent MgS04), and sufficiently diluted with water, coagulates after a certain
time, and at the same time becomes acid, and a typical myosin-clot separates.
This coagulation, which is accelerated by warming or by the addition of myosin
ferment, is, according to HALLIBURTON, a process analogous to the coagulation
of the muscle-plasma. According to this same investigator, myosin when dis-
solved in water by the aid of a neutral salt is reconverted into myosinogen, while
after diluting with water myosin is again produced from the myosinogen. The
musculin (paramyosinogen) is carried down, according to HALLIBURTON, with the
myosin-clot, but has nothing to do with the coagulation, as the myosin-clot also
forms in the absence of musculin, and this last is not changed into myosin.

Besides the traces of globulin and albumin, which perhaps do not
belong to the muscle-plasma, there occur in mammals, according to
v. FURTH, two proteins, namely, musculin (myosin according to v. FURTH)
and myogen.

MUSCULIN (NASSE) = paramyosinogen (HALLIBURTON) = myosin (v.
FURTH) forms about 20 per cent of the total proteins of the muscle-
plasma of rabbits. Its properties have already been given, and it is
sufficient to remark that its solutions become cloudy on standing, and
a precipitate of myosin fibrin occurs, which is insoluble in salt solutions.

Myogen, or MYOSINOGEN (HALLIBURTON), forms the chief mass,
75-80 per cent, of the proteins of rabbit muscle-plasma. It does not
separate from its solutions on dialysis and is not a true globulin, but
a protein sui generis. It coagulates at 55-65° C. and is precipitated
in the presence of 26-40 per cent ammonium sulphate. Myogen solu-
tions are precipitated by acetic acid only in the presence of some salt.
It is converted into an albuminate by alkalies, this albuminate being
precipitable by ammonium chloride. Myogen passes spontaneously,
especially with higher temperatures as well as in the presence of salt,
into an insoluble modification, myogen fibrin. A protein, coagulating
at 30-40° C., soluble myogen fibrin, is produced as a soluble intermediate
step. This substance occurs to a considerable extent in native frog-
muscle plasma. It does not always occur in the muscle-plasma of
warm-blooded animals, and when it does it is present only to a slight
extent. It can be separated by precipitating with salt or by diffusion.
HALLIBURTON'S assumption as to the action of a special myosin ferment
has not sufficient basis, according to v. FURTH, nor has the often-admitted
analogy with the coagulation of the blood. The difference between
the musculin and the myogen in their becoming insoluble is that the
musculin passes into myosin fibrin without any soluble intermediate
steps.

Myogen may be prepared, according to v. FURTH, by heating, for a
short time, the dialyzed and filtered plasma to 52° C., separating it in

PROTEINS OF THE MUSCLES. 571

this way from the rest of the musculin. The myogen exists in the new
nitrate and can be precipitated by ammonium sulphate. The musculin
may also be removed by adding 28 per cent ammonium sulphate and then
precipitating the myogen from the nitrate by saturating with the salt.

STEWART and SOLLMANN admit of only two soluble proteins in the" muscles.
One is the paramyosinogen, which is the same as v. FURTH'S myosin+the soluble
myogen fibrin. The other they call myosinogen, which corresponds to v. FURTH'S
myogen or to HALLIBURTON'S myosinogen + my oglobulin. It is a typical globulin
which coagulates at 50-60° C. The paramyosinogen as well as the myosinogen
is readily converted into an insoluble modification, myosin. The myosin of the
above investigators is the same as v. FURTH'S myosin fibr in + myogen fibrin, and
corresponds, it seems, also to myosin mixed with paramyosinogen (HALLIBURTON) .
STEWART and SOLLMANN differ from HALLIBURTON in considering that paramy-
osinogen also coagulates and is converted into myosin. According to them
myosin is also insoluble in a NaCl solution.

The views of the various investigators differ so essentially and the
nomenclature is so complicated (three different things are designated
by the name myosin) that it is extremely difficult to give any correct
review of the various opinions.1 Thorough investigations on this subject
are very necessary.

Myoproteid is a protein found by v. FURTH in the plasma from fish-muscles.
It does not coagulate on boiling, is precipitated by acetic acid, and is considered
as a compound protein by v. FURTH.

In connection with v. FURTH'S work, PRZIBRAM has carried on ivestiga-
tions on the occurrence of muscle-proteins in various classes of animals. The
myosin (v. FURTH) and myogen occur in all classes of vertebrates; the myogen
is always absent in the invertebrates. Myoproteid occurs, at least in considerable
quantity, only in fishes. In the muscle after cutting the nerve, STEYRER 2 found
somewhat more musculin and less myogen in the muscle-juice than in the normal
muscle.

Muscle-pigments. There is no question that the red color of the
muscles, even when completely freed from blood, depends in part on
haemoglobin. K. MORNER has shown that muscle-haemoglobin is not
quite identical with blood-haemoglobin. The statement of MAcMuNN
that in the muscles another pigment occurs which is allied to haemo-
chromogen, and called myohcematin by him, has not been substantiated,
at least for muscles of higher animals (LEVY and MORNER 3) . MACMUNN
claims that myohaematin occurs in the muscles of insects, which do not
contain any haemoglobin. The reddish-yellow coloring-matter of the
muscles of the salmon has been little studied.

1 For these reasons the author is not sure whether he has understood and correctly
given the work of the different investigators.

2 Przibram, Hofmeister's Beitrage, 2; Steyrer, ibid., 4.

8 See MacMunn, Phil. Trans, of Roy. Soc., 177, part 1, Journ. of Physiol., 8 and
Zeitschr. f. Physiol. Chem., 13; Levy, ibid., 13; K. Morner, Nord. Med. Archiv. Fest-
band., 1897, and Maly's Jahresber., 27.

572 MUSCLES.

Various enzymes have been found in the muscles. To these belong
(besides traces of fibrin ferment and myosin ferment?) the catalases and
oxidases, which occur only to a slight extent and the glycolytic enzyme
(Chapter VII). An amylolytic and a proteolytic enzyme (HEDIN and
ROWLAND 1) have also been found, and the hydrolytic and oxidizing
enzymes (Chapter XIV) active in the formation and destruction of uric
acid are also present.

Extractive Bodies of the Muscles.

The nitrogenous extractives in the muscles of higher animals con-
sist chiefly of creatine and creatinine (especially in fishes) and carnosine.
To these also belong inosinic acid (and the closely related carnine), phos-
phocarnic acid, carnitine and purine bases, especially hypoxanihine. The
purine bases occur partly .free (which is especially the case with hypoxan-
thine) and partly combined.

Among the extractive substances is also found the acid noticed by LIMPRICHT
in the flesh of certain cyprinidea, namely, the nitrogenized protic acid, while the
isocreatinine found by J. THESBN in fish-flesh is nothing but impure creatinine,
according to POULSSON, SCHMIDT and KoRNDORFER.2 The following have also
been found in the muscles, in certain cases only, of a few varieties of animals:
uric acid (especially in alligators), taurine (in cephalopoda and oysters), glycocpll
(in gasteropoda), betaine and methyl guanidine, in fish meat, several monamino
acids and also the three hexone bases histidine, lysine and arginine.3 Urea occurs
in large quantities in the muscle of the shark and ray. The reports are very
contradictory in regard to the occurrence of urea in the muscles of higher animals.
According to the investigations of KAUFMANN and SCHONDORFF, confirmed by
BRUNTON-BLAiKiE,4 urea is a regular constituent of the muscles, although M.
NENCKI and KOWARSKI dispute this.

In regard to the division of the nitrogenous extractives of the muscles, v.
FURTH and SCHWARZ found the following in 1000 grams of the moist extremity
musculature of the horse and dog (after subtracting the proteoses derived by
secondary cleavage processes), 3.27-3.82 gram extractive nitrogen. Of this
4.5-7 per cent was ammonia, 6.1-11.1 per cent purine bodies, 26.5-37.1 per cent
creatine and creatinine, 30.3-36.3 per cent carnosine fraction, 8.2-15.3 per cent
base residue (carnitine, methylguanidine, etc.) and 6.3-16 per cent urea, poly-
peptides and amino-acids. The quantity of purine base nitrogen, according
to BURIAN and HALL in fresh meat of the horse, ox and calf, was 0.55 p. m., 0.63

1 Zeitschr. f. physiol. Chem., 32.

2 See Limpricht, Annal. d. Chem. u. Pharm., 127, and Thesen, Zeitschr. f . physiol.
Chem., 24; Poulsson, Arch. f. exp. Path. u. Pharm., 51; Schmidt and Korndorfer,
ibid., 51.

3 In regard to the extractives of the muscles see besides the specially cited works,
Kurkenberg and Wagner, Zeitschr. f. biol. 21; U. Suzuki and collaborators, Zeitschr. f.
physiol. Chem. 62 and Chem. Centralbl. 1913, 1; Suwa, Pfluger's Arch. 128 and 129;
Zunz, Centralbl. f. Physiol., 18.

4Kaufmann, Arch, de Physiol. (5), 6; Schondorff, Pfluger's Arch., 62; Nencki
and Kowarski., Arch. f. exp. Path. u. Pharrn., 36; Brunton-Blaikie, Journ. of Physiol.,
23, Supplement.

CREATINE. 573

p. m. and 0.71 p. m. respectively, which corresponds closely to the results found
by SCAFFIDI, BUGLIA and COSTANTINO for the striated muscle of the calf, namely,
0.58-0.68 p. m. According to RINALDI and SCAFFIDI 1 the lowest values for the
purine nitrogen occur in the striated muscles of the covering of polype, 0.436
p. m., then in fishes 0.595-0.82 p. m. and the highest 1.061 p. m. in birds. BUGLIA
and COSTANTINO have determined the nitrogen titratable with formol, and from
this determined the amount of monamino-acid nitrogen as well as diamino-acid
nitrogen in various animals. In oxen they found in the moist, striated muscle
0.18 p. m. monamino- and 0.40 p. m. diamino-nitrogen. In the heart the cor-
responding figures were 0.18 and 0.18 p. m. In percentage of the total nitrogen
the total amino-acid nitrogen in the striated muscle was 1.70 per cent and in
the heart 1 .48 per cent.

The most extensively occurring nitrogenous extractives in the muscle
are creatine and carnosine.

</NH2

Creatine, C4H9N3O2, C==NH , or methyl-guanidine-

\N(CH3).CH2COOH

acetic acid, occurs in the striated as well as smooth muscles. In the
striated muscle of vertebrates the amount varies between 2.5 and 7 p. m.
It is also found in the brain, blood, transudates, amniotic fluid, and
sometimes also in the urine.

Creatine may be prepared synthetically from cyanamide and sar-
cosine (methylglycocoll) . On boiling with baryta-water it decomposes,
with the addition of water, and yields urea, sarcosine, and certain other
products. Because of this behavior several investigators consider
creatine as a step in the formation of urea in the organism. On boiling
with acids, creatine is easily converted, with the elimination of water
into the corresponding anhydride, creatinine, C^yNsO, which is retrans-
formed into creatine by the action of alkali.

The question as to the mutual relation of creatine to creatinine in
metabolism will be treated in Chapter XIV (urine). In this chapter,
besides the properties and reactions, we will discuss the question as to
the origin of creatine and its relation to the metabolism of the muscles.

Of special interest in this regard, besides the relation between creatine
and muscle work which will be discussed below, is the question as to
the occurrence of free or combined creatine in the muscle. URANO by
the aid of dialysis experiments has shown the probability that the crea-
tine does not exist free in the muscle, but as a labile, non-dialyzable
combination. Nevertheless GOTTLIEB and STANGASSINGER claim by
various researches to have shown in the autolysis of muscles and other
organs,. that .creatine is first formed and then first changed into creatinine
by special bodies of an enzymotic nature, and then destroyed. SEEMANN

1 v. Furth and Schwarz, Bioch. Zetschr., 30; Scaffldi, ibid., 33; Burian and Hall,
Zeitschr. f. physiol. Chem. 38; Buglia and Costantino, ibid., 81 and 82; Rinaldi and
Scaffidi, Bioch. Zeitschr., 41.

574 MUSCLES. ,

claims, by an autolysis of three months' duration, to have obtained two
to three times as much creatinine, directly from the muscle, and after
the addition of creatinine-free-gelatin four times as much, which is an
argument against the enzymotic destruction of creatinine in autolysis,
and he admits of the formation of creatine (or creatinine) from protein.
The autoiytic experiments of ROTHMANN also indicate the formation of
creatine from a preliminary body, and the recent experiments of VAN
HOOGENHUYZE and VERPLOEGH make the enzymotic transformation
of creatine and creatinine probable. MELLANBY positively denies the
re-formation of creatine as well as its destruction in autolysis entirely
free from bacteria. It is hard to draw positive conclusions from exper-
iments with autolysis. The transfusion experiments of GOTTLIEB and
STANGASSINGER, with the kidneys and livers of dogs, not only point to
the ability of these organs to decompose creatine, but also for a re-forma-
tion of creatine in the liver. Further investigations are still very nec-
essary, especially as the conditions are probably not the same in all animals.
Thus NOEL-PATON and MACKIE 1 found that the exclusion of the liver
in birds is without influence upon the creatine metabolism.

As will be discussed in Chapter XIV, no certain relationship
exists between the quantity of food protein and the extent of creatine
and creatinine elimination. On the contrary, several observations speak
for a relation between creatine formation and catabolism of organ pro-
tein, especially muscle protein and according to NOEL-PATON 2 the
elimination of creatine in bird urine, which here corresponds to the
creatinine in the mammalian urine, is a measure of the protein catabo-
lism of the muscles.

Under all circumstances the proteins, and especially the guanidine
groups contained therein, are the mother-substance for the creatine or
creatinine. The guanidine occurs in the protein molecule as arginine;
but according to OTORI it is not improbable that in the protein also
other guanidine groups exist. Nevertheless the observations of JAFFE a
speak against the assumption as to a creatine formation from argin-
ine as he found that arginine subcutaneously injected did not cause
any increase in the elimination of creatine substances. But as the
introduced arginine was probably decomposed by the enzyme arginase,
because the urea elimination was greatly increased,4 does not exclude

1 Urano, Hofmeister's Beit rage, 9; Gottlieb and Stangassinger, Zeitschr. f. physiol.
Chem., 52 and 55; Stangassinger, ibid., 55; Seemann, Zeitschr. f. Biol., 49; Roth-
mann, Zeitschr. f. physiol. Chem., 57; v. Hoogenhuyze and Verploegh, ibid., 57;
Mellanby, Journ. of Physiol., 36; Noel-Paton and Mackie, Journ. of Physiol. 45.

2Journ. of Physiol., 39.

»Otori, Zeitschr. f. physiol. Chem., 42, 43; Jaffe, ibid., 48.

4 See Thompson, Journ. of Physiol., 32 and 33.

CREATINE. 575

the possibility that in the muscles, which according to KOSSEL and DAKIN
contain only little arginase, the arginine was decomposed in other ways.
In autolysis as well as perfusion experiments with livers, INOUYE l has
recently shown that an increase in the creatine occurs at the expense of
the arginine added.

Starting with the observation of Jaff6 2 that glycocyamine (guanidine acetic
acid) in rabbits is transformed with a methylation into creatine, we can consider
the cleavage of arginine into creatine in the following manner, basing this con-
ception upon the ruling conception on the cleavage of ammo-acids and fatty acids
in the animal body.

NH2

NH2

NH2

NH2

HN=C<^ HI

*=C/

HN=C^ iff

N=C/

NH

NH

NH

N(CH3)

CH2

CH2

CH2

CH2

CH2

1
(CH2)2

> COOH

COOH

CH2

COOH

CJuaniHine acetic
acid (glycocyamine).

Creatine.

|

•y-guanidine

CH(NH2)

butyric acid.

COOH

Arginine.

The opinions are not unanimous in regard to the organ producing
creatine or creatinine. Based upon several investigations it is generally
admitted that the liver here plays an important r61e. Several other
organs may also be considered and in the first place, the muscles. Accord-
ing to MELLANBY the creatinine is probably formed in the liver, trans-
formed into creatine in the muscles and there stored up as such. Other
observations still speak for the fact that the creatine is formed in the
muscles and transformed into creatinine in the liver, while according
to NOEL-PATON and MACKIE the exclusion of the liver in birds is without
effect upon the creatinine metabolism.

Creatine crystallizes in hard, colorless, monoclinic prisms which
lose their water of crystallization at 100° C. It is soluble in 74 parts
of water at the ordinary temperature, and in 9419 parts absolute alcohol.
It dissolves more easily with the aid of heat. Its watery solution has
a neutral reaction. Creatine is not dissolved by ether. If a creatine
solution is boiled with precipitated mercuric oxide, this is reduced,
especially in the presence of alkali, to mercury and oxalic acid, and the
foul-smelling methyluramine (methylguanidine) is developed. A solu-

1 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41 and 42; Inouye, ibid., 81.

2 Zeitschr. f. physiol. Chem., 48; see also Dorner, ibid., 52.

576 MUSCLES.

tion of creatine in water is not precipitated by basic lead acetate, but
gives a white, flaky precipitate with mercurous nitrate if the acid reac-
tion is neutralized. When boiled for an hour with dilute hydrochloric
acid, creatine is converted into creatinine, and may be identified by its
reactions. On boiling with formaldehyde it can be transformed into
dioxymethylenecreatinine, which crystallizes readily (JAFFE l).

The preparation and detection of creatine is best accomplished by the
following method of NEUBAUER,2 which was first used in the preparation
of creatine from muscles: Finely cut meat is extracted with an equal
weight of water at 50-55° C. for 10-15 minutes, pressed, and extracted
again with water. The proteins are removed from the united extracts
so far as possible by coagulation at boiling heat, the filtrate precipitated
by the careful addition of basic lead acetate, the lead removed from this
filtrate by H^S, and the solution then carefully concentrated to a small
volume. The creatine, which crystallizes in a few days, is collected on a
filter, washed with alcohol of 88 per cent, and purified, when necessary,
by recrystallization. In the preparation of large quantities of creatine
we can especially start with meat extracts. The quantitative estimation
of creatine is performed by transforming it into creatinine (see Chapter
XIV).

Carnosine, CgHuN^a, is a base first isolated by GULEWITSCH and
AMIRADZIBI from meat extracts and which subsequently was also pre-
pared directly from meat. The quantity seems to be relatively consider-
able, as according to the above-mentioned determination of v. FURTH
and SCHWARZ, the carnosine fraction from the horse and dog muscles
was just as large or indeed greater than the creatine-creatinine fraction
of the extractive nitrogen. KRIMBERG found 1.3 p. m. and SKWcmzow,3
1.76 p. m. (as nitrate) in fresh meat.

Carnosine, which according to GULEWITSCH is identical with the base
ignotine isolated from meat extracts by KUTSCHER while both bases are
isomeric bodies according to KuTSCHER,4 is a histidine derivative accord-
ing to GULEWITSCH which on cleavage yields /3-alanine besides histidine.

Carnosine is a base readily soluble in water, which is precipitated
as stellar warts of short delicate needles from the concentrated watery
solution by the addition of alcohol. The specific rotation for the light
X = 546 is according to GULEWITSCH in watery solution where c = 12.925
per cent and 20.1° C. = +25.3°. The base is precipitated by phospho-

1 Ber. d. d. Chem. Gesellsch., 35.

2 Zeitschr. f. analyt. Chem., 2 and 6.

3 Gulewitsch and Amiradiibi, Zeitschr. f. physiol. Chem., 30; Gulewitsch, ibid.,
50, 51, 52 and 73; Krimberg. ibid., 48; Skworzow, ibid., 68.

4 Gulewitsch and Amirad&bi, Zeitschr. f. physiol. Chem., 30; Gulewitsch, ibid., 50,
61, 52 and 73; Krimberg, ibid., 68; Skworzow, ibid., 68; Kutscher, ibid., 50, 51.

CARNITINE. CARNINE. 577

tungstic acid, by mercuric nitrate and by silver nitrate with an excess of
barium hydrate. Carnosine-silver is soluble with difficulty in cold water
but readily soluble in hot water. Carnosine nitrate melts at 211-212° C.
Carnosine also gives a crystalline copper salt.

The principle in preparing this base consists in precipitating with
phosphotungstic acid, separating the free base with barium hydrate,
conversion into the nitrate, precipitating with silver nitrate and barium
hydrate, decomposing the salt with H^S and conversion into nitrate.
From the latter, which is readily obtained as crystals, the base is precip-
itated by phosphotungstic acid and then set free by barium hydrate.

Carnitine, C7Hi6N03 (or C7Hi6N03), another base isolated by GULEWITSCH
and KRIMBERG from meat extracts, has a strong alkaline reaction, is very
readily soluble in water, and was also found by KRIMBERG in fresh meat. SKWORZOW
found 0.19 p. m. carnitine in calf's muscles. Carnitine according to KRIMBERG
is probably 7-trimethyl-j3-oxybutyrobetaine with the formula

,0 CO

(CH3)3N< | . According to ENGELAND it is on the contrary

\CH2— CH-(OH)— CH2

a T-trimethyl-a-oxybutyrobetaine (CH3)3-Nr~~ I j, .

CH2 • CH2 . CH (OH)— CO

according to KRIMBERG and .ENGELAND1 identical with novaine prepared by KOSSEL
from meat extracts. It gives crystalline double compounds with platinum, gold
and mercuric chlorides, among which the following, C7Hi5N032HgCl2, with a
melting-point of 196-197° C., is especially used in the isolation of the base.
The hydrochloride and the nitrate are readily soluble and the solution of the first
is laBvo-rotatory, about («)D =—21°.

The inosinic acid has been discussed in Chapter II. In close relation to this
stands probably the carnine.

Carnine, CyHsN^+ILO, is one of the substances found by WEIDEL in American
meat extract. It has also been found by KRUKENBERG and WAGNER in frog
muscles and in the flesh of fishes, and by POUCHET in the urine. Carnine is, accord-
ing to HAISER and WENZEL,2 probably only an equimolecular mixture of hypo-
xanthine and the crystalline pentoside (hypoxanthin-riboside) inosine, which is
readily split by acid into hypoxanthine and pentose.

Carnine has been obtained as a white crystalline mass. It dissolves with
difficulty in cold water, but more readily in warm. It is insoluble in alcohol
and ether. It dissolves in warm hydrochloric acid and yields a salt crystallizing
in shining needles, which gives a double compound with platinum chloride. Its
watery solution is precipitated by silver nitrate, but this precipitate is dissolved
neither by ammonia nor by warm nitric acid. Its watery solution is precipitated
by basic lead acetate ; but the lead compound may be dissolved on boiling.

Phosphocarnic acid8 is a complicated substance, first isolated by SIEGFRIED

1 Gulewitsch and Krimberg, Zeitschr. f. physiol. Chem. 45; Krimberg, ibid.; 49,
50, 53 and 56, Ber. d. d. Chem. Gesellsch. 42; Engeland, ibid., 42; Skworzow, 1. c.

2 Weidel, Annal. d. Chem. u. Pharm., 158; Krunkenberg and Wagner, Sitzungsber.
d. Wurzb. phys.-med. Gesselsch., 1883; Pouchet, cited from Neubauer-Huppert,
Analyse des Harnes, 10. Aufl., 335; Haiser and Wenzel, Monatsch. f. Chem., 29.

3 In regard to carnic acid and phosphocarnic acid, see the works of Siegfried, Arch,
f. (Anat. u.) Physiol., 1894, Ber. d. deutsch. chem. Gesellsch., 28, and Zeitschr. f.
physiol. Chem., 21 and 28; M. Miiller, ibid., 22; Kriiger, ibid., 22 and 28; Balke and

578 MUSCLES.

from meat extracts, which yields as cleavage products succinic acid, paralactic
acid, carbon dioxide, phosphoric acid, and a carbohydrate group, besides the
previously mentioned carnic acid, which is identical with or nearly related to
antipeptone. It stands, according to SIEGFRIED, in close relation to the nucleins,
and as it yields peptone (carnic acid), it is designated as a nucleon by SIEGFRIED.
Phosphocarnic acid may be precipitated as an iron compound, carniferrine, from
the extract of the muscles free from proteins. The quantity of phosphocarnic
acid, calculated as carnic acid, can be determined by multiplying the quan-
tity of nitrogen in the compound by the factor 6.1237 (BALKE and IDE). In
this way SIEGFRIED found 0.57-2.4 p. m. carnic acid in the resting muscles
of the dog, and M. MULLER 1-2 p. m. in the muscles of adults and a maximum
of 0.57 p. m. in those of new-born infants. According to CAVAZZANI nucleon
occurs to a much greater extent in oysters, namely, an average of 3.725 p. m.
It also occurs, as he and MANICARDI found, in the plant kingdom. Phospho-
carnic acid has not been prepared in the pure state and possesses on this account
a variable composition; according to SIEGFRIED it serves as a source of energy
in the muscles and is consumed during work. Besides, by means of its property
of forming soluble salts with the alkaline earths, as also an iron combination
soluble in alkalies, it acts as a means of transportation for these bodies in the
animal body.

Phosphocarnic acid is prepared from the extract free from protein by first
removing the phosphate by CaCl2 and NH3. The acid is precipitated as carnifer-
rine by ferric chloride from the filtrate while boiling.

From LIEBIG'S extract of beef KUTSCHER has isolated besides the above-
mentioned ignotine and novaine, several other bodies, neosine, C6Hi7N02, which
according to KUTSCHER and ACKERMANN is a homologue of choline, vitiatine
(as gold salt, C6Hi4N6.2HC1.2AuCl3), carnomuscarine, methylguanidine (also found
by GULEWITSCH), oblitine, CisHssNaOs. which probably contains two novaine
groups, which corresponds well with KRIMBERG'S view, and also choline and
neurine. From dog muscles ACKERMANN * has isolated a platinum compound,
CiiH3oN204PtCl6, of a base called myocynine, which seems to be a hexamethyl-
ornithine. MICRO 2 found in meat extracts small quantities of alanine, glutamic
acid, taurine and inosite, but no dipeptides. In crab extract KUTSCHER and ACK-
ERMANN found no creatine and creatinine, but among others betaine and two new
bases, crangitine, Ci3H2oN204, and crangonine, Ci3H26N203. In crab muscles SUZUKI 3
and collaborators found a base, canirine which although it has the same composi-
tion, C6Hi4N202, as lysine, is not identical therewith.

The base musculamine, isolated by ETARD and VILA on the hydrolysis of veal,
is nothing but cadaverine, according to POSTERNAK.*

We must also include among the nitrogenous extractives those bodies which
were first discovered by GAUTiER,5 and which occur only in very small quantities,
namely, the leucomaines, xanthocreatinine, C5Hi0H40, crusocreatinine, C6H8N40,
amphicreatine, C9Hi9N704, and pseudoxanthine, C4HsN50.

Ide, ibid., 21, and Balke, ibid., 22; Macleod, ibid., 28; E. Cavazzani, Centralbl. f.
Physiol., 18, 666; Panella, Maly's Jahresber., 34.

1 Kutscher, Zeitschr. f. Unters. d. Nahrungs- u. Genussmittel, 10, 11, Centralbl.
f. Physiol., 19 and 21, Zeitschr. f. physiol. Chem., 48, 49, 50, 51, with Ackermann,
ibid., 56; Gulewitsch. ibid., 47; Krimberg, ibid., 56; Ackermann (on myocynine).
Zeitschr. f. biol. 59.

2 Zeitschr. f. physiol. Chem., 56.

3 Kutscher and Ackermann, Zeitschr. f. Unters. d. Nahrungs- u. Genusmittel, 13
and 14; Suzuki, Chem. Centralbl. 1913, 1.

4Etard and Vila, Compt. Rend., 135; Posternak, ibid., 135.
6 See Maly's Jahresber., 16, 523.

INOSITE. 579

In the analysis of meat, and for the detection and separation of the various
extractive bodies of meat, we make use of the systematic method as suggested
by GAUTiER,1 for details of which the reader is referred to the original article
as well as for the Kutscher method for working the meat extracts.

The non-nitrogenous extractive bodies of the muscles are inosite ^ gly-
cogen, sugar, and lactic acid.

Inosite, C6Hi206+H20 = C6H6(OH)6+H20. This body, discovered
by SCHERER, is not a carbohydrate, but belongs to the hydroaromatic
compounds, and is a hexahydroxybenzene (MAQUENNE2). That it
stands in certain relation to the carbohydrates follows from the fact that
NEUBERG obtained some furfurol from inosite by distillation with phos-
phoric anhydride, and also that P. MEYER 3 found fermentation lactic
acid in the urine of rabbits after the introduction of inosite per os. It
has been known for some time that inosite undergoes lactic acid fermenta-
tion. The acid formed thereby is sarcolactic acid according to HILGER
and fermentation lactic acid according to VoHL.4

Inosite is found in the muscles, liver, spleen, leucocytes, kidneys,
suprarenal capsule, lungs, brain, testicles, and in the urine in pathological
cases, and as traces in normal urine. It is found very widely dis-
tributed in the vegetable kingdom, especially in the unripe fruit of green
beans (Phaseolus vulgaris), and therefore it is also called PHASEOMANNITE.
In the plant kingdom another substance occurs which is called phytin
and which is the Mg and Ca compound of inosite and phosphoric acid
and which was first isolated by POSTERNAK. WINTERSTEIN identified this
as an inosite-phosphoric acid. This inosite-phosphoric acid can be split
into phosphoric acid and inosite by the plant enzyme phytase (SUZUKI,
YOSHIMURA and TAKAISHI) as well as by enzymes of the animal tissues
(STARKENSTEIN) . Inosite is found in plants, especially in the develop-
ing organs (MEILLERE), and according to STARKENSTEIN 5 it occurs to a
greater extent in the organs of young animals as compared with those of
older animals. From this it follows that inosite is probably not a decom-
position product of metabolism, but rather a body necessary for the devel-
opment of the cells (MEILLERE); but according to STARKENSTEIN the
facts are different.

1 Maly's Jahresb., 22.

2 Bull. SOG. chem. (2), 47 and 48; Compt. Rend., 104.

3 Neuberg, Bioch. Zeitschr., 9; P. Meyer, ibid., 9.

4 Hilger, Annal. d. Chem. u. Pharm., 160; Vohl, Ber. d. d. Chem. Gesellsch., 9.

6 Winterstein, Ber. d". d. chem. Gesellsch., 30; and Zeitschr. f. physiol. chem., 58;
Posternak, Contribution a l'6tude chim. de I'assimilation chlorophyllienne. Revue
generate botanique, Tome 12 (1900), and Compt. Rend., 137; Suzuki, Yoshimura and
Takaishi, Bull, agric. Univers. Tokio, 7; Starkenstein, Bioch. Zeitschr., 30.

580 MUSCLES.

According to STAKKENSTEIN the free inosite is without importance
and is only a decomposition product of metabolism; of importance,
especially for young, growing individuals is according to this worker
only the phytin, which is decomposed in the intestine by bacteria, and in
the tissues by enzymes, and correspondingly supplies phosphoric acid and
lime to the organism while the inosite is excreted as a valueless cleavage
product. The free inosite in the animal body originates according to
STARKENSTEIN from the inositephosphoric acid and in this sense the
assumption of RosENBERGER1 as to the occurrence of an inositogen in the
animal body, is substantiated.

Inosite, which almost without exception is inactive mesoinosite,
crystallizes in large, colorless, rhombic crystals of the monoclinic sys-
tem, or, if not pure and if only a small quantity crystallizes, it forms
groups of fine crystals similar to cauliflower. It loses its water of crys-
tallization at 110° C., also if exposed to the air for a long time. Such
exposed crystals are non-transparent and milk-white. The crystals
melt at 225° C. when dry. Inosite dissolves in 7.5 parts of water at
ordinary temperature, and the solution has a sweetish taste. It is insoluble
in, strong alcohol and in ether. It dissolves cupric hydrate in alkaline
solutions, but does net reduce on boiling. It gives negative results with
MOORE'S test and with BOTTGER-ALMEN'S bismuth test. It does not
ferment with beer-yeast, but may undergo lactic- and butyric-acid fer-
mentation. With an excess of nitric acid inosite is oxidized to rhodizonic
acid, and the following reaction depends upon this.

If inosite is evaporated to dryness on paltinum-foil with nitric acid
and the residue treated with ammonia and a drop of calcium chloride
solution and carefully re-evaporated to dryness, a beautiful rose-red
residue is obtained (SHERER'S inosite test). If we evaporate an inosite
solution to incipient dryness and moisten the residue with a little mer-
curic nitrate solution, there is obtained a yellowish residue on drying
which becomes a beautiful red on strongly heating. The coloration
disappears on cooling, but it reappears on gently warming (GALLOIS'
inosite test) . Other inosite reactions have been suggested by DENIGES 2
and others.3

To prepare inosite from a liquid or from a watery extract of a tissue,
the proteins are first removed by coagulation at boiling heat. The filtrate

1 Meillere, Journ. d. Chim. et Pharm. (6) 28; Starkenstein, Zeitschr. f. exp. Path,
u. Therap. 5, Bioch. Zeitschr. 30 and Zeitschr. f. physiol. Chem. 58; Rosenberger,
ibid., 56, 57 and 58.

2 Compt. rend. soc. biol., 62.

3 In regard to the salts of phytin and compounds of inosite see Anderson, Journ.
of biol. Chem. 11 and 12.

GLYCOGEN. 581

is precipitated by sugar of lead, this filtrate boiled with basic lead acetate
and allowed to stand 24-48 hours. The precipitate thus obtained,
which contains all the inosite, is decomposed in water by H^S. The
filtrate is strongly concentrated, treated with 2-4 vols. hot alcohol, and
the liquid removed as soon as possible from the tough or flaky masses
which ordinarily separate. If no crystals separate from the liquid within
twenty-four hours, then treat with ether until the liquid has a milky
appearance and allow it to stand. In the presence of a sufficient quantity
of ether, crystals of inosite separate within twenty-four hours. The
crystals thus obtained, as also those which are directly obtained from the
alcoholic solution, are recrystallized by redissolving in very little boiling
water and adding 2-4 yols. of alcohol. MEILLERE 1 and others have
suggested modifications in the methods for detecting and quantitatively
estimating inosite.

Scyllite is a body which is isomeric with inosite, according to JOH. MULLER,*
and which was found long ago in the kidneys, liver and spleen of Plagiostomata
and also in the plant kingdom as cocosite and quercinite. Scyllite crystallizes
in shining prisms, is soluble in water 1:100 at 18° C., is similar to inosite in its
reactions, but has a much higher melting-point, namely about 360° C. From
the adductor muscles of the Mytilus JANSSEN 3 has isolated a substance, called
mytilite which is crystalline, soluble with , difficulty in cold water and readily sol-
uble in hot water, and having the formula C6Hi205.2H20. He claims that it is
stereisometric with the alcohol quercite.

Glycogen is a constant constituent of the living muscle, while it may
be absent in the dead muscle. The quantity of glycogen varies in the
different muscles of the same animal and according to MAIGNON this
is not only true for the same muscles in both halves of the body but also
for different parts of the same muscle. BOHM found 10 p. m. glycogen
in the muscles of cats, and moreover he found a smaller amount in the
muscles of the extremities than in those of the rump. MOSCATI found an
average of 4 p. m. in human muscles, and ScnoNDORFF4 has found a
maximum of 37.2 p. m. in the dog-muscle. Reports as to the quantity
of glycogen in the heart are conflicting; although the heart is considered
as somewhat poorer in glycogen than the other muscles, still this difference
is not very great, and can be explained by the ready disappearance of
glycogen from the heart after death, as well as after starvation and
after strong work (BORUTTAU, JENSEN5). Work and food have a great
influence upon the quantity of glycogen. BOHM found 1-4 p. m.
glycogen in the muscles of fasting animals, and 7-10 p. m. after partak-

1 Compt. rend. soc. biol., 60, and Journ. d. Chim. et Pharm. (6), 24; see also
Starkenstein, Zeitschr. f. exp. Path. u. Ther., 5.

2 Ber. d. d. chem. Gesellsch., 40.

3 Zeitschr. f. physiol. Chem., 85.

4 Maignon, Journ. de physiol. et d. path. 10 Bohm, Pfliiger's Arch., 23, 44; Schon-
dorff, ibid., 99; Moscati, Hofmeister's Beitrage, 10.

6 Boruttau, Zeitschr. f. physiol. Chem., 18; Jensen, ibid., 35.

582 MUSCLES.

ing of food. As stated in Chapter VII, work, starvation, and lack of
carbohydrates in the food cause the glycogen to disappear earlier from
the liver than from the muscles.

The sugar of the muscles, of which only traces occur in the living mus-
cle, and which is probably formed after the death of the muscle from
the muscle-glycogen, is, according to the investigations of PANQRMOFF, in
part glucose, but consists principally of maltose (OSBORNE and ZOBEL l)
with some dextrin. ,

Lactic Acids. Of the oxypropionic acids with the formula CsHeOa
there is one, ethylene lactic acid, CH2(OH).CH2.COOH, which is not
found in the animal body, and therefore has no physiological chemical
interest.

CH3
Indeed only a-oxypropionic acid or ethylidene lactic acid, CH(OH), of

COOH

which there are two physical isomers, namely, the dextrorotatory PAR-
ALACTIC or SARCOLACTIC ACID, and the LEVOLACTIC ACID obtained by
SCHARDINGER by the fermentation of cane-sugar by means of a special
bacillus. This levolactic acid, which is formed by the typhoid bacillus
and various vibriones 2 need not be discussed here, and we will only treat
here the d-Z-lactic acid (the inactive fermentation lactic acid) and the
dextrolactic acid.

The fermentation lactic acid, which is formed from lactose by allow-
ing milk to sour, and by the acid fermentation of other carbohydrates,
is considered to exist in small quantities in the muscles (HEINTZ), in the
gray matter of the brain (GSCHEIDLEN), and in diabetic urine. The
occurrence of fermentation lactic acid in the brain and other organs
is still very improbable and has been disputed by MORI Y A.3 During
digestion this acid is also found in the contents of the stomach and intestine,
and as alkali lactate in the chyle. The paralactic acid, is at all events,
the true acid of meat extracts, and this alone has been found with certainty
in dead muscle. The lactic acid which is found in the brain, spleen,
lymphatic glands, thymus, thyroid gland, blood, bile, pathological
transudates, osteomalacial bones, in perspiration in puerperal fever,
in the urine after fatiguing marches, in acute yellow atrophy of the liver,

1 Panormoff, Zeitschr. f. physiol. Chem., 17; Osborne and Zobel, Journ. of Physiol.,
29.

2 See Schardinger, Monatshefte f. Chem., 11 ; Blachstein, Arch, des sciences biol.
de St. Petersbourg, 1, 199; Kuprianow, Arch. f. Hygiene, 19, and Gosio, ibid., 21;
Herzog and Horth, Zeitschr. f. physiol. Chem., 60.

3 Heintz, Annal. d. Chem. u. Pharm., 157, and Gscheidlen, Pfliiger's Arch., 8,
171; Moriya, Zeitschrift f. physiol. Chem.. 43.

LACTIC ACIDS. 583

in poisoning by phosphorus, and especially after extirpation of the liver
seems to be paralactic acid.

The origin of paralactic acid in the animal organism has been sought
by several investigators, who took for basis the researches of GAGLIO,
MINKOWSKI, and ARAKI, in a decomposition of protein in the- tissues
GAGLIO claims a lactic-acid formation by passing blood through the sur-
viving kidneys and lungs. He also found 0.3-0.5 p. m. lactic acid in the
blood of a dog after protein food, and only 0.17-0.21 p. m. after fast-
ing for forty-eight hours. According to MINKOWSKI the quantity of lactic
acid eliminated by the urine in animals with extirpated livers is increased
with protein food, while the administration of carbohydrates has no
effect. ARAKI has also shown that if we produce a scarcity of oxygen
in animals (dogs, rabbits, and hens) by poisoning with carbon monoxide,
by the inhalation of air deficient in oxygen, or by any other means, a
considerable elimination of lactic acid (besides sugar and also often
albumin) takes place through the urine, an observation which has been
confirmed by SAITO and KATSUYAMA.1 As a scarcity of oxygen, accord-
ing to the ordinary statements, produces an increase of the protein
catabolism in the body, the increased elimination of lactic acid in these
cases must be due in part to an increased protein destruction and in parti
to a diminished oxidation.

ARAKI has not drawn such a conclusion from his experiments, but
he considers the abundant formation of lactic acid to be due to a cleavage
of the sugar formed from the glycogen. He found that in all cases where
lactic acid and sugar appeared in the urine the quantity of glycogen
in the liver and muscles was always diminished. Without denying
the possibility of a formation of lactic acid from protein, he states that
with lack of oxygen we have to deal with an incomplete combustion
of the lactic acid derived by a cleavage of the sugar. Although the
abundant formation of lactic acid under these circumstances can be
explained in different ways, still there are other conditions which make
the formation of lactic acid from proteins very probable. To this
belongs the lactic acid formation from alanine, in the liver, as mentioned
in a previous chapter, and recently further substantiated by EMBDEN
and F, KRAUS.2

The carbohydrates are also considered as the mother-substance
of the lactic acid, as it is now generally admitted that the cleavage of the

1 Gaglio, Arch. f. (Anat. u.) Physiol., 1886; Minkowski., Arch exp. Path, u. Pharm.,
21 and 31; Araki, Zeitschr. f. physiol. Chem., 15, 16, 17, and 19; Saito and Katsuyama,
ibid., 32.

2 Neuberg and Langstein, Arch. f. (Anat. u.) Physiol. 1903; Embden and F. Kraus,
Bioch. Zeitschr. 45.

584 MUSCLES.

sugar in the animal body occurs, or at least can occur, with lactic acid as
an intermediary step. The views are indeed different 1 as to the closer
mechanism of this cleavage, but there does not exist any doubt that a
formation of lactic acid, and in fact paralactic acid, can take place from
carbohydrates in the animal body. HOPPE-SEYLER 2 held the view that
the formation of lactic acid, in the absence of free oxygen, from gly-
cogen or glucose was probably a function of all living protoplasm and in
the anaerobic metabolism of the animal cells, according to the investiga-
tions of STOKLASA3 and his collaborators on alcoholic fermentation in
the tissues, a formation of alcohol and carbon dioxide takes place from
the sugar with lactic acid as intermediary step. The correctness of these
statements is now disputed from many sides, but we have direct observa-
tions which speak positively for a lactic acid formation from glycogen
or sugar. Thus EMBDEN 4 and co-workers have found that on transfus-
ing blood through the liver rich in glycogen, a formation of lactic acid
takes place, and an abundance of lactic acid is formed when blood rich in
sugar is transfused through a glycogen free liver, while a blood poor in
sugar led only to a very inconsiderable formation of lactic acid.

Certain investigators (see page 333) admit of the occurrence of glyceric
aldehyde (and also dioxyacetone) as intermediary products in the forma-
tion of lactic acid from sugar. Another intermediary product in the
lactic acid formation has been shown by recent thorough investigations
to be methylglyoxal, CHs.CO.CHO. An abundant formation of lactic
acid from methylglyoxal has been obtained by certain investigators,
such as DAKIN and DUDLEY, and by NEUBERG, in experiments with
tissues, organ extracts and organ pulp, and by LEVENE and MEYER 5 in
experiments with leucocytes or kidney tissue. The process is of an
enzymotic nature and the active enzyme, which also converts phenyl-
glyoxal into mandelic acid has been called glyoxylase by DAKIN and
DUDLEY. The process is reversible according to these experimenters,
in that they have been able to show a retransformation of lactic acid
into methylglyoxal. They also found that lactic acid as well as methyl-
glyoxal could form glucose in diabetic animals. The detailed procedure
in the cleavage of sugar to lactic acid is still undecided.

The carbohydrates, as well as the proteins, it seems, must be con-
sidered as the material from which the lactic acid is formed in the body.

1See Embden und Oppenheimer, Bioch. Zeitschr., 45; Parnas and Baer, ibid., 41.
2 Virchow's Festschrift, also Eer. d. deutsch. chem. Gesellsch., 25, Referatb., 685.
3Simdcek, Centralbl. f. Physiol., 17; Stoklasa, Jelinek, and Cerny, ibid., 16. In
regard to opposed statements see Harden and Mac Lean, Journ. of Physiol., 42.

4 Embden and Almagia with F. Kraus, Bioch. Zeitschr. 45; S. Oppenheimer, ibid., 45.

5 Dakin and Dudley, Journ. of biol. Chem., 14; Neuberg, Bioch. Zeitschr., 49;
Levene and Meyer, Journ. of biol. Chem., 14.

LACTIC ACIDS. 585

The phosphocarnic acid (SIEGFRIED) and the inosite are also considered
as possible mother-substances for sarcolactic acid. Further research
will show whether also other mother-substances for this acid occur. The
autolytic experiments of TURKEL 1 with livers and the formation of lactic
acid in the muscles, not from carbohydrates, inosite or alanine, as observer!
by EMBDEN 2 and his collaborators seem to indicate this.

The lactic acids are amorphous. They have the appearance of
colorless or faintly yellowish, acid-reacting syrups, which mix in all pro-
portions with water, alcohol, or ether. The salts are soluble in water,
and most of them also in alcohol. The two acids are differentiated from
each other by therr different optical properties — paralactic acid being
dextrogyrate, while fermentation lactic acid is optically inactive — also
by their different solubilities and the different amounts of water of crys-
tallization cf the calcium and zinc salts. The zinc salt of fermentation
lactic acid dissolves in 58-63 parts of water at 14-15° C., and contains
18.18 per cent water of crystallization, corresponding to the formula,
Zn(C3H503)2+3H2O. The zinc salt of paralactic acid dissolves in 17.5
parts of water at the above temperature and contains ordinarily 12.9
per cent water, corresponding to the formula, Zn(C3H5O3)2+2H20.
The calcium salt of fermentation lactic acid dissolves in 9.5 parts water
and contains 29.22 per cent ( = 5 molecules) water of crystallization,
while calcium paralactate dissolves in 12.4 parts water and contains 24.83
or 26.21 per cent ( = 4 or 4J molecules) water of crystallization. Both
calcium salts crystallize, not unlike tyrosine, in spears or tufts of very
fine microscopic needles. HOPPE-SEYLER and ARAKI, who have closely
studied the optical properties of the lactic acids and lactates, consider
the lithium salt as best suited for the preparation and quantitative estima-
tion of the lactic acids. The lithium salt contains 7.29 per cent Li. For
further information as to the salts and specific rotation of the lactic acids
see HOPPE-SEYLER-THIERFELDER'S Handbuch, 8. AufL, 1909 .3

Lactic acids may be detected in organs and tissues in the following
manner: After complete extraction with water, the protein is removed
by coagulation at boiling temperature and the addition of a small quan-
tity of sulphuric acid. The liquid is then exactly neutralized, while
boiling, with caustic baryta, and then evaporated to a syrup after filtra-
tion. The residue is precipitated with absolute alcohol, and the pre-
cipitate completely extracted with alcohol. The alcohol is entirely
distilled from the united alcoholic extracts, and the neutral residue is

1Tiirkel, Bioch. Zeitschr., 20. The statements on the formation of lactic acid
in the muscle autolysis are rather conflicting; see Fletcher, Journ. of Physiol., 43.

2Embden, Kalberlah and Engel, Bioch. Zeitschr. 45; Kondo, ibid., 45.

3 See also E. Jungfleisch, Compt. Rend., 139, 140, and 142; Herzog and Slansky,
Zeitschr. f. physiol. Chem., 73.

586 MUSCLES.

shaken with ether to remove the fat. The residue is dissolved in water
and phosphoric acid is added, and the solution repeatedly shaken with fresh
quantities of ether, which dissolves the lactic acid. The ether is now
distilled from the united ethereal extracts, the residue dissolved in water,
and this solution carefully warmed on the water-bath to remove the last
traces of ether and volatile acids. A solution of zinc lactate is prepared
from this filtered solution by boiling with zinc carbonate, and this is
evaporated until crystallization commences, and is then allowed to stand
over sulphuric acid. An analysis of the salts is necessary in careful
work. In regard to methods for the detection and quantitative estima-
tion of lactic acid we must refer to larger hand-books.

Fat is never absent in the muscles. Some fat is always found in the
intennuscular connective tissue; but the muscle-fibers themselves also
contain fat. The quantity of fat in the real muscle substance is always
small, usually amounting to about 10 p. m. or somewhat more. A con-
siderable quantity of fat in the muscle-fibers is found only in fatty degenera-
tion. A part of the muscle-fat can be readily extracted, while another
part can be extracted only with the greatest difficulty. This latter
part, it is claimed, exists finely divided in the contractile substance
itself and is richer in free fatty acids, standing, according to ZUNTZ and
BoGDANOW,1 in close relation to the activity of the muscles because
it is consumed during work. Lecithin is a regular constituent of the
muscles, and it is quite possible that the fat which is difficult of extrac-
tion and which is rich in fatty acids depends in part on a decomposition
of the lecithin and the phosphatides. ERLANDSEN has shown that
phosphatides of various kinds occur in the muscles, the quantities
varying in different muscles. According to him the ox-heart muscle
is richer in phosphatides than the muscle of the thigh, and RuBow2
claims that the heart of the dog is richer in phosphatides than the striated
muscle. ERLANDSEN found lecithin and diamino-phosphatide in the
heart as well as the thigh-muscle, while the monoamido-phosphatide
cuorin, which occurs abundantly in the heart, is found as traces in the
thigh-muscle. CosTANTiNO3 has carried on investigations on the divi-
sion of the inorganic and organic phosphorus in striated and smooth
muscles.

The Mineral Bodies of the Muscles. The ash remaining after burning
the muscle, which amounts to about 10-15 p. m., calculated on the moist
muscle, is acid in reaction. The largest constituent of the ash is potas-
sium, whose occurrence, according to MACALLUM,* is restricted to the dark

1 Arch, f . (Anat. u.) Physiol., 1897.

2 Erlandsen, Zeitschr. f. physiol. Chem., 51; Rubow, Arch. f. exp. Path. u. Pharm.,
52.

'Bioch. Zeitschr., 43.
4 Journ. of Physiol., 32.

MINERAL BODIES. 587

diagonal bundles, and phosphoric acid. Next in amount we have sodium
and magnesium, and lastly calcium, chlorine, and iron oxide. Sulphates
exist only as traces in the muscles, but are formed by the burning of the
proteins of the muscles, and therefore occur in abundant quantities in the
ash. The muscles contain such a large quantity of potassium and phos-
phoric acid, that potassium phosphate seems to be, unquestionably, the
predominating salt. Chlorine is found in such insignificant quantities
that it is perhaps derived from a contamination with blood or lymph.
The quantity of magnesium is, as a rule, considerably greater than that
of calcium. Iron occurs only in very small amounts. The water of the
muscle occurs in part free and partly as imbibition water of the colloids.
According to the investigations of JENSEN and FiscHEB1 only a small part,
a few per cent, of the total water exists in this condition.

URANO 2 has removed the salts of the intermediary fluid (blood,
lymph) from frogs' muscles by treating them with an isotonic cane-sugar
solution (of 6 per cent) and in this manner found tKat the sodium did
not belong to the muscle substance itself, but to the intermediary fluid,
while at least a small part of the chlorine is a true muscle constituent.
He also calculated, from the quantity of sodium, that the intermediary
fluid, if it- has about the same composition as the muscle plasma, makes
up about one-sixth of the volume of the muscle. According to further
investigations of URANO the possibility of a disturbance in the osmotic
properties of the muscle-fibers by the sugar solution is not entirely excluded,
and the question whether the muscle-fibers are free from sodium or not
has therefore not been positively decided. FAHR'SS researches make
the absence of sodium in frog's muscle very probable.

The importance of the various mineral bodies for the function of the
muscles has been the subject of numerous investigations and by many
of these we have obtained further proof, as mentioned in a previous
chapter, of the ion action of the electrolytes and the antagonism of
different ions. These researches also indicate that each of the ions
Na, Ca, and K plays a certain part in the maintenance of the excitability,
in the contraction and in the fatigue of the muscle (heart); still these
investigations have not led to concordant results, so that we are not yet
clear as to the action of these ions. Nevertheless it seems to be estab-
lished that the combined action of various ions is a necessity for the nor-
mal function of the muscles. It has also been shown that it is possible
to maintain the muscle (the heart) in regular activity for a long time by
means of a transfusion of liquid saturated with oxygen, and which con-

1 Jensen and Fischer, Bioch. Zeitschr., 20.

2 Zeitschr. f. Biol., 50.

3 Urano, ibid., 51; Fahr., ibid., 52.

588 MUSCLES.

tained about 7 p. m. Nad, besides small amounts of CaCk (0.2 p. m.),
KC1 (0.1 p. m.), and NaHCO3 (0.1 p. m.).

The gases of the muscles consist of large quantities of carbon dioxide
besides traces of nitrogen.

In regard to the permeability of the muscles for various bodies there
are the complete investigations of OvERTON.1 The different sheaths of
the muscles, the sarcolemma and perimysium internum, offer no very
great resistance to the diffusion of the most soluble crystalloid com-
pounds, while the muscle-fibers, on the contrary (exclusive of the sar-
colemma), are almost if not entirely impervious to most inorganic com-
pounds and to many organic compounds. The muscle-fibers themselves
are actually semipermeable structures which are permeable to water
but not to the molecules or ions of sodium chloride and of potassium phos-
phate. The muscle-fibers, as well as the various sheaths, are impermeable
to colloids.

*• The behavior of the numerous bodies investigated cannot be discussed
in this work. The general rule is as follows: All compounds which,
besides having a marked solubility in water, are readily soluble in ethyl
ether, in the higher alcohols, in olive-oil and in similar organic solvents,
or are not much less soluble in the last-mentioned solvents than in water,
pass through the living muscle-fibers with great ease. The greater the
difference between the solubility of a compound in water and in the other
solvents mentioned, the slower does the passage into the muscle-fibers
take place. The permeability changes essentially on the death of the
muscle.

The living muscle-fibers are readily permeable to oxygen, carbon
dioxide, and ammonia, while the hexoses and disaccharides do not readily
pass into them. It is very remarkable that a great portion of those
compounds which take part in the normal metabolism of plants and
animals belongs to those bodies to which the muscle-fibers (and also other
cells) are entirely or at least nearly impermeable. On the contrary,
derivatives can be prepared from these bodies which pass into the cells
very readily, and OVERTON finds that it is not impossible that the organ-
ism in part makes use of a similar artifice in order to regulate the concen-
tration of the nutritive bodies within the protoplasm. (See Chapter I.)

Rigor Mortis of the Muscles. If the influence of the circulating
oxygenated blood is removed from the muscles, as after the death of
the animal or by ligature of the aorta or the muscle-arteries (STENSON'S
test), rigor mortis sooner or later takes place. The ordinary rigor
appearing under these circumstances is called the spontaneous or the

1Pfliiger's Arch., 92. See also Hober, ibid., 106, and Hamburger, Osmotischer
Druck und lonenlehre. Bd. 3.

RIGOR MORTIS. 589

fermentative rigor, because it seems to depend in part on the action of
an enzyme. A muscle may also become stiff or other reasons. The
muscles may become momentarily stiff by warming, in the case of frogs
to 40°, in mammalia to 48-50°, and in birds to 53° C. Distilled water
may also produce a rigor in the muscles (water-rigor). Acids, even very
weak ones, such as carbon dioxide, may quickly produce a rigor (acid-
rigor), or hasten its appearance. A number of chemically different
substances, such as chloroform, ether, alcohol, ethereal oils, caffeine,
and many alkaloids, produce a similar effect.

When the muscle passes into 'rigor mortis it becomes shorter and
thicker, harder and non-transparent, and less ductile. The acid part
of the amphoteric reaction becomes stronger, which is explained by most
investigators by the assumption of a formation of lactic acid. There is
hardly any doubt that this increase in acidity may at least in part be
due to a transformation of a part of the diphosphate into monophosphate
by the lactic acid. The statements as to whether in the rigor mortis
muscles, besides acid phosphate also free lactic acid exists or not are
rather contradictory;1 that an acid formation precedes the rigor is gen-
erally admitted and this acid formation is now accepted as being in close
relation to the rigor. While we used to consider the appearance of a clot
consisting of myosin (KUHNE) or of myogen- and myosin fibrin (v. FURTH)
as the essential moment for the rigor, we now admit, based upon the
investigations of MEIGS, v. FURTH and LENK,2 that the most essential
factor is the imbibition of the disdiaclasts, which become broader or
shorter, by their taking up of water from the sarcolemma fluid and this
action produced by the acid formation. This view stands in accord with
the experience on the imbibition of colloids and muscles in water or salt
solutions, in the presence and absence of acid, as well as the fact that the
rigor can be retarded by the artificial circulation of blood or by the action
of salt solutions, namely by those which contain small amounts of
NaHCOs. This also agrees well with the old experience, that the muscle
work, which is also connected with a formation of acid, accelerates the
appearance of rigor.

On further post-mortal changes, namely by a further accumulation
of acid, a progressive coagulation of the proteins gradually occurs. In
this coagulation the ability of the colloid systems to imbibe water

1 It is impossible to enter into the details of the disputed theories as to the reac-
tion of the muscles, etc. We shall only refer to the works of Rohmann, Pfliiger's
Arch., 50 and 55, and HefTter, Arch. f. exp. Path. u. Pharm., 31 and 38. These
works contain also the researches of the earlier investigators more or less completely.

2 Meigs, Journ. of Physiol. 39 and especially, Amer. Journ. of Physiol., 24 and
26; v. Fiirth and Lenk, Bioch. Zeitschr., 33, and Wien. klin. Wochenschr., 24 (1911).

590 MUSCLES.

diminishes, water is given off, and a re-imbibition takes place and the
so-called " solution of the rigor " appears (v. FURTH and LENK).

The ordinary rigor is an acid rigor and the same applies, according
to MEiGS,1 to the water rigor as a shortening of the muscles takes place
when placed in distilled water, by a formation of lactic acid, and because
when such a muscle is placed in RINGER'S solution the acid is removed
and the muscle again expands.

The views are rather contradictory in regard to the production of heat
rigor. According to v. FURTH this rigor depends upon the coagulation
of certain proteins, and its occurrence at lower temperatures in cold-
blooded as compared with warm-blooded animals is due, accojrding to
v. FURTH, to the fact that in the first a soluble myogen fibrin occurs
preformed in the muscle which coagulates at 30-40° C., while in the
warm-blooded animals the coagulating substance is musculin (myosin
of v. FURTH) which coagulates at a higher temperature. According
to INAGAKI 2 the various stages in contractions occurring on heating a
muscle (frog) do not correspond to those of the coagulation of the pro-
tein which would occur on heating the muscle plasma, and MEIGS has
arrived at a similar view. It must be remarked that also a lactic acid
formation takes place on heating a muscle, and this prevents an exact
comparison of the coagulation of the proteins within and outside
of the muscle. The observations of VERNON that the striated and
the smooth muscles on heating to between 40 and 50° behave differently,
in that the striated become shorter and the smooth become longer,
while both kinds become shorter at higher temperatures, indicates against
a coagulation at these low temperatures. According to MEIGS 3 we must
here also admit of an imbibition rigor, due to the formation of lactic acid,
and the different behavior of the two kinds of muscle depends upon a
different arrangement of their anatomical elements.

The chemical rigor produced by different chemically active substances
is also produced, according to MEIGS as well as to v. FURTH and LENK,
upon a formation of acid, causing a chemical damage of the muscles, and
is to be considered as an imbibition rigor.

As it is now generally admitted that the formation of lactic acid dur-
ing the death of the muscle is the cause of the muscle rigor, the question
arises, from what constituents of the muscle is this acid derived? The
most probable explanation is that the lactic acid is produced from the
glycogen, as certain investigators, such as NASSE and WERTHER, have
observed a decrease in the quantity of glycogen in rigor of the muscle.

1 Journ. of Physiol., 39.

2 Inagaki, Zeitschr. f. Biol., 48; Meigs, Journ. of Physiol., 24.

* Vernon, Journ. of Physiol., 24; Meigs, Amer. Journ. of Physiol., 24.

METABOLISM IN THE MUSCLES. 591

On the other side, BOHM has observed cases in which no consumption
of glycogen took place in rigor of the muscle, and he also found that the
quantity of lactic acid produced is not proportional to the quantity of
glycogen. According to MOSCATI l the diminution in the glycogen is
independent of the appearance of rigor. It is therefore possible that the
consumption of glycogen and the formation of lactic acid in the muscles
are two processes independent of each other, and, as above stated in regard
to the formation of paralactic acid, the origin of the lactic acid in the
muscle is still not positively known. The phosphocarnic acid must
also be considered as a mother-substance of the lactic acid, and of the
carbon dioxide, also formed in the rigor, as it yields lactic acid as well as
carbon dioxide on its cleavage.

Metabolism in the Inactive and Active Muscles. It is admitted
by a number of prominent investigators, PFLUGER and COLASANTI,
ZUNTZ and RoHRiG2, and others, that the metabolism in the muscles
is regulated by the nervous system. When at rest, when there is no
mechanical exertion, there exists a condition which ZUNTZ and ROHRIG
have designated " chemical tonus." This tonus seems to be a reflex
tonus, for it may be reduced by discontinuing the connection between the
muscles and the central organ of the nervous system by cutting through
the spinal cord or the muscle-nerves. The possibility of reducing the
chemical tonus of the muscles in various ways offers an important means
of deciding the extent and kind of chemical processes going on in the
muscles when at rest. In comparative chemical investigation of the
processes in the active and the inactive muscles several methods of pro-
cedure have been adopted. The same active and inactive muscles have
been compared after removal, also the arterial and venous muscle-blood
in rest and activity, and lastly the total exchange of material, the receipts
and expenditures of the organism, have been investigated under these
two conditions.

By investigations according to these several methods it was found
that the resting muscle takes up oxygen from the blood and returns to
it carbon dioxide, and also that the quantity of oxygen taken up is greater
than the oxygen contained in the carbon dioxide eliminated at the same
time. The muscle, therefore, holds in some form of combination a part
of the oxygen taken up while at rest. During activity the exchange of
material in the muscle, and therewith the exchange of gas, is increased.

'Nasse, Beitr. z. Physiol. der kontrakt. Substanz, Pfluger's Arch., 2; Werther,
ibid., 46; Bohm, ibid., 23 and 46; Moscati, Hofmeister's Beitrage, 10.

2 See the works of Pfliiger and his pupils in Pfluger's Arch., 4, 12, 14, 16, and 18;
Rohrig, ibid., 4. See also Zuntz, ibid., 12. In regard to the metabolism after curare
poisoning, see also Frank and Voit, Zeitschr. f. Biologic, 42, and Frank and Geb-
hard, ibid., 43.

592 MUSCLES.

The animal organism takes up much more oxygen in activity than
when at rest, and eliminates also considerably more carbon dioxide.. The
quantity of oxygen which leaves the body as carbon dioxide during
activity is much larger than the quantity of oxygen taken up at the same
time; and the venous muscle-blood is poorer in oxygen and richer in
carbon dioxide during activity than during rest. The exchange of gases
in the muscles during activity is the reverse of that at rest, for the active
muscle gives up a quantity of carbon dioxide which does not correspond
to the quantity of oxygen taken up, but is considerably greater. It
follows from this that in muscular activity not only does oxidation take
place, but also splitting processes occur. This also results from the fact
that removed blood-free muscles when placed in an atmosphere devoid of
oxygen can labor for some time and still yield carbon dioxide (HERMANN1).

During muscular inactivity, in the ordinary sense, a consumption
of glycogen takes place. This is inferred from the observations of sev-
eral investigators, that the quantity of glycogen is increased and its cor-
responding consumption reduced in those muscles whose chemical tonus
is reduced either by cutting through the nerve or for other reasons
(BERNARD, CHANDELON, VAY,2 and others). In activity this consump-
tion of glyccgen is increased, and it has been positively proved by the
researches of numerous investigators3 that the quantity of glycogen
in the muscles in activity decreases quickly and freely. The sugar is
removed from the blood and consumed during activity.4 The recent
investigations of JOH. MULLER, LOCKE and ROSENHEIM and CAMIS 5
have given direct proof of the consumption of sugar during muscular
activity. In experiments on surviving hearts of different animals through
which was perfused a salt solution containing sugar, they could detect
an undoubted consumption of sugar which was quite considerable and
which to all appearances was used as material for muscle work.

The amphoteric reaction of the inactive muscles is changed during

1L. Hermann, Unters. tiber d. Stoffwechsel der Muskeln, etc., Berlin, 1867.
In regard to gas exchange in removed muscles, see also J. Tissot, Arch, de Physiol.
(5), 6 and 7, and Compt. Rend., 120.

*Chandelon, Pfluger's Arch., 13; Vay, Arch. f. exp. Path. u. Pharm., 34, which
also contains the pertinent literature.

»Nasse, Pfluger's Arch., 2; Weiss, Wien. Sitzungsber., 64; Kiilz, in Ludwig's
Festschrift, Marburg, 1890; Marcuse, Pfluger's Arch., 39; Manche, Zeitschr. f. Bio-
logie, 25; Moral and Dufour, Arch, de Physiol. (5), 4.

4Chauveau and Kaufmann, Compt. Rend., 103, 104, and 105; Quinquaud, Maly's
Jahresber., 16; Morat and Dufour, 1. c.; Cavazzani, Centralbl. f. Physiol., 8; Seegen,
" Die Zuckerbildung im Thierkorper," Berlin, 1890, Centralbl. f. Physiol., 8, 9, and
10; Arch. f. (Anat. u.) Physiol., 1895 and 1896; Pfluger's Arch., 50.

6 Joh. Miiller, Zeitschr. f allgem. Physiol., 3; Camis, ibid., 8; Locke and Rosen-
heim, Joura. of Physiol., 36.

LACTIC ACID FORMATION IN ACTIVE MUSCLES. 593

activity to an acid reaction (Du BOIS-REYMOND and others), and the
acid reaction increases, to a certain point, with the work. The quickly
contracting pale muscles produce, according to GLEISS/ more acid dur-
ing activity than the more slowly contracting red muscles. Numerous
investigations have been carried out on the cause of this increased acid
reaction, using the muscles in situ and also upon removed muscles and
rather contradictory results have been obtained. Some have found a
diminution in the amount of lactic acid in the active muscle while others
have found an increase.2 The work of FLETCHER and HOPKINS 3 is of
great importance in this disputed question, in which they show that in
the removal of the muscle, and in its preparation for the testing for lactic
acid several sources of error are possible. The mechanical irritation
as well as warming or treating the muscle with alcohol (not ice-cold)
can lead to a formation of lactic acid. It was also shown that the
absence of oxygen accelerated the formation or accumulation of lactic
acid, while an abundance of oxygen had the opposite effect.

It is evident that the experiments with the muscles in situ — in other
words, with muscles through which blood is passing — cannot yield any
conclusion to the above question, as the lactic acid formed during work
may perhaps be removed by the blood. The following objections can
be made against those experiments in which lactic acid has been found,
after moderate work, in the blood or the urine, as also especially against
the experiments with removed active muscles, namely, that in these cases
the supply of oxygen to the muscles was not sufficient, and that the
lactic acid formed thereby is not, in accordance with the views of HOPPE-
SEYLER, a perfectly normal process. The same is probably true also for
the formation of lactic acid with excessive work during life, and ZILLESSEN 4
has found that the artificial cutting off of the oxygen supply in the
muscles during life, that more lactic acid was formed than under normal
conditions. Other observations indicate a formation of lactic acid dur-
ing activity. Thus SPIRO and recently also H. FRIESS found an increase
in the quantity of lactic acid in the blood during work. COLAS ANTI
and MOSCATELLI found small quantities of lactic acid in human urine
after strenuous marches, and WERTHER 6 observed an abundance of lactic
acid in the urine of frogs after tetanization.

iPfliiger'sArch., 41.

* Astaschewsky, Zeitschr. f. physiol. Chem., 4; Warren, Pfliiger's Arch., 24,
Monari, Maly's Jahresber., 19; Heffter, Arch. f. exp. Path. u. Pharm., 31; Marcuse,
1. c.; Werther, Pfliiger's Arch., 46; Spiro, Zeitschr. f. physiol. Chem., 1; Colasanti.

3 Journ. of Physiol., 35.

4 Hoppe-Seyler, 1. c. and Zeitschr. f. physiol. Chem., 19, 476; Zillessen, ibid., 15.
6 Spiro, Zeitschr. f. physiol. Chem. 1; Fries, Bioch. Zeitschr., 35.

6 Colasanti and Moscatelli; Maly's Jahresb.. 17, 212; Werther, Pfliiger's Arch., 46.

594 MUSCLES.

According to SIEGFRIED the amount of phosphocarnic acid is dimin-
ished during activity. MACLEOD l claims that this is true only for
intense muscular activity, and the mother-substance of lactic acid can
at least in part be phosphocarnic acid. The question as to the forma-
tion of lactic acid during activity, and the origin of the phosphocarnic
acid is certainly in many points somewhat undecided; the general
view seems to be, that during work lactic acid is formed, which transforms
a part of the diphosphates into monophosphates.

The amount of proteins in the removed muscles is, according to the
earlier investigators, decreased by work. The correctness of this state-
ment is, however, disputed by other investigators. Earlier reports in
regard to the nitrogenous extractive bodies of the muscle in rest and
in activity are likewise uncertain. According to the recent researches
of MoNARi*2 the total quantity of creatine and creatinine is increased by
work, and indeed the amount of creatinine is especially augmented
by an excess of muscular activity. The creatinine is formed essentially
from the creatine. The investigations of GRAHAM BROWN and CATHCART
on removed nerve-muscle preparations of frogs, and those of S. WEBER 3
on hearts, indicate an increase in the formation of creatine and creatinine
during work. WEBER found that the working heart gave up creatine
(and creatinine) to RINGER'S solution, and indeed much more when
strongly active than during a lesser activity. An increased creatinine
elimination after work does not occur according to several investigators
(see Chapter XIV) and according to PEKELHARING and v. HOOGENHTJYZE
with ordinary muscle activity neither an increased creatine formation
nor an increased creatinine elimination takes place. In the tonic
contraction the creatine is formed from the proteins, and correspond-
ingly according to PEKELHARING and HARKiNK4 the creatinine elimina-
tion is increased under the influence of the muscle tonus. The purine
bases are produced, according to BURIAN, in the muscles themselves,
also in activity, and an increased formation takes place during work
due to a re-formation. ScAFFiDi5 found on the contrary, with frogs and
tortoise, during work that a diminution of the total quantity of purine
bases occurred and indeed not the free but the combined purines.

Attempts have been made to solve the question relative to the
behavior of the nitrogenized constituents of the muscle at rest and during

1 Siegfried, Zeitschr. f. physiol. Chem., 21; Macleod, ibid., 28.

2 Maly's Jahresber., 19, 296.

3Cathcart and Graham Brown, Journ. of Physiol., 37; Weber, Arch. f. exp. Path,
u. Pharm., 58.

4Pekelharing and v. Hoogenhuyze, Zeitschr. f. physiol. Chem., 64, with Harkink,
ibid., 75.

5Burian, Zeitschr. f. physiol. Chem., 43; Scaffidi; Bioch. Zeitschr., 30.

NITROGEN CATABOL1SM IN ACTIVE MUSCLES. 595

activity by determining the total quantity of nitrogen eliminated under
these different conditions of the*body. While formerly it was held with
LIEBIG that the elimination of nitrogen by the urine was increased by
muscular work, the researches of several experimenters, especially those
of VOIT on dogs, and PETTENKOFER and VOIT on men, have led to quite
different results. They have shown, as has also lately been confirmed
by other investigators, especially I. MUNK and HrascHFELD,1 that during
work no increase, or only a very insignificant increase, in the elimination
of nitrogen takes place.

We should not omit to mention the fact that a series of experiments
has been made showing a significant increase in the metabolism of pro-
teins during or after work. There are for example the observations
of FLINT and of PAVY on a pedestrian, v. WOLFF, v. FUNKE, KREUZHAGE,
and KELLNER on a horse, and DUNLOP and his collaborators on working
human beings, and of KRUMMACHER, PFLUGER, ZUNTZ and his pupils,2
and others. The researches on the elimination of sulphur during rest
and activity also belong to this category. The elimination of nitrogen
and sulphur runs parallel with the metabolism of proteins in resting and
active persons, and the quantity of sulphur excreted by the urine is there-
fore also a measure of the protein catabolism. The earlier researches
of ENGELMANN, FLINT, and PAVY, as well as the more recent ones of BECK
and BENEDICT,3 and DUNLOP and his collaborators, show an increased
elimination of sulphur during or after work, and this indicates an increased
protein metabolism because of muscular activity.

That an increased destruction of protein is not necessarily produced
by work follows from the observations of CASPARI, BORNSTEIN, KAUP,
WAIT, A. LOEWY, ATWATER and BENEDICT,4 that a retention of nitrogen
and a deposition of protein occur during work. The discordant observa-
tions on the protein destruction during, and caused by, work are not
directly in opposition to each other, because the extent of protein
metabolism is dependent upon many conditions, such as the quantity

1 Voit, Untersuchungen iiber den Einfluss des Kochsalzes, des Kaffees und der
Muskelbewegungen auf den Stoffwechsel (Munchen, 1860), and Zeitschr. f. Biologic,
2; J. Munk, Arch. f. (Anat. u.) Physiol., 1890 and 1896; Hirschfeld, Virchow's Arch.,
121.

2 Flint, Journ. of Anat. and Physiol., 11 and 12; Pavy, The Lancet, 1876 and 1877;
v, Wolff, v. Fimke, Kellner, cited from Voit, Hermann's Handb., 86, 197; Dunlop
Noel-Paton, Stockman, and Maccadam, Journ. of Physiol., 22; Krummacher, Zeitschr.
f. Biologic, 33; Pfliiger, Pfluger's Arch., 50; Zuntz, Arch. f. (Anat. u.) Physiol., 1894.

3Engelmann, Arch. f. (Anat. u.) Physiol., 1871; Beck and Benedict, Pfluger's
Arch., 54, and also footnote 2.

4Caspari, Pfluger's Arch., 83; Bornstein, ibid.; Kaup, Zeitschr. f. Biologic, 43;
Wait, U. S. Depart. Agricult. Bulletin, 89; (1901) Atwater and Benedict, ibid., Bull.,
69 (1899); Loewy.Arch. f. (Anat. u.) Physiol., 1901.

596 MUSCLES.

and composition of the food, the condition of the adipose tissue of the
body, the action of the work upon the respiratory mechanism, etc., all
of which have an influence on the results of the experiments.

What has been said above in regard to the protein catabolism dur-
ing muscular activity only applies for the metabolism experiments
carried on in the generally accepted manner. THOMAS l has made an
experiment, under RUBNER'S direction, on the action of work upon the
nitrogen elimination upon a person when the nitrogen minimum was
reduced to the wear and tear quota (see Chapter XVII), and this experi-
ment seems to indicate a small increase in the nitrogen elimination due
to work.

The older investigations on the amount of fat in muscles removed
after activity and after rest have not led to any definite results. Accord-
ing to the investigations of ZUNTZ and BoGDANOW,2 the fat belonging
to the muscle-fibers, which is extracted with difficulty, takes part in work.
Besides these there are several researches by VOIT, PETTENKOFER and
VOIT, J. FRENTZEL,3 and others which make an increased destruction
of fat during work probable or proved.

If the results of the investigations thus far made of the chemical
processes going on in the active and inactive muscles were collected,
we would find the following characteristics for the active muscle: The
active muscle takes up more oxygen and gives off more carbon dioxide
than the inactive muscle ; still the elimination of carbon dioxide is increased

considerably more than the absorption of oxygen. The respiratory
c^(~\

quotient, ~yr~, is found to be regularly raised during work; yet this rise,

which will be explained in detail in a following chapter on metabolism,
can hardly be conditioned on the kind of processes going on in the muscle
during activity with a sufficient supply of oxygen. In work a consump-
tion of carbohydrates, glycogen, and sugar takes place. The acid reac-
tion of the muscle becomes greater with work. In regard to the extent
of a re-formation of lactic acid opinion is divided. An increased con-
sumption of fat has occasionally been observed. On the behavior of
creatine (or creatinine) and purine bodies the statements are some-
what divergent. Protein metabolism has been found increased in cer-
tain series of experiments and not in others; but an increased elimina-
tion of nitrogen as a direct consequence of muscular exertion has thus
far not been positively proved.

In close connection with the above-mentioned facts there is the

1 Arch. f. (Anat, u.) Physiol. 1910, Supplelbd-

2 Ibid., 1897.

3 Pfliiger's Arch., 68.

MUSCLE WORK. 597

question as to the material basis of muscular activity so far as it has
its origin in chemical processes. In the past the generally accepted
opinion was that of LIEBIG, that the source of muscular action con-
sisted of a catabolism of the protein bodies; to-day another generally
accepted view prevails. FICK and WISLICENUS 1 climbed the Faulhorn
and calculated the amount of mechanical force expended in the attempt.
With this they compared the mechanical equivalent transformed in the
same time from the proteins, calculated from the nitrogen eliminated in
the urine, and found that the work really performed was not by any
means compensated by the consumption of protein. It was, therefore,
proved by this that proteins alone cannot be the source of muscular
activity and that this depends in great measure on the metabolism of
non-nitrogenous substances. Many other observations have led to the
same result, especially the experiments of VOIT, of PETTENKOFER and VOIT,
and of other investigators, whose observations show that while the
elimination of nitrogen remains unchanged, the elimination of carbon
dioxide during work is very considerably increased. It is also gen-
erally considered as positively proved that muscular work is produced,
at least in greatest part, by the catabolism of non-nitrogenous substances.
Nevertheless there is no warrant for the statement that muscular activity
is produced entirely at the cost of the non-nitrogenous substances,
and that the protein bodies are without importance as a source of
energy.

The investigations of PFLUGER 2 are of great interest in this connec-
tion. He fed a bulldog for more than seven months with meat which
alone did not contain sufficient fat and carbohydrates even for the pro-
duction of heart activity, and then let him work very hard for periods
of 14, 35, and 41 days. The positive result obtained by these series
of experiments was that " complete muscular activity may be effected
to the greatest extent in the absence of fat and carbohydrates," and the
ability of proteins to serve as a source of muscular energy cannot be
denied.

The nitrogenous as well as the non-nitrogenous nutriments may serve
as a source of energy; but the views are divided in regard to the relative
value of these. PFLUGER claims that no muscular work takes place
without a decomposition of protein, and the living cell-substance prefers
always the protein and rejects the fat and sugar, contenting itself with
these only when proteins are absent. Other investigators, on the con-
trary, believe that the muscles first draw on the supply of non-nitrogenous

1 Vierteljahrsschr. d. Zurich, naturf. Gesellsch., 10, cited from Centralbl. f. d. med.
Wiss., 1866, 309.

2 Pfliiger's Arch., 50.

598 MUSCLES.

nutriments, and according to SEEGEN, CHAUVEAU, and LAULANIE 1
the sugar is the only direct source of muscular force. The last-men-
tioned investigator holds that the fat is not directly utilized for work,
but only after a previous conversion into sugar. ZUNTZ and his collabora-
tors have made strong objections to the correctness of such a view. If
according to ZUNTZ, the fat must be first transformed into sugar before
it can serve as the source of muscular work, a definite expenditure of
force must require about 30 per cent more energy with fatty food than
it does with carbohydrates; but this is not the case. The investiga-
tions of ZUNTZ (together with), LOEB, HEINEMANN, FRENTZEL and REACH
show that all foodstuffs have nearly the same power of serving as the
material for the work of the muscles. The extensive metabolism investi-
gations of ATWATER and BENEDICT2 have also led to similar results
as to the fats being a source of muscular energy. The law of the sub-
stitution of the foodstuffs, according to their combustion equivalents,
is also true for muscular work, and fat correspondingly acts with its full
amount of energy without previously being transformed into sugar.
The question which of the foodstuffs the muscle prefers is dependent upon
the relative quantities of the same at the disposal of the muscle. A
direct substitution of the body material by the bodies supplied as food
does not take place in the muscular activity in the ordinary nutritive
condition. According to JOHANSSON and KORAENS the CC>2 excretion
produced by certain work is not influenced by the supply of foodstuffs
(protein or sugar).

SIEGFRIED considers, as above stated, the phosphocarnic acid as a source of
energy. According to his and KRUGER'S* researches, phosphocarnic acid, which
yields on cleavage, among other bodies, carbon dioxide, occurs in part preformed
in the muscle, and in part as a hypothetical aldehyde compound of the same —
a compound which forms phosphocarnic acid on oxidation. SIEGFRIED therefore
makes the suggestion that in the resting muscle, which requires more oxygen
than exists in the carbon dioxide eliminated, this reducing aldehyde substance is
gradually oxidized to phosphocarnic acid, which is used in the activity of the
muscle with the splitting off of carbon dioxide.

Quantitative Composition of the Muscle. A large number of analyses
have been made of the flesh of various animals for purely practical
purposes, in order to determine the nutritive value of different varieties

1 See Seegen, footnote 4, page 592. The works of Chauveau and his collaborators
are found in Compt. Rend., 121, 122, and 123; Laulanie, Arch, de Physiol. (5), 8.

2 Loeb, Arch. f. (Anat. u.) Physiol., 1894; Heinemann, Pfluger's Arch., 83; Frentzel
and Reach, ibid.-, Atwater and Benedict, U. S. Dept. of Agric., Bull. 136, and Ergeb-
nisse der Physiologie, 3.

3 Skand. Arch. f. Physiol., 13.

4 Zeitschr. f. physiol. Chem., 22.

QUANTITATIVE COMPOSITION OF THE MUSCLES. 599

of meat; but there are no exact scientific analyses with sufficient regard
to the quantity of different protein bodies and the remaining muscle
constituents, that is, these analyses are incomplete or of little value.
We will only give a few of the results of the work of various investigators.
The figures are parts per 1000.

Muscles of Muscles of Muscles of

Mammals. Birds. Cold-boolded

Animals.

Solids 217-278 225-282 200

Water 722-783 717-773 800

Organic bodies 207-263 217-263 180-190

Inorganic bodies 10-15 10-19 10-20

Myosin 30-106 29.8-110 29.7-87

Stroma substance (DA.NILEWSKI) 78-161 88.0-184 70.0-121

Creatine 2-4.5 3-4.9 2.3-7

Carnosine 1 . 3-4

Carnitine 0. 19 —

Purinebases 1.3-1.7 0.7-1.3 0.53-0.88

Inosinic acid (barium salt) 0.1 0. 1-0 . 3

Phosphocarnic acid 0 . 57-2 . 4

Inosite 0.03 — —

Glycogen 1-37

Lactic acid 0.4-0.7 — —

Of the mineral substances the largest part consists of phosphoric
acid 3.4-4.8 p. m. and potassium 3-4 p. m. The amount of sodium
is ordinarily only J-J of that of the potassium. Pork, according to
KATZ,1 who has carried out complete investigations as to the quantity
of mineral constituents of the human muscle and of other animals, is
considerably richer than other varieties of meat, in sodium than potas-
sium. The quantity of chlorine, which is also variable, was found by
MAGNUS-LEVY to be 2.4 p. m. (calculated as NaCl) for the human heart
muscle and 1.004 p. m. in other muscles. The amount of Ca and Mg
was found by him to be equal to 0.019 and 0.174 p. m. respectively in the
heart muscle and 0.065 and 0.215 p. m. respectively in other muscles,
v. MORACZEWSKI obtained higher results for the Ca content of the human
heart muscle, namely 0.07 p.m. GLEY and RICHAUD 2 found 0.25-0.26
p. m. Ca in the heart muscle of the dog, and 0.089-0.248 p. m. Ca in that
from the rabbit. The magnesium content of the muscles seems, with
the exception of the haddock, eel and pike (KATZ), to be greater than the
calcium content. The statements differ very considerably in regard
to the iron content. Thus ScHMEY3 found 0.0793 p. m. iron in the human
muscle, while MAGNUS-LEVY found 0.253 p. m., and in the human heart

1Katz, Pfliiger's Arch., 63; see also Schmey, Zeitschr. f. physiol. Chem., 39.

2 Magnus-Levy, Bioch. Zeitschr. 24; v. Moraczewski, Zeitschr. f. physiol. Chem.
23; Gley and Richaud, Journ. de Physiol. et de Path., 12.

3 Zeitschr. f. physiol. Chem. 39; Magnus-Levy, 1. c.

600 MUSCLES.

muscle only 0.067 p. m. iron. Other investigators have only found <0.014-
0.035 p. m. iron in the muscle.

In the table which is given above, no results are given as to the estimates
of Jot. Owing to the variable quantity of fat in meat it is hardly possible
to quote a positive average for this substance. After most careful
efforts to remove the fat from the muscles without chemical means, it
has been found that a variable quantity of intermuscular fat, which does
not really belong to the muscular tissue, always remains. The smallest
quantity of fat in the muscles from lean oxen is 6.1 p. m. according to
GROUVEN, and 7.6 p. m. according to PETERSEN. This last observer
also regularly found a smaller quantity of fat, 7.6-8.6 p. m., in the
fore quarters of oxen, and a greater amount, 30.1-34.6 p. m., in the hind
quarters of the animal, but this could not be substantiated by STEiL.1
A small quantity of fat has also been found in the muscle of wild animals.
B. KONIG and FARWICK found 10.7 p. m. fat in the muscles of the extrem-
ities of the hare, and 14.3 p. m. in the muscles of the partridge. The
muscles of pigs and fattened animals are, when all the adherent fat is
removed, very rich in fat, amounting to 40-90 p. m. The muscles of
certain fishes also contain a large quantity of fat. According to ALMEN,
in the flesh of the salmon, the mackerel, and the eel there are contained
respectively 100, 164, and 329 p. m. fat.2

The quantity of water in the muscle is liable to considerable variation.
The quantity of fat has a special influence on the quantity of water, and
one finds, as a rule, that the flesh which is deficient in water is correspond-
ingly rich in fat. The quantity of water does not depend upon the amount
of fat alone, but upon many other circumstances, among which must
be mentioned the age of the animal. In young animals, the 'organs
in general, and therefore also the muscles, are poorer in solids and richer
in water. In man the quantity of water decreases until mature age,
but increases again toward old age. Different muscles have also- a
different water content and the uninterruptedly active heart is the
richest muscle in water. In man, MAGNUS-LEVY found 748 p. m. water
in the heart, and 722 p. m. in the other muscles. That the quantity
of water may vary independently of the amount of fat is strikingly shown
by comparing the muscles of different species of animals. In cold-blooded
animals the muscles generally have a greater quantity of water, in birds
a lower. The comparison of the flesh of cattle and fish shows very strik-
ingly the different amounts of water (independent of the quantity of fat)

1 See Steil, Pfliiger's Arch., 61.

2 In regard to the literature and complete reports on the composition of flesh of
various animals, see Konig, Chemie der menschlichen Nahrungs- und Genussmittel,
5. Aufl.

SMOOTH MUSCLES. 601

in the flesh of different animals. According to the analysis of ALMEN l
the muscles of lean oxen contain 15 p. m. fat and 767 p. m. water; the
flesh of the pike contains only 1.5 p. m. fat and 839 p. m. water.

For certain purposes, as, for example, in experiments on metabolism, it is
important to know the elementary composition of flesh. In regard to the quan-
tity of nitrogen we generally accept VOIT'S figure, namely, 3.4 per cent, as an
average for fresh lean meat. According to NOWAK and HUPPERT 2 this quantity
may vary about 0.6 per cent, and in more exact investigations it is therefore
necessary to specially determine the nitrogen. Complete elementary analyses
of flesh have been made with great care by ARGUTiNSKy. The average for ox-
flesh dried in vacuo and free from fat and with the glycogen deducted was as fol-
lows: C 49.6; H 6.9; N 15.3; 0+S 23.0; and ash 5.2 per cent. KOHLER
found as an average for water and fat-free beef C 49.86; H 6.78; N 15.68; 0+S
22.3 per cent, which are very similar results. This investigator also made similar
analyses of the flesh of various animals and determined the calorific value of the
ash- and fat-free dried meat substance. This value was, per gram of substance,
5509-5677 cal. The relation of the carbon to nitrogen, which ARGUTiNSKy calls
the "flesh quotient," is on an average 3.24 : 1. From KOHLER' s analyses the
average for beef is 3.15 : 1 and for horse-flesh 3.38: 1. MAX MULLER has shown
with experiments on dogs, that the flesh of the same individual shows some varia-
tion in this quotient after different foods. According to SALKOWSKI, of the total
nitrogen of beef 77.4 per cent was insoluble proteins, 10.08 per cent soluble pro-
teins, and 12.52 per cent other soluble bodies. FRENTZEL and SCHREUER 3 find
that about 7.74 per cent of the total nitrogen belongs to the nitrogenous
extractives.

Smooth Muscles.

The smooth muscles have a neutral or alkaline reaction (Du Bois-
REYMOND) when at rest. During activity they are acid, which is inferred
from the observations of BERNSTEIN, who found that the almost con-
tinually contracting sphincter muscle of the Anodonta is acid during
life. The smooth muscles may also, according to HEIDENHAIN and
KtiHNE,4 pass into rigor mortis and thereby become acid. A spontaneous
but slowly coagulating plasma has also been observed in several cases.

In regard to the proteins of the smooth muscles we have the earlier
accounts of HEIDENHAIN and HELL WIG ;5 but they were first carefully
studied according to newer methods by MUNK and VELicm.6 These

1 Nova Act. Reg. Soc. Scient. Upsal., Vol. extr. ord., 1877; also Maly's Jahresber., 7.

2 Voit, Zeitschr. f. Biologic, 1; Huppert, ibid., 7; Nowak, Wien. Sitzungsber., 64,
Abt. 2.

'Argutinsky, Pfluger's Arch., 55; Kohler, Zeitschr. f. physiol. Chem., 31; Sal-
kowski, Centralbl. f. d. med. Wissensch., 1894; Frentzel and Schreuer, Arch. f. (Anat.
u.) Physiol., 1902; Miiller, Pfluger's Arch., 116.

4 Du Bois-Reymond in Nasse, Hermann's Handb., 1, 339;. Bernstein, ibid., Heiden-
hain, ibid., 340, with Hellwig, ibid., 339; Kiihne, Lehrbuch, 331.

5 Heidenhain in Nasse, Hermann's Handb., 1, 340, with Hellwig, ibid., 339; Kuhne,
Lehrbuch, 331.

6 Munk and Velichi, Centralbl. f. Physiol., 12.

602 , MUSCLES.

experimenters prepared a neutral plasma from the gizzard of geese,
according to v. FURTH'S method. This plasma coagulated spon-
taneously at the temperature of the room, although slowly. It con-
tained a globulin, precipitated by dialysis, which coagulated at 55-60°
C. and also showed certain similarities with KUHNE'S myosin. A spon-
taneously coagulating albumin, which differed from myogen (v. FURTH)
by coagulating at 45-50° C., and which passes by spontaneous coagula-
tion into the coagulated modification without a soluble intermediate
product, exists in still greater quantities in this plasma. Alkali albu-
minates do not occur, but a nucleoprotein is found, which exists in about
five times the quantity as compared with striated muscles. Nucleon
is, according to PANELLA,1 a normal constituent of smooth muscles and
occurs in larger amounts than in striated muscles.

Recent investigations of BOTTAZZI and CAPPELLI, VincENT and
LEWIS, VINCENT and v. FuRTH,2 some on the muscles of warm-blooded
and some on those of lower animals, have led to dissimilar results, but
they substantiate, as a whole, the observations of MUNK and VELICHI.
Besides the nucleoproteins the smooth muscles contain two bodies
corresponding in coagulation temperature to musculin and myosinogen
(myogen, v. FURTH), but they are not identical therewith. Hcemo-
globin occurs in the smooth muscles of certain animals, but is absent in
others. In the smooth muscles (in certain varieties of animals) creatine,
creatinine, hypoxanthine, taurine, inosite, glycogen, and lactic acid have
been found. Purine bases, especially xanthine also occur accord-
ing to BUGLIA and COSTANTINO but the quantity is smaller than in
striated muscles. This applies at least to the total quantity while the
amount of free purine bases, according to ScAFFiDi,3 in the smooth
muscles is greater than in the striated muscles. Creatine and carnosine
are less abundant in the smooth muscles than in the striated muscles.
The first are richer in diamino-acid than in monamino-acid-nitrogen
than the striated muscles (BUGLIA and COSTANTINO) .

In regard to the mineral constituents, COSTANTINO has found that
the smooth muscles are richer in chlorine, namely 0.84-1.3 p. m., than
the striated muscles with 0.25-0.46 p. m. . According to older state-
ments the sodium compounds exceed the potassium compounds but
COSTANTINO 4 could not substantiate this. He found, namely, no general

1 Maly's Jahresber., 34.

2 Bottazzi, Centralbl. f. Physiol., 15; Vincent and Lewis, Journ. of Physiol., 26;
Vincent, Zeitschr. f. physiol. Chem., 34; v. Fiirth, ibid., 31.

3 Scaffidi, Bioch. Zeitschr. 33; Buglia and Costantino, Zeitschr. f. physiol. Chem.
83, 81 and 82.

4 Costantino, Bioch. Zeitschr. 37; See also Meigs and Ryan, Journ. of biol.
Chem. 11.

SMOOTH MUSCLES. 603

difference in the proportion K : Na in the smooth and striated muscles.
According to SAIKI 1 magnesium does not occur to a greater extent
than calcium in the smooth muscles of the stomach or the bladder of
pigs. The same investigator found 801-811 p. m. water and 199-189
p. m. solids in these muscles.

HENZE found abundance of taurine in the muscles of Octopods, 5 p. m., but
no creatine, which, according to FREMY and VALENCIENNES, 2 occurs in the muscles
of Cephalopods. He also found no glycogen and no paralactic acid, but, on the
contrary, small amounts of fermentation lactic acid. The muscles of Octopods
are richer in mineral bodies than the muscles of vertebrates, and are nearly twice
as rich in sulphur as these.

1 Journ. of Biol. Chem., 4.

2 Henze, ibid., 43; Fre"my and Valenciennes, cited from Kuhne's Lehrbuch, p. 333.

CHAPTER XI.
BRAIN AND NERVES.

ON account of the difficulty in making a mechanical separation
and isolation of the different tissue-elements of the central nervous
organ and the nerves, we must resort to a few microchemical reactions,
principally to qualitative and quantitative investigations of the different
parts of the brain, in order to study the varied chemical composition
of the cells and the nerve-axes. This study is accompanied with the
greatest difficulty, and although our knowledge of the chemical com-|
position of the brain and nerves has been somewhat extended by the
investigations of modern times, still it must be admitted that this sub-
ject is as yet one of the most obscure and complicated in physiological
chemistry.

Proteins of different kinds have been shown to be chemical constit-
uents of the brain and nerves, and these are representatives of the same
chief groups as occur in the protoplasm. In the brain there occur some
proteins which are insoluble in water and neutral salt solutions, and
which resemble the stroma substances of the muscles and cells, while
other proteins are soluble in water and neutral salt solutions. Among
the latter we find mainly nudeoproteins and globulins. The nucleo-
protein found by HALLIBURTON and also by LEVENE 1 in the gray substance
contains 0.5 per cent phosphorus and coagulates as 55-60°. LEVENE
obtained adenine and guanine but no hypoxanthine as cleavage prod-
ucts. According to HALLIBURTON there are two globulins, namely,
the neuroglobulin a, which coagulates at 47°, or as in the case of
birds, 50-53°, and the neuroglobulin /3, whose coagulation temperature
is 70-75°, but which varies somewhat in different animals. In the frog
still another protein body occurs, which coagulates at a still lower tem-
perature, about 40°. It must be remarked that the coagulation tempera-
ture of a-globulin corresponds with the temperature of the first heat
contraction of the nerves of different classes of animals (HALLIBURTON).

1 Halliburton, On the Chemical Physiology of the Animal's Cell, King's College,
London, Physiological Laboratory, Collected Papers No. 1, 1893, and Ergebnisse der
Physiologic, 4; Levene, Arch, of Neurology and Psychopathology, 2 (1899).

604

CONSTITUENTS OF THE NERVOUS SYSTEM. 605

The gray substance is only slightly richer in proteins than the white
substance; but as the neurokeratin, which forms the neurolgia, and as
a double sheath envelops the outside of the nerves, belongs in great
part, or according to KOCH, entirely, to the white substance (KUHNE,
and CHITTENDEN, BAUMSTARK x), the gray substance is actually richer
in protein. The same is true also for the nucleoprotein or at least for
the nudeins which v. JAKSCH found in large amounts in the gray sub-
stance. The mixture of amino-acids obtained from the proteins of
the gray and white substances has about the same composition (ABDER-
HALDEN and WEIL 2) . Glycocoll could not be detected in this mixture.

The so-called protagon has been considered as one of the chief con-
stituents, perhaps the only constituent (BAUMSTARK), of the white
substance. This protagon, according to most investigators, is only a
mixture of phosphatides with cerebron or with a mixture of cerebrosides
(see below). Protagon belongs to the so-called brain lipoids, which
include three chief groups, phosphatides, cerebrosides and cholesterin and
which are contained to a greater extent in the white than in the gray
substance. Among the closely studied phosphatides the cephalin
seems to occur to the greatest extent in the brain. The lecithin, accord-
ing to FRANKEL,3 does not occur in the human brain and only in very
small quantities in other brains (of sheep and beef). Other brain
phosphatides especially described by THUDICHUM and by FRANKEL,4
have not been positively proved as chemical individuals. The same
is true for the jecorin and the sulphurized lipoids isolated from the
human brain and from ox brains. Cholesterin occurs chiefly in the
white substance. Fatty acids and neutral fats may be prepared from
the brain and nerves; but as these may be readily derived from a decom-
position of phosphatides, which exist in the fatty tissue between the
nerve-axes, it is difficult to decide what part the fatty acids and neutral
fats play as constituents of the real nerve-substance.

By allowing water to act on the contents of the medulla, round or oblong
double-contoured drops or fibers, not unlike double-contoured nerves, are formed.
These remarkable formations, which can also be seen in the medulla of the dead
nerve, have been called " myeline forms/' and they were formerly considered as
produced from a special body, " myeline." Myeline forms may, however, be
obtained from other bodies, such as impure protagon, lecithin, and impure choles-
terin, and they depend upon a decomposition of the constituents of the medulla.

1 Koch, Amer. Journ. of Physiol., 11; Kiihne and Chittenden, Zeitschr. f. Biologic,
26; Baumstark, Zeitschr. f. physiol. Chem., 9.

2 v. Jaksch, Pfliiger's Arch. 13; Abderhalden and Weil, Zeitschr. f. physiol. Chem.
81 and 83.

3 Bioch. Zeitschr., 24.

4 Thudichum, Die chemische Konstitution des Gehirns des Menschen und der
Tiere, Tubingen, 1901; S. Frankel, and collaborators, Bioch. Zeitschr., 24 and 28.

606 BRAIN AND NERVES.

The extractive bodies seem to be almost the same as in the muscles.
One finds creatine, which may, however, be absent (BAUMSTARK), purine
bases, inosite, choline, paralactic acid (MORIYA), phosphocarnic acid, uric
acid, and the diamine neuridine, C5Hi4N2, discovered by BRIEGER l
and which is most interesting because of its appearance in the putrefac-
tion of animal tissues or in cultures of the typhoid bacillus. Among
the enzymes we must mention catalases, peroxidases, Upases and amylases
( WR6BLEWSKi) . According to the autolytic experiments of SIMON 2
a proteolytic enzyme, and an enzyme acting upon the organic phos-
phorized substance with the splitting off of phosphoric acid also occur.
Under pathological conditions leucine and urea have been found in the
brain. Urea is also a physiological constituent of the brain of cartilagi-
nous fishes.

Several of the lipoids occurring in the brain have been discussed in
previous chapters, and we will here only speak of the protagon and the
cerebrosides.

Protagon. Under this name LIEBREICH described a crystalline,
nitrogenous and phosphorized substance, which has been found in the
brain of man, mammalia and also birds (ARGIRIS) but not in the brain
of fishes (ARGIRIS). Its elementary composition, according to GAMGEE
and BLANKENKORN, is C 66.39, H 10.69, N 2.39 and P 1.07 per cent.
The results obtained by CRAMER correspond well with these figures and
he found that protagon also contained sulphur which had previously been
found by RUPPEL and by KOSSEL. Recently WILSON and CRAMER 3 have
reported more recent analyses and they find for protagon, recrystallized
4-5 times, almost the same figures as GAMGEE and BLANKENHORN, namely,
C 66.53, H 10.97, N 2.37, P 0.95 and S 0.73 per cent. They consider
protagon as a unit substance.

GIES, POSNER and ROSENHEIM and TEBB4 dispute the unit nature
of protagon. They have found, on fractional precipitation or on recrys-
tallization, that protagons can be obtained from the various solvents,
having variable composition, especially different P and N contents. They

1 Brieger, Ueber Ptomaine, Berlin, 1885 and 1886.

2 Wr6blewski, Compt. Rend., 152; Fr. Simon, Zeitschr. f. physiol. Chem., 72.

3 Liebreich, Annal. d. Chem. u. Pharm., 134; Argiris, Zeitschr. f. physiol. Chem.,
57; Gamgee and Blankenhorn, ibid., 3; Kossel and Freytag, ibid., 17; Ruppel,
Zeitschr. f. BioL, 31; Cramer, Journ. of Physiol., 31, with R. A. Wilson, Journ. of
exp. Physiol., 1, with Lockhead, Bioch. Journ., 2; also Cramer, Quarterly Journ. of
exp. Physiol. 3, and Bioch. Handlexikon (Abderhalden) Bd. 3, which contains the
literature.

4 Gies and Lesem, Amer. Journ. of Physiol., 8; Posner and Gies, Journ. of biol.
Chem., 1; Gies, ibid., 3; Rosenheim and Tebb, Journ. of Physiol., 36 and 37, Quarterly
Journ. of exp. Physiol., 2, and Bioch, Zeitschr., 25.

PROTAGON. 607

are, therefore, as are LESEM, THUDICHUM, WORNER and TmERFELDER,1
and others, of the opinion that protagon does not exist as a chemical
individual, but as a mixture of cerebrosides and phosphatides. It is
not easy to come to any decision on this disputed question. On the
one hand it must be recalled that several investigators call the impure
mixture of brain lipoids, protagon, which they obtain from the solution
in warm alcohol on cooking, and which is not purified, and this mixture
is claimed to be identical, without sufficient basis, with the substance
isolated and analyzed by GAMGEE and CRAMER. On the other hand
it cannot be denied that certain investigations, especially those of ROSEN-
HEIM arid TEBB speak against the chemical individuality of protagon.
These investigations do not exclude the possibility that protagon is a
loose chemical combination between cerebroside and phosphatide, which
like other readily dissociable combinations, exist only under certain
conditions or in certain solvents. It is difficult to understand how a
mixture of amorphous or only difficultly crystallizable bodies can be
so easily crystallized and yield a product, which with proper care, can be
recrystallized repeatedly without changing its composition, and physical
properties. According to ROSENHEIM and TEBB if the proper quantity
is used in solution, a crystalline product can be obtained from the decom-
position products of protagon, which has the same specific rotation as
protagon and can be repeatedly recrystallized without changing its
composition or its optical activity.2 A further study of these con-
ditions would naturally be of great interest.

As we are not decided whether protagon is only a mixture or is a body
contaminated with other substances, it is difficult to decide as to how
far the so-called decomposition products exist as preformed constituents
of the mixture or whether they are true decomposition products. On
boiling with baryta-water protagon yields cerebrosides (see below) and
the decomposition products of lecithin, namely, fatty acids, glycerophos-
phoric acid, and choline. KOSSEL and FREYTAG found three cerebro-
sides, namely, CEREBRIN, KERASIN (homocerebrin), and ENCEPHALIN.
According to KOCH 3 the protagon molecule contains cerebroside, lecithin
and sulphuric acid (in ester-like combination with the cerebroside)
besides excess of cerebroside. Of interest is the finding of KITAGAWA
and TmERFELDER4 that protagon dissolved in methyl alcohol contain-
ing chloroform, deposits crusts of cerebron (not pure) after a time at

1Lesem, 1. c.; Thudichum, 1. c.; Worner and Thierfelder, Zeitschr. f. physiol.
Chem., 30.

2 Journ. of Physiol., 37; Proc. physiol. Soc., January, 1908, p. 3.

8 Zeitschr. f. physiol. Chem., 53.

4 Kitagawa and Thierfelder, ibid., 49; Rosenheim and Tebb, Journ. of Physiol., 37,
341 and 348.

608 BRAIN AND NERVES.

ordinary temperature, and that as shown by ROSENHEIM and TEBB,
on dissolving in pyridine at 30° C. and heating or cooling the solution
deposits a precipitate of a substance rich in phosphorus. Although we
generally consider the phosphorized component of protagon as lecithin,
still, according to ROSENHEIM and TEBB, it is probably a diamido-
phosphatide, called sphingomyelin by THUDICHUM. On boiling protagon
with dilute mineral acids it yields galactose, due to the decomposition
of the cerebrosides.

Protagon appears, when dry, as a loose white powder. It dissolves
in alcohol of 85 vols. per cent at 45° C., but separates on cooling as a
snow-white, flaky precipitate, consisting of globules or groups of fine
crystalline needles. On heating to 150° it becomes yellowish, softens
at 180° and melts sharply at 200° forming a brown, oily liquid (CRAMER).
It is difficultly soluble in cold alcohol or ether, but dissolves, at least
when freshly precipitated, in ether on warming. It dissolves in methyl
alcohol containing chloroform and, as above stated, separates cerebron.
Protagon is soluble in pyridine at 30° C., yielding a clear solution, and
this solution has a specific rotation («)D==+6.9 to 7.7° according to
the concentration of the solution (WILSON and CRAMER). On warm-
ing or cooling according to ROSENHEIM and TEBB, the rotation changes
with the separation of sphingomyelin so that it first diminishes in rota-
tion, then is zero, and then becomes strongly levorotatory until it reaches
—242°, and finally, when nearly all the sphingomyelin has separated
out it becomes constant at about —13.3°. The strong levorotation
depends upon the accumulations of doubly refracting spheroid crystals
of sphingomyelin. With little water protagon swells up and is partly
decomposed. With more water it forms a jelly or pasty-like mass which,
with the addition of considerable water, forms an opalescent liquid.

Protagon can be prepared in the following way: The finely ground
brain-mass, as free as possible from blood and membrane, is dehydrated,
which is best done by cold acetone or by grinding with burned plaster-of-
paris or anhydrous sodium sulphate, and then extracted with ether.
The mass is then extracted at 45° C. with 85 vol. per cent alcohol until
the filtrate when cooled to 0° C. gives no more precipitate. All the
precipitates obtained on cooling to 0° C. are extracted with ether and
recrystallized from alcohol. Further details can be found in the cited
works of CRAMER, WILSON, GIES, ROSENHEIM and TEBB.

Among the phosphatides occurring in the brain we must mention besides the
lecithin and cephalin, the following substances.

Myelin, C^H^NPOio, according to THUDICHUM, is not well'known but is char-
acterized by the fact that its alcoholic solution is not precipitated by CdCl2 or
PtCl4. On the contrary an alcoholic solution of lead acetate gives a precipitate.
The existence of a second monaminomonophosphatide, paramyelin, C38
according to THUDICHUM, is very improbable.

CEREBROSIDES. 609

Sphingomyelin, is a diaminomonophosphatide which THUDICHUM prepared
from the brain and is the chief phosphatide obtainable from the impure protagon
mixtures. ROSENHEIM and TEBB obtained it, as above mentioned, from the
protagon. It has been given the formula CsaHNnNaPOg-fH^O. As cleavage
products an alcohol, sphingol, neurin, cholin, according to ROSENHEIM and TEBB,
the base sphingosin (see cerebron) and sphingostearic acid have been' obtained.
Sphingomyelin is soluble with difficulty in cold alcohol but readily soluble in hot
alcohol and crystallizes therefrom in needles. It is insoluble in ether. In regard
to the specific rotation see above in reference to protagon. Amidomyelin (Thud-
ichum) is another diaminomonophosphatide of an unknown constitution and of
an uncertain composition. Its existence is uncertain.

Sahidin was found by FRANKED in the brain, and is a triaminodiphosphatide,
whose cadmium compound has the formula CsoHieyNsPzOio.SCdCL. It is a crys-
talline powder which is insoluble in water, cold ethyl or methyl alcohol and in
ether. It is soluble with difficulty in warm alcohol but readily soluble in chloro-
form and hot benzene. It yields saturated and unsaturated fatty acids, choline
and glycerophosphoric acid.

Leucopoliin is an unsaturated phosphatide found by FRANKEL and ELIAS 2 in
the brain and which is a decaminodiphosphatide or a pentaminomonophosphatide.
It crystallizes from boiling alcohol on cooling. It does not contain any methylated
base but does contain a carbohydrate group.

Sulphatide is the name given by KOCH 3 to a sulphurized and phosphorized
product obtained from the human brain which separates from warm pyridine
on cooling as a crystalline, granular mass. It contains phosphatide, sulphuric
acid and cerebroside and is claimed to be phosphatidesulphuric acid cerebroside.

Cerebrosides.

On decomposing protagon (or the protagons), or the brain substance
by the gentle action of alkalies we obtain, as cleavage products, as above
stated, one or more bodies which THUDICHUM has embraced under the
name cerebrosides. The cerebrosides are nitrogenous substances free
from phosphorus, which yield galactose on boiling with dilute mineral
acids. With concentrated sulphuric acid they first give a yellow and
then a purple-red coloration. With sulphuric acid and cane-sugar
they give a purple coloration directly. The cerebrosides isolated from
the brain are cerebrin, homocerebrin, phrenosin, kerasin, encephalin,
and cerebron, but it must be remarked that there is no doubt that
sometimes the same body of varying purity has received different names.
According to LEVENE and JACOBS 4 it must be admitted that the cere-
brosides are mixtures of stereoisomeric substances.

Cerebrin. Under this name W. MuLLER5 first described a nitrog-
enous substance, free from phosphorus, which he obtained by extracting,
with boiling alcohol, a brain-mass which had been previously boiled with

1 Bioch. Zeitschr. 24.

2 Frankel and Elias, Bioch. Zeitschr. 28.

3 Zeitschr. f. physiol. Chem. 70.

4 Journ. of biol. Chem. 12.

6 Annal. d. Chem. u. Pharm., 105.

610 BRAIN AND NERVES.

baryta-water. Following a method essentially the same, but differing
slightly, GEOGHEGAN prepared, from the brain, a cerebrin with the
same properties as MULLER' s, but containing less nitrogen. Accord-
ing to PARCUS l the cerebrin isolated by GEOGHEGAN, as well as by
MULLER, consists of a mixture of three bodies, " cerebrin," " homo-
cerebrin," and " encephalin." KOSSEL and FREYTAG isolated two
cerebrosides from protagon which were identical with the cerebrin and
homocerebrin of PARCUS. According to these investigators, the two
bodies phrenosin and kerasin, as described by THUDICHUM, seem to be
identical with cerebrin and homocerebrin.

Cerebrin, according to PARCUS, has the following composition: C
69.08, H 11.47, N 2.13, 0 17.32 per cent, which corresponds with the
analyses made by KOSSEL and FREYTAG. No formula has been given to
this body. In the dry state it forms a pure white, odorless, and tasteless
powder. On heating it melts, decomposes gradually, smells like burned
fat, and burns with a luminous flame. Melting-point is 170-176° C. It
is insoluble in water, dilute alkalies, or baryta-water; also in cold alcohol
and in cold or hot ether. On the contrary, it is soluble in boiling alcohol
and separates as a flaky precipitate on cooling, and this is found to con-
sist of a mass of globules or grains on microscopical examination. Cere-
brin forms a compound with baryta, which is insoluble in water and is
decomposed by the action of carbon dioxide. The variety of sugar
split off on boiling with mineral acids — the so-called brain-sugar — is,
as THIERFELDER 2 first showed, galactose. On cleavage with nitric acid
fatty acids (stearic acid) were obtained.

Kerasin (THUDICHUM), or homocerebrin (PARCUS), has the following
composition: C 70.06, H 11.60, N 2.23, and 0 16.11 per cent. Enceph-
alin has the composition C 68.40, H 11.60, N 3.09, and 0 16.91 per cent.
Both bodies remain in the mother-liquor after the impure cerebrin has
precipitated from the warm alcohol. These bodies have the tendency
of separating as gelatinous masses. Kerasin is similar to cerebrin, but
dissolves more easily in warm alcohol and also in warm ether. It may
be obtained as extremely fine needles. Encephalin is, PARCUS thinks,
a transformation product of cerebrin. In the perfectly pure state it
crystallizes in small lamellae. It swells in warm water into a pasty mass.

As the purity and the chemical individuality of the above-mentioned bodies
is questionable, it is perhaps sufficient in regard to their preparation to simply
call attention to the cited works of MULLER, GEOGHEGAN, KOSSEL and FREYTAG.
All these methods split with barium hydroxide and purify the cerebroside by
solution in hot alcohol and a precipitation by cooling.

1 Geoghegan, Zeitschr. f. physiol. Chem., 3; Parcus, Ueber einige neue Gehrinstoffe,
Inaug.-Diss. Leipzig, 1881.

2 Zeitschr. f. physiol. Chem., 14.

CEREBRON. 611

Whether the above-described cerebrosides are chemical individuals or
mixtures, i. e., impure substances, is still undecided. The purest cere-
broside thus far investigated is undoubtedly THIERFELDER'S cerebron,
and there is hardly any doubt that the above-mentioned cerebrosides
consist essentially of this body.

Cerebron. This cerebrin, isolated by THIERFELDER and WORNER
and then especially studied by THIERFELDER, was first isolated by GAM-
GEE and called pseudocerebrin by him. THUDICHUM'S phrenosin is,
according to GIES/ identical with cerebron. Cerebron can be prepared
directly from the brain without saponification with baryta, by treat-
ment with alcohol containing benzene or chloroform at a temperature
of 50°, and hence it is considered as existing preformed in the brain.
According to THIERFELDER, cerebron has the formula C48H9sN09; it
melts at 212°, dissolves in warm alcohol, and separates out on cooling.
From proper solvents (acetone or methyl alcohol containing chloroform)
it may be separated as small needles or plates. If cerebron is suspended
in 85-per cent alcohol at a temperature of 50° C. it balls together in
amorphous masses, and from these needle- and leaf-shaped crystals
gradually form. It is dextrorotatory, and in about a 5-per cent solu-
tion in methyl alcohol (containing 75 per cent chloroform) is (Q;)D = +7.6°
(KITAGAWA and THIERFELDER). According to THIERFELDER it yields
as cleavage products, galactose, cerebronic add (THUDICHUM " neuro-
stearic acid ") and sphingosin which is in part obtained as such and part
as dimethylsphingosin. The base sphingosin, CiyHssNC^, discovered
by THUDICHUM, is, according to THIERFELDER and to LEVENE. and JACOBS,2
an unsaturated, diatomic, monoamino-alcohol which is readily soluble
in alcohol, ether, acetone and petroleum ether but insoluble in water,
nas an alkaline reaction and has not been obtained in a crystalline
state. The sulphate of dimethylsphingosin crystallizes, on the contrary,
from alcohol. Cerebronic acid is an oxyacid with the formula C2sH5oO3,
which is crystalline and which gives a crystalline methyl ester which
melts at 65° C. It has been obtained by LEVENE and JACOBS 3 in part
in a dextrorotatory and in part as an inactive form. The first melts
at 106-108° and the other at 82-85° C.

Cerebron can best be prepared, according to THIERFELDER and
KITAGAWA, by decomposing the protagon in methyl alcohol containing

1 Thierfelder and Worner, Zeitschr. f. physiol. Chem., 30; Thierfelder, ibid., 43,
44, 46, with Kitagawa, ibid., 49; with H. Loening, ibid., 68, 74, 77; Gamgee, Text-
book of Physiol. Chem., London, 1880; Thudichum, 1. c.; Gies, Journ. of Biol.
Chem., 1 and 2.

2 Thierfelder and O. Riesser and K. Thomas, Zeitschr. f. physiol. Chem., 77;
Levene and Jacobs, Journ. of biol. Chem. 11.

3 Journ. of biol. Chem., 12.

612 BKAIN AND NERVES.

chloroform (see page 607), and purifying the separated cerebron from
contaminating phosphatides by precipitating these with an ammoniacal
solution of zinc hydroxide in methyl alcohol, and recrystallizing the cere-
bron from methyl alcohol containing chloroform. THIERFELDER and
LOENING have devised another method of purification and at the same
time they have suggested another method for preparing cerebron. This
method is based upon the resistance of the cerebrosides to baryta and
their solubility in hot acetone. It consists in boiling the impure protagon
mixture with baryta-water and boiling the insoluble residue with acetone.

Neuridine, C6Hi4N2, is a non-poisonous diamine discovered by BRIEGER, and
obtained by him in the putrefaction of meat and gelatin, and from cultures of
the typhoid bacillus. It also occurs under physiological conditions in the brain,
and as traces in the yolk of the egg.

Neuridine dissolves in water and yields on boiling with alkalies a mixture
of dimethylamine and trimethylamine. It dissolves with difficulty in amyl
alcohol. It is insoluble in ether or absolute alcohol. In the free state, neuridine
has a peculiar odor, suggesting semen. With hydrochloric acid it gives a compound
crystallizing in long needles. With platinic chloride or gold chloride it gives
crystallizable double compounds which are valuable in its preparation and detec-
tion.

The so-called CORPUSCULA AMYLACEA, which occur on the upper surface of the
brain and in the pituitary gland, are colored more or less pure violet by iodine
and more blue by sulphuric acid and iodine. They perhaps consist of the same
substance as certain prostatic calculi, but they have not been closely investigated.

Quantitative Composition of the Brain. The quantity of water is
greater in the gray than in the white substance, and greater in new-born
or young individuals than in adults. The brain of the foetus contains
879-926 p. m. water. The observations of WEiSBACH1 show that the
quantity of water in the several parts of the brain (and in the medulla)
varies at different ages. The following figures are in 1000 parts — A for
men and B for women:

20-30 years. 30-50 years. 50-70 years. 70-94 yeara.

A. B. A. B. A. B. A. B.

White brain-substance.. 695.6 682.9 683.1 703.1 701.9 689.6 726.1 7220

Gray 833.6 826.2 836.1 830.6 838.0 838.4 847.8 839 5

Gyri 784.7 792.0 795.9 772.9 796.1 796.9 802.3 8017

Cerebellum 788.3 794.9 778.7 789.0 787.9 784.5 803.4 797.9

PonsVarolii 734.6 740.3 725.5 722.0 720.1 714.0 727.4 724.4

Medulla oblongata 744.3 740.7 732.5 729.8 722.4 730.6 736.2 733.7

The recent investigations of K. LiNNERT2 correspond to the above
in that the pons and the medulla were found to be next to the white sub-
stance, the poorest in water, of the human brain.

Quantitative analyses of human brains at different ages, namely
6 weeks, 2 and 19 years, have been made by KOCH and MANN.S These
analyses show that with increasing age the water, proteins, extractives

1 Cited from K. B. Hoffmann's Lehrbuch d. Zioch., Wien, 1877, p. 121.

2 Wien. klin. Wochenschr., 23.

» Journ. of Physiol., 36, Proc. physiol. Soc., 1907.

COMPOSITION OF THE BRAIN. -613

and salts diminish relatively, while the phosphatides, cerebrosides and
especially cholesterin strikingly increase. The sulphur of the lipoids
increased to the second year, but then existed in the same amounts as at
nineteen years.

BAUMSTARK claims to have found that a part pfuthe cholesterin in the brain
occurs in a combined state, perhaps as ester; this view has been found to be
incorrect by the recent investigations of BUNZ. He obtained from the brain
neither esters of cholesterin with higher fatty acids nor other compounds of
cholesterin which split on saponification. TEBB * Jias also found only free
cholesterin.

According to FRANKEL,2 who has fractionally extracted the human
brain with various solvents, found 230 p. m. solids in the brain and this
consisted of f lipoids and J proteins. Of the lipoids about 17 per cent
was cholesterin, 34.482 per cent saturated and 48.293 per cent unsat-
urated compounds. The amount of cholesterin in the different parts
of the brain was as follows, according to FRANKEL, KIRSCHBAUM and
LINNERT. In the cortex 11.5 p. m., in the white substance 24.7 p. m.,
in the cerebellum 13.1 p. m., and in the bridge and medulla 40.3 p. m.,
all calculated in the moist substance.

The analysis of the brain of an epileptic made by KOCH 3 is of very great
interest. As the protagon is considered by KOCH as a mixture, no results for
the quantity of protagon are given. As no accurate methods for the estima-
tion of the little known bodies cephalin, myelin, phrenosin and kerasin are
available, the figures given for these are of little value. The following results
are calculated to 1000 parts :

Corpus Cortex

Callosum (pref rental).

Water 679.7 841 .3

Protein 32.0 50.0

Nucleoproteins 37.0 30.0

Neurokeratin 27.0 (CHITTENDEN) 4.0 (CHITTENDEN)

Extractives (water-soluble) 15.1 15.8

Lecithins 51.9 31.4

Cephalin and myelin 34 . 9 7.4

Phrenosin and kerasin 45 . 7 15.5

Cholesterin 48.6 7.0

Sulphurized substance 14 . 0 14 . 5

Mineral bodies 8.2 8.7

PIGHINI and CARBONE found that the brains of paralytics were richer in water,
considerably richer in cholesterin, but poorer in cephalin than healthy brains.
This last corresponds, to the observations of KOCH and MANN 4 that the quan-
tity of lipoid phosphorus was diminished in paralytics.

^aumstark, Zeitschr. f. physiol. Chem. 9; R. Biinz ibid., 46; Tebb, Journ. of
Physiol., 34.

2 Bioch. Zeitschr., 19, with Kirschbaum and Linnert, ibid., 46.

3 Amer. Journ. of Physiol., 11.

4 Pighini and Carbone, Bioch. Zeitschr., 46; Koch and Mann, Arch, of Neurol.
and Psychol., 1910.

614 BRAIN AND NERVES.

According to FR. FALK l the cerebrosides occur in the medullary
nerve fibers as well as in the nerves without medullas. These latter
yielded much less substance on extraction than the medullary, namely,
11.51 per cent extract as compared to 46.59 per cent. The extract of
the first was poorer in cerebrosides, but richer in cholesterin, cephalin
and lecithin, as shown by the following figures.

Non-medullary fibers Medullary fibers in

in p. m. of the total p. m. of the total

extract extract

Cholesterm 470 250

Cephalin 237 124

Cerebrosides 60 182

Lecithins 98 29

S. FRANKEL and L. DIMITZ 2 find that the spinal marrow contains on
an average 740 p. m. water, 180 p. m. lipoids and 80 p. m. protein. The
quantity of cholesterin (in the fresh, spinal marrow containing water)
is 40 p. m., the unsaturated phosphatide 120 p. m., and the saturated
15 p. m. The spinal marrow is the richest part of the nervous system
in unsaturated phosphatides and it contains abundance of cephalin.

According to NOLL the white substance of the spinal marrow^ is some-
what richer in protagon than the brain, and in nerve degeneration the
quantity of protagon diminishes. The method used by him would not
allow of an exact determination of the disputed substance protagon.
MOTT and HALLIBURTON 3 have also shown that in degenerative diseases
of the nervous system, the quantity of substances containing phosphorus
diminishes, and that in these cases, especially in general paralysis, choline
passes into the cerebrospinal fluid and the blood. In degenerated nerves,
the quantity of water increases, and the phosphorus decreases. On
comparative investigations of the central nervous system of normal
persons, and those afflicted with dementia pracox (5 cases), Kocn4 found
that the variation from the normal composition was not great enough
nor so constant that positive conclusions could be drawn therefrom.

The quantity of neurokeratin in the nerves and the different parts
of the brain has been carefully determined by KUHNE and CniTTENDEN.5
They found 3.16 p. m. in the plexus brachialis, 3.12 p. m. in the cortex
of the cerebellum, 22.434 p. m. in the white substance of the cerebrum,
25.72-29.02 p. m. in the white substance of the corpus callosum, and
3.27 p. m. in the gray substance of the cortex of the cerebrum (when

1 Bioch. Zeitschr., 13.
» Ibid., 28.

8 Noll, Zeitschr. f. physiol. Chem., 27; Mott and Halliburton, Philos. Transactions,
Ser. B., 191 (1899), and 194 (1901).
* Arch, of Neurology, 3.
6 Zeitschr. f . Biologic, 26.

VISUAL PURPLE. 615

free as possible from white substance). The white is decidedly richer
in neurokeratin than the peripheral nerves or the gray substance. Accord-
ing to GRIFFITHS,1 neurochitin replaces neurokeratin in insects and crus-
tacea, the quantity of the first being 10.6-12 p. m.

The quantity of mineral constituents in the brain amounts to 2.95-
7.08 p. m. according to GEOGHEGAN. He found in 1000 parts of the
fresh, moist brain 0.43-1.32 Cl, 0.956-2.016 P04, 0.244-0.796 C03,
0.102-0.220 S04, 0.01-0.098 Fe2(P04)2, 0.005-0.022 Ca, 0.016-0.072
Mg, 0.58-1.778 K, and 0.450-1.114 Na. The gray substance yields an
alkaline ash, the white an acid ash. MAGNUS-LEVY 2 found in fresh brain
substance 1.305 p. m. Cl, 0.166 p. m. Ca, 0.139 p. m. Mg, and 0.083 p.m.
Fe.

Appendix.

THE TISSUES AND FLUIDS OF THE EYE.

The retina contains in all 865-899.9 p. m. water, 57.1-84.5 p. m.
protein bodies — myosin, albumin, and mucin (?), 9.5-28.9 p. m. lecithin,
and 8.2-11.2 p. m. salts (HOPPE-SEYLER and CAHN 3). The mineral bodies
consist of 422 p. m. Na2HP04 and 352 p. m. NaCl. The retina con-
tains, according to BARBiERi,4 also cholesterin but no cerebrosides and in
fact none of the specific constituents of the brain substance.

Those bodies which form the different segments of the rods and cones
have not been closely studied, and the greatest interest is therefore con-
nected with the coloring-matters of the retina.

Visual purple, also called rhodopsin, erythfopsin, or VISUAL RED, is
the pigment of the rods. BOLL,S in 1876, observed that the layer of rods
in the retina during life had a purplish-red color which was bleached
by the action of light. KUHNE 6 later showed that this red color might
remain for a long time after the death of the animal if the eye was pro-
tected from daylight or investigated by a sodium light. Under Jthese
conditions it was also possible to isolate and closely study this substance.

Visual red (BOLL) or visual purple (KUHNE) has become known mainly by
the investigations of KUHNE. The pigment occurs mainly in the rods and only
in their outer parts. In animals whose retina has no rods the visual purple is

1 Compt. Rend., 115.

2 Geoghegan, Zeitschr. f. physiol Chem.. 1; Magnus-Levy, Bioch. Zeitschr. 24.

3 Zeitschr. f. physiol. Chem., 5.

4 Compt. Rend., 154.

6 Monatsber. d. Kgl. Preuss. Akad., 12. Nov., 1876.

6 The investigations of Kiihne and his pupils, Ewald and Ayres, on the visual purple
will be found in Untersuchungen aus dem physiol. Institut der Universitat Heidel-
berg, 1 and 2, and in Zeitschr. f. Biologic, 32.

616 BRAIN AND NERVES.

absent, and is also necessarily absent in the macula lutea. In a variety of bat
(Rhinolophus hipposideros), in hens, pigeons and new-born rabbits, no visual
purple has been found in the rods.

A solution of visual purple in water which contains 2-5 per cent crys-
tallized bile, wrhich is the best solvent for it, is purple-red in color, quite
clear, and not fluorescent. On evaporating this solution in vacuo we
obtain a residue similar to ammonium carminate which contains violet
or black grains. If the above solution is dialyzed with water, the bile
diffuses and the visual purple separates as a violet mass. Under all
circumstances, even when still in the retina, the visual purple is quickly
bleached by direct sunlight, and with diffused light with a rapidity cor-
responding to the intensity of the light. It passes from red and orange
to yellow. Red light bleaches the visual purple slowly; the ultra-red
light does not bleach it at all. A solution of visual purple shows no special
absorption bands, but only a general absorption which extends from the
red side, beginning at D and extending to the G line. The strongest
absorption is found at E.

KOETTGEN and ABELSDORF » have shown that there are, in accordance with
KUHNE'S views, two varieties of visual purple, the one occurring in mammals,
birds, and amphibians, and the other, which is more violet-red, in fishes. The
first has its [maximum absorption in the green and the other in the yellowish-
green.

Visual purple when heated to 52-53° C. is destroyed after several
hours, and almost instantly when heated to 76° C. It is also destroyed
by alkalies, acids, alcohol, ether, and chloroform. On the contrary,
it resists the action of ammonia or alum solution.

As the visual purple is easily destroyed by light, it must therefore also be
regenerated during life. KUHNE has also found that the retina of the eye of the
frog becomes bleached when exposed for a long time to strong sunlight, and that
its color gradually returns when the animal is placed in the dark. This regenera-
tion of the visual purple is a function of the living cells in the layer of the pigment
epithelium of the retina. This may be inferred from the fact that a detached
piece of the retina which has been bleached by light may have its visual purple
restored if it is carefully laid on the choroid having layers of the pigment-epithe-
lium attached. The regeneration has, it seems, nothing to do with the dark
pigment, the melanin or fuscin, in the epithelium cells. A partial regeneration
seems, according to KUHNE, to be possible in the retina which has been completely
removed. On account of this property of the visual purple of being bleached
by light during life we may, as KUHNE has shown, under special conditions and
by observing special precautions, obtain after death, by the action of intense
light or more continuous light, the picture of bright objects, such as windows
and the like — so-called optograms.

The physiological importance of visual purple is unknown. It follows
that the visual purple is not essential to sight, since it is absent in certain
animals and also in the cones.

1 Centralbl. f. Physiol., 9; also Maly's Jahresber., 25, 351.

CRYSTALLINE LENS. 617

Visual purple must always be prepared exclusively in a sodium light. It is
extracted from the net membrane by means of a watery solution of crystallized
bile. The filtered solution is evaporated in vacuo or dialyzed until the visual
purple is separated. To prepare a visual-purple solution perfectly free from
haemoglobin, the solution of visual purple in cholates is precipitated by saturating
with magnesium sulphate, washing the precipitate with a saturated solution of
magnesium sulphate, and then dissolving in water by the aid of the cholates sim-
ultaneously precipitated.1

The Pigments of the Cones. In the inner segments of the cones of birds, rep-
tiles, and fishes a small fat-globule of varying color is found. KUHNE 2 has
isolated from this fat a green, a yellow, and a red pigment called respectively
chlorophan, xanthophan, and rhodophan.

The dark pigment of the epithelium-cells of the net membrane, which was
formerly called melanin, but has since been named fuscin by KUHNE and MAYS,*
contains iron, dissolves in concentrated caustic alkalies or concentrated sul-
phuric acid on warming, but, like the melanins in general, has been little studied.
The pigment occurring in the pigment-cells of the choroid will be discussed with
the melanins in Chapter XV.

The vitreous humor is often considered as a variety of gelatinous
tissue. The membrane consists, according to C. MORNER, of a gelatin-
forming substance. The fluid contains a little proteid and a mucoid,
hyalomucoid, which was first showTn by MORNER, and which is precipitated
by acetic acid. This contains 12.27 per cent N, and 1.19 per cent S.
Among the extractives we find a little urea — according to PICARD 5 p. m.,
according to RAHLMANN 0.64 p. m. PAUTz4 found besides some urea,
paralactic acid, and, in confirmation of the claims of CHABBAS, JESNER,
and KUHN, also glucose in the vitreous humor of oxen. The reaction
of the vitreous humor is alkaline, and the quantity of solids amounts
to about 9-11 p. m. The quantity of mineral bodies is about 6-9 p. m.,
and the proteins 0.7 p. m. In regard to the aqueous humor see page
361.

The Crystalline Lens. That substance which forms the capsule of
the lens has been investigated by C. MORNER. It belongs, according
to him, to a special group of proteins, called membranins. The mem-
branin bodies are insoluble at the ordinary temperature in water, salt
solutions, dilute acids, and alkalies, and, like the mucins, yield a reducing
substance on boiling with dilute mineral acids. They contain lead-
blackening sulphur. The membranins are colored a very beautiful red
by MILLON'S reagent, but give no characteristic reaction with concentrated
hydrochloric acid or ADAMKIEWICZ'S reagent. They are dissolved with

1 Kuhne, Zeitschr. f . Biologic, 32.

2 Kuhne, Die nichtbestandigen Farben der Netzhaut, Untersuch. aus dem physiol.
Institut Heidelberg, 1, 341.

3 Kuhne, ibid., 2, 324.

4 Morner, Zeitschr. f. physiol. Chem., 18; Picard, cited from Gamgee, Physiol.
Chem., 1, 454; Rahlmann, Maly's Jahresber., 6; Pautz, Zeitschr. f. Biologic, 31. A
complete review of the literature will also be found here.

618 BRAIN AND NERVES.

great difficulty by pepsin-hydrochloric acid or trypsin solution, but are
soluble in dilute acids and alkalies in the warmth. Membranin of the
capsule of the lens contains 14.10 per cent N and 0.83 per cent S, and is
a little less soluble than that from DESCEMET'S membrane.

The principal mass of the solids of the crystalline lens consists of
proteins, whose nature has been investigated by C. M6RNER.1 Some of
these proteins dissolve in dilute salt solution, while others remain
insoluble in this solvent.

The Insoluble Protein. The lens fibers consist of a protein sub-
stance which is insoluble in water and in salt solution and to which
MORNER has given the name albumoid. It dissolves readily in very dilute
acids or alkalies. Its solution in caustic potash of 0.1 per cent is very
similar to an alkali-albuminate solution, but coagulates at about 50°
C. on nearly complete neutralization and the addition of 8 per cent NaCl.
Albumoid has the following composition: C 53.12, H 6.8, N 16.62, and
S 0.79 per cent. The lens fibers themselves contain 16.61 per cent N
and 0.77 per cent S. The inner parts of the lens are considerably richer
in albumoid than the outer. The quantity of albumoid in the entire
lens amounts on an average to about 48 per cent of the total weight of
the proteins of the lens.

The Soluble Protein consists, exclusive of a very small quantity of
albumin, of two globulins, a- and p-crystallin. These two globulins differ
from each other in this manner: a-crystallin contains 16.68 per cent N
and 0.56 per cent S; jS-crystallin, on the contrary, 17.04 per cent N and
1.27 per cent S. The first coagulates at about 72° C. and the other at
63° C. Besides this, j8-crystallin is precipitated from a salt-free solu-
tion with greater difficulty and less completely by acetic acid or carbon
dioxide. These globulins are not precipitated by an excess of NaCl at
either the ordinary temperature or 30° C. Magnesium or sodium sul-
phate in substance precipitates both globulins, on the contrary, at 30° C.
These two globulins are not equally divided in the mass of the lens. The
quantity of a-crystallin diminishes in the lens from without inward;
j8-crystallin, on the contrary, from within outward.

A. JESS 2 has found that the different proteins of the crystalline lens
behave differently with ARNOLD'S protein reaction with sodium nitro-
prusside (page 100). The albumoid gives negative results with this
reagent. The a-crystallin gives it faintly, while the /3-crystallin gives a
strong reaction. The absence of this reaction, as observed by WEISS
in senile cataract, is connected with the fact as JESS has shown by his
investigations on the senile cataract in oxen, that the crystallin con-

1 Zeitschr. f . physiol. Chem., 18. This contains also the pertinent literature.
« Zeitschr. f. Biol., 61.

PROTEINS OF THE LENS. 619

taining cysteine, disappears in part from the lens and is partly transformed
into albumoid. The relation between albumoid and crystallins is changed
with increasing age, so that the albumoid increases. In normal lens
the relation of the crystallins to the albumoid changes correspondingly
from 82:18 in youth to 41:59 in old age; in senile cataract the relation
can be changed to 25 : 75. The amount of fat, cholesterin and lecithin
is on the contrary not changed.

The average results of four analyses made by LAPTSCHINSKY 1 of the
lens of oxen are here given, calculated in parts per 1000:

Proteins 349.3

Lecithin 2.3

Cholesterin 2.2

Fat 2.9

Soluble salts 5.3

Insoluble salts 2.4

In cataract the amount of proteins is diminished and the amount of
cholesterin increased. This statement requires further substantiation.2

The quantity of the different proteins in the fresh moist lens of oxen
is, as follows, according to MORNER:

Albumoid (lens fibers) 170 p. m.

/3-Crystallin 110 "

a-Crystallin *. 68 "

Albumin 2 "

The corneal tissue has been previously considered (page 550). The
sclerotic has not been closely investigated, and the choroid coat is princi-
pally of interest because of the coloring-matter (melanin) it contains
(see Chapter XV).

Tears consist of a water-clear, alkaline fluid of a salty taste. Accord-
ing to the analyses of LERCHS they contain 982 p. m. water, 18 p. m. solids
with 5 p. m. albumin and 13 p. m. NaCl.

THE FLUIDS OF THE INNER EAR.

The perilymph and endolymph are alkaline fluids, which, besides
salts, contain — in the same amounts as in transudates — traces of protein,
and in certain animals (codfish) also mucin. The quantity of mucin
is greater in the perilymph than in the endolymph.

Otoliths contain 745-795 p. m. inorganic substance, which consists
chiefly of crystallized calcium carbonate. The organic substance is very
similar to mucin.

1 Pfliiger's Arch., 13.

2 See Gross, Arch. f. Augenheilk., 55 and 58.

3 Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4 Aufl., 401.

CHAPTER XII.
ORGANS OF GENERATION.

(a) Male Generative Secretions.

The testes have been little investigated chemically. In the testes of
animals we find protein bodies of different kinds — seralbumin, alkali
albuminate (?), and an albuminous body related to ROVIDA'S hyaline
substance; also leudne, tyrosine, creatine, purine bases, cholesterin, lecithin,
inosite, and fat. In regard to the occurrence of glycogen the reports are
conflicting. DARESTE 1 found, in the testes of birds, starch-like granules,
which were colored blue with difficulty by iodine.

In the autolysis of the testes LEVENE 2 found tyrosine, alanine, leucine,
aminovaleric acid, ammobutyric acid, a-proline, phenylalanine, aspartic acid,
glutamic acid, and hypoxanthine. Pyrimidine and hexone bases could not be
detected.

The semen as ejected is a white or whitish-yellow, viscous, sticky
fluid of a milky appearance, with whitish, non-transparent lumps. The
milky appearance is due to spermatozoa. Semen is heavier than water,
contains proteins, has a neutral or faintly alkaline reaction and a peculiar
specific odor. Soon after ejection semen becomes gelatinous, as if it
were coagulated, but afterward becomes more fluid. When diluted
with water white flakes or shreds separate (HENLE'S fibrin). According
to the analyses of SLOWTZOFF,3 human semen contains on an average
96.8 p. m. solids with 9 p. m. inorganic and 87.8 p. m. organic substance.
The amount of protein substances was, on an average, 22.6 p. m. and 1.69
p. m. of bodies soluble in ether. The protein substances consist of nucleo-
proteins, traces of mucin, albumin, and a substance similar to proteose
(found earlier by POSNER). According to CAVAZZANI semen contains
relatively considerable nucleon, more than any organ, v. HOFFMANN*

1 Compt. Rend., 74.

2 Amer. Journ. of Physiol., 11.

* Zeitschr. f. physiol. Chem., 35.

1 Posner, Berl. klin. Wochenschr., 1888, No. 21, and Centralbl. f. d. med. Wissensch.,
1890; Cavazzani, Biochem. Centralbl., 1, 502, and Centralbl. f. Physiol., 19; v. Hoff-
mann, cited in Bioch. Centralbl., 9, 206.

620

SEMEN. SPERMINE. 621

has found a protamine in human semen which yielded arginine and
perhaps also lysine on cleavage. The mineral bodies consist mainly of cal-
cium phosphate and considerable NaCl. Potassium occurs only in
smaller amounts.

The semen in the vas deferens differs chiefly from the ejected semen
in that it is without the peculiar odor. This last depends on the admixture
with the secretion of the prostate. This secretion, according to IVERSEN,
has a milky appearance and ordinarily an alkaline reaction, very rarely
a neutral one, and contains small amounts of proteins, especially nucleo-
proteins, besides a substance similar to fibrinogen and to mucin (STERN *),
and mineral bodies, especially NaCl. Besides this it contains an enzyme
vesiculase (see below), lecithin, choline (STERN), and a crystalline com-
bination of phosphoric acid with a base, C2HsN. This combination has
been called B DITCHER'S spermine crystals, and it is claimed that the
specific odor of the semen is due to a partial decomposition of these
crystals.

The crystals which appear on slowly evaporating the semen, and
which are also observed in anatomical preparations kept in alcohol, are
not identical with the CHARCOT-LEYDEN crystals found in the blood and
in the lymphatic glands in leucaemia (Tn. COHN, B. LEWY2). They are,
according to SCHREINER,S as above stated, a combination of phosphoric
acid with a base, spermine, C2H5N, which he discovered.

Spermine. Opinions in regard to the nature of this base are not unanimous
According to the investigations of LADENBURG and ABEL, it is not improbable
that spermine is identical with ethylenimine; but this identity is disputed by
MAJERT and A. SCHMIDT, and also by POEHL. The compound of spermine with
phosphoric acid — BOTTCHER'S spermine crystals — is insoluble in alcohol, ether,
and chloroform, soluble with difficulty in cold water, but more readily in hot
water, and easily soluble in dilute acids or alkalies, also alkali carbonates and
ammonia. The base is precipitated by tannic acid, mercuric chloride, gold
chloride, platinic chloride, potassium-bismuth iodide, and phosphotungstic acid.
Spermine has a tonic action, and, according to POEHL, 4 it has a marked action on
the oxidation processes of the animal body.

On the addition of a solution of potassium iodide and iodine to spermatozoa,
characteristic dark-brown or bluish-black crystals are obtained — FLORENCE'S
sperm reaction, which is considered by many as a reaction for spermine. Accord-
ing to BocARius,5 this reaction is due to choline.

1 Iversen, Nord. med. Ark., 6; also Maly's Jahresber., 4, 358; Stern, Biochem.
Centralbl., 1, 748.

2Th. Cohn, Centralbl. f. allg. Path. u. path. Anat., 10 (1899), and Zeitschr.f.Urolog.,
1908; B. Lewy, Centralbl. f. d. med. Wissensch., 1899, 479.

8 Annal. d. Chem. u. Pharm., 194.

4Ladenburg and Abel, Ber. d. deutsch. chem. Gesellsch., 21; Majert and A.
Schmidt, ibid., 24; Poehl, Compt. Rend., 115, Berlin, klin. Wochenschr., 1891 and
1893, Deutsch. med. Wochenschr., 1892 and 1895, and Zeitschr. f. klin. Med., 1894.

6 In regard to Florence's sperm reaction, see Posner, Berl. klin. Wochenschr.,
1897, and Richter, Wien. klin. Wochenschr., 1897; Bocarius, Zeitschr. f. physiol.
Chem,, 34.

622 ORGANS OF GENERATION.

CAMUS and GLEY » have found that the prostate fluid in certain rodents has
the property of coagulating the contents of the seminal vesicles. This property
is due to a special ferment substance (vesiculase) of the prostate fluid.

The spermatozoa show a great resistance to chemical reagents in
general. They do not dissolve completely in concentrated sulphuric
acid, nitric acid, acetic acid, or in boiling-hot soda solutions. They
are soluble in a boiling-hot caustic-potash solution. They resist putre-
faction, and after drying they may be obtained again in their original
form by moistening them with a 1-per cent common-salt solution. By
careful heating and burning to an ash the shape of the spermatozoa may
be seen in the ash. The quantity of ash is about 50 p. m. and consists
mainly (three-quarters) of potassium phosphate.

The spermatozoa show well-known movements, but the cause of this
is not known. These movements may continue for a very long time,
as under some conditions they may be observed for several days in the
body after death, and in the secretion of the uterus longer than a week.
Acid liquids stop these movements immediately; they are also destroyed
by strong alkalies, especially ammoniacal liquids, also by distilled water,
alcohol, ether, etc. The movements continue for a longer time in faintly
alkaline liquids, especially in alkaline animal secretions, and also in
properly diluted neutral salt solutions.2

Spermatozoa are nucleus formations and hence are rich in nucleic
acid, which exists in the heads. The tails contain protein, and are besides
this rich in lecithin, cholesterin, and fat, which bodies occur only to a
small extent (if at all) in the heads. The tails seem by their composi-
tion to be closely allied to the non-medullated nerves or the axis-cylinders.
In the various kinds of animals investigated, the head contains nucleic
acid, which in fishes is partly combined with protamines and partly
with histones. In other animals, such as the bull and boar, protein-
like substances occur with the nucleic acid, but no protamine.

Our knowledge of the chemical composition of spermatozoa has
been greatly enhanced by the important investigations of MiESCHER3
on salmon milt. The intermediate fluid of the spermatozoa of Rhine
salmon is a dilute salt solution containing 1.3-1.9 p. m. organic and
6.5-7.6 p. m. inorganic bodies. The last consist principally of sodium
chloride and carbonate, besides some potassium chloride and sulphate.
The fluid contains only traces of protein, but no peptone. The tails consist
of 419 p. m. protein, 318.3 p. m. lecithin, and 262.7 p. m. cholesterin and

1 Compt. rend, de soc. biolog., 48, 49.

2 See G. Giinther, Pfluger's Arch., 118.

3 See Miescher, " Die histochemischen und physiologischen Arbeiten von Friedrich
Miescher, gesammelt und herausgegeben von seinen Freunden," Leipzig, 1897.

OVARIES. 623

fat. The heads extracted with alcohol-ether contain on an average
960 p. m. protamine nucleate, which nevertheless is not uniform, but is
so divided that the outer layers consist of basic protamine nucleate,
while the inner layers, on the contrary, consist of acid protamine nucleate.
Besides the protamine nucleate there are present in the heads, although
to a very slight extent, organic substances. Of these we must mention
a nitrogenous substance containing iron which gives MILLON'S reaction
and which MIESCHER calls karyogen. The unripe salmon spermatozoa,
while developing, also contain nucleic acid, but no protamine, with a
protein substance, " albuminose," which probably is a step in the forma-
tion of protamine. According to KOSSEL and MATHEWS/ in the herring
as in the salmon, the heads of the spermatozoa consist of protamine
nucleate but no free protein.

The chemical investigations on the spermatozoa have not given
us any information as to the condition for fertilization and the develop-
ment of the egg.

Spermatin is a name which has been given to a constituent similar to alkali
albuminate, but it has not been closely studied.

Prostatic concrements are of two kinds. One is very small, generally oval
in shape, with concentric layers. In young but not in older persons they are
colored blue by iodine (IVERSEN 2). The other kind is larger, sometimes the size
of the head of a pin, consisting chiefly of calcium phosphate (about 700 p.m.), with
only a very small amount (about 160 p. m.) of organic substance.

(b) Female Generative Organs.

The stroma of the ovaries is of little interest from a physiologico-
chemical standpoint, and the most important constituents of the ovaries,
the Graafian follicles with the ovum, have not thus far been the subject
of a careful chemical investigation. The fluid in the follicles (of the
cow) does not contain, as has been stated, the peculiar bodies, paral-
bumin or metalbumin, which are found in certain pathological ^ovarial
fluids, but seems to be a serous liquid. The corpora lutea are colored
yellow. Earlier investigators (PICCOLO and LIEBEN, KUHNE and EWALD 3)
have found a crystalline pigment in the . corpora lutea. In recent
investigations EscHER4 has shown that this substance is a crystalline
hydrocarbon (C^Hse) which seems to be identical with the carotin of
the carrot and green leaves. The color of the crystals as well as the con-
centrated solution is reddish-orange. Carotin differs from the yellow
pigment of the yolk of the egg, the lutein, in having another formula

1 Zeitschr. f. physiol. Chem., 23.^

2 Nord. med. Ark., 6.

» See Chapter V, p. 301.

* Zeitschr. f . physiol. Chem., 83, 198 (1912).

624 ORGANS OF GENERATION.

(page 631) and being soluble with difficulty in alcohol and readily soluble
in petroleum ether.

The cysts often occurring in the ovaries are of special pathological
interest, and these may have essentially different contents, depending
upon their variety and origin.

The serous cysts (HYDROPS FOLLICULORUM GRAAFII), which are
formed by a dilation of the Graafian follicles, contain a serous liquid
which has a specific gravity of 1.005-1.022. A specific gravity of 1.020
is less frequent. Generally the specific gravity is lower, 1.005-1.014,
with 10-40 p. m. solids. As far as is known, the contents of these cysts
do not essentially differ from other serous liquids.

The proliferous cysts (MYXOID CYSTS, COLLOID CYSTS), which are
developed from PFLUGER'S epithelium-tubes, may have a content of a
decidedly variable composition.

We sometimes find in small cysts a semi-solid, transparent, or some-
what cloudy or opalescent mass which appears like solidified glue or
quivering jelly, and which has been called colloid because of its physical
properties. In other cases the cysts contain a thick, tough mass which
can be drawn out into long threads, and as this mass in the different
cysts is more or less diluted with serous liquids their contents may have
a variable consistency. In still other cases the small cysts may also
contain a thin, watery fluid. The color of the contents is also variable.
Sometimes they are bluish-white, opalescent, and again they are yellow,
yellowish-brown, or yellowish with a shade of green. They are often
colored more or less chocolate-brown or red-brown, due to the decom-
posed blood-coloring matters. The reaction is alkaline or nearly neutral.
The specific gravity, which may vary considerably, is generally 1.015-
1.030, but may occasionally be 1.005-1.010 or 1.050-1.055. The amount
of solids is very variable. In rare cases it amounts to only 10-20 p. m.;
ordinarily it varies from 50-70-100 p. m. In a few instances 150-200
p. m. solids have been found.

As form-elements one finds red and white blood-corpuscles, granular
cells, partly fat-degenerated epithelium and partly large so-called GLTJGE'S
corpuscles, fine granular masses, epithelium-cells, cholesterin crystals, and
colloid corpuscles — large, circular, highly refractive formations.

Though the contents of the proliferous cyst may have a variable
composition, still it may be characterized in typical cases by its slimy
or ropy consistency; by its grayish-yellow, chocolate-brown, sometimes
whitish-gray color; and by its relatively high specific gravity, 1.015-
1.025. Such a liquid does not ordinarily show a spontaneous fibrin
coagulation.

We consider colloid, metalbumin, and paralbumin as characteristic
constituents of these cysts.

COLLOID. PSEUDOMUCIN. 625

Colloid. This name does not designate any particular chemical
substance, but is given to the contents of tumors with certain physical
properties similar to gelatin jelly. Colloid is found as a pathological
product in several organs.

Colloid is a gelatinous mass, insoluble in water and acetic acid'; it is
dissolved by alkalies and gives a liquid which is not precipitated by
acetic acid or by acetic acid and potassium ferrocyanide. According to
PFANNENSTIEL l such a colloid is designated /3-pseudomucin. Some-
times a colloid is found which, when treated with a very dilute alkali,
gives a solution similar to a mucin solution. Colloid is very closely
related to mucin and is considered by certain investigators as a modified
mucin. An ovarial colloid analyzed by PANZER contained 931 p. m.
water, 57 p. m. organic substance, and 12 p. m. ash. The elementary
composition was C 47.27, H 5.86, N 8.40, S 0.79, P 0.54, and ash 6.43
per cent. A colloid found by WURTZ 2 in the lungs contained C 48.09,
H 7.47, N 7.00, and 0(+S) 37.44 per cent. Colloids of different origin
seem to be of varying composition.

Metalbumin. This name SCHERER 3 gave to a protein substance
found by him in an ovarial fluid. The metalbumin was considered by
SCHERER to be an albuminous body, but it belongs to the muein group,
and it is for this reason called pseudomudn by HAMMARSTEN.*

Pseiidomucin. This body, which, like the mucins, gives a reducing
substance when boiled with acids, is a mucoid of the following com-
position: C 49.75, H 6.98,. N 10.28, S 1.25, 0 31.74 per cent (HAMMAR-
STEN). With water pseudomucin gives a slimy, ropy solution, and it is
this substance which gives the fluid contents of the ovarial cysts their
typical ropy property. Its solutions do not coagulate on boiling, but
only become milky or opalescent. Unlike mucin, pseudomucin solutions
are not precipitated by acetic acid. With alcohol they give a coarse
flocculent or thready precipitate which is soluble even after having been
kept under water or alcohol, for a long time.

Paralbumin is another substance discovered by SCHERER, which occurs
in ovarial liquids, and also in ascitic fluids, with the simultaneous presence
of ovarial cysts and rupture of the same. It is therefore only a mixture
of pseudomucin with variable amounts of protein, and the reactions of
paralbumin are correspondingly variable.

1 Arch. f. Gynak., 38.

2 Panzer, Zeitschr. f . physiol. Chem., 28; Wiirtz, see Lebert, Beitr. zur Kenntnis
des Gallertkrebses, Virchow's Arch., 4.

8 Verh. d. physik.-med. Gesellsch. in Wiirzburg, 2, and Sitzungsb.er. der physik.-
med. Gesellsch. in Wiirzburg fur 1864-1865; Wiirzburg med. Zeitschr., 7, No. 6.
4 Zeitschr. f . physiol. Chem., 6.

626 ORGANS OF GENERATION.

MITJUKOFF * has isolated and investigated a colloid from an ovarial cyst. It
had the following composition: C 51.76, H 7.76, N 10.7 S 1.09, and 0 28.69 per
cent, and differed from mucin and pseudomucin by reducing FEHLING'S solu-
tion before boiling with acid. It must be remarked that pseudomucin, on boiling
sufficiently long with alkali, or by the use of a concentrated solution of caustic
alkali,- also splits and causes a reduction. This reduction is nevertheless weak
as compared with that produced after boiling with an acid. The body isolated
by MITJUKOFF is called paramucin.

The pseudomucin as well as colloid are mucoid substances, and the
carbohydrate obtained from them is glucosamine (chitosamine), as espe-
cially shown by FK. MULLER, NEUBERG and HEYMANN.2 From pseudo-
mucin ZANGERLE 3 obtained 30 per cent glucosamine, and NEUBERG and
HEYMANN have shown that the glucosamine is the only carbohydrate
regularly taking part in the structure of these substances. Still there are
reports as to the occurrence of chondroitin-sulphuric acid (or an allied
acid) in pseudomucin or colloid (PANZER), but this is not constant
according to the experience of HAMMARSTEN.

As hydrolytic cleavage products of pseudomucin OTORI obtained,
besides carbohydrate derivatives such as levulinic acid and humus sub-
stances, leucine, tyrosine, glycocoll, aspartic acid, glutamic acid, valeric
acid, arginine, lysine, and guanidine. The quantity of guanidine, it
seems, was greater than that which could be derived from the arginine,
hence this body probably originated from another complex. PREGL*
obtained on the hydrolysis of a colloid, which behaved like paramucin,
no glycocoll and only traces of diamino acids, but otherwise the same
amino-acids as OTORI found, besides alanine, proline, phenylalanine and
tryptophane.

The detection of metalbumin and paralbumin is naturally connected
with the detection of pseudomucin. A typical ovarial fluid containing
pseudomucin is, as a rule, sufficiently characterized by its physical proper-
ties, and a special chemical investigation is necessary only in cases where a
serous fluid contains very small amounts of pseudomucin. The pro-
cedure is as follows: The protein is removed by heating to boiling with
the addition of acetic acid; the filtrate is strongly concentrated and pre-
cipitated by alcohol. The precipitate, a transformation product of
pseudomucin, is carefully washed, with alcohol and then dissolved in water.
A part of this solution is digested with saliva at the temperature of the
body and then tested for glucose (derived from glycogen or dextrin).
If glycogen is present, it will be converted into glucose by the saliva;
precipitate again with alcohol and then proceed as in the absence of

1 K. Mitjukoff, Arch. f. Gynakol., 49.

2 Miiller, Verh. d. Naturf. Gesellsch. in Basel. 12, part 2; Neuberg and Heymann;
Hofmeister's Beitrage, 2. See also Leathes, Arch. f. exp. Path. u. Pharm., 43.

3 Munch, med. Wochenschr., 1900.

4Otori, Zeitschr. f/physiol. Chem., 42 and 43; Pregl, ibid., 58.

OVARIAL CYSTS. 627

glycogen. In this last-mentioned case, first add acetic acid to the solu-
tion of the alcohol precipitate in water so as to precipitate any existing
mucin. The precipitate produced is filtered off, the filtrate treated with
2 per cent HC1 and warmed on the water-bath until the liquid is deep
brown in color. In the presence of pseudomucin this solution gives
TROMMER'S test.

The other protein bodies which have been found in cystic fluids are
serglobulin and seralbumin, peptone (?), mucin, and mucin-peptone (?).
Fibrin occurs only in exceptional cases. The quantity of mineral bodies
on an average amounts to about 10 p. m. The amount of extractive
bodies (cholesterin and urea) and/ai is ordinarily 2-4 p. m. The remaining
solids, which constitute the chief mass, are protein bodies and pseudo-
mucin.

The intraligamentary, papillary cysts contain a yellow, yellowish-
green, or brownish-green, liquid which contains either no pseudomucin
or very little. The specific gravity is generally rather high, 1.032-1.036,
with 90-100 p. m. solids. The principal constituents are the simple
proteins of blood-serum.

The rare tubo-ovarial cysts contain as a rule a watery, serous fluid
containing no pseudomucin.

The parovarial cysts or the CYSTS of the LIG AMENTA LATA may attain
a considerable size. In general, and when quite typical, the contents are
watery, mostly very pale-yellow-colored, water-clear or only slightly
opalescent liquids. The specific gravity is low, 1.002-1.009, and the
solids only amount to 10-20 p. m. Pseudomucin does not occur as a
typical constituent; protein is sometimes absent, and when it does occur
the quantity is very small. The principal part of the solids consists of
salts and extractive bodies. In exceptional cases the fluid may be rich
in protein and may show a higher specific gravity.

In regard to the quantitive composition of the fluid from ovarial
cysts we refer the reader to the work of OERUM.1

E. LUDWIG and R. y. ZEYNEK have investigated the fat from dermoid cysts.
Besides a little arachidic acid, they found oleic, stearic, palmitic, and myristic
acids, cetyl alcohol, and a cholesterin-like substance. In regard to the occurrence
of cetyl alcohol see the work of AMESEDER,2 page 239.

The colloid from a uterine fibroma analyzed by STOLLMANNS contained a
pseudomucin soluble in water, and a colloid (paramucin) insoluble in water, both
of which behaved differently with alcohol as compared with the corresponding
substances from ovarial cysts.

1 Kemiske Studier over Ovariecystevaedsker, etc., Koebenhavn, 1884. See also
Maly's Jahresber., 14; 459.

2Ludwig and v. Zeynek, Zeitschr. f. physiol. Chem., 23; Ameseder, ibid., 52;
Salkowski, Bioch. Zeitschr., 32.

3 Amer. Gynecology, 1903.

628 ORGANS OF GENERATION.

The Ovum.

The small ova of man and mammals cannot, for evident reasons, be
the subject of a searching chemical investigation. Up to the present
time the eggs of birds, amphibians, and fishes have been investigated,
but above all the hen's egg. We will here occupy ourselves with the con-
stituents of this last.

The Yolk of the Hen's Egg. In the so-called white yolk, which
forms the germ with a process reaching to the center of the yolk (latebra),
and forming a layer between the yolk and yolk-membrane, there occurs
protein, nuclein, lecithin, and potassium (LIEBERMANN 1). The occur-
rence of glycogen is doubtful. The yolk-membrane consists of an albu-
minoid similar in certain respects to keratin (LIEBERMANN).

The principal part of the yolk — the nutritive yolk or yellow — is a
viscous, non-transparent, pale-yellow or orange-yellow alkaline emulsion
of a mild taste. The yolk contains vitellin, lecithin, cholesterin, fat, color-
ing-matters, traces of neuridine (BniEGER2), purine bases (MESERNiTZKi3),
glucose in very small quantities, and mineral bodies. The occurrence of
cerebrin and of granules similar to starch (DARESTE 4) has not been posi-
tively proved.

Several enzymes have been found in the yolk, especially a diastatic
enzyme (MULLER and MASUYAMA), a glycolytic enzyme (STEPANEK)
which in the absence of air brings about an alcoholic fermentation of
sugar and in the presence of air forms carbon dioxide and lactic acid,
and finally a proteolytic, a lipolytic, and a chromolytic (?) enzyme

(WOHLGEMUTH 5).

Ovovitellin. This body, which is often considered as a globulin
is in reality a nucleoalbumin. The question as to what relation other
protein substances, which are related to ovovitellin, like the aleuron
grains of certain p ^ds, and the yolk spherules of the eggs of certain fishes
and amphibians, bear to this substance is one which requires further
investigation.

The ovovitellin which has been prepared from the yolk of eggs is not a
pure protein body, but always contains lecithin. HOPPE-SEYLER found
25 per cent lecithin in vitellin. The lecithin may be removed by boiling
alcohol, but the vitellin is changed thereby, and it is therefore probable

1 Pfliiger's Arch., 43.

2 Ueber Ptomaine, Berlin, 1885.

8 Mesernitzki, Biochem. Centralbl., 1, 739.
4 Compt. Rend., 72.

6 Miiller and Masuyama, Zeitschr. f. Biologic, 39; Stepanek, Centralbl. f. Physiol.,
18, 188; Wohlgemuth in Salkowski's Festschrift and Zeitschr. f. physiol. Chem., 44.

OVOVITELLIN. 629

that the lecithin is chemically united with the vitellin (HOPPE-SEYLER 1).
According to OSBORNE and CAMPBELL, the so-called ovovitellin is a mix-
ture of various vitellin-lecithin combinations, with 15 to 30 per cent of
lecithin. The protein substance freed from lecithin is the same in all
these compounds and has the following composition: C 51.24, H 7.16,
N 16.38, S 1.04, P 0.94, 0 23.24 per cent. These figures differ somewhat
from those obtained by GROSS for vitellin prepared by another method
(precipitation with [NH4]2S04), namely, C 48.01, H 6.35, N 14.91-16.97,
P 0.32-0.35, S 0.88, and the composition of ovovitellin is therefore not
positively known. Besides the vitellin GROSS found a globulin coagulat-
ing at 76-77° C. in a solution containing salt, and PLiMMER2 found a
protein which he calls livetin which only contained 0.1 per cent phos-
phorus and which gave more monamino acids but less amide and diamino
nitrogen than vitellin.

On the pepsin digestion of ovovitellin, OSBORNE and CAMPBELL.
obtained a pseudonuclein with varying amounts of phosphorus, 2.52-
4.19 per cent. BUNGES prepared a pseudonuclein by digesting the yolk
with gastric juice, and his pseudonuclein, he claims, is of great impor-
tance in the formation of the blood, and on these grounds he called it
hcematogen. This haematogen has the following composition: C 42.11,
H 6.08, N 14.73, S 0.55, P 5.19, Fe 0.29, and O 31.05 per cent. The
composition of this substance may vary considerably even on using the
same method of preparation.

Vitellin is similar to the globulins in that it is insoluble in water, but
on the contrary soluble in dilute neutral-salt solutions (although the solu-
tion is not quite transparent). It is also soluble in hydrochloric acid of
1 p. m. and in very dilute solutions of alkalies or alkali carbonates. It
is precipitated from its salt solution by diluting with water, and when
allowed to stand some time in contact with water the vitellin is gradually
changed, forming a substance more like the albuminates. The coagu-
lation temperature for the solution containing salt (NaCl) lies between
70 and 75° C., or, when heated very rapidly, at about 80° C. Vitellin
differs from the globulins in yielding pseudonuclein by peptic digestion.
It is not always completely precipitated by NaCl in substance. The
ovovitellin isolated by GROSS gave MOLISCH'S reaction. NEUBERG4
has also split off glucosamine from the yolk and has identified it as nori-

1 Med. chem. Untersuch., 216.

2 Osborne and Campbell, Connecticut Agric. Exp. Station, 23d Ann. Report, New
Haven, 1900; Gross, Zur Kenntn. d. Ovovitellins, Inaug.-Diss. Strassburg, 1899;
Plimmer, Journ. Chem. Soc., London, 93.

3 Zeitschr. f. physiol. Chem., 9, 49. See also Hugounenq and Morel, Compt. Rend.r
140 and 141.

4 Ber. d. d. chem. Gesellsch., 34.

630 ORGANS OF GENERATION.

sosaccharic acid. It is difficult to state whether this glucosamine was
derived from the vitellin or from some other constituent of the yolk.

The principal points in the preparation of ovovitellin are as follows:
The yolk is thoroughly agitated with ether; the residue is dissolved in
a 10-per cent common-salt solution, filtered, and the vitellin precipitated
by adding an abundance of water. The vitellin is now purified by repeat-
edly redissolving in dilute common-salt solutions and precipitating with
water.

Ichthulin, which occurs in the eggs of the carp and other, fishes is, accord-
ing to KOSSEL and WALTER, an amorphous modification of the crystalline body
ichthidin, which occurs in the eggs of the carp. Ichthulin is precipitated on
diluting with water. It was formerly considered as a vitellin. According to
WALTER it yields a pseudonuclein on peptic digestion; and this pseudonuclein
gives a reducing carbohydate on boiling with sulphuric acid. Ichthulin has the
following composition; C 53.42, H 7.63, N 15.63, 0 22.19, S 0.41, P 0.43 percent.
It also contains iron. The ichthulin investigated from codfish eggs by LEVENE
had the composition C 52.44, H 7.45, N 15.96, S 0.92, P 0.65, Fe+0 22.58
per cent, and yielded no reducing substances on boiling with acids. The pure
vitellin isolated by HAMMARSTEN from perch eggs had a similar behavior and
was very readily changed by a little hydrochloric acid so that it was converted
into a typical pseudonuclein. The codfish ichthulin yielded a pseudonucleic acid
with 10.34 per cent phosphorus, but this acid still gave the protein reactions.
MCCLENDEN l has prepared a vitellin from frogs' eggs which he calls batrachiolin.

The yolk also contains albumin, besides vitellin and the above-men-
tioned proteins.

The fat of the yolk of the egg, LiEBERMANN2 claims, is a mixture of
a solid and a liquid fat. The solid fat consists principally of tripalmitin
with some tristearin. On the saponification of the egg-oil LIEBERMANN
obtained 40 per cent oleic acid, 38.04 per cent palmitic acid, and 15.21
per cent stearic acid. The fat of the yolk of the egg contains less carbon
than other fats, which may depend upon the presence of monoglycerides
and diglycerides, or upon a quantity of fatty acid deficient in carbon
(LIEBERMANN). The composition of yolk fat is dependent upon the
food, as HENRI QUES and HANSEN 3 have shown that the fat of the food
passes into the egg.

The phosphatides of the yolk seem to be of various kinds. THIER-
FELDER and STERN have found three different phosphatides. One of
these, which was soluble in alcohol-ether, behaved like lecithin. The
second was soluble with difficulty in alcohol, but readily soluble in ether,
contained 1.37 per cent N and 3.96 per cent P. The third was a diamino

1 Walter, Zeitschr. f. physiol. Chem., 15; Levene, ibid., 32; Hammarsten, Skand.
Arch. f. Physiol., 17; McClenden, Amer. Journ. of Physiol. 25; see also .Plimmer
and Scott, Journ. Chem. Soc., 93.

2 Pfliiger's Arch., 43.

* Skand. Arch. f. Physiol., 14.

LUTEIN. 631

phosphatide, soluble with difficulty in ether, but obtained in crystalline
needles from hot alcohol, and contained 2.77 per cent N and 3.22 per cent
P, and had a melting-point of 160-170° C. FRANKEL and BOLAFFIO l
also found a substance crystallizing from hot alcohol and insoluble in
ether with 2.78 per cent N and 2.18 per cent P. They call this body
neottin and claim that it is a triamino-monophosphatide having the formula
Cs4Hi72N3POi5. BARBIERI has obtained a sulphurized phosphatide
called ovin, containing 1.35 per cent P, 3.66 per cent N and 0.4 per cent S.
The relation of all these bodies to each other must be further studied.

Lutein. With the name lutein we in the past have included several
yellow or orange-red amorphous coloring-matters which occur in the
yellow of the egg, and in several other places in the animal organism;
for instance, in the blood-serum and serous fluids, fatty tissues, milk-
fat, corpora lutea, and in the fat-globules of the retina as well as in dif-
ferent plants (THUDICHUM). Among these bodies belong the crys-
talline substance obtained by ESCHER from the corpora lutea (page 623).
It was difficultly soluble in alcohol but readily soluble in petroleum ether
and showed itself isomeric or perhaps identical with the plant pigment
carotin (C^Hse) analyzed by WILLSTATTER and MIEG. The lutein of
the egg yolk, which is more readily soluble in alcohol and less soluble in
petroleum ether than carotin has also been obtained by WILLSTATTER and
ESCHER in a pure, crystalline form. On analysis it gave the formula
C4oH5e02. As shown by C. A. SCHUNCK the yolk lutein stands in
close relation to the yellow plant pigment, xanthophyll. The formula
given by WILLSTATTER and ESCHER for lutein was in fact the same as
for the xanthophyll, as previously found by WILLSTATTER and MIEG.
These two substances are also similar in other respects; still the melting-
points of the two are different. The carotin and the yolk lutein differ
also by the absorption spectra, which is different in different solvents
as well as by their formulae and different solubilities.2

The relation of the other substances called luteins to each other and
to the yolk lutein is unknown. All are soluble in alcohol, ether, and chloro-
form. They differ from the bile-pigment, bilirubin, in that they are not
separated from their solution in chloroform by water containing alkali,
and also in that they do not give the characteristic play of colors with
nitric acid containing a little nitrous acid, but give a transient blue color.
The luteins withstand the action of alkalies so that they are not changed
when we remove the fats present by means of saponification.

1 Thierfelder and Stern, Zeitschr. f. physiol. Chem., 53; Frankel and Bolaffio,
Bioch. Zeitschr., 9; Barbieri, Compt. Rend., 145.

2Thudichum, Centralbl. f. d. med. Wiss. 1869; Willstatter and Mieg. Ann. d.
Chem., 355 (1907); Willstatter and Escher, Zeitschr. f. physiol. Chem., 64 (1909);
76 (1911); Schunck, see Chem. Centralbl., 1903.

632 ORGANS OF GENERATION.

MALY l found two pigments free from iron in the eggs of a water-spider (Maja
squinado) — one a red (vitellorubiri) and the other a yellow pigment (vitelloluteiri) .
Both of these pigments are colored blue by nitric acid containing nitrous acid
and a beautiful green by concentrated sulphuric acid.

The mineral bodies of the yolk of the egg consist, according to PoLECK,2
of 51.2-65.7 parts soda, 80.5-89.3 potash, 122.1-132.8 lime, 20.7-21.1
magnesia, 11.90-14.5 iron oxide, 638.1-667.0 phosphoric acid, and 5.5-
14.0 parts silicic acid in 1000 parts of the ash. We find phosphoric acid
and lime the most abundant, and then potash, which is somewhat greater
in quantity than the soda. These results are not, however, .quite cor-
rect: first, because no dissolved phosphate occurs in the yolk (LIEBER-
MANN), and secondly, in burning, phosphoric and sulphuric acids are
produced, and these drive away the chlorine, which is not accounted
for in the above analyses.

The yolk of the hen's egg weighs about 12-18 grams. The quan-
tity of water and solids amounts, according to PARKED to 471.9 p. m.
and 528.1 p. m. respectively. Among the solids he found 156.3 p. m.
protein, 3.53 p. m. soluble and 6.12 p. m. insoluble salts. The quantity
of fat, according to PABKE, is 228.4 p. m.; the lecithin, calculated from
the amount of phosphorus in the organic substance of the alcohol-ether
extract, was 107.2 p. m. and the cholesterin 17.5 p. m.

The white of the egg is a faintly yellow albuminous fluid inclosed
in a framework of thin membranes; and this fluid is in itself very liquid,
but seems viscous because of the presence of these fine membranes. That
substance which forms the membranes, and of which the chalaza con-
sists, seems to be a body closely related to horn substances (LIEBER-
MANN) .

The white of egg has a specific gravity of 1.038-1.045, and always
has an alkaline reaction toward litmus. It contains 850-880 p. m. water,
100-130 p. m. protein bodies, and 7 p. m. salts. LEHMANN found a fer-
mentable variety of sugar which SALKOWSKI showed was glucose. C. TH.
MORNER could not find any other sugar in egg-white; the quantity of
glucose as found by MORNER 4 was 3-5 p. m. Besides these one finds
in the white of egg traces of fats, soaps, lecithin and cholesterin.

The white of egg of the Insessores becomes transparent on boiling and acts
in many respects like alkali albuminate. This albumin TARCHANOFF 5 called
" tatcdbumin."

1 Monatshefte f. Chem., 2.

2 Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4, Aufl., 740.

3 Hoppe-Seyler, Med. chem. Untersuch., Heft 2, 209.

4Lehmann, Lehrb. d. physiol. Chem. 2 Aufl. 1855, Bd. 1, s. 271; Bd. 2, s. 312.
Salkowski, Centralbl. f. d. med. Wiss., 31 (1893); Morner, Zeitschr. f. physiol. Chem;
80 (1912).

5 Pfliiger's Arch., 31, 33, and 39.

OVOGLOBULIN. OVALBUMIN. 633

The protein substances of the white of egg behave like glycoproteins,
as they all yield glucosamine. For the globulin and albumin it has not
been proved, nor is it probable, that the glucosamine belongs to the pro-
tein molecule (see page 84). According to the solution and precipita-
tion properties they are similar to the globulins, albumins or proteoses.
The representatives of the first two groups, are ovoglobulin and ovalbumin.
The proteose-like body is ovomucoid.

Ovoglobulin separates in part on diluting the egg-white with water.
It is precipitated upon saturation with magnesium sulphate, or upon
one-half saturation with ammonium sulphate, and coagulates at about
75° C. By repeated solution in water and precipitation with ammonium
sulphate a part of the globulin becomes insoluble (LANGSTEIN). This
also occurs on precipitation by diluting with water or by dialysis, and
it is quite possible that the globulin is a mixture. That portion which
readily becomes insoluble seems to be identical with EICHHOLZ'S gly-
coprotein or OSBORNE and CAMPBELL'S ovomucin. LANGSTEIN obtained
11 per cent of glucosamine from the soluble ovoglobulin. The total
quantity of globulins, according to DILLNER, is about 6.7 per cent of
the total protein substances, and this corresponds with the recent deter-
minations of OSBORNE and CAMPBELL. In regard to the probable occur-
rence of several globulins in the white of the egg there are the determina-
tions of CORIN and BERARD as well as of LANGSTEIN/ but they have
not led to any positive conclusions.

Ovalbumin. The so-called albumin of the egg-white is undoubtedly
a mixture of at least two albumin-like proteins. Opinions differ con-
siderably in regard to the number of these proteins (BONDZYNSKI and
ZOJA, GAUTIER, BECHAMP, CORIN and BERARD, PANORMOFF, and others).
Since HOFMEISTER has been able to prepare ovalbumin in a crystalline
form, and since HOPKINS and PINKUS 2 have shown that not more than
one-half of the ovalbumin can be obtained in such a form, OSBORNE and
CAMPBELL have isolated two different ovalbumins or principal fractions;
the crystallizable they call ovalbumin and the non-crystallizable con-
albumin. The two fractions have only a slight variation in elementary
composition; the conalbumin coagulates between 50-60° C., nearer to
60° C., and the ovalbumin at 64° C. or at a higher temperature. There
are no conclusive investigations as to whether the non-crystallizable

1 Langstein, Hofmeister's Beitrage, 1; Eichholz, Journ. of Physiol., 23; Osborne
and Campbell, Connecticut Agric. Exp. Station., 23d Ann. Report, New Haven, 1900;
Dillner, Maly's Jahresber., 15; Corin and Berard, ibid., 18.

2 Hofmeister, Zeitschr. f. physiol. Chem., 14, 16, and 24, Gabriel, ibid., 15; Bond-
zynski and Zoja, ibid., 19; Gautier, Bull. Soc. chim., 14; Be"champ, ibid., 21; Corin
and Berard, 1. c.; Hopkins and Pinkus, Ber. d. d. chem. Gesellsch., 31, and Journ. of
Physiol., 23; Osborne and Campbell, 1. c.; Panormoff, Maly's Jahresber., 27 and 28.

634 ORGANS OF GENERATION.

conalbumin is a mixture or not, and the question concerning the unity
of the crystallizable ovalbumin is also disputed. According to BOND-
ZYNSKI and ZOJA, crystallizable ovalbumin is a mixture of several albumins
having somewhat different coagulation temperatures, solubilities, and
specific rotations, while HOFMEISTER and LANGSTEIN on the contrary
believe that crystallizable ovalbumin is a unit. The reports as to the
specific rotation of the different fractions unfortunately differ, and the
elementary analyses have also given no positive results, as a variation
of 1.2-1.7 per cent has been observed in the quantity of sulphur. Accord-
ing to the consistent analyses of OSBORNE and CAMPBELL and of LANG-
STEIN, the conalbumin contains about 1.7 per cent sulphur and about
16 per cent nitrogen, while the ovalbumin contains on an average about
15.3 per cent nitrogen. LANGSTEIN 1 obtained 10-11 per cent glucosa-
mine from ovalbumin and about 9 per cent from conalbumin. The
ovalbumin, like the conalbumin, has the properties of the albumins in
general, but differs from seralbumin in that the specific, rotation
is lower. It is quickly made insoluble by alcohol and is precipitated
by a sufficient quantity of HC1, but dissolves in an excess of acid with
greater difficulty than the seralbumin. The products isolated by
ABDERHALDEN and PREGL 2 on the hydrolysis of ovalbumin do not show
anything of special interest.

As in the past certain doubts have existed as to the purity and chem-
ical unity of the ovalbumins, or also of the crystalline ovalbumin, so now
this doubt has become still stronger since ovalbumin has been pre-
pared partly free from phosphorus and partly with a variable phos-
phorus content of 0.1-3.06 per cent (KAAS, WILLCOCK and HARDY3).

In preparing crystalline ovalbumin, mix, according to HOFMEISTER,
the beaten white of egg free from foam with an equal volume of a saturated
ammonium-sulphate solution, filter off the globulin, and allow the filtrate
to evaporate slowly in thin layers at the temperature of the room. After
a time the masses which separate out are dissolved in water, treated
with ammonium sulphate-solution until they begin to get cloudy, and
are allowed to stand. After repeated recrystallization the mass is either
treated with alcohol, which makes the crystals insoluble, or they are
dissolved in water and purified by dialysis. From these solutions the
proteid does not crystallize again on spontaneous evaporation. (See also
page 633, footnote 2, for the HOPKINS and PINKUS method.) WILL-
COCK 4 has recently found that magnesium sulphate can also be used in
the crystallization of ovalbumin.

1 Zeitschr. f. physiol. Chem., 31.

2 Ibid., 46.

3Kaas, Monatsh. f. Chem., 27; Willcock and Hardy, cited from Chem. Centralbl..
1907, 2, 821.

4 Journ. of Physiol., 37.

OVOMUCOID. 635

Conalbumin can be removed from the filtrate, after the complete
crystallization of the ovalbumin, by removing the sulphate by means of
dialysis and coagulating by heat.

GAUTIER x found a fibrinogen-like substance in the white of egg, which was
changed into a fibrin-like body by the action of a ferment.

Ovomucoid. This substance, first observed by NEUMEISTER and
considered by him as a pseudopeptone, and then later studied by SALKOW-
SKI, is, according to C. TH. MORNER^ a mucoid with 12.65 per cent
nitrogen and 2.20 per cent sulphur. Ovomucoid exists in hens' eggs
to the extent of about 12 per cent of the total solids.

A solution of ovomucoid is not precipitated by mineral acids nor by
organic acids, with the exception of phosphotungstic acid and tannic
acid. It is not precipitated by metallic salts, but basic lead acetate and
ammonia render it insoluble. Ovomucoid is thrown down by alcohol,
but sodium chloride, sodium sulphate, and magnesium sulphate give
no precipitates either at the ordinary temperature or when the salts are
added to saturation at 30° C. Its solutions are not precipitated by an
equal volume of a saturated solution of ammonium sulphate, but are
precipitated on adding more salt thereto. The substance is not pre-
cipitated on boiling, but the part which has become insoluble in cold
water and which has been dried, is dissolved by boiling water. ZANETTI
has prepared glucosamine on splitting ovomucoid with concentrated
hydrochloric acid, and SEEMANN found that the quantity of glucosamine
in ovomucoid was 34.9 per cent.3

Ovomucoid may be prepared by removing all the proteins by boil-
ing with the addition of acetic acid, and then concentrating the filtrate
and precipitating with alcohol. The substance is purified by repeated
solution in water and precipitation with alcohol.

PANORMOW believes that the eggs of other birds, such as the pigeon and duck,
contain a special protein in the egg-white, which is not identical with that of the
hen's egg. WORMS 4 has prepared a crystalline albumin from the white of the
turkey eggs which contained 15.37 per cent N, 1.6 per cent S and had a specific
rotation of (<*)D = -34.9°.

The mineral bodies of the white of egg have been analyzed by
POLECK and WEBER.S They found in 1000 parts of the ash: 276.6-

1 Compt. Rend., 135.

2R. Neumeister, Zeitschr. f. Biologic, 27; Salkowski. Centralbl. f. d. med. Wis-
sensch., 1893, 513 and 706; C. Morner, Zeitsch. f. physiol. Chem., 18 and 80. See
also Langstein, Hofmeister's Beitrage, 3 (literature).

3 Zanetti, Chem. Centralbl., 1898, 1; Seemann, cited from Langstein, Ergebnisse
der Physiol., 1, Abt. 1, 86.

4 Panormow, see Bioch. Centralbl., 5; Worms, cited from Chem. Centralbl., 1906,
2, 1508.

5 Cited from Hoppe-Seyler, Physiol. Chem., 778.

636 ORGANS OF GENERATION.

284.5 grams potash, 235.6-329.3 soda, 17.4-29.0 lime, 17-31.7 magnesia,
4.4-5.5 iron oxide, 238.4-285.6 chlorine, 31.6-48.3 phosphoric acid (P2O5),
13.2-26.3 sulphuric acid, 2.8-20.4 silicic acid, and 96.7-116.0 grams carbon
dioxide. Traces of fluorine have also been found (NicKLES1). The
white of egg contains, as compared with the yolk, a greater amount of
chlorine and alkalies and a smaller amount of lime, phosphoric acid, and
iron.

The Shell-membrane and the Egg-shell. The shell-membrane con-
sists, as above stated (page 112), of a keratin substance. The shell con-
tains very little organic substance, 36-65 p. m. The principal mass, more
than 900 p. m., consists of calcium carbonate; besides this there are very
small amounts of magnesium carbonate and earthy phosphates.

The diverse coloring of birds' eggs is due to several different coloring-matters.
Among these we find a red or reddish-brown pigment called " oorodein " by
SoRBY,2 which is perhaps identical with hsematoporphyrin. The green or blue
coloring-matter, SORBY'S oocyan, seems, according to LIEBERMANN 3 and KRUKEN-
BERG,4 to be partly biliverdin and partly a blue derivative of the bile-pigments.

The eggs of birds have a space at their blunt end filled with gas; this
gas contains on an average 18.0-19.9 per cent oxygen (HuFNER5).

The weight of a hen's egg varies between 40-60 grams and may some-
times reach 70 grams. The shell and shell-membrane together, when
carefully cleaned, but still in the moist state, weigh 5-8 grams. The
yolk weighs 12-18 and the white 23-34 grams, or about double. The
entire egg contains 2.8-7.5, or average 4.6, milligrams of iron oxide, and
the quantity of iron can be increased by food rich in iron (HARTUNG 6) .

The white of the egg of cartilaginous and bony fishes contains only traces of
true albumin, but consists, at least in many fishes, of mucin substance; and the
cover of the frog's egg also consists, according to GIACOSA, of mucin. The eggs
of the river-perch contain, HAMMARSTEN 7 claims, mucin in the envelope in the
unripe state and only mucinogen in the ripe state. The crystalline formations
(yolk-spherules, or dotterpldttchen) which have been observed in the egg of the
tortoise, frog, ray, shark, and other fishes, and which are described by VALEN-
CIENNES and FREMY under the names emydin, ichthin, ichthidin, and ichthulin,
seem, as above stated in connection with ichthulin, to consist mainly of phos-
phoglycoproteins. The klupeovin obtained by HUGOUNENQ 8 from the herrings'
eggs and from which he obtained the three so-called hexone bases and abundant

1 Compt. Rend., 43.

2 Cited from Krukenberg, Verb. d. phys.-chem. Gessellsch. in Wiirzburg, 17.
8 Ber. d. deutsch. chem. Gesellsch., 11.

•1. c.

6 Arch. f. (Anat. u). Physiol., 1892.
«Zeitschr. f. Biol., 43.

7Giacosa, Zeitschr. f. physiol. Chem., 7; Hammarsten, Skand. Arch. f. Physiol., 17.
8 Valenciennes and Fr&ny. cited from Hoppe-Seyler, Physiol. Chem., p. 77;
Hugounenq, Bull. soc. chim. (3), 33, and Compt. Rend., 143.

THE EGG. 637

monamino-acids, especially leucine, but not glycocoll or glutamic acid, is to
all appearances not a unit body. The eggs of the river-crab and the lobster
contain the same pigment as the shell of the animal. This pigment, called
cyanocrystallin, becomes red on boiling in water.

C. MORNER l has isolated a substance which he calls percaglobulin, from the
unripe eggs of the river-perch. It is a globulin and has a strong astringent taste.
Especially striking is its property of precipitating certain glycoproteins, such as
ovomucoid and ovarial mucoids, and polysaccharides, such as glycogen, gum,
tragacanth and starch-paste, and of being precipitated by them. Percaglobulin
could not be obtained by MORNER from the eggs of the sea-bass.

In fossil eggs (of APETNODYTES, PELECANUS, and HALL.EUS) in old guano deposits,
a yellowish-white, silky, laminated compound has been found which is called
guanovulit, (NH4)2S04-h2K2S04+3KHS04+4H20, and which is easily soluble in
water, but is insoluble in alcohol and ether.

Those eggs which develop outside of the mother-organism must con-
tain all the elements necessary for the young animals. One finds, there-
fore, in the yolk and white of the egg an abundant quantity of protein
bodies of different kinds, and especially phosphorized proteins in the
yolk. Further, we also find abundance of phosphatides in the yolk,
which seem to occur habitually in all developing cells. KATO and BLEIB-
TREU 2 found glycogen in the eggs of the frog which during the spawning
season increased at the cost of the liver glycogen. Besides this the egg
is very rich in fat, which doubtless is important as a source of supply
for nourishment and in maintaining respiration for the embryo. The
cholesterin or at least the lutein can hardly have a direct influence on
the development of the embryo. The egg also seems to contain the
mineral bodies necessary for the development of the young animal.
The lack of phosphoric acid is compensated by an abundant amount of
phosphorized organic substance, and the nucleoalbumin containing
iron, from which the hsematogen (see page 629) is formed, is doubtless, as
BUNGE claims, of great importance in the formation of the haemoglobin
containing iron. The silicic acid, necessary for the development of the
feathers, is also found in the egg.

During the period of incubation the egg loses weight, due chiefly to
loss of water. The quantity of solids, especially the fat and the proteins,
diminishes, and the egg gives off carbon dioxide, but TANGL disproves
the older claim of LIEBERMANN 3 that nitrogen or a nitrogenous substance
is given off. On the contrary a corresponding absorption of oxygen
takes place, and it is found that during incubation a respiratory exchange
of gases occurs.

As BOHR and HASSELBALCH have shown by exact investigations, the
elimination of carbon dioxide is very small in the first days of incuba-

1 Zeitschr. f. physiol. Chem., 40 and 58.

*Kato, Pfluger's Arch. 132; Bleibtreu, ibid., 132 (1910).

* Tangl and v. Mituch, Pfluger's Arch., 121; Liebermann, ibid., 43.

638 ORGANS OF GENERATION.

tion; on the fourth day the carbon-dioxide production gradually increases,
and after the ninth day it augments in the same proportion as the weight
of the foetus. Calculated upon 1 kilogram weight for one hour it is,
from the ninth day on, about the same as in the full-grown hen. HASSEL-
BALCH 1 has also shown that the fertilized hen's egg not only gives off
nitrogen the first five or six hours of incubation, but also some oxygen,
and that we are here dealing with an oxygen production which runs
parallel with the cell-division. It is not known whether this oxygen
formation connected with the life of the cell is a fermentative or a so-
called vital process.

While the quantity of dry substance in the egg during this period
always decreases, the quantity of mineral bodies, protein, and fat always
increases in the embryo. The increase in the amount of fat in the
embryo depends, in great part upon a taking up of the nutritive yolk
in the abdominal cavity. PLIMMER and SCOTT 2 have observed in the incu-
bation of the hen's egg, that a rapid diminution of phosphorized substances
soluble in ether takes place, while at the same time an increase in the
inorganic phosphorus is found in the chick.

The weight of the shell and the quantity of lime-salts contained therein
do not remain unchanged, according, to the recent investigations of
TANGL.S The egg-shell (lime shell; and shell-membrane) of a hen's egg
weighing 60 grams loses (calculated on the dry) during incubation about
0.4 gram, of which 0.15 gram is calcium and 0.2 gram is organic substance.

A very complete and careful chemical investigation on the develop-
ment of the embryo of the hen has been made by LiEBERMANN.4 From
his researches we may quote the following: In the earlier stages of the
development, tissues very rich in water are formed, but upon the con-
tinuation of the development the quantity of water decreases. The
absolute quantity of the bodies soluble in water increases with the develop-
ment, while their relative quantity, as compared with the other solids,
continually decreases. The quantity of the bodies soluble in alcohol
quickly increases. A specially important increase is noticed in the fat,
whose quantity is not very great even on the fourteenth day, but after
that it becomes considerable. The quantity of protein bodies and albu-
minoids soluble in water grows continually and regularly in such a way
that their absolute quantity increases, while their relative quantity
remains nearly unchanged. LIEBERMANN found no gelatin in the

1 Bohr and Hasselbalch, Maly's Jahresber., 29; Hasselbalch, Skand. Arch. f.
Physiol., 13.

2 Journ. of Physiol., 38.

3 Tangl with Hammerschlag, Pfliiger's Arch., 121.
M. c.

DEVELOPMENT OF THE CHICK EMBRYO. 639

embryo of the hen. The embryo does not contain any gelatin-forming
substance until the tenth day, and from the fourteenth day on it contains
a body which, when boiled with water, gives a substance similar to chon-
drin. A body similar to mucin occurs in the embryo when about six
days old, but then disappears. The quantity of haemoglobin shows a
continual increase compared with the weight of the body. LIEBERMANN
found that the relation of the haemoglobin to the body weight was 1:728
on the eleventh day and 1:421 on the twenty-first day.

By means of BERTH BLOT'S thermometric methods TANGL l has
determined the chemical energy present at the beginning and end of
the development of the embryo of the sparrow's and hen's eggs. The
difference was considered as work of development. He found that the
chemical energy necessary for the development of each gram of ripe hen's
embryo (Plymouth) was equal to 0.805 Cal. This energy originated
chiefly from the fat. Of the total chemical energy utilized, about 70
per cent was used for the embryo and about 30 per cent remained in the
yolk. Of the utilized energy about two-thirds was used in the con-
struction of the embryo and about one-third transformed into other
forms of energy as work of development.

By their investigations on the development of the trout egg, TANGL
and FARKAS 2 have found that the loss in weight of each egg which had
an average weight of 88 milligrams was 4.9 milligrams during the 42
days of incubation, of which 4.11 milligrams was water and 0.722 milli-
gram dry substance with 0.367 milligram C. The eggs lose no nitro-
gen and no fat. The fat content increases a little, and indeed, as these
authors believe, at the expense of the proteins. The chemical energy
used during development was 6.68 gram-calories.

The highly interesting investigations made by LOEB upon the fer-
tilization of the eggs of lower sea-animals will be discussed in this con-
nection. According to these experiments after the fertilization of the
egg by means of a sort of cytolysis small drops of a colloid substance
form on the surface of the egg. These drops enlarge in volume and
conglomerate to a continuous mass, while its surface hardens to a tight,
continuous membrane — the fertilization membrane. The process of
membrane formation is in fact the essential step in the fertilization.
Besides, by spermatozoa, the membrane formation is caused by different
actions. For many eggs all that is necessary is the artificial calling
forth of the processes for the membrane formation in order that the
egg shall develop to normal larvse (for example the eggs of the star
fish and of certain worms) . In other cases, for example the sea-urchin,
Strongylocentrotus, a second action is necessary for the production of

i Pfliiger's Arch., 93 and 121. 2 Ibid., 104.

640 ORGANS OF GENERATION.

normal larvae. The principal points in the treatment of such eggs are
the following.

The formation of the fertilization membrane can be brought about
by placing the eggs in sea water which has been faintly acidified with
a fatty acid, for example with butyric acid, and after 1J to 2 minutes
placed again in sea-water. The formation of the membrane now takes
place. The oxyacids and especially the inorganic acids are less active,
than the fatty acids. The H-ions are without effect in this acid action
and LOEB explains the action by the introduction of the undissociated
molecules into the egg. Parallel with the membrane formation chemical
processes begin, among which we must especially mention oxidations.
These processes, if they proceed undisturbed, especially at 15° or above,
lead quickly to the death of the egg. This can, nevertheless, be prevented
if the oxidation processes are inhibited 40-60 minutes after the mem-
brane formation by removing the oxygen or by the addition of some
potassium cyanide. In this process probably certain injurious substances
for the egg are destroyed. If eggs treated in this way are placed in
sea-water after 2-3 hours they develop in a normal manner.

The membrane formation can also be brought about in other ways
besides by the action of acids, for example by treating the egg with saponin,
solanin, digitalin, soaps and fat dissolving substances such as amylene,
benzene, toluene, chloroform, ether and alcohol. The sea-urchin egg is
also excited to membrane formation by the serum of certain animals.
Alkalies and elevation of temperature can also cause the formation of
membrane.

On the other hand the chemical processes, which, when not prevented,
lead to the death of the egg, can also be inhibited by placing the eggs in
a hypertonic solution (50 cc. sea-water and 8 cc. 2.5 normal NaCl)
about one hour after the artificial membrane has been formed and then
after 20-50 minutes placing them in sea-water again.

According to LOEB the artificial fertilization of the sea-urchin's egg
depends upon two special actions, of which the first brings about the for-
mation of membrane with oxidation processes by means of cytolysis
while the second gives the direction of these oxidation processes necessary
for the maintenance of life.

The non-fertilized, ripe egg, as the investigations of LOEB on star-
fish have shown, dies in 4-6 hours at sufficiently high temperatures. The
death of the egg can, nevertheless, be prevented if oxygen is removed
from the egg or the oxidation inhibited by the addition of traces of potas-
sium cyanide. If the ripe egg is fertilized by spermatozoa then it remains
alive although the process of fertilization, as WARBURG 1 found, causes

1 Zeitschr. f. physiol. Chem., 57, 60, 66.

PLACENTA. 641

a considerable rise in the oxidation. For this reason LOEB believes that
the spermatozoa save the life of the egg by bringing membrane forming
substances to the egg, but also other substances, which remove or make
inert a harmful substance or condition complex of the unfertilized egg,
so that even now the increased oxidation cannot have any harmful effect.1

The enzymes of the sea-urchin suffer an increase in natural as well
as in artificial fertilization as JACOB Y2 has shown that glycyltryptophane
is split after fertilization but not before.

The placenta has recently been the subject of several investigations.
This tissue contains a protein which coagulates at 60-65° C. (BOTTAZZI
and DELFINO) whose relation to the nudeoprotein, found by others, is
not clear. The protein found by SAVAR£ contained 0.45 per cent phos-
phorus. The nucleic acid studied by KiKKOJi,3 which is very similar to
the thymus nucleic acid, originates from this nudeoprotein. Glycogen
occurs regularly in the placenta, and MOSCATI believes the human pla-
centa contains 5 p. m. glycogen. After removal the glycogen diminishes,
and after 24 hours it has disappeared. According to LOCHHEAD and
CRAMER4 the quantity of glycogen in the placenta is not increased by
food rich in carbohydrate. In the fcetus (rabbits) the above authors
found that the placenta is a storage organ for glycogen until the second
half of the gestation period, when the liver begins to functionate in this
direction. From this time on the quantity of glycogen in the placenta
diminishes.

Enzymes of various kinds, proteolytic as well as lipolytic (mono-
butyrase), amylases and oxidases have been found in the placenta.5
In the edges of the placenta of the bitch and of cats, an orange-colored,
crystalline pigment (bilirubin) and a green, amorphous pigment, whose
relation to biliverdin is not clear, have been found.6

From the cotyledons of the placenta in ruminants a white or faintly rose-colored
creamy fluid, the uterine milk, can be obtained by pressure. It is alkaline in

1 A complete review of the investigations of Loeb and his collaborators, with the
literature can be found in Vorlesungen iiber die Dynamik der Lebenserscheinungen,
Leipzig, 1906, s. 239. See also liber den chemischen Charakter des Befruchtungsvor-
ganges, Leipzig, 1908; Zeitschr. f. physik. Chem. 70, 220 (1910), Arch. f. Entwickelungs-
mech., 31, 658 (1910).

2Bioch. Zeitschr., 26, 333 (1910).

8 Bottazzi and DelGno, Centralbl. f. Physiol., 18, 114; Savare, Hofmeister's, Beitrage,
11; Kikkoji, Zeitschr. f. physiol. Chem., 53.

4 Moscati, Zeitschr, f. physiol. Chem., 53; Lochhead and Cramer, Proc. Roy. Soc.,
80 B. (1908).

6Ascoli, Centralbl. f. Physiol., 16; Raineri, Bioch. Centralbl., 4, 428; Bergell and
Liepmann, Munch, med. Wochenschr., 1905; Savare, Hofmeister's Beitrage, 9; Bergell
and Falk, Munch, med. Wochenschr., 55.

6 See Etti, Maly's Jahresber., 2, 287, and Preyer, Die Blutkristalle, Jena, 1871.

642 ORGANS OF GENERATION.

reaction, but quickly becomes acid. Its specific gravity is 1.033-1.040. It con-
tains as form-elements fat-globules, small granules, and epithelium-cells. There
have been found 81.2-120.9 p. m. solids, 61.2-105.6 p. m. protein, about 10 p. m.
fat, and 3.7-8.2 p. m. ash in the uterine milk.

The fluid occurring in the so-called GRAPE-MOLE (MOLA racemosa) has a low
specific gravity, 1.009-1.012, and contains 19.4-26.3 p. m. solids with 9-10 p. m.
proteia bodies and 6-7 p. m. ash.

The amniotic fluid in women is thin, whitish, or pale yellow; some-
times it is somewhat yellowish-brown and cloudy. White flakes separate.
The form-elements are mucus-corpuscles, epithelium-cells, fat-drops, and
lanugo hair. The odor is stale, the reaction neutral or faintly alkaline.
The specific gravity is 1.002-1.028.

The amniotic fluid contains the constituents of ordinary transudates.
The amount of solids at birth is scarcely 20 p. m. In the earlier stages of
pregnancy the fluid contains more solids, especially proteins. Among
the protein bodies, WEYL found one substance similar to vitellin, and with
great probability also seralbumin, besides small quantities of mucin.
Enzymes of various kinds (pepsin, diastase, thrombin, lipase) occur,
according to BONDI. Sugar is regularly found in the amniotic fluid of
cows, but not in human beings. In the ox, pig, and goat GURBER and
GRUNBAUM also found fructose. The human amniotic fluid also contains
some urea, uric acid, allantoin and creatinine (AMBERG and ROWNTREE).
The quantity of these may be increased in hydramnion (PROCHOWNICK,
HARNACK), which depends on an increased secretion by the kidneys and
skin of the foetus. Lactates are doubtful constituents of the amniotic
fluid. The quantity of urea in the amniotic fluid, is, according to PRO-
CHOWNICK, 0.16 p. m. In the fluid in hydramnion PROCHOWNICK and
HARNACK found, respectively, 0.34 and 0.48 p. m. urea. The principal
mass of the solids consists of salts. The quantity of chlorides (NaCl) is
5.7-6.6 p. m. The molecular concentration of the amniotic fluid is some-
what lower than that of the blood, which is no doubt due to a dilution
by the fcetal urine (ZANGEMEISTER and MEISSL *).

l, Arch. f. (Anat. u.) Physiol., 1876; Bondi, Centralbl. f. GynakoL, 1903;
Prochownick, Arch. f. Gynak., .11, also Maly's Jahresber., 7, 155; Harnack, Berlin.
klin. Wochenschr., 1888, No. 41; Zangemeister and Meissl, Munch, med. Wochenschr.
1903; Giirber and Grtinbaum, ibid., 1904; Amberg and Rowntree, cited from Bioch.
Centralbl., 10, 237.

CHAPTER XIII.
MILK.

THE chemical constituents of the mammary glands have been little
studied. The cells are rich in protein and nucleoproteins. Among the
latter we have one that yields pentose and guanine, on boiling with dilute
mineral acids, but no other purine base. This compound protein, inves-
tigated by ODENIUS, contains as an average the following: 17.28 per
cent N, 0.89 per cent S, and 0.277 per cent P. Besides this compound
proteid we have at least one other, as MANDEL and LEVENE and LOEBISCH l
have isolated a nucleic acid from the mammary gland, which, like the
thymonucleic acids, yielded adenine, guanine, thymine, and cytosine.
This nucleic acid also gave the pentose reactions and yielded an abundance
of levulinic acid. Besides this nucleic acid, MANDEL and LEVENE isolated
from the glands a glucothionic acid with 2.65 per cent S and 4.38 per
cent N. Among the cleavage products of the nucleoprotein MANDEL 2
obtained no glycocoll, and the products of hydrolysis show a great cor-
respondence with those of casein. We cannot state what relation the
above-mentioned nucleic acids and the glucothionic acid bear to the
not well-known constituent of the glands found by BERT and by THIER-
FELDER and which yields a reducing substance when boiled with dilute
acids.

It is to be expected that these bodies are steps in the formation of
milk-sugar; still we have no point of support for such an assumption,
and recent investigations seem to indicate that the milk-sugar is produced
in the glands by a transformation of the sugar of the blood. Fat seems,
at least in the secreting glands, to be a never-failing constituent of the
cells, and this fat may be observed in the protoplasm as large or small
globules similar to milk-globules. The extractive bodies of the mam-
mary glands have been little investigated, but among them are found
considerable amounts of purine bases. The mammary glands also
contain enzymes, among which we especially mention : catalase,

1 Odenius, Maly's Jahresber., 30; Mandel and Levene, Zeitschr. f. physiol. Chem.,
46; Loebisch, Hofmeister's Beitrage, 8.

'Mandel and Levene, Zeitschr. f. physiol. Chem., 45, Mandel, Bioch. Zeitschr., 23.

643

644 MILK.

peroxidase and a proteolytic enzyme which, according to HILDEBRANDT/
occurs to a much greater extent in the active gland as compared with
the inactive one.

As human milk and the milk of animals are essentially of the same
constitution, it seems best to speak first of the one most thoroughly
investigated, namely, cow's milk, and then of the essential properties
of the remaining important kinds of milk.2

Cow's Milk.

Cow's milk, like every- other kind, forms an emulsion which consists
of very finely divided fat suspended in a solution consisting principally
of protein bodies, milk-sugar, and salts. Milk is non-transparent, white,
whitish-yellow, or in thin layers somewhat bluish-white, of a faint, insipid
odor and mild, faintly sweetish taste. The specific gravity is 1.028
to 1.0345 at 15° C. The freezing-point is -0.54-0.59° C., average
—0.563° C., and the molecular concentration 0.298.

The reaction of perfectly fresh milk is generally amphoteric toward
litmus. The extent of the acid and alkaline part of this amphoteric
reaction has been determined by different investigators, especially
THORNER, SEBELIEN, and COURANT.S The results differ with the indi-
cators used, and moreover the milk from different animals, as well as
that from the same animal at different times during the lactation period,
varies slightly. COURANT determined the alkaline part by N/10 sul-
phuric acid, using blue lacmoid as indicator, and the acid part by N/10
caustic soda, using phenolphthalein as indicator. He found, as an average
for the first and last portions of the milking of twenty cows, that 100
cc. milk had the same alkaline reaction toward blue lacmoid as 41 cc.
N/10 caustic soda, and the same acid reaction toward phenolphthalein
as 19.5 cc. N/10 sulphuric acid. The actual reaction of cow's milk,
which follows from the electrometric estimation, is, on the contrary,
FoA*4 claims, nearly neutral, like the reaction of animal fluids and
tissues in general.

Milk gradually changes when exposed to the air, and its reaction
becomes more and more acid. This depends on a gradual transforma-
tion of the milk-sugar into lactic acid, caused by micro-organisms.

1 Bert, Compt. Rend., 98; Thierfelder, Pfliiger's Arch., 34, and Maly's Jahresber.,
13; Hildebrandt, Hofmeister's Beitrage, 5.

2 A very complete reference to the literature on milk may be found in Raudnitz's
" Die Bestandteile der Milch/' in Ergebnisse der Physiol., 2, Abt. 1. The literature
of the last few years may be found in the references by Raudnitz, Monatsschrift f.
Kinderheilkunde.

8 Thorner, Maly's Jahresber., 22; Sebelien, ibid., Courant; Pfliiger's Arch., 50.
4 Compt. rend. soc. biolog. (58), 59, 51.

COW'S MILK. 645

Perfectly fresh amphoteric milk does not coagulate on boiling, but
forms a pellicle consisting of coagulated casein and lime-salts, which
rapidly re-forms after being removed. After a sufficiently strong spon-
taneous formation of acid it coagulates on boiling, and lastly, when the
formation of lactic acid is sufficient, it coagulates spontaneously at the
ordinary temperature, forming a solid mass. It may also happen, espe-
cially in the warmth, that the casein-clot contracts and a yellowish or
yellowish-green acid liquid (acid whey) separates.

Milk may undergo various fermentations. Lactic-acid fermentation, brought
about by HUPPE'S lactic-acid bacillus and also other varieties, takes first place.
In the spontaneous souring of milk we generally consider the formation of lactic
acid as the most essential product, but a formation of succinic acid may also take
place, and in certain bacterial decompositions of milk, succinic acid and no lactic
acid is formed. The materials from which these two acids are formed are lactose
and lactophosphocarnic acid. Besides the lactic acids, the optically inactive
as well as the dextro and levo acids, and succinic acid, volatile fatty acids, such
as acetic acid, butyric acid, and others, may be formed in the bacterial decompo-
sition of milk.

Milk sometimes undergoes a peculiar kind of coagulation, being converted
into a thick, ropy, slimy mass (thick milk). This conversion depends upon a
peculiar change in which the milk-sugar is made to undergo a slimy transforma-
tion. This transformation, which requires further investigation, is caused by
special micro-organisms.

If the milk is sterilized by heating, and contact with micro-organisms
prevented, the formation of lactic acid may be entirely stopped. The
production of acid may also be prevented, at least for sometime, by many
antiseptics, such as salicylic acid, thymol, boric acid, and other bodies.

If freshly drawn amphoteric milk is treated with rennet, it coagulates
quickly, especially at the temperature of the body, to a solid mass (curd)
from which a yellowish fluid (sweet whey) is gradually pressed out. This
coagulation occurs without any change in the reaction of the milk, and
therefore it is distinct from the acid coagulation.

In cow's milk we find as form-elements a few colostrum corpuscles
(see Colostrum) and a few pale nucleated cells. The number of these
form-elements is very small compared with the immense amount of the
most essential form-constituents, the milk-globules.

The Milk-globules. These consist of extremely small drops of fat
whose number is, according to WoLL,1 1.06-5.75 millions in 1 c.mm.,
and whose diameter is 0.0024-0.0046 rnm. and 0.0037 mm. as an average
for different kinds of animals. It is unquestionable that the milk-globules
contain fat, and we consider it as positive that all the milk-fat exists in
them. Another disputed question is whether the milk-globules consist
entirely of fat or whether they also contain protein.

xOn the Conditions Influencing the Number and Size of Fat-globules in Cow's
Milk, Wisconsin Exp. Station, 6, 1892.

646 MILK.

The observations of AscHERSON1 show that drops of fat, when dropped in an
alkaline protein solution, are covered with a fine albuminous coat, a so-called haptogen-
membrane. As milk on shaking with ether does not give up its fat, or only very
slowly in the presence of a great excess of ether, and as this takes place very readily
after the addition of acids or alkalies, which dissolve proteins, it was formerly
thought that the fat-globules of the milk were enveloped in a protein coat. A
true membrane has not been detected; and since, when no means of dissolving
the protein is resorted to— for example, when the milk is precipitated by carbon
dioxide after the addition of very little acetic acid, or when it is coagulated by
rennet — the fat can be very easily extracted by ether, the theory of a special albu-
minous membrane for the fat-globule has been generally abandoned. The observa-
tions of QUINCKE 2 on the behavior of the fat-globules in an emulsion prepared
with gum have led, at the present time, to the conclusion that each fat-globule
in the milk is surrounded by a stratum of casein solution held by molecular attrac-
tion, and this prevents the globules from uniting with each other. Everything
that changes the physical condition of the casein in the milk or precipitates it
must necessarily help the solution of the fat in ether, and it is in this way that the
alkalies, acids, and rennet act.

V. STORCH has shown, in opposition to these views, that the milk-
globules are surrounded by a membrane of a special slimy substance.
This substance is very insoluble, contains 14.2-14.79 per cent nitrogen,
and yields a sugar, or at least a reducing substance, on boiling with
hydrochloric acid. It is neither casein nor lactalbumin, but it seems to
all appearances to be identical with the so-called " stroma substance "
detected by RADENHAUSEN and DANILEWSKY. STORCH was able to
show, by staining the fat-globules with certain dyes, that this substance
enveloped them like a membrane. Recently VOLTZ has given further
proofs of the view that the fat-globules probably have a membrane,
which in his opinion is a very labile formation of variable composition,
and BAUER has also given further proofs for the assumption of a mem-
brane. DROOP-RICHMOND and BONNEMA,S on the other hand, present
several deductions conflicting with STORCH'S theory. If STORCH'S observa-
tion that the purified fat-globules contain a special protein substance
differing from the dissolved proteins of the milk is correct, then the
assumption as to a special body forming a membrane or stroma of the
fat-globules becomes very probable. The correctness of STORCH'S view
has been substantiated very recently by ABDERHALDEN and VOLTZ .4
On the acid hydrolysis of the fat-globules they obtained glycocoll, which
is absent in the casein as well as in the lactalbumin, and this shows that the

1 Arch, f , Anat. u. Physiol., 1840.

2 Pfliiger's Arch., 19.

3 V. Storch, see Maly's Jahresber., 27; Radenhausen and Danilewsky, Forschungen
auf dem Gebiete der Viehhaltung (Bremen, 1880), Heft 9; Voltz, Pfliiger's Arch., 102;
Bauer, Bioch. Zeitschr. 32; Droop-Richmond, see Chem. Centralbl., 1094, 2, 356;
Bonnema, ibid., 1243.

4 Zeitschr. f. pbysiol. Chem., 59.

MILK FAT. CASEIN. 647

fat-globules at least cannot contain these two proteins alone. They
must contain another protein, and it is still a question whether besides this
they also contain casein and lactalbumin.

The milk-fat which is obtained under the name of butter consists
mainly of olein and palmitin. Besides these it contains, as triglycerides,
myristic acid, stearic acid, small amounts of lauric acid, arachidic acid,
and dioxystearic acid, besides butyric acid and caproic acid, traces of
caprylic acid and capric acid. RIEGEL claims that triglycerides of vola-
tile fatty acids do not occur, but rather mixed triglycerides of volatile
and non-volatile fatty acids. Milk-fat also contains small quantities
of phosphatides (lecithin), and cholesterin and a yellow coloring-matter.
The quantity of volatile fatty acids in butter is, according to DUCLAUX,
on an average about 70 p.m., of which 37-51 p.m. is butyric acid and
30-33 p. m. is caproic acid. The non-volatile fat consists of /<$— A olein
and the remainder is principally palmitin. The composition of butter
is not constant, but varies considerably under different circumstances.1
The question whether the small fat-globules have a different composition
from the large ones is still disputed.

The milk-plasma, or that fluid in which the fat-globules are suspended,
contains several different proteins, the statements as to the number and
nature of which are somewhat at variance. The three following, casein,
lactalbumin, and lactoglobulin, have been most closely studied and are
well characterized. The milk-plasma contains at least two carbohy-
drates, of which the one, lactose, is of great importance. It also contains
extractive bodies, traces of urea, creatine, creatinine, or otic acid, hypoxan-
thine (?), cholesterin, citric acid (SOXHLET and HENKEL2), and lastly also
mineral bodies and gases.

Casein. This protein substance, which thus far has been detected
positively only in milk, belongs to the nucleoalbumins, and differs from
the albuminates chiefly by its content of phosphorus and by its behavior,
with the rennet enzyme. Casein from cow's milk has about the follow-
ing composition: C 53.0, H 7.0, N 15.7, S 0.8, P 0.85, and O 22.65 per
cent. Its specific rotation is, according to HOPPE-SEYLER, rather variable;
in neutral solution it is (o:)D=— 80°; its faintly alkaline solution has a
stronger rotation, namely, — 97.8 to —111.8°, in a solution of N/10-N/5

1 Riegel, Maly's Jahresber., 34; Duclaux, Compt. Rend., 104. Various statement.
as to the composition of milk-fat can be found in Koefoed, Bull. d. 1'Acad. Roys
Danoise, 1891, and Wanklyn, Chemical News, 63; Browne, Chem. Centralbl., 1899,
2, 883. In regard to the elementary composition of milk-fat see Fleischmann and
Warmbold, Zeitschr., f. Biol., 50.

2 Cited from Soldner, Die Salze der Milch, etc., Landwirthsch. Versuchsstation,
35, Separatabzug, 18.

648 MILK.

NaOH (LONG1). The question whether the casein from different kinds
of milk is identical or whether there are several caseins cannot be decided
by the elementary analysis. According to TANGL and CSOK!S, 2 mare's
and ass's casein seem to be somewhat richer in nitrogen (16.44 and 16.28
per cent, respectively) but poorer in sulphur (0.528 and 0.588 per cent)
and carbon (52.36 and 52.27 per cent) than the casein from cud chewers.
The ass's casein was richer in phosphorus (1.057 per cent) than the mare's
or cow's casein (both with 0.887 per cent).

Casein when dry appears like a fine white powder, which has no
measurable solubility in pure water (LAQUEUR and SACKUR). Casein
is only very slightly soluble in the ordinary neutral-salt solutions. Accord-
ing to ARTHUS it dissolves rather easily in a 1-per cent solution of sodium
fluoride, ammonium or potassium oxalate. ROBERTSON thinks that it
is more soluble in potassium cyanide and the alkali salts of certain vola-
tile fatty acids such as butyric acid and valeric acid, than in solutions
of the ordinary neutral salts. It is at least a tetrabasic acid, whose
equivalent weight is 1135, according to LAQUEUR and SACKUR, and 1250
according to ROBERTSON. The statements as to the molecular weight
are disputed (LAQUEUR and SACKUR, L. and D. VAN SLYKE3).

It dissolves readily in water with the aid of alkali or alkaline earths,
also calcium carbonate, from which it expels carbon dioxide and it
thus forms caseinates of variable composition. If casein is dissolved
in lime-water and the solution carefully treated with very dilute phos-
phoric acid until it is neutral in reaction (to litmus), the casein appears
to remain in solution, but is probably only swollen as in milk, and the
liquid contains at the same time a large quantity of calcium phosphate
without any precipitate or any suspended particles being visible. The
casein solutions containing lime are opalescent, and have on warming
the appearance of milk deficient in fat (which is also true for the salts
of casein with the alkaline earths). Therefore it is not impossible that
the white color of the milk is due partly to the casein and calcium phos-
phate. SOLDNER and others have prepared two calcium compounds
of casein with 1.5 p. c. CaO (the neutral caseinate according to SOLDNER)
and 2.4 p. c. CaO (the basic caseinate). The first is neutral to litmus
while the other is neutral to phenolphthalein.

According to ROBERTSON 4 the alkali equivalent of casein at neutrality
toward litmus =53x10 ~5 equivalent-grm.-mol. per gram and at neutrality toward

1 Hoppe-Seyler, Handb. d. physiol. u. pathol. chem. Analyse, 8. Aufl., 489; Long,
Journ. Amer. Chem. Soc., 27.
2Pfliiger's Arch., 121.

3 Laqueur and Sackur, ' Hofmeister's Beitrage, 3; M. Arthus, Theses presentees
& la faculte" des sciences de Paris, 1893; Robertson, Journ. of biol. Chem., 2; L. and
D. van Slyke, Amer. Chem. Journ., 38.

4 See Ergebnis. d. Physiol. 10 and Journ. of physical Chem., 13.

CASEIN. 649

phenolphthalein=80XlO~5 equivalent-grm.-mol. per gram. On saturation (with
monacidic bases) the alkali equivalent is = HX10~5 grm.-mol. per gram. On
saturating (with monobasic acids) the acid equivalent is=32XlO~6 grm.-mol.
per gram.

Besides the rather earlier investigations on the salts of casein By SOLD-
NER, COURANT, ROHMANN, LAQUEUR, RAUDNiTZ l and others we have
the recent observations and theoretical discussion of ROBERTSON 2 on the
composition, nature and dissociation of the caseinates. We can here
only refer to this and the earlier investigations.

Casein solutions do not coagulate on boiling, but solutions of casein-
lime are covered, like milk, with a pellicle. They are precipitated by
very little acid, but the presence of neutral salts retards the precipitation.
A casein solution containing salt or ordinary milk requires, therefore,
more acid for precipitation than a salt-free solution of casein of the same
concentration. The precipitated casein dissolves very easily again in
a small excess of hydrochloric acid, but less readily in an excess of acetic
acid. The combination between casein and acid, like other protein
and acid compounds, is precipitated by neutral salts. These acid solu-
tions are precipitated by mineral acids in excess.3 Casein is precipitated
from neutral solutions or from milk by common salt containing calcium,
or magnesium sulphate in substance, without changing its properties.4
Metallic salts, such as alum, zinc sulphate and copper sulphate, com-
pletely precipitate the casein from neutral solutions.

On drying at 100° C., casein, according to LAQUEUR and SACKUR, decomposes
and splits into two bodies. One of these, called caseid, is insoluble in dilute alkalies,
while the other, the isocasein, is soluble therein. The isocasein is a stronger acid
and has other precipitation limits and a rather lower equivalent weight than the
casein.

The property which is the most characteristic of casein is that it
coagulates with rennet in the presence of a sufficiently large amount
of lime-salts. In solutions free from lime-salts the casein does not coagu-
late with, rennet, but it is changed so that the solution (even if the enzymes
are destroyed by heating) yields a coagulated mass, having the properties
of a curd, if lime-salts are added. The rennet enzyme, rennin, has there

1 Soldner, Die Salze der Milch, etc., and Maly's Jahresber., 25; Courant, 1. c.;
Rohmann, Berlin, klin. Wochenschr., 1895; Laqueur, 1. c.; and Hofmeister's Beitrage,
7; Raudnitz, Ergebn. d. PhysioL, 2, Abt. 1.

2 Journ. of physical Chem., 11 and 12; Journ. of biol. Chem., 5.

3 In regard to the acid combinations of casein and the ability to take up acid, see
Laxa, Milch wirthsch. Centralbl., 1905; Long, Journ. Amer. Chem. Soc., 29; L. and
D. van Slyke, Amer. Chem. Journ., 38; Robertson, Journ. of biol. Chem., 4.

4 See the works of Hammarsten and Schmidt-Nielsen, Hammarsten's Festschrift,
1906.

650 MILK.

fore an action on casein even in the absence of lime-salts. These last
are only necesary for the coagulation or the separation of the curd,
and the process of coagulation is hence a two-phase process. The first
phase is the transformation of the casein by the rennin, the second is
the visible coagulation caused by the lime-salts. This fact, which was
first proved by HAMMARSTEN, was later confirmed by ARTHUS and PAGES
and recently closely studied by FULD, SPIRO, and LAQUEUR and others.1

The curd formed on the coagulation of milk contains large quantities of
calcium phosphate. According to SOXHLET and SOLDNER, the soluble lime-salts
are of essential importance only in coagulation, while the calcium phosphate
is without importance. COURANT believes that the calcium-casein on coagula-
tion may carry down with it, if the solution contains dicalcium phosphate, a part
of this as tricalcium phosphate, leaving mono-calcium phosphate in the solution.
A solution of calcium casein is not coagulated by rennin alone but only when soluble
lime-salts are added. Contrary to the generally accepted view that the soluble
lime-salts are of importance in the coagulation, VAN DAM 2 claims that it is the
quantity of lime combined with the casein which is of importance in the coagula-
tion process. The role of the lime-salts in coagulation is not clear, and this fol-
lows from the chemical procedure in rennin coagulation.

If one makes use of a pure solution of casein and as pure rennin as
possible, then after coagulation it is always found that the filtrate con-
tains very small amounts of a protein, the whey protein, which is probably
formed in the coagulation. This behavior, which was first shown by
HAMMARSTEN, has been substantiated by many others and recently by
FULD, SPIRO and SCHMIDT-NIELSEN. Whey protein is generally con-
sidered as a proteose substance, and KOSTER 3 found 13.2 per cent nitro-
gen therein. In correspondence with these observations casein coagula-
tion with rennin is considered as a cleavage process, in which the principal
mass of the casein, sometimes more than 90 per cent, is split off as para-
casein^ a body closely related to casein, and in the presence of sufficient

1 See Maly's Jahresber., 2 and 4; also Hammarsten, Zur Kenntniss des Kasei'ns und
der Wirkung des Labfermentes, Nova Acta Reg. Soc. Sclent. Upsala, 1877, Fest-
schrift; Zeitschr. f. physiol. Chem., 22; Arthus et Pages, Arch, de Physiol. (5), 2,
and M<§m. soc. biol., 43; Fuld, Hofmeister's Beitrage, 2, and Ergebnisse der Physiol.,
1, Abt. 1, where a good review of the literature may be found, Spiro, Hofmeister's
Beitrage, 6 and 7, with Reichel, ibid., 7 and 8; Laqueur, ibid., 7.

2 Zeitschr. f. physiol. Chem., 58.

8 Hammarsten, 1. c. ; Fuld. Bioch. Zeitschr., 4, and Hofmeister's Beitrage, 10;
Spiro, Hofmeister's Beitrage, 8; Schmidt-Nielsen, Hammarsten's Festschrift, 1906;
Koster, see Maly's Jahresber., 11, 14.

4 It has been proposed to designate the ordinary casein as caseinogen and the curd
as casein. Although such a proposition is theoretically correct, it leads in practice
to confusion. On this account the author calls the curd paracasein, according to
Schulze and Rose (Landwirthsch. Versuchsstat., 31). A summary of the literature on
the casein coagulation may be found in E. Fuld, Ergebnisse der Physiol., 1; Raudnitz,
ibid., 2; and Laqueur, Biochem. Centralbl., 4, 344.

CASEIN. 651

amounts of lime-salts the paracasein-lime precipitates out while the
proteose-like substance (whey protein) remains in solution. In the
coagulation in an acid medium the conditions are entirely different and
proteoses and peptones are hereby formed to a considerable extent.

The paracasein is very similar to casein, but cannot be recoagulated
by rennin. A solution of alkali-paracaseinate is much more readily
precipitated by CaCb than an alkali-caseinate solution of the same con-
centration, and the precipitation limits for saturated ammonium-sul-
phate solution, the upper as well as the lower limit, lie, according to
LAQUEUR, lower with paracasein than with casein. The internal friction
of paracasein solutions is also, in his opinion, less than that of casein
solutions and indeed even to 20 per cent.

By continued action of rennin upon paracasein a further transformation
has been found in many cases (PETRY, SLOWTZOFF, v. HERWERDEN *). This
is explained by the presence of another proteolytic enzyme in the (impure) rennin
preparation. This assumption seems to be plausible, and we are here probably
dealing only with a secondary process which has nothing whatever to do with the
true formation of paracasein. Whey protein is also formed after the very short
action of rennin, and the continued cleavage occurs with varying speed. Thus
SCHMIDT-NIELSEN found that the quantity of whey protein was even 3 per cent
of the casein nitrogen after the action of rennet for 15 minutes, and only 4.25
per cent after 6 hours' action. These and other recent investigations favor
the assumption that the casein coagulation by rennet is a hydrolytic cleavage,
but the conditions are not so clear that this can be considered as proved.2

Fresh, unchanged milk does not, as is known, coagulate on boiling; but in
not too rapid action of rennin a state may be observed in which the milk coagu-
lates on heating (metacasein reaction). A solution of paracasein lactate, accord-
ing to LAX A,3 coagulates with rennin the same as a solution of casein lactate,
which indicates, he believes, that the paracasein is transformed into casein again
by the lactic acid. But as a precipitation of the paracasein from the acid solu-
tion is perhaps a pepsin action, the transformation of the paracasein into casein
by the lactic acid must not be considered as proved.

In the digestion of casein with pepsin-hydrochloric acid primarily a
phosphorized proteose is formed, from which then the pseudonuclein is
split off (SALKOWSKI). The quantity thus split off is variable, as shown
by the researches of SALKOWSKI, HAHN, MOBACZEWSKI, SEBELIEN,
and ZAiTSCHEK.4 The amount of phosphorus in the pseudonucleins
obtained also varies considerably. SALKOWSKI considers that the quan-
tity of pseudonuclein split off is dependent upon the relation between
the casein and the digestion fluid, e.g., the quantity of the pseudonu-

, Hofmeister's Beitrage, 8; Slowtzoff, ibid., 9; v. Herwerden, Zeitschr. f.
physiol. Chem., 52; W. van Dam, ibid., 61.

2 See also Werncken, Zeitschr., f . Biol., 52.

3 Laxa, 1. c.

4Salkowski, Zeitschr. f. physiol. Chem., 27; Salkowski and Hahn, Pfliiger's Arch.,
59; Salkowski, ibid., 63; v. Moraczewski, Zeitschr. f. physiol. Chem., 20; Sebelien
ibid., 20; Zaitschek, Pfluger's Arch., 104.

652 MILK.

clems diminishes as the pepsin-hydrochloric acid increases. In the
presence of 500 grams of pepsin-hydrochloric acid to I gram of casein,
SALKOWSKI digested the latter completely without obtaining any
pseudonuclein.

In peptic as well as tryptic digestion a part of the organic phosphorus
is split off as orthophosphoric acid, the quantity increasing as the diges-
tion progresses. Another part of the phosphorus is retained in organic
combination in the proteoses as well as in the true peptones (SALKOWSKI,
BIFFI, ALEXANDER, ADERS-PLIMMER and BAYLISS l).

From the products of peptic digestion of casein, after the separation of the
pseudonuclein, SALKOWSKI 2 has isolated an acid rich in phosphorus. He con-
siders this a paranudeic acid. This acid which gives the biuret test and a
faint xanthoproteic reaction, contains 4.05-4.31 per cent phosphorus. A still
richer product in phosphorus, with 6.9 per cent P, has been isolated by REH
from the peptic digestive products of casein. He calls this body polypeptid
phosphoric acid. This product, which also gives the above-mentioned protein re-
actions, and is not comparable with the nucleic acids, is characterized by a remark-
ably high content of ami no-nitrogen, namely. 23.8 per cent. Among the products
obtained by REH, DIETRICH 3 found a mixture of at least four different lime-salts
of a peptone character, and which he considers as polypeptide-like combination
with P2O5, caseonphosphoric acids. The amount of phosphorus was, respectively,
10.0, 4.1, 3.84 and 3.88 per cent.

Casein may be prepared in the following way: The milk is diluted
with 4 vols. of water and the mixture treated with acetic acid to 0.75-
1 p. m. Casein thus obtained is purified by repeatedly dissolving in water
with the aid of the smallest quantity of alkali possible, by filtering and
reprecipitating with acetic acid and thoroughly washing with water.
Most of the milk-fat is retained by the filter on the first filtration, and the
casein contaminated with traces of fat is purified by treating with alcohol
and ether.

Lactoglobulin was obtained by SEBELIEN from cow's milk by saturating
it with NaCl in substance (which precipitated the casein) and saturating
the filtrate with magnesium sulphate. As far as it has been investigated
it had the properties of serglobulin; the globulin isolated by TiEMANN4
from colostrum had, nevertheless, a markedly low content of carbon,
namely, 49.83 per cent.

Lactalbumin was first prepared in a pure state from milk by SEBELIEN.
He gives its composition as, C 52.19, H 7.18, N 15.77, S 1.73, O 23.13
per cent. Lactalbumin has the properties of the albumins, and WICH-

^alkowski, 1. c.; Biffi, Virchow's Arch., 152; Alexander, Zeitschr. f. physiol.
Chem., 25; Plimmer and Bayliss, Journ. of Physiol., 33; See also Kiittner, Pfliiger'a
Arch. 129.

2 Zeitschr. f. physiol. Chem., 32.

3 Reh. Hofmeister's Beitrage 11; Dietrich, Bioch. Zeitschr. 22.

4 Zeitschr. f. physiol. Chem., 25.

LACTALBUMIN. ENZYMES. 653

MANN found that it crystallizes in forms similar to ser- or ovalbumin.
It coagulates, depending on the concentration and the amount of salt
in solution, at 72-84° C. It is similar to seralbumin, but differs from
it in having a considerably lower specific rotatory power: (a)p = — 37°.
According to FASAL1 it is especially rich in tryptophane, namely, 3.07
per cent.

The principle of the preparation of lactalbumin is the same as for the
preparation of seralbumin from serum. The casein and the globulin
are removed by MgS04 in substance, and the filtrate treated as previously
stated (page 263).

The occurrence of other proteins, such as proteases and peptones, in milk has
not been positively proved. These bodies are easily produced as laboratory
products from the other proteins of the milk. Such a laboratory product is
MILLON'S and COMAILLE'S lactoprotein, which is a mixture of a little casein with
changed albumin, and proteose 2 which is formed by chemical action. In regard
to opalisin, see Human Milk, p. 662.

Milk also contains, SIEGFRIED 3 claims, a nucleon related to phos-
phocarnic acid, which yields fermentation lactic acid (instead of para-
lactic acid) and a special carnic acid, orylic acid (instead of muscle carnic
acid), as cleavage products. Lactophosphocarnic acid may be precipitated
as an iron compound from the milk freed from casein and coagulable
proteins as well as from earthy phosphates.

Milk also contains enzymes of various kinds. Of these we must men-
tion catalases, peroxidases, and redudases, but the statements as to their
occurrence in the milk from different animals as well as the question
how much of their action is due to micro-organisms are conflicting.
Among these enzyme actions a special interest has been given to the
SCHARDINGER reaction, which consists in the fact that milk at 70° C.
in the presence of formaldehyde or acetaldehyde reduces certain dyes,
such as methylene blue, to leucobases. An amylolytic enzyme which
converts starch into maltose occurs, especially, in human milk, while
it is absent in cow's milk or occurs only to a slight extent. A fermenta-
tion enzyme which in the absence of micro-organisms decomposes the
lactose into lactic acid, alcohol, and C02, occurs, according to STOKLASA 4
and his co-workers, in cow's milk as well as in human milk. Human
milk, as well as cow's milk, contains a lipase which has the property
at least of acting upon monobutyrin. BABCOCK and RUSSEL have
found in these two kinds of milk, as well as certain others, a proteolytic

1Sebelien, Zeitschr., f. physiol. Chem., 9; Wichmann, ibid., 27; Fasal,- Bioch.
Zeitschr., 44.

8 See Hammarsten, Maly's Jahresber., 6, 13.
8 Zeitschr. f. physiol. Chem., 21 and 22.
4 See Chem. Centralbl., 1905, 1, 107.

654 MILK.

enzyme which they call galactase, which is allied to trypsin, but differs
therefrom in that it develops ammonia from milk even in the early stages
of digestion. The occurrence of such an enzyme is denied by ZAITSCHEK
and v. SZONTAGH, but on the other hand VANDEVELDE, DE WAELE, and
SUGG 1 confirm the occurrence of a proteolytic enzyme in milk.

Orotic acid, C5HnN204.2H20, is the name given by BISCARO and BELLONI 2
to a new constituent of milk which they have discovered. This acid, which can.
be precipitated by basic lead acetate from whey free from protein, is slightly
soluble in water, crystalline, and gives several crystalline salts. The mono-
methyl and ethyl esters of this acid are also known. It yields urea on treatment
with potassium permanganate.

Lactose, MILK-SUGAR, Ci2H220n -f-H^O. This sugar, on hydrolysis,
can be split into two hexoses, glucose and galactose. It yields mucic acid
besides other organic acids, by the action of dilute nitric acid. Levulinic
acid is formed, besides formic acid and humin substances, by the stronger
action of acids. By the action of alkalies, among other products we
find lactic acid and pyrocatechin.

Milk-sugar occurs, as a rule, only in milk, but it has also been found
in the urine of pregnant women, on stagnation of milk, as well as in the'
urine after partaking of large quantities of the same sugar.

Lactose occurs ordinarily as colorless rhombic crystals with 1 mole-
cule of water of crystallization, which is driven off by slowly heating to
100° C., but more easily at 130-140° C. On quickly boiling down a milk-
sugar solution, anhydrous milk-sugar separates out. Milk-sugar dissolves
in 6 parts cold or in 2.5 parts boiling waiter; it has a faintly sweetish
taste. It does not dissolve in ether or absolute alcohol. Its solutions
are dextrogyrate. The rotatory power, which on heating the solution to
100° C. becomes constant, is (a)i>=+52.50. Milk-sugar combines with
bases; the alkali combinations are insoluble in alcohol.

Milk-sugar is not fermentable with pure yeast. It undergoes, on the
contrary, alcoholic fermentation by the action of certain schizomycetes,
and E. FISCHER 3 found that the milk-sugar is first split into glucose
and galactose by an enzyme, lactase, existing in the yeast. The prep-
aration of milk- wine, " kumyss," from mare's milk and " kephir " and
" yoghurt " from cow's milk is based upon this fact. Other micro-organ-
isms also take part in this change, causing a lactic-acid fermentation of
the milk-sugar.

^abcock and Russel, Centralbl. f. Bakt. u. Parisitenkunde (II), 6, and Maly's
Jahresber., 31; Zaitschek and v. Szontagh, Pfliiger's Arch., 104; Vandevelde, de
Waele, and Sugg, Hofmeister's Beitrage, 5.

2 See Chem. Centralbl., 1905, 2, 63.

1 Ber. d. d. Chem. Gesellsch., 27.

LACTOSE. 655

Lactose responds to the reactions of glucose, such as MOORE'S,*
TROMMER'S and RUBNER'S, and the bismuth test. It also reduces mer-
curic oxide in alkaline solutions. After warming with phenylhydrazine
acetate it gives on cooling a yellow crystalline precipitate of- phenyl
lactosazone, C24H32N4Og. It differs from cane-sugar by giving positive
reactions with MOORE'S or TROMMER'S and the bismuth test, and also in
that it does not darken when heated to 100° C. with anhydrous oxalic
acid. It differs from glucose and maltose by its solubility and crystalline
form, but especially, by its not fermenting with yeast, and by yielding
mucic acid with nitric acid.

• The osazone obtained with phenylhydrazine acetate, which melts at
200° C., differs from the other osazOnes by being inactive when 0.2 gram
is dissolved in 4 cc. of pyridine and 6 cc. of absolute alcohol and viewed
through a layer 10 centimeters long (NEUBERG2).

For the preparation of milk-sugar we make use of the by-product
in the preparation of cheese, the sweet whey. The protein is removed
by coagulation with heat, and the filtrate evaporated to a syrup. The
crystals which separate after a certain time are recrystallized from water
after decolorizing with animal charcoal. A pure preparation may be
obtained from the commercial milk-sugar by repeated recrystallization.
The quantitative estimation of milk-sugar may be performed either by
the polaristrobometer or by means of titration with FEHLING'S solution.
Ten cc. of FEHLING'S solution are reduced by 0.0676 gram of milk-sugar
in 0.5-1.5 per cent solution after boiling for six minutes. (In regard to
FEHLING'S solution and the titration of sugar see larger hand-books.)

From the non-correspondence between the quantity of sugar in the
milk as determined by polarization and gravimetrically, when the polar-
ization results are always higher, SEBELIENS has concluded that the
milk must contain a second reducing substance which polarizes stronger
than lactose. This substance is probably a pentose and occurs to a very
slight extent in ordinary milk, 0.25-0.35 p. m. (SEBELIEN and SUNDE),
and more in colostrum, 0.5 p. m.

RITTHAUSEN found another carbohydrate in milk which is soluble in water,
non-crystallizable, which has a faint reducing action, and which yields, on boiling
with an acid, a body having a greater reducing power. BECHAMP 4 considers
this as dextrin.

AThe well-known beautiful red color, which milk produces after the addition of
alkali, at the room temperature and to which attention has been called recently by
Gautier, Morel, and Monod (Compt. rend. soc. biol., 60 and 62), and Kriiger (Zeitschr.
f. Physiol. Chem., 50) is a Moore's reaction modified by the presence of protein and
perhaps also other milk constituents.

2 Ber. d. d. Chem. Gesellsch., 32.

'Sebelien, Hammarsten's Festschrift, 1906; with Sunde, Zeitschr. f. angew.
Chem., 21.

4 Ritthausen, Journ. f. prakt. Chem. (N. F.), 15; Bechamp, Bull. Soc. Chim. (3), 6.

656 MILK.

The mineral, bodies of milk will be treated in connection with its quan-
titative composition.

The methods for the quantitative analysis of milk are very numerous,
and as all cannot be treated here, we will give the principal points of a
few of the methods considered most trustworthy and most frequently
employed.

In determining the solids a carefully weighed quantity of milk is mixed with
an equal weight of heated quartz sand, fine glass powder, or asbestos. The
evaporation is first done on the water-bath and finished in a current of carbon
dioxide or 'hydrogen not above 100° C.

The mineral bodies are determined by incinerating the milk, using the pre-
cautions mentioned in the text-books. The results obtained for the phosphoric
acid are incorrect on account of the burning of phosphorized bodies, such as
casein and lecithin. We must, therefore, according to SOLDNER, subtract in round
numbers 25 per cent from the total phosphoric acid found in the milk. The
quantity of sulphate in the ash also depends on the combustion of the proteins.

In the determination of the total amount of proteins RITTHAUSEN'S method
is employed, namely, the precipitation of the milk with copper sulphate according
to the modification suggested by MuNK.1 He precipitates all the proteins by
means of cupric hydroxide at boiling heat, and determines the nitrogen in the
precipitate by means of KJELDAHL'S method. This modification gives more
exact results.

According to SEBELIEN'S method, three to four grams of milk are diluted
with an equal volume of water, a little common-salt solution added, and the
proteins precipitated with an excess of tannic acid. The precipitate is washed
with cold water, and then the quantity of nitrogen determined by KJELDAHL'S
method. The total nitrogen found when multiplied by 6.37 (casein and lactal-
bumin contain both 15.7 per cent nitrogen) gives the total quantity of proteins.
This method, which is readily performed, gives very good results. I. MUNK
used this method in the analysis of woman's milk. In this case the quantity
of nitrogen found must be multiplied by 6.34. G. SIMON 2 found that the
precipitation with tannic acid, also with phosphotungstic acid, is the simplest
and most accurate. The objection to this and other methods in which the pro-
teins are precipitated is that perhaps other bodies (extractives) may be carried
down at the same time (CAMERER and SOLDNER 3) . It is not known to what
extent this takes place.

A part of the nitrogen in the milk exists as extractives, and this nitrogen is
calculated as the difference between the total nitrogen and the protein nitrogen.
According to MUNK'S analyses about -j^ of the total nitrogen belongs tp'the extract-
ives in cow's milk. CAMERER and SOLDNER determine the nitrogen in the filtrate
from the tannic-acid precipitate by KJELDAHL'S method, and also according to
HUFNER'S method (hypobromite). In this way they found 18 milligrams of
nitrogen according to HUFNER (urea, etc.) in 100 grams of cow's milk.

To determine the casein and albumin separately we may make use of the
method first suggested by HOPPE-SEYLER and TOLMATSCHEFF/ in which the casein
is precipitated by magnesium sulphate. According to SEBELIEN the milk is diluted
with its own volume of a saturated magnesium-sulphate solution, then saturated
with the salt in substance, and the precipitate then filtered and washed with a
saturated magnesium-sulphate solution. The nitrogen is determined in the pre-

1Ritthausen, Journ. f. prakt. Chem. (N. F.), 15; I. Munk, Virchow's Arch., 134.

2Sebelien, Zeitschr. f. physiol, Chem., 13; Simon, ibid., 33.

8 Zeitschr. f. Biologic, 33 and 36.

4 Hoppe-Seyler, Med. chem. Untersuch., 272.

MILK ANALYSIS. 657

cipitate by KJELDAHL'S method, and the quantity of casein (+globulin) determined
by multiplying the result by 6.37. The quantity of lactalbumin may be calculated
as the difference between the casein and the total proteins found. The lactal-
bumin may also be precipitated by tannic acid from the filtrate from the casein
precipitate containing MgS04, after diluting with water, the nitrogen determined
by KJELDAHL'S method and the result multiplied by 6.37.

SCHLOSSMANN 1 suggests an alum solution, which precipitates the casein,
in order to separate the casein from the other proteins, and the albumin is then
precipitated from the filtrate by tannic acid. The nitrogen in the precipitate
is determined by the KJELDAHL method. This method has recently been tested
by SIMON and he recommends it highly.

The fat is gravimetrically determined by thoroughly extracting the dried
milk with ether, evaporating the ether from the extract, and weighing the residue.
The fat may be determined by aerometric means by adding alkali to the milk,
shaking with ether, and determining the specific gravity of the fat solution by means
of SOXHLET'S apparatus. In determining the amount of fat in a large number of
samples the lactocrit of DE LAVAL may be used with success. There are numer-
ous other methods for estimating milk-fat, but they cannot be considered here.

In determining the milk-sugar the proteins are first removed. For this pur-
pose we precipitate either with alcohol, which must be evaporated from the filtrate,
or by diluting with water, and removing the casein by the addition of a little acid,
and the lactalbumin by coagulation at boiling heat. The sugar is determined by
titration with FEHLING'S or KNAPP'S solution (see Chapter XIV). The principle
of the titration is the same as for the titration of sugar in the urine; 10 cc. of
FEHLING'S solution correspond to 0.0676 gram of milk-sugar; 10 cc. of KNAPP'S
solution correspond to 0.0311-0.0310 gram of milk-sugar, when the saccharine
liquid contains about J-l per cent of sugar. In regard to the modus operandi
of the titration we must refer the reader to more extensive works.

Instead of these volumetric determinations other methods of estimation, such
as ALLIHN'S method, the polariscope method, and others, may be used. In calcu-
lating the analysis or in determining the solids it is of importance to remember,
as suggested by CAMERER and SOLDNER, that the milk-sugar in the residue is
anhydrous. Many other methods for determining the milk-sugar have been
suggested and recommended.

The quantitative composition of cow's milk is naturally very variable.
The average obtained by KONIG 2 is as follows in 1000 parts:

Water. Solids. Caaein. Albumin. Fata. Sugar. Salts.

871.7 128.3 30.2 5.3 36.9 48.8 7.1

35.5

The quantity of mineral bodies in 1000 parts of cow's milk is, accord-
ing to the analyses of SOLDNER, as follows: K20 1.72, Na20 0.51, CaO
1.98, MgO 0.20, ?205 1.82 (after correction for the pseudonuclein) ,
C1JX98 grams. BUNGE found 0.0035 gram Fe2Oa, and EDELSTEIN and
CsoNKA3 found 0.0007-0.001 gm. Fe2Os. According to SOLDNER the
K, Na, and Cl are found in the same quantities in whole milk as in milk-
serum. Of the total phosphoric acid 36-56 per cent, and of the lime

1 Zeitschr. f. physiol. Chem., 22.

2 Chemie der menschlichen Nahrungs- und Genussmittel, 4. Aufl.

3 Bunge, Zeitschr. f. Biol. 10; Edelstein and Csonka, Bioch. Zeitschr., 38.

658 MILK.

53-72 per cent is not in simple solution. A part of this lime is combined
with the casein; the remainder is found united with the phosphoric acid
as a mixture of dicalcium and tricalcium phosphates which is kept dis-
solved or suspended by the casein. RONA and MICHAELIS 1 found that
about 40-50 per cent of the total quantity of lime was diffusable ; accord-
ing to them nearly one-half of the calcium is contained in the milk as a
non-dissociable casein compound, while the milk only contains the very
smallest amounts of suspended calcium phosphate.

The bases are in excess of the mineral acids in the milk-serum.
The excess of the first is combined with organic acids, which correspond
to 2.5 p. m. citric acid (SOLDNER).

The gases of the milk consist mainly of CO2, besides a little N and
traces of O. PFLUGER 2 found 10 vols. per cent CO2 and 0.6 vol. per cent
N calculated at 0° C. and 760 mm. pressure.

The variation in the composition of cow's milk depends on several
circumstances.

The colostrum, or the milk which is secreted before calving and in
the first few days after, is yellowish, sometimes alkaline, but often acid,
of higher specific gravity, 1.046-1.080, and richer in solids than ordinary
milk. The colostrum contains, besides fat-globules, an abundance of
colostrum-corpuscles — nucleated granular cells 0.005-0.025 mm. in di-
ameter with abundant fat-granules and fat-globules. The fat of colos-
trum has a somewhat higher melting-point and is poorer in volatile fatty
acids than the fat from ordinary milk (NILSON 3) . The iodine equivalent
of the colostrum-fat is higher than that of milk-fat. The quantity of
cholesterin and lecithin is generally greater. The most apparent dif-
ference between it and ordinary milk is that colostrum coagulates on heat-
ing to boiling because of the absolutely and relatively greater quantities
of globulin and albumin that it contains.4 The composition of colostrum
varies considerably. KONIG gives as average the following figures in
1000 parts:

Water. Solids. Casein. Albumin and Globulin. Fat. Sugar. Salts.

746.7 253.3 40.4 136.0 35.9 26.7 15.6

The influence which food exercises upon the composition of milk will
be discussed in connection with the chemistry of the milk secretion.

1 Bioch. Zeitschr., 21.

2Pfluger'sArch., 2.

•See Maly's Jahresber., 21. See also Engel and Bode, Zeitschr. f. physiol. Chem.,
74.

4 See Sebelien, Maly's Jahresber., 18, and Tiemann, Zeitschr. f. physiol. Chem.,
25. See also Simon, ibid., 33; Winterstein and Strickler, ibid., 47.

MILK OF OTHER ANIMALS. 659

In the following table is given the average composition of skimmed milk and
certain other preparations of milk :

Water. Proteins. Fat. Sugar. Lactic Acid. Salts.

Skimmed milk 906.6 31.1 7.4 47.5 ... 7.4

Cream 655.1 36.1 267.5 35.2 ... 6.1

Buttermilk 902.7 40.6 9.3 37.3 3.4 6.7

Whey 932.4 8.5 2.3 47.0 3.3 6.5

KUMYSS, KEPHIR and YOGHURT are obtained, as above stated, by the alcoholic
and lactic-acid fermentation of the milk-sugar, the first from mare's milk and
the other from cow's milk. Large quantities of carbon dioxide are formed thereby,
and besides this the protein bodies of the milk are partly converted into proteoses
and peptones, which increase the digestibility. The quantity of lactic acid in
these preparations may be about 10-20 p. m. The quantity of alcohol varies
from 10 to 35 p. m.

Milk of Other Animals. GOAT'S milk has a more yellowish color and a
more specific odor than cow's milk. The coagulum obtained by acid or rennet
is more solid and is harder than that from cow's milk. SHEEP'S milk is similar
to goat's milk, but has a higher specific gravity and contains a greater amount
of solids.

MARE'S milk is alkaline and contains a casein which is not precipitated, by
acids, in lumps or solid masses, but, like the casein from woman's milk, in fine
flakes. This casein is only incompletely precipitated by rennet, and it is very
similar also in other respects to the casein of human milk. In BEIL'S l opinion
the casein from mare's and cow's milk is the same, and the different behavior
of the two varieties of milk is due to varying amounts of salts and to a different
relation between the casein and the albumin. This does not agree with the
analyses of casein by TANGL and CSOAKS given above nor with the investiga-
tions of ZAITSCHEK and v. SZONTAGH, who find that the casein from mare's milk,
like that from human and ass's milk, is digested by pepsin-hydrochloric acid
without leaving a residue. According to ENGEL and DENNEMARK 2 the colostrum
from the mare differs from that from the ass by being richer in casein than the milk.
The milk of the ASS is claimed by earlier authorities to be similar to human milk'
but SCHLOSSMANN finds it considerably poorer in fat. The researches of ELLEN-
BERGER give similar results, and show great similarity between ass's milk and
human milk. The average results were 15 p. m. protein with 5.3 p. m. albumin
and 9.4 p. m. casein. This latter, like human casein, does not yield any pseudo-
nuclein on pepsin digestion, which agrees well with the above-mentioned investiga-
tions of ZAITSCHEK. The quantity of nucleon was about the same as in woman's
milk. The quantity of fat was 15 p. m., and the sugar was 50-60 p. m. REINDEER
milk is characterized, according to WERENSKIOLD,S by being very rich in fat,
144.6-197.3 p. m., and casein, 80.67 86.9 p. m.

The milk of CARNIVORA (the bitch and cat) is acid in reaction and very rich
in solids. The composition of the milk of these animals varies with the com-
position of the food.

To illustrate the composition of the milk of other animals the following figures,
the compilation of KONIG, are given. As the milk of each kind of animal may
have a variable composition, these figures should only be considered as examples
of the composition of milk of various kinds:4

1 Studein iiber die Eiweissstoffe des Kumys imd Kefirs, St. Petersburg, 1886 (Ricker).

2 Zeitschr. f. physiol. Chem., 76.

3 Zaitschek, 1. c.; Schlossmann, Zeitschr. f. physiol. Chem., 22; Ellenberger, Arch.
f. (Anat. u.) Physiol., 1899 and 1902; Werenskiold, Maly's Jahresber., 25.

4 Details in regard to the milk of different animals may be found in Proscher,
Zeitschr. f. physiol. Chem., 24; Abderhalden, ibid., 27. In regard to pig milk, see
Zuntz and Ostertag, Landw. Jahresb., 37.

660 MILK.

Milk of the Water. Solids Proteins. Fat. Sugar. Salts.

Dog ............. 754.4 245.6 99.1 95.7 31.9 7.3

Cat ............. 816.3 183.7 90.8 33.3 49.1 5.8

Goat ............ 869.1 130.9 36.9 40.9 44.5 8.6

Sheep ........... 835.0 165.0 57.4 61.4 39.6 6.6

Cow ............. 871.7 128.3 35.5 36.9 48.8 7.1

Horse ........... 900.6 99.4 18.9 10.9 66.5 3.1

Ass .............. 900.0 100.0 21.0 13.0 63.0 3.0

Pig .............. 823.7 176.3 60.9 64.4 40.4 10.6

Elephant ......... 678.5 321.5 30.9 195.7 88.5 6.5

Dolphin ......... 486.7 513.3 ____ 437.6 ____ 4.6

Whale1 .......... 698.0 302.0 94.3 194.0 9.9

Human Milk.

Woman's milk is amphoteric in reaction. According to COURANT
its reaction is relatively more alkaline than cow's milk, but it has, never-
theless, a lower absolute reaction for alkalinity as well as for acidity. He
found between the tenth day and the fourteenth month after confinement
practically constant results. The alkalinity, as well as the acidity, was
a little lower than in childbed. One hundred cc. of the milk had the
same average alkalinity as 10.8 cc. N/10 caustic soda, and the same
acidity as 3.6 cc. N/10 acid. The relation between the alkalinity and the
acidity in woman's milk was as 3:1, and in cow's milk as 2.1:1. The
actual reaction determined electrometrically is, according to Fol,2 still
nearly neutral, like the other kinds of milk. ALLARIA has also arrived
at similar results, according to whom the tendency of human milk toward
alkaline reaction even in the most prominent cases never corresponds

to a NaOH solution-

Human milk also contains fewer fat-globules than cow's milk, but
they are larger in size. The specific gravity of woman's milk varies
between 1.026 and 1.036, generally between 1.028 and 1.034. It is highest
in well-fed and lowest in poorly-fed women. The freezing-point is lowered
on an average 0.589° C., according to WINTER and PARMENTIERS con-
stant at 0.55°, and the molecular concentration is 0.318.

The fat of woman's milk has been investigated by RUPPEL. It forms
a yellowish- white mass, similar to' ordinary butter, having a specific gravity
of 0.966 at 15°. It melts at 34.0° C. and solidifies at 20.2° C. The fol-
lowing fatty acids can be obtained from the fat, namely, butyric, caproic,
capric, myristic, palmitic, stearic, and oleic acids. The fat from woman's
milk is-, according to RUPPEL and LAVES,4 relatively poor in volatile
fatty acids. The non-volatile fatty acids consist of one-half oleic acid,

1 Scheibe, cited in Bioch. Centralbl., 7, 553.

2 Compt. rend. soc. biol. 58; Allaria, Maly's Jahresb., 39, 242.

3 See Maly's Jahresber., 34.

4Ruppel, Zeitschr. f. Biologic, 31; Laves, Zeitschr. f. physiol. Chem., 19.

HUMAN MILK. 661

while among the solid fatty acids myristic and palmitic acids are found
to a greater extent than stearic acid.

The essential qualitative difference between woman's and cow's milk
seems to lie in the proteins or in the more accurately determined casein.
A number of both the earlier and more recent investigators l claim that
the casein from woman's milk has other properties than that from cow's
milk. The essential differences are the following: The casein from
woman's milk is precipitated with greater difficulty with acids or salts.
It does not coagulate uniformly in the milk after the addition of rennet,
which depends, essentially, upon the low amount of lime-salts and casein
contained in the milk.2 It may be precipitated by gastric juice, but
dissolves completely and easily in an excess of gastric juice; the casein pre-
cipitate produced by an acid is more easily soluble in an excess of the acid;
and lastly, the clot formed from the casein of woman's milk does not
appear in such large and coarse masses as in the casein from cow's
milk, but is more loose and flocculent. This last-mentioned fact is of
great importance, since it explains the generally admitted fact of the
easy digestibility of the casein from woman's milk.

The question as to whether the above-mentioned variations depend
on a decided difference in the two caseins, or only on an unequal relation
between the casein and the salts in the two kinds of milk, or upon other
circumstances, has not as yet been decided. According to SZONTAGH
and- ZAITSCHEK and also WR6BLEWSKY, the casein from human milk
does not yield any pseudonuclein on peptic digestion, and hence it cannot
be a nucleoalbumin. According to KOBRAK, woman's casein yields
some pseudonuclein, and with repeated solution in alkali and precipitation
by an acid it becomes more and more like cow's casein. He therefore
suggests the possibility that woman's casein is a compound between a
nucleoalbumin and a basic protein. WR6BLEWSKY found the follow-
ing for the composition of casein from woman's milk: C 52.24, H 7.32,
N 14.97, P 0.68, S 1.117 per cent. LANGSTEIN and BERGELL obtained
much lower figures for N, S and especially P, namely, 14.34, 0.85 and 0.27
per cent, respectively. According to LANGSTEIN and EDELSTEIN the
phosphorus content is only 0.22-0.29 per cent. On hydrolysis ABDER-
HALDEN and LANGSTEIN3 could not find any difference between cow
and human casein.

^ee Biedert, Untersuchungen iiber die chemischen Unterschiede der Menschen-
und KuhmiHh (Stuttgart), 1884; Langgaard, Virchow's Arch., 64; Makris, Studien
iiber die Eiweisskorper der Frauen- und Kuhmilch, Inaug.-Diss. Strassburg, 1876.

2 See among others Bienenfeld, Bioch. Zeitschr., 7, and Fuld and Wohlgemuth,
ibid., 8.

3 Szontagh, Maly's Jahresber., 22; Zaitschek, 1. c.; Wr6blewsky, Beitrage zur
Kenntniss des Frauenkaseins, Inaug.-Diss. Bern. 1894, and Ein neuer eiweissartiger

662 MILK.

Woman's milk also contains lactalbumin, besides the casein, and a protein
substance, very rich in sulphur (4.7 per cent) and relatively poor in carbon, which
WROBLEWSKY calls opalisin. The statements as to the occurrence of proteoses and
peptones are conflicting as in many other cases. No positive proof as to the
occurrence of proteoses and peptones in fresh milk has been given.

Because of the properties and low amount of casein in human milk
it is often difficult to precipitate it, with acid, and to prepare it, but
this can easily be accomplished by dialysis. A number of methods
have been suggested for the preparation of human casein. FULD and
WOHLGEMUTH recommend the freezing of the milk previous to pre-
cipitation, so that the casein masses become larger to a certain extent
and the precipitation becomes easier. ENGEL l recommends dilution
with water to 5 volumes, and the addition of 60-80 cc. N/10 acetic acid
for each 100 cc. milk. The mixture is first cooled for 2-3 hours and then,
after shaking, warmed on the water-bath to 40° for a few minutes.

Even after those differences are eliminated which depend on the imper-
fect analytical methods employed, the quantitative composition of woman's
milk is variable to such an extent that it is impossible to give any average
results. The numerous analyses, especially those made on a large number
of samples by PFEIFFER, ADRIANCE, CAMERER and SoLDNER,2 have posi-
tively shown that woman's milk is essentially poorer in proteins but
richer in sugar than cow's milk. The quantity of protein varies between
10-20 p. m., often amounting to only 15-17 p. m. or less, and is dependent
upon the length of lactation (see below). The quantity of fat also varies
considerably, but ordinarily amounts to 30-40 p. m. The quantity of
sugar should not be below 50 p. m., but may rise to even 80 p. m. About
60 p. m. may be considered as an average, but it should be borne in mind
that the quantity of sugar is also dependent upon the length of lactation,
as it increases with duration. The amount of mineral bodies varies
between 2 and 4 p. m.

The division of the total nitrogen in human milk is, according to A.
FREHN,S very variable. As approximate average figures we can say
that 40-45 per cent of the total nitrogen is casein, 35-40 per cent remain-

Bestandteil der Milch, Anzeiger der Akad. d. Wiss. in Krakau, 1898; Kobrak, Pfluger's
Arch., 80; Langstein and Bergell, cited in Bioch. Centralbl., 8, 323; Langstein and
Edelstein, Maly's Jahresber, 40, 254; Abderhalden and Langstein. Zeitschr., f. physiol.
Chem., 66.

1 Fuld and Wohlgemuth, Bioch. Zeitschr., 5; Engel, ibid., 14.

2 Pfeiffer, Jahrb. f. Kinderheilkunde, 20, also Maly's Jahresber., 13; V. Adriance
and J. Adriance, A Clinical Report of the Chemical Examination, etc., Archives of
Pediatrics, 1897; Camerer and Soldner, Zeitschr. f. Biologic, 33 and 36. In regard
to the composition of Woman's milk, see also Biel, Maly's Jahresber., 4; Christenn,
ibid., 7; Mendes de Leon, ibid., 12; Gerber, Bull. soc. chim., 23; Tolmatscheff,
Hoppe-Seyler's Med.-chem. Untersuch., 272.

3 Zeitschr. f. physioi. Chem., 65; see also Engel and Frehn, Maly's Jahresber., 40.

HUMAN MILK. 663

ing proteins and about 20 per cent for rest nitrogen. The principal part
of the rest nitrogen is considered as urea.

From a quantitative standpoint, the most essential differences between
woman's and cow's milk are the following: As compared with the quan-
tity of albumin, the quantity of casein is not only absolutely but also
relatively smaller in woman's milk than in cow's milk, while the latter is
poorer in milk-sugar. Human milk is richer in lecithin, at least relatively
to the amount of protein. BUROW found 0.49-0.58 p. m. lecithin in cow's
milk and 0.58 p. m. in woman's milk, which corresponds to 1.40 per cent
for the first milk and 3.05 per cent for the second, calculated on the per-
centage of protein. NERKING and HAENSEL found as average for lecithin
in cow's milk 0.63 p. m. and in woman's milk 0.50 p. m. GLIKIN found
0.765 p. m. lecithin (phosphatides) as average for cow's milk and 1.329
p. m. for human milk. KOCH found that both human milk and cow's
milk contain lecithin as well as cephalin. The total quantity of both
bodies in human milk was 0.78 p. m. and in cow's milk 0.72-0.86 p. m.
The quantity of nucleon is greater in woman's milk. WITTMAACK claims
that cow's milk contains 0.566 p. m. nucleon, and woman's milk 1.24
p. m., and according to VALENTI the quantity of nucleon in human milk
is indeed still higher. SIEGFRIED finds that the nucleon phosphorus
amounts to 6.0 per cent of the total phosphorus in cow's milk and 41.5
per cent in woman's milk, and also that in human milk the phosphorus
is almost all in organic combination. This does not agree with the results
of SIKES who found on an average of only 42 per cent of the total P205
in organic combination. Because of the large amount of casein (and
calcium phosphate) cow's milk is much richer in phosphorus than human
milk. The relation P205:N, according to SCHLOSSMANN/ is equal to
1 : 5.4 in human milk and 1 : 2.7 in cow's milk. Woman's milk is poorer
in mineral bodies, especially lime, and it contains only one-sixth of the
quantity of lime as compared with cow's milk. The mineral constituents
of human milk are better assimilated by the organism of the nursing
child than those of cow's milk. Human milk is also claimed to be poorer
in citric acid (ScHEiBE2), although this is not an essential difference.

Another difference between woman's milk and other varieties of milk is
UMIKOFF'S reaction, which seems to depend upon the quantitative composition,
especially the relation between the milk-sugar, citric acid, lime, and iron (SIBBER 3).
This reaction consists in treating 5 cc. of woman's milk with 2.5 cc. ammonia

, Zeitschr. f. physiol. Chem., 30; Koch, ibid., 47; Wittmaack, ibid., 22;
Siegfried, ibid., 22; Nerking andHaensel, Bioch. Zeitschr., 13; G\ikm,ibid., 21; Valenti,
Biochem. Centralbl., 4; Schlossmann, Arch. f. Kinderheilkunde, 40; Sikes, Journ.
of Physiol., 34.

2 Maly's Jahresber., 21.

* Zeitschr. f. physiol. Chem., 30.

664 MILK.

(10 per cent) and heating to 60° C. for 15—20 minutes, when the mixture becomes
violet-red. Cow's milk gives a yellowish-brown color when thus treated.

According to RUBNER woman's milk contains about 3 p. m. soaps, but this
could not be substantiated by CAMERER and SOLDNER. They conclude that
woman's milk contains no soaps, or at least only very small amounts. They also
found the quantity of urea nitrogen in woman's milk to be 0.11-0.12 p. m.,
although SCHONDORFF l found nearly twice this amount, namely, 0.23 p. m.

, In regard to the quantity of mineral bodies in woman's milk we have
the analyses of several investigators, especially of BUNGE (analyses A
and B) and of SOLDNER and CAMERER (analysis C) .2 BUNGE analyzed the
milk of a woman, fourteen days after delivery, whose diet contained
very little common salt for four days previous to the analysis (A), and again
three days later after a daily addition of 30 grams of NaCl to the food
(B). The figures are in 1000 parts of the milk-

ABC

K20 0.780 0.703 0.884

Na20 0.232 0.257 0.357

CaO 0.328 0.343 0.378

MgO 0.064 0.065 0.053

Fe2O3 0.004 0.006 0.002

P2O6 0.473 0.469 0.310

Cl 0.438 0.445 0.591

The relation of the two bodies potassium and sodium to each other
may, BUNGE believes, vary considerably (1.3-4.4 equivalents of potash
to 1 of soda). By the addition of salt to the food, the quantity of
sodium and chlorine in the milk increases, while the quantity of potas-
sium decreases. DE LANGE found more Na than K in the milk at the
beginning of lactation. JOLLES and FRIEDJUNG found on an average
5.9 milligrams of iron per liter of woman's milk. CAMERER and SOLDNER 3
find about the same amount, namely, 10-20 milligrams Fe2Oa = 3.5-7
milligrams iron in 1000 grams human milk.

The gases of woman's milk have been investigated by KtJLZ.4 He
found 1.07 -1.44 cc. of oxygen, 2.35-2.87 cc. of carbon dioxide, and 3.37-
3.81 cc. of nitrogen in 100 cc. of milk.

The proper treatment of cow's milk by diluting it with water and by
certain additions in order to render it a proper substitute for woman's
milk in the nourishment of children cannot be determined before the
difference in the protein bodies of these two kinds of milk has been com-
pletely studied.

JRubner, Zeitschr. f. Biologie, 36; Camerer and Soldner, ibid., 39; Schondorff,
Pfliiger's Arch., 81.

2 Bunge, Zeitschr. f . Biologie, 10; Camerer and Soldner, ibid., 39 and 44.

3 De Lange, Maly's Jahresber., 27; Jolles and Friedjung, Arch. f. exp. Path. u.
Pharm., 46; Camerer and Soldner, Zeitschr. f. Biologie, 46.

4 Zeitschr. f. Biologie, 32.

HUMAN COLOSTRUM. 665

The colostrum has a higher specific gravity, 1.040-1.060, a greater
quantity of coagulable proteins, and a deeper yellow color than ordinary
woman's milk. Even a few days after delivery the color becomes less
yellow, the quantity of albumin less, and the number of colostrum-cor-
puscles diminishes.

We have the older analyses of CLEMM 1 and the recent investigations
of PFEIFFER, V. and J. ADRIANCE, CAMERER and SOLDNER on the changes
in the composition of milk after delivery. It follows, as a unanimous
result from these investigations, that the quantity of protein, which
amounts to more the first two days, sometimes to more than 30 p. m.
at first, rather quickly and then more generally diminishes as long as the
lactation continues, so that in the third week it equals about 10-18 p. m.
Like the protein substances, the mineral bodies also gradually decrease.
The quantity of fat shows no regular or constant variation during lacta-
tion, while the lactose, especially according to the observations of V.
and J. ADRIANCE (120 analyses), increases rather quickly the first days
and then only slowly until the end of lactation. The analyses of PFEIFFER,
CAMERER and SOLDNER also show an increase in the quantity of milk-sugar.

The two mammary glands of the same woman may yield somewhat different
milk, as shown by SOURDAT and later by BBUNNER.2 Likewise the different
portions of milk from the same milking may have varying composition. The
first portions are always poorer in fat.

According to L'HERITIER and to VERNOIS and BECQUEREL, the milk of blondes
contains less casein than that of brunettes, a difference which TOLMATSCHEFF 3
could not substantiate. Women of delicate constitutions yield a milk richer in
solids, especially in casein, than women with strong constitutions (V. and B.).

According to VERNOIS and BECQUEREL, the age of the woman has an effect on
the composition of the milk, so that we find a greater quantity of proteins and
fat in women 15^20 years old and a smaller quantity of sugar. The smallest
quantity of proteins and the greatest quantity of sugar are found at 20 or from
25 to 30 years of age. VERNOIS and BECQUEREL, consider that the milk with the
first-born is richer in water — with a proportionate diminution of casein, sugar,
and fat — than after several deliveries.

The influence of menstruation seems to diminish slightly the milk-sugar and
to increase considerably the fat and casein (VERNOIS and BECQUEREL).

Witch's milk is the secretion of the mammary glands of new-born children
of both sexes immediately after birth. This secretion has from a qualitative
standpoint the same constitution as milk, but may show important differences and
variations from a quantitative point of view. SCHLOSSBERGER and HAUFF,
GUBLER and QUEVENNE, and v. CENSER/ have made analyses of this milk and
give the following results: 10.5-28 p. m. proteins, 8.2-14.6 p. m. fat, and 9-60
p. m. sugar.

1 See Hoppe-Seyler, Physiol. Chem., 734.

2Sourdat, Compt. Rend., 71; Brunner, Pfliiger's Arch., 7.

3 1'Heritier, cited from Hoppe-Seyler, Physiol. Chem., 738; Vernois and Becquerel,
Du lait chez la femme dans 1'etat de sante, etc., (Paris, 1853) ; Tolmatscheff, Hoppe-
Seyler, Med.-chem. Untersuch., 272.

4 Schlossberger and Hauff, Annal. d. Chem., u. Pharm., 96; Gubler and Quevenne,
cited from Hoppe-Seyler 's Physiol. Chem., 723; v. Censer, ibid.

666 MILK.

As milk is the only form of nourishment during a certain period of
the life of man and mammals, it must contain all the nutriment necessary
for life. This fact is shown by the milk containing representatives of
the three principal groups of organic nutritive substances — proteins,
carbohydrates, and fat, and the last two groups can here also in part
mutually substitute each other. Besides this all milk seems to contain,
without doubt, some lecithin and nucleon. The mineral bodies in milk
must also occur in proper proportions, and on this point the experiments
of BUNGE on dogs are of special interest. He found that the mineral
bodies of the milk occur in about the same relative proportion as they
do in the body of the sucking animal. BUNGE l found in 1000 parts of
the ash the following results (A represents results from the new-born
dog, and B the milk from the bitch);

K2O.. 114.2 149.8

Na2O 106.4 88.0

CaO 295.2 272.4

MgO 18.2 15.4

FeaOs 7.2 1.2

P2O6 394.2 342.2

Cl 83.5 169.0

BUNGE explains the fact that the milk-ash is richer in potash and
poorer in soda than the new-born animal by saying that in the growing
animal the ash of the muscles rich in potash relatively increases and the
cartilage rich iti soda relatively decreases. In regard to the amount
of iron we find an unexpected condition, the ash of the new-born animal
containing six times as much as the milk-ash. This condition BUNGE
explains by the fact founded on his and ZALESKY'S experiments, that the
quantity of iron in the entire organism is highest at birth. The new-born
has therefore its own supply of iron for the growth of its organs even at
birth.

The investigations of HUGOUNENQ, DE LANGE, CAMERER and SOLDNER 2
have shown that in man the conditions are different from those in animals,
as the ash of the child has an entirely different composition as compared
with the milk. As an example the following analyses are given (of
CAMERER and SOLDNER). (A, the ash of the sucking infant, and B, the
ash of the milk.) The results are in 1000 parts of the ash.

K2O..

A

78

B
314

Na2O

91

119

CaO

361

164

MgO

9

26

Fe2O3

8

6

p,O5

389

135

Cl

77

200

1 Zeitschr. f. physiol. Chem., 13.

2Hugounenq, Compt. Rend., 128; de Lange, Zeitschr., f. Biologic, 40; Camerer
and Soldner, ibid., 39, 40, and 44.

INFLUENCE OF THE FOOD. 667

We cannot therefore state as a definite fact that the composition of
the ash of the sucking young and the ash of the corresponding milk coin-
cide. BUNGE 1 nevertheless claims that the composition of the ash of
the sucking young of various mammals is nearly the same, but that the
ash of the milk differs from the ash of the young in so far as the slower
the young grows the richer it is in alkali chlorides and relatively poorer
in phosphates and lime-salts. The constituents of the ash have two
functions to perform, namely, the building up of the tissues and secondly
the preparation of the excreta, especially the urine. The faster the
young grows the more is the first in evidence, while the slower it develops,
the more prominent is the second.

The quantity of mineral bodies in the milk, and especially the amount
of lime and phosphoric acid, as shown by BUNGE and PROSCHER and
PAGES, stands in close relation to the rapidity of growth, because the
amount of these mineral constituents in the milk is greater in animals
which grow and develop quickly than in those which grow only slowly.
A similar relation also exists, as shown by the researches of PROSCHER,
and especially of AfiDERHALDEN,2 between the quantity of protein in
the milk and the rapidity of development of the sucking young. The
amount of protein is greater in the milk the quicker the animal develops.

The influence of the food on the composition of the milk is of interest
from many points of view and has been the subject of many investigations.
From these we learn that in human beings as well as in animals an insuffi-
cient diet decreases the quantity of milk and the quantity of solids, while
abundant food increases both. From the observations of DECAiSNE3
on nursing women during the siege of Paris in 1871, the amount of casein,
fat, sugar, and salts, but especially the fat, was found to decrease with
insufficient food, while the quantity of lactalbumin was found to be some-
what increased. Food rich in proteins increases the quantity of milk,
and also the solids contained, especially the fat, according to most
reports. The quantity of sugar in woman's milk is found by certain
investigators to be increased after food rich in proteins, while others
claim it is diminished. A diet rich in fat may, as the researches of SOXHLET
and many others4 have shown, cause a marked increase in the fat of
the milk when the fat partaken is in a readily digestible and assimilable
form. The presence of large quantities of carbohydrates in the food

1 Bunge, " Die zimehmende Unfahigkeit der Frauen ihre Kinder zu stillen," Miin-
chen, 1900, cited by Camerer, Zeitschr. f. Biologic, 40.

2 Proscher, Zeitschr. f. physiol. Chem., 24; Abderhalden, ibid., 27; Pages, Arch,
de Physiol. (5), 7.

3 Cited from Hoppe-Seyler, 1. c., 739.

4 See Maly's Jahresber., 26. See also Basch, Ergebnisse der Physiologic, 2, Abt. 1.

668 MILK.

seems to cause no constant, direct action on the quantity of the milk
constituents.1 From feeding experiments with different foods we come
to the conclusion that the character of the food is of comparatively little
influence, while the race and other conditions play an important role.
Watery food gives a milk containing an excess of water and having little
value. In the milk from cows which were fed on distillers' grain COM-
MAILLE 2 found 906.5 p. .m. water, 26.4 p. m. casein, 4.3 p. m. albumin,
18.2 p. m. fat, and 33.8 p. m. sugar. Such milk has sometimes a peculiar
sharp after-taste, although not always. TANGL and ZAixscHEK3 could
not find any difference in the average composition of the milk produced
after feeding with dry and with moist fodder.

Chemistry of Milk-secretion. That the constituents which occur
actually dissolved in milk pass into the secretion and not alone by filtra-
tion or diffusion, but more likely are secreted by a specific secretory
activity of the granular elements, is shown by the fact that milk-sugar,
which is not found in the blood, is to all appearances formed in the glands
themselves. A further proof lies in the fact that the lactalbumin is not
identical with seralbumin; and lastly, as BuNGE4 has shown, the mineral
bodies secreted by the milk are in quite different proportions from those
in the blood-serum.

Little is known in regard to the formation and secretion of the specific
constituents of milk. The older theory, that the casein was produced
from the lactalbumin by the action of an enzyme, is incorrect, and prob-
ably originated from mistaking an alkali albuminate for casein. Better
founded is the theory that the casein originates from the protoplasm
of the gland-cells. According to BASCH'S researches, the casein is formed
in the mammary gland by the nucleic acid of the nucleus being set
free and uniting intra-alveolar with the transudated serum, thus form-
ing a nucleoalbumin, the casein. The untenableness of this view has
been shown by LOBISCH, and the investigations, of HILDEBRANDT 5
upon the proteolytic enzyme of the mammary gland, and the autolysis

1 In regard to the literature on the action of various foods on woman's milk, see
Zalesky, " Ueber die Einwirkung der Nahrung auf die Zusammensetzung und Nahr-
haftigkeit der Frauenmilch," Berlin, klin. Wochenschr., 1888, which also contains the
literature on the importance of diet on the composition of other kinds of milk. In
regard to the extensive literature on the influence of various foods on the .milk pro-
duction of animals, see Konig, Chem. d. menschl. Nahrungs und Genussmittel. 3. Aufl.,
1, 298. See also Maly's Jahresber., 29-40, and Morgen, Beger and Fingerling, Landw.
Versuchsst., 61, and Raudnitz, Monatschr. f. Kinderheilk.

2 Cited from Konig, 2, 235.

3 Landwirt. Vers. St. 1911.

4Lehrbuch d. physiol. und pathol. Chem., 3. Aufl., 93.

6 Basch, Jahrb. f. Kinderheilkunde, 1898; Hildebrandt, Hofmeister's Beitrage, 5;
Lobisch. ibid., 8.

CHEMISTRY OF MILK-SECRETION. . 669

of the gland have not given any clue as to the mode of formation of
casein. The findings of MANDEL l that the hydrolytic cleavage products
of the nucleoprotein from the mammary glands occur approximately
quantitatively in the same proportions as in casein, are important in this
connection.

That the milk-fat is produced by a formation of fat in the protoplasm,
and that the fat-globules are set free by their destruction, is a generally
admitted opinion, which, however, does not exclude the possibility that
the fat is in part taken up by the glands from the blood and eliminated
with its secretion. That the fats of the food can pass into the milk
follows from the investigations of WINTERNITZ, as he has been able to
detect the passage of iodized fats in the milk, and these observations
have been substantiated by the investigations of CASPARI and PARASCHT-
SCHUK.2 The abundant quantities of iodized fat which were eliminated
with the milk in these cases without doubt depend, at least in great part,
upon the iodized fat of the food, hence it cannot be said that all of the
milk-fat containing iodine was unchanged iodized fat of the food. The
previously-mentioned older investigations of LEBEDEFF and ROSENFELD
and also the recent ones of SPAMPANI and DADDI, PARASCHTSCHUK, GOGI-
TIDSE and others on the passage of foreign fats into the milk also indicate
the passage of the fat of the food into the milk, although we are still uncer-
tain on this point. According to SOXHLET the fat of the food does not
pass into the milk directly, but is destroyed in place of the body-fat,
which then becomes available and is, as it were, pushed into the milk.
HENRIQUES and HANSEN could not detect any mentionable quantity of
linseed-oil in the milk after feeding with this oil; the milk-fat was not
normal, but had a higher iodine equivalent and a higher melting-point,
from which they also concluded that a transformation of the food-f^t
in the glandular cells is possible. The results of the experiments of
GOGITIDSE 3 with soaps also indicate that the mammary glands have the
property of forming fats by synthesis from their components. As a
formation of fat from carbohydrates in the animal organism is at the
present day considered as positively proved, it is likewise possible that
the milk-glands also produce fats from the carbohydrates brought to
them by the blood. It is a well-known fact that an animal gives off
for a long time, daily, considerably more fat in the milk than it receives

1 Bioch. Zeitschr., 22.

2 Winternitz, Zeitschr. f. physiol. Chem., 24; Caspari, Arch. f. (Anat. u.) Physiol.,
1899, Supplbd. and Zeitschr. f. Biologic, 46; with Winternitz, ibid., 49; Paraschtschuk,
Chem. Centralbl., 1903, 1.

3 Lebedeff, Pfliiger's Arch. 31; Rosenfeld, Ergebn. d. Physiol. 1 and 2; Spampani
and Daddi, Maly's Jahresber., 26; Henriques and Hansen, ibid., 29; Gogitidse, Zeitschr.
f. Biologic, 45, 46, and 47. See also Basch/Ergebnisse d. Physiol., 2, Abt. 1.

670 MILK.

as food, and this proves that at least a part of the fat secreted by the
milk is produced from proteins or carbohydrates, or perhaps from both.
The question as to how far this fat is produced directly in the milk-
glands, or from other organs and tissues, and brought to the gland by
means of the blood, cannot be decided.

The origin of milk-sugar is not known. MUNTZ calls attention to the
fact that a number of very widely diffused bodies in the vegetable king-
dom— vegetable mucilage, gums, pectin bodies — yield galactose as a
product of decomposition, and he believes, therefore, that milk-sugar
may be formed in herbivora by a synthesis from glucose and galactose.
This origin of milk-sugar does not apply to carnivora, as they produce
milk-sugar when fed on food consisting entirely of lean meat. The
observations of BERT and THIERFELDER l that a mother-substance of
the milk-sugar, a saccharogen, occurs in the glands, does not explain
the formation of milk-sugar, as the nature of this mother-substance
is still unknown. As the animal body has undoubtedly the power of
converting one variety of sugar into another, the origin of the milk-
sugar can be sought simply in the glucose introduced as food or formed
in the body. Certain observations of PORCHER indicate such an origin
as he found in sheep, cows, and goats whose mammary glands "were
extirpated, that glucose appeared in the urine after delivery. He also
found that milk secreting animals 'became glycosuric on the removal
of the mammary glands, and he explains this glycosuria by the fact that
the lactose-forming action of the gland was removed at the time of delivery,
when large amounts of glucose were being produced. The experiments
of KAUFMANN and MAGNE upon cows also indicate a formation of lactose
from glucose. They found that during secretion the glands took sugar
from the blood, so that the venous gland-blood was poorer in sugar than
otherwise. NOEL-PATON and CATHCART2 have carried on experiments
on phlorhinized dogs which show a lactose formation from glucose.

The passage of foreign substances into the milk stands in close connec-
tion with the chemical processes of milk secretion.

It is a well-known fact that milk acquires a foreign taste from the
food of the animal, which is in itself a proof that foreign bodies pass into
the milk. This fact becomes of special importance in reference to such
injurious substances as may be introduced into the organism of the nurs-
ing child by means of the milk.

Among these substances may be mentioned opium and morphine,
which after large doses pass into the milk and act on the child. Alcohol

1 Muntz, Compt. Rend., 102; Bert and Thierf elder, footnote 1, p. 644.
2Porcher, Compt. Rend. 138 and 141 and Bioch. Zeitschr., 23; Kaufmann and
Magne, Compt. Rend., 143; Noel-Paton and Cathcart, Journ. of Physiol., 42.

MILK IN DISEASES. 671

may also pass into the milk, but probably not in such quantities as to
have any direct action on the nursing child.1 Alcohol is claimed to have
been detected in the milk after feeding cows with brewer's grains.

Among inorganic bodies, iodine, arsenic, bismuth, antimony, zinc,
lead, mercury, and iron have been found in milk. In icterus neither
bile-acids nor bile-pigments pass into the milk.

Under diseased conditions no constant change has been found in woman's
milk. In isolated cases SCHLOSSBERGER, JOLY and FILHOL 2 have indeed observed
a markedly abnormal composition, but no positive conclusion can be derived
therefrom.

The changes in cow's milk in disease have been little studied. In tuber-
culosis of the udder, STORCH 3 found tubercle bacilli in the milk, and he also noted
that the milk became more and more diluted, during the disease, with a serous
liquid similar to blood-serum, so that that the glands finally, instead of yielding
milk, gave only blood-serum or a serous fluid. HUSSON 4 found that milk from
murrain cows contained more proteins but considerably less fat and (in severe
cases) less sugar than normal milk.

The milk may be blue or red in color, due to the development of micro-organisms.

The formation of concrements in the exit-passages of the cow's udder is often
observed. These consist chiefly of calcium carbonate, or of carbonate and phos-
phate with only a small amount of organic substances.

1 See Klingemann, Virchow's Arch., 126, and Rosemann, Pfliiger's Arch., 78.

2 Schlossberger, Annal. d. Chem. u. Pharm., 96; Joly and Filhol, cited from v.
Gorup-Besanez, Lehrb., 4, Aufl., 438.

3 See Bang, Om Tuberkulose i Koens Yver og om tuberkulos Malk, Nord. Med.
Arkiv, 16, and also Maly's Jahresber., 14, 170; Storch, Maly's Jahresber., 14.

4 Compt. Rend., 73.

CHAPTER XIV.
URINE.

URINE is the most important excretion of the animal organism; it
is the means of eliminating the nitrogenous metabolic products, also
the water and the soluble mineral substances; and in many cases it
furnishes important data relative to the metabolism, quantitatively
by its variation, and qualitatively by the appearance of foreign bodies
in the excretion. Moreover, in many cases we are able, from the chemical
or morphological constituents which the urine abstracts from the kidneys,
ureter, bladder, and urethra, to judge of the condition of these organs; and
lastly urinary analysis affords an excellent means of deciding the question
as to how certain medicinal agents or other foreign substances intro-
duced into the organism are absorbed and chemically changed. In this
respect, urinary analysis has furnished very important particulars especially
in regard to the nature of the chemical processes taking place within
the organism, and it is therefore not only an important aid to the
physician in diagnosis, but it is also of the greatest importance to the
toxicologist and the physiological chemist.

In studying the secretions and excretions, the relation must be
sought between the chemical structure of the secreting organ and the
chemical composition of its secreted products. Investigations with
respect to the kidneys and the urine have led to very few results from
this standpoint. Although the anatomical relation of the kidneys has
been carefully studied, their chemical composition has not been the sub-
ject of thorough analytical research. In cases in which a chemical
investigation of the kidneys has been undertaken, it has been in general
only of the organ as such, and not of the different anatomical parts.
An enumeration of the chemical constituents of the kidneys known at
the present time can, therefore, only have a secondary value.

In the kidneys we find proteins of different kinds. According to
HALLIBURTON the kidneys do not contain any albumin, but only a
globulin and a nucleoprotein. The globulin coagulates at about 52° C.,
and the nucleoprotein contains 0.37 per cent phosphorus. LIEBER-
MANN claims that the kidneys contain a lecithalbumin, and he ascribes
to this body a special importance in the secretion of acid urines. The

672

THE KIDNEYS. 673

kidneys also contain, according to LONNBERG, a mucin-like substance.
This substance yields no reducing body on boiling with acids, and belongs
chiefly to the papillae, and is, this author says, a nucleoalbumin
(nucleoproteid?) . The cortical substance is richer in another nucleoal-
bumin (nucleoproteid) unlike mucin. It has not been decided what
relation this last substance bears to HALLIBURTON' s nucleoprotein.
Chondroitin sulphuric acid also occurs as traces. MANDEL and LEVENB
have also obtained glucothionic acid from the kidneys, and the question
as to the relation of this to the renosulphuric acid described by MANDEL
and NEUBERG l is still undecided. This renosulphuric acid to all appear-
ances is not a unit substance but a sulphuric acid ester, and a com-
ponent related to glucuronic acid which contained 2.63 p. c. S., 4.53
p. c., N., and 1.34 p. c. P.

Fat occurs only in very small amounts and this fat, like the organ
fat in general, is relatively rich in unsaturated fatty acids. The phos-
phatides seem to be of different kinds. FRANKEL and NOGUEIRA 2 found
a cephalin-like substance, a triaminodiphosphatide and a diamino-
monophosphatide. DUNHAM and JACOBSONS found in beef-kidneys a
substance which they called carnaubon which is soluble in alcohol but
insoluble in ether, and which is a triaminomonophosphatide with the
formula Cy^isoNaPOis. Carnaubon does not contain any glycerin
but an amino-sugar, two choline groups and a molecule of each of the
following acids: stearic, palmitic and carnaubic (C24H4sO2) acids. Among
the extractive bodies of the kidneys one finds purine bases, betaine* urea,
uric acid (traces), glycogen, leucine, inosite, taurine, and cystine (in ox-
kidneys). The quantitative analyses of the kidneys thus far made
possess little interest. In the kidney of a healthy suicide MAGNUS-
LEVY 5 found in 1000 parts of the fresh substance 756 p. m. water, 244
p. m. solids, 52.7 p. m. fat, 2.08 p. m. CL, 0.192 p. m. Ca., 0.207 p. m.
Mg and 0.158 p. m. Fe. *

The fluid collected under pathological conditions, as in hydronephrosis, is
thin with a variable but generally low specific gravity. Usually it is straw-yellow
or paler in color, and sometimes colorless. Most frequently it is clear, or only
faintly cloudy from white blood-corpuscles and epithelium-cells; in a few cases
it is so rich in form-elements that it appears like pus. Protein generally occurs
in small amounts; occasionally it is entirely absent, but in a few rare cases the

1 Halliburton, Journ. of Physiol., 13, Suppl., and 18; Liebermann, Pfliiger's Arch.,
50 and 54; Lonnberg, see Maly's Jahresber., 20; Mandel and Levene, Zeithschr. f.
physiol. Chem., 47; Mandel and Neuberg, Bioch. Zeitschr., 13; Morner, Skand.
Arch. f. Physiol., 6.

2 Bioch. Zeitschr., 16.

3 Zeitschr. f. physiol. Chem., 64.
4Bebeschin, Zeitschr., f. physiol. Chem., 72.
3 Bioch. Zeitschr., 24.

674 URINE. *

amount is nearly as large as in the blood-serum. Urea occurs sometimes in
considerable amounts when the parenchyma of the kidneys is only in part atro-
phied; in complete atrophy the urea may be entirely absent.

I. PHYSICAL PROPERTIES OF URINE.

Consistency, Transparency, Odor, and Taste of Urine. Under
physiological conditions urine is a thin liquid and gives, when shaken
with air, a froth which quickly subsides. Human urine, or urine from
earnivora, which is habitually acid, appears clear and transparent, often
faintly fluorescent, immediately after voiding. When allowed to stand for
a little while human urine shows a light cloud (nubecula), which consists of
the so-called '''mucus," and generally also contains a few epithelium
cells, mucus-corpuscles, and urate-granules. The presence of a larger
quantity of urates renders the urine cloudy, and a clay-yellow, yellowish-
brown, rose-colored, or often brick-red precipitate (sedimentum lateri-
tium) settles on cooling, because of the greater insolubility of the urates
at the ordinary temperature than at the temperature of the body.
This cloudiness disappears on gently warming. In new-born infants
the cloudiness of the urine during the first 4-5 days is due to epithelium,
mucus-corpuscles, uric acid, and urates. The urine of herbivora, which
is habitually neutral or alkaline in reaction, is very cloudy on account
of the carbonates of the alkaline earths present. Human urine may
sometimes be alkaline under physiological conditions. In this case it
is cloudy, due to the earthy phosphates, and this cloudiness does not
disappear on warming, differing in this respect from the sedimentum
lateritium. Urine has a salty and faintly bitter taste produced by sodium
chloride and urea. The odor of urine is peculiarly aromatic; the bodies
which produce this odor are unknown.

The color of urine is normally pale yellow when the specific gravity
is 1.020. The color otherwise depends on the concentration of the urine
and varies from pale straw-yellow, when the urine contains small amounts
of solids, to a dark reddish-yellow or reddish-brown in stronger con-
centration. As a rule the intensity of the color corresponds to the con-
centration, but under pathological conditions, exceptions occur such as
are found in diabetic urine, which contains a large amount of solids and
has a high specific gravity and a pale-yellow color.

The reaction of urine depends essentially upon the composition of the
food. The earnivora, as a rule, void an acid, the herbivora, a neutral
or alkaline urine. If a carnivore is put upon a vegetable diet, its urine
may become less acid or neutral, while the reverse occurs when an herbi-
vore is starved, that is, when it lives upon its own tissues, as then the
urine voided is acid.

The urine of a healthy man on a mixed diet has an acid reaction,

PHYSICAL PROPERTIES OF THE URINE. 675

and the sum of the acid equivalents is greater than the sum of the basic
equivalents. This depends upon the fact that in the physiological
combustion of neutral substances (proteins and others) within the
organism, acids are produced, chiefly sulphuric acid, but also phosphoric
and organic acids, such as hippuric, uric, and oxalic acids, aromatic
oxyacids, oxyproteic acids and others. From this it follows that the
acid reaction is not due to one acid alone. The various acids take part
in the acid reaction in proportion to their dissociation, since, according
to the ion theory, the acid reaction of a mixture is dependent upon the
number of hydrogen ions present. Hence the theory that the acidity
is due entirely to dihydrogen phosphate is incorrect although this salt
takes such a great part in the acid reaction that its quantity is often
taken as a measure of the degree of acidity of the urine.1

The composition of the food is not the only influence which affects the degree
of acidity of human urine. For example, after taking food at the beginning of
digestion, when a larger amount of gastric juice containing hydrochloric acid
is secreted, the urine may be neutral or even alkaline.2 As to the time of the
appearance of the maximum and minimum of acidity, the various investigators
do not agree, which may in part be explained by the varying individuality and
conditions of life of the persons investigated. It has not infrequently been
observed that perfectly healthy persons in the morning void a neutral or alkaline
urine which is cloudy from earthy phosphates. The effect of muscular activity
on the acidity of urine has not been positively determined. According to HOFF-
MANN, RINGSTEDT, ODDI, and TARULLI and VOZARIK muscular work raises the
degree of acidity, but ADUCCO 3 claims that it decreases it. Abundant perspira-
tion reduces the acidity (HOFFMANN).

In man and especially in carnivora it seems that the degree of acidity
of the urine cannot be increased above a certain point, even though
mineral acids or organic acids which are burned up with difficulty are
ingested in large quantities. Under such conditions a different behavior
has been repeatedly observed between carnivora and herbivora. In the
first (and also in man) it has been found that the acids are in part neu-
tralized by the alkalies and alkaline earths of the body, but that the
excess of acid is combined with ammonia, split off from the proteins or
their cleavage products, and eliminated in the urine as ammonium salt.
In herbivora such a combination of the excess of acid with ammonia

1 In regard to the acidity of the urine see the recent works of Ringer, Zeitschr. f .
physiol. Chem. 60; Henderson, Bioch. Zeitschr. 24, with Spiro, ibid., 15; De Jager,
Maly's Jahresb. 39 and Bioch. Zeitschr. 38; v. Skramlik, Zeitschr. f. physiol. Chem.
71; Klein and Moritz, Deutsch. Arch. f. klin. Med. 99; Quagliariello, Chem. Cen-
tralbl. 1912, 1, 506.

2 Contradictory statements are found in Linossier, Maly's Jahresber., 27.

3 Hoffmann, see Maly's Jahresber., 14; Ringstedt, ibid., 20; Oddi and Tarulli,
ibid., 24; Aducco, ibid., 17; Vozdrik, Pfliiger's Arch., 111.

676 URINE. •

seems not to take place, or not to the same extent,1 and this is given as
a reason why herbivora soon die when acids are given. This is true at
least for rabbits, while according to BAER this power of increasing the
elimination of ammonia exists also in the goat, monkey, and pig, hence
no definite difference in this regard exists between herbivora and carnivora.
The differences which have been observed are, according to EPPINGER,
not of a special kind, and they may be caused, he says, from a different
amount of protein in the food which yields ammonia. Thus dogs with
food poor in protein behave like rabbits while, according to EPPINGER,
in herbivora (rabbits) a de-toxification of the acid can be brought about
by the abundant supply of proteins or their cleavage products. The
correctness of this statement is still disputed (POHL) or has only been
partly confirmed (BOSTOCK). The point is disputed and it must not be
forgotten that, as A. LoEWY2 found, the sensitiveness toward the action
of acids varies very much in different individuals.

Although one cannot raise the degree of acidity of the urine above a
certain limit by the introduction of acid, still it may be easily diminished,
so that the reaction becomes neutral or alkaline. This occurs after the
taking of carbonates of the .fixed alkalies or of such alkali salts of vege-
table acids — citric acid, and malic acid — as are easily burned into car-
bonates in the organism. Under pathological conditions, as in the
absorption of alkaline transudates, or the alkaline fermentation within
the bladder, the urine may become alkaline.

A urine with an alkaline reaction caused by fixed alkalies has a very
different diagnostic value from one whose alkaline reaction is caused by
the presence of ammonium carbonate. In the latter case we have to
deal with a decomposition of the urea of the urine by the action of micro-
organisms.

If one wishes to determine whether the alkaline reaction of the urine
is due to ammonia or to fixed alkalies, a piece of red litmus paper is dipped
into the urine and allowed to dry exposed to the air or to a gentle heat.
If the alkaline reaction is due to ammonia, the paper becomes red again;
but if it is caused by fixed alkalies, it remains blue.

Determination of the Acidity. As the quantity of phosphoric acid
present as dihydrogen salt, as above stated, cannot be used as a measure
of the acidity, none of the older methods suggested for the. estimation
of this portion of the phosphoric acid is suited for acidity determinations.

1 See Winterberg, Zeitschr. f. physiol. Chem., 25, and J. Baer, Arch. f. exp. Path. u.
Pharm., 64.

2 Eppinger, Zeitschr. f. exp. Path. u. Therap., 3; with Tedesko, Bioch. Zeitschr.,
16; Pohl. ibid., 18; Staal, Zeitschr. f. physiol. Chem., 58; Bostock, ibid., 84; A.
Loewy, Centralbl. f. Physiol., 20; 337.

ACIDITY OF THE URINE. 677

We now determine the acidity simply by acidimetric methods, titrat-
ing with N/10 caustic alkali, using phenolphthalein as an indicator
(NAEGELI, HOBER, FOLIN). On account of the color of the urine and the
presence of ammonium salts and alkaline earths, this method cannot
yield entirely exact results. The greatest error is due to the alkaline
earths, which, on titration with caustic alkali, precipitate as earthy
phosphates in variable amounts and of variable composition. This
error can be prevented, according to FOLIN, by the addition of neutral
potassium oxalate, which precipitates the lime, and in this way the dis-
turbing action of the ammonium salts is also inhibited. Perfectly
accurate results are not obtained by this method, but it is the best of
those which have been suggested.

It is performed as follows: 25 cc. of urine are placed in an Erlenmeyer
flask (about 200 cc. capacity), treated with 1-2 drops of J-per cent
phenolphthalein solution, and shaken with 15-20 grams of powdered
potassium oxalate and immediately titrated with N/10 caustic soda
with constant shaking until a pronounced pale-rose color appears.
VOZARIK l titrates the diluted urine without the addition of oxalate
and uses phenolphthalein as indicator.

The acidity, as determined by titration, varies considerably under
physiological conditions, but calculated as hydrochloric acid it amounts
in man to about 1.5-2.3 grams in the twenty-four hours.

By titration we learn the amount of hydrogen present which can
be substituted by a metal, i.e., the acidity in the ordinary older sense,
but not the true acidity, the ion acidity, which is given by the concentra-
tion of the hydrogen ions of the urine. For similar reasons, as previously
indicated in treating of the alkalinity of the blood-serum (page 272),
the ion acidity cannot be determined by titration, while it can be deter-
mined according to the principle of the electrometric gas-chain method
as there given. Such estimations have been made by v. RHORER and by
HOBER. For normal urine v. RHORER found as a minimum 4X10""7,
as a maximum 76X10""7, and as an average 30X10"7. HOBER found
4.7X10"7, 100X10"7, and 49X10"7, respectively. On an average the
urine therefore contains 30-50 grams of hydrogen ions in 10 million
liters. HENDERSON2 has obtained much lower values, namely 10.10""7
as the average of 50 investigations, and has rather great differences
for different persons. From the comparative estimation of the titration

1 In regard to the degree of acidity and its estimation see Naegeli, Zeitschr. f .
physiol. Chem., 30; Hober, Hofmeister's Beitrage, 3; Folin, Amer. Journ. of Physiol.,
9; Vozarik, 1. c.; de Jager, Zeitschr. f. physiol. Chem., 55; and Ringer, ibid., 60; Grim-
bert and Morel, Compt. Rend., 154.

2v. Rhorer, Pfliiger's Arch., 86; Hober, 1. c. See also Jolles, Bioch. Zeitschr.,
13; Henderson, Bioch. Zeitschr., 24.

678 URINE.

acidity and the ion acidity it follows that no direct relation exists between
these and that the extent of these two acidities may be independent of
each other.

The osmotic pressure of the urine varies considerably even under
physiological conditions. The limit for the freezing-point depression
has been found by a number of investigators to be A 1.3° to 2.3° C. After
partaking of considerable water it may be markedly lower, and on
diminished supply of water it may be considerably higher.

In regard to the further physical-chemical imvestigations of the urine
and as to the conclusions drawn from a combination of the chemical
and the physico-chemical investigations of the urine, we must refer to
the extensive work of CARL NEUBERG.1

The specific gravity of urine, which is dependent upon the relation
existing between the quantity of water secreted and the solid urinary
constituents, especially the urea and sodium chloride, may vary con-
siderably, but is generally 1.017-1.020. After drinking large quantities
of water it may fall to 1.002, while after profuse perspiration or after
drinking very little water it may rise to 1.035-1.040. In new-born
infants the specific gravity is low, 1.007-1.005. The determination
of the specific gravity is an important means of learning the average
amount of solids eliminated from the organism in the urine, and on this
account the determination becomes of true value only when at the same
time the quantity of urine voided in a given time is determined. The
different portions of urine voided in the course of the twenty-four hours
are collected, mixed together, the total quantity measured, and then the
specific gravity taken.

The -determination of the specific gravity is most accurately obtained
with the pycnometer. For ordinary cases the specific gravity may be
determined with sufficient accuracy by means of areometers. The
areometers found in the trade, or urinometers, are graduated from 1.000
to 1.040; for exact observations it is better to use two urinometers, one
graduated from 1.000 to 1.020, and the other from 1.020 to 1.040.

To determine the specific gravity of urine, if necessary filter the
urine, or if it contains a urate sediment, first dissolve it by gentle heat,
then pour the clear urine into a dry cylinder, avoiding the formation of
froth. Air bubbles or froth, when present, must be removed with a glass
rod or filter-paper. The cylinder, which should be about four-fifths full,
must be wide enough to allow the urinometer to swim freely in the liquid
without touching the sides. The cylinder and urinometer should both
be dry or previously washed with the urine. On reading, the eye is
brought on a level with the lower meniscus — which occurs when the sur-
face of the liquid and the lower limb of the meniscus coincide; the read-

1 Der Harn sowie die iibrigen Ausscheidungen und Korperflussigkeiten von Mensch
und Tier. Teil. 2, Berlin, 1911.

ORGANIC PHYSIOLOGICAL CONSTITUENTS. 679

ing is then made from the point where this curved line coincides with
the scale of the urinometer. If the eye is not in the same horizontal
plane with the convex line of the meniscus, but is too high or too low,
the surface of the liquid assumes the shape of an ellipse, and the reading
in this position is incorrect. Before reading, press the urinometer gently
down into the liquid and then allow it to rise, and wait until it is at rest.

Each urinometer is graduated for a certain temperature, which,
at least in the case of the better ones, is marked on the instrument.
If the urine is not at the proper temperature, the following corrections
must be made: For every three degrees above the normal temperature
one unit of the last order is added to the reading, and for every three
degrees below the normal temperature one unit (as above) is subtracted
from the specific gravity observed. For example, when a urinometer
graduated for 15° C. shows a specific gravity of 1.017 at 24° C., then the
specific gravity at 15° C. = 1.017+0.003 = 1.020.

When great exactitude is required, as, for instance, a determina-
tion to the fourth decimal point, we make use of a urinometer constructed
by LoriNSTEiN.1 JOLLES 2 has also devised a small urinometer for the
determination of the specific gravity of small amounts of urine, 20-25
cc. The specific gravity may also be determined by the WESTPHAL
hydrostatic balance.

H. ORGANIC PHYSIOLOGICAL CONSTITUENTS OF URINE.

+ XNH2

Urea, Ur, CO^H* = CO^ , has been synthetically prepared in sev-

NNH2

eral ways, especially, as WOHLER showed in 1828, by the metameric
transformation of ammonium isocyanate: CO.N.NH4 = CO(NH2)2- It
is also produced by the decomposition or oxidation of certain bodies
found in the animal organism, such as purine bodies, creatine, arginine,
other amino-acids, and other substances.

Urea is found most abundantly in the urine of carnivora and man,
but in smaller quantities in that of herbivora. In carnivora (dog) the
urea nitrogen by abundant protein feeding may amount to 97-98 per cent
of the total nitrogen of the urine (SCHONDORFF 3) . The quantity in human
urine is ordinarily 20-30 p. m. It has also been found in small quantities
in the urine of amphibians, fishes, and certain birds. Urea occurs in
the perspiration in small quantities, and as traces in the blood and in
most of the animal fluids. It also occurs in rather large quantities in the
blood, liver, muscle,4 and bile 5 of sharks, even in rather large quantities.
Urea is also found in certain tissues and organs of mammals, especially

1 Pfliiger's Arch., 59; Chem. Centralbl., 1895, 1, and 1896, 2.

2 Wien. med. Presse, 1897, No. 8
8 Pfluger's Arch., 117.

4 v. Schroeder, Zeitschr. f. physiol. Chem., 14.

5 Hammarsten, ibid., 24.

680 URINE.

in the liver, spleen, muscles and others, although only in small amounts.
Under pathological conditions, as in obstructed excretion, urea may
appear to a considerable extent in the animal fluids and tissues.

The quantity of urea which is voided in twenty-four hours on a mixed
diet is in a grown man about 30 grams, in women somewhat less. While
children void less, the excretion relative to their body weight is greater
than in grown persons. The physiological significance of urea lies in
the fact that this body forms in man and carnivora, from a quantitative
standpoint, the most important nitrogenous end-product of the metabolism
of protein bodies. On this account the elimination of urea varies to a
great extent with the catabolism of the protein, and above all with the
quantity of absorbable proteins in the food ingested. The elimination
of urea is greatest after an exclusive meat diet, and lowest, indeed less
than during starvation, after the consumption of non-nitrogenous sub-
stances, since these diminish the metabolism of the proteins of the body.

If the consumption of the proteins of the body is increased, then
the elimination of nitrogen is correspondingly increased. This is found to
be the case in fevers, after poisoning with arsenic, antimony, phosphorus,
and other protoplasmic poisons, and when there is a diminished supply
of oxygen — as in severe and continuous dyspnoea, poisoning with carbon
monoxide, hemorrhage, etc. In these cases it used to be considered that
the rise in the excretion of nitrogen was due to an increased elimination
of urea, because no exact difference was made between the quantity
of urea and of total nitrogen in the urine. Recent researches have con-
clusively demonstrated the untrustworthiness of these observations.
Since PFLUGER and BORLAND have shown that 16 per cent of the total
nitrogen of the urine exists under physiological conditions in other com-
pounds, not urea, attention has been called to the relation of the dif-
ferent nitrogenous constituents of the urine to each other, and it has
been found, under pathological conditions, that this relation may vary
considerably, especially in regard to the urea. We have numerous
determinations by different investigators,1 on the relation of the different
nitrogenous constituents to each other in the normal urine of adults.

1 Pfluger and Bohland, Pfliiger's Arch., 38 and 43; Bohland, ibid., 43; Schultze,
ibid., 45; Camerer, Zeitschr. f. Biologic, 24, 27, and 28; Voges, Ueber die Mischung
der stickstoffhaltigen Bestandtheile im Harn. etc. (Inaug.-Diss. Berlin. 1892), cited
from Maly's Jahresber., 22; K. Morner and Sjoqvist, Skand. Arch. f. Physiol., 2.
See also Sjoqvist, Nord. Med. Arkiv., 1892, No. 36, and 1894, No. 10; Gumlich, Zeitschr.
f. physiol. Chem., 17; Bodtker, see Maly's Jahresber., 26; Folin, Amer. Journ. of
Physiol., 13; Osterberg and Wolff, Journ. of biol. Chem., 3; Haskins, ibid., 2; Bonze"
et Lambling, Journ. de Physiol. et de Path., 5; Bouchet, ibid., 14; Lambling et
Bouchet, Compt. rend. soc. biol., 71; Long and Gephart, Journ. Amer. Chem.
Soc., 34.

UREA. 681

Thus LONG and GEPHART found in the urine of six healthy men to whom
the same qualitative diet was fed for a long time, the following division
of the nitrogen in percentage of the total nitrogen: urea 79.87-84.34,
creatinine 5.21-6.87, ammonia 3.6-4.74, uric acid 1.57-1.99, purine
0.33-0.96 and rest nitrogen 4.23-6.01 per cent. SJOQVIST has made
similar determinations on new-born babes from 1 to 7 days old. From
all these analyses we obtain the following figures (A for adults and B for
new-born babes). Of the total nitrogen there exists:

A. B.

Per Cent. Per Cent.

Urea 84-91 73-76

Ammonia 2-5 7.8-9.6

Uric acid 1-3 3.0-8.5

Remaining nitrogenous substances 7-12 7 . 3-14 . 7

The variable relation between uric acid, ammonia, and urea nitro-
gen in children and adults is remarkable, since the urine of children
is considerably richer in uric acid and ammonia, and considerably poorer
in urea, than the urine of adults. A much larger number of analyses
of children's urine is necessary to explain the division of the nitrogen
therein. The absolute quantity of urea nitrogen in adults amounts to
about 10-16 grams per day. In disease the proportion of the nitroge-
nous substances may be markedly changed, and a decrease in the quan-
tity of urea and an increase in the quantity of ammonia have been observed
in certain diseases of the liver. This will be considered in detail in
connection with the formation of urea in the liver. It is natural that
there should be a diminished formation of urea after a decrease in the
ingestion of proteins or in a lowered catabolism. In diseases of the
kidneys which disturb or destroy the integrity of the epithelium of
the convoluted urinary tubules, the elimination of urea is considerably
diminished.

Recently by means of PFAUNDLER'S * method, by precipitating the urine with
phosphotungstic acid and closely studying the precipitate as well as the filtrate,
it has been possible to learn further about the division of the nitrogen of the urine.
We determine a, the total nitrogen; b, the nitrogen of the phosphotungstate pre-
cipitate; and c, the nitrogen in the filtrate from the phosphotungstate pre-
cipitate. This last contains the urea, hippuric acid, oxyproteic acids, and other
bodies whose nitrogen is ordinarily designated as monaminp-acid nitrogen. The
urea nitrogen is especially determined. The bodies precipitated by phospho-
tungstic acid are not all known; but uric acid and purine bases, ammonia,
creatinine, pigments, diamino-acids, diamines and ptomaines (if they occur), sul-
phocyanides, carbamic acid, urine mucoid, and proteid belong to this group.
Special methods have been suggested for the determination of several of these
substances (see below).

The urea nitrogen is always the greatest part of the total nitrogen,
but otherwise the division of the nitrogen undergoes considerable varia-

1 Zeitschr. f. physiol. Chem., 30.

682 URINE.

tion and very great variations seem to occur not only in the healthy
individual, but also and to a greater degree in diseased conditions.1

Formation of Urea in the Organism. The older statements of BE CHAMP
that urea is directly formed from proteins by oxidation has been denied
by several investigators but according to recent statements of FOSSE 2
this is correct. On the hydrolysis of proteins arginine is found among
other products, and as it is also produced in tryptic digestion, it is possible
that a small portion of the urea is produced in this manner, varying
according to the kind of protein. DRECHSEL claims that about 10 per
cent of the urea can be accounted for in this way.

The possibility of a formation of urea from arginine has gained in
interest since KOSSEL and DAKIN have discovered the presence of an
enzyme, arginase, in the liver and other organs, which has the power
of splitting arginine with the formation of urea. THOMPSON 3 has given
a direct proof for the formation of urea from arginine. The introduc-
tion of arginine into the body of a dog either per os or subcutaneously
has in his experiments led to an elimination of urea. While outside of
the body only one-half of the nitrogen of arginine is split off as urea
and the other half as ornithine, in the above experiments the increase
in urea in several instances corresponded to the greater part if not the
whole of the nitrogen of the arginine introduced. This increased forma-
tion of urea makes it probable that also ornithine is deamidized and the
urea is formed from the ammonia split off.

By the action of alkalies, as above mentioned (Chapter X), urea may
be formed from creatinine; still such an origin of urea in the animal body
has not thus far been proved.

The amino-acids are considered as special mother-substances of urea.
By numerous, generally older experiments with these acids, it has been
proved that the amino-acids of the animal body are transformed in part
into urea. The investigations by 'SALASKIN with the three amino-acids,
glycocoll, leucine, and aspartic acid, have unmistakably shown that the
surviving dog-liver, supplied with arterial blood, has the property of
transforming the above amino-acids into urea or a closely allied sub-
stance.4 Like the amino-acids the polypeptides are also transformed into

1 See Satta, Hofmeister's Beitrage, 6, which also gives the literature, and Erben,
Zeitschr. f. Heilkunde, 25.

2 Compt. Rend., 154.

3 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41; Thompson, Journ. of PhysioL,
32 and 33.

4Schultzen and Nencki, Zeitschr. f. Biologic, 8; v. Knieriem, ibid., 10; Salkowski,
Zeitschr. f. physiol. Chem., 4; Salaskin, ibid., 25; Stolte, Hofmeister's Beitrage, 5;
Levene and Meyer, Amer. Journ. of PhysioL, 25; see also Loewi, Zeitschr. f. physiol.
Chem., 25; Richet, Compt. Rend., 118, and Compt. rend. Soc. biol., 49; Ascoli,
Pfliiger's Arch., 72.

FORMATION OF UREA. 683 v

urea in the animal body, as shown by the investigations of ABDERHALDEN
and his collaborators.1

There is no doubt that the ammonia formation is of great importance
in the production of urea in the animal body.

A great number of older investigations2 on the behavior of ammo-
nium salts in the animal body have shown that not only ammonium car-
bonate, but also those ammonium salts which are burned into carbonate
in the organism, are transformed into urea by carnivora as well as her-
bivora. v. ScHROEDER,3 by irrigating the surviving dog's liver with
blood treated with ammonium carbonate or ammonium formate, has
shown that the formation of urea takes place, at least in part, in this organ.
NENCKI, PAWLOW, ZALESKI and SALASKIN 4 have also found that, in dogsr
the quantity of ammonia in the blood from the portal vein is considerably
greater than that from the hepatic vein, and they claim that the liver
retains in great part the ammonia thus supplied. The formation of urea
from ammonia in the liver is a positively proved fact.

The assumption of a splitting off of ammonia from amino-acids
stands in agreement with the experience that a deamidation of the amino-
acids takes place in the animal body. The ammonia split off finds, in
the blood and tissues, the carbon dioxide necessary for the formation
of carbonate, and the investigations of NOLF, as well as those of MACLEOD
and HASKiNS,5 on the equilibrium of carbonate and carbamate solutions
and the conditions for the formation of both salts, must also be abundant
evidence of a carbamate formation.

Important observations have been made which give support to the
views of SCHULTZEN and NENCKI, 6 namely, that the amino-acids are
transformed into urea with ammonium carbamate, EUN.O.CO.NH^,
as an intermediate step. DRECHSEL has shown that the amino-acids
yield carbamic acid by oxidation in alkaline fluid outside of the organism,
and he obtained urea from ammonium carbamate by alternate oxidation
and reduction. Carbamate has also been found in the blood (DRECHSEL)
as well as in the urine (DRECHSEL, ABEL and MUIRHEAD} 7 and NENCKI

1 Abderhalden with Teruuchi and with Babkin, Zeitschr. f. physiol. Chem., 47,
with Schittenhelm, ibid., 51.

2v. Knieriem, Zeitschr. f. Biologie, 10; Feder, ibid., 13; Salkowski, Zeitschr. f.
Biologie, 1; Munk, ibid., 2; Coranda, Arch. f. exp. Path. u. Pharm., 12; Schmiede-
berg and Walter, ibid., 7; Hallervorden, ibid., 10; Pohl and Miinzer, Arch., f. exp.
Path. u. Pharm., 43.

3 Arch. f. exp. Path. u. Pharm., 15. See also Salomon, Virchow's Arch., 97.

4 Arch, des sciences biol. de St. Petersbourg, 4; see also Chapter V, p. 336.

6 Nolf, Zeitschr. f. physiol. Chem., 23; Macleod and Haskins, Journ. of biol. Chem., 1.

6 Zeitschr. f . Biologie, 8.

7 Drechsel, Ber. d. sachs. Gesellsch. d. Wissensch., 1875. See also Journ. f. prakt.
Chem. (N. F.), 12, 16, and 22; Abel, Arch. f. (Anat. u.) Physiol., 1891; Abel and
Muirhead, Arch. f. exp. Path. u. Pharm., 31.

684 URINE.

and HAHN have made further observations on dogs with Eck's fistula,
which substantiate this view. In such fistula dogs, they observed that
when meat was fed, violent poisonous symptoms developed which were
almost identical with those produced when carbamate was introduced
into the blood. The same symptoms also appeared on the introduction
of carbamate into the stomach of the fistula animal, while the intro-
duction of carbamate into the stomach of a normal dog had no action.1
As these observers also found that the urine of the dog on which the
operation was made was richer in carbamate than that of the normal
dog, they concluded that the symptoms were due to the non-transforma-
tion of the ammonium carbamate into urea in the liver, and they consider
the ammonium carbamate as the substance from which the urea is derived
in the mammalian liver.

Besides the above view of the formation of urea from ammonium
carbonate and carbamate, which has been called the anhydride theory,
we also have the oxidation theory of HOFMEISTER.

F. HOFMEISTER 2 found in the oxidation of different members of
the fatty series, as well as in amino-acids and proteins, that urea was
formed in the presence of ammonia, and he therefore suggests the pos-
sibility that urea may be formed by an oxidation-synthesis. Accord-
ing to him, in the oxidation of nitrogenous substances a radical CONH2,
containing the amide group, unites at the moment of formation with the
radical NH2 remaining on the oxidation of ammonia, forming urea.

Besides the above-mentioned theories as to the formation of urea,
there are others which will not be given, because the only theory which
has thus far been positively demonstrated is the formation of urea in
the liver from ammonium compounds and amino-acids.

The liver is the only organ in which, up to the present time, a forma-
tion of urea has been directly detected;3 and the question arises, what
importance has this urea formation which takes place in the liver? Is
the urea wholly or chiefly formed in the liver?

If the liver is the only organ capable of forming urea, it is to be
expected, on the extirpation or atrophy of that organ, that a reduced

1 Hahn, Massen, Nencki et Pawlow, La fistule d'Eck de la veine cave inferieure et
de la veine porte, etc. Arch, des sciences biol. de St. P£tersbourg, 1, No. 4, 1892.
In regard to certain differences between the symptoms with carbamate poisoning and
after meat feeding with Eck fistula dogs, see Rothberger and Winterberg, Zeitschr.
f . exp. Path. u. Therap., 1; Hawk, Amer. Journ. of Physiol., 21.

2 Arch. f. exp. Path. u. Pharm., 37.

3 In regard to the investigations of Prevost and Dumas, Meissner, Voit, Grehant,
Gscheidlen and Salkowski, and others, on the role of the kidneys in the formation of
urea, see v. Schroeder, Arch. f. exp. Path. u. Pharm., 15 and 19, and Voit, Zeitschr.
f. Biologic, 4.

FORMATION OF UREA. 685

or, in short experiments, at least a strongly diminished elimination of
urea should occur. As at least a part of the urea is formed in the liver
from ammonium compounds, a simultaneous increase in the elimination
of ammonia is to be expected.

The extirpation and atrophy experiments made on animals by dif-
ferent methods 1 have shown that sometimes a rather marked increase
of ammonia and a diminished elimination of urea takes place after the
operation, but that there are also cases in which, irrespective of the pro-
nounced atrophy, an abundant formation of urea occurs, and no appre-
ciable, if any, change in the proportion of ammonia to the total nitrogen
and urea is observed. After shutting out from the circulation the organs
of the posterior part of the body, especially the liver and kidneys, KAUF-
MANN 2 also found an important increase in the urea of the blood, and these
different observations show that the liver is not the only organ, in the
various animals experimented upon, in which urea is formed.

The observations made by numerous investigators 3 on human beings
with cirrhosis of the liver, acute yellow atrophy of the liver, and phos-
phorus poisoning have led to the same result. These investigations
teach that in certain cases the proportion of the nitrogenous substances
may be so changed that urea is only 50-60 per cent of the total nitrogen,
while in other cases, on the contrary, even in very extensive atrophy
of the liver-cells, the formation of urea is not diminished, neither is the
proportion between the total nitrogen, urea, and ammonia essentially
changed. Even in the cases in which the formation of urea was relatively
diminished and the elimination of ammonia considerably increased, fur-
ther investigation must be instituted before it will be possible to assume
a reduced ability of the organism to produce urea. An increased elimi-
nation of ammonia may, as shown by MUNZER in the case of acute
phosphorus poisoning, be dependent upon the formation of abnormally
large quantities of acids, caused by abnormal metabolism, and these acids
require a greater quantity of ammonia for their neutralization according
to the law of elimination of ammonia. That an abnormal formation

1 Nencki and Hahn, 1. c.; Slosse, Arch. f. (Anat. u.) Physiol., 1890; Lieblein, Arch,
f. exp. Path. u. Pharm., 33; Nencki and Pawlow, Arch, des science, biol. de St. Pe"ters-
bourg, 5. See also v. Meister, Maly's Jahresber., 25; Salaskin and Zaleski, Zeitschr. f .
physiol. Chem., 29; Fischler and Bardach, ibid., 78.

2 Compt. rend. soc. biol., 46, and Arch, de Physiol. (5), 6.

3 See Hallervorden, Arch. f. exp. Path. u. Pharm., 12; Weintraud, ibid., 31; Miinzer
and Winterberg, ibid., 33; Stadelmann, Deutsch. Arch. f. klin. Med., 33; Fawitzki,
ibid., 45; Munzer, ibid., 52; Frankel, Berlin, klin. Wochenschr., 1878; Richter, ibid.,
1896; Morner and Sjoqvist, Skand. Arch. f. Physiol., 2, and Sjoqvist, Nord. Med.
Arkiv, 1892; Gumlich, Zeitschr. f. physiol. Chem., 17; v. Noorden, Lehrb. d. Pathol.
des Stoffwechsels, 2. Aufl., Bd. 1, 104.

686 UKINE.

of acid occurs after the cutting out of the liver has been especially shown
by SALASKIN and ZALESKi.1

For the present we are not justified in the statement that the liver
is the only organ in which urea is formed, and only continued investiga-
tion can yield further information as to the extent and importance of the
formation of urea, from ammonium compounds, in the liver.

Properties and Reactions of Urea. Urea crystallizes in needles or in
long, colorless, four-sided, often hollow, anhydrous rhombic prisms.
It has a neutral reaction, and produces a cooling sensation on the tongue
like saltpeter. It melts at 132° C. At ordinary temperatures it dis-
solves in an equal weight of water and in five parts alcohol; it requires
one part boiling alcohol for solution; it is insoluble in alcohol-free anhy-
drous ether, and also in chloroform. If urea in substance is heated in a
test-tube, it melts, decomposes, gives off ammonia, and finally leaves a
non-transparent white residue which, among other substances, contains
cyanuric acid and biuret, which latter dissolves in water, giving a beautiful
reddish-violet liquid with copper sulphate and alkali (biuret reaction).
On heating with baryta-water or caustic alkali, also in the so-called
alkaline fermentation of urine caused by micro-organisms, urea splits
into carbon dioxide and ammonia with the addition of water. The
same decomposition products are produced when urea is heated with
concentrated sulphuric acid. An alkaline solution of sodium hypo-
bromite decomposes urea into nitrogen, carbon dioxide, and water accord-
ing to the equation

With a concentrated solution of furfuroi and hydrochloric acid, urea
in substance gives a coloration passing from yellow, green, blue, to violet,
and then after a few minutes beautiful purple-violet (SCHIFF'S reaction).
According to HuppERT2 the test is best performed by taking 2 cc. of a
concentrated furfuroi solution, 4-6 drops of concentrated hydrochloric
acid, and adding to this mixture, which must not be red, a small crystal
of urea. A deep violet coloration appears in a few minutes.

Urea forms crystalline compounds with many acids. Among these
the one with nitric acid and the one with oxalic acid are the most
important.

UREA NITRATE, CO(NH2)2.HN03. On crystallizing quickly, this
compound forms thin rhombic or six-sided overlapping tiles, or colorless

1 Zeitschr. f. physiol. Chem., 29.

2 Huppert-Neubauer, Analyse des Hams, 10. Aufl., 296.

PROPERTIES AND REACTIONS OF UREA. 687

plates, with an angle of 82°. When crystallizing slowly, larger and
thicker rhombic pillars or plates are obtained. This compound is rather
easily soluble in pure water, but is considerably less soluble in water
containing nitric acid; it may be obtained by treating a concentrated
solution of urea with an excess of strong nitric acid free from nitrous
acid. On heating this compound it volatilizes without leaving a residue.

This compound may be employed with advantage in detecting small amounts
of urea. A drop of the concentrated solution is placed on a microscope slide and
the cover-glass placed upon it; a drop of nitric acid is then placed on the side
of the cover-glass and allowed to flow under. The formation of crystals begins
where the solution and the nitric acid meet. Alkali nitrates may crystallize
very similarly to urea nitrate when they are contaminated with other bodies;
therefore, in testing for urea, the crystals must be identified • as urea nitrate by
heating and by other means.

UREA OXALATE, 2.CO(NH2)2-H2C204. This compound is more
sparingly soluble in water than the nitric-acid compound. It is obtained
in rhombic or six-sided prisms or plates on adding a saturated oxalic-
acid solution to a concentrated solution of urea.

Urea also forms combinations with mercuric nitrate in variable
proportions. If a very faintly acid mercuric-nitrate solution is added
to a 2 per cent solution of urea and the mixture carefully neutralized,
a compound is obtained of a constant composition which contains for
every 10 parts of urea 72 parts of mercuric oxide. This compound serves
as the basis of LIEBIG'S titration method. Urea also combines with
salts, forming mostly crystallizable combinations, as, for instance, with
sodium chloride, with the chlorides of the heavy metals, etc. An alka-
line but not a neutral solution of urea is precipitated by mercuric chloride.

If urea is dissolved in dilute hydrochloric acid and then an excess of formal-
dehyde is added, a thick, white, granular precipitate is obtained which is dif-
ficultly soluble and whose composition is somewhat disputed.1 With phenyl-
hydrazine, urea in strong acetic acid gives a colorless crystalline compound of
phenylsemicarbazid, CeHeNH.NHiCONHU, which is soluble with difficulty in cold
water and melts at 172° C. (JAFF£ 2).

The method of preparing urea from urine is in the main as follows:
Concentrate the urine, which has been faintly acidified with sulphuric
acid, at a low temperature, add an excess of nitric acid, at the same time
keeping the mixture cool, press the precipitate well, decompose it in
water with freshly precipitated barium carbonate, dry on the water-
bath, extract the residue with strong alcohol, decolorize when necessary
with animal charcoal, and filter while warm. The urea which crystallizes

*See Tollens and his pupils, Ber. d. deutsch. chem. Gesellsch., 29, 2751; Gold-
schmidt, ibid., 29; and Chem. Centralbl., 1897, 1, 33; Thorns, ibid., 2, 144 and 737.
2 Zeitschr. f. physiol. Chem., 22.

688 URINE.

on cooling is purified by recrystallization from warm alcohol. A fur-
ther quantity of urea may be obtained from the mother-liquor by con-
centration. The urea is purified from contaminating mineral bodies
by redissolving in alcohol-ether. If it is only necessary to detect the
presence of urea in urine, it is sufficient to concentrate a little of the
urine on a watch-glass and, after cooling, treat it with an excess of nitric
acid. In this way we obtain crystals of urea nitrate.

Quantitative Estimation of the Total Nitrogen and Urea in Urine.
Among the various methods proposed for the estimation of the total
nitrogen, that suggested by KJELDAHL is to be recommended. LIEBIG'S
method for the estimation of urea is really a method for determining
the total nitrogen, but as it is very seldom used now, we can refer to
larger works in regard to details.

KJELDAHL'S method consists in transforming all the nitrogen of the
organic substances into ammonia by heating with a sufficiently con-
centrated sulphuric acid. The ammonia is distilled off, after super-
saturating with alkali, and collected in standard sulphuric acid. The
following reagents are necessary:

1. Sulphuric Acid. Either a mixture of equal volumes of pure con-
centrated and fuming sulphuric acid, or else a solution of 200 grams
phosphoric anhydride in 1 liter of pure concentrated sulphuric acid.
2. Caustic soda free from nitrates, 30-40 per cent solution. The quantity
of this caustic-soda solution necessary to neutralize 10 cc. of the acid
mixture must be determined. 3. Metallic mercury or pure yellow mercuric
oxide. (The addition of this facilitates the destruction of the organic
substances.) 4. A potassium-sulphide solution of 4 per cent, whose
object is to decompose any mercuric amide combination which might
not evolve il£ ammonia completely during the distillation with caustic
soda. 5. 1/5 normal sulphuric acid and 1/5 normal caustic soda solution.

In performing the determination 5 cc. of the carefully measured and
filtered urine are placed in a long-necked KJELDAHL flask, a drop of mercury
or about 0.3 gram of mercuric oxide added, and then treated with 10-15
cc. of the strong sulphuric acid. The contents are heated very care-
fully, placing the flask at an angle, until they just begin to boil gently;
this is continued for about half an hour after the mixture becomes color-
less. On cooling, the contents are transferred to a voluminous distilling-
flask, carefully washing the KJELDAHL flask with water and the greater
part of the acid is neutralized by caustic soda. A few zinc shavings are
added to prevent too rapid ebullition on distillation, and then an excess
of caustic-soda solution which has previously been treated with 30-40
cc. of the potassium-sulphide solution. The flask is quickly connected
with the condenser-tube and all the ammonia distilled off. In order
to prevent loss of ammonia it is best to lower the end of the exit-tube
below the surface of the acid, the regurgitation of the acid being prevented
by having a bulb blown on the exit-tube. Not less than 25-30 cc. of
the standard acid is used for every 5 cc. of urine, and on completion
of the distillation the acid is retitrated with 1/5 normal caustic soda
using rosolic acid, tincture of cochineal, or lacmoid as indicator. Each
cubic centimeter of the acid corresponds to 2.8 milligrams nitrogen. As a
control and in order to test the purity of the reagents, or to eliminate
any error caused by an accidental quantity of ammonia in the air, we
always make a blank determination with the reagents.

METHODS FOR THE DETERMINATION OF UREA. 689

Recently FOLIN and FARMER 1 have suggested a method for the estima-
tion of the total nitrogen in very small quantities of urine, 1 cc. dilute
urine. After hydrolysis with acid the ammonia formed is colorimetrically
determined by means of NESSLER'S reagent.

Among the methods suggested for the special estimation of urea,
that of MORNER-SJOQVIST, in combination with FOLIN'S method, is the
one that is generally used.

Principle of Mvrner-Sjdqyist's Method.2 According to this method
the nitrogenous constituents of the urine, with the exception of urea,
ammonia, hippuric acid, creatinine, and traces of allantoin,3 are pre-
cipitated by a mixture of alcohol and ether after the addition of a solu-
tion of barium chloride and barium hydroxide, or in the presence of sugar
with solid barium hydroxide. The urea is determined in the concentrated
filtrate, after driving off the ammonia, by KJELDAHL'S nitrogen estima-
tion. The slight error due to the presence of hippuric acid and creatinine
can be prevented according to MORNER by a combination of his method
with FOLIN'S method.

Principle of Folin's Method.4 On heating urea with hydrochloric
acid and crystalline magnesium chloride, which melts in its water of
crystallization at 112-115° C. and then boils at about 150-155° C., the
urea is completely decomposed, while no appreciable decomposition
of the hippuric acid and creatinine takes place. The ammonia produced
from the urea is distilled off and determined by titration. The amount
of ammonia previously existing in the urine must be specially determined.

Determination of Vrea by the Morner-Sjoqvist and Folin Method.5
Five cc. of the urine are treated with 1.5 grams of powdered barium
hydroxide, and when as much of this is dissolved as possible by gently
mixing, it is precipitated by 100 cc. of the alcohol and ether mixture
(J vol. ether). On the following day it is filtered and the precipitate
washed with the alcohol and ether mixture. The alcohol and ether
are distilled off from the filtrate at about 55° C. (not above 60° C.). The
remaining liquid is treated with 2 cc. of hydrochloric acid of sp.gr.
1.124 (for 5 cc. urine), and carefully transferred to a flask of 200 cc.
capacity, and evaporated to dryness on the water-bath. Then add 20
grams of crytalline magnesium chloride to the contents of the flask
and 2 cc. of concentrated hydrochloric acid, and boil on a wire gauze
over a small flame for two hours, making use of a proper return cooler.

1 Journ. of biol. Chem., 11.

2Skand. Arch. f. Physiol., 2, and Morner, ibid., 14, where the recent literature
may also be found.

3 According to Wiechowski, Hofmeister's Beitrage, 11, the quantity of allantoin
is so great iji urine that it must be considered in this method.

4 Zeitschr. f. physiol. Chem., 32, 36 and 37.
6 See Morner, Skand. Arch, f . Physiol., 14.

690 URINE.

After cooling it is diluted to about f to 1 liter with water, the ammonia
completely distilled off, after making it alkaline with caustic soda, and
the ammonia collected in standard acid. After boiling in order to drive
off the C02 and cooling, the acid is retitrated.

In recent years objections of various kinds have been made against
these methods, which are directed towards their exactness and which have
led to changes in several directions (BENEDICT and GEPHART, LEVENE
and MEYER, GILL, ALLISON and GRINDLEY). These changes are:
precipitation of the other nitrogenous substances (nearly all the ammonia)
with phosphotungstic acid, decomposition of the urea in the nitrate by
heating with acid in an autoclave to 150,° and distilling off the ammonia
from the solution, made alkaline, not by boiling with alkali, but by the
aid of a vacuum or by means of a current of air. These changes have
been carefully studied by HENRIQUES and GAMMELTOFT 1 and they have
suggested the following method:

Henriques and Gammeltoft Method. First determine in 5 cc. urine
how much of a 10 per cent phosphotungstic acid solution (in N/2 H^SCU)
is necessary to exactly cause a complete precipitation. Then place 10 cc.
of the urine in a 100 cc. flask, add the determined quantity of phospho-
tungstic acid solution and fill the flask up to the 100 cc. mark with N/2
H2S04. The liquid is allowed to stand after mixing until it has settled
and it is then filtered. Two portions of 10 cc. each are placed in test-
tubes of Jena glass, covered with tin-foil and placed in the autoclave at
150° C. for 1J hours. The contents of the test-tubes are now placed in a
flask, and the ammonia determined either by passing a current of air
through it (after the addition of sodium carbonate) or by distillation in a
vacuum (after the addition of barium hydrate dissolved in methyl alcohol) .
FOLIN and PETTIBONE 2 have suggested a method, according to which the
ammonia is determined colorimetrically with Nessler reagent.

KNOP-HUPNER'S method 3 is based on the fact that urea, by the action of
sodium hypobromite, splits into water, carbon dioxide (which dissolves in the
alkali), and nitrogen, whose volume is measured (see page 686). This method
is less accurate than the preceding ones, and therefore in scientific work it is dis-
carded. It is of value to the physician and for practical purposes, because of
the ease and rapidity with which it may be performed, even though it may not
give very accurate results. For practical purposes a number of different appa-
ratus have been constructed to facilitate the use of this method.

1 Benedict and Gephart, Journ. of Amer. Chem. Soc., 30; Levene and Meyer,
ibid., 31; Gill, Allison and Grindley, ibid., 31; Henriques and Gammeltoft, Skand.
Arch. f. Physiol., 25.

2 Folin and Pettibone, Journ. of Biol. Chem., 11.

3 Knop, Zeitschr. f. analyt. Chem., 9; Hiifner, Journ. f. prakt. Chem. (N. F.), 3.
In regard to the extensive literature, see Huppert-Neubauer, 10. Aufl., 304, and follow-
ing. See also Keogh, Zeitschr. f. physiol. Chem., 84.

CARBAMIC ACID. 691

In regard to other methods such as BUNSEN'S method with its many
modifications as suggested by PFLUGER, BORLAND and BLEIBTREU,
we refer to more complete handbooks.

For the quantitative estimation of urea in blood or other animal
fluids, as well as in the tissues, SCHONDORFF has proposed a method
where the proteins and extractives are first precipitated by a mixture
of phosphotungstic acid and hydrochloric acid, and then the filtrate
made alkaline with lime. The quantity of ammonia formed on heating
a part of this filtrate to 150° C. with phosphoric acid and the amount
of carbon dioxide produced by heating the other part to 150° C. are
determined. In regard to the principles of this method, as well as to
the details, we refer to the original article (PFLUGER'S Arch., 62). SAL-
KOWSKI 1 has recently suggested a method for estimating the urea in
tissues.

Urein is the name given by OVID MOOR to a product which he obtained by
extracting urine, which had been evaporated to a syrup, with absolute alcohol
and precipitating the urea with alcohol containing oxalic acid, or by cooling and
treatment with alcohol. Urein is a golden-yellow oil which is poisonous; it
reduces permanganate in the cold, and it forms the chief portion of the nitro-
genous extractives of urine. There is no doubt that urein is a mixture of several
substances. According to Moon,2 the amount of urea in the urine is only about
one-half that ordinarily given, and he has suggested a new method for the deter-
mination of the true quantity of urea. The possibility that in the urine we have
other bodies besides urea which have been determined with the urea cannot be
denied a priori. From the investigations published so far it must be said that
MOOR'S assertions are not sufficiently grounded.3

yNH2

Carbamic Acid, CH3N02=CO\ . This acid is not known in the free

state, but only as salts. Ammonium carbamate is produced by the action of
dry ammonia on dry carbon dioxide, but also after the addition of Na2C03 to a
solution which contains an ammonium salt (MACLEOD and HASKINS). Carbamic
acid is also produced by the action of potassium permanganate on protein and
several other nitrogenous organic bodies.

The occurrence of carbamic acid in human and animal urines has already
been considered in connection with the formation of urea. The calcium salt
which is soluble in water and ammonia but insoluble in alcohol, is the most impor-
tant in the detection of this acid. The solution of the calcium salt in water
becomes cloudy on standing, but much more quickly on boiling, and calcium car-
bonate separates. NOLF, MACLEOD and HASKINS have made experiments as to
the method of formation of carbamic acid. The latter have indicated a new
method for the quantitative estimation of carbamates.4

1 Arbeiten aus dem pathol. Institute, Berlin, 1906.

2 O. Moor, BuM. Acad. de St. Petersbourg, 14 (also Maly's Jahresber., 31, 415),
and Zeitschr. f. Biologic, 44 and 45, and Zeitschr. f. physiol. Chem., 40 and 48.

3 See Kuliabko, Maly's Jahresber., 31, 415; Erben, Zeitschr. f. physiol. Chem.,
38; Folin, ibid., 37; Gies, Journ. Amer. Chem. Soc., 25; Haskins, Amer. Journ. of
Physiol., 12; Lippich, Zeitschr. f. physiol. Chem., 48 and 52.

4 Nolf, Zeitschr. f. physiol. Chem., 23; Macleod and Haskins, Amer. Journ. of
Physiol., 12, and Journ. of biol. Chem., 1.

692 URINE.

Carbamic-acid ethylester (urethane), as shown by JAFFE/ may pass, by the
mutual action of alcohol and urea, into the alcoholic extract of urine when one
is working with large quantities.

FOLIN 2 claims that all human urine contains a body which is probably methyl-
urea.

XNH CO

Creatinine, C4H7N3O, or NH:C<; | , is the anhydride of

XN(CH3).CH2

xNH2
Creatine, NH:C\ , which occurs in the muscles,

XN(CH3).CH2.COOH
bird urine and sometimes also in human urine.

Creatinine occurs in human urine and in that of certain mammalia.
It has also been found in ox-blood, milk, though in very small amounts,
in meat extracts, and in the flesh of certain fishes.

The quantity of creatinine in human urine is, in a grown man voiding
a normal quantity of urine in the course of a day, 0.6-1.3 grams (NEU-
BAUEE), or on an average 1 gram. JOHNSON 3 found 1.7-2.1 grams per
day, and similar results have been obtained by v. HOOGENHUYZE and
VERPLOEGH.4 The quantity of creatinine with a diet free from meat
is, FOLIN 5 says, variable for different individuals, but is constant for the
same person. He never found the quantity below 1 gram and often
between 1.3 and 1.7 grams. Nurslings also eliminate creatinine, although
the quantity is small (v. HOOGENHUYZE and VERPLOEGH). The quantity
of creatinine nitrogen in per cent of the total nitrogen varies under
different conditions, but is on an average about 4.5-6.9 per cent, as
determined by several experimenters.

Creatine occurs especially in the urine of birds and also in the urine
of nurslings, but also in older children (ROSE, FOLIN and DENIS). It
has also been found in the urine of pregnant women (KRAUSE and CRAMER)
but otherwise only in starvation, in diabetes, diseases of the liver, fevers
and diseases accompanied by a destruction of the body proteins, espe-
cially muscle-proteins. Between creatine and creatinine elimination a
relation exists it seems, at least for certain cases, namely with a decrease
in the quantity of creatinine eliminated the quantity of creatine increases
(LEVENE and KRISTELLER) .6

1 Zeitschr. f . physiol. Chem., 14.

2 Journ. of biol. Chem., 3.

* Huppert-Neubauer, Harnanalyse, 10. Aufl., 387.

4 Zeitschr. f . physiol. Chem., 46.

6 Amer. Journ. of Physiol. 13; af. Klercker, Hofmeister's Beitrage, 8.

6 Rose, Journ. of biol. Chem., 10; Folin and Denis, ibid., 11; Krause and Cramer,
Journ. of Physiol., 40 (Proc. physiol. Soc., July, 1910, LXI); Schaffer, Amer. Journ.
of Physiol., 23; Levene and Kristeller, ibid., 24.

CREATININE. 693

As the two bodies, creatine and creatinine, can easily be transformed
into each other, it has been considered for a long time that the urinary
creatinine is formed from the creatine of the muscles and other organs.
Unfortunately the authorities disagree on this question. FOLIN. in his
investigations found that about 80 per cent of the creatinine intro-
duced was again eliminated, while the creatine taken did not appear in
the urine as creatinine, but was partly retained by the body and in
part eliminated as such. An intravital transformation of creatine into
creatinine is disputed by v. KLERCKER, MELLANBY and LEFMANN/ while
it is accepted by GOTTLIEB, STANGASSINGER, S. WEBER, v. HOOGEN-
HUYZE and VERPLOEGH and ROTHMANN. The observations of MYERS
and FINE indicate a production of urinary creatinine from creatine, that is
they found that the creatinine elimination by the urine in rabbits was
greater according to the total creatine content of the respective animal.
The investigations of PEKELHARING and v. HOOGENHUYZE on the behavior
of parenterally introduced creatine in rabbits and dogs, show without
any doubt that a part of the creatine is actually transformed into creatinine.
TOWLES and VoEGTLiN2 have also observed that the subcutaneously
injected creatine increases somewhat the creatinine elimination, while this
is not the case with creatine taken per os. The condition of the digestive
apparatus also seems to be of importance here. PEKELHARING and v.
HOOGENHUYZE found that in dogs of the parenterally introduced creatine
always a smaller part (as creatine and creatinine) passed into the urine
during the digestion than during rest of the digestive organs. They
explain this by the accepted ability of the liver to partly destroy the
creatine and partly by an anhydride formation of transforming the creatine
into creatinine.

As mentioned in Chapter X the proteins and the guanidine groups
therein are considered as the mother-substance of these two bodies.
If the creatinine (creatine) originates from the protein it is evident that
we must differentiate between food-protein and body-protein. The quan-
tity of creatinine is, inasmuch as it is increased by meat diet, dependent
upon the food; but otherwise, as found by FOLIN and in chief substantiated
by others, is rather independent of the food. Its elimination does not
run parallel with the urea and the total nitrogen, and consequently is
not in general greater with food rich in protein than with food poor therein.
Oh the contrary, its extent, as shown by other conditions, is dependent
upon the intensity of the metabolism in the cells, especially the muscle

1 Folin, Hammarsten's Festschrift, 1906; v. Klercker, Bioch. Zeitschr., 3; Mellanby,
Journ. of Physiol., 36; Lefmann, Zeitschr. f. physiol. Chem., 57.

2 See footnote 1, page 574, and v. Hoogenhuyze and Verploegh, Zeitschr. f. physiol.
Chem., 59; Pekelharing and v. Hoogenhuyze, ibid., 69; Towles and Voegtlin, Journ.
of biol. Chem., 10; Myers and Fine, ibid., 14.

694 URINE.

tissue, and the creatinine, according to FOLIN, is a product of the endo-
genous protein metabolism.

Reports as to the behavior of the creatinine elimination with work
are conflicting, v. HOOGENHUYZE and VERPLOEGH, who made use of
a much more trustworthy method of quantitative estimation than their
predecessors, find that muscular activity as a rule does not cause any
rise in the creatinine elimination, and that in man such a rise with work
occurs only when the body is obliged to live upon its own tissues. S.
WEBER 1 also finds an absolute increase in the elimination of creatinine
only in starving dogs. Other investigators could not find any increase
in the elimination of creatinine by work, although such a rise was found
as shown by PEKELHARING and HARKiNK,2 by the muscle tonus.

In starvation a decrease in the creatinine but a simultaneous increase
in the elimination of creatine has been found in man (v. HOOGENHUYZE
and VERPLOEGH, CATHCART, BENEDICT and MYERS 3). Such an increase
in the creatinine elimination only occurs in those conditions which are
accompanied by acidosis, and correspondingly it can be prevented by
the introduction of carbohydrates (CATHCART, MENDEL and ROSE)
The creatinine elimination in certain cases has therefore been explained
by a disturbed carbohydrate metabolism. This is neverthless on the
other hand disputed by WOLF and OESTERBERG 4 who find that the crea-
tinine elimination in starvation can be arrested by the introduction of
proteins alone.

Little is known about the behavior of creatinine in disease, nor are
the observations in accord. In anaemia and cachexia the elimination
of creatinine is diminished, and when the metabolism is increased the
elimination is also increased. That this is the case, at least in fevers,
seems to be borne out by several concurrent observations.5 In diseases
of the liver a diminished elimination of creatinine may occur, and in cases
of carcinoma of the liver considerable creatine has been found in the urine
(v. HOOGENHUYZE and VERPLOEGH, MELLANBY). The role of the liver
in the creatine-creatinine metabolism, is, as has already been mentioned
in Chapter X, not clear. The exclusion of the liver from the metabolism
of a dog with Eck fistula had no result in the experiments of TOWLES

1 Arch. f. exp. Path. u. Pharm., 58. Further literature may be found in v. Hoeg-
enhuyze and Verploegh, Zeitschr. f . physiol. Chem., 46.

2 Maillard and Clausmann, Journ. de Physiol. et de Path., 12; Prayon, Maly'a
Jahresb., 40; Pekelharing and Harkink, Zeitschr. f. physiol. Chem., 75.

3v. Hoogenhuyze and Verploegh, Zeitschr. f. physiol. Chem., 57; Cat heart, Bioch.
Zeitschr., 6; Benedict and Myers, Amer. Journ. of Physiol., 18; Jaffe, 1. c.

4Cathcart, Jour, of Physiol., 39; Mendel and Rose, Journ. of biol. Chem., 10;
Wolf and Osterberg, Bioch. Zeitschr., 35; Wolf, Journ. of biol. Chem., 10.

6 See O. af Klercker, Zeitschr. f. klin. Med., 68.

PROPERTIES OF CREATININE. 695

and VOEGTLIN. The dogs, after feeding with creatine and creatinine,
behaved like normal dogs, and the observations of other investigators
such as LONDON and BOLJARSKI, FOSTER and FISHER 1 upon dogs with
Eck fistula have not had any unanimous results or they are hard to explain.

Properties of Creatinine. Creatinine crystallizes in colorless, shining
monoclinic prisms which differ from creatine crystals in not becoming
white with loss of water when heated to 100° C. It dissolves in 11 parts
cold water, but more easily in warm water. It is difficultly soluble in
cold alcohol, but the reports in regard to its solubility differ widely.2
It is more soluble in warm alcohol and nearly insoluble in ether. In
alkaline solution creatinine is very easily converted into creatine on
warming.

Creatinine gives an easily soluble crystalline compound with hydro-
chloric acid. A solution of creatinine acidified with mineral acids gives
crystalline precipitates with phosphotungstic and phosphomolybdic
acids even in very dilute solutions (1:10000), (KERNER, HOFMEISTER 3) .
It is precipitated, like urea, by mercuric-nitrate solution and also by
mercuric chloride. On treating a dilute creatinine solution with sodium
acetate and then with mercuric chloride a precipitate of glassy globules
having the composition 4(C4H7N30.HCl.HgO)3HgCl2 separates on
standing some time (JOHNSON). Among the compounds of creatinine,
that with zinc chloride, creatinine-zinc chloride, (C-iHyNsO^ZnC^, is
of special interest. This combination is obtained when a sufficiently
concentrated solution of creatinine in alcohol is treated with a concentrated,
faintly acid solution of zinc chloride. Free mineral acids dissolve the com-
pound, hence they must not be present; this, however, may be prevented
by an addition of sodium acetate. In the impure state, as from urine,
creatinine-zinc chloride forms a sandy, yellowish powder which under
the microscope appears as fine needles, forming concentric groups,
mostly complete rosettes or yellow balls or tufts, or grouped as brushes.
On slowly crystallizing or when very pure, more sharply defined prismatic
crystals are obtained. The compound is slightly soluble in water.

Creatinine acts as a reducing agent. Mercuric oxide is reduced to
metallic mercury, and oxalic acid and methylguanidine (methyluramine)
are formed. Creatinine also reduces cupric hydroxide in alkaline solution,
forming a colorless soluble compound, and only after continued boiling
with an excess of copper salt is free suboxide of copper formed. Creat-

lTowles and Voegtlin, 1. c.; London and Boljarski, Zeitschr. f. physiol. Chem.,
62; Foster and Fischer, Journ. of biol. Chem., 9.

2 See Huppert-Neubauer, 10. Aufl., and Hoppe-Seyler-Thierfelder's Handbuch.
8. Aufl.

3 Kerner, Pfliiger's Arch., 2; Hofmeister, Zeitschr. f. physiol. Chem., 5.

696 URINE.

inine interferes with TROMMER'S test for sugar, partly because it has a
reducing action, and partly by retaining the copper suboxide in solution.
The compound with copper suboxide is not soluble in a saturated soda
solution, and if a little creatinine is dissolved in a cold saturated soda
solution and then a few drops of FEHLING'S reagent added, a white flocculent
compound separates after heating to 50-60° C. and then cooling (v.
MASCHKE's1 reaction). An alkaline bismuth solution (see Sugar Tests)
is not reduced by creatinine.

An aqueous solujbion of creatinine is precipitated by picric acid.
The precipitate consists on recrystallization from hot water, of thin,
silky, pale yellow needles (JAFFE). If the urine is treated with picric
acid (20 cc. of a 5 per cent solution in alcohol for each 100 cc. urine),
then a double picrate of creatinine and potassium is precipitated (JAFFE).
If a solution of creatinine in w&ter (or urine) is treated with a watery
solution of picric acid and a few drops of a dilute caustic-soda solution,
a red coloration, lasting several hours, immediately occurs at the ordinary
temperature, which turns yellow on the addition of acid (JAFFE'S 2 reac-
tion). Acetone gives a more reddish-yellow color. Glucose gives
with this reagent a red coloration only after heating. If we add a few
drops of a freshly prepared very dilute sodium-nitroprusside solution
(sp.gr. 1.003) to a dilute creatinine solution (or to the urine) and then a
few drops of caustic soda, a ruby-red liquid is obtained which quickly
turns yellow again (WEYL'SS reaction). If the cold yellow solution is
neutralized and treated with an excess of acetic acid, a crystalline pre-
cipitate of a nitroso-compound (C^eN^C^) of creatinine separates on
stirring (KRAMM) or creatininoxim (SCHMIDT 4). If, on the contrary,
the yellow solution is treated with an excess of acetic acid and heated,
the solution becomes first green and then blue (SALKOWSKI 5) ; finally
a precipitate of Prussian blue is obtained.

A reaction which in description is similar and which, although not solely
(ARNOLD) but at least partially (HOLOBUT), appears after partaking of protein
food or meat soup is ARNOLD'S reaction.6 This reaction is due to an unknown
endogenous metabolism product. If 10-20 cc. urine are treated with a few drops
of a 4 per cent sodium nitroprusside solution and then with 5-10 cc. of a 5 per
cent sodium or potassium hydroxide solution, at first a strong and pure violet
color is obtained with an absorption band between D and E, then it becomes
purple-red and then brown-red and finally yellow. On the addition of acetic

1 Zeitschr. f. analyt. Chem., 17.

2 Zeitschr. f. physiol. Chem., 10.

3 Ber. d. deutsch. chem. Gesellsch., 11.

4Kramm, Centralbl. f. d. med. Wissensch., 1897; Schmidt, cited from Chem.
Centralbl., 1912, 2.

5 Zeitschr. f. physiol. Chem., 4.

6 Arnold, Zeitschr. f. physiol. Chem., 49 and 83; Holobut, ibid., 56.

ESTIMATION OF CREATININE. 697

acid the violet or purple-red color passes into' blue, which soon becomes pale
and finally a pale yellow color. It differs from the creatinine in color and
the absorption band as well as in that the creatinine reaction requires more
sodium nitroprusside.

The best method for preparing creatinine is the following, suggested
by FoLiN.1 The creatinine is first precipitated as the double picrate
of creatinine and potassium by means of picric acid according to JAFFE'S
method, and then this precipitate, while still moist, is decomposed by
KHCOs and water. The solution, which contains the creatinine besides
potassium carbonate and small amounts of impurities, is neutralized
with sulphuric acid and the sulphate precipitated by alcohol. The
creatinine is now converted into the double zinc-chloride salt and this
last treated with moist lead hydroxide. After the removal of the lead,
the solution contains a mixture of creatinine and creatine, which last is
completely transformed into creatinine by heating for forty-eight hours
with normal sulphuric acid. After exact neutralization with barium-
hydroxide solution it is concentrated to the point of crystallization.

According to recent work of FOLIN and BLANCK the creatinine-zinc chloride
can be dissolved in warm 10 per cent sulphuric acid when creatinine-zinc alum
(CJIrNsO^SCXZnSO^ 8H20 is obtained and from this the creatinine can be
obtained by decomposing with barium acetate and removing the zinc by H2S.
Creatine can, according to FOLIN and DENIS, 2 be transformed into creatinine by
heating in an autoclave for 3 hours under a pressure of 4-5 kg. per qcm.

The quantitative estimation of creatinine used to be performed accord-
ing to NEUBAUER'S method for the preparation of creatinine, or more
simply by SALKOWSKi's3 modification of this method. As this method
is now seldom used we refer the reader to other hand-books.

FOLTN 4 has suggested a colorimetric method for determining creatinine
which is based upon JAFFE'S picric-acid reaction and is as follows: 10
cc. of the urine are treated in a graduated flask of 500 cc. capacity
with 15 cc. of a 1.2 per cent solution of picric acid and 5 cc. of a 10
per cent NaOH solution. After shaking and allowing to stand for five
minutes it is diluted with water to 500 cc. and mixed. This solution
is now compared in a DUBOSCQ colorimeter Jwith a 1/2 normal potassium-
dicromate solution. The latter solution has in a layer 8 mm. thick
exactly the same intensity of color as a layer 8.1 mm. thick of a solution
of 10 milligrams creatinine after the addition of 15 cc. picric-acid solu-
tion and 5 cc. NaOH solution and dilution to 500 cc. The calculations
are simple. For example, in case the urine tested in a layer 7.2 mm.
thick has the same color as the dichromate solution in a layer 8 mm.
thick, then the quantity of creatinine in 10 cc. of the urine will be

8 1
= =^X10, or 11.25 milligrams. This method has been tried by many

authorities and found to be trustworthy.

1 Zeitschr. f. physiol. Chem., 41.

2 Folin and Blanck, Journ. of biol. Chem., 8, with Denis, ibid., 8.

3 Zeitschr. f. physiol. Chem., 10 and 14.

., 41.

698 URINE.

The same method is used in the determination of creatine, which
for this purpose is first converted into creatinine by wanning with dilute
mineral acid. The quantity of creatine is the difference obtained between
the values for creatinine before and after treatment with acid. More
detailed directions can be found in the cited works of FOLIN, v. HOOGEN-
HUYZE and VEKPLOEGH, GOTTLIEB and STANGASSINGER.

In regard to other methods, see the works of KOLISCH and

Xanthocreatinine, CsHio^O. This body, which was first prepared from
meat extract by GAUTIER, has been found, by MONARI, in dog's urine after the
injection of creatinine into the abdominal cavity, and in human urine after several
hours of exhaustive marching. According to COLASANTI it occurs to a relatively
greater extent in lion's urine. STADTHAGEN 2 considers the xanthocreatinine
isolated from human urine after strenuous muscular activity as impure creatinine.

Xanthocreatinine forms thin sulphur-yellow plates, similar to cholesterin,
which have a bitter taste. It dissolves in cold water and in alcohol, and gives
a crystalline compound with hydrochloric acid and a double compound with
gold and platinum chloride. It gives a compound with zinc chloride, which
^crystallizes in fine needles. Xanthocreatinine has a poisonous action.

Methylguanidine occurs, according to ACHELIS, KUTSCHER and LOHMANN,
to a slight extent as a regular constituent of the urine of man, horse and dog.
It has been found in urines associated with dimethylguanidine by ENGELAND.*

HN— CO

I i
Uric Acid, Ur, C5H4N403; 2, 6, 8-trioxypurine, OC C— NHV , has

I II >CO

vr n ISJTT/

HN— C—

been prepared synthetically by HORBACZEWSKI by fusing urea anu
glycocoll, or by heating trichlorlactic-acid amide with an excess of urea.
BEHREND and ROOSEN prepared it from isodialuric acid and urea; it
is also readily produced from isouric acid on boiling with hydrochloric
acid (E. FISCHER and TULLNER) and finally E. FISCHER and ACH 4 have
prepared uric acid from pseudouric acid by heating with oxalic acid to
145° C.

On strongly heating uric acid it decomposes with the formation of
urea, hydrocyanic acid, cyanuric acid, and ammonia. On heating with
concentrated hydrochloric acid in sealed tubes to 170° C. it splits into
glycocoll, carbon dioxide, and ammonia. By the action of oxidizing
agents splitting and oxidation take place, and either monoureides or
diureides are produced. By oxidation with lead peroxide, carbon dioxide,

i J Kolisch, Centralbl. f. innere Med., 1895; Gregor, Zeitschr. f. physiol. Chem., 31.

2 Gautier, Bull, de 1'acad. de m6d. (2), 15, and Bull, de la soc. chim. (2), 48; Monari,
Maly's Jahresber., 17; Colasanti, Arch. ital. d. Biologie, 15, Fasc. 3; Stadthagen,
Zeitschr. f . klin. Med., 15.

3 Achelis, Centralbl. f . Physiol., 20, 455, and Zeitschr. f. physiol. Chem., 50; Kut-
r scher and Lohmann, ibid., 49; Engeland, ibid., 57.

4 Horbaczewski, Monatshefte f. Chem., 6 and 8; Behrend and Roosen, Ber. d. d.
chem. Gesellsch., 21; Fischer and Tiillner, ibid., 35; Fischer and Ach, ibid., 28.

URIC ACID. 699

oxalic acid, urea, and allantoin, which last is glyoxyldiureide, are pro-
duced (see below). By oxidation with nitric acid in the cold, urea and
a monoureide, the mesoxalyl urea, or alloxan, are obtained, CsH^N^a-f
O-f H2O = C4H2N2O4+(NH2)2CO. On warming with nitric acid, alloxan
yields carbon dioxide and oxalyl urea, or parabanic acid, CiH^^Oa.
By the addition of water the parabanic acid passes into oxaluric acid,
CaEU^CU, traces of which are found in the urine and which easily splits
into oxalic acid and urea. In alkaline solution uric acid may, by taking
up water and oxygen, be transformed into a new acid, uroxanic acid,
CsHgN^e, which may then be changed into oxonic acid, C4H5N304.1
On the oxidation of uric acid by hydrogen peroxide in alkaline solution
SCHITTENHELM and WIENER 2 have obtained urea with carbonyl diurea
as intermediary product. Uric acid may, as F. and L. SESTINI as well
as GERARD have shown, undergo bacterial fermentation with the forma-
tion of urea. According to ULPIANI and CiNGOLANi,3 uric acid is quan-
titatively split into urea and carbon dioxide, according to the equation

Uric acid occurs most abundantly in the urine of birds and of scaly
amphibians, in which animals the greater part of the nitrogen of the urine
appears in this form. Uric acid frequently occurs in the urine of carniv-
orous mammalia, but is sometimes absent; in urine of herbivora it is
habitually present, though only as traces; in human urine it occurs in
greater but still small and variable amounts. Traces of uric acid are
also found in several organs and tissues, as in the spleen, lungs, heart,
pancreas, liver (especially in birds), and in the brain. It always occurs
in the blood of birds. Traces have been found in human blood under
normal conditions. Under pathological conditions it occurs to an
increased extent in the blood, as in pneumonia and nephritis, but espe-
cially in leucffimia and sometimes also in arthritis. Uric acid also occurs
in large quantities in " chalk-stones," certain urinary calculi, and in
guano. It has also been detected in the urine of insects and certain
snails, as also in the wings (which it colors white) of certain butterflies
(HOPKINS 4).

The amount of uric acid eliminated with human urine is subject to
considerable individual variation, but amounts on an average to 0.7

1 See Sundwik, Zeitschr. f. physiol. Chem., 20 and 41; also Behrend, Annal. d.
Chem. u. Pharm., 333.

2 Zeitschr. f. physiol. Chem., 62.

3 See Chem. Centralbl., 1903, where the other investigators are cited, and Centralbl.
f. Physiol., 19.

4 Philos. Trans. Roy. Soc., 186, B, 061.

700 URINE.

gram per day on a mixed diet. The ratio of uric acid to urea varies con-
siderably with a mixed diet, but is on an average 1 : 50-1 : 70. In new-
born infants and in the first days of life the elimination of uric acid is
relatively increased, and the relation between uric acid and urea has been
found to be 1:6.42-17.1.

We used to ascribe an increasing action upon the elimination of uric
acid to protein food, but the investigations of HIRSCHFELD, ROSEN-
FELD and ORGLER, SIVEN, BURIAN, and SnuR,1 and many others have
positively proven that a diet rich in protein does not itself increase the
elimination of uric acid, but only according to the amount of nucleins
or purine bodies contained therein. The common assumption that the
elimination of uric acid is smaller with a vegetable diet than with an ani-
mal diet, when the quantity may be 2 grams or more per twenty-four
hours, is explained by this.2

Still a purine-free diet is not without some influence upon the elimina-
tion of uric acid, as the quantity of uric acid eliminated with a '

free diet is considerably greater than in starvation and can be increased
by protein feeding. The action of the food-protein is here probably an
indirect one, consisting in that the proteins raise the work of the digestive
glands and the metabolism of their cells and thereby also raise the endo-
genous uric acid formation (see below) somewhat.3 Work and rest do
not seem to have any special influence upon the uric acid elimination,
although according to the confirmed statement of SIVEN and LEATHES*
the elimination in the night is less than in the morning hours.

The reports in regard to the influence of other circumstances, as well
as of different substances, on the elimination of uric acid are diverse.
This is in part due to the fact that the earlier investigators used an
inaccurate method (HEINTZ), and also that the extent of uric-acid elimina-
tion is dependent in the first place upon the individuality. Thus the
investigators are not in accord in regard to the action of drinking-

1 See the extensive review of the literature in Wiener, " Die Harnsaure," in Ergeb-
nisse der Physiologic, 1, Abt. 1, 1902.

2 J. Ranke, Beobachtungen und Versuche iiber die Ausscheidung der Harnsaure,
etc. (Miinchen, 1858); Mares, Centralbl. f. d. med. Wissensch., 1888; Horbaczewski,
Wien. Sitzungsber., 100, Abt. 3, 1891. In regard to the action of various diets the
reader is referred to the above-cited authors, and especially to A. Hermann, Arch. f.
klin. Med., 43, and Camerer, Zeitschr. f. Biologic, 33, and Folin, Amer. Journ. of Physiol.,
13.

3 See Hirschstein Arch. f. exp. Path. u. Pharm., 57; Smetanka, Pfliiger's Arch.,
138 and 149 ; Mares, ibid., 134 and 149. Contrary views, Brugsch and Schitten-
helm, Zeitschr. f. exp. Path. u. Therp., 4, and Siven, Pfliiger's Arch., 146.

4Siv6n, Skand. Arch. f. Physiol., 11; Leathes, Journ. of Physiol, 35; see also Ken-
naway, Journ. of Physiol., 38.

FORMATION OF URIC ACID. 701

water l and of alkalies.2 Certain medicines, such as quinine and atro-
pine, diminish, while others, such as pilocarpine and, as it seems, salicylic
acid,3 increase the elimination of uric acid.

There is much diversity of opinion regarding the elimination of uric
acid in disease,4 although it is known that it is increased after an abun-
dant destruction of nucleated cells as in pneumonia, after the crisis,
and in leucaemia. In the latter in most cases not only is the elimina-
tion to the urea increased absolutely, but also relatively; and the relation
between uric acid and urea (total nitrogen calculated as urea) may in
lineal leucaemia even be 1:9, while under normal conditions, accord-
ing to different investigators, it is 1:50 to 70 to 100. As to the behavior
of uric acid in gout, authorities are by no means agreed. That the
blood contains uric acid in gout has been repeatedly shown, and
it is also found in this disease with a purine-free diet (BRUGSCH and
SCHITTENHELM) . According to these investigators a diminished enzy-
motic decomposition of uric acid occurs in the body in gout and this
causes the occurrence of uric acid in the blood and its accumulation in
certain tissues. Strong arguments against this view have been presented
by others such as WELLS and CORPER, MILLER and JoNES.5

Formation of Uric Acid in the Organism. Since HORBACZEWSKI
first showed that uric acid could be produced by oxidation from the
nuclein-rich spleen-pulp or nucleins outside of the body, he also showed
that nucleins when introduced into the animal body caused an increase
in the elimination of uric acid. These observations have been confirmed,
and at the same time developed by the work of a great number of investi-
gators, and we are sure that uric acid can be produced from purine
bases either outside or inside the animal body, and also that food rich
in nucleins (especially the thymus gland) increases the elimination of
uric acid. It is nevertheless true that a few investigators after intro-
ducing pure purine bases into the organism could not observe any essential
rise in the uric acid or its transformation products; still we have a large
number of recent investigations which positively show that nucleic acids,
as well as purine bases, when introduced into the animal body are trans-
formed in abundant quantities into uric acid in the body.c At present

1 See Schondorff , Pfliiger's Arch., 46, which contains the pertinent literature.

2 See Clar, Centralbl. f. d. rned. Wissensch., 1888; Haig, Journ. of Physiol., 8; and
A. Hermann, Arch. f. klin. Med., 43.

3 See Bohland, cited from Maly's Jahresber., 26; Schreiber and Zaudy, ibid., 30.

4 In regard to the extensive literature on the elimination of uric acid in disease
we must refer to special works on internal diseases.

6 Brugsch and Schittenhelm, Zeitschr. f. exp. Path. u. Therp., 4; Wells and Corper,
Journ. of biol. Chem., 6; Miller and Jones, Zeitschr. f. physiol. Chem., 61.

6 As it is not within the scope of this book to enter into a discussion of the numer-
ous researches on this subject, we will refer to Wiener, " Die Harnsaure," Ergebnisse

702 URINE.

we consider the formation of uric acid from the purine bases of the nuclein
substances as a positively proven fact.

According to the original view of HORBACZEWSKI the nucleins do
not directly (by their purine bases) cause an increased elimination of uric
acid, but indirectly by causing a leucocytosis with a consequent destruc-
tion of leucocytes. This view has been justly discarded on account,
of the above-mentioned conditions; still on the other hand it cannot
be denied that the formation of uric acid is als'o in certain regards related
to the formation or the destruction of leucocytes and to the metabolism
in the cells as a whole.1

The uric acid, in so far as it is produced from nuclein bases, is in part
derived from the nucleins of the destroyed cells of the body and in part
from the nucleins or free purine bases introduced with the food. It
is therefore possible to admit, with BURIAN and ScnuR,2 of a double origin
for the uric acid as well as the urinary purines (all purine bodies of the
urine, including the uric acid), namely, an endogenous and an exogenous
origin. BURIAN and SCHUR attempted to determine the quantity of
endogenous urinary purines by feeding with sufficient food, but as free
as possible from purine bodies, and they found that this quantity was
constant for every individual, while it was variable for different persons.
The observations of many other investigators have led to similar con-
clusions, and we are now unanimous in our opinion that the uric acid
originating from the nucleins is partly endogenous and partly exogenous,
and that the amount of endogenous uric acid is only very slightly dependent
upon the protein content of the food.

The formation of uric acid from the nucleins or the purine bases seems
at least in great part to be of an enzymotic kind. After it was shown that
certain organs, such as the liver and spleen, had the power of converting
oxypurines into uric acid in the presence of oxygen (HORBACZEWSKI,
SPITZER and WIENER 3), recently SCHITTENHELM, BURIAN, JONES and
co-workers4, by more careful investigations have shown that enzymes

der Physiol., 1, Abt. 1, 1902. See also Schittenhelm, Zeitschr. f. physiol. Chem., 62,
with Frank, ibid., 63, with Seisser, Zeitschr. f. exp. Path. u. Ther., 7; Abderhalden,
London and Schittenhelm, Zeitschr. f. physiol. Chem., 61; Mendel and Lyman., Journ.
of biol. Chem., 8.

1 See Plimmer, Dick and Lieb, Journ. of Physiol., 39; Mares Pfliiger's Arch., 134,
and Smetanka, ibid., 138.

2 Pfliiger's Arch., 80, 87, and 94.
1 See footnote, 6, page 701.

4 Schittenhelm, Zeitschr. f. physiol. Chem., 42, 43, 45, 46, 57, 63, 66, with Schmid,
ibid., 50, and Zeitschr. f. exp. Path. u. Therap., 4; Burian, Zeitschr. f. physiol.
Chem., 43; Jones and Partridge, ibid., 42; Jones with Winternitz, ibid., 44 and 60;
Jones, ibid., 45, 65, with Austrian, ibid., 48, with Miller, ibid., 61; Jones, Journ. of
biol. Chem., 9; Wells, ibid., 7; Mendel and Mitchell, Amer. Journ. of Physiol., 20.

FORMATION OF URIC ACID. 703

of different kinds act together. By means of the two deamidizing enzymes
adenase and guanase, the adenine and guanine are transformed into
hypoxanthine and xanthine respectively, and from the latter by means
of an oxidizing enzyme, called xanthine oxidase by BURIAN, the uric
acid is formed. In the formation of uric acid from the nucleoproteins
we must admit of a gradual decomposition of these by the aid of different
enzymes, proteases, nucleases and deamidases. The deamidases seem
to be present in most organs, and we have numerous investigations upon
their distribution, especially those of JONES and SCHITTENHELM and
his collaborators.1 The distribution is not the same in all animals and
the reports regarding it are unfortunately conflicting (SCHITTENHELM,
JONES and MILLER). We must exercise the greatest caution in drawing
conclusions as to the occurrence of these enzymes, and from experiments
made with the extracts of organs, because it seems as if also other
unknown factors must be considered in the formation of uric acid.
Thus JONES has with ROHDE 2 shown that in rats the organs do not con-
tain any xanthine oxidase, and that nevertheless the urine of this animal
contains uric acid. On the other hand deamidases occur in the organs
of monkeys (and xanthine oxidase in the liver) but the urine does not
contain any uric acid and only traces of allantoin ( WELLS) .3 The pos-
sibility of a uric acid formation in man and mammalia in another way
from the enzymotic destruction of the purines cannot, for several reasons,
be denied.

In birds the conditions are different, v. MACH4 has shown that in
the bird family a part of the uric acid may be formed from the purine
bodies. The chief quantity of uric acid, however, is undoubtedly formed
in birds by synthesis.

The formation of uric acid in birds is increased by the administra-
tion of ammonium salts (v. SCHRODER), and urea acts in a similar
manner (MEYER and JAFFE). MINKOWSKI observed, in geese with
extirpated livers, a very significant decrease in the elimination of uric
acid, while the elimination of ammonia was increased to a corresponding
degree. This indicates a participation of ammonia in the formation
of uric acid in the organism of birds; and as MINKOWSKI has also found,
after the extirpation of the liver, that considerable amounts of lactic
acid occur in the urine, it is probable that the uric acid in birds is pro-
duced in the liver by synthesis, perhaps from lactic acid and ammonia;

1 See footnote 4, page 703.

2 Jones, Zeitschr. f . physiol. Chem., 60, with Alice Rohde, Journ. of biol. Chem., 7;
see also Voegtlin and Jones, Zeitschr. f. physiol. Chem., 66.

3 Journ. of biol. Chem., 7.

4 Arch. f. exp. Path. u. Pharm., 24.

704 UKINE.

although, as SALASKIN and ZALESKI and LANG have shown, after the
extirpation of the liver, and increase in the formation of lactic acid pri-
marily occurs, and this causes an increase in the elimination of ammonia
(neutralization ammonia). The direct proof for the uric-acid formation
from ammonia and lactic acid in the liver of birds has been given by
KOWALEWSKY and SALASKIN1 by means of blood-transfusion experiments
on geese with extirpated livers. -They observed a relatively abundant
formation of uric acid after the addition of ammonium lactate and a
still greater formation after arginine. They not only consider ammonium
lactate but also amino-acids as substances from which the uric acid can
be produced in the liver by synthesis. That these, for example, leucine,
glycocoll, and aspartic acid, increase the elimination of uric acid in
birds was first shown by v. KNiERiEM.2

The possibility of a formation o«f uric acid from lactic acid has been
shown in another manner by WIENER,S namely, by feeding birds with
urea and lactic acid and different non-nitrogenous substances, oxy-,
keto-, and dibasic acids of the aliphatic series. The dibasic acids, with a
chain of 3 carbon atoms or their ureides, showed themselves most active
as uric-acid formers, and WIENER is therefore of the opinion that the
active substances must first be converted into dibasic acids. By the
attachment of a urea residue the corresponding ureide is produced,
according to WIENER, and from this the uric acid is derived by the attach-
ment of a second urea residue.

Among the substances tested, only tartronic acid and its ureide, dialuric acid,
have shown themselves active in the experiments with the isolated organs, and
WIENER therefore also considers that the other acids must be first converted into
tartronic acid by oxidation or reduction. From lactic acid, CH3.CH(OH).COOH,
we first obtain tartronic acid, COOH.CH(OH).COOH, which by the attachment

/NH— C0\
of a urea residue forms dialuric acid, C0\ /CHOH, and from this, by

\NH— CO/
the attachment of a second urea residue, uric acid is formed.

Recently IzAR4 has shown on perfusing blood containing urea and
dialuric acid through the liver of a dog and at the same time saturating
the blood with carbon dioxide, that an abundant formation of uric acid
occurred, and that a combined action between an enzyme occurring

1 v. Schroder, Zeitschr. f. physiol. Chem., 2; Meyer and Jaffe, Ber. d. f. Chem.
Gesellsch., 10; Minkowski, Arch. f. exp. Path. u. Pharm., 21 and 31; Salaskin and
Zaleski, Zeitschr. f. physiol. Chem., 29; Lang, ibid., 32; Kowalewsky and Salaskin,
ibid., 33.

2 Zeitschr. f . Biologic, 13.

3 Hofmeister's Beitrage, 2. See also Arch. f. exp. Path. u. Pharrn., 42, and Ergeb-
nisse d. Physiol., 1, Abt. 1, 1902.

4 Zeitschr. f. physiol. Chem., 73, see also ibid., 65.

FORMATION OF UKIC ACID. 705

in the blood and an alcohol-soluble co-enzyme occurring in the liver
and spleen took place. He has besides this also given further proof of
the formation of uric acid in the bird-liver from urea and ammonium
carbonate.

We cannot give any positive answer as to the question whether uric
acid is formed by synthesis in man and other mammalia. WIENER
has reported experiments which seem to indicate a synthetic uric-acid
formation in the isolated mammalian liver, and he has also obtained
an increase in the uric-acid elimination, although only a slight one, after
feeding lactic acid and dialuric acid to man. In opposition to these
experiments PFEIFFER 1 could find no increase in the elimination of
uric acid after feeding malonamide and tartronamide to monkeys as
well as tartronic acid and pseudouric acid to monkeys or human beings,
and he finds that a synthesis of uric acid in mammalia and man is very
doubtful. According to BuRiAN2 we have for the present no proof
of a synthetical formation of uric acid in the mammalian liver; in
view of the above-mentioned experiments of IZAR, we cannot deny the
possibility of a synthetical formation of uric acid also in mammalia and
man even if we do not know to what extent this occurs.

The liver seems to be the organ in birds where the synthetical forma-
tion of uric acid occurs, and the fact that it was possible for MINKOWSKI 3
to arrest the uric-acid formation by the extirpation of the liver, apparently
shows that the liver is the only organ taking part in this synthesis. If a
synthesis of uric acid also occurs in man and other mammalia, we must
consider the liver as at least one of the organs taking part in the work,
as shown by WIENER'S and IZAR'S investigations. The liver is considered
as the most important organ in the oxidative formation of uric acid from
nucleins and purine bases. That this organ, at least in the dog, is not
the only or at least not the most important follows from the investiga-
tions of ABDERHALDEN, LONDON and SCHITTENHELM 4 on dogs with Eck
fistula. They found that, on excluding the liver in this manner, that the
transformation of the nucleic acid fed, the deamidation of the purine
bases and the oxidation of these into uric acid and allantoin was
undisturbed. In the dog also other organs must be considered in
this connection. It is not known how other animals behave in this
regard.

'Uric acid when introduced into the mammalian organism is, as first
shown by WOHLER and FRERICHS, in the dog, and later substantiated

1 Hofmeister's Beitrage, 10.

2 Zeitschr. f. physiol. Chem., 43.

3 I.e.

4 Zeitschr. f. physiol. Chem., 61.

706 URINE.

by several experimenters,1 in great part destroyed and more or less com-
pletely changed into urea. As shown by WOHLER and FRERICHS for the
dog and by later investigators 2 also for cats, rabbits and other animals,
that allantoin is the most essential or indeed the chief decomposition
product is now considered as positively proven. In man, on the con-
trary, the conditions are different. According to WIECHOWSKIS probably
also a formation of allantoin from uric acid takes place in man, but it
is only of such an extent as to be without consideration, while in the dog
for example about 96 per cent of the purine base nitrogen may appear
as allantoin in the urine. According to the investigations of FRANK and
SCHITTENHELM 4 the uric acid in man is in part transformed into urea.
This different behavior of uric acid in the metabolism of man and
animals depends, as numerous investigations5 have shown, upon the
occurrence of a urocolytic enzyme in the liver and also other organs of
animals, which transforms the uric acid into allantoin with the taking up
oxygen and splitting off of carbon dioxide. This enzyme, which has been
called uricolase and also uricase and whose occurrence in the organs of
different animals varies, is absent in the organs of man. The results
obtained in regard to the enzymotic transformation of uric acid by
experiments with organ extracts must be judged with the greatest care.
Thus according to the statements of WIECHOWSKI, BATTELLI and STERN
and ScmTTENHELM,6 in dogs, the liver is the only organ which in a test-
tube shows a positive . uricoly sis; still in dogs, with excluded livers
(Eck fistula) such an abundant formation of allantoin from uric acid
occurs so that only 10-20 per cent of the uric acid escapes this trans-
formation.7

1 Wohler and Frerichs, Annal. d. Chem. u. Pharm., 65. See also Wiener, Ergeb-
nisse der Physiologie, 1, Abt. 1.

2Salkowski, Zeitschr. f. physiol. Chem., 35, and Ber. d. d. Chem. Gesellsch., 9;
Mendel and Brown, Amer. Jour, of Physiol., 3; Mendel and White, ibid., 12; Wie-
chowski, Arch. f. exp. Path. u. Pharm., 60, and Bioch. Zeitschr., 19 and 25, with Wiener,
Hofmeister's Beitrage, 9; Schittenhelm, Zeitschr. f. physiol. Chem., 62, with Seisser,
Zeitschr. f. exp. Path. v. Ther., 7; Abderhalden, London and Schittenhelm, Zeitschr.
f. physiol. Chem., 61.

8 Bioch. Zeitschr., 25.

4 Zeitschr. f. physiol. Chem., 63.

5Chassevant and Richet, Comp. rend. soc. biolog., 49; Ascoli, Pfliiger's Arch., 72;
Jacoby, Virchow's Arch., 157; Wiener, Arch. f. exp. Path. u. Pharm., 42, and Centralbl.
f. Physiol., 18; Schittenhelm, Zeitschr. f. physiol. Chem., 43, 45, and 63; Burian,
ibid., 43; Almagia, Hofmeister's Beitrage, 7; Pfeiffer, ibid., 7; Wiechowski and Wiener,
ibid., 9; Galeotti, Bioch. Zeitschr., 20; Battelli and Stern, ibid., 19; Scaffidi, ibid.,
18; Miller and Jones, Zeitschr. f. physiol. Chem., 61; Wells, Journ. of biol. Chem., 7,
with Corper, ibid., 6.

6 See Schittenhelm, Zeitschr. f. physiol. Chem., 63, 256.

7 Abderhalden, London and Schittenhelm, 1. c.

PROPERTIES AND REACTIONS OF URIC ACID. 707

ASCOLI, IZAR, BEZZOLA and PRETI x have studied the remarkable ability of
the liver of destroying uric acid in the blood by transfusing the arterial blood
through this organ and on transfusing the blood, saturated with C02, they have
regenerated the uric acid. It is not known what becomes of the uric acid in
these cases and from what substance the regeneration occurs. PRETI v has shown
that in the regeneration a combined action of an enzyme in the blood with a
co-enzyme of the liver, takes place.

From this power of the various organs of destroying uric acid it
follows that the quantity of uric acid eliminated is not a sure indication
of the amount of the acid formed. We must, therefore, admit that a
part of the uric acid formed in the body is destroyed in a manner similar
to that introduced from without. BURIAN and ScnuR2 have indeed
suggested a factor, the so-called " integral factor," with which the quan-
tity of uric acid eliminated in the twenty-four hours must be multiplied
in order to find the quantity of uric acid formed during this time. Such
calculations are necessarily very uncertain and are for the present not
admissible.

Properties and Reactions of Uric Acid. Pure uric acid is a white,
odorless, and tasteless powder consisting of very small rhombic prisms
or plates. Impure uric acid is easily obtained as somewhat larger,
colored crystals.

In rapid crystallization, small, thin, four-sided, apparently colorless,
rhombic prisms are formed, which can be seen only by the aid of the
microscope, and these sometimes appear as spools because of the round-
ing of their obtuse angles. The plates are sometimes six-sided, irregularly
developed; in other cases they are rectangular with partly straight and
partly jagged sides; and in other cases they show still more irregular
forms, the so-called dumb-bells, etc. In slow crystallization, as when
the urine deposits a sediment or when treated with acid, large, invariably
colored crystals separate. Examined with the microscope these crystals
always appear yellow or yellowish brown in color. The most common
type is the whetstone shape, formed by the rounding off of the obtuse
angles of the rhombic plate. The whetstones are generally connected,
two or more crossing each other. Besides these forms, rosettes of pris-
matic crystals, irregular crosses, brown-colored rough masses of broken-
up crystals and prisms occur, as well as other forms.

Uric acid is insoluble in alcohol and ether; it is rather easily soluble
in boiling glycerin, but very insoluble in cold water, in 39480 parts at
18° C. (His and PAUL), and in 15505 parts at 37° (GUDZENT). At
this temperature, according to His and PAUL, 9.5 per cent of the uric
acid is dissociated in the saturated solution. Because of the reduction

1 See Zeitschr. f . physiol. Chem., 58, 62, 64 and 65.

2 Pfluger's Arch., 87.

708 URINE.

in the dissociation on the addition of strong acids, uric acid is soluble
with difficulty in the presence of mineral acids. It is soluble in a warm
solution of sodium diphosphate, and in the presence of an excess of uric
acid, monophosphate and acid urate are produced. It is ordinarily
assumed that sodium diphosphate forms a solvent for the uric acid in
the urine, while according to GUDZENT this is not dissolved by the mono-
phosphate. RUDEL * believes that urea is an important solvent, but
this view has not been confirmed by the observations of His and PAUL.
Uric acid is not only dissolved by alkalies and alkali carbonates, but also
by several organic bases, such as ethylamine and propylamine, piperidine
and piperazine. Uric acid can form supersaturated solutions with alkalies
and these, according to SCHADE and BoDEN2 contain colloidal uric acid
and they may gelatinize on cooling as well as under other conditions.
Uric acid dissolves, without decomposing, in concentrated sulphuric
acid. It is completely precipitated from the urine by picric acid (JAFFES).
Uric acid gives a chocolate-brown precipitate with phosphotungstic
acid in the presence of hydrochloric acid.4

Uric acid is dibasic and consequently forms two series of salts, neu-
tral and acid. Of the alkali urates the lithium salts are the most soluble
and the acid ammonium salt is the most insoluble. The acid alkali
urates are very insoluble and separate as a sediment (sedimentum later-
itium) from concentrated urine on cooling. According to GUDZENT
1 liter of water at 18° C. dissolves (as primary salts) 1.5313 grams
potassium, 0.8328 gram sodium, and 0.4141 gram ammonium urate,
and at 37° C. 2.7002, 1.5043 and 0.7413 grams of the respective urates.5
The salts of the alkaline earths are soluble with great difficulty. The
above solubilities apply only, in GuDZENT's6 experience, to the freshly
prepared solution, as the solubility to a certain limit gradually dimin-
ishes, due to intramolecular transposition (change of the uric acid from
the lactam-form into the lactim-f orm) .

Besides the mono- and diurates also " quadriurates " have been described
and these occur in the excrement of snakes and birds and in the sedimentum

1 His, Jr., and Paul, Zeitschr. f. physiol. Chem., 31; Smale, Centralbl. f. physiol.,
9; Riidel, Arch. f. exp. Path. u. Pharm., 30; Gudzent, Zeitschr. f. physiol. Chem.,
60 and 63.

2 Zeitschr. f. physiol. Chem., 83.
a Ibid., 10.

4 In regard to the combinations of formaldehyde and uric acid, see Nicolaier,
Deutsch. Arch. f. klin. Med., 89 (1906).

6 Determinations of the solubility of the monourates in serum have been made by
Gudzent, Zeitschr. f. physiol. Chem., 63. See also Bechhold and Ziegler, Bioch. Zeitschr.,
20.

6 Zeitschr. f. physiol. Chem., 56 and 60.

PROPERTIES OF URIC ACID. 709

lateritium. Whether these quadriurates, which have recently been studied
by RINGER, KOHLER and ScHMUTZER,1 are chemical combinations of 2 molecules
uric acid and 1 atom of K or Na or are mixtures, so-called solid solutions of uric
acid in monourates, is still a disputed question.

If a little uric acid in substance is treated on a porcelain dish with
a few drops of nitric acid, the uric acid dissolves on warming, with a
strong development of gas, and after thoroughly drying on the water-
bath a beautiful red residue is obtained, which turns a purple-red (ammo-
nium purpurate or murexide) on the addition of a little ammonia. If
instead of the ammonia we add a little caustic soda (after cooling), the
color becomes deeper blue or bluish violet. This color disappears quickly
on warming, differing from certain purine bodies. This reaction is called
the murexide test.

A solution of phosphotungstic acid, prepared according to certain
directions, gives with a solution of uric acid, when treated with an excess
of sodium carbonate, a beautiful blue solution. This extremely delicate
reaction (1:500,000) was suggested by FOLIN and DENis.2

Uric acid does not reduce an alkaline solution of bismuth, while, on
the contrary, it reduces an alkaline cupric-hydroxide solution. In the
presence of only a little copper salt we obtain a white precipitate consist-
ing of cuprous urate. In the presence of more copper salt red cuprous
oxide separates. The compound of uric acid with cuprous oxide is formed
when copper salts are reduced by glucose or a bisulphite in alkaline
solution in the presence of a sufficient amount of urate.

If a solution of uric acid in water containing alkali carbonate is treated
with magnesium mixture and then a silver-nitrate solution added, a
gelatinous precipitate of silver-magnesium urate is formed. If a drop
of uric acid dissolved in sodium carbonate is placed on a piece of filter-
paper which has been previously treated with silver-nitrate solution,
a reduction of silver oxide occurs, producing a brownish-black or, in the
presence of only 0.002 milligram of uric acid, a yellow spot (SCHIFF'S
test).

If a weak alkaline solution of uric acid in water is treated with a soluble zinc
salt, a white precipitate is produced, which on the filter in the presence of alkali
is oxidized by the air, and becomes sky-blue in color, especially in sunlight.
Potassium persulphate causes a blue coloration immediately (GANASSINI'S
reaction 3).

The precipitation of free uric acid from its alkali salts by means of acids can
be .prevented to some extent by the presence of thymic acid or nucleic acid (GOTO).
According to SEO we are here dealing with combinations of 1 molecule nucleic

1 Ringer, ibid., 67 (literature) and 75; Kohler, ibid., 70 and 72; Ringer and Schmut-
zer, ibid., 82.

2 Journ. of biol. Chem., 12.

3 Cited f. Bioch. Centralbl., 8, 250.

710 URINE.

acid and 2 molecules uric acid, which protects the uric acid within the body against
destruction or transformation into allantoin. This view is incorrect, accord-
ing to SCHITTENHELM and SEISSER.1 According to them no constant combina-
tion between nucleic acid and uric acid exists, and in rabbits the nucleic acid does
not protect the uric acid from transformation to allantoin.

Preparation of Uric Add from Urine. Filtered normal urine is treated
with 20-30 cc. of 25-per cent hydrochloric acid for each liter of urine.
After forty-eight hours collect the crystals and purify them by redis-
solving in dilute alkali, decolorizing with animal charcoal and repre-
cipitating with hydrochloric acid. Large quantities of uric acid are
easily obtained from the excrement of serpents by boiling it with dilute
caustic potash (5-per cent) until no more ammonia is developed. A
current of carbon dioxide is passed through the filtrate until it barely
has an alkaline reaction; dissolve the separated and washed acid potas-
sium urate in caustic potash, and precipitate the uric acid in the filtrate
by addition of an excess of hydrochloric acid.

Quantitative Estimation of Uric Acid in the Urine. As the older
method suggested by HEINTZ, even after recent modifications, gives
inaccurate results, it will not be considered here.

Salkowski and Ludwig's2 method consists in precipitating the uric
acid, by silver nitrate, from the urine previously treated with magnesium
mixture, and weighing the uric acid obtained from the silver precip-
itate. Uric acid determinations by this method are often performed
according to the suggestion of E. LUDWIG, which requires the follow-
ing solutions:

1. An AMMONIACAL SILVER-NITRATE SOLUTION, which contains in 1 liter 26
grams of silver nitrate and a quantity of ammonia sufficient to redissolve com-
pletely the precipitate produced by the first addition of ammonia. 2. MAGNE-
SIA MIXTURE. Dissolve 100 grams of crystallized magnesium chloride in water,
add ammonia until the liquid smells strongly of it, and enough ammonium
chloride to dissolve the precipitate; then dilute the solution to 1 liter. 3. SODIUM
SULPHIDE SOLUTION. Dissolve 10 grams of caustic soda which is free from nitric
acid and nitrous acid in 1 liter of water. One half of this solution is completely
saturated with sulphuretted hydrogen and then mixed with the other half.

The concentration of the three solutions is so arranged that 10 cc.
of each is sufficient for 100 cc. of the urine.

100-200 cc., according to concentration, of the filtered urine, freed
from protein (by boiling after the addition of a few drops of acetic acid),
are poured into a beaker. In another vessel mix 10-20 cc. of the silver
solution with 10-20 cc. of the magnesia mixture and add ammonia,
and when necessary also some ammonium chloride, until the mixture
is clear. This solution is added to the urine while stirring, and the mix-
ture allowed to stand quietly for half an hour. The precipitate isjcol-
lected on a filter, washed with ammoniacal water, and then returned to

lGoto, Zeitschr. f. physiol. Chem., 30; Seo, Arch. f. exp. Path. u. Pharm., 58;
Schittenhelm and Seisser, Zeitschr. f. exp. Path. u. Ther., 7.

2 Salkowski, Virchow's Arch., 52; Pfliiger's Arch., 5; Salkowski, Laboratory
Manual of Physiol. and Path. Chem., translated by Orndorff, 1904; Ludwig, Wien.
med. Jahrbuch, 1884, and Zeitschr. f. anal Chem., 24.

ESTIMATION OF URIC ACID. 711

the same beaker by the aid of a glass rod and a wash-bottle, without
destroying the filter. Now heat to boiling 10-20 cc. of the alkali- sulphide
solution, which has previously been diluted with an equal volume of
water, and allow this solution to flow through the above filter into the
beaker containing the silver precipitate; wash with boiling water, and
warm the contents of the beaker on a water-bath for a time, stirring
constantly. After cooling, filter into a porcelain dish, wash the filter
with boiling water, acidify the filtrate with hydrochloric acid, evaporate
it to about 15 cc., add a few drops more of hydrochloric acid, and allow
it to stand for twenty-four hours. The uric acid which has crystallized
is collected on a small weighed filter, washed with water, alcohol, ether,
and carbon disulphide, dried at 100-110° C., and weighed. For each
10 cc. of aqueous filtrate we must add 0.00048 gram uric acid to the
quantity found directly. Instead of the weighed filter-paper a glass
tube filled with glass wool as described in other handbooks may be sub-
stituted (LUDWIG). Too intense or too long continued heating with
the alkali sulphide must be prevented, otherwise a part of the uric acid
may be decomposed.

SALKOWSKI deviates from this procedure by first precipitating the
urine with a magnesium mixture (50 cc. to 200 cc. urine), filling up to
300 cc., and filtering. Of the filtrate, 200 cc. are precipitated by 10-15
cc. of a 3-per cent silver-nitrate solution. The silver precipitate is shaken
with 200-300 cc. of water acidified with a few drops of hydrochloric
acid, decomposed by sulphuretted hydrogen, heated to boiling, the
silver-sulphide precipitate boiled with fresh water, filtered, the filtrate
concentrated to a few cubic centimeters, treated with 5-8 drops of hydro-
chloric acid, and allowed to stand until the next day. According to
SALKOWSKI and KASHIWABARA 1 the precipitation with zinc salts can also
be used in the estimation of uric acid.

Hopkins' methcd is based on the fact that the uric acid is com-
pletely precipitated from the urine as ammonium urate on saturating
with ammonium chloride. The uric acid can either be weighed after
being set free by hydrochloric acid or it can be determined in several
ways — by titration with potassium permanganate or by the KJELDAHL
method. Several modifications of this method have been worked out
by FOLIN, FOLIN and SCHAFFER, WORNER, and JoLLES.2 Of these methods
we shall describe only that suggested by FOLIN-SCHAFFER.

Folin-Schaffer Method. Treat 300 cc. urine with 75 cc. of a solu-
tion containing 500 grams of ammonium sulphate, 5 grams of uranium
acetate, and 60 cc. of 10 per cent acetic acid in a liter, and filter after
five minutes. This removes an unknown constituent of the urine (a
protein substance) which would otherwise contaminate the uric acid.
Take 125 cc. of the filtrate (corresponding to 100 cc. of the urine) and
add 5 cc. of concentrated ammonia. After twenty-four hours the pre-
cipitate is filtered off and washed free from chlorine on the filter by means
of an ammonium-sulphate solution. The precipitate is washed off the

1 Zeitschr. f. physiol. Chem., 4.

, 8 Hopkins, Journ. of Path, and Bact., 1893, and Proceed. Roy. Soc., 52; Folin,
Zeitschr. f. physiol. Chem., 24; Folin and Schaffer, ibid., 32; Worner, ibid,, 29; Jolles,
ibid., 29; and Wien. med. Wochenschr., 1903.

712 URINE.

filter by water (total 100 cc.) into a flask, treated with 15 cc. of con-
centrated sulphuric acid, and titrated at 60-63° C. with N/20 potassium-
permanganate solution. Each cubic centimeter of this solution cor-
responds to 3.75 milligrams uric acid. Because of the solubility of the
ammonium urate a correction of 3 milligrams must be added for every
100 cc. of the urine.

In regard to the numerous other methods for estimating uric acid,
we must refer to special works on the subject, and especially to HUPPERT-
NEUBAUER. FOLIN with MACULLUM JR. and with DENIS 1 have sug-
gested a colorimetric method for estimating uric acid, making use of
phosphotungstic acid.

Purine Bases ( ALLOXURIC BASES) . The purine bases found in human
urine are xanthine, (guanine), hypoxanthine, adenine, paraxanthine,
heteroxanthine, episarkine, epiguanine, l-methylxanthine. The occur-
rence of guanine and carnine (POUCHET) is, according to KRUGER and SALO-
MON,2 not positively shown. The quantity of these bodies in the urine
is extremely small and varies in different individuals. FLATOW and REIT-
ZENSTEIN 3 found 15.6-45.1 milligrams in the urine voided during twenty-
four hours. The quantity of alloxuric bases in the urine is regularly
increased after feeding with nucleins or food rich in nucleins, and after an
abundant destruction of leucocytes. The quantity is especially increased
in leucaemia. We have a number of observations on the elimination of
these bodies in different diseases, but they are hardly trustworthy on
account of the inaccuracy of the methods used in the determinations.
It must also be remarked that the three purine bases, heteroxanthine,
paraxanthine, and l-methylxanthine, which form the chief mass of the
purine bases of the urine, are derived, according to numerous investiga-
tions4 from the theobromine, caffeine, and theophylline which occur
in the food. With the purine bases we must also differentiate between
those of endogenous and those of exogenous origin,5 and the same factors
apply as for the uric acid, viz., the endogenous purine formation represents
a value which is somewhat variable for different individuals and relatively

1 Journ. of biol. Chem., 13 and 14.

2 Zeitschr. f. physiol. Chem., 24; Pouchet, " Contributions a la connaissance des
matieres extractives de 1'urine." These, Paris, 1880. Cited from Huppert-Neubauer,
333 and 335.

3 Deutsch. med. Wochenschr., 1897.

4Albaneee, Ber. d. d. chem. Gesellsch., 32; Arch. f. exp. Path. u. Pharm., 35;
Bondzynski and Gottlieb, ibid., 36, and Ber. d. deutsch. chem. Gesellsch., 28; E.
Fischer, ibid., 30, 2405; Kriiger and Salomon, Zeitschr. f. physiol. Chem., 26; Kriiger
and Schmidt, Ber. d. d. chem. Gesellsch., 32, and Arch. f. exp. Path. u. Pharm., 45;
Kotake, Zeitschr. f. physiol. Chem., 57.

5 See Burian and Schur, footnote 2, page 702, and Kaufmann and Mohr, Deutsch.
Arch. f. klin. Med., 74.

PURINE BASES. 713

constant for the same individual. According to SiVEN,1 with purine-free
diet the elimination of purines is lowest at night and highest in the morn-
ing hours. Rest and work do not show any positive difference. As
the four true nuclein bases have been treated in Chapter II, it only
remains to describe the special urinary purine bodies.

HN— CO

I I
Heteroxanthine, C6H6N402, 7-monomethylxanthine, OC C.N.CH3, was first

CH

HN— C.N

detected in the urine by SALOMON. It is identical with the monomethylxan-
thine which passes into the urine after feeding with theobromine or caffeine.
SALOMON and NEUBURG 2 found heteroxanthine in the urine of a dog fed entirely
upon meat, and this was probably formed by a methylation in the body.

Heteroxanthine crystallizes in shining needles and dissolves with difficulty
in cold water (1592 parts at 18° C.). It is readily soluble in ammonia and alkalies.
The crystalline sodium salt is insoluble in strong caustic alkali (33-per cent) and
dissolves with difficulty in water. The chloride crystallizes beautifully, is rela-
tively insoluble, and is readily decomposed into the free base and hydrochloric
acid by water. Heteroxanthine is precipitated by copper sulphate and bisul-
phite, mercuric chloride, basic lead acetate and ammonia, and by silver nitrate.
The silver compound dissolves rather easily in dilute, warm nitric acid; it crystal-
lizes in small rhombic plates or prisms, often grown together, forming charac-
teristic crosses. Heteroxanthine does not give the xanthine reaction, but does
give WIEDEL'S reaction, according to FISCHER (see Chapter II).

CH3.N— CO

! I

l-Methylxanthine, C«H6N402, OC C.NH , was first isolated from the

urine and studied by KRUGER, and then by KRUGER and SALOMON.3 It is diffi-
cultly soluble in cold water, but readily soluble in ammonia and caustic soda,
and does not give an insoluble sodium compound. It is readily soluble in dilute
acids, and it crystallizes from its acetic-acid solution in thin, generally hexagonal
plates. The chloride is decomposed into the base and hydrochloric acid by
water. 1-methylxanthine gives crystalline double salts with platinum and gold.
It is not precipitated by basic lead acetate, nor when pure by basic lead acetate
and ammonia. With ammonia and silver nitrate it gives a gelatinous precipitate.
The silver-nitrate compound crystallized from nitric acid forms rosettes of united
needles. With the xanthine test with nitric acid it gives an orange coloration
on the addition of caustic soda. It gives WEIDEL'S reaction (according to FISCHER)
beautifully.

CH..N— CO

I I
Paraxanthine, C7H8N402, 1.7-dimethylxanthine, OC C.NCH3, urotheo-

HN— C.

bromine (THUDICHUM), was first isolated from the urine by THUDICHUM and

1 Skand. Arch. f. Physiol., 18.

2 Salkowski's Festschrift, Berlin, 1904.

3Kriiger, Arch. f. (Anat. u.) Physiol., 1894; Kriiger and Salomon, Zeitschr. f.
physiol. Chem., 24.

714 URINE.

SALOMON.1 It crystallizes beautifully in six-sided plates or in needles. The
sodium compound crystallizes in rectangular plates or prisms and, like the hetero-
xanthine-sodium compound, is insoluble in 33-per cent caustic-soda solution.
The sodium compound separates in a crystalline state on neutralizing its solution
in water. The chloride is readily soluble and is not decomposed by water. The
chloroplatinate crystallizes very beautifully. Mercuric chloride precipitates it
only when added in excess and after a long time. The silver-nitrate compound
separates as White silky crystals from hot nitric acid on cooling. It gives WEIDEL'S
reaction, but not the xanthine test, with nitric acid and alkali.

Episarkine is the name given by BALKE to a purine body occurring in human
urine. The same body has been observed by SALOMON 2 in pigs' and dogs' urine,
as well as in urine in leucaemia. BALKE gives C4H«N80 as the probable formula
for episarkine. It is nearly insoluble in cold water, dissolves with difficulty in
hot water, but may be obtained therefrom as long fine needles. Episarkine does
not give the xanthine reaction with nitric acid, or WEIDEL'S reaction. With
hydrochloric acid and potassium chlorate it gives a white residue which turns
violet with ammonia. It does not form any insoluble sodium coimoound. The
silver compound is difficultly soluble in nitric acid.

HN— CO

Epiguanine, C6H7N60, 7-methylguanine, H2N.C C.N.CH3, was first pre-

N— C.

pared from the urine by KRUGER.3 It is crystalline and difficultly soluble in
hot water or ammonia. It crystallizes from a hot 33-per cent caustic-soda solu-
tion on cooling in broad shining crystals and dissolves readily in hydrochloric or
sulphuric acid. It gives a characteristic chloroplatinate crystallizing in six-sided
prisms. It is precipitated neither by basic lead acetate nor by basic lead ace-
tate and ammonia. Silver nitrate and ammonia give a gelatinous precipitate,
It responds to the xanthine test with nitric acid and alkali. It acts like episarkine
with WEIDEL'S test according to FISCHER.

In preparing alloxuric bases from the urine, the fluid is supersaturated with
ammonia and precipitated by a silver-nitrate solution. The precipitate is then
decomposed with sulphuretted hydrogen. The boiling-hot filtrate is evaporated
to dryness and the dried residue treated with 3-per cent sulphuric acid. The
purine bases are dissolved, while the uric acid remains undissolved. This filtrate
is saturated with ammonia and precipitated by silver-nitrate solution. If instead
of precipitating with silver solution we desire to precipitate, according to KRUGER
and WULFF, with copper suboxide, the urine may be heated to boiling, and imme-
diately are added, successively, 100 cc. of a 50-per cent sodium-bisulphite solu-
tion and 100 cc. of a 12-per cent copper-sulphate solution for every liter of urine.
The thoroughly washed precipitate is decomposed with hydrochloric acid and
sulphuretted hydrogen. The uric acid remains in great part on the filter. Further
details in regard to the treatment of the solution of the hydrochloric-acid com-
pounds may be found in KRUGER and SALOMON.4

iThudichum, " Grundzuge d. anal. med. klin. Chemie " (Berlin, 1886); Salomon,
Arch. f. (Anat. u.) Physiol., 1882, and Ber. d. deutsch. chem. Gesellsch., 16 and 18.

2Balke, " Zur Kenntniss der Xanthinkorper " (Inaug.-Diss., Leipzig, 1893); Salo-
mon, Zeitschr. f. physiol. Chem., 18.

3 Arch. f. (Anat. u.) Physiol., 1894; Kriiger and Salomon, Zeitschr. f. physiol.
Chem., 24 and 26.

4 Zeitschr. f . physiol. Chem., 26, and also Hoppe-Seyler-Thierfelder's Handbuch,
8. Aufl., 188.

ESTIMATION OF PURINE BASES. 715

Quantitative Estimation of Purine Bases according to SALKOWSKi.1
400-600 cc. of the urine free from protein are first precipitated by mag-
nesia mixture, and then by a 3-per cent silver-nitrate solution as described
on page 710. The thoroughly washed silver precipitate is decomposed
by sulphuretted hydrogen after being suspended in 600-800 cc. of water
with the addition of a few drops of hydrochloric acid. It is heated to
boiling and filtered hot, and finally evaporated to dryness on the water-
bath. The residue is extracted with 20-30 cc. of hot 3-per cent sul-
phuric acid and allowed to stand twenty-four hours; the uric acid is
filtered off, washed, the filtrate made ammoniacal, and the purine bodies
again precipitated by silver nitrate, the precipitate collected on a small
chlorine-free filter, washed thoroughly, dried, carefully incinerated,
the ash dissolved in nitric acid, and titrated with ammonium sulpho-
cyanide according to VOLHARD'S method. The ammonium-sulphocyanide
solution should contain 1.2-1.4 grams per liter, and its strength should
be determined by a silver-nitrate solution: 1 part silver corresponds
to 0.277 gram nitrogen X)f purine bases, or to 0.7381 gram purine bases.
By this method the uric-acid and purine bases can be simultaneously
determined in the same portion of urine.2

MALFATTI 3 determines the nitrogen of the purine bases in the hydrochloric-
acid filtrate from the separated uric acid. This filtrate is evaporated with mag-
nesia until all the ammonia has been expelled and the residue used for the KJEL-
DAHL determination.

The nitrogen of the purine bases is also determined as the difference between
the uric-acid nitrogen and the total nitrogen of the purine bodies of the silver
precipitate (CAMERER, ARNSTEiN4). Certain objections have been raised
against this method but they can be overcome by using the modified method
as suggested by KENNAWAY.5

According to the method of KRUGER and SCHMID 6 the uric acid and the
purine bases are precipitated as a cuprous compound by copper-sulphate solu-
tion and sodium bisulphite. The precipitate is decomposed in sufficient water
by sodium sulphide, and the uric acid precipitated from the concentrated filtrate
with hydrochloric acid, and the purine bases again precipitated from this filtrate
as cuprous or silver compounds. Finally, the nitrogen in the uric-acid part and
the part containing the mixture of purine bases is estimated.

Oxaluric Acid, C3H4N2O4 = (CON2H3).CO.COOH. This acid, whose relation
to uric acid and urea has been spoken of above, does not always occur in the
urine, and then only in traces as the ammonium salt. This salt is not directly
precipitated by CaCl2 and NH3, but on boiling it is decomposed into urea and
oxalate. In preparing oxaluric acid from urine the latter is filtered through
animal charcoal. The oxalurate retained by the charcoal may be obtained by
boiling with alcohol.

1 Pfliiger's Arch., 69.

2 In regard to the details we refer the reader to the original paper.

3 Centralbl. f. innere Med., 1897.

4 Camerer, Zeitschr. f. Biologic, 26 and 28; Arnstein, Zeitschr. f. physiol. Chem., 23.
6 Journ. of Physiol., 39.

6 Zeitschr. . f. physiol. Chem., 45, and Hoppe-Seyler-Thierfelder's Handbuch, 8.
Aufl., 590.

716 URINE.

f^OOTT
Oxalic Acid, C2H2O4, or • , occurs under physiological conditions

in very small amounts in the urine, about 0.02 gram in twenty-four hours
(FuRBRiNGER l). According to the generally accepted view it exists in
the urine as calcium oxalate, which is kept in solution by the acid phos-
phates present. Calcium oxalate is a frequent constituent of uninary
sediments, and also occurs in certain urinary calculi.

The origin of the oxalic acid in the urine is not well known. Oxalic
acid when administered is eliminated unchanged, at least in part, by
the urine;2 and as many vegetables and fruits, such as cabbage, spinach,
asparagus, sorrel, apples, grapes, etc., contain oxalic acid, it is possible
that a part of the oxalic acid of the urine originates directly from the
food. That oxalic acid may be formed in the animal body as a metabolic
product from proteins or fats follows from the observations of MILLS
and LUTHJE and others, who found that in dogs on an exclusively meat
and fat diet, as also in starvation, oxalic acid was eliminated by the urine.
The oxalic acid which is eliminated in increased quantity with a diminished
oxygen supply and an increased protein catabolism, as found by REALE
and BOERI, and also by TERRAY, is supposed to be derived partly from
the greater destruction of proteins. Pure protein does not, accord-
ing to SALKOWSKI and WEGRZYNOWSKI 3 increase the quantity of oxalic
acid eliminated; on the contrary, after meat feeding the amount of this
acid is increased, due in part to the meat containing oxalic acid (SAL-
KOWSKI). Gelatin and gelatin-yielding tissues seem to increase the
excretion of oxalic acid, and the same is also true for fats or at least
glycerin (WEGRZYNOWSKI). After feeding nucleins no constant increase
in the elimination of oxalic acid has been observed. The statements
as to the action of carbohydrates are contradictory. The production of
oxalic acid due to an incomplete combustion of the carbohydrates has also
been suggested, and the work of HILDEBRANDT and P. MAYER seems to
indicate this under abnormal conditions. According to DAKiN,4 in rabbits
an increased elimination of oxalic acid occurs after the introduction of
glycollic or glyoxylic acids, and the oxalic acid seems in many cases to
be an intermediary product of metabolism, which is further burnt. We
cannot exclude the possibility of the formation of oxalic acid in the
oxidation of uric acid in the animal body, yet we have no positive proof

1 Deutsch. Arch, f . klin. Med., 18. See also Dunlop, Journ. Path, and Bacteriol., 3.

2 In regard to the behavior of oxalic acid in the animal body, see page 773.
3Reale and Boeri, Wien. med. Wochenschr., 1895; Terray, Pfluger's Arch., 65;

Salkowski, Berl. klin. Wochenschr., 1900; Wegrzynowski. Zeitschr. f. physiol. Chem.,
83 which contains the literature.
4 Journ. of biol. Chem., 3, 57.

ALLANTOIN. 717

of such a formation.1 An endogenous as well as an exogenous origin
of oxalic acid has also been suggested.

Oxalic acid is best detected and quantitatively determined according
to the method suggested by SALKOWSKI : Shaking out the oxalic ^acid from
the acidified urine by means of ether. Detailed account of this can
be found in WEGRZYNOWSKi.2

xNH.CH.HN.CO.NH2,

Allantoin (GLYOXYLDIUREIDE) , C4H6N4O.3, OOC |

NNH.CO

occurs, it is claimed by earlier writers, in the urine of children within the
first eight days after birth, and in very small amounts also in the urine
of adults (GUSSEROW, ZIEGLER and HERMANN). It is found in rather
abundant quantities in the urine of pregnant women (GUSSEROW).
According to WIECHOWSKI the urine of adults, if it contains any allan-
toin at all, has only traces, and he could not detect any in the urine of
nurslings or in the amniotic fluid, which does not agree with previous
reports. Allantoin has also been found in the urine of suckling calves
(WOHLER), in urine of oxen (SALKOWSKI), and sometimes in the urine of
other animals (MEISSNER). WIECHOWSKI has found it in relatively
large quantities in the urine of the dog, cat, rabbit and monkey, and he
considers that allantoin is a terminal metabolic product in these ani-
mals. It is also found, as first shown by VAUQUELIN and LASSAIGNE,S
in the allantoic fluid of the cow (hence the name). That allantoin is
formed from the uric acid in mammalia is almost certain, and the inves-
tigations on which this is based have already been given in discussing the
decomposition of uric acid.4 The allantoin thus originates from the
purine bodies, and consequently in dogs and other animals the excretion
of allantoin is considerably increased, according to MINKOWSKI, COHN,
SALKOWSKI, and MENDEL and BROWN,S after feeding thymus or pan-
creas. A strong allantoin excretion is also found in dogs after poisoning
with hydrazine (BoRissow), hydroxylamine, semicarbazide, and amino-
guanidine (POHL), and this increase in the excretion of allantoin is

1 See Wiener, Ergebn. d. Physiol., 1; Tomaszewski, Zeitschr. f. exp. Path. u. Ther.,
7; Pohl, ibid., 8; Jastrowitz, Bioch. Zeitschr., 28.

2 Zeitschr. f . physiol. Chem., 83.

3 Ziegler and Hermann, see Gusserow, Arch, f . Gynakol., 3 — both cited from Huppert-
Neibauer, Harn-Analyse, 10. Aufl., 377; Wohler, Annal. d. Chem. u. Pharm., 70;
Salkowski, Zeitschr. f. physiol. Chem., 42; Meissner, Zeitschr. f. rat. Med. (3), 31;
Lassaigne, Annal. de Chim. et Phys., 17; Wiechowski, Hofmeister's Beitrage, 11, and
Arch. f. exp. Path. u. Pharm., 60, and Bioch. Zeitschr., 19 and 25.

4 See footnote 2, page 706.

6 Minkowski, Arch. f. exp. Path. u. Pharm., 41, and Centralbl. f. innere Med., 1898;
Cohn, Zeitschr. f. physiol. Chem., 25; Salkowski, Centralbl. f. d. med. Wissensch.,
1898; Mendel and Brown, Amer. Journ. of Physiol., 3.

718 URINE.

connected with the nuclein metabolism. POHL l has found, in dogs on
poisoning with hydrazine, that the liver contained allantoin and that other
organs contained traces, while it does not exist in the organs of normal
dogs, and he has also detected the formation of allantoin in the autolysis
of the intestinal mucosa, liver, thymus, spleen and pancreas. It is very
probable that in these cases we are dealing with a destruction of cells
and an enzymotic uric acid formation with a subsequent uricolysis
with the formation of allantoin. Certain food-stuffs such as milk, wheat
bread, peas and beans contain, according to ACKROYD, small amounts of
allantoin, which are introduced into the body. Nothing is known about
how these traces of allantoin behave in the body. According to Po-
DUSCHKA and MiNKOWSKi,2 allantoin introduced into dogs appears almost
entirely in the urine, while in man only a small portion of the ingested
substance is eliminated in the urine and seems in the last case to be chiefly
burned.

Allantoin is a colorless substance often crystallizing in prisms, dif-
ficultly soluble in cold water, easily soluble in boiling water, and also in
warm alcohol, but not soluble in cold alcohol or ether. A watery alla-
toin solution gives no precipitate with silver nitrate alone, but by the
careful addition of ammonia a white flocculent precipitate is formed,
C4H5AgN4Os, which is soluble in an excess of ammonia and which con-
sists after a certain time of very small, transparent microscopic globules.
The dry precipitate contains 40.75 per cent silver. A watery allantoin
solution is precipitated by mercuric nitrate. On continued boiling
allantoin reduces FEHLING'S solution. It gives SCHIFF'S furfurol reac-
tion less rapidly and less intensely than urea. Allantoin does not give
the murexide test.

Allantoin is most easily prepared by the oxidation of uric acid with
lead peroxide or potassium permanganate. In preparing allantoin from
urine we must proceed differently according to whether we are using the
urine of animals comparatively rich in allantoin or whether we are using
human urine, which is very poor in allantoin. The same applies to the
quantitative estimation of allantoin. As the methods in both cases are
complicated and require certain percautions we cannot here enter into a
detailed description of them, and we refer to the works of LOEWI and
WiECHOWSKi3 and to the complete handbooks for details. The pre-

1 Borissow, Zeitschr. f. physiol. Chem., 19; Pohl. Arch. f. exp. Path. u. Pharm.,
46; Poduschka, ibid., 44. According to Underbill and Kleiner, Journ. of biol. Chem.,
4, hydrazine has no other action on the excretion of allantoin than that caused by
the refusal to take food brought about by the poison.

2 Ackroyd, Bioch. Journ., 5; Poduschka, Arch. f. exp. Path. u. Pharm., 44; Min-
kowski, ibid., 41.

3 Loewi, ibid., 44; Wiechowski, Hofmeister's Beitrage, 11, and Arch, f. exp. Path,
u. Pharm., 60; and Bioch. Zeitschr., 19 and 25.

HIPPURIC ACID. 719

cipitation of allantoin from the urine can be accomplished by mercuric
nitrate and by mercuric acetate solutions, in the presence of sodium
acetate.

Glyoxylic Acid, C2H404, QQQTT , is produced on boiling allantoin as well as

uric acid with alkalies, and also on the oxidation of many substances, among
which we can mention creatine and creatinine. It is also of interest that allantoin
can be prepared synthetically from glyoxylic acid and urea and that glyoxylic
acid yields oxalic acid when introduced into the body. The reports in regard
to its occurrence in the urine conflict,1 as it is readily destroyed in the body, and
its passage into the urine is very improbable, or at least only seldom occurs.

Hippuric Acid (BENZOYL-AMINO ACETIC ACID),

C9H9NO3 = (C6H5CO)HN • CH2COOH.

This acid decomposes into benzoic acid and glycocoll on boiling with
mineral acids or alkalies, and also in the putrefaction of the urine. The
reverse of this occurs if these two components are heated in a sealed
tube, according to the following equation: C6H5COOH+NH2.CH2.COOH
= C6H5.CO.NH.CH2.COOH+H2O. This acid may be synthetically
prepared from benzamide and monochloracetic acid, CeH5.CO.NH2
+CH2Cl.COOH = C6H5.CO.NH.CH2.COOH-r-HCl, and in various other
ways, but most simply from glycocoll and benzoyl chloride in the presence
of alkali.

Hippuric acid occurs in large amounts in the urine of herbivora,
but only in small quantities in that of carnivora. The quantity of hip-
puric acid eliminated in human urine on a mixed diet is usually less than
1 gram per day; as an average it is 0.7 gram. After eating freely of vege-
tables and fruit, especially such fruit as plums, the quantity may be
more than 2 grams. Hippuric acid is also found in the perspiration, the
blood, the suprarenal capsule of oxen, and in ichthyosis scales. Noth-
ing is positively known in regard to the quantity of hippuric acid in the
urine in disease.

The Formation of Hippuric Acid in the Organism. Benzoic acid and
also the substituted benzoic acids are converted into hippuric acid and
substituted hippuric acids within the body. Moreover, those bodies
are transformed into hippuric acid which by oxidation (toluene, cinnamic
acid, hydrocinnamic acid) or by reduction (quinic acid) are converted
into benzoic acid. The question of the origin of hippuric acid is there-
fore connected with the question of the origin of benzoic acid; the for-
mation of the second component, glycocoll, from the protein substances
in the body is unquestionable.

1 The literature on the occurrence and detection of glyoxylic acid in the urine can
be found in Granstrom, Hofmeister's Beitrage, 11.

720 URINE.

Hippuric acid is found in the urine of starving dogs (SALKOWSKI),
also in dog's urine after a diet consisting entirely of meat (MEISSNER
and SHEPARD, SALKOWSKI, and others1). It is evident that the benzoic
acid originates in these cases from the proteins, and it is generally admitted
that it is produced by the putrefaction of proteins in the intestine.
Among the products of the putrefaction of protein outside of the body
SALKOWSKI found phenylpropionic acid, C6H5.CHo.CH2.COOH, which
is oxidized in the organism to benzoic acid and eliminated as hippuric
acid after combining with glycocoll. Phenylpropionic acid seems to be
formed from the phenylalanine. The supposition that the phenylpro-
pionic acid is produced from tyrosine by putrefaction of the intestine
has not been substantiated by the researches of BAUMANN, SCHOTTEN,
and BAAS.2 The importance of putrefaction in the intestine in pro-
ducing hippuric acid is evident from the fact that after thoroughly dis-
infecting the intestine of dogs with calomel the hippuric acid disappears,
from the urine (BAUMANNS) .

The large quantity of hippuric acid present in the urine of herbivora
is partly explained by the specially active processes of putrefaction going
on in the intestine of these animals. According to VASiLiu4 this can
hardly be correct, because, as he has found, by feeding sheep with casein,
this would require a too intense putrefaction of the protein (indeed 40
per cent of it). This author's explanation lies in part that in the her-
bivora only a small part of the phenylalanine is burnt, and is used to a
greater extent in the formation of hippuric acid than in man and car-
nivora, and in part by the fact that the food of herbivora contains larger
quantities of a non-nitrogenous mother-substance of the benzoic acid.
There is hardly any doubt that the hippuric acid in human urine after
a mixed diet, and especially after a diet of vegetables and fruits, orig-
inates in part from the aromatic substances, e. g., quinic acid.

The view proposed by WEISS and others that a parallelism exists between
the excretion of hippuric acid and uric acid in that an increase in the first is
followed by a diminution in the second, and that, for example, quinic acid pro-
duces a diminution in the excretion of uric acid corresponding to the increased
formation of hippuric acid (WEISS, LEWIN), cannot be considered as sufficiently
proven (HupFER).5

1Salkowski, Ber. d. deutsch. chem. Gesellsch., 11; Meissner and Shepard,
Untersuch. iiber das Entstehen der Hippursaure im thierschen Organismus. Hanover,
1886.

2 E. and H. Salkowski, Ber. d. deutsch. Chem. Gesellsch., 12; Baumann, Zeitschr.
f. physiol. Chem., 7; Schotten, ibid., 8; Baas, ibid., 11.

3 Ibid., 10, 131.

4 Vasiliu, Mitt. d. landwirt. Inst. Breslau, Bd. 4, 1907.

6 Weiss, Zeitschr. f. physiol. Chem., 25, 27, 38; Lewin, Zeitschr. f. klin. Med., 42;

FORMATION OF HIPPURIC ACID. 721

As the thorough investigations of WIECHOWSKI teach, the synthesis
of hippuric acid does not stand in any direct relation to the extent of
protein metabolism; it varies, on the contrary, with the duration of
circulation of benzoic acid and the quantity of glycocoll present in
the body. The amount of the latter in intermediary metabolism is so
great that in rabbits, on the administration of benzoic acid, more than
one-half of the total urine nitrogen may exist as glycocoll. MAGNUS-
LEVY l found in rabbits and sheep up to 27.8 per cent of the total nitro-
gen as hippuric-acid nitrogen, and both investigators have found so much
hippuric-acid nitrogen that it could not be accounted for by the glycocoll
preformed from the proteins, which amounts to about 4-5 per cent of
the total nitrogen of the protein of the food and body.

In carnivora (dog) and man the conditions are different, according
to BBUGSCH and R. HIRSCH, FEIGIN and BRUGSCH, as in these cases there
is no more glycocoll available for hippuric acid formation than is split
off from the proteins en hydrolysis. According to the investigations of
LEWINSKI 2 this does not seem to be correct, at least not for man. After
abundant introduction of benzoic acid in man about 34 per cent of the
total nitrogen may be excreted as hippuric acid and in a recent investiga-
tion he was able to obtain 50.5 grams pure crystalline hippuric acid
from the 24-hour urine of a man after feeding sodium benzoate.

The abundant production of hippuric acid in herbivora induced ABDER-
HALDEN, GIGON and STRAUSS to investigate the comparative supply of
certain amino-acids in carnivora and herbivora, and they found in cats,
rabbits and hens that the percentage quantity of glycocoll split off from
the entire organism (with the exception of the intestinal contents and
fat and feathers) by hydrolysis was the same, namely 2.33 to 3.34 per
cent of the proteins. In order to account for the large quantity of
glycocoll which can be eliminated as hippuric acid, we must admit of a
formation of glycoccll. That this occurs in animals fed with benzoic
acid has been recently proved by ABDERHALDEN and HIRSCH by very
conclusive experiments. It can be assumed that the benzoic acid com-
bines with higher amino-acids and that the hippuric acid is formed from
this combination. The investigations of MAGNUS-LEVY to prove this
assumption, where he used benzoylated higher amino-acids, have not

Hupfer, Zeitschr. f. physiol. Chem., 37. See also Wiener, " Die Harnsaure," Ergeb-
nisse der Physiol., 1, Abt. 1.

1 Wiechowski, Hofmeister's Beitrage, 7 (literature); A. Mangus-Levy, Munch,
med. Wochenschr., 1905; Ringer, Journ. of biol. Chem., 10; Epstein and Bookman,
ibid., 10.

2 Brugsch and Hirsch, Zeitschr. f. exp. Path. u. Therap., 3; Brugsch, Maly's
Jahresber., 37, 621, and Bioch. Centralbl., 8, 336; Feigin, Maly's Jahresber., 36,
631; Lewinski, Arch. f. exp. Path. u. Pharm., 58 and 61.

722 URINE.

given support to this assumption; EPSTEIN and BOOKMAN l found never-
theless in experiments with rabbits after feeding with benzoyl-leucine
that a great elimination of hippuric acid occurred which they consider
as a formation of glycocoll from this leucine. Free leucine on the con-
trary does not increase the hippuric acid elimination.

The kidneys may be considered in dogs as special organs for the syn-
thesis of hippuric acid (SCHMIEDEBERG and BUNGE 2). In other animals
as in rabbits, the formation of hippuric acid seems to take place in other
organs, such as the liver and muscles. The synthesis of hippuric acid is
therefore not exclusively limited to any special organ, though perhaps
in some species of animals it may be more abundant in one organ than in
another.

Properties and Reactions of Hippuric Acid. This acid crystallizes in
semi-transparent, long, four-sided, milk-white, rhombic prisms or columns,
or in needles by rapid crystallization. They dissolve in 600 parts cold
water, but more easily in hot water. They are easily soluble in alcohol,
but with difficulty in ether. The acid dissolves more easily (about 12
times) in acetic ether than in ethyl ether. Petroleum-ether does not
dissolve hippuric acid.

On heating hippuric acid it first melts at 187.5° C. to an oily liquid
which crystallizes on cooling. On continued heating it decomposes,
producing a red mass and a sublimate of benzoic acid, with the genera-
tion, first, of a peculiar pleasant odor of hay and then an odor of hydro-
cyanic acid. Hippuric acid is easily differentiated from benzoic acid
by this behavior, also by its crystalline form and its insolubility in
petroleum ether. Hippuric acid and benzoic acid both give LUCRE'S
reaction, namely, they generate an intense odor of nitrobenzene when
evaporated to dryness with nitric acid and when the residue is heated
with sand in a glass tube. Hippuric acid in most cases forms crystal-
lizable salts, with bases. The combinations with alkalies and alkaline
earths are soluble in water and alcohol. The silver, copper, and lead
salts are soluble with difficulty in water; the ferric salt is insoluble.

Hippuric acid is best prepared from the fresh urine of a horse or cow.
The urine is boiled a few minutes with an excess of milk of lime. The
liquid is filtered while hot, concentrated and then cooled, and the hippuric
acid precipitated by the addition of an excess of hydrochloric acid. The
crystals are pressed, dissolved in milk of lime by boiling, and treated as
above; the hippuric acid is precipitated again from the concentrated

1 Abderhalden, Gigon and Strauss, Zeitschr. f. physiol. Chem., 51; Abderhalden
and Hirsch, ibid., 78; Mangus-Levy, Bioch. Zeitschr., 6; Epstein and Bookman,
Journ. of biol. Chem., 13.

2 Arch. f. exp. Path. u. Pharm., 6; also A. Hoffmann, ibid., 7, and Kochs, Pfliiger's
Arch., 20; Bashford and Cramer, Zeitschr. f. physiol. Chem., 35.

PHENACETURIC ACID. BENZOIC ACID. 723

filtrate by hydrochloric acid. The crystals are purified by recrystalliza-
tion and decolorized, when necessary, by animal charcoal.

The quantitative estimation of hippuric acid in the urine may be
performed by the following method (BUNGE and SCHMIEDEBERG):
The urine is first made faintly alkaline with soda, evaporated nearly to
dryness, and the residue thoroughly extracted with strong alcohol.
After the evaporation of the alcohol the residue is dissolved in water,
the solution acidified with sulphuric acid, and completely extracted by
agitating (at least five times) with fresh portions of acetic ether. The
acetic ether is then repeatedly washed with water, which is removed by
means of a separatory funnel, then evaporated at a medium temperature
and the dry residue treated repeated!}' with petroleum-ether, which
dissolves the benzoic acid, oxyacids, fats, and phenols, while the hippuric
acid remains undissolved. This residue is now dissolved in a little warm
water and evaporated at 50-60° C. to crystallization. The crystals are
collected on a small weighed filter. According to HENRIQUES and
SORENSEN the acidified urine can be directly shaken out with acetic ether,
the residue after evaporation of the acetic ether boiled with hydrochloric
acid in order to split the hippuric acid into benzoic acid and glycocoll
and the quantity of nitrogen in the latter determined by a formol titra-
tion. Other methods have recently been suggested by FOLIN and
FLANDERS, by STEENBOCK and by HRYNTSCHAK.1

Phenaceturic Acid, C1oHnN03=C6H5.CH2.CO.NH.CH2.COOH. This acid,
which is produced in the animal body by a combination of glycocoll with the phenyl-
acetic acid, C6H6.CH2.COOH, formed in the putrefaction of the proteins, has
been prepared from horse's urine by SALKOWSKi,2 but it probably also occurs in
human urine. According to VASILIU 3 it is just as important a constituent of the
urine of herbivora as hippuric acid is.

Benzoic Acid, C7H602 or C6H5.COOH, is found in rabbit's urine and sometimes,
though in small amounts, in dog's urine (WEYL and v. ANREP). According to
JAARSVELD and STOKVIS and to KRONECKER it is also found in human urine in
diseases of the kidneys. The occurrence of benzoic acid in the urine seems to
be due to a fermentative decomposition of hippuric acid. Such a decomposi-
tion may very easily occur in an alkaline urine or in one containing proteid(VAN
DE VELDE and STOKVIS). In certain animals — pigs and dogs — the kidneys,
according to SCHMIEDEBERG and MiNKOwsKi,4 contain a special enzyme, SCHMIEDE-
BERG'S histozym, which splits the hippuric acid with the separation of benzoic
acid.

Ethereal Sulphuric Acids. In the putrefaction of proteins in the
intestine, phenols — whose mother-substance is considered to be tyrosine —
and also indol and skatol are produced. These phenols directly, and the
two last-named bodies after they have been oxidized respectively into

1 Bunge and Schmiedeberg, Arch. f. exp. Path. u. Pharm., 6; Henriques and
Sorensen, Zeitschr. f. physiol. Chem., 64; Folin and Flanders, Journ. of biol. Chem., 11;
Steenbock, ibid., 11; Hryntschak, Bioch. Zeitschr., 43.

2 Zeitschr. f. physiol. Chem., 9.

3 Mitteil. d. landw. Inst., Breslau, 4.

4 Weyl and v. Anrep, Zeitschr. f. physiol. Chem., 4; Jaarsveld and Stokvis, Arch,
f. exp. Path. u. Pharm., 10; Kronecker, ibid., 16; Van de Velde and Stokvis, ibid.,
17; Schmiedeberg, ibid., 14, 379; Minkowski, ibid., 17.

724 UKINE.

indoxyl and skatoxyl, pass into the urine as ethereal sulphuric acids
after uniting with sulphuric acid. The most important of these ethereal
acids are phenol- and cresol-sulphuric acids — which were formerly also
called phenol-forming substances — indoxyl- and skatoxyl-sulphuric acids.
To this group also belong pyrocatechin-sulphuric acid, which occurs
only in very small amounts in human urine, and hydroquinone-sulphuric
acid, which appears in the urine after poisoning with phenol, and under
physiological conditions perhaps other ethereal acids occur which have
not been isolated. The ethereal sulphuric acids of the urine were dis-
covered and specially studied by BAUMANN.X The quantity of these
acids in human urine is small, while horse's urine contains larger quan-
tities. According to the determinations of v. D. VELDEN the quantity
of ethereal sulphuric acid in human urine in twenty-four hours varies
between 0.094 and 0.620 gram. C. TOLLENS found an average of 0.18
gram. The relation of the sulphate-sulphuric acid A to the conjugated
sulphuric acid B, in health, is on an average 10:1. It undergoes such
great variations, as found by BAUMANN and HERTER,2 and after them
by many other investigators, that it is hardly possible to consider the
average figures as normal. After taking phenol and certain other
aromatic substances, as well as when putrefaction within the organism
is general, the elimination of ethereal sulphuric acid is greatly increased.
On the contrary, it is diminished when the putrefaction in the intestine
is reduced or prevented. For this reason it may be greatly diminished
by carbohydrates and exclusive milk diet.3 The intestinal putrefaction
and the elimination of ethereal sulphuric acid have also been diminished
in some cases by certain therapeutic agents which have an antiseptic
action; still the investigators do not agree in their reports.4

Great importance has been given to the relation between the total
sulphuric acid and the conjugated sulphuric acid, or between the con-
jugated sulphuric acid and the sulphate-sulphuric acid, in the study
of the intensity of the putrefaction in the intestine under different con-
ditions. Several investigators, F. MULLER, SALKOWSKI, and v. NOORDEN,S

ipfluger's Arch., 12 and 13.

2 v. d. Velden, Virchow's Arch., 70; Tollens, Zeitschr. f. physiol. Chem., 67; Herter,
Zeitschr. f. physiol. Chem., 1.

3 See Hirschler, Zeitschr. f. physiol. Chem., 10; Biernacki, Deutsch. Arch. f. klin.
Med., 49; Rovighi, Zeitschr. f. physiol. Chem,, 16; Winternitz, ibid., and Schmitz,
ibid., 17 and 19.

4 See Baumann and Morax, Zeitschr. f. physiol. Chem., 10; Steiff, Zeitschr. f.
klin. Med., 16; Rovighi, 1. c.; Stern, Zeitschr. f. Hyg., 12; and Bartoschewitsch,
Zeitschr. f. physiol. Chem., 17; Mosse, ibid., 23.

5 Miiller, Zeitschr. f. klin. Med., 12; v. Noorden, ibid., 17; Salkowski, Zeitschr.
f. physiol. Chem., 12.

PHENOL- AND CRESOL-SULPHURIC ACIDS. 725

consider correctly that this relation is only of secondary value, and that
it is more correct to consider the absolute value. It must be remarked
that the absolute values for the conjugated sulphuric a,cid also undergo
great variation, so that it is at present impossible to give the upper or
lower limit for the normal value.

Phenol- and p-Cresol-sulphuric Acids, C6H5.O.SO2.OH and

O.S02.OH
CeH4<^ These acids are found as alkali salts in human urine,

CH3

in which also orthocresol has been detected. The quantity of cresol-
sulphuric acid is considerably greater than of phenol-sulphuric acid.
In the quantitative estimation the phenols are set free from the two
ethereal acids and determined together as tribromphenol. The quan-
tity of phenols which are separated from the ethereal-sulphuric acids
of the urine amounts to 17-51 milligrams in the twenty-four hours
(MUNK) . In nine case investigated by 'SIEGFRIED and ZIMMERMANN 1
they found in the urine of healthy students in 1500 cc. urine an average
of 44.6 milligrams phenols, of which 26 milligrams was cresol and 18.6
milligrams was phenol. After the ingestion of carbolic acid, which is
in great part converted by synthesis within the organism into phenol-
sulphuric acid, also into pyrocatechin- and hydroquinon-sulphuric acid 2
or when the amount of sulphuric acid is not sufficient to combine with
the phenol, it forms phenol-glucuronic acid,3 the quantity of phenols
and ethereal-sulphuric acids in the urine is considerably increased at the
expense of the sulphate-sulphuric acid. The same is also true of other
phenols. The cresol is in great part changed into phenol in dogs, accord-
ing to SIEGFRIED and ZIMMERMANN .4

An increased elimination of phenol-sulphuric acids occurs in active
putrefaction in the intestine with stoppage of the contents of the intes-
tine, as in ileus, diffused peritonitis with atony of the intestine, or tuber-
culous enteritis, but not in simple obstruction. The elimination is also
increased by the absorption of the products of putrefaction from
purulent wounds or abscesses. An increased elimination of phenol has
been observed in a few other cases of diseased conditions of the body.6

1 Munk, Pflviger's Arch., 12; Siegfried and Zimmermann, Bioch. Zeitschr ., 34.

2 See Baumann, Pfliiger's Arch., 12 and 13, and Baumann and Preusse, Zeitschr.
f. physiol. Chem., 3, 156.

3 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 14; C. Tollens, Zeitschr. f. physiol.
Chem., 67.

4 Bioch. Zeitschr., 46.

5 See G. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 12 (this contains also all refer-
ences to the literature on this subject); Fedeli, Moleschott's Untersuch., 15.

726 UKINE.

The alkali salts of phenol- and cresol-sulphuric acids crystallize in
white plates, similar to mother-of-pearl, which are rather freely soluble
in water. They ,are soluble in boiling alcohol, but only slightly soluble
in cold alcohol. On boiling with dilute mineral acids they are decom-
posed into sulphuric acid and the corresponding phenol.

Phenol-sulphuric acids have been synthetically prepared by BAUMANN
from potassium pyrosulphate and potassium phenolate or p-cresolate.
For the method of their preparation from urine, which is rather compli-
cated, and also for the known phenol reactions, the reader is referred
to other text-books. The quantitative estimation of the phenols from
these etheral sulphuric acids is now ordinarily done by the following
methods :

KOSSLER and PENNY'S method with NEUBERG'S l modification. The
liquid containing phenol is treated with N/10 caustic soda until strongly
alkaline, warmed on the water-bath in a flask with a glass stopper, and
then treated with an excess of N/10 iodine solution, the quantity being
exactly measured. Sodium iodide is first formed and then sodium
hypoiodite, which latter forms tri-iodophenol with the phenol accord-
ing to the following equation:

On cooling, acidify with sulphuric acid and determine the excess of iodine
by titration with N/10 sodium thiosulphate solution. This process is
also available for the estimation of paracresol. Each cubic centimeter
of the iodine solution used is equivalent to 1.5670 milligrams of phenol
or 1.8018 milligrams of cresol. As the determination does not give any
idea as to the variable proportions of the two phenols, the quantity of
iodine used must be calculated as one or the other of the two phenols.
Before such a determination is carried out, the concentrated urine is
first distilled after acidification with sulphuric acid and the distillate
purified by precipitation with lead, and distilled again (NEUBERG).
MOOSER has raised objections against the use of sulphuric acid and rec-
ommends instead the use of phosphoric acid. In regard to the dispute
which has arisen between NEUBERG and MOOSER 2 as well as to the details
of NEUBERG'S method we must refer to the original publications and to
larger handbooks.

For the separate estimation of phenol and p-cresol in the urine a
special method has been suggested by SIEGFRIED and ZIMMERMANN.S
The principle of the method consists in the two following estimations:
1. The quantity of bromine necessary to convert the phenol and cresol
into tribromphenol and tribromcresol is determined. 2. The quantity
of bromine necessary to convert the phenol into tribromphenol and the

1 Kossler and Penny, Zeitschr. f. physiol. Chem., 17; Neuberg, ibid., 27.

2 Mooser, Zeitschr. f. physiol. Chem., 63, with Liechti, ibid., 73; Neuberg and
Hildesheimer, Bioch. Zeitschr., 28; Marie Hensel, Zeitschr. f. physiol. Chem., 78.

8 Siegfried and Zimmermann, Bioch. Zeitschr., 29, 34 and 38; see also Ditz and Bar-
dach, ibid., 37 and 42.

PYROCATECHIN-SULPHURIC ACID. 727

cresol into dibromcresol under exactly observed conditions is deter-
mined and from the quantities of bromine by weight (Bi and 62)
quantities by weight of phenol and cresol can be calculated. In regard to
the procedure as well as to the necessary solutions we refer to the original
publication.

The methods for the separate determination of the conjugated sul-
phuric acid and the sulphate-sulphuric acid will be spoken of later in
connection with the determination of the sulphuric acid of the urine.

Pyrocatechin-sulphuric Acid. This acid was first found in horse's urine in
rather large quantities by BAUMANN. It occurs in human urine only in the very
smallest amounts, and perhaps not constantly, but it is present abundantly in
the urine after taking phenol, pyrocatechin, or protocatechuic acid.

With an exclusively meat diet this acid does not occur in the urine, and it
therefore must originate from vegetable food. It probably originates from the
protocatechuic acid, which, according to PREUSSE, passes in part into the urine
as pyrocatechin-sulphuric acid. This acid may also perhaps be formed by the
oxidation of phenol within the organism (BAUMANN and PREUSSE l).

Pyrocatechin, or O-DIOXYBENZENE, C6H4(OH)2, was first observed in the urine
of a child (EBSTEIN and J. MULLER). The reducing body ALCAPTON, first found
by BODEKER 2 in human urine and which was considered for a long time as iden-
tical with pyrocatechin, is in most cases probably homogentisic acid (see below).

Pyrocatechin crystallizes in prisms which are soluble in alcohol, ether, and
water. It melts at 102-104° C., and sublimes in shining plates. The watery
solution becomes green, brown, and finally black in the presence of alkali
and the oxygen of the air. If very dilute ferric chloride is treated with tartaric
acid and then made alkaline with ammonia, and this added to a watery solution of
pyrocatechin, we obtain a violet or cherry-red liquid which becomes green on
adding excess of acetic acid. Pyrocatechin is precipitated by lead acetate. It
reduces an ammoniacal silver solution at the ordinary temperature, and with
heat reduces alkaline copper-oxide solutions but does not reduce bismuth oxide.

A urine containing pyrocatechin, if exposed to the air, especially when alkaline,
quickly becomes dark and reduces alkaline copper solutions when heated. In
detecting pyrocatechin in the urine it is concentrated when necessary, filtered,
boiled with the addition of sulphuric acid to remove the phenols, and repeatedly
shaken, after cooling, with ether. The ether is distilled from the several ethereal
extracts, the residue neutralized with barium carbonate and shaken again with
ether. The pyrocatechin which remains after evaporating the ether may be
purified by recrystallization from benzene.

Hydroquinone, or P-DIOXYBENZENE, C6H4(OH)2, often occurs in the urine after
the use of phenol (BAUMANN and PREUSSE). The dark color which certain urines,
so-called " carbolic urines/' assume in the air is due to decomposition products.
Hydroquinone does not occur as a normal constituent of urine, but only after
the administration of hydroquinone; and according to LEWiN,3 it may be found
in the urine of rabbits as an ethereal-sulphuric acid, being a decomposition product
of arbutin.

Hydroquinone forms rhombic crystals which are readily soluble in water,
alcohol, and ether. It melts at 169° C. Like pyrocatechin, it easily reduces
metallic oxides. It acts like pyrocatechin with alkalies, but is not precipitated
by lead acetate. It is oxidized into quinone by ferric chloride and other oxidiz-

1 Baumann and Herter, Zeitschr. f. physiol. Chem., 1; Preusse, ibid., 2; Baumann,
ibid., 3.

2 Ebstein and Muller, Virchow's Arch., 62; Bodeker, Zeitschr. f. rat. Med. (3), 7.
J Lewin, Virchow's Arch., 92; Bass, Zeitschr. f. exp. Path. u. Ther., 10.

728 URINE.

ing agents, and quinone can be detected by its peculiar odor. Hydroquinone-
sulphuric acid is detected in the urine by the same methods as pyrocatechin sul-
phuric acid.

C.O.S02.OH
Indoxyl-sulphuric Acid, C8H7NS04, C6H</\CH , also called

NH

URINE INDICAN, formerly called UKOXANTHINE (HELLER), occurs as an
alkali-salt in the urine. This acid is the mother-substance of a great
part of the indigo of the urine. The quantity of indigo which can be
separated from the urine is considered as a measure of the quantity of
indoxyl-sulphuric acid (and indoxyl-glucuronic acid) contained in the
urine. This amount, according to JAFFE, for man is 5-20 milligrams
per twenty-four hours, and 0.9-37.6 milligrams according to MAiLLARD.1
Horse's urine contains about twenty-five times as much indigo-forming
substance as human urine.

Indoxyl-sulphuric acid is derived, as previously mentioned (page 515),
from indol, which is first oxidized in the body into indoxyl and is then
conjugated with sulphuric acid. After subcutaneous injection of indol
the elimination of indican is considerably increased (JAFFE, BAUMANN
and BRIEGER, and others). It is also increased by the introduction
in the animal organism of orthonitrophenolpropiolic acid (G. HOPPE-
SEYLER2). Indol is formed by the putrefaction of proteins. The
putrefaction of secretions rich in protein in the intestine also explains the
occurrence of indican in the urine during starvation. Gelatin, on the
contrary, does not increase the elimination of indican.

An abnormally increased elimination of indican occurs in those
diseases where the small intestines are obstructed, causing an increased
putrefaction and thus producing an abundance of indol. Such an increased
elimination of indican occurs on tying the small intestine of a dog, but
not the large intestine (JAFFE), an observation which has been recently
confirmed by ELLINGER and PRUTZ.3 They removed an intestine loop
in dogs and replaced it in a reversed position, the distal end of the loop
being attached to the proximal end of the intestine, and in this manner,
by the inverted peristalsis so obtained, they effected a disturbance in
the movement of the intestinal contents. It was shown that this obstruc-
tion in the small intestine caused an increased elimination of indican,
while an obstruction in the large intestine showed no such action.

1 Jaffe, Pfliiger's Arch., 3; Maillard, Journ. de Physiol. et de Pathol., 12.

2 Jaffe, Centralbl. f. d. med. Wissensch., 1872; Baumann and Brieger, Zeitschr. f.
physiol. Chem., 3; G. Hoppe-Seyler, ibid., 7 and 8. See also Porcher and Hervieux,,
Journ. de Phyisol., 7.

3 Jaffe, Virchow's Arch., 70; Ellinger and Prutz, Zeitschr. f. physiol. Chem., 38.

INDOXYL-SULPHUKIC ACID. 729

The putrefaction of proteins in other organs and tissues besides the
intestine may also cause an increase in the indican of the urine. Cer-
tain investigators, BLUMENTHAL, ROSENFELD, and LEWIN, claim to
have shown that an increased excretion of indican can also be 'brought
about without putrefaction by an increased destruction of tissue in starva-
tion and also after phlorhizin poisoning; but these statements are vehe-
mently opposed by other investigators, such as P. MAYER, SCHOLZ, and
ELLINGER, and are improbable. The indol, it seems, is not formed from
the tryptophane (indolaminopropionic acid) as intermediary step in the
demolition of the proteins in the animal body, but rather from the
putrefaction of the tryptophane in the intestine. GENTZEN l has also
shown that tryptophane introduced subcutaneously or per os into the
body does not lead to an indicanuria, but only when it is exposed to bac-
terial decomposition in the large intestine. The reports as to the elimina-
tion of indican after oxalic-acid poisoning are conflicting. After poison-
ing with oxalic acid HARNACK and v. LEYEN found an increased indican
elimination, and MORACZEWSKI believes he has proven a certain par-
allelism between the quantity of indican and the quantity of oxalic acid
in diabetes. ScHOLZ,2 on the contrary, obtained no increase in the
excretion of indican after oxalic-acid poisoning.

The excretion of indican is, as above stated, increased by the introduction of
indol, but also by indoxyl or indoxyl-carboxylic acid. Indol-carboxylic acid,
on the contrary, does not yield indican, but, according to PORCHER and HER-
VIEUX, another chromogen. BENEDICENTI has also shown that indigo blue or
analogous blue or green pigments are produced only from those derivatives

CH CH

of indol which, like n-methyl indol C6H4<^%CH, a-naphtindol, Ci0H6/\CH or

N.CH3 NH

CH2

tt-methylindolin, C6H4<. >CH2, do not have the hydrogen atoms of the two methine

N.OH,

groups substituted by alkyl. From those derivatives in which one or two hydro-
gen atoms are substituted by alkyl, such as skatol, a-methyl indol, dimethyl indol,
C.CH3 CH

C6H4<(j>C.CH3, and bz. 3, p. 2-dimethyl indol, CH3.C6H3<^\C.CH3, red pig-
NH NH

1 Blumenthal, Arch. f. (Anat. u.) Physiol., 1901, Suppl., and 1902, with Rosenfeld,
Charite annalen, 27, and Hofmeister's Beitrage, 5; Lewin, Hofmeister's Beitrage, 1;
Mayer, Arch. f. (Anat. u.) Physiol., 1902, Zeitschr. f. klin. Med., 47, and Zeitschr. f.
physiol. Chem., 29, 32; Scholz, ibid., 38; Ellinger, ibid.t 39; Gentzen, " Ueber die Vor-
etufen des Indols bei der Eiweissfaulnis im Thierkorper," Inaug. -Dissert.. Konigsberg,
1904.

2 Harnack, Zeitschr. f. physiol. Chemie, 29; Scholz, 1. c., Moraczewski, Centralbl.
f. innere Med.. 1903.

730 URINE.

ments are produced, a behavior which PORCHER and HERVIEUX l have observed in
several alkyl-substituted indols.

An increased elimination of indican has been observed in many
diseases,2 and in these cases the quantity of phenol eliminated is also
generally increased. A urine rich in phenol is not always rich in indican.

The potassium salt of indoxyl-sulphuric acid, which was prepared
pure by BAUMANN and BRIEGER from the urine of dog fed on indol,
has subsequently been prepared synthetically by BAUMANN and THESEN,S
by fusing phenyl-glycine-orthocarboxylic acid with alkali and then from
this producing the indoxylsulphate by means of potassium pyrosulphate.
It crystallizes in colorless, shining plates or leaves which are easily soluble
in water, but less readily in alcohol. It is solit by mineral acids into
sulphuric acid and indoxyl. The latter without access of air passes into
a red compound, indoxyl red, but in the presence of oxidizing reagents
is converted into indigo blue: 2C8H7NO+2O = Ci6HioN2O2+2H2O. The
detection of indican is based on this last fact.

For the rather complicated preparation of indoxyl-sulphuric acid as
potassium salt from urine the reader is referred to other text-books.
For the detection of indican in urine in ordinary cases the following
method of JAFFE-OBERMAYER, which also serves as an approximate test
for the quantity of indican, is sufficient.

JAFFE-OBERMAYER'S Indican Test. JAFFE uses chloride of lime as
the oxidizing agent, while OBERMAYER employs ferric chloride. Other
oxidizing agents have been suggested, such as potassium permanganate,
potassium dichromate, alkali chlorate, and hydrogen peroxide (the
latter suggested by PORCHER and HERVIEUX 4). With OBERMAYER'S
reagent the test is performed as follows :

The acid urine (if alkaline it must be acidified with acetic acid) (ELLIN-
GER) is precipitated with basic lead acetate, 1 cc. for every 10 cc. of
the urine. 20 cc. of the filtrate are treated in a test-tube with an equal
volume of pure concentrated hydrochloric acid (specific gravity 1.19)
which contains 2-4 grams ferric chloride to the liter, and 2-3 cc. chloro-
form are added and the mixture immediately thoroughly shaken. The
chloroform is thereby colored more or less blue, depending upon the
amount of indican. Besides indigo blue we may also have indigo red
produced, whose formation has been explained in various ways. The

1 The work of Porcher and Hervieux can be found in Compt. Rend., 145, Compt.
rend. soc. biol., 62, and Bull. soc. chim. (4), 1; Benedicenti, Zeitschr. f. physiol. Chem.,
53 and Arch. f. exp. Path. u. Pharm., 1908, Suppl. (Schmiedeberg's Festschr.).

2 See Jaff£, Pfluger's Arch., 3; Senator, Centralbl. f. d. med. Wissensch., 1877;
G. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 12 (contains older literature; also Berl.,
klin. Wochenschr., 1892.

3 Baumann with Brieger, Zeitschr. f. physiol. Chem., 3; with Thesen, ibid., 23.

4 Jaffe, Pfluger's Arch., 3; Obermeyer, Wien. klin. Wochenschr., 1890; Salkowski,
Zeitschr. f. physiol. Chem., 67; Porcher and Hervieux, Zeitschr. f. physiol. Chem., 39.

INDICAN TESTS. 731

quantity of indigo red becomes greater the more slowly the oxidation
takes place, and especially when the decomposition takes place in the
warmth (see the works of ROSIN, BOUMA, WANG, MAILLARD, ELLINGER
and HERVIEUX l).

According to ELLINGER the source of the indigo-red formation may be the
isatin that is produced by the superoxidation of the indoxyl by the action of
the reagent, and this isatin forms indigo red with the indoxyl in the hydrochloric-
acid solution. MAILLARD, on the contrary, is of the view that the blue substance
which is taken up by the chloroform from the urine mixed with hydrochloric
acid is not indigotin (indigo-blue), but another substance, called by him hemi-
indigotin, which in alkaline solution polymerizes immediately into indigotin,
while in acid reaction it is converted into indirubin (indigo red).

The chloroform solution of indigo obtained in the indican test may be
used in the quantitative colorimetric determination by comparison with
a solution of indigo in chloroform of known strength (KRAUSS and ADRIAN) .
WANG and others convert the indigo into indigo-sulphonic acid by con-
centrated sulphuric acid and titrate with potassium permanganate.
There is still doubt as to the surest and most trustworthy method for
the determination of indican, and especially as to the question how the
indigo residue is to be washed (see WANG, BOUMA, ELLINGER, and SAL-
KOWSKI 2), and for this reason we shall refer only to the works cited above.

Because of the difficulty arising from the production of indirubin
in addition to indigotin, BOUMA has recommended the conversion of
all the indoxyl into indirubin by boiling the urine with hydrochloric
acid containing isatin. The indirubin can be taken up by chloroform
and determined by titration with potassium permanganate and sul-
phuric acid after purification of the chloroform residue. OERUM 3 has
also worked out a colorimetric method of estimation based upon BOUMA'S
method.

Indol seems also to pass into the urine as a glucuronic acid, indoxyl-
glucuronic acid (SCHMIEDEBERG) . Such an acid has been found in the
urine of animals after the administration of the sodium-salt of o-nitro-
phenylpropiolic acid (G. HOPPE-SEYLER). PORCHER and HERVIEUX 4
have obtained indoxyl sulphuric acid in dogs and asses under similar
conditions.

Free indigo, and in fact indirubin as well as indigotin, occur in rare cases in
the undecomposed urine. GROBER and WANG have recently observed such cases.
According to STEENSMA 5 traces of free indol occur always in the urine.

1 Rosin, Virchow's Arch., 123; Bouma, Zeitschr. f. physiol. Chem., 27, 30, 32,
39; Wang, ibid., 25, 27, 28; Ellinger, ibid., 38 and 41; Maillard, Bull. soc. chim.,
Paris (3), 29, and Compt. Rend., 136; also L'indoxyle urinaire et les couleurs qui en
derivent, Paris, 1903, and Zeitschr. f. physiol. Chem., 41; Hervieux, see Bioch.
Centralbl., 8, 54:

2 Krauss, Zeitschr. f. physiol. Chem., 18; Adrian, ibid., 19; Wang, ibid., 25; Sal-
kowski, ibid., 42.

3 Bouma, Zeitschr. f. physiol. Chem., 32; Oerum, ibid., 45.

4 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 14; G. Hoppe-Seyler, ' Zeitschr. f.
physiol. Chem., 7 and 8; Porcher and Hervieux, Journ. de Physiol., 7.

5Grober, Munch, med. Wochenschr., 1904; Wang, Salkowski's Festschrift, 1904;
Steensma, Maly's Jahresb., 40, 314.

732 URINE.

C.CH3

Skatoxyl-sulphuric Acid, C9H9NS04, C6H4<^C.O.S02.OH, has not

NH

been positively prepared as a constituent of normal urine, but OTTO
has once prepared its alkali salt from diabetic urine. Perhaps skatoxyl
occurs in normal urine as a conjugated glucuronate (MAYER and NEU-
BERG *), and it is believed that the urine contains a skatol-chromogen
from which red and reddish-violet coloring-matters are obtained by
decomposition with strong acids and an oxidizing agent.

Skatoxyl-sulphuric acid originates, if it exists in the urine, from
skatol, which is formed by putrefaction in the intestine, and which is
then conjugated with sulphuric acid after oxidation into skatoxyl. That
skatol introduced into the body passes partly as an ethereal-sulphuric
acid into the urine has been shown by BRIEGER. Indol and skatol act
differently, at least in dogs, indol producing a considerable amount
of ethereal-sulphuric acid, while skatol gives only a small quantity (Mss-
TER 2). Reports on this subject are at variance.

The conditions for the formation of indol and skatol by the putrefaction of
proteins in the intestine are decidedly different, according to HERTER, as skatol
is produced by other putrefaction bacteria than indol. For example, bacillus coli
communis produces indol, but only traces of skatol, while skatol is formed by
certain anaerobic putrefactive bacteria. An important intermediary step in the
formation of skatol is the indol acetic acid (skatol carboxylic acid, according to
SALKOWSKI) and this can also pass into the urine and is the chromogen of the
urorosein, according to HERTER.3

The potassium salt of skatoxyl-sulphuric acid is crystalline; it dis-
solves in water, but with difficulty in alcohol. A watery solution becomes
deep violet with ferric chloride. The solution becomes red with con-
centrated hydrochloric acid with the separation of a red precipitate.
This precipitate (skatol red) is, after washing with water, insoluble in
ether but soluble in amyl alcohol. On distillation with zinc-dust the
red pigment gives a strong odor of skatol.

Urines containing skatoxyl are colored dark red to violet by JAFFE'S
indican test even on the addition of hydrochloric acid alone; with nitric
acid they are colored cherry red, and red on warming with ferric chloride
and hydrochloric acid. A red coloration of the urine can also be brought
about by the appearance of indigo red (indirubin) and a confusion of
this pigment can also take place. ROSIN 4 is of the opinion that no

1 Otto, Pfliiger's Arch., 33; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29.
* Brieger, Ber. d. deutsch. chem. Gesellsch., 12, and Zeitschr. f. physiol. Chem.,
4, 414; Mester, ibid., 12.

3 Journ. of biol. Chem., 4.

4 Rosin, Virchow's Arch., 123.

INDOL ACETIC ACID. 733

skatol chromogen exists in human urine, and that the observations made
heretofore were due to a confusion of skatol red with indigo red or uroro-
sein. It cannot be disputed that derivatives of skatol sometimes occur in
human urine, and to prevent confusion with indigo red it musfr be borne
in mind that indigo red is soluble in chloroform as well as in ether, while
skatol red is insoluble in these solvents. On the contrary skatol red
is soluble in amyl alcohol, and this solution shows absorption bands close
to the line D between it and E, corresponding to X = 577-550 (PORCHER
and HERVIEUX x).

In regard to a confusion of skatol red for urorosein it must also be
remarked that urorosein may also be a skatol red. The chromogen of
urorosein, as HERTER has shown in a case, is identical with indol acetic
acid, which passes into skatol on splitting off carbon dioxide. According
to HERTER2 urorosein is not identical with skatol red, although the
investigations of STAAL, GROSSER, PORCHER and HERVIEUX 3 indicate
that they are identical, and the last two investigators consider them
identical, because they both have the same spectrum and the same
chemical behavior.

C.CH2COOH

Indol Acetic Acid (skatol-carboxylic acid), Ci0H9N02, C6H4<^\CH

NH

This acid, whose occurrence in the urine was first shown by SALKOWSKI, is found
in the urine in special putrefactive processes in the intestine (HERTER) and in
various diseases, especially in cachectic conditions. This is of course dependent
upon the fact whether indol acetic acid is the actual chromogen of urorosein, and
also whether the experience obtained as to the occurrence of urorosein can be
applied to the indol acetic acid. According to WECHSELMANN 4 it occurs (more
correctly as urorosein) as traces in normal urine, abundantlyiin horse urine, and
in especially large quantities in cow urine. When introduced into the animal
body it appears unchanged in the urine.

This acid crystallizes in leaves which melt at 165°, and on strongly heating
it yields skatol with the splitting off of carbon dioxide. The solution, acidified
with hydrochloric acid, when treated with a little ferric chloride solution, becomes
cherry red on boiling. With some acid and a little nitrite as well as with hydro-
chloric acid and chloride of lime the solution becomes red, then cloudy, and a
red pigment precipitates. This pigment is soluble in amyl alcohol and gives the
above-mentioned absorption bands between D and E. This red pigment is
urorosein.

Urorosein is the name given by NENCKIS to a red pigment which occurs in
the urine under the conditions mentioned under indol acetic acid. This pig-

1 Zeitschr. f. physiol. Chem., 45.

2 Journ. of biol. Chem., 4.

3 Staal, Zeitschr. f. physiol. Chem., 46; Grosser, ibid., 44; Porcher and Hervieux,
ibid., 45; Compt. Rend., 138, and Journ. de Physiol., 7.

4 Salkowski, Zeitschr. f. physiol. Chem., 9; Wechselmann, cited in Bioch. Centralbl.,
5, 784.

5 Nencki and Sieber, Journ. f. prakt. Chem., (N. F.), 26.

734 URINE.

ment is not preformed in the urine, but is produced from its chromogen (indol
acetic acid) when the urine is treated with hydrochloric acid alone. The urine
becomes red. Urorosein differs from indirubin essentially by the same properties
as skatol, with which, according to some, it is identical (see above).

Nephrorosein is a pigment described by V. ARNOLD 1 which is closely'related to
urorosein and which, like this, is produced from a chromogen when the urine is
treated with nitric acid or with concentrated hydrochloric acid and a little sodium
nitrite solution. Nephrorosein is soluble in amyl alcohol and gives a spectrum
with a band between 6 and F, reaching from 6 to a little beyond the middle between
b and F. It is changed by the action of light and finally gives a band between
D and E, near E. The new pigment thus obtained is called (3-urorosein to dif-
ferentiate it from the ordinary urorosein, a-urorosein. The nephrorosein has not
been observed in normal urines but only in certain pathological cases.

The pigment obtained by DE JAGER by precipitating the urine with HC1 and
formol seems to be related to urorosein and nephrorosein. According to ELLINGER
and FLAMAND 2 urorosein belongs probably, like skatol-red, to the group of tri-
indyl methane pigments prepared by them from /3-indol aldehyde by boiling in
acid solution. Probably the leucobase HC.(C8H6N)3, which gives the red pigment,
HO.C 1 (C8H6N)3 is produced by condensation.

Aromatic Oxyacids. In the putrefaction of proteins in the intes-
tine, paraoxyphenyl-acetic acid and paraoxyphenyl-propionic acid are
formed from tyrosine as an intermediate step, and these in great part
pass unchanged into the urine. The quantity of these acids is usually
very small. They are increased under the same conditions as the phenols,
especially in acute phosphorus poisoning, in which the increase is con-
siderable. A small portion of these oxyacids is also combined with
sulphuric acid.

Besides these two oxyacids which regularly occur in human urine
we sometimes have other oxyacids in urines. To these belong homo-
gentisic acid in alcaptonuria, oxyhydroparacoumaric acid, found by BLENDER- .
MANN in the urine on feeding rabbits with tyrosine, gallic acid, which,
according to BAUMANN,S sometimes appears in horse's urine, and kynu-
renic acid (oxyquinolincarboxylic acid), which up to the present time
has been found only in dog's urine. Although all these acids do not
belong to the physiological constituents of the urine, still they will be
treated in connection with these.

Paraoxyphenylacetic Acid, C8H803, C6H4\ , and p-Oxyphenyl-

XCH2.f~

are

/OH

.COOH

/OH

propionic Acid (Hydroparacoumaric Acid), C9Hi003, C6H/ ,

\CH2.CH2COOH

crystalline and are both soluble in water and in ether. The one melts at 148° C.
and the other at 125° C. Both give a beautiful red coloration on being warmed
with MILLON'S reagent.

1 Zeitschr. f. physiol. Chem., 61 and 71.

2de Jager, Zeitschr. f. physiol. Chem., 61; Ellinger and Flamand, ibid., 62.

3 Blendermarm, Zeitschr. f. physiol. Chem., 6, 267; Baumann, ibid., 6, 193.

HOMOGENTISIC ACID. 735

To detect the presence of these oxyacids proceed in the following way (BAU-
MANN): Warm the urine for a while on the water-bath with hydrochloric acid
in order to drive off the volatile phenols. After cooling shake three times with
ether, and then shake the ethereal extracts with dilute soda solution, which dis-
solves the oxyacids, while the residue of the phenols which are soluble in ether
remains. The alkaline solution of the oxyacids is now faintly acidified with sul-
phuric acid, shaken again with ether, the ether removed and allowed to evaporate
the residue dissolved in a little water, and the solution tested with MILLON'S
reagent. The two oxyacids are best differentiated by their different melting-
points. The reader is referred to other works for the method of isolating and
separating these two oxyacids.

Homogentisic Acid (Dioxyphenylacetic Acid), CgHgCU =
This acid, which was discovered by MARSHALL1

.
\CH2COOH(5)

and calle:l by him glycosuric acid, was isolated in larger quantities by
WOLKOW and BAUMANN in a case of alcaptonuria and carefully studied
by them. They called it homogentisic acid because it is a homologue
of gentisic acid, and they showed that the peculiar properties of so-called
alcaptonuric urine in this case were due to this acid. This acid has later
been found in many cases of alcaptonuria. Glycosuric c cid, isolated from
alcaptonuric urine by GEYGER,2 seems to be identical with homogentisic
acid.

The quantity of acid eliminated, which varies in most cases between
3 and 7 grams per twenty-four hours, and which is higher — 14-16 grams —
in exceptional cases, is increased by food rich in protein. On the inges-
tion of tyrosine by persons with alcaptonuria, WOLKOW and BAUMANN
and EMBDEN observed a greater quantity of homogentisic acid in the
urine and this has been substantiated by other observers. Since LANG-
STEIN and E. MEYER showed in a case of alcaptonuria that the quantity
of tyrosine in the protein, even when calculated to a maximum, was
not sufficient to account for the quantity of homogentisic acid, and that
therefore we must admit of another source (the phenylalanine) for the
alcapton, FALTA and LANGSTEIN 3 have given a direct proof that homo-
gentisic acid can also be formed from phenylalanine. ABDERHALDEN,
BLOCK and RONA 4 have shown that in alcaptonurics the excretion of
homogentisic acid is increased by the introduction of tyrosine or phenyl-

1 The Medical News, Philadelphia, January 8, 1887.

2 Wolkow and Baumann, Zeitschr. f. physiol. Chem., 15; Geyger, cited from Emb-
den, 1. c., 18. The literature can be found in Fromherz, Ueber Alkaptonurie, Inaug.-
Dis. Freiburg, 1908.

3 Langstein and Meyer, Deutsch. Arch. f. klin. Med., 78; Falta and Langstein,
Zeitschr. f. physiol. Chem., 37; Falta, Der Eiweiss-Stoffwechsel bei der Alkaptonurie,
Habilitationsschrift, Naumburg, a. S., 1904.

4 Zeitschr. f. physiol. Che:::., 52.

736 URINE.

alanine in the form of polypeptides, from dipeptides as well as tripeptides.
The p-tyrosine and phenylalanine are quantitatively converted into homo-
gentisic acid, in alcaptonuria (FALTA). The m- and o-tyrosine, on the
contrary, are not converted, according to BLUM/ into homogentisic acid
in alcaptonurics, and the dibromtyrosine yields just as little homogentisic
acid as the bromine or iodine derivatives of protein bodies (FALTA).
According to the investigations of LANGSTEIN and MEYER, and especially
of FALTA, different proteins yield varying quantities of homogentisic
acid in alcaptonuria, and accordingly larger amounts in proportion as
the protein is rich in tyrosine and phenylalanine.

On this account the quotient H ( = homogentisic acid) : N (nitrogen)
is variable on the introduction of different proteins. For example, with
casein H : N is on an average much higher than with white of egg. In
most of the cases of alcaptonuria examined the H : N was equal to
40-50: 100, and with the same alcaptonuric, when no essential change
in the diet occurs, the quotient is relatively constant.

WOLKOW and BATJMANN explain the formation of homogentisic acid
from tyrosine by an abnormal fermentation in the upper parts of the
intestine, but this view has now been generally rejected. The observa-
tions of ABEEHALDEN, BLOCK and RONA 2 that glycyl-/-tyrosine on
subcutaneous injection causes an increased formation of homogentisic
acid, disproves this theory, and indicates a formation of homogentisic
acid in the tissues. This acid is also burnt in the healthy organism if
not too large quantities of the acid are introduced at one time, and it is
the general view that alcaptonuria is an anomaly in the protein metabolism.

In order to understand this anomaly and the origin of the homogentisic
acid we must call attention to the fact that the investigations of O.
NEUBAUER and FALTA, LANGSTEIN and others3 show that only such
aromatic acids are converted, in the body, into homogentisic acid, which
have a three-membered side-chain which is substituted by NEb, OH
or O in the a-position to the carboxyl group and not in the /3-position.
p-tyrosine, phenylalanine, phenyl-a-lactic acid and phenyl-pyroracemic
acid are such acids. It can be admitted with FALTA that the phenyl-
alanine in the body by deamidation is converted into phenyl-a-lactic
acid, C6H5.CH2.CHOH.COOH, from which by taking up two hydroxyl
groups, dioxyphenyl-a-lactic acid (uroleucic acid), (OH^CeHs.CH^.
CHOH.COOH, is formed, and then from this by oxidation dioxyphenyl-
acetic acid (homogentisic acid), (OH^CeHa.CH^.COOH, is produced.
Tyrosine is also supposed to undergo an analogous transformation

1 Arch. f. exp. Path. u. Pharm., 59.

2 Zeitschr. f. physiol. Chem., 52.

3 Ibid., 42; Fromherz, 1. c.

HOMOGENTISIC ACID. 737

whereby a removal of the OH group in the para position must be
admitted.

According to NEUBAUER,1 on the contrary, the tyrosine, as well as
the other ammo-acids, is first transformed into the corresponding keto-
acid, p-oxyphenyl pyroracemic acid, OH.C6H4.CH2.CO.COOH, which
is then oxidized into the corresponding chinol and transformed into
hydroquinone pyroracemic acid, (OH^CeHs.CH^.CO.COOH. The homo-
gentisic acid is derived from this latter by the splitting off of carbon
dioxide by oxidative means. Phenylalanine is either changed into
phenyl pyroracemic acid or into p-oxyphenyl pyroracemic acid with
tyrosine as intermediary body and then changed as above stated.

According to the accepted hypothesis the demolition of tyrosine
and phenylalanine takes place into homogentisic acid, and the anomaly
in the metabolism of alcaptonurics consists in that in these the demoli-
tion stops at this point and that the ability to rupture the benzene ring
is absent, in the organism, in alcaptonuria.

,The difficulties in accepting the assumption of a transformation of tyrosine
into homogentisic acid due to the different positions of the hydroxyl groups in

the side chain of the two bodies, as shown by the formulae HO^ yOH (homo-

\ /

CH,COOH
OH

gentisic acid) and \^ "/ (tyrosine) do not exist now, since we have

CH2CHNH2COOH
learnt of other analogous processes. For example, the oxidation, by KUMAGAI and

WoLFFENSTEiN,2 of paracresol H3C<f /OH with potassium persulphate in

OH
acid solution. In this manner the expected 3.4 dioxytoluene H3C<f y>OH

was not obtained, but instead homohydroquinone HO<T /OH, and hence a

CH3

transference of the alkyl group must have occurred.

ABDERHALDEN 3 has also shown in healthy human beings that tyrosine
may cause an elimination of homogentisic acid, as he positively detected
a small quantity of homogentisic acid in the urine of a man who had
taken 50 grams Z-tyrosine per os (of which 44 grams were absorbed).
In the urine of another man he could not detect either homogentisic

1 Cited from Centralbl. f . Physiol, 23, 76.

2 Ber. d. d. Chem. Gesellsch., 41.
8 Zeitschr. f. physiol. Chem., 77.

738 URINE.

acid or any other intermediary product of the cleavage of tyrosine in
the urine after taking 150 grams /-tyrosine (of which 141 grams were
absorbed) .

DAKIN 1 has recently opposed the above-mentioned view that in the
cleavage of tyrosine and phenylalanine, homogentisic acid is always
produced, and that the condition of alcaptonuria consists in an inability
of the body to burn this intermediary product of metabolism. He has
found that tyrosin-methyl ether, which cannot form any quinone-like
intermediary product, can in cats be just as completely burnt as tyrosine,
and the same is true for p-methylphenylalanine and for p-methoxy-
phenylalanine, which cannot form any quinone derivatives. Still
these substances can be completely burnt by alcaptonurics and accord-
ing to DAKIN the body in alcaptonuria has still the ability to completely
burn the aromatic nucleus of tyrosine and phenylalanine when the
transformation into homogentisic acid is prevented by a proper sub-
stitution in the para-groups. DAKIN therefore considers alcaptonuria
as a condition in which partly the formation of an abnormal metabolic
product — the homogentisic acid — takes place and where partly the ability
of the body to burn this product is diminished.

GARROD,2 who has observed several cases of alcaptonuria, has also
tabulated a large number of cases of alcaptonuria which he finds in the
literature, and he shows that the anomaly of the protein metabolism
occurs oftener in males than in females, and also that blood relationship
of the parents (first cousins) predisposes to alcaptonuria.

On fusing homogentisic acid with alkali it yields gentisic acid (hydro-
quinone-carboxylic acid) and hydroquinone. When introduced into the
intestine of the dog a part is converted into toluhydroquinone, which
is eliminated in the form of an ethereal sulphuric acid. Homogentisic
acid has also been prepared synthetically by BAUMANN and FRANKEL,
starting with gentisic aldehyde, and by NEUBAUER and FLATOW 3 from
o-oxyphenylglyoxylic acid with hydroquinone glyoxylic acid and hydro-
quinone glycollic acid as intermediary bodies.

Homogentisic acid crystallizes with 1 mol. of water in large, trans-
parent prismatic crystals, which become non-transparent at the tem-
perature of the room with the loss of water of crystallization. They
melt at 146.5-147° C., and are soluble in water, alcohol, and ether, but
nearly insoluble in chloroform and benzene. Homogentisic acid is

1 Journ. of Biol. Chem., 8 and 9, with Wakeman, ibid., 9.

2 Med. chirurg. Transact., 1899 (where all cases up to that time are tabulated);
also The Lancet, 1901 and 1902; Garrod and Hele, Journ. of Physiol., 33.

3 Baumann and Frankel, Zeitschr. f. physiol. Chem., 20; Neubauer and Flatow.,
ibid., 52.

HOMOGENTISIC ACID. 739

optically inactive and non-fermentable. Its watery solution has the
properties of so-called alcaptonuric urine. It becomes greenish brown
from the surface downward on the addition of very little caustic soda
or ammonia with access of oxygen, and on shaking it quickly becomes
dark brown or black.

If alcaptonuric urine or a homogentisic acid solution is treated with 10-40
per cent ordinary ammonia, a beautiful, intensive reddish-violet coloration is
produced on access of air according to C. MoRNER,1 and two beautiful pigments,
a- and 0-alcaptochrome, are formed. The first, a-alcaptochrome, is crystalline and
has a beautiful violet color in alkaline solution and is without fluorescence. The
/3-alcaptochrome is not crystalline and its alkaline solution has a more reddish
color with strong fluorescence in the yellowish-red.

Homogentisic acid reduces alkaline copper solutions with even slight
heat, and ammoniacal silver solutions immediately in the cold. It
does not reduce alkaline bismuth solutions. It gives a lemon-colored
precipitate with MILLON'S reagent, which becomes light brick-red on
warming. Ferric chloride gives to the solution a blue color which soon
disappears. On boiling with concentrated ferric-chloride solution an
odor of quinone develops. With benzoyl chloride and caustic soda in
the presence of ammonia we obtain the amide of dibenzoylhomogentisic
acid, which melts at 204° C., and which can be used in the isolation of
the acid from the urine, and also for its detection (ORTON and GARROD).
Among the salts of this acid must be mentioned the lead salt containing
water of crystallization and 34.79 per cent Pb. This salt melts at
214-215° C.

In order to prepare the acid, heat the urine to boiling, add 5 grams
of lead acetate for every 100 cc., filter as soon as the lead acetate has
dissolved, and allow the filtrate to stand in a cool place for twenty-four
hours until it crystallizes (GARROD). The dried, powdered lead salt
is suspended in ether and decomposed by H^S. After the spontaneous
evaporation of the ether the acid is obtained in almost colorless crystals
(ORTON and GARROD 2) .

In regard to the quantitative estimation we proceed according to the sug-
gestion of BAUMANN by titrating the acid with a N/10 silver solution. For details
of this method the reader is referred to the works of BAUMANN, C. TH. MORNER,
MITTELBACH, GARROD and HuRTLEY. DENiGEs 3 has suggested another method.

Uroleucic acid, C9Hi005, is, according to HUPPERT, probably a dioxyphenyl-
lactic acid, C6H3(pH)2.CH2.CH(OH).COOH. This acid was first prepared by
KIRK from the urine of children with alcaptonuria, which also contained homo-
gentisic acid. LANGSTEIN and MEYER 4 have also found a small amount of this
acid in a case of alcaptonuria studied by them. It melts at 130-133° C. Other-

1 Zeitschr. f. physiol. Chem., 69.

2Orton and Garrod, Journ. of Physiol., 27; Garrod, ibid., 23.

3 Mittelbach, Deutsch. Arch. f. klin. Med., 71 (which contains the work of Baumann
and Morner); Garrod and Hurtley, Journ. of Physiol., 33; DenigSs, Chem. Centralbl.,
1897, 1, 338.

4 Huppert, Zeitschr. f. physiol. Chem., 23; Kirk, Brit. Med. Journ., 1886 and 1888;
Langstein and Meyer, 1. c.

740 URINE.

wise, in regard to its behavior with alkalies, with access of air, and also with
alkaline copper solutions and ammoniacal silver solutions, and also MILLON'S
reagent, it is similar to homogentisic acid.

NEUBAUER and FLATOW, who have prepared dioxyphenyl-a-lactic acid syn-
thetically, find that this acid has entirely different properties from the so-called
uroleucic acid. GARROD and HURTLEY 1 have also shown that an impure homo-
gentisic acid with a low melting-point is easily obtained, and they suggest the
possibility that the earlier reports in regard to uroleucic acid are due to an error.

CH COH

HC C C.COOH

Kynurenic acid (Y-oxy-/3-quinolincarboxylic acid), CioH7N03, | || |

HC C CH

CH N

has only been found thus far in dog's urine, but not always; its quantity is increased
by meat feeding. It does not occur in the urine of cats. ELLINGER 2 has been
able to show positively that tryptophane is the mother-substance of this acid.
By the introduction of tryptophane in the organism he has shown the formation
of a kynurenic acid not only in dogs but also in rabbits.

The acid is crystalline, does not dissolve in cold water, rather well in hot alcohol,
and yields a barium salt which crystallizes in triangular, colorless plates. On
heating it melts and decomposes into C02 and kynurin. On evaporation to dry-
ness on the water-bath with hydrochloric acid and potassium chlorate a reddish
residue is obtained which on adding ammonia becomes first brownish green and
then emerald green (JAFFE'S reaction 3).

Urinary Pigments and Chromogens. The yellow color of normal
urine depends perhaps upon several pigments, but in greatest part upon
urochrome. Besides this the urine seems to contain a very small quantity
of hcematoporphyrin as a regular constituent. Uroerythrin is also of
frequent occurrence in normal urine, but not always. Finally, the
excreted urine when exposed to the action of light regularly contains a
yellow pigment, urobilin, which is derived from a chromogen, urobilinogen,
by the action of light (SAILLET) and air (JAFFE, DisquE4) and others.

Besides this chromogen, urine contains various other bodies from which color-
ing matters may be produced by the action of chemical agents. Humin sub-
stances (perhaps in part from the carbohydrates of the urine) may be formed
by the action of acids (v. UDRANSZKY) without regard to the fact that such sub-
stances may sometimes originate from the reagents used, as from impure amyl

1 Journ. of Physiol., 36.

2Ellinger, Ber. d. d. chem. Gesellsch., 37, 1804, and Zeitschr. f. physiol. Chem.,
43. The older literature on kynurenic acid may be found in Josephsohn, Beitrage zur
Kenntnis der Kynurensaure ausscheidung beim Hunde, Inaug.- Dissert., Konigsberg,
1898.

3 Zeitschr. f. physiol. Chem., 7. In regard to kynurenic acid, see also Huppert-
Neubauer, 10. Aufl., and Mendel and Jackson, Amer. Journ. of Physiol., 2; Mendel
and Schneider, ibid., 5; Camps, Zeitschr. f. physiol. Chem., 33.

4Jaffe", Centralbl. f. d. med. Wissensch., 1896 and 1869, and Virchow's Arch., 47;
Disque, Zeitschr. f. physiol. Chem., 2; Saillet, Revue de medecine, 17, 1897.

UROCHROME. 741

alcohol (v. UDRANSZKY *). To these humin bodies developed by the action of
acid in normal urine when exposed to the air must be added the urophain of HELLER,
the various uromelanins and other bodies described by different investigators
(PLOSZ, THUDICHUM, SCHUNCK, DOMBROWSKI 2). Indigo blue (uroglaudn of
HELLER, urocyanin, cyanurin, and other coloring matters of earlier investigators 3)
is Split off from the indoxyl-sulphuric acid or indoxyl-glucuronic acid. Red
coloring matter may be formed from the conjugated indoxyl and skatoxyl acids,
and urohodin (HELLER), urorubin (PLOSZ), urohcematin (HARLEY), and perhaps
also urorosein (NENCKI and SIEBER 4) probably have such an origin.

We cannot discuss more in detail the different coloring matters obtained
as decomposition products from normal urine. Hsematoporphyrin has
already been referred to in a previous chapter (V) and will best be
described in connection with the pathological pigments. It only remains
to describe urochrome, urobilin, and uroerythrin.

Urochrome is the name given by GARKOD to the yellow pigment of
the urine. THUDICHUM 5 had previously given the same name to a less
pure pigment isolated by himself. The accounts as to the composi-
tion and properties of urochrome differ so considerably that it is ques-
tionable whether anybody has ever had this pigment in a pure form.
Urochrome is free from iron, but contains nitrogen. DOMBROWSKI
found 11.15 per cent nitrogen, HOHLWEG found 9.89 per cent nitrogen,
and KLEMPERER found only 4.2 per cent nitrogen. According to
DOMBROWSKI urochrome contains about 5 per cent sulphur, while
other investigators like HOHLWEG, SALOMONSEN, and MANCINI found
that it was free from sulphur.6 According to GARROD it stands in
close relation to urobilin and is transformed into urobilin by the action
of'" active " acetaldehyde, while RivA7 claims to have obtained a body
similar to urochrome by the oxidation of urobilin by permanganate.
This relation of the two pigments is denied by DOMBROWSKI. On the
contrary it is the unanimous opinion that urochrome under certain con-,
ditions may yield the pyrrol reaction. Certain investigators such as
BONDZYNSKI and DOMBROWSKI consider urochrome as a member of the
oxyproteic acid group (see further on), a view which does not seem to

1 v. Udrdnszky, Zeitschr. f. physiol. Chem., 11, 12, and 13.

2P16sz, Zeitschr. f. physiol. Chem., 8; Thudichum, Brit. Med. Journ., 201, and
Journ. f. prakt. Chem., 104; Schunck, cited from Huppert-Neubauer, 10. Aufl., 509;
Dombrowski, Zeitschr. f. physiol. Chem., 62.

3 See Huppert-Neubauer, 161.

*In regard to this and other red pigments, see Huppert-Neubauer, 593 and 597;
Nencki and Sieber, Journ. f. prakt. Chem. (2). 26.

6 Garrod, Proc. Roy. Soc., 55; Thudichum, 1 c.

6 Dombrowski, Zeitschr. f. physiol. Chem., 54 and 62; Hohlweg, Bioch. Zeitschr.,
13; Salomonsen. ibid., 13; Mancini, ibid., 13; Klemperer, Berl. klin. Wochenschr.,
40.

7 Garrod, Journ. of Physiol., 21 and 29; Riva, cited from Huppert-Neubauer, 524.

742 URINE.

have sufficient basis and in fact is denied by others such as WEisz.1 The
above disputed statements as to the presence or absence of sulphur in
urochrome as well as the nitrogen content of urochrome make it very
probable that the preparation of pure urochrome has not thus far been
accomplished.

Urochrome, as obtained thus far, is amorphous, brown, readily solu-
ble in water and ordinary alcohol, but less soluble in absolute alcohol.
It dissolves but slightly in acetic ether, amyl alcohol, and acetone, while
it is insoluble in ether, chloroform, and benzene. Urochrome is pre-
cipitated by copper acetate, lead acetate, silver nitrate, mercuric acetate,
phosphotungstic and phosphomolybdic acids. On saturating the urine
with ammonium sulphate a great part of the urochrome remains in solu-
tion. It does not show any absorption-bands and does not fluoresce
after the addition of ammonia and zinc chloride. Urochrome is very
readily decomposed, by the action of acids, with the formation of brown
substances.

Urochrome can be prepared according to a rather complicated method which
is based upon the fact that the substance remains in great part in solution on
saturating the urine with ammonium sulphate. If the proper quantity of alcohol
is added to the filtrate, a clear, yellow alcoholic layer forms on the salt solution,
which contains the urochrome and which can be used for the further preparation
of the latter (GARROD, 0. BOCCHI 2). KLEMPERER, on the contrary, removes the
pigment from the urine by means of animal charcoal, washes it with water to
remove the indican and other bodies, and then extracts with alcohol and uses
this alcoholic extract for the further purification according to GARROD. HOHLWEG,
SALOMOSEN and MANCINI also remove the pigment from the urine, which has
previously been precipitated by calcium or barium salts, by means of animal
charcoal. DOMBROWSKI uses an entirely different method which is based upon
the precipitation of the urochrome by copper acetate. In regard to the details
of these different methods we refer to the original works.

DOMBROWSKI, BROWINSKI and DOMBROWSKI 3 have worked out a
quantitative method for estimating urochrome, but its value is dependent
upon a further investigation as to the purity and composition of the
urochrome obtained by them. On this account the results found by these
investigators will not be given. The urochrome can be quantitatively
estimated, according to KLEMPERER, by a colorimetric method, using
a solution of true yellow G. If 0.1 gram of this dye is dissolved in 1 liter
of water and 5 cc. of this solution diluted to 50 cc. with water, then
this solution has the same color and shade as a 0.1 per cent urochrome
solution. The urine must be diluted with water until it has the same
depth of color. The comparison is performed in vessels with parallel
walls. The value of this method cannot be judged at the present time.

1 Dombrowski, 1. c.; Bondzynski, Chem. Centralbl., 1910, Bd. II; Weisz, Bioch.
Zeitschr., 30.

2 Garrod, 1. c.; Bocchi, Hofmeister's Beitrage, 11.

3 Dombrowski, Zeitschr. f. physiol. Chem., 54, with Browinski, Bull. Acad. d.
d. scien. Cracovie, 1908; Klemperer, 1. c.

UROBILIN. UROBILINOIDS. 743

Urobilin is the pigment first isolated from the urine by jAFFis,1 and
which is characterized by its strong fluorescence and by its absorption-
spectrum. Various investigators have prepared, from the urine, by dif-
ferent methods, pigments which differed slightly from each other but
behaved essentially like JAFFA'S urobilin. Thus different urobilins have
been suggested, such as normal, febrile, physiological, and pathological
urobilins.2 The possibility of the occurrence of different urobilins in
the urine cannot be denied; but as urobilin is a readily changeable body
and difficult to purify from other urinary pigments, the question as to the
occurrence of different urobilins must still be considered open.

In the perfectly fresh urine of healthy human beings no urobilin
occurs, as first suggested by SAiLLET,3 but only the chromogen, uribilino-
gen, from which the urobilin is readily formed by the action of light
or by weak oxidizing agents. Pathological urines contain on the contrary
preformed urobilin.

Urobilinoids, i.e., bodies which are similar to urobilin in that they fluoresce
and show the same absorption spectrum have been prepared from bile-pig-
ments (by MALY and STOKVIS) and from hsematin or haematoporphyrin (by
HOPPE-SEYLER, LE NOBEL, NENCKI and SIBBER, MAcMuNN4) by reduction
as well as by oxidation. According to H. FISCHER and MEYER-BETZ 5 also non-
stable pyrrols, which contain a non-substituted hydrogen atom in a ring carbon
atom, pass readily in the animal body into substances which give the character-
istic urobilin reactions. These reactions are also given by bodies of different
constitution, but which probably contain the same chromophore groups, and it is
these conditions which cause the above-mentioned uncertainty as to the occur-
rence of different urobilins.

That urobilin is identical with the hydrobilirubin of MALY (see page
428) has been considered for a long time. In opposition to this view
we find that both bodies, not to mention other small differences, have an
essentially different composition. While the hydrobilirubin contains
9.22 per cent nitrogen, according to MALY, the urobilin contains only
4.09 per cent nitrogen, according to HOPKINS and GARROD, and 5.93 per
cent nitrogen, according to FROMHOLDT. In the urobilin of the feces,
stercobilin, which is identical with urobilin. HOPKINS and GARROD found

1 Centralbl. f. d. med. Wissensch., 1868 and 1869, and Virchow's Arch., 47.

2 See MacMunn, Proc. Roy. Soc., 31 and 35; Ber. d. deutsch. chem. Gesellsch., 14,
and! Journ. of Physiol., 6 and 10; Bogomoloff, Maly's Jahresber., 22; Eichholz, Journ.
of Physiol., 14; Ad. Jolles, Pfluger's Arch., 61.

3 Revue de medecine, 1 7, 1897.

4 Maly, Ann. d. Chem. u. Pharm., 163; Disque, Zeitschr. f. physiol. Chem., 2;
Stokvis, Centralbl. f. d. med. Wissensch., 1873, 211 and 449; Hoppe-Seyler, Ber. d.
deutsch. chem. Gesellsch., 7; Le Nobel, Pfluger's Arch., 40; Nencki and Sieber,
Monatshefte f. Chem., 9, and Arch. f. exp. Path. u. Pharm., 24; MacMunn, Proc. Roy.
Soc., 31.

5 H. Fischer and Meyer-Betz, Zeitschr. f. physiol. Chem., 75.

744 URINE.

4 per cent nitrogen. Still H. FISCHER l has now found that the stercobilin
has a lower nitrogen content because of a contamination with cholesterin
or bile acids, and it is possible that also the low nitrogen content of the
urine-urobilin may be caused by contamination with non-nitrogenous
substances.

These possibilities bane, it is sure, have not been tested; but the
unequal nitrogen content of the two pigments does not positively exclude
the identity of urobilin and hydrobilirubin. FISCHER and MYER-BETZ
have in fact shown that the hemibilirubin, which forms about one-half
of the mixture called hydrobilirubin (see page 429) is identical with the
urobilinogen from human urine.

The possibility of the formation of urobilinogen and of urobilin from
bile pigments is assured and many physiological as well as clinical observa-
tions2 support the view that this transformation of the bile pigments
occurs by means of putrefactive processes in the intestine. Of these
observations we must mention the regular appearance in the intestinal
tract of stercobilin, undoubtedly derived from the bile-pigmets; the
absence of urobilin in the urine of new-born infants, as well as on the com-
plete exclusion of bile from the intestine, and also the increased elimina-
tion of urobilin with strong intestinal putrefaction. On the other hand
there are investigators who, basing their opinion on clinical observations,
deny the enterogenous origin of urobilin and claim that the urobilin is
derived from a transformation of the bilirubin elsewhere than in the
intestine, by an oxidation of the bile-pigment or by a transformation of
the blood-pigments.3

Urobilin or urobilinogen dees not occur in the urine of all animals,
and according to FROMHOLDT it is absent in the urine of rabbits. The
correctness of this statement is denied by GAUTIER and Rosso. In nor-
mal human blood they seem to be absent, while according to BiFFi4
urobilin and urobilinogen occur sometimes in disease and in cadaver blood.

1 Hopkins and Garrod, Journ. of Physiol., 22; Fromholdt, Zeitschr. f. exp. Path.,
u. Ther., 7; H. Fischer, Zeitschr. f. physiol. Chem., 73.

2 See Fr. Miiller, Schles. Gesellsch. f. vaterl. Kultur, 1892; D. Gerhardt, " Ueber
Hydrobilirubin und seine Bezieh. zum Ikterus " (Inaug.-Diss., Berlin, 1889); Beck,
Wien. klin. Wochenschr., 1895; Harley, Brit. Med. Journ., 1896; Fischler, Zeitschr.
f. physiol. Chem., 48.

3 In regard to the various theories as to the formation of urobilin, see Harley,
Brit. Med. Journ., 1896; A. Katz, Wien. med. Wochenschr., 1891, Nos. 28-32; Grimm,
Virchow's Arch., 132; Zoja, Conferenze cliniche italiane, Ser. la, 1; Hildebrandt,
Zeitschr. f. klin. Med., 59; Biffi, Boll. d. scienc. med. di Bologna (8), anno 78, 7; Troisier,,
Compt. rend. soc. biol., 66 and Tsuschija, Zeitschr. f. exp. Path. u. Ther., 7; Fromholdt
and Nersessoff, ibid., 11.

4 Fromholdt, Zeitschr. f. physiol. Chem., 53; Cl. Gautier and Russo, Compt. rend.
BOC. biol., 64; Biffi, Folia hsematol., 4, and 1. c. Boll., 78.

UROBILIN. 745

The quantity of urobilin in the urine under physiological conditions
varies widely. SAILLET found 30-130 milligrams and G. HOPPE-SEYLER
80-140 milligrams in one day's urine.

There are numerous observations on the elimination of urobilin or
urobilinogen in disease, especially by JAFF£, DISQU£, GERHARDT, G.
HOPPE-SEYLER,1 and others. The quantity is increased in hemorrhage
and in disease where the blood-corpuscles are destroyed, as is the case
after the action of certain blood-poisons, such as antifebrin and anti-
pyrine. It is also increased in fevers, cardiac diseases, lead colic, atrophic
cirrhosis of the liver, and is especially abundant in so-called urobilin
icterus.

The properties of urobilin may vary, depending upon the method
of preparation and the character of the urine used; therefore only the
most important properties will be given. Urobilin is amorphous, brown,
reddish brown, red, or reddish yellow, depending upon the method
of preparation. It dissolves readily in alcohol, amyl alcohol, and
chloroform, but less readily in ether or acetic ether. It is less soluble
in water, but the solubility is augmented by 'the presence of neutral
salts. It may be completely precipitated from the urine by saturating
with ammonium sulphate, especially after the addition of sulphuric acid
(MEHU 2). It is soluble in alkalies, and is precipitated from the alkaline
solution by the addition of acid. It is partly dissolved by chloroform
from an acid (watery-alcoholic) solution; alkali solutions remove the
urobilin from the chloroform. The neutral or faintly alkaline solutions
are precipitated by certain metallic salts (zinc and lead), but not by others,
such as mercuric sulphate. Urobilin is precipitated from the urine by
phosphotungstic acid. It does not give GMELIN'S test for bile-pigments.
It gives, on the contrary, a reaction which may be mistaken for the biuret
test, by the action of copper sulphate and alkali.3

Neutral alcoholic urobilin solutions are, in strong concentration,
brownish yellow, in great dilution yellow or rose-colored. They have a
strong green fluorescence. The acid alcoholic solutions are brown,
reddish yellow, or rose-red, according to concentration. They are not
fluorescent, but show a faint absorption-band, X, between b and F, which
borders on F. The absorption maximum lies according to LEWIN and
STENGER4 at 7 = 494-497. The alkaline solutions are brownish yellow,

1 In regard to the literature on this subject we refer the reader to D. Gerhardt,
" Ueber Hydrobilirubin und seine Beziehungen zum Ikterus " (Berlin, 1889), and
also G. Hoppe-Seyler, Virchow's Arch., 124.

2 Journ. de Pharm. et Chim., 1878, cited from Maly's Jahresber., 8.

3 See Salkowski, Berlin, klin. Wochenschr., 1897, and Stokvis, Zeitschr. f. Biologic,
34.

4 Pfliiger's Arch., 144.

746 URINE.

yellow, or (the ammoniacal) yellowish green, according to concentration.
They show a dark band 7, which is moved somewhat toward the red
end of the spectrum and lies between E and F. The absorption max-
imum lies at X = 506-510. If some zinc-chloride solution is added to
an ammoniacal solution of the pigment it becomes red and shows a
beautiful green fluorescence and gives the same absorption bands. If
a sufficiently concentrated solution of urobilin alkali is carefully acidified
with sulphuric acid it becomes cloudy and shows a second band exactly
at E, and connected with 7 by a shadow (GARROD and HOPKINS, SAILLET 1).
Urobilinogen is colorless or only faintly colored, but is very quickly
changed in the air and by the action of light and is transformed into urobilin.
The urobilinogen, which is identical with hemibilirubin, can be obtained
as colorless prisms by solution in hot acetic-ether and treating this with
ligroin, and evaporating. Urobilinogen is soluble in ether, acetic ether,
amyl alcohol and in chloroform, and can in part be removed from the
urine after adding sodium bicarbonate and shaking with chloroform
(FISCHER and MEYER-BETZ). It can also be obtained directly from the
urine or from the acidified urine by shaking with chloroform or ether,
although it is less pure. In a chloroform solution of urobilin and urobilino-
gen, according to GRiMBERT2, only urobilin and not urobilinogen is
taken up by a sodium diphosphate solution, which is not colored red by
phenolphthalein. Like urobilin, it is precipitated from the urine on
saturating with ammonium sulphate. When free from urobilin it does
not give any absorption bands and no fluorescence with ammonia and
zinc salt. For the detection and identification of urobilinogen we make
use of EHRLICH'S reagent (p-dimethylamino-benzaldehyde). This
reagent consists of dissolving 2 grams p-dimethylaminobenzaldehyde
in 50 cc. concentrated, fuming hydrochloric acid and diluting to 100
cc. with water. To 10 cc. of the urine add 1 cc. of the reagent and
thoroughly shake. According to the amount of urobilinogen, the solution
becomes pink colored or intensely red, and in the spectrum we find a
band between D and E. The red color can be taken up by amyl alcohol.
Urobilin does not give this reaction, which is common to certain haematin
and pyrrol derivatives.

The preparation of urobilin from the urine can be done according to the
original method of JAFFE, or according to the method suggested by MEHU,
which has been modified somewhat by GARROD and HOPKINS 3 (precipita-
tion with ammonium sulphate) or according to CHARNAS' suggestion.

According to CHARNAS* the preparation is best from urobilinogen,

1 Garrod and Hopkins, Journ. of Physiol., 20; Saillet, 1. c.

2 Fischer and Meyer-Betz, 1. c.; Grimbert, Compt. rend. soc. biol., 70.
8 Jaffe\ 1. c.; Me"hu, 1. c.; Garrod and Hopkins, Journ. of Physiol., 20.
4Charnas, Bioch. Zeitschr., 20.

DETECTION AND ESTIMATION OF UROBILIN. 747

and if the urine contains urobilin, it is first allowed to undergo alkaline
fermentation, when the urobilin is converted into urobilinogen. The
urine is acidified with tartaric acid and extracted with ether. The
foreign pigments are precipitated from the ethereal solution by petroleum
ether; the ether solution is washed with water, evaporated, and the residue
allowed to stand with water for several hours at 38° C., when the urobilino-
gen is transformed into urobilin. The urobilin can now be precipitated
with ammonium sulphate, and the dried precipitate extracted with absolute
alcohol. This urobilin has about three times as much extinction ability
as HALT'S urobilin (hydrobilirubin) . Other methods of preparation
have been suggested.

The urobilinogen is prepared by shaking the urine, directly after
adding sodium bicarbonate, with chloroform (FISCHER and MEYER-
BETZ). In regard to details we must refer to the original publication.

The detection of urobilin can sometimes be done directly on the urine.
Otherwise the urine is shaken with ether, amyl alcohol or chloroform and
these solutions tested. According to ScHLESiNGER1 the urine can also be
precipitated by an equal volume of a saturated solution of zinc acetate in
alcohol and the filtrate directly tested for the fluorescence and absorption.
GRIMBERT 2 has suggested a method for the separate testing for urobilin and
urobilinogen by using the chloroform, after shaking the urine therewith.
For the detection of urobilin we always make use of the color of the acid
or alkaline solutions, the absorption spectrum and the beautiful fluorescence
of the ammoniacal solution containing zinc chloride. For the detection
of urobilinogen we make use of ERHLICH'S reagent, and the property of
the colorless solution of being changed into urobilin in the air and light.

In the quantitative estimation of urobilin we proceed as follows,
according to G. HOPPE-SEYLER :3 100 cc. of the urine are acidified with
sulphuric acid and saturated with ammonium sulphate. The precipitate
is collected on a filter after some time, washed with a saturated solu-
tion of ammonium sulphate, and repeatedly extracted with equal parts
of alcohol and chloroform after pressing. The filtered solution is treated
with water in a separatory funnel until the chloroform separates well
and becomes clear. The chloroform solution is evaporated on the water-
bath in a weighed beaker, the residue dried at 100° C., and then extracted
with ether. The ethereal extract is filtered, the residue on the filter dis-
solved in alcohol, and transferred to the beaker and evaporated, then
dried and weighed. According to this method G. HOPPE-^EYLER found
0.08-0.14 gram of urobilin in one day's urine of a healthy person, or an
average of 0.123 gram.

The urobilin can also be determined according to the method sug-
gested by CHARNAS for its preparation and urobilin can also be deter-
mined spectroscopically by the method suggested by SAiLLET.4 Further
details will be found in the original publications and in larger handbooks.

The quantitative estimation of urobilinogen can be accomplished
spectroscopically by means of EHRLICH'S reagent, as suggested by CHARNAS.

1 Deutsch. med. Wochenschr., 1903.

* Compt. rend. soc. biol., 70.

• Virchow's Arch., 124.

4Charnas, 1. c.; Saillet, 1. c.; see also Tsuschija, Zeitschr. f. exp. Path. u. Ther., 7.

748 UKINE.

Uroerythrin is the pigment which often gives the beautiful red color to
the urinary sediments (sedimentum lateritium). It also frequently occurs
although only in very small quantities, dissolved in normal urines. The
quantity is increased after great muscular activity, after profuse perspira-
tion, immoderate eating, or partaking of alcoholic drinks, as well as after
digestive disturbances, fevers, circulatory disturbances of the liver, and
in many other pathological conditions.

Uroerythrin, which has been especially studied by ZOJA, RIVA, and
GARROD,1 has a pink color, is amorphous, and is very quickly destroyed
by light, especially when in solution. The best solvent is amyl alcohol;
acetic ether is not so good, and alcohol, chloroform, and water are even
less valuable. The very dilute solutions show a pink color; but on greater
concentration they become reddish orange or bright red. They do not
fluoresce either directly or after the addition of an ammoniacal solution
of zinc chloride; but they have a strong absorption, beginning in the
middle between D and E and extending to about F, and consisting of two
bands which are connected by a shadow between E and b. Concentrated
sulphuric acid colors a Uroerythrin solution a beautiful carmine red;
hydrochloric acid gives a pink color. Alkalies make its solution grass
green, and often a play of colors from pink to purple and blue is observed.
PORCHER and HERVIEUX 2 claim that Uroerythrin is a skatol pigment.

In preparing Uroerythrin according to GARROD, the sediment is dissolved
in water at a gentle heat and saturated with ammonium chloride, which pre-
cipitates the pigment with the ammonium urate. This is purified by repeated
solution in water and precipitation with ammonium chloride until all the urobilin
is removed. The precipitate is finally extracted on the filter in the dark with
warm water, filtered, then diluted with water, any hsematoporphyrin remaining
being removed by shaking with chloroform; the precipitate is then faintly acidi-
fied with acetic acid and shaken with chloroform, which takes up the Uroerythrin.
The chloroform is evaporated in the dark at a gentle heat.

Volatile fatty adds, such as formic acid, acetic acid, and perhaps also butyric
acid, occur under normal conditions in human urine (y. JAKSCH), also in that of
dogs and herbivora (SCHOTTEN). The acids poorest in carbon, such as formic
acid and acetic acid, are more stable in the body than those richer in carbon,
and therefore the relatively greater part of these pass unchanged into the urine
(SCHOTTEN). Normal human urine contains besides these bodies others which
yield acetic acid when oxidized by potassium dichromate and sulphuric acid
(v. JAKSCH). The quantity of volatile fatty acids in normal urine calculated as
acetic acid is, according to v. JAKSCH, 0.008-0.009 gram per twenty-four hours;
according to v. ROKITANSKY, 0.054 gram; and according to MAGNUS-LEVY
0.060 gram. The quantity is increased by exclusively farinaceous food (ROKI-
TANSKY), in fever and in certain diseases, while in others it is diminished (v.
JAKSCH, ROSENFELD). Large amounts of volatile fatty acids are produced in the
alkaline fermentation of the urine, and the quantity is 6-15 times as large as in

1 Zoja, Arch. ital. di clinica med., 1893, and Centralbl. f. d. med. Wissensch., 1892;
Riva, Gaz. med. di Torino, Anno 43, cited from Maly's Jahresber., 24; Garrod, Journ.
of Physiol., 17 and 21.

2 Journ. de Physiol., 7.

CARBOHYDRATES AND REDUCING SUBSTANCES. 749

normal urine (SALKOwsKi).1 Non-volatile fatty acids have been detected as
normal constituents of urine by K. MORNER and HYBBiNETTE.2

Paralactic Acid. It is claimed that this acid occurs in the urine of healthy
persons after very fatiguing marches (COLASANTI and MOSCATELLI). It is found
in larger amounts in the urine in acute phosphorus-poisoning or acute yellow
atrophy of the liver (SCHULTZEN and RIESS), in pregnancy (UNDERBILL) , and
especially abundant in eclampsia (ZWEIFEL and others). According to the
investigations of HOPPE-SEYLER, ARAKI, and v. TERRAY, lactic acid passes into
the urine as soon as the supply of oxygen is decreased in any way, and this probably
explains the occurrence of lactic acid in the urine after epileptic attacks (!NOUYE
and SAIKI). MINKOWSKI 3 has shown that lactic acid occurs in the urine in large
quantities on the extirpation of the liver of birds.

Glycerophosphoric acid occurs as traces in the urine,4 and it is probably a
decomposition product of lecithin. The occurrence of succinic acid in normal
urine is a subject of discussion.

Carbohydrates and Reducing Substances in the Urine. The occurrence
of glucose, as traces, in normal urine is highly probable, as the investiga-
tions of BRUCKE, ABELES, and v. UDRA^NSZKY show. The last investigator
has also shown the habitual occurrence of carbohydrates in the urine,
and their presence has been positively proven by the investigations of
BAUMANN and WEDENSKI, and especially by BAISCH. Besides glucose
normal urine contains, according to BAISCH, another not well-studied
variety of sugar, according to LEMAIRE, probably isomaltose, and besides
this a dextrin-like carbohydrate (animal gum), as shown by LANDWEHR,
WEDENSKI, and BAISCH. The quantity of carbohydrates eliminated under
normal conditions in the twenty-four hours' urine and determined by
the benzoylation method, which- is perhaps Dot sufficiently trustworthy,
varies considerably between 1.5 and 5.09 grams.5

The precipitate obtained from concentrated urine by the aid of alcohol and
whose nitrogen (colloidal nitrogen according to SALKOWSKI) in normal urine
amounts to 2.34-4.08 per cent of the total nitrogen, and in pathological urines to
8-9 per cent, and in a case of acute yellow atrophy of the liver to 21.8 per cent
contains, SALKOWSKI 6 claims, a nitrogenous carbohydrate which has strong

1 v. Jaksch, Zeitschr. f. physiol. Chem., 10; Schotten, ibid., 7; Rokitansky, Wien.
med. Jahrbuch, 1887; Salkowski, Zeitschr. f. physiol. Chem., 13; Magnus-Levy,
Salkowski's Festschrift, 1904; Rosenfeld, Deutsch, med. Wochenschr., 29.

2 Skand. Arch. f. Physiol., 7.

3 Colasanti and Moscatelli, Moleschott's Untersuch., 14; Schultzen and Reiss,
Chem. Centralbl., 1869; Underbill, Journ. of biol. Chem., 2; Zweifel, Arch. f. Gynakol.,
76; Araki, Zeitschr. f. physiol. Chem., 15, 16, 17, 19. See also Irisawa, ibid., 17; v.
Terray, Pfliiger's Arch., 65; Schiitz, Zeitscbr. f. physiol. Chem., 19; Inouye and
Saiki, ibid., 37; Minkowski, Arch. f. exp. Path. u. Pharm., 21 and 31.

4 See Pasqualis, Maly's Jahresber., 24.

5 Lemaire, Zeitschr. f. physiol. Chem., 21; Baisch, ibid., 18, 19, and 20. In these
as well as in Treupel, ibid., 16, the works of other investigators are cited. See also
v. Alfthan, Deutsch. med. Wochenschr., 26.

6 Berlin, klin. Wochenschr., 1905. In regard to urinary colloids see also Lichtwitz,
Zeitschr. f. physiol. Chem., 61 and 72, with Rosenbach, ibid., 61.

750 URINE.

reducing action upon alkaline copper solutions after cleavage with hydrochloric
acid.

Besides traces of sugar and the reducing substances previously men-
tioned, uric acid and creatinine, the urine contains still other bodies of this
character. These latter are partiy conjugated compounds of glucuronic
acid, CeNioO?, which is closely allied to sugar. The reducing power of
normal urine corresponds, according to various investigators, to 1.5-5.96
p. m. glucose. That portion of the reduction belonging to glucose
alone is equal to 0.1-0.6 p. m. LAVESON 1 believes that of the total
reduction 17.8 per cent is due to sugar, 26.3 per cent to creatinine, 7.8
per cent to uric acid, and the remainder, nearly 50 per cent, is caused
by chiefly unknown bodies.

Conjugated glucuronates occur, as indicated by FLUCKIGER and first
positively shown by MAYER and NEUBERG, in an exact manner, in very
small amounts in normal urine. They occur chiefly as phenol- and only
very small amounts of indoxyl- or skatoxylglucuronates. The quantity
of glucuronic acid obtained from the conjugated glucuronates is estimated
as 0.04 p. m. by MAYER and NEUBERG, and by C. TOLLENS and FR. STERNA
on the contrary it was found to be 2.5 p. m. or 0.37 gram per day. Besides
these conjugated glucuronates perhaps the urine sometimes contains the
urea glucuronic acid, the ureidoglucuronic acid prepared synthetically
by NEUBERG and NIEMANN.S

Very large amounts of these conjugated glucuronates occur in the
urine, on the other hand, after partaking of various therapeutic agents
and other substances, such as chloral hydrate, camphor, naphthol, borneol,
turpentine, morphine, and many other substances. The elimination
of glucuronic acid may be markedly increased in severe disturbances of
the respiration, severe dyspnoea, in diabetes mellitus, and by the direct
introduction of large amounts of glucose. According to P. MAYER,
in the oxidation of glucose a part of it forms glucuronic acid, hence it is
to be expected that the glucuronic acid can in part be derived from the
glucose. As a conjugation of the glucuronic acid with other bodies,
such as aromatic atomic complexes, prevents the combustion of this
acid in the animal body, it ought to follow that after the introduction
of such an atomic complex in the body during a glycosuria a correspond-
ing reduction of the glucose elimination would take place with the
increased excretion of conjugated glucuronates. In order to prove
this possibility, O. LoEWi4 fed dogs with camphor during phlorhizin

1 Fliickiger, Zeitschr. f. physiol. Chem., 9; Laveson, Bioch. Zeitschr., 4.
2Fluckiger, 1. c.; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Tollens and
Stern, Zeitschr. f. physiol. Chem., 67.

3 Zeitschr. f. physiol. Chem., 44.

4 Arch. f. exp. Path. u. Pharm., 47.

CONJUGATED GLUCURONIC ACIDS. 751

diabetes and found that the above expectation was not realized. Although
large quantities of campho-glucuronic acid were excreted, the sugar
excretion was only slightly diminished and not in proportion to the
quantity of conjugated glucuronate excreted. These negative results
are contradicted by the positive results obtained by PAUL MAYER.1
Rabbits normally convert almost all the camphor introduced into con-
jugated glucuronic acid. MAYER claims that if we allow a rabbit to
starve several days, the animal becomes so poor in the mother-substance
(glycogen) yielding the glucuronic acid that the introduction of camphor
only brings about an elimination of small quantities of glucuronic acid.
By the simultaneous administration of camphor and glucose while
starvation is going on, the elimination of glucuronic acid rises again to
the same height as it was before the starvation period. This shows that
the sugar had conjugated itself with the camphor as glucuronic acid.
HiLDEBRANDT2 has also made experiments showing that glucuronic acid
can very likely be formed from sugar. The observations of MAYER are
not substantiated by the recent investigations of FENYVESSY,S and the
observers do not agree on this question.

The conjugated glucuronic acids are formed, based upon the investi-
gations of SUNDWIK, FISCHER and PILOTY,* by a combination taking
place first between the conjugator and the glucose by means of the alde-
hyde group, and then the end alcohol group, CH^OH, is oxidized to COOH.
The conjugated glucuronic acids, at least in most cases, seem to be con-
structed after the glucoside type, a view which has received further
support by the synthesis of phenolglucuronic acid and euxanthonglu-
curonic acids by NEUBERG and NEIMANN.S Based upon their cleavage
(as far as they have been investigated) by kephir lactase and emulsin,
but not by yeast lactase (NEUBERG and WOHLGEMUTH 6), the conjugated
glucuronic acids must belong to the /3-series of glucosides. We also
know of certain conjugated glucuronates that are constructed upon the
ester type, namely, the dimethylaminobenzoicglucuronate, discovered
by JAFFE and also the benzoicglucuronic acid, after feeding benzoic
acid (MAGNUS-LEVY).7

According to the body with which they are conjugated the glucuronates
vary in behavior. On taking up water they split into glucuronic acid

1 Zeitschr. f . klin. Med., 47.

2 Arch. f. exp. Path. u. Pharm., 44.

3 See Maly's Jahresber., 34.

4E. Sundwik, Akademische Abhandlung Helsingfors, 1886; see also Maly's Jahr-
esber., 16, 76; Fischer and Piloty, Ber. d. d. chem. Gesellsch., 24.
6 Zeitschr. f. physiol. Chem., 44.

6 See Neuberg, Ergebnisse der Physiologic, Bd. 3, Abt. 1, 444.

7 Jaffe, Zeitschr. f. physiol. Chem., 43; Magnus-Levy, Bioch. Zeitschr., 6.

752 URINE.

and the conjugated group and this is brought about by boiling with
a dilute mineral acid. They are precipitated by basic lead acetate or
by basic lead acetate and ammonia. Most of the conjugated glucuronic
acids do not have a direct reducing action but are reducing after hydrolysis.
Certain of them, and to this group belong especially those acids of the
ester type, reduce copper oxide and certain other metallic oxides in alkaline
solution directly, and hence cause errors in the investigations of the
urine for sugar. The conjugated acids of the glucoside type rotate the
plane of polarized light to the left, while the glucuronic acid itself is dextro-
rotatory. The conjugated acids of the ester type, which as a rule are
less stable, rotate the ray of polarized light to the right. As the detection
of conjugated glucuronic acids is connected with the tests for sugar in
the urine, we will treat of this in connection with these tests.

Organic combinations containing sulphur of unknown kind, which may
in small part consist of sulphocyanides, 0.04 (GSCHEIDLEN) to 0.11 p. m.
(I. MuNK),1 cystine or bodies related to it, taurine derivatives, chon-
droitin-sulphuric acid and protein bodies, but in greater part are made up
of antoxyproteic acid, oxyproteic acid, alloxyproteic acid, and uroferric
acid, are found in human as well as in animal urines. The sulphur of
these mostly unknown combinations has been called " neutral," to dif-
ferentiate it from the " acid " sulphur of the sulphate and ethereal-sul-
phuric acid (SALKOWSKi2). The neutral sulphur in normal urine is
13-24 per cent of the total sulphur.3 In anaemia, cachetic conditions,
pulmonary tuberculosis and especially in carcinoma the quantity is
greatly increased (WEISS). In general it can be said that the quantity
is increased by an increased catabolism of protein and therefore an increase
in the neutral sulphur has been found in starvation (FR. MULLER), with
insufficient oxygen supply (REALE and BOERI, HARNACK and KLEINE)
and after chloroform narcosis (KAST and MESTER). After the introduc-
tion of free sulphur the quantity of neutral sulphur is increased, accord-
ing to PRESCH and YVON and to MAILLARD* The quantity of neutral
sulphur varies, according to BENEDICT, within rather narrow limits
and especially, according to FOLIN, it is dependent to a less degree than
the sulphate excretion upon the extent of the protein metabolism. The
relation between the neutral and acid sulphur depends in the first" place

1 Gscheidlen, Pfliiger's Arch., 14; Munk, Virchow's Arch., 69.

2 Ibid., 58, and Zeitschr. f. physiol. Chem., 9.

»Salkowski, 1. c.; Stadthagen, Virchow's Arch., 100; Le*pine, Compt. Rend., 91
and 97; Harnack and Kleine, Zeitschr. f. Biologic, 37; Mor. Weiss, Bioch. Zeitschr., 27.

4 Weiss, 1. c.; Fr. Miiller, Berl. klin. Wochenschr., 1887; Reale and Boeri, Maly's
Jahresber., 24; Harnack and Kleine, 1. c.; Kast and Mester, Zeitschr. f. klin. Med.,
18; Preach., Virchow's Arch., 119; Yvon, Arch, de Physiol. (5), 10; Maillard, Compt.
Rend., 152.

ORGANIC SULPHUR COMBINATIONS. 753

upon the extent of the sulphuric-acid excretion. According to HARNACK
and KLEINE/ the relation of the oxidized sulphur to the total sulphur
changes always in the same way as the relation of the nitrogen of the
urea to the total nitrogen. The more unoxidized sulphur is eliminated
the more abundant are the nitrogen compounds, not urea, in the urine —
a statement which coincides with recent observations showing that the
neutral sulphur originates chiefly from the different proteic acids, and
the uroferric acid.

According to LEPINE, a part of the neutral sulphur is more readily oxidized
(directly with chlorine or bromine) into sulphuric acid than the other, which is
only converted into sulphuric acid after fusing with potash and saltpeter. The
investigations of W. SMITH 2 show that it is probable that the difficultly pxidizable
part of the neutral sulphur occurs as sulpho-acids. An increased elimination of
neutral sulphur has been observed in various diseases, such as pneumonia, cystin-
uria, and especially where the flow of bile into the intestine is prevented.

The total quantity of sulphur in the urine is determined by fusing the solid
urinary residue with saltpeter and caustic alkali or sodium peroxide, or by oxida-
tion with nitric acid.3 The quantity of neutral sulphur is determined as the
difference between the total sulphur and the sulphur of the sulphate and ethereal-
sulphuric acids. The readily oxidizable part of the neutral sulphur is determined
by oxidation with bromine or potassium chlorate and hydrochloric acid (LEPINE,
JEROME 4).

Sulphuretted hydrogen occurs in the urine only under abnormal conditions
or as a decomposition product. This compound may be produced from the
neutral sulphur of the organic substances of the urine by the action of certain
bacteria (Fn. MULLER, SALKOWSKI 5). Other investigators have given hypo-
sulphites as the source of the sulphuretted hydrogen. The occurrence of hypo-
sulphites in normal human urine, which is asserted by HEFFTER, is disputed by
SALKOWSKI and PRESCH.6 Hyposulphites occur constantly in cat's urine and,
as a rule, also in dog's urine.

Antoxyproteic acid is a nitrogenous acid containing sulphur which
BONDZYNSKI, DOMBROWSKI, and PANEK 7 have isolated from human
urine. The composition of the acid was: C 43.21, H 4.91, N 24.4, S 0.61,
and O 26.33 per cent. A part of the sulphur can be split off by alkali.
This acid is soluble in water, is dextrorotatory, and is only precipitated
from concentrated solution by phosphotungstic acid. It does not give
the protein color reactions, but gives EHRLiCHrs diazo reaction (see below) .

Benedict, Zeitschr. f. klin. Med., 36; Harnack and Kleine, 1. c.; Folin, Amer.
Journ. of Physiol., 13.

2 Lepine, 1. c.; Smith, Zeitschr. f. physiol. Chem., 17.

3 See Abderhalden and Funk, Zeitschr. f. physiol. Chem., 58 and 59, which also
cites other methods. See S. R. Benedict, Journ. of biol. Chem., 6 and 8; Denis, ibid.,
8; Gill and Grindley, Journ. Amer. Chem. Soc., 31; Folin, ibid., 31.

4 Jerome, Pfliiger's Arch., 60.

6Fr. Muller, Berlin, klin. Wochenschr., 1887; Salkowski, ibid., 1888.

6 Heffter, Pfluger's Arch., 38; Salkowski, ibid., 39; Presch, Virchow's Arch., 119.

7 Zeitschr. f. physiol. Chem., 46.

754 URINE.

The salts with the alkalies, barium, calcium, and silver are soluble in
water, and of these salts that with barium and, to a still higher degree,
the silver salt are soluble with difficulty in alcohol. The free acid and
its salts are precipitated by mercuric nitrate and acetate, and by this
last reagent even from solutions strongly acidified with acetic acid. Basic
lead acetate does not precipitate the pure acid.

Oxyproteic acid is the name given by BONDZYNSKI and GOTTLIEB l
to a nitrogenous acid containing sulphur, and which they prepared from
human urine, which has recently been further studied by BONDZYNSKI,
DOMBBOWSKI and PANEK. This acid contained C 39.62, H 5.64, N
18.08, S 1.12, and 0 35.54 per cent, and also contains sulphur which could
be split off. On cleavage it yields no tyrosine, nor does it give EHRLICH'S
diaao reaction, the xanthoproteic nor the biuret reaction. It gives a
faint indication of a MILLON reaction and is not precipitated by phos-
photungstic acid, hence it leads to an error in the PFLUGER-BOHLAND
method for estimating urea. The acid soluble in water is precipitated
by mercuric nitrate and acetate in neutral solutions, but is not precipitated
by basic lead acetate. The salts of this acid are readily soluble in water
and more soluble in alcohol than the corresponding salts of antoxy-
proteic acid.

The acid which is found in large quantities, especially in the urine of
dogs poisoned with phosphorus (BONDZYNSKI and GOTTLIEB), is considered
like the preceding acid as an intermediary oxidation product of the pro-
teins, and oxyproteic acid seems to represent a higher state of oxidation
or a demolition of the proteins than the antoxyproteic acid.

The acid called uroproteic acid by CLOETTA is probably a mixture of several
bodies, according to the recent investigations of BONDZYNSKI; DOMBROWSKI, and
PANEK. The same applies also to the barium oxyproteate prepared by PREGL 2
from the urine.

Alloxyproteic acid is a third acid related to the above, which was
first isolated by BONDZYNSKI and PANEK 3 from the urine and then care-
fully studied with DOMBROWSKI. The composition is: C 41.33, H
5.70, N 13.55, S 2.19, and 0 37.23 per cent, based upon new investigations.
The free acid is soluble in water. It gives neither the biuret reaction
nor EHRLICH'S reaction, and is not precipitated by phosphotungstic
acid. Differing from the other acids, it is precipitated by basic lead
acetate, and its salts are only slightly soluble in alcohol. According to

iCentralbl. f. d. med. Wissensch., 1897, No. 33.

2 Cloetta, Arch, f . exp. Path. u. Pharm., 40; Pregl, Pfliiger's Arch., 75.

8 Ber. d. d. chem. Gesellsch., 35.

OXYPROTEIC ACIDS. 755

LIEBERMANN * this acid is not a unit substance, and contains a part of
its sulphur as ethereal sulphuric acid, and it also contains uroferric acid.

The urochrome, which has been specially studied by DoMBROWSKi,2 is con-
sidered by him and BONDZYNSKI as belonging to the oxyproteic acid ' group. I
contains about 5 per cent sulphur, is precipitated by copper acetate and yields
melanin-like substances on its decomposition. No positive proofs are at hand in
regard to the purity of this urochrome and the reports as to its composition,
which are very contradictory, do not exclude the possibility that this is a mixture
of a yellow pigment with another substance (see page 741).

BROWINSKI and DOMBROWSKI 3 have carried out investigations on the nitrogen
titratable with formol on the oxyproteic acids before and after acid hydrolysis.
They found that the antoxyproteic acid and the oxyproteic acid did not contain
any nitrogen split off as NH3 by MgO before hydrolysis, while the alloxyproteic
acid as well as the urochrome yielded about 3 per cent of the total nitrogen in this
form. After acid hydrolysis all gave about the same quantity of ammonia. The
two first-mentioned acids, especially oxyproteic acid, were before hydrolysis
considerably richer in amino groups, titratable with formol, than the others.
This indicates that these two acids are produced from the proteins by a deeper
cleavage than is the alloxyproteic acid. The large amount of free amino groups,
which occur especially in the oxyproteic acid and which amount to 38.8 per cent
of the total nitrogen, is nevertheless remarkable.

The preparation of the three above-mentioned acids is based in part
upon the fact that alloxyproteic acid alone is precipitated by basic lead
acetate and that the two other acids can be precipitated from the ni-
trate by mercuric acetate, the antoxyproteic acid in acetic acid solution
and the oxyproteic acid in neutral solution. The preparation is never-
theless very tedious and complicated and therefore we must refer to the
original works for details.

Uroferric acid is an acid isolated by THIELE 4 from the urine, according to
SIEGFRIED'S method for preparing pure peptone. It also contains sulphur, 3.46
per cent, and has the formula CasHseNsSOig. The acid forms a white powder
which is readily soluble in water, saturated ammonium-sulphate solution, and
methyl alcohol. It is soluble with difficulty in absolute alcohol, insoluble in benzene,
chloroform, ether, and acetic ether. About one-half of the sulphur can be split
off as sulphuric acid on boiling with hydrochloric acid. The acid gives neither
the biuret test nor MILLON'S or ADAMKIEWICZ'S reactions. It is precipitated by
mercuric nitrate and sulphate, and also by phosphotungstic acid. This acid is
hexabasic, and its specific rotation at 18° C. (QJ)D = —32.5°. On cleavage it
yields melanine substances, sulphuric acid, aspartic acid, but no hexone bases.
The existence of this acid is disputed by BONDZYNSKI, DOMBROWSKI and PANEK.
The investigations of GINSBERG also contradict the occurrence of such an acid,
because no sulphuric acid could be split off from the mixture of the oxyproteic
acids by hydrolysis.

Methods for the quantitative estimation of the total oxyproteic
acids have been suggested by GINSBERG and by GAWINSKI.S Accord-

1 Zeitschr. f. physiol. Chem., 52.

2 Ibid., 46 and 62.

3 Ibid., 77.

4 Zeitschr. f. physiol. Chem., 37.

8 Ginsberg, Hofmeister's Beitrage, 10; Gawinski, Zeitschr. f. physiol. Chem., 58.

756 URINE.

ing to their determinations, in man with a mixed diet, the nitrogen of the
oxyproteic acids represented 3-6.8 per cent of the total nitrogen, and with
a milk diet it sinks to about one-half of this (GAWINSKI). In dogs it
amounts to 2 per cent of the total nitrogen (GINSBERG). In disease it
may rise, and in typhoid cases it may rise to 14.69 per cent of the total
nitrogen (GAWINSKI). In phosphorus poisoning this nitrogen fraction
is also markedly increased according to several observations. The
oxyproteic acids are considered, as above remarked, as intermediary
products of the protein metabolism, and GAWINSKI holds that the
elimination of their nitrogen runs parallel with the elimination of neutral
sulphur, so that this latter may serve as an approximate measure of the
elimination of these acids.

ABDERHALDEN and PREGL l have shown that human urine normally contains
compounds which stand, perhaps, in close relation to the polypeptides, and
which on hydrolysis with acids yield at least a part of the moities existing in the
protein molecule. In the case investigated they obtained abundant glycocoll,
also leucine, alanine, glutamic acid, phenylalanine, and probably also aspartic
acid. The relation between these polypeptide-like bodies and the above-men-
tioned proteic acids and uroferric acid has not been investigated.

HENRIQUES and SORENSEN 2 have given further proof for the occurrence of
nitrogen in peptide combinations in the urine. They have shown by formol
titration that in normal urine amino-acid nitrogen occurs. It must be remarked
that they consider as amino-acid nitrogen not only the nitrogen occurring as amino-
acids but also the urine nitrogen directly titratable with formol, therefore also the
titratable amino-nitrogen in the oxyproteic acids, polypeptides or more com-
plicated protein derivatives. They have further shown that after boiling with
acid that the quantity of titratable nitrogen increases, and this increase which
in man may be 8.9-28.3 per cent of the amino-acid nitrogen, they consider as pep-
tide-like nitrogen. We have abundant literature 3 on the ways and means of
carrying out the formol titration in urines, considering the presence of ammonia.

Ammo-acids may, when they are introduced into the body in large amounts,
also pass in part into the urine. This has been shown for r-alanine by R. HIRSCH
in the dog, and by PLAUT and REESE in dog and man, and for r-leucine by
ABDERHALDEN and SAMUELY4 in rabbits and by others using different amino-acids.
EMBDEN and REESE, FORSSNER, ABDERHALDEN and SCHITTENHELM, SAMUELY,
EMBDEN and MARX 5 were able, by means of the naphthalene sulpho-
chloride method to detect glycocoll in normal human urine, and this glycocoll
must occur in the urine in a combination which is readily split by alkali. Although

1 Zeitschr. f. physiol. Chem., 46.

2 Henriques, Zeitschr. f. physiol. Chem., 60; Henriques and Sorensen, ibid., 63
and 64.

3 See Henriques and S6rensen, 1. c.; Malfatti, Zeitschr. f. physiol. Chem., 61 and
66; de Jager, ibid., 62 and 65; Fry and Gigon, Bioch. Zeitschr., 22.

4R. Hirsch, Zeitschr. f. exp. Path. u. Therap., 1; Plaut and Reese, Hofmeister's
Beitrage, 7; Abderhalden and Samuely, Zeitschr. f. physiol. Chem., 47.

6Forssner, Zeitschr. f. physiol. Chem., 47; Abderhalden and Schittenhelm, ibid.,
47; Samuely, ibid., 47; Embden and Reese, Hofmeister's Beitrage, 7, with Marx,
ibid., 11, which also cites the rather conflicting deductions of Neuberg and Wolgemuth
and of Hirschstein.

ORGANIC PHOSPHORUS COMBINATIONS. 757

there have been numerous investigations, no amino-acids besides glycocoll could
be detected in normal human urine, while, on the contrary, in pathological con-
ditions other amino-acids have been found several times. The amino-acid frac-
tion of the urine seems to be increased in starvation and in high altitudes (LOEWY *).
The conclusions of various investigators 2 in regard to the behavior of amino-
acids in diseases such as gout, disagree.

Non-dialyzable substances, the so-called adialyzdble bodies, or bodies that
dialyze with difficulty, also occur in the urine. They consist in part of chon-
droitin-sulphuric acid whose daily amount, according to PONS, is 0.08-0.09 gram,
and also of nucleic acid, mucoids, the colloidal nitrogenous bodies (see SALKOWSKI,
page 749) and unknown bodies. SASAKI found 0.218-0.68 gram of such bodies
per liter of normal urine, and EBBECKE found 1.44 grams in men. In pregnant
women SAVARE found somewhat higher results (0.6 gram per liter) than in non-
pregnant women (0.4 gram). The quantity is increased in fevers, in pneumonia
(EBBECKE), in nephritis, and especially in eclampsia, where SAVARE 3 indeed in
one case found 13.84 grams per liter. The adialyzable bodies occurring in eclampsia
are toxic.

Organic combinations containing phosphorus such as glycerophosphoric acid,
phosphocarnic acid (ROCKWOOD), etc., which yield phosphoric acid on fusing with
saltpeter and caustic alkali, are also found in urine (LEPINE and EYMONNET,
OERTEL). With a total elimination of about 2.0 grams total P205, OERTEL found
on an average about 0.05 gram P205 as phosphorus in organic combination. Accord-
ing to KONDO the quantity of organic phosphorus is increased by taking phos-
phatides and nucleins but not to the same extent as the quantity of phosphoric
acid. According to SYMMERS 4 the organic combined phosphorus may in many
pathological conditions be 25-50 per cent of the total phosphoric acid. In lym-
phatic leucaemia, and especially in degenerative diseases of the nervous system,
the quantity may increase.

Enzymes of various kinds have been isolated from the urine. Among these
may be mentioned pepsin, diastatic enzyme and lipase.5

Mucin. The nubecula consists, as shown by K. M6RNER,6 of a mucoid which
contains 12.74 per cent N and 2.3 per cent S. This mucoid, which apparently
originates in the urinary passages, may pass to a slight extent into solution in
the urine. In regard to the nature of the mucins and nucleoalbumins otherwise
occurring in the urine we refer the reader to the pathological constituents of the
urine.

Ptomaines and leucomaines, or poisonous substances of an unknown kind,
which are often described as alkaloidal substances, occur in normal urine, as shown
by earlier investigations (POUCHET, BOUCHARD, ADUCCO and others 7) and also
by recent researches of KUTSCHER, LOHMANN and ENGELAND. The trimethyl-

1 Deutsch. med. Wochenschr., 1905; see also Signorelli, Bioch. Zeitschr., 39.

2 See Jastrowitz, Arch. f. exp. Path. u. Pharm., 59; Walker Hall, Bioch. Journ.,
1; Brugsch and Schittenhelm, Zeitschr. f. exp. Path. u. Therap., 4.

3 Pons, Hofmeister's Beitrage, 9; Sasaki, ibid., 9; Savard, ibid., 9 and 11; Ebbecke,
Bioch. Zeitschr., 13.

4Rockwood, Arch. f. (Anat. u.) Physiol., 1895; Oertel, Zeitschr. f. physiol. Chem.,
26, which cites the other works. See also Keller, Zeitschr. f. physiol. Chem., 29;
Mandel and Oertel, N. Y. Univ. Bull. Med. Sciences, 1; and Maly's Jaljresber., 31;
Symmers, Journ. of Path, and Bact., 10.

5 In regard to the literature on enzymes in the urine, see Huppert-Neubauer, 599.
In regard to trypsin in the urine see Johansson, Zeitschr. f. physiol. Chem., 85.

6 Skand. Arch, f . Physiol., 6.

7 A complete bibliography on the ptomaines and leucomaines of the urine is found
in Huppert-Neubauer, .403.

758 URINE.

amine, which originates from the phosphatides, and was first detected by DE FILIPPI
and later by K. BAUER, belong to the leucomanines and also the bases found by
KUTSCHER and by KUTSCHER and LOHMANN, namely, methyl guanidine (also
found by ACHELIS), dimethylguanidine, novain (previously found by DOMBROWSKI),
reductonovain, C7Hi7N02, gynesin, C^HosNsOs (from female urine) mingin, CiiHigNjOj,
mtiatin (Chapter X) and methylpyridine chloride, which is not a leucomaine, but
is probably derived from smoking tobacco or from drinking coffee. The imidazole
derivatives hislidine and imidazolamino-acetic acid found by KUTSCHER and
ENGELAND also belong to this group and the urohyperlensin and urohypotensin
of ABELOUS and BARDiER.1

Under pathological conditions the quantity of leucomaines and other bodies
may be increased (BOUCHARD, LEPINE and GEURIN, VILLIERS, GRIFFITHS, ALBU,
and others). Within the last few years the poisonous properties of urine have
been the subject of more thorough investigation, especially by BOUCHARD. He
found that the night urine is less poisonous than the day urine, and that the
poisonous constituents of the day and night urine have not the same action.
In order to be able to compare the toxic power of the urine under different con-
ditions, BOUCHARD determines the UROTOXIC COEFFICIENT, which is the weight
of rabbit in kilos that is killed by the quantity of urine excreted in twenty-four
hours by 1 kilo of the person experimented upon.2

Many substances have been observed in animal urine which are not found in
human urine. To these belong the above-described kynurenic acid, urocanic acid,
which according to HUNTER is imidazolacrylic acid, also found in dog's urine;
damaluric acid and damolic acid (according to SCHOTTEN,S probably a mixture
of benzoic acid with volatile fatty acids), obtained by the distillation of cow's
urine; and lastly lithuric acid, found in the urinary concrements of certain
animals.

m. INORGANIC CONSTITUENTS OF URINE.

Chlorides. The chlorine occurring in the urine is undoubtedly com-
bined with the bases contained in this excretion; the chief part is in
combination with sodium. In accordance with this, the quantity of
chlorine in the urine is generally expressed as NaCl.

The question as to whether a part of the chlorine contained in the
urine exists as organic combinations, as considered by BERLIOZ and
LEPINOIS, is still disputed, although recently BAUMGARTEN4 has supported
this view.

The quantity of chlorine combinations in the urine is subject to con-
siderable variation. In general the amount from a healthy adult on a

1 de Filippi, Zeitschr. f. physiol. Chem., 49; Bauer, Hofmeister's Beitrage, 11;
Kutscher, Zeitschr. f. physiol. Chem., 51, with Lohmann, ibid., 48 and 49; Achelis,
ibid., 50; Engeland, ibid., 57, and Munch, med. Wochenschr., 55; Abelous and Bardier,
Maly's Jahresb., 39 and 40.

2 See footnote 7, page 757.

3 Hunter, Journ. of biol. Chem., 11; Schotten, Zeitschr. f. physiol. Chem., 7.

4 Berlioz and Lepinois, see Chem. Centralbl., 1894, 1, and 1895, 1; also Petit and
Terrat, ibid., 1894, 2, and Vitali, ibid., 1897, 2; Ville and Moitessier, Maly's Jahres-
ber., 31; Meillere, ibid.; Bruno, ibid., 452; Baumgarten, Zeitschr. f. exp. Path. u.
Therap., 5.

CHLORIDES. 759

mixed diet is 10-15 grams of NaCl per twenty-four hours. The quantity
of common salt in the urine depends chiefly upon the amount of salt in the
food, with which the elimination of chlorine increases and decreases.
The free drinking of water also increases the elimination of chlorine,
which is greater during activity than during rest (at night). Certain
organic chlorine combinations, such as chloroform, may increase the
elimination of inorganic chlorides by the urine (ZELLER, KAST1).

In diarrhoea, in quick formation of large transudates and exudates,
also in specially marked cases of acute febrile diseases at the time of the
crisis, the elimination of NaCl is materially decreased. The excretion
of chlorine may vary considerably in disease, but still the NaCl taken with
the food has here, as in physiological conditions, a great influence on the
NaCl excretion.2

The quantitative estimation of chlorine in the urine is most simply per-
formed by titration with silver-nitrate solution. The urine must not
contain either proteid (which if present must be removed by coagulation)
or iodine or bromine compounds.

In the presence of bromides or iodides evaporate a measured quantity of the
urine to dryness, fuse the residue with saltpeter and soda, dissolve the fused
mass in water, and remove the iodine or bromine by the addition of dilute sul-
phuric acid and some nitrite, and thoroughly shake with carbon disulphide.
The liquid thus obtained may now be titrated with silver nitrate according to
VOLHARD'S method. The quantity of bromide or iodide is calculated as the
difference between the quantity of silver-nitrate solution used for the titration
of the solution of the fused mass and the quantity used for the coresponding
volume of the original urine.

The otherwise excellent titration method of MOHR, according to which
we titrate with silver nitrate in neutral liquids, using neutral potassium
chromate as an indicator, cannot be used directly on the urine in careful
work. Organic urinary constituents are also precipitated by the silver
salt, and the results are therefore somewhat high for the chlorine. If
this method is to be employed, the organic urinary constituents must be
destroyed, by incineration after the addition of saltpeter free from
chlorine.

According to BANG and LARSSON 3 the disturbing substances which react
with AgN03 can be removed by shaking with blood-charcoal. The value of this
suggestion is essentially diminished, because every blood-charcoal cannot be used,
and therefore a special testing of the blood-charcoal must be done.

teller, Zeitschr. f. physiol. Chem., 8; Kast, ibid., 11; Vitali, Chem. Centralbl,
1899, 2.

2 On the elimination of chlorine in disease, see Albu and Neuberg, Physiol. u. Pathol.
des Mineralstoffwechsels, Berlin, 1906.

3 Bioch. Zeitschr., 49.

760 URINE.

The silver-nitrate solution may be a N/10 one. It is often made of
such a strength that each cubic centimeter corresponds to 0.006 gram
Cl or 0.01 gram NaCl. This last-mentioned solution contains 29.075
grams of AgNOs in 1 liter.

FEEUND and TOEPFER, as well as BODTKER/ have suggested modifica-
tions of MOHR'S method.

VOLHARD'S METHOD. Instead of the preceding determination, VOL-
HARD'S method, which can be performed directly on the urine, may be
employed. The principle is as follows: All the chlorine from the urine
acidified with nitric acid is precipitated by an excess of silver nitrate,
filtered, and in a measured part of the filtrate the quantity of silver added
in excess is determined by means of a sulphocyanide solution. This
excess of silver is completely precipitated by the sulphocyanide, and a
solution of some ferric salt, which, as is well known, gives a blood-red
reaction with the smallest quantity of sulphocyanide, is used as an indicator.

We require the following solutions for this titration: 1. A silver-
nitrate solution which contains 29.075 grams of AgNOs per liter, and of
which each cubic centimeter corresponds to 0.01 gram NaCl or 0.00607
gram Cl. 2. A saturated solution at the ordinary temperature of chlorine-
free iron alum or ferric sulphate. 3. Chlorine-free nitric acid of a specific
gravity of 1.2. 4. A potassium-sulphocyanide solution which contains
8.3 grams KCNS per liter, and of which 2 cc. corresponds to 1 cc. of the
silver-nitrate solution.

About 9 grams of potassium sulphocyanide are dissolved in water and diluted
to 1 liter. The quantity of KCNS contained in this solution is determined by the
silver-nitrate solution in the following way: Measure exactly 10 cc. of the silver
solution and treat it with 5 cc. of nitric acid and 1-2 cc. of the ferric-salt solu-
tion and dilute with water to about 100 cc. Now the sulphocyanide solution
is added from a burette, constantly stirring until a permanent faint-red colora-
tion of the liquid takes place. The quantity of sulphocyanide found in the solu-
tion by this means indicates how much it must be diluted to be of the proper
strength. Titrate once more with 10 cc. of AgN03 solution and correct the sul-
phocyanide solution by the careful addition of water until 20 cc. exactly cor-
responds to 10 cc. of the silver solution.

The determination of the chlorine in the urine is performed by this
method in the following way: Exactly 10 cc. of the urine are placed in a
flask which has a mark corresponding to 100 cc. and which is provided
with a stopper; 5 cc. of nitric acid are added; dilute with about 50 cc.
of water and then allow exactly 20 cc. of the silver-nitrate solution to
flow in. Close the flask with the stopper and shake well, remove the stop-
per and wash it with distilled water into the flask, and fill the flask to the
100-cc. mark with distilled water. Close again with the stopper, care-
fully mix by shaking, and filter through a dry filter. Measure off 50
cc. of the filtrate by means of a dry pipette, add 3 cc. of ferric-salt solu-
tion, and allow the sulphocyanide solution to flow in until the liquid
above the precipitate has a permanent red color. The calculation is
very simple. For example, if 4.6 cc. of the sulphocyanide solution

1Freund and Toepfer, see Maly's Jahresber., 22; Bodtker, Zeitschr. f. physioL
Chem., 20.

PHOSPHATES. 761

was necessary to produce the final reaction, then for 100 cc. of the filtrate
( = 10 cc. urine) 9.2 cc. of this solution are necessary. 9.2 cc. of the
sulphocyanide solution corresponds to 4.6 cc. of the silver solution,
and since 20—4.6 = 15.4 cc. of the silver solution was necessary to com-
pletely precipitate the chlorine in 10 cc. of the urine, then 10 cc. con-
tains 0.154 gram of NaCl. The quantity of sodium chloride in the urine
is therefore 1.54 per cent, or 15.4 p. m. If we always use 10 cc. for the
determination, and always 20 cc. of AgNOa solution, and dilute with
water to 100 cc., the quantity of NaCl in 1000 parts of the urine is found
by subtracting from 20 the number of cubic centimeters of sulphocyanide
(R) required with 50 cc. of the filtrate. The quantity of NaCl p. m.
therefore under these circumstances = 20 — R, and the percentage of

XT „, 20-R
NaC1=__

If it is necessary to destroy the organic urinary constituents before titration,
this can be best performed, according to DEHN,1 by evaporating the urine (10 cc.)
to dryness on the water-bath after the addition of a small amount of sodium per-
oxide, then faintly acidifying with nitric acid and then titrating according to
VOLHARD. Incineration is unnecessary.

For the approximate estimation of chlorine in the urine EKEHORN has made
use of VOLHARD'S titration method by using for the determination a glass tube
closed at one end and divided into half cubic centimeters and called the chlorom-
eter. The reagents necessary are: (a) a mixture of 20 cc. silver-nitrate solution
(according to VOLHARD), 5 cc. nitric acid and water to 100 cc.; (6) 40 cc. sul-
phocyanide solution and 60 cc. of a ferric alum, chlorine free and saturated at
the ordinary temperature. The silver-nitrate solution, of which each cubic
centimeter corresponds to 0.002 gm. NaCl, is equivalent to the iron sulphocyanide
solution. First 2 cc. of the urine are placed in the graduated tube and then 0.5
cc. sulphocyanide solution, and the silver-nitrate solution gradually added (shak-
ing the tube closed with a rubber stopper) until the coloration of the sulphocyanide
just disappears. 0.5 cc. is subtracted from the silver solution for the 0.5 cc. of
the sulphocyanide; the tube is so graduated that the quantity of NaCl in the
urine in parts per thousand is read off directly on the tube. The difference
between these results and those obtained by VOLHARD'S titration method amounts
only, according to C. TH. MORNER,* to 0.25 to at most 0.5 p. m.

The approximate estimation of chlorine in the urine (which must be free from
protein) is made by strongly acidifying with nitric acid and then adding to it,
drop by drop, a concentrated silver-nitrate solution (1:8). In a normal quantity
of chlorides the drop sinks to the bottom as a rather compact cheesy lump. In
diminished quantity of chlorides the precipitate is less compact and coherent,
and in the presence of very little chlorine a fine white precipitate or only a
cloudiness or opalescence is obtained.

Phosphates. Phosphoric acid occurs in acid urines partly as dihydro-
gen, MH2PO4, and partly as monohydrogen, M2HP04, phosphates,
both of which may be found in acid urines at the same time. The
proportion of these may vary considerably; acid urines contain chiefly
dihydrogen phosphate and in many cases the urine seems to contain only
dihydrogen phosphate and sometimes indeed only a small quantity of phos-

1 Zeitschr. f. physiol. Chera., 44.

2Ekehorn, Hygiea, Stockholm, 1906; Morner, Upsala Lakaref. Forh. (N. F.), 11.

762 URINE.

phoric acid. The total quantity of phosphoric acid varies and depends on
the character and the quantity of food. The average quantity of P2Os
is in round numbers 2.5 grams, with a variation of 1-5 grams per day.
A small part of the phosphoric acid of the urine originates from the
burning of organic compounds, such as nuclein, and phosphatides
within the organism; on exclusive feeding with substances rich in nuclein
or pseudonuclein the quantity of phosphates is essentially increased;
still it is undecided to what extent the excretion of phosphoric acid is
a measure of the absorption and decomposition of these bodies.1 The
greater part originates from the phosphates of the food, and the quan-
tity of phosphoric acid eliminated is greater when the food is rich in
alkali phosphates in proportion to the quantity of lime and magnesium
phosphates. If the food contains much lime and magnesia, large quan-
tities of earthy phosphates are eliminated by the excrement; and even
though the food contains considerable amounts of phosphoric acid in these
cases, the quantity excreted by the urine is small. This is especially
true of herbivora, in which the kidneys are the chief organs for the
excretion of alkali phosphates. In man, according to EHRSTROM, the
content of lime in the food seems to play no important role, as in his exper-
iments about one-half of the phosphoric acid taken as CaHPC>4 was
absorbed, still the extent of phosphoric-acid excretion through the urine
depends in man not only upon the total quantity of phosphoric acid in
the food, but also upon the relative amounts of the alkaline earths and the
alkali salts of the food. In carnivora, in which phosphate injected sub-
cutaneously is eliminated by the intestine (BERGMANN), the urine is
habitually poor in phosphates.2

As the extent of the elimination of phosphoric acid is mostly dependent
upon the character of the food and the absorption of the phosphates
in the intestine, it is apparent that the relation between the nitrogen and
phosphoric-acid excretion cannot run parallel. This is in fact so, and,
according to EHRSTROM, the organism has the power of accumulating
large quantities of phosphorus for a relatively long time independent of
the condition of the nitrogen balance. With a certain regular food the
relation between nitrogen and phosphoric acid in the urine can be kept
almost constant. Thus on feeding with an exclusive meat diet, as
observed by Vorr3 in dogs, when the nitrogen and phosphoric acid

1 gee A. Gumlich, Zeitschr. f. physiol. Chem., 18; Roos, ibid., 21; Weintraud,
Arch. f. (Anat. u.) Physiol., 1895; Milroy and Malcolm, Journ. of Physiol., 23; Roh-
mann and Steinitz, Pfliiger's Arch., 72; Loewi, Arch. f. exp. Path. u. Pharm., 44 and 45.

2 Ehrstrom, Skand. Arch. f. Physiol., 14; Bergmann, Arch. f. exp. Path. u. Pharm.,
47.

3 Physiologie des allgemeinen Stoffwechsels und der Ernahrung in L. Hermann's
Handbuch, 6, Thul. 1, 79.

PHOSPHATES. 763

of the food exactly reappeared in the urine and feces, the rela-
tion was 8.1:1. In starvation, as shown by the compilation of R. TIGER-
STEDT,1 the phosphorized constituents of the body are destroyed to a
much greater extent than when food very poor in phosphorus is given.
In starvation this relation is changed, namely, relatively more phosphoric
acid is eliminated, which seems to indicate that besides flesh and related
tissues another tissue rich in phosphorus is largely destroyed. The
starvation experiments show that this is the bone-tissue. According to
PREYSZ, OLSAVSZKY, KLUG, I. MUNK and MAILLARD 2 the elimination
of phosphoric acid is considerably increased by intense muscular work.
As the phosphoric acid is in part derived from the nucleins, it would
be expected that in those diseases in which the excretion of purine
bodies was increased the phosphoric acid would also be augmented.
This is not the case, and indeed we have observed cases with an increased
elimination of purine bodies with a diminution in the phosphoric-acid
excretion. Cases of leucaemia have been observed in which the phos-
phoric-acid excretion was reduced, although there was a pronounced
increase in the number of leucocytes. In these cases there may be a
subsequent excretion or a retention of phosphoric acid. This last condition
also occurs in inflammatory and renal diseases. The earthy phosphates
of the urine sometimes have the tendency of precipitating either spon-
taneously or after warming, and this has been called phosphaturia. We
are here dealing with a diminished acidity and, it seems, with a dimin-
ished excretion of phosphoric acid and an increased elimination of lime,
or at least an essentially different relation between the phosphoric acid
and the alkaline earths of the urine, as compared with the normal (PANEK

IWANOFF, SOETBER and KRIEGER 3) .

Quantitative Estimation of the Total Phosphoric Add in the Urine.
This estimation is most simply performed by titrating with a solution
of uranium acetate. The principle of the titration is as follows: A
warm solution of phosphates containing free acetic acid gives a whitish-
yellow precipitate of uranium phosphate with a solution of a uranium
salt. This precipitate is insoluble in acetic acid, but dissolves in mineral
acids, and on this account there is always added, in titrating, a certain
quantity of sodium-acetate solution. Potassium ferrocyanide is used
as the indicator, which does not act on the uranium-phosphate precipitate,
but gives a reddish-brown precipitate or coloration in the presence of the

1 Skand. Arch. f. Physiol., 16.

2Preysz, see Maly's Jahresber., 21; Olsavszky and Klug, Pfliiger's Arch.. 54;
Munk, Arch. f. (Anat. u.) Physiol., 1895; Maillard, Journ. de Physiol. et de Path.
10 and 11.

3Panek, see Maly's Jahresber., 30, 112; Iwanoff, Biochem. Centralbl., 1, 710;
Soetber^and Krieger, Deutsch. Arch. f. klin. Med., 72; Campani, Biochem. Centi&lbl.,
3, 616; "Tobler, Arch. f. exp. Path. u. Pharm., 52.

764 URINE.

smallest amount of soluble uranium salt. The solutions necessary for
the titration are: 1. A solution of a uranium salt of which each cubic
centimeter corresponds to 0.005 gram P2Os and which contains 20.3
grams of uranium oxide per liter. 20 cc. of this solution corresponds
to 0.100 gram P2O5. 2. A solution of sodium acetate. 3. A freshly
prepared solution of potassium ferrocyanide.

The uranium solution is prepared from uranium nitrate or acetate. Dissolve
about 35 grams uranium acetate in water, add some acetic acid to facilitate solu-
tion, and dilute to 1 liter. The strength of this solution is determined by titrat-
ing withasolution of sodium phosphate of known strength (10.085 grams crystallized
salt in 1 liter, which corresponds to 0.010 gram P205 in 50 cc.). Proceed in the
same way as in the titration of the urine (see below), and correct the solution by
diluting with water, and titrate again until 20 cc. of the uranium solution cor-
responds exactly to 50 cc. of the above phosphate solution.

The sodium-acetate solution should contain 10 grams sodium acetate and
10 grams cone, acetic acid in 100 cc. For each titration 5 cc. of this solution
is used with 50 cc. of the urine.

In performing the titration, mix 50 cc. of filtered urine in a beaker
with 5 cc. of the sodium acetate, cover the beaker with a watch-glass,
and warm over the water-bath. Then allow the uranium solution to
flow in from a burette, and when the precipitate does not seem to increase,
place a drop of the mixture on a porcelain plate with a drop of the potas-
sium-ferrocyanide solution. If the amount of uranium solution added
has not been sufficient, the color will remain pale yellow and more
uranium solution must be added; but as soon as the slightest excess of
uranium solution has been used the color becomes a faint reddish brown.
When this point has been obtained, warm the solution again and add
another drop. If the color remains of the same intensity, the titration
is ended; but if the color varies, add more uranium solution, drop by
drop, until a permanent coloration is obtained after warming, and now
repeat the test with another 50 cc. of the urine. The calculation is so
simple that it is unnecessary to give an example.

In the above manner one determines the total quantity of phosphoric
acid in the urine. If we wish to know the phosphoric acid combined
with alkaline earths and with alkalies, we first determine the total phos-
phoric acid in a portion of the urine and then remove the earthy phos-
phates in another portion by ammonia. The precipitate is collected on
a filter, washed, transferred into a beaker with water, treated with acetic
acid, and dissolved by warming. This solution is now diluted to
50 cc. with water, and 5 cc. sodium-acetate solution added, then
titrated with uranium solution. The difference between the two deter-
minations gives the quantity of phosphoric acid combined with the
alkalies. The results obtained are not quite accurate, as a partial trans-
formation of the monophosphates of the alkaline earths and also calcium
diphosphate into triphosphates of the alkaline earths and ammonium
phosphate takes place on precipitating with ammonia, and the method
gives too high results for the phosphoric acid combined with alkalies and
remaining in solution.

Sulphates. The sulphuric acid of the urine originates only to a very
small extent from the sulphates of the food. A disproportionately

SULPHATES. 765

greater part is formed by the burning within the body of the proteins
which contain sulphur, and it is chiefly this formation of sulphuric acid
from the proteins which gives rise to the previously mentioned excess
of acids over the bases in the urine. The quantity of sulphuric acid
eliminated by the urine amounts to about 2.5 grams H^SCU per day.
As the sulphuric acid chiefly originates from the proteins, it follows that
the elimination of sulphuric acid and the elimination of nitrogen runs
almost parallel, and the relation N:H2SO4 is about 5:1. A complete
parallelism can hardly be expected, as in the first place a part of the sul-
phur is always eliminated as neutral sulphur, and secondly because the
small proportion of sulphur in different protein bodies undergoes greater
variation as compared with the large proportion of nitrogen contained
therein. In general the elimination of nitrogen and sulphuric acid under
normal and under diseased conditions seems to run parallel. Sulphuric
acid occurs in the urine partly preformed (sulphate-sulphuric acid) and
partly as ethereal-sulphuric acid. The first is designated as A- and the
other as J3-sulphuric acid.

The quantity of total sulphuric acid is determined in the following
way, but at the same tin^e the precautions described in other works
must be observed. 100 cc. of filtered urine is treated with 5 cc. of con-
centrated hydrochloric acid and boiled for fifteen minutes. While
boiling precipitate with 2 cc. of a saturated BaC^ solution, and warm for
a little while until the barium sulphate has completely settled. The
precipitate must then be washed with water and also with alcohol and
ether (to remove resinous substances), and then treated according to
the usual method.

The separate determination of the sulphate-sulphuric acid and the
ethereal-sulphuric acid may be accomplished, according to BAUMANN'S
method, by first precipitating the sulphate-sulphuric acid by BaCb from
the urine acidified with acetic acid, then decomposing the ethereal-
sulphuric acid by boiling after the addition of hydrochloric acid, and
finally determining the sulphuric acid set free as barium sulphate. A
still better method is the following, suggested by SALKOWSKI l :

200 cc. of urine are precipitated by an equal volume of a barium solu-
tion, which consists of 2 vols. barium hydrate and 1 vol. barium chloride
solution, both saturated at the ordinary temperature. Filter through
a dry filter, measure off 100 cc. of the filtrate which contains only the
ethereal-sulphuric acid, treat with 10 cc. of hydrochloric acid of a specific
gravity 1.12, boil for fifteen minutes, and then warm on the water-bath
until the precipitate has completely settled and the supernatant liquid
is entirely clear. Filter and wash with warm water and with alcohol
and ether, and proceed according to the generally prescribed method.
The difference between the ethereal-sulphuric acid found and the total
quantity of sulphuric acid as determined in a special portion of urine
is taken to be the quantity of sulphate-sulphuric acid.

1 Baumann, Zeitschr. f. physiol. Chem., 1; Salkowski, Virchow's Aich., 79.

766 URINE.

FOLIN l has suggested a method for estimating the sulphate-sul-
phuric acid as well as the ethereal-sulphuric acid, and also the total
sulphur, which is somewhat different from the ordinary methods.

Nitrates occur in small quantities in human urine (SCHONBEIN), and they
probably originate from the drinking-water and the food. According to WEYL
and CITRON, 2 the quantity of nitrates is smallest with a meat diet and greatest
with vegetable food. The average amount is about 42.5 milligrams per liter.

Potassium and Sodium. The quantity of these bodies eliminated
by the urine by a healthy adult on a mixed diet is, according to SALKOW-
SKi,3 3-4 grams K2O and 5-8 grams Na20, with an average of about 2-3
grams K2O and 4-6 grams Na2O. The proportion of K to Na is ordinarily
3:5. The quantity depends above all upon the food. In starvation
the urine may become richer in potassium than in sodium, which results
from the lack of common salt and the destruction of tissue rich in potas-
sium. The quantity of potassium may be relatively increased during
fever, while after the crisis the reverse is the case.

The quantitative estimation of these bodies is made by the gravi-
metric methods as described in works on quantitative analysis. In
the ' determination of the total alkalies new methods have been devised
by PRIBRAM and GREGOR, and for the potassium alone a method by

AUTENRIETH and BERNHEIM.4

Ammonia. Some ammonia is habitually found in human urine and
in that of carnivora. The quantity in human urine on a mixed diet is
an average of 0.7 gram, according to NEUBAUER. MAILLARD 5 found
higher values for soldiers, namely 1.11 grams. The ammonia nitrogen
relative to the total nitrogen is, on a mixed diet, 3.6-5.8 per cent.

As above stated (page 685), on the formation of urea from ammonia,
this quantity may represent the small amount of ammonia which is
excluded from the synthesis to urea by being combined with acids formed
in excess by combustion and not united with the fixed alkalies. This
view is confirmed by the observation that the elimination of ammonia was
smaller on a vegetable diet and larger on a rich meat diet than on a mixed
diet. After abundant meat feeding BOUCHEZ found, for example, 1.35-
1.67 gram NH? in twenty-four hours. The relationship of the ammonia
elimination to the acid formation in the animal body corresponds also
to the unquestioned relation between the hydrochloric acid content of the

1 Journ. of Biol. Chem., 1, and Amer. Journ. of Physiol., 13.

8 Schonbein, Journ. f. prakt. Chem., 92; Weyl, Virchow's Arch., 96, with Citron,
ibid., 101.

» Ibid., S3.

4 Pribram and Gregor, Zeitschr. f. analyt. Chem., 38; Autenreith and Bernheim,
Zeitschr. f. physiol. Chem., 37.

6 Journ. de Physiol. et de Path., 10.

AMMONIA. 767

gastric juice and the ammonia elimination. Thus SCHITTENHELM found
that with a rise in the hydrochloric acid content the percentage of ammonia
in the urine was raised and also the reverse. A. LOEB and GAMMELTOFT l
have also observed a fall in the ammonia elimination a few hours after a
meal, although no satisfactory explanation of this behavior has been given.
That ammonia plays the role of a neutralization medium for the acids
produced in the body or introduced therein has been shown by various
observations.

In man and certain animals the elimination of ammonia is increased
by the introduction of mineral acids; and, as shown by JoLiN,2 organic
acids, such as benzoic acid, which are not destroyed in the body act in a
similar manner. The ammonia set free in the protein destruction is in
part used in the neutralization of the acids introduced, and in this way
a destructive removal of fixed alkalies is prevented.

Acids formed in the destruction of proteins in the body act on the
elimination of ammonia like those introduced from without. For this
reason the quantity of ammonia in human urine is increased under such
conditions and in such diseases where an increased formation of acid
takes place, because of an increased metabolism of proteins. This is the
case with a lack of oxygen in fevers and diabetes. In the last-mentioned
disease, organic acids — /3-oxybutyric acid and acetoacetic acid — are pro-
duced, which pass into the urine combined with ammonia.3

The liver forms urea from the ammonia supplied to it by the blood
and it would therefore be expected that in certain diseases of the liver
or with insufficient liver function that a diminished urea formation and an
increased ammonia elimination should take place. This condition has
already been mentioned above (page 685), and as there remarked we
must consider whether the abnormal production of acid with increased
elimination of neutralization ammonia is primary or whether it is a
diminished synthetic activity of the liver.

In close relation to what has been said stands the question whether all
of the ammonia occurring in the urine under normal conditions is to be
considered as neutralization ammonia. If this were so then probably
by introducing large amounts of alkali it would be possible to cause the
disappearance of ammonia from the urine. In STADELMANN and BECK-

1 Bouchez, Journ. de Physiol. et de Path., 14; Schittenhelm, Deutsch. Archiv. f.
klin. Med., 77; Adam Loeb, Zeitschr. f. klin. Med., 56, and Zeitschr. f. Biol., 55; Gam-
meltoft, Zeitschr. f. physiol. Chem., 75.

2 Jolin, Skand. Arch. f. Physiol., 1. In regard to the behavior of ammonium salts
in the animal body, see Rumpf and Kleine, Zeitschr. f. Biologic, 84; Kowalewski and
Markewicz, Bioch. Zeitschr., 4, and the works cited on pages 682, 683.

3 On the elimination of ammonia in disease, see the works of Rumpf, Vir chow's
Arch., 143; Hallervorden, ibid. .

768 URINE.

MANN'S experiments this was not possible, still in recent experiments of
JANNEY 1 it was possible, by introducing large quantities of sodium citrate,
which was burned in the body into carbonate, to reduce the ammonia
elimination to very insignificant quantities.

The detection and quantitative estimation of ammonia used to be performed
according to the method suggested by SCHLOSING. The principle of this method
is that the ammonia from a measured amount of urine is set free by lime-water
in a closed vessel and absorbed by a measured amount of N/10 sulphuric acid.
After the absorption of the ammonia the quantity is determined by titrating the
remaining free sulphuric acid with a N/10 caustic-alkali solution. This method
gives low results, and in exact work we must proceed as suggested by BORLAND. 2

The recent methods for estimating the ammonia are all based upon
the distillation of the ammonia, after the addition of lime, magnesia,
or alkali carbonate, at low temperatures either by the aid of vacuum
(NENCKI and ZALESKI, WURSTER, KRUGER, REICH and SCHITTENHELM
and SCHAFFER) or by the aid of a current of air (FOLIN) and then collect-
ing it in a standard acid.

According to the methods suggested by KRUGER, REICH and SCHITTEN-
HELM 3 25 cc. of the urine are placed in a distillation-flask with about
10 grams of NaCl and 1 gram of Na2COs, and this distilled at 43° C.
and a pressure of 30-40 millimeters Hg with the aid of an air-pump.
Alcohol is added to prevent foaming. The ammonia is absorbed in
N/10 acid contained in a PELIGOT tube surrounded by ice- water, and
when the distillation is finished the acid is retitrated, making use of
rosolic acid as indicator. In regard to details, see the original publica-
tions. Instead of alkali carbonate a one-half normal solution of barium
hydrate in methyl alcohol can be used. According to FOLIN'S 4 method,
25-50 cc. of the urine are treated in a wash-bottle with 1-2 grams soda and
8-10 grams sodium chloride and some petroleum, in order to prevent
frothing, and then a current of air is passed through and this passed
through a second . wash-bottle containing N/10 acid. It has also been
suggested (RONCHESE, MALFATTI and others) to determine the ammonia
by the formol titration. This method is based upon the fact that an
ammonium salt yields hexamethylentetramine and free acid with for-
maldehyde according to the equation 4NH4Cl+6HCOH = C6Hi2N4
-J-6H2O+4HC1. This acid is determined by titration after the addition
of formol. FOLIN 5 also recently suggested a method for the quantitative
colorimetric estimation of ammonia by the use of NESSLER'S reagent.

Calcium and Magnesium occur in the urine chiefly as phosphates.
The quantity of earthy phosphates eliminated daily is somewhat more

1 N. Janney, Zeitschr. f. physiol. Chem., 76, which also contains the literature.

2 Pfluger's Arch., 43, 32.

3 Zeitschr. f. physiol. Chem., 39; Schaffer, Amer. Journ. of Physiol., 8, which contains
the literature. Henriques and S6rensen, Zeitschr. f. physiol. Chem., 64.

4 Folin, Zeitschr. f. physiol. Chem., 37, and Journ. of biol. Chem., 8; Steel, ibid., 8.
6Ronchese, see Maly's Jahresber., 38, 321; Malfatti, Zeitschr. f. anal. Chem., 47;

H. Bjorn-Andersen and M. Lauritzen, Zeitschr. f. physiol. Chem., 64; L. de Jager,
ibid., 62; Folin and Maccallum, Journ. of biol. Chem. ,.11.

CALCIUM AND MAGNESIUM. 769

than 1 gram, and of this amount f is magnesium and J calcium phos-
phate. This statement, as found by RENWALL and GROSS, is not correct,
or at least is not true in general, as they found more calcium than mag-
nesium in the urine. LONG and GEPHART l obtained similar results.
In acid urines the mono- as well as the dihydrogen earthy phosphates
are found, and the solubility of the first, among which the calcium salt
CaHPO4 is especially insoluble, is particularly augmented by the presence
in the urine of dihydrogen alkali phosphates and sodium chloride (OTT 2) .
The quantity of alkaline earths in the urine depends on the composi-
tion of the food. The lime-salts absorbed are in great part excreted
again into the intestine, and the quantity of lime-salts in the urine is
therefore no measure of their absorption. The introduction of readily
soluble lime-salts or the addition of hydrochloric acid to the food may
therefore cause an increase in the quantity of lime in the urine, while
the reverse takes place on adding alkali phosphate to the food. Accord-
ing to GRANSTROM starvation in rabbits or the introduction of food which
yields an acid ash and causes an acid urine produces the same effect as
the introduction of acid. The observation of DE JAGERS is significant,
namely, he found that the partaking of CaSCU and to a higher degree
of MgSO4 causes an increase in the urine ammonia and of acid. Noth-
ing is known with certainty in regard to the constant and regular change
in the elimination of calcium and magnesium salts in disease, and in these
conditions the excretion is chiefly dependent upon the diet and the forma-
tion and introduction of acid.4

The quantity of calcium and magnesium is determined according to
the ordinary well-known methods.

Iron occurs in the urine only in small quantities, and it does not exist as a
salt, but as an organic combination of a colloidal nature. The earlier reports in
regard to the iron present seem to show that the quantity ranges from 1 to 11
milligrams per liter of urine. HOFFMANN, NEUMANN and MAYER found lower
results — an average of 1.09 and 0.983 milligrams and according to recent determi-
nations of WOLTER and REICH 5 the quantity is about 1 milligram. The quantity
of silicic acid is ordinarily stated to amount to about 0.3 p. m. H. SCHULZ 6 found

1 Renwall, Skand. Arch. f. Physiol., 16; Gross, Biochem. Ccntralbl., 4, 189; Long
and Gephart, Journ. Amer. Chem. Soc., 34.

2 Zeitschr. f. physiol. Chem., 10.

8 Granstrom, Zeitschr. f. physiol. Chem., 58; de Jager, Bioch. Zeitschr., 38.

4 See page 758, Albu and Neuberg, 1. c., and E. Zak, Ueber Harn bei Lungenent-
ziindung, Wien. klin. Wochenschr., 21. '

6Kunkel, cited from Maly's Jahresber., 11; Giacosa, ibid., 16; Robert, Arbeiten
des Pharm. Inst. zu Dorpat, 7; Magnier, Ber. d. deutsch. chem. Gesellsch., 7; Gott-
lieb, Arch. f. exp. Path. u. Pharm., 26; Jolles, Zeitschr. f. anal. Chem., 36; Hoff-
mann, Zeitschr. f. anal. Chem., 40; Neumann and Mayer, Zeitschr. f. physiol.
Chem., 37; Wolter, Bioch. Zeitschr., 24; Reich, ibid., 36.

8 Pfluger's Arch., 144.

770 URINE.

0.1046 to 0.2594 grams per day on a mixed diet. Traces of hydrogen peroxide
also occur in the urine.

The gases of the urine are carbon dioxide, nitrogen, and traces of
oxygen. The quantity of nitrogen is not quite 1 vol. per cent. The
carbon dioxide varies considerably. In acid urines it is hardly one-half
as great as in neutral or alkaline urines.

IV. THE QUANTITY AND QUANTITATIVE COMPOSITION OF URINE.

The quantity and composition of urine are liable to great variation.
The circumstances which under physiological conditions exercise a great
influence are the following: the blood-pressure, and the rapidity of the
blood-current in the glomeruli. The quantity of urinary constituents,
especially water in the blood; and, lastly, the condition of the secretory
glandular elements. Above all, the quantity and concentration of the
urine depend on the quantity of water which is introduced into the blood
or which leaves the body in other ways. The excretion of urine is increased
by drinking freely or by reducing the quantity of water otherwise removed ;
and it is decreased by a diminished ingestion of water or by a greater loss
of water in other ways. Ordinarily in man just as much water is elimi-
nated by the kidneys as by the skin, lungs, and intestine together. At
lower temperatures and in moist air, since under these conditions the
elimination of water by the skin is diminished, the excretion of urine
may be considerably increased. Diminished introduction of water or
increased elimination of water by other means — as in violent diarrhoea or
vomiting, or in profuse perspiration — greatly diminishes the amount of
urine excreted. For example, the urine may sink as low as 500-400 cc.
per day in intense summer heat, while after copious draughts of water
the elimination of 3000 cc. of urine has been observed during the same
time. The quantity of urine voided in the course of twenty-four hours
varies considerably from day to day, the average being ordinarly cal-
culated as 1500 cc. for healthy adult men and 1200 cc. for women. The
minimum elimination occurs during the early morning between 2 and 4
o'clock; the maximum, in the first hours after waking and from 1-2
hours after a meal.

The quantity of solids excreted per day is nearly constant, even though the
quantity of urine may vary, and it is quite constant when the manner of living
is regular. Therefore the percentage of solids in the urine is naturally in inverse
proportion to the quantity of urine. The average amount of solids per twenty-
four hours is calculated as 60 grams. The quantity may be calculated with approx-
imate accuracy from the specific gravity if the second and third decimals of this
factor be multiplied by HASER'S coefficient, 2.33. The product gives the amount
of solids in 1000 cc. of urine, and if the quantity of urine eliminated in twenty-
four hours be measured, the quantity of solids in twenty-four hours may be
easily calculated. For example, 1050 cc. of urine of a specific gravity 1.021 was

QUANTITATIVE COMPOSITION. 771

eliminated in twenty-four hours; therefore the quantity of solids excreted was

48 9 X 1050

21 X 2.33 =48.9 and — "Tonn — =51.35 grams. LONG l has made a new determina-
tion of the coefficient for the specific gravity taken at 25° C. and finds that it is
equal to 2.6, which almost corresponds to HASER'S coefficient at 15° C.

Those bodies which, under physiological conditions, affect the density
of the urine are common salt and urea. The specific gravity of the first
is 2.15 and the last only 1.32, so it is easy to understand, when the relative
proportion of these two bodies essentially deviates from the normal,
why the above calculation from the specific gravity is not exact. The
same is true when a urine poor in normal constituents contains large
amounts of foreign bodies, such as albumin or sugar.

As above stated, the percentage of solids in the urine generally decreases
with a greater elimination, and a very considerable excretion of urine
(polyuria) has therefore, as a rule, a lower specific gravity. An important
exception to this rule is observed in urine containing sugar (diabetes
mellitus), in which there is a copious excretion with a very high specific
gravity due to the sugar. In cases where very little urine is excreted
(oliguria), e.g., during profuse perspiration, in diarrhoea, and in fevers,
the specific gravity of the urine is as a rule very high; the percentage of
solids is also high and the urine has a dark color. Sometimes, as for
example, in certain cases of albuminuria, the urine may have a low specific
gravity notwithstanding the oliguria, and be poor in solids and light in
color. i

In certain cases it is interesting to know the relation between the
carbon and the nitrogen, or the quotient C/N. This factor may vary
between 0.6 and 1 ; as a rule, it amounts on an average to 0.87, but changes
according to the nature of the food and is higher after a diet rich in carbo-
hydrates than after food rich in fat (PREGL, TANGL, LANGSTEIN and
STEINITZ). According to MAGNUS- ALSLEBEN it rises after body exer-
tion, but in healthy individuals the variation is independent of the
kind of food. In the urine analyses of BOUCHEZ 2 a variation between
0.62 and 0.90 was observed which showed no regular relation to the food.

On account of the great variations which the composition of the urine
shows it is difficult and of little value to give a tabular review of the
composition of the urine. The following table contains only approximate
values and it must not be overlooked that the results are not given for
1000 parts of urine, but only approximate figures for the quantities

1 Journ. Amer. Chem. Soc., 25.

2 Pregl, Pfliiger's Arch., 75, which contains the earlier literature. Tangl, Arch. f.
(Anat. u.) Physiol., 1899, Suppl.; Langstein and Steinitz, Centralbl. f. Physiol., 19;
Magnus-Alsleben, Zeitschr. f. klin. Med., 68, Bouchez, footnote 1, page 767.

772 URINE.

of the most important constituents which are eliminated during the
course of twenty-four hours in a volume of 1500 cc. of urine. These
figures apply only to a diet which corresponds to VOIT'S standard figures,
namely 118 grams protein, 56 grams fat, and 500 grams carbohydrate
per day, and to a man of average weight.

Daily quantity of solids = 55-70 grams.

Organic constituents 35-45 grams. Inorganic constituents 20-25 grams.

Urea 25-35.0 grams. Sodium chloride (NaCl) . 10-15.0 grams.

Uric acid 0.7 ' ' Sulphuric acid (H2SO4). . 2.5 ' '

Creatinine 1.5 ' * Phosphoric acid (P2O5) . . 2.5 "

Hippuric acid 0.7 " Potash (K2O) 3.3 "

Ammonia (NH3) 0.7 "

Magnesia (MgO) \ OR „

Lime (CaO) / ' ' ' ' U'b

Urine contains on an average 40 p. m. solids. The quantity of urea is
about 20 p. m., and common salt about 10 p. m.

The physico-chemical methods are being used in urinary analysis even to a
greater extent than in the analysis of other animal fluids. A great number
of cryoscopic determinations, but fewer conductivity determinations, have been
made. A constant relation between the values found by physico-chemical methods
and the analytical methods has been sought, for example, between the freezing-
point depression and the specific gravity or the common salt content and others; or
have been made to find certain constants in the composition of the urine based
upon the results of various methods, and in this way to obtain an explanation
as to the mechanism of the excretion of urine in order to apply them for diag-
nostic purposes. The results obtained are, as is to be expected, so variable and
dependent upon so many conditions which cannot be controlled that definite
conclusions must be drawn with the greatest caution. In regard to the value and
usefulness of the various constants and relations which are based upon theoretical
considerations, opinions are unfortunately still too divergent and as the plan and
scope of this book do not allow of more detailed description of these facts we
must refer to larger works on the urine and diseases of the kidneys.

V. CASUAL URINARY CONSTITUENTS.

The casual appearance, in the urine, of medicinal agents or of urinary
constituents resulting from the introduction of foreign substances into
the organism is of practical importance, because such compounds may
interfere in certain urinary investigations; they also afford a good means
of determining whether certain substances have been introduced into the
organism or not. From this point of view a few of these bodies will be
spoken of in a following section (on the pathological urinary constituents).
The presence of these foreign bodies, in the urine, is of special interest
in those cases in which they serve to elucidate the chemical transformations
which certain substances undergo within the organism. As inorganic
substances generally leave the body unchanged,1 they are of very little

1 In regard to the behavior of certain of these bodies, see Heffter, Die Ausscheidung
korperfremden Substanzen im Ham, Ergebnisse d. Physiol., 2, Abt. 1.

CASUAL URINARY CONSTITUENTS. 773

interest from this standpoint; but the changes which certain organic
substances undergo when introduced into the animal body may be studied
by the transformation products as found in the urine.

The bodies belonging to the fatty series undergo, though not without
exceptions, a combustion leading toward the final products of metab-
olism; still, often a greater or smaller part of the bodies in question
escape oxidation and appear unchanged in the urine. A part of the acids
belonging to this series, which are otherwise decomposed into water and
carbonates, and render the urine neutral or alkaline, may act in this manner.
The volatile fatty acids poor in carbon are less easily oxidized than those
rich in carbon, and they therefore pass unchanged into the urine in
large amounts. This is especially true of formic and acetic acids
(SCHOTTEN, GREHANT and QUINQUAUD 1). In birds, according to GAGLIO
and GIUNTI, oxalic acid is not oxidized. Opinions on the behavior of
oxalic acid in mammalia and man, are conflicting; the investigations
of SALKOWSKI and especially of HILDEBRANDT and DAKIN 2 show
that oxalic acid, when introduced in medium amounts, is in part
oxidized in the animal body. Racemic acid, d-l tartaric acid, passes
(in dogs) in part into the urine, and this unburned part is optically inactive
according to NEUBERG and SANEYOSHI. The statement of BraoN3
that Z-tartaric acid is more readily burned than d-tartaric acid is accord-
ingly incorrect, and the d-/-tartaric acid therefore does not belong to
those substances which are asymmetrically attacked in the animal
body. Malic acid and citric acid belong to those acids which are in great
part burned in the body.4

The destruction of normal fatty acids with several membered chains
takes place, our belief being based upon the work of KNOOP and DAKIN 5
especially, in an oxidation in the /3-position, i.e.', in the group which is in
the /3-position to the carboxyl group at the end. The conversion into an

1Schotten, Zeitschr. f. physiol. Chem., 7; Grehant and Quinquaud, Compt. Rend.,
104.

2Gaglio, Arch. f. exp. Path. u. Pharm., 22; Giunti, Chem. Centralbl., 1897, 2;
Marfori, Maly's Jahresber., 20 and 27; Pohl, Arch. f. exp. Path. u. Pharm., 37; Sal-
kowski, Berl. klin. Wochenschr., 1900; Pierallini, Virchow's Arch., 160; Stradomsky,
ibid. ,163; Klemperer and Tritschler, Zeitschr. f. klin. Med., 44; Hildebrandt, Zeitschr.
f. physiol. Chem., 35; Dakin, Journ. of biol. Chem., 3.

3 Biron, Zeitachr. f. physiol. Chem., 25; Neuberg and Saneyoshi, Bioch. Zeitschr.,
36. O

4 Pohl, Arch. f. exp. Path. u. Pharm., 37, which also contains reports on the inter-
mediary products formed in the oxidation of the fatty bodies; K. Ohta, Bioch.
Zeitschr., 44.

5 F. Knoop, Hofmeister's Beitrage, 6 and Habilit.-Schrift, Freiburg, 1904; Dakin,
Journ. of biol. Chem., 4, 5/6 and 9.

774 URINE.

acid having two carbon atoms less takes place according to this assump-
tion according to the formula:

R.CH2.CH2.COOH-^R.CH(OH).CH2.COOHR.CO.CH2.COO^H->

R.COOH.

The animal body has therefore the ability to transform oxyacids (alcohol
acids) into keto-acids by oxidation as well as the reverse, the conversion
of keto-acids into oxyacids, and this behavior, which is indicated by the
above formula, makes it difficult to state which products are primary
and which are secondary. As example of such a reversible process we
will mention the following; the 0-oxybutyric acid CH3.CH(OH) .CH2COOH
is transformed by oxidation into the keto-acid, acetoacetic acid,
CH3.CO.CH2COOH, and this latter by reduction is changed into /3-
oxybutyric acid. Both processes may take place, as FRIEDMANN and
MAASE, DAKIN and WAKEMAN l have shown, in the liver, and as these
two so-called acetone bodies have great importance in diabetes, they
may serve also as an example of the first stages of a /?-oxidation (of
n. butyric acid).

Most of the investigations on the demolition of fatty acids have been
carried out by KNOOP, DAKIN, FRTEDMANN and others upon substituted,
especially phenyl-substituted fatty acids, and in speaking of the behavior
of the cyclic compounds we will discuss the behavior of these.

The amino-acids are, when large amounts are introduced into the
animal body, eliminated unchanged, and even under physiological con-
ditions traces of the amino-acids formed in the animal body can pass
into the excretions — glycocoll in the urine and serine in the perspiration.
Otherwise they are as a rule decomposed and a deamidation takes place,
the ammonia split off serving for material for the formation of urea.
The two components of a racemic a-amino-acid behave differently in that
the alien component is burned with greater difficulty and less completely
than the component occurring in the body protein, which is burned
more readily and more completely.

In the demolition of the a-amino-acids, fatty acids, poorer in carbon,
are formed; the detailed manner of this demolition has been explained
in various ways.

According to a long-accepted view it was believed that a hydrolytic
splitting off of ammonia with the formation of the corresponding oxyacid
(alcohol acid) took place, according to the formula R.CHNH2.COOH+
H2O = R.CH(OH).COOH+NH3, and then a further demolition to

1 Friedmann and Maase, Bioch. Zeitschr., 27; Dakin and Wakeman, Journ. of biol.
Chem., 8.

CASUAL UKINARY CONSTITUENTS. 775

R.COOH. The appearance of lactic acid in the urine of rabbits after
feeding alanine is an example of such deamidation.1 The possibility
is not excluded that in the first place the keto-acid, pyruvic acid,
CH3.CO.COOH, is formed from the alanine and then the lactic acid,
CHs.CHOH.COOH, formed from this as a secondary reduction product.

In agreement with the views of NEUBAUER2 it is now rather
generally conceded that the hydrolytic deamidation is not as important
as the oxidative deamidation, with the formation of keto-acids
R.CH(NH2).COOH+O = R.CO.COOH+NH3, although this is not the
only possibility. The proofs for the correctness of this view have been
obtained essentially by experiments with aromatic amino-acids and
will be given as examples of such deamidation.

DAKIN and DUNDLEY 3 have shown that all a-amino-acids investi-
gated by them can be decomposed under special conditions so that they
to a certain degree yield ammonia and an a-keto-aldehyde.

R.CH.NH2.COOH^R.CO.CHO+NH3.

Thus, with alanine, and as the reaction to all appearances is reversible,
they consider the relationship between alanine and lactic acid is as follows :

CHs.CH.NHa.COOH^CHa.CO.CHO^CHsCHOH.COOH.

They also found it probable, that the a-keto-aldehydes represent the
first step in the demolition of the a-amino-acids whereby the regular
demolition of these acids takes place over the a-keto-acids and not over
oxyacids, which explains also the formation of sugar from certain amino-
acids (over methylglyoxal as intermediary step).

The deamidation after previous oxidation with the formation of
keto-acids has awakened special interest because recently in perfusion
experiments on dog-livers the reverse process, namely a synthesis of amino-
acids from keto-acids (in part also from oxyacids) and ammonia has been
performed (KNOOP, EMBDEN and SCHMITZ, KoNDO4). Among such
syntheses we can here call attention to the synthesis in the dog-liver
of alanine, phenylalanine and tyrosine from pyruvic acid (also lactic acid),
phenylpyruvic acid and p-oxyphenyl pyruvic acid, or of a-amino-n-butyric
acid from a-keto-butyric acid (all as ammonium salts).

1 See Langstein and Neuberg, Arch. f. (Anat. u.) Physiol., 1903. Suppl. Bd.
« Deutsch. Arch. f. klin. Med., 95, and Habilit. Schrift., Leipzig, 1908. See also
further on in regard to the literature on the demolition of the aromatic amino-acids.

3 Journ. of biol. Chem., 14. *

4 Knoop, Zeitschr. f. physiol. Chem., 67 and 71; Embden and Schmitz, Bioch.
Zeitschr., 29 and 38; Kondo, ibid., 38.

776 URINE.

The residue of the amino-acids remaining after deamidation can
naturally, according to the rule governing the fatty acids, be burned
and in certain cases this combustion takes place with the formation of
acetone bodies (which see). The fatty acid residue can also be used, be-
sides in the synthesis of amino-acids, also in the synthesis of other
substances, and in Chapter VII the formation of carbohydrates from
amino-acids has been mentioned.

Among the amino-acids the cystine, or better the cysteine,

CH2.(SH).CH(NH2).COOH,

show a special behavior. On oxidation in the SH group and splitting
off of CO2 (see page 149) it is transformed into a new amino-acid, taurine
(H2N)CH2.CH2(S02OH) . Taurine,iwhich when conjugated with cholic acid
forms taurocholic acid, occurring in the bile and which is habitually decom-
posed in the intestine or other parts of the animal body, can when intro-
duced as such into the human body, at least in part, be eliminated in
the urine as such or as tauro-carbamic acid, H2N.CO.NH.C2H4.SO20H
(SALKOWSKI l). Otherwise as end-products of the demolition of cystine
and taurine an increased elimination of urinary sulphur, sulphuric acid and
thiosulphate, have been observed (BLUM, ABDERHALDEN and SAMUELY2).
The sulphydryl group of cysteine also serves in the formation of sulpho-
cyanide, which is formed from the nitriles, introduced into the animal
body, by the HCN (LANG). The loosely combined sulphur of the pro-
teins, according to the observations of PASCHELES, in alkaline reaction and
body temperature, can be readily transformed, with the cyan alkali into
sulphocyanide alkali. The alkali sulphocyanides when ingested are
almost quantitatively eliminated in the urine, according to POLLAK.S

By substituting one of the hydrogen atoms in the NH2 group of
normal a-amino-acids by an alkyl radical (methyl) the combustion of the
acids of the series C2 and C4 is considerably retarded and almost entirely
prevented in the members of the €5 and CG series (FRIEDMANN) .4 Sar-
cosine (methyl glycocoll), (CH3)NH.CH2.COOH, is not readily burnt, and
therefore passes in great part unchanged into the urine, but perhaps also
passes in small part into the corresponding uramino-acid, methylhydan-
toic add, NH2.CO.N(CH3).CH2.COOH (SCHULTZEN 5), is an example

1 Ber. d. d. Chem. Gesellsch., 6, and Virchow's Arch., 58.

2 Blum, Hofmeister's Beitrage, 5; Abderhalden and Samuely, Zeitschr. f. physiol.
Chem., 46.

3 Lang, Arch. f. exp. Path. u. Pharm., 34; Pascheles, ibid.} Pollak, Hofmeister's
Beitrage, 2.

4 Hofmeister's Beitrage, 11.

6 Ber. d. d. Chem. Gesellsch., 5. See also Baumann and v. Mering, ibid., 8, and
E. Salkowski, Zeitschr. f. physiol. Chem., 4.

CONJUGATION WITH SULPHURIC AND GLUCURONIC ACIDS. 777

of this kind. Substitution of both hydrogen atoms of the amino-group
by methyl groups seems to make the demolition of the amino-acids still
more difficult (FRIEDMANN). Ordinary betaine (trimethyl glycocoll)
passes, according to KOHLRAUCH/ in part unburned into the urine in
carnivora as well as herbivora.

The combustion of the aliphatic bodies can be retarded or prevented
also by substitutions of other kinds and by combining with other sub-
stances.

By substitution with halogens, bodies otherwise readily oxidizable are
converted into difficultly oxidizable ones. While the aldehydes are readily
and completely burnt like the primary and secondary alcohols of the
fatty series, the halogen-substituted aldehydes and alcohols, are, on the
contrary, difficultly oxidizable. The halogen-substitution products of
methane (chloroform, iodoform, and bromoform) are at least in part
destroyed, and the corresponding alkali compounds of the halogen pass
into the urine.2

By conjugation with sulphuric acid, the alcohols which are otherwise
readily oxidizable may be protected against combustion, and conse-
quently the alkali salt of ethyl-sulphuric acid is not burnt in the body

(SALKOWSKI3).

Conjugation with other substances can prevent the combustion of
the aliphatic bodies as shown in the conjugation of glycocoll with benzoic
acid into hippuric acid. A conjugation can also be a mutual protection
against the combustion of two bodies as in the case of glucuronic acid
and certain substances,

Conjugation with glucuronic acid occurs, according to the investiga-
tions of SUNDVIK and especially of 0. NEUBAUER, in many substituted
as well as non-substitued alcohols, aldehydes, and ketones. Chloral
hydrate, CCl3CH(OH)2, passes, after it has been converted into tri-
chlorethyl-alcohol by a reduction, into a levogyrate reducing acid, uro-
chloralic acid or trichlorethylglucuronic acid, CCla.C^.CeHgO? (MUSCULUS
and v. MERINO). Of the primary alcohols investigated by NEUBAUER 4
(upon rabbits and dogs) methyl alcohol gave no conjugated glucuronic
acid, and ethyl alcohol only a small amount. Isobutyl alcohol and active

1 Zeitschr. f. Biol., 57.

2 See Harnack and Griindler, Berlin, klin. Wochenschr., 1883; teller, Zeitschr. f.
physiol. Chem., 8; Kast, ibid., 11; Binz, Arch. f. exp. Path. u. Pharm., 28; Zeehuisen,
Maly's Jahresber., 23.

3 Pfliiger's Arch., 4.

4 Sundvik, Maly's Jahresber., 16; Musculus and V. Mering, Ber. d. deutsch. chem.
Gesellsch., 8; also v. Mering, ibid., 15; Zeitschr. f. physiol. Chem., 6; Kiilz, Pfliiger's
Arch., 28 and 33; O. Neubauer, Arch. f. exp. Path. u. Pharm., 46; Saneyoshi, Bioch.
Zeitschr., 36.

778 URINE.

amyl alcohol yielded relatively large quantities. Secondary alcohols
produced conjugated glucuronic acids, and indeed to a greater extent than
the primary alcohols, especially in rabbits. The ketones are reduced in
part into secondary alcohols and are partly excreted as the conjugated
acid. This could be shown for acetone with rabbits but not with dogs.

The homo- and heterocyclic compounds pass, as far as is known,
into the urine as such, or after a previous partial oxidation or synthesis
with other bodies, and appear as so-called aromatic compounds. This
applies at least to foreign substances that are introduced into the body.

The fact that benzene may be oxidized outside of the body into carbon
dioxide, oxalic acid, and volatile fatty acids has been known for a long
time; and as in these cases a rupture of the benzene ring must take place,
so also, it must be admitted, when aromatic substances undergo a com-
bustion in the animal body, a splitting of the benzene nucleus with the
formation of fatty bodies must be the result. If this does not occur, then
the benzene nucleus is eliminated with the urine as an aromatic compound
of one kind or another. The manner in which this benzene ring is opened
is not known. Still JAFF£ l has detected muconic acid in the urine of
dogs and rabbits which had been fed for a long time with benzene, and
suggest one way in which the benzene can be split in the animal body.

CH CH

HCX CH HCX COOH

| 1 1 — » | . That the demolition of the benzene nucleus

HC CH HC COOH

CH CH

occurs in certain cases, as in tyrosine and phenylalanine according to
the present view, over homogentisic acid, has already been mentioned.
The most striking example of a complete combustion of the benzene
nucleus is given by tyrosine, which as previously mentioned (page 737)
can be absorbed even in large quantities and decomposed without the
observer being able to detect any of the cleavage products of it in the urine.
Other examples of readily and at least in greatest part combustible aroma-
tic substances are phenyl-a-lactic acid, p-oxyphenylpyruvic acid and a-amino
cinnamic acid. According to JUVALTA and PORCHER phthalic acid is
also burnt in the animal body. The last investigator found that the three
phthalic acids have varying effects, as the o-acid is almost completely
burnt in dogs, while about 75 per cent of the m- and p-acids are excreted
unconsumed. This corresponds with the rule found by R. CoHN,2 that
among the di-derivatives of benzene the ortho-compounds are more

1 Zeitschr. f. physiol. Chem., 62.
s Ibid., 17.

OXIDATION IN THE NUCLEUS AND SIDE CHAIN. 779

readily destroyed than the corresponding meta- and para-compounds.
The claims of JUVALTA and PORCHER are unfortunately disputed by
PRIBRAM and PoHL.1

An oxidation in the side chain of aromatic compounds is often found,
and may also occur in the nucleus itself. As an example, benzene is first
oxidized to oxybenzene (SCHULTZEN and NAUNYN), and this is then
further in part oxidized into dioxybenzenes (BAUMANN and PREUSSE).
Naphthalene appears to be converted into oxy naphthalene, and probably
a part also into dioxynaphthalene (LESNIK and M. NENCKI). The hydro-
carbon with an amino- or imino-group may also be oxidized by a sub-
stitution of hydroxyl for hydrogen, especially when the formation of a
derivative in the para-position is possible (KLINGENBERG). For example,'
aniline, Cells. NH2, passes into paraminophenol, which latter passes into
the urine as its ethereal-sulphuric acid, H^N.CeH^O.SC^.OH (F. MULLER).'
Acetanilid is in part converted into acetyl paraminophenol (JAFFE and
HILBERT, K. MORNER), and carbazol into oxycarbazol (KLINGENBERG) ?

An oxidation of the side chain may occur by the hydrogen atoms being
replaced by hydroxyl, or may also take place with the formation of carboxyl;
thus, for example, toluene, CeHs.CHs (SCHULTZEN and NAUNYN), ethyl-
benzene, CeHs^Hs, and propylbenzene, CeH^CaHr (NENCKI and GiAco&A)3
besides many other bodies, are oxidized into benzoic acid. Cymene is
oxidized to cumic acid, xylene to toluic acid, methylpyridine to pyridine-
carboxylic acid in the same way.

If several side chains are present in the benzene nucleus, then only one
is always oxidized into carboxyl. Thus xylene, CeH^CHs^, is oxidized
into toluic acid, C6H4(CH3)COOH (SCHULTZEN and NAUNYN); mesitylene,
C6H3(CH3)3, into mesitylenic acid, C6H3(CH3)2.COOH (L. NENCKI);
cymene, (CH3)2CH.C6H4.CH3, into cumic acid, (CH3)CH.C6H4.COOH
(M. NENCKI and ZIEGLER 4).

If the side-chain has several members, then the behavior is dif-
ferent and in these cases the demolition of aromatic amino-acids and
fatty acids is especially to be considered.

1 Juvalta, Zeitschr. f. physiol. Chem., 13; Pribram, Arch. f. exp. Path. u. Pharm.,
51; Porcher, Bioch. Zeitschr., 14; Pohl, ibid., 16.

2 Schultzen and Naunyn, Arch. f. (Anat. u.) Physiol., 1867; Baumann and Preusse,
Zeitschr. f. physiol. Chem., 3, 156. See also Nencki and Giacosa, ibid., 4; Lesnik and
Nencki, Arch. f. exp. Path. u. Pharm., 24; F. Miiller, Deutsch. med. Wochenschr.,
1887; Jaffe and Hilbert, Zeitschr. f. physiol. Chem., 12; Morner, ibid., 13; Klingen-
berg, " Studien iiber die Oxydation aromatischer Substanzen," etc., Inaug.-Diss.,
Rostock, 1891.

3 Zeitschr. f. physiol. Chem., 4.

4 Nencki, Arch. f. exp. Path. u. Pharm., 1; Nencki and Ziegler, Ber. d. d. Chem.
Gesellsch.. 5; see also O. Jacob sen, ibid., 12.

780 URINE.

The aromatic amino-adds are, like the amino-acids in general, decom-
posed to fatty acids and have one carbon atom less. For example phenyl-
amino-acetic add is in part converted into benzoic acid (O. NEUBAUER);
o- and w-tyrosine yield o- and m-oxyphenylacetic acid respectively
(BLUM, FLATOW); p-chlorphenylalanine passes according to FRIEDMANN
and MAASE into p-chlorphenylacetic acid, and phenyl-a-aminobutyric
acid is changed, as KNOOP 1 showed, into phenylpropionic acid. As
intermediary steps in this demolition we have, as in the other amino-acids,
part the hydrolytic cleavage of NH2 groups and part the demolition by
way of the corresponding keto-acid.

As an example of a demolition of the first kind we have for a long
time considered the finding by SCHOTTEN, of mandelic acid

C6H5.CH(OH).COOH

in the urine after feeding phenylaminoacetic acid, Cells. CH(NH2).COOH.
According to O. NEUBAUER 2 the process is nevertheless of another kind,
namely, mandelic acid is produced secondarily by reduction from the
intermediarily formed keto-acid, phenylglyoxylic add, CeHs.CO.COOH.
As example of a hydrolytic deamidation we will use the production, as
first observed by BLENDERMANN, of p-oxyphenyl-lactic add,

HO.C6H4.CH2.CH(OH).COOH

from tyrosine (in rabbits). This acid has also been found in the urine
by SCHULTZEN and RIESS in acute yellow atrophy of the liver, and by
BAUMANN in phosphorus poisoning, although the earlier investigators
incorrectly considered the acid as oxymandelic acid. That this acid,
which was considered as oxymandelic acid, is Z-p-oxypbenyl-lactic acid
has been proved by ELLINGER and KOTAKE and FROMHERZ.S

As shown especially by the investigations of O. NEUBAUER the demoli-
tion of the aromatic amino-acids passes ordinarily by way of the cor-
responding keto-acid. As stated above (page 737) in regard to the forma-
tion of homogentisic acid, the demolition of tyrosine, according to NEU-
BAUER, passes over the p-oxyphenylpyruvic acid, HO.CeH^CH^.CO.COOH.
According to him phenylamino-acetic add also yields phenylglyoxylic acid;

1 Neubauer, Deutsch. Arch. f. klin. Med.,95; L. Blum, Arch.f. exp. Path. u. Pharm.,
59; Flatow, Zeitschr. f. physiol. Chem., 64; F. Knoop, ibid., 67; Friedmann and
Maase, Bioch. Zeitschr., 27.

2 Schotten, Zeitschr. f. physiol. Chem., 8; O. Neubauer, 1. c.

3 Blendemann, Zeitschr. f. physiol. Chem., 6; Schultzen and Riess, Chem. Cdntralbl,
1869; Baumann, Zeitschr. f. physiol. Chem., 6; Ellinger and Kotake, ibid., 65; From-
herz, ibid., 70.

DEMOLITION OF AROMATIC FATTY ACIDS. 781

the w-tyrosine passes according to FLATOW 1 in part as ra-oxyphenyl
pyruvic acid in the urine. The keto-acids give also the same end products
as the corresponding amino-acids. Thus o-tyrosine, like o-oxyphenyl-
pyruvic acid, yields o-oxyphenylacetic acid (FLATOW) as end product;
the p-chlorphenylalanine and the p-chlorphenylpyruvic add pass into the
p-chlorphenylacetic acid, which is not the case with the oxyacid, the p-chlor-
phenyl-lactic acid (FRIEDMANN and MAASE 2) . This last-mentioned case is
an example of the more ready combustibility of the keto-acids as com-
pared to the oxy acids. Another such example is the p-oxy phenyl pyruvic
acid, which is in great part burned, while the p-oxyphenyl-lactic acid is
hardly burned at all (KOTAKE, SUWA). A correspondingly different
behavior is shown by these two acids in perfusion experiments with
the excised liver of the dog. The oxyphenylpyruvic acid, like tyrosine,
shows itself to be an acetone former while oxyphenyl-lactic acid, on
the contrary, does not (NEUBAUER and GROSS, E. ScHMiTz3). The
ready combustibility of the keto-acids indicate that these acids and
not the oxyacids are the important intermediary cleavage products.

In regard to the demolition of aromatic fatty acids, KNOOP 4 has found
that the acids with even carbon chains, such as phenyl butyric acid and
phenyl caproic acid, are converted into phenylacetic acid, which con-
jugates with glycocoll to form phenaceturic acid, while the acids with
uneven carbon chains, like phenylpropionic and phenylvaleric acid,
yield benzoic acid, which then is eliminated as hippuric acid. This
behavior is in close agreement with the generally accepted oxidation of
fatty at the /3-group, for which DAKIN has also given important support.
Thus DAKIN found after feeding phenylpropionic acid to cats, that
phenyl-@-oxypropionic acid, benzoylacetic acid and acetophenone, the latter
passing into benzoic acid or hippuric acid, were formed, which pre-
supposes an oxidation in the /3-position. According to the investigations
of DAKIN and FRIEDMANN 5 the conditions are still very complicated.
Certain of the processes are reversible, oxidations as well as reductions
occur, and a-0-unsaturated acids may also be formed as intermediary
products. DAKIN as well as FRIEDMANN have obtained cinnamic acid
as intermediary product in the demolition of phenylpropionic acid, and

1 Neubauer, Deutsch. Arch. f. klin. Med., 95; Flatow, Zeitschr.f. physiol. Chem., 64.

2 Flatow, 1. c.; Friedmann and Masse, Bioch. Zeitschr., 27.

8 Kotake, Zeitschr. f. physiol. Chem., 69; Suwa, ibid., 72; Neubauer and Gross,
ibid., 67; Schmitz, Bioch. Zeitschr., 28.

4 Hofmeister's Beitrage, 6, and Habilit.-Schrift, Freiburg, 1904.

6Dakin, Journ. of biol. Chem., 4, 5, 6, 8, and 9; Friedmann, see Med. Klinik,
No. 28, 1911, and Bioch. Zeitschr., 35.

782

URINE.

this is probably formed from the phenyl-/3-oxypropionic acid by the with-
drawal of water:

C6H5.CH(OH) .CH2.COOH - H20 = C6H5.CH :CH.COOH.

FRIEDMANN has also (in part with SASAKI) 1 studied the decomposition, of
furfurpropionic acid and found that pyromucic acid with furfuracrylic
acid as intermediary step, was formed:

The above-mentioned investigators are therefore of the opinion that the
demolition takes place in part over the a-/3-unsaturated acids and in part
over the 0-keto-acids or /3-alcohol acids.

According to the investigations of DAKIN and FRIEDMANN and to the schematic
illustration which they give, we can consider the demolition of pheaylpropionic
acid as follows:

C6H5.CH2.CH2COOH (Phenylpropionic acid)

(Olnnamlcacid) C6H6.CH: CH.COOH

X
C6H5.CO. CH2.COOH (Benzoylaceficacid)

C6H6.CH (OH). CH?.COOH

' (Phenyl-/3-oxypropionic acid)

C6H6.COOH

(Benzoic acid;

C6H5.CO .CH3

' ( Acetophenone)

C6H5.COOH

(Benzole acid)

C6H5.CO. NH.CH2.COOH

(Hippuric acid)

Reductions may also occur and besides the examples of the reduction
of keto-acids to alcohol-acids, we will mention as further examples the
conversion, as observed by E. MEYER,2 of nitrobenzene, CeHsNC^, or of
nitrophenol, HO.CeH4.NO2 into aminophenol, HO.CeH4.NH2, and also the
behavior of m-nitro-benzaldehyde in the animal body as mentioned below.

Syntheses of aromatic substances with other atomic groups occur
frequently. To these syntheses belongs, in the first place, the conjugation
of benzoic acid with glycocoll to form hippuric acid, the discovery of which
is generally ascribed to WOHLER, but according to HEFFTER 3 more cor-

1 Sasaki, Bioch. Zeitschr., 25; Friedmann, ibid., 35.

2 Zeitschr. f. physiol. Chem., 46.

1 Die AuBscheidung korperfremder Substanzen im Ham, Ergebnisse der Physiol.,
4, 252.

SYNTHESES OF AROMATIC SUBSTANCES. 783

rectly to KELLER and URE. All the numerous aromatic substances which
are converted into ben zoic acid in the body are voided partly as hippuric
acid. This statement is not true for all species of animals. According
to the observations of JAFFE/ benzoic acid does not pass into hippuric
acid in birds, but after conjugation with ornithin, into the correspond-
ing acid, ornithuric acid, (a-S-dibenzoyldiamino valeric acid). Not only
are the oxybenzoic acids and the substituted benzoic acids conjugated with
glycocoll, forming corresponding hippuric acids, but also the above-
mentioned acids, toluic, mesitylenic, cumic, and phenylacetic acids. These
acids are voided as toluric, mesitylenuric, cuminuric, and phenaceturic
acids.

It must be remarked in regard to the oxybenzoic acids that a conjugation with
glycocoll has been shown only with salicylic and p-pxybenzoic acid (BERTAGNINI,
and others), while BAUMANN and HERTER 2 find it only very probable for m-
oxybenzoic acid. According to BALDONi,3 in dogs, the salicylic acid does not pass
into salicyluric acid, and he indeed found two acids which he calls ursalicylic
acid, CisHuOg and uramin-salicylic acid, CieHieNOg. The oxybenzoic acids are
also in part eliminated as conjugated sulphuric acids, which is especially true for
m-oxybenzoic acid. The three aminobenzoic acids, according to the experiments
of HILDEBRANDT, on rabbits, appeared at least in part unchanged in the urine.
SALKOWSKI found, as was later confirmed by R. CoHN,4 that in rabbits m-amino-
benzoic acid passes in part into uraminobenzoic acid, H2N.CO.HN.C6H4.COOH.
It is also in part eliminated as aminohippuric acid.

The behavior of the halogen-substituted compounds of toluene varies in
different animals according to HILDEBRANDT'S experiments. In dogs they are
converted into the corresponding substituted hippuric acid. In rabbits o-brom-
toluene is completely changed to hippuric acid, the ra- and p-bromtoluene only
partly. The three chlortoluenes are converted in rabbits into the corresponding
benzoic acid and are eliminated as such and not as hippuric acid.

The substituted aldehydes are of special interest as substances which
may undergo conjugation with glycocoll. According to the investiga-
tions of R. CoHN5 on this subject, o-nitrobenzaldehyde when introduced
into a rabbit is only in a very small part converted into nitrobenzoic
acid, and the chief mass, about 90 per cent, is destroyed in the body.
According to SIEBER and SMiRNOW6 m-nitrobenzaldehyde passes in dogs
into m-nitrohippuric acid, and according to COHN into urea-m-nitro-
hippurate, but in rabbits a different action results. In this case not only
does an oxidation of the aldehyde into benzoic acid take place, but the

1 Ber. d. d. chem. Gesellsch., 10 and 11.

2 Zeitschr. f. physiol. Chem., 1, where Bertagnini's work is also cited. See also
Dautzenberg, Maly's Jahresber., 11, 231.

3 Arch. f. exp. Path. u. Pharm., 1908, Suppl. Bd. (Schmiedeberg's Festschrift).
4Salkowski, Zeitschr. f. physiol. Chem., 7; Cohn, ibid., 17; Hildebrandt, Hof-

meister's Beitrage, 3.

6 Zeitschr. f. physiol. Chem., 17.
6 Monatshefte f. Chem., 8.

784 URINE.

nitrogroup is also reduced to an amino-group, and finally acetic acid
attaches itself to this with the expulsion of water, so that the final product
is m-acetylaminobenzoic acid, (CHs.COXNH.CeH^COOH. The p-nitro-
benzaldehyde acts in rabbits in part like the ra-aldehyde and passes in
part into p-acetylaminobenzoic add. Another part is converted into
p-nitrobenzoic acid, and the urine contains a chemical combination of
equal parts of these two acids. According to SIEBER and SMIRNOW p-
nitrobenzaldehyde yields only urea p-nitrohippurate in dogs. The above-
mentioned pyridine-carboxylic acid, formed from methylpyridine
(a-picoline) passes into the urine after conjugation with glycocoll as
a-pyridinuric acid.1

To those substances which undergo a conjugation with glycocoll
belongs also furfural (the aldehyde of pyromucic acid, C^sO.CHO),
which, when introduced into rabbits and dogs, as shown first by JAFFE
and CoHN,2 and then further shown by SASAKI and FRIEDMANN, is elimi-
nated in two ways from the body. The furfurol can, by a similar synthesis
to PERKIN'S reaction, be transformed into the unsaturated Sicidfurfuracrylic
acid C4H3O.CH:CH.COOH, and also into pyromucic acid C4H3O.COOH.
These two acids pass, after conjugation with glycocoll, into the urine as
furfuracryluric acid and pyromucuric acid. In birds the pyromucic acid
is conjugated with ornithine and is eliminated as pyromucinornithuric acid.

It has not been proved how thiophene, C4H4S, behaves in the animal
body. Of methylthiophene (thiotolene), C^aS.CHs, a very small part is
oxidized to thiophenic acid, C^aS.COOH (LEVY). This acid, as shown
by JAFFE and LEVY,S is conjugated with glycocoll in the body (rabbits)
and eliminated as thiophenuric acid.

Another very important synthesis of aromatic substances is that of
the ethereal-sulphuric acids. Phenols, and in particular the hydroxylated
aromatic hydrocarbons and their derivatives are voided as ethereal-sul-
phuric acids, according to BAUMANN, HERTER and others.4

A conjugation of aromatic acids with sulphuric acid occurs less often.
The two previously mentioned aromatic acids, p-oxyphenylacetic and
p-oxyphenylpropionic acid, are in part eliminated in this form. Gentisic
acid (hydroquinone-carboxylic acid) also increases, according to LIK-
HATSCHEFF,5 the quantity of ethereal-sulphuric acid in the urine, while

1 In regard to the extensive literature on glycocoll conjugations we refer the reader
to O. Kuhling, Ueber Stoffwechselprodukte aromatischer Korper. Inaug.-Diss.,
Berlin, 1887.

2 Ber. d.d. Chem. Gesellsch., 20 and 21; Sasaki and Friedmann, footnote 1, page 782.

3 Levy, Ueber das Verhalten einiger Thiophenderivate, etc., Inaug.-Diss., Konigs-
berg, 1889; Jaffe and Levy, Ber. d. d. chem. Gesellsch., 21.

4 In regard to the literature, see O. Kuhling, 1. c.
6 Zeitschr. f. physiol. Chem., 21.

CONJUGATION OF AROMATIC SUBSTANCES. 785

ROST asserts, contrary to earlier claims, that the same occurs with gallic
acid (trioxybenzoic acid) and tannic acid.1

Although NENCKI and REKOWSKI 2 have shown that acetophenone (phen-
ylmethylketone), CeHs.CO.CHs, is oxidized to benzoic acid and eliminated
as hippuric acid, the aromatic oxyketones with hydroxyl groups, such as
resacetophenone, 2, 4 dioxacetophenone (HO^.CeHs.CO.CHs, pass into
the urine as ethereal-sulphuric acids and in part after conjugation with
glucuronic acid. Euxanthon, which is also an aromatic ketone, namely

CO
dioxyxanthon, HO.Celis^ yCeHa.OH, passes into the urine as eux-

XCT
anthic acid after conjugation with glucuronic acid.

A conjugation of other aromatic substances with glucuronic acid,
which last is protected from combustion, occurs rather often. The phenols,
as above stated (page 725), pass in part as conjugated glucuronic acids
into the urine. The same is true for the homologues of the phenols, for
certain substituted phenols, and for many aromatic substances, also
hydrocarbons after previous oxidation and hydration. Thus HILDE-
BRANDT and FROMM and CLEMENS 3 have shown that the terpenes and cam-
phors, by oxidation or hydration, or in certain cases by both, are converted
into hydroxyl derivatives when the body in question is not previously
hydroxylized, and that these hydroxyl derivatives are eliminated as con-
jugated glucuronic acids. Conjugated glucuronic acids are detected in
the urine after the introduction of various substances into the organsim,
e.g., therapeutic agents, such as terpenes, borneol, menthol, camphor (cam-
phoglucuronic acid was first observed by SCHMIEDEBERG), naphthalene,
oil of turpentine, oxyquinolines, antipyrine, and many other bodies.4
Orthonitrotoluene in dogs passes first into o-nitrobenzyl alcohol and then

1 In regard to the behavior of gallic and tannic acids in the animal body, see C.
Morner, Zeitschr. f. physiol. Chem., 16, which also contains the earlier literature; also
Harnack, ibid., 24, and Rost, Arch. f. exp. Path. u. Pharm., 38, and Sitzungsber. d.
Gesellsch. zur Beford. d. ges. Naturwiss. zu Marburg, 1898.

2 Arch. d. scienc. biol. de St. Petersbourg, 3, and Ber. d. deutsch. chem.
Gesellsch., 27.

3 Hildebrandt, Arch. f. exp. Path. u. Pharm., 45, 46; Zeitschr. f. physiol. Chem.,
36; with Fromm, ibid., 33; and with Clemens, ibid., 37; Fromm and Clemens, ibid.,
34. Extensive investigations on the behavior of alicylic compounds with the glucuronic
acid conjugation in the organism have been carried out by Hamalainen, Skand. Arch,
f. Physiol., 27.

4 See O. Kiihling, 1. c., which gives the literature up to 1887; also E. Kiilz, Zeitschr.
f. Biologic, 27; the works of Hildebrandt, Fromm and Clemens, see footnote 3;
Brahm, Zeitschr. f. physiol. Chem., 28; Fenyvessy, ibid., 30; Bonanni, Hofmeister's
Beitrage, 1; Lawrow, Ber. d. d. chem. Gesellsch., 33.

786 URINE. .

into a conjugated glucuronic acid, uronitrotoluolic acid (JAFFE1). The
glucuronic acid split off from this conjugated acid is levogyrate and hence
is not identical, but only isomeric, with the ordinary glucuronic acid.
Dimethylaminobenzaldehyde, according to JAFFE, is converted in part
into dimethylaminobenzoglucuronic acid in rabbits. The same conjugated
glucuronic acid is also produced, according to HiLDEBRANDT,2 from p-
dimethyltoluidine, which is first changed into p-dimethylaminobenzoic
acid. Indol and skatol seem, as above stated (page 731), to be eliminated
in the urine partly as conjugated glucuronic acids. The mercapturic
acids, which will be mentioned below, also belong to those substances
which are conjugated with glucuronic acid, and after this conjugation
appear in the urine.

A conjugation of carbamic acid, NEbCOOH, with amino-acids to form
uramino-acids, R.CH.NH.(CONH2)COOH, or their anhydrides, the
hydantoins, have also been observed in several cases, as after feeding
sarcosin, amino-benzoic acid, phenylalanine, taurine, tyrosine. It must
be remarked that according to LIPPICH and DAKIN,S the uramino-acids
can be easily produced as transformation products from the urea in the
concentration of the urine by the aid of heat.

Syntheses with a simultaneous acetylation are of great interest.
Such a synthesis is the formation of the mercapturic acids. These acids,
which are produced in the body of dogs after the introduction of brom-
and chlorbenzene (BAUMANN and PREUSSE, JAFFE, FniEDMANN4) are
acetylated derivatives of the protein cystine, and the acetylated brom-
phenylcysteine is CH2.S (C6H4Br) .CH.NH(COCH3) .COOH. Another
example of a synthesis with acetylation is the phenylaminoacetic acid,
which, as NEUBAUER and WARBURG 5 have shown, in perfusion exper-
iments with dog's livers, gives among other products also acetylated
phenylaminoacetic acid, C6H5.CHNH(COCH3).COOH.

The synthesis of amino-acids, with simultaneous acetylation, as
recently observed by KNOOP, are of specially great interest. After the
introduction of 7-phenyl-a-keto butyric acid into the body of a dog,
the formation of the corresponding acetylated amino-acid,

C6H5.CH2.CH2.CHNH(COCH3).COOH

1 Zeitschr. f. physiol. Chem., 2.

2 Jaffe", Zeitschr. f. physiol. Chem., 43; Hildebrandt, Hofmeister's Beitrage, 7.

3 Lippich, Ber. d. d. chem. Gesellsch., 41; Dakin, Journ. of biol. Chem., 8; Weiland,
Bioch. Zeitschr., 38.

4 Baumann and Preusse, Zeitschr. f. physiol. Chem., 5; Jaffe", Ber. d. deutsch.
chem. Gesellsch., 12; Friedmann, Hofmeister's Beitrage, 4.

6 Neubauer and O. Warburg, Zeitschr. f. physiol. Chem., 70.

METHYLATION. 787

was observed. In perfusion experiments with dog livers, EMBDEN and
SCHMITZ 1 have shown the formation of phenylalanine, tyrosine and other
amino-acids, as has already been mentioned (pages 530 and 775) by
synthesis from the ammonium salts of the corresponding keto-acids and
also in part from the oxy-acids.

Methylation also often occurs, and as an example we will mention that
His has shown that pyridine, CsH5N, is transformed in dogs into methyl-
pyridine, and then passes into the urine as methylpyridylammonium
hydroxide. Pyridine behaves similarly in hens (HosniAi), pigs and goats,
(TOTANI and HOSHIAT) while according to ABDERHALDEN 2 and collabora-
tors, in rabbits it passes unchanged into the urine. Further examples
of methylation, although not aromatic substances, are the conversion
of guanidine acetic acid into creatine (JAFFE) and the observation of
TAKEDA 3 of the appearance of aminobutyrobetaine in the urine of dogs
with phosphorus poisoning.

Several alkaloids, such as quinine, morphine, and strychnine, may pass into
the urine. After the ingestion of turpentine, balsam of copaiba, and resins, these
may appear in the urine as resin acids. Different kinds of coloring-matters, such
as alizarin, crysophanic acid, after rhubarb or senna, and the coloring-matter of
the blueberry, etc., may also pass into the urine. After rhubarb, senna, or santonin
the urine assumes a yellow or greenish-yellow color, which is transformed into
a beautiful red by the addition of alkali. Phenol produces, as above mentioned,
a dark-brown or dark-green color which depends mainly on the decomposition
products of hydroquinone and humin substances. After naphthalene the urine
has a dark color, and several other medicinal agents produce a special coloration.
Thus after antipyrine it becomes yellow or blood-red. After balsam of copaiba
the urine becomes, when strongly acidified with hydrochloric acid, gradually
rose- and purple-red. After naphthalene or naphthol the urine gives with con-
centrated sulphuric acid (1 cc. of concentrated acid and a few drops of urine)
a beautiful emerald-green color, which is probably due to naphthol-glucuronic
acid. Odoriferous bodies also pass into the urine. After asparagus the urine
acquires a digusting odor which is probably due to methylmercaptan. After
turpentine the urine may have a peculiar odor similar to that of violets.

VI. PATHOLOGICAL CONSTITUENTS OF URINE.

Proteid. The appearance of slight traces of proteid in normal urine
has been observed by many investigators, such as POSNER PL6sz, v.
NOORDEN, LEUBE, and others. According to K. MORNER* proteid
regularly occurs as a normal urinary constituent to the extent of 22-78

1 Knoop, Zeitschr. f. physiol. Chem., 67; Embden and Schmitz, Bioch. Zeitschr., 29
and 38.

2 His, Arch. f. exp. Path. u. Pharm., 22; Cohn, Zeitschr. f. physiol. Chem., 18;
Hoshiai, ibid., 62; with Totani, ibid., 68; Abderhalden and collaborators, ibid., 59
and 62.

3 Jaffe, Zeitschr. f. physiol. Chem., 48; Takeda, Pfluger's Arch., 133.
4Skand. Arch. f. Physiol., 6 (literature).

788 URINE.

milligrams per liter. Frequently traces of a substance similar to a
nucleoalbumin, which is easily mistaken for mucin, and whose nature
will be treated of later, appears in the urine. In diseased conditions
proteid occurs in the urine in a variety of cases. The albuminous bodies
which most often occur are serglobulin and seralbumin. Proteoses (or
peptones) are also sometimes present. The quantity of proteid in the
urine is in most cases less than 5 p. m., rarely 10 p. m., and only very rarely
does it amount to 50 p. m. or over. Cases are known, however, where it
was even more than 80 p. m.

Among the many reactions proposed for the detection of proteid in
urine, the following are to be recommended:

The Heat Test. Filter the urine and test its reaction. An acid
urine may, as a rule, be boiled without further treatment, and only in
especially acid urines is it necessary to first treat with a little alkali.
An alkaline urine is made neutral or faintly acid before heating. If the
urine is poor in salts, add 1/10 vol. of a saturated common-salt solution
before boiling; then heat to the boiling-point, and if no precipitation,
cloudiness, or opalescence appears, the urine in question contains no
coagulable proteid, but it may contain proteoses or peptones. If a pre-
cipitate is produced on boiling, this may consist of proteid, or of earthy
phosphates,1 or of both. The monohydrogen calcium phosphate decom-
poses on boiling, and the normal phosphate may separate out. The
proper amount of acid is now added to the urine, so as to prevent any
mistake caused by the presence of earthy phosphates, and to give a better
and more flocculent precipitate of the proteid. If acetic acid is used
for this, then add 1-3 drops of a 25 per cent acid to each 10 cc. of the
urine and boil after the addition of each drop. On using nitric acid,
add 1-2 drops of the 25 per cent acid to each cubic centimeter of the
boiling-hot urine.

On using acetic acid, when the quantity of proteid is very small,
and especially when the urine was originally alkaline, the proteid may
sometimes remain in solution on the addition of the above quantity of
acid. If, on the contrary, less acid is added, the precipitate of calcium
phosphate, which forms in amphoteric or faintly acid urines, is liable
not to dissolve completely, and this may cause it to be mistaken for a
proteid precipitate. If nitric acid is used for the heat test, the fact must
not be overlooked that after the addition of only a little acid a combina-
tion between it and the proteid is formed which is soluble on boiling and
which is only precipitated by an excess of the acid. On this account the
large quantity of nitric acid, as suggested above, must be added, but in
this case a small part of the proteid is liable to be dissolved by the excess
of the nitric acid. When the acid is added after boiling, which is absolutely
necessary, the liability of a mistake is not so great. It is on these grounds
that the heat test, although it gives very good results in the hands of
experts, is not recommended to physicians as a positive test for proteid.

1 In regard to the cause of the phosphate precipitation on boiling the urine, see
Malfatti, Hofmeister's Beitrage, 8.

PROTEIDS IN THE URINE. 789

A confounding with mucin, when this body occurs in the urine, is
easily prevented in the heat test with acetic acid by acidifying another
portion with acetic acid at the ordinary temperature. Mucin and
nucleoalbumin substances similar to mucin are hereby precipitated. If
in the performance of the heat and nitric-acid test, a precipitate first
appears on cooling or is strikingly increased, then this shows the presence
of proteoses in the urine, either alone or mixed with coagulable proteid.
In this case a further investigation is necessary (see below). In a urine
rich in urates a precipitate consisting of uric acid separates on cooling.
This precipitate is colored and granular, and is hardly to be mistaken
for a proteose or proteid precipitate.

HELLER'S test is performed as follows (see page 99): The urine is
very carefully floated on the surface of nitric acid in a test-tube, or the
urine is placed in a test-tube and then the acid is slowly added by means
of a funnel, drawn out to a point, and extending to the bottom. In the
presence of albumin a white disk, or as we ordinarily say a white ring or
at least a sharply denned cloudiness, appears at the point of contact of
the two fluids. With this test a red or reddish-violet transparent ring
is always obtained with normal urine; it depends upon the indigo color-
ing-matters and can hardly be mistaken for the white or whitish proteid
ring. In a urine rich in urates, another complication may occur, due
to the formation of a ring produced by the precipitation of uric acid.
The uric-acid ring does not lie, like the proteid ring, between the two
liquids, but somewhat higher. For this reason two simultaneous rings
may exist in urines which are rich in urates and do not contain very much
proteid. The disturbance caused by uric acid is easily prevented by
diluting the urine with 1-2 vols. of water before performing the test.
The uric acid now remains in solution, and the delicacy of HELLER'S
test is so great that after dilution only in the presence of insignificant
traces of proteid does this test give negative results. In a urine very
rich in urea a ring-like separation of urea nitrate may also appear. This
ring consists of shining crystals, and it does not appear in urine previously
diluted. A confusion with resinous acids, which also give a whitish
ring with this test, is easily prevented, since these acids are soluble on the
addition of ether. Stir, add ether, -and carefully shake the contents of
the test-tube. If the cloudiness is due to resinous acids, the urine gradually
becomes clear, and on evaporating the ether a sticky residue of resinous
acids is obtained. A liquid which contains true mucin does not .give
a precipitate with this test, but it gives a more or less strongly opalescent
ring, which disappears on stirring. The liquid does not contain any
precipitate after stirring, but is somewhat opalescent. If a faint, not
wholly typical reaction is obtained with HELLER'S test after some time
with undiluted urine, while the diluted urine gives a pronounced reaction,
the presence is shown of the substance which used to be called mucin
or nucloealbumin. In this case proceed as described below for the detec-
tion of nucleoalbumin.

If the above-mentioned possible errors and the means by which they
may be prevented are borne in mind, there is hardly another test for
proteid in the urine which is at the same time so easily performed, so
delicate, and so positive as HELLER'S. With this test even 0.002 per
cent of albumin may be detected without difficulty. Still the student
must not be satisfied with this test alone, but should apply at least a

790 URINE.

second one, such as the heat test. In performing this test the (primary)
proteoses are also precipitated.

The reaction with metaphosphoric acid is very convenient and easily
performed. It is not quite so delicate and positive as HELLER'S test.
The proteoses are also precipitated by this reagent.

Reaction with Acetic Acid and Potassium Ferrocyanide. Treat the
urine first with acetic acid until it contains about 2 per cent, and then
add drop by drop a potassium-ferrocyanide solution (1:20), carefully
avoiding an excess. This test is very good, and in the hands of experts
it is even more delicate than HELLER'S. In the presence of a very small
quantity of proteid it requires more practice and dexterity than HEL-
LER'S, as the relative quantities of reagent, proteid, and acetic acid influence
the result of the test. The quantity of salts in the urine likewise seems
to have an influence. This reagent also precipitates proteoses.

SPIEGLER'S Test. SPIEGLER recommends a solution of 8 parts mercuric
chloride, 4 parts tartaric acid, 20 parts glycerin, and 200 parts water as a very
delicate reagent for proteid in the urine. A test-tube is half filled with this
reagent and the urine is allowed to flow upon its surface drop by drop from a
pipette along the wall of the test-tube. In the presence of proteid a white ring is
obtained at the point of contact between the two liquids. The delicacy of this
test is 1 :350,000. JOLLES l does not consider this reagent suited for urines very
poor in chlorine, and for this reason he has changed it as follows: 10 grams mer-
curic chloride, 20 grams succinic acid, 10 grams NaCl, and 500 cc. water.

Reaction with sulphosalicylic acid. Treat the urine either with a 20 per cent
watery solution of sulphosalicylic acid or a few crystals of the acid. This reagent
does not precipitate the uric acid or the resin acids. (Rocn's2 test.)

As every normal urine contains traces of proteid, it is apparent that
very delicate reagents are to be used only with the greatest caution. For
ordinary cases HELLER'S test is sufficiently delicate. If no reaction is
obtained with this test within 2J to 3 minutes, the urine tested contains
less than 0.003 per cent of proteid, and is to be considered free from pro-
teid in the ordinary sense.

The use of precipitating reagents presumes that the urine to be investi-
gated is perfectly clear, especially in the presence of only very little
proteid. The urine must first be filtered. This is not easily done with
urine containing bacteria, but a clear urine may be obtained, as suggested
by A. JOLLES, by shaking the urine with infusorial earth. Although
a little proteid is retained in this procedure and lost, it does not seem to
be of any importance (GRUTZNER, SCHWEISSINGERS).

The different color reactions cannot be directly used, esspecially in deep-colored
urines which contain only little proteid. The common salt of the urine has a
disturbing action on MILLON'S reagent. To prove more positively the presence

^piegler, Wien. klin. Wochenschr., 1892, and Centralbl. f. d. klin. Med., 1893;
Jolles, Zeitschr. f. physiol. Chem., 21.

2 Pharmaceut. Centralbl., 1889, and Zeitschr. f. anal. Chem., 29.

'Jolles, Zeitschr. f. anai. Chem., 29; Grutzner, Chem. Centralbl., 1901, 1;
Schweissinger, ibid.

PROTEIDS IN THE URINE. 791

of protein, the precipitate obtained in the boiling test may be filtered, washed,
and then tested with MILLON'S reaction. The precipitate may also be dissolved
in dilute alkali and the biuret test applied to the solution. The presence of pro-
teoses or peptones in the urine is directly tested for by this last-mentioned test.

In testing the urine for proteid one should never be satisfied with one
reaction alone, but must apply the heat test and HELLER'S, or the potas-
sium-ferrocyanide test. In using the heat test alone the proteoses may
be easily overlooked, but these are detected, on the contrary, by HELLER'S
or the potassium-ferrocyanide test. If only one of these tests is employed,
no sufficient intimation of the kind of proteid present can be obtained,
whether it consists of proteoses or coagulable proteid.

For practical purposes several dry reagents for proteid have been recommended.
Besides the metaphosphoric acid may be mentioned STUTZ'S or FURBRINGER'S
gelatin capsules, which contain mercuric chloride, sodium chloride, and citric
acid; and GEISSLER'S albumin-test papers, which consist of strips of filter-paper
some of which have been dipped in a solution of citric acid, and some into a
solution of mercuric-chloride and potassium-iodide solution, and then dried.

If the presence of proteid has been positively proved in the urine by
the above tests, it then remains necessary to determine its character.

The Detection of Globulin and Albumin. In detecting serglobulin
the urine is exactly neutralized, filtered, and treated with magnesium
sulphate in substance until it is completely saturated at the ordinary
temperature, or with an equal volume of a saturated neutral solution of
ammonium sulphate. In both cases a white, flocculent precipitate is
formed in the presence of globulin. In using ammonium sulphate with
a urine rich in urates, a precipitate consisting of ammonium urate may
separate. This precipitate does not appear immediately, but only after
a certain time, and it must not be mistaken for the globulin precipitate.
In detecting seralbumin heat the filtrate from the globulin precipitate
to boiling-point, or add about 1 per cent acetic acid to it at the ordinary
temperature.

For the detection and also for the quantitative estimation of the various
globulins (fibringlobulin, euglobulin, and pseudoglobulin) OSWALD l has pro-
posed the fractional precipitation with ammonium sulphate.

Proteoses and peptones have been repeatedly found in the urine in
different diseases. Reliable reports are at hand on the occurrence of
proteoses in the urine. The statements in regard to the occurrence of
peptones date from a time when the conception of proteoses and pep-
tones was different from that of the present day, and in part they are
based upon investigations using untrustworthy methods. According
to ITO 2 true peptones are sometimes found in the urine in cases of pneu-

1 Munch, med. Wochenschr., 1904. See also Zak and Necker, Deutsch. Arch. f.
klin. Med., 88.

2 In regard to the literature on proteoses and peptones in urine, see Huppert-
Neubauer, Harn- Analyse, 10. Aufl., 466 to 492; also A. Stoffregen, Ueber das Vorkom-
men von Pepton iin Harn, Sputum, und Eiter (Inaug.-Diss., Dorpat, 1891); E. Hirsch-
feldt, Ein Beitrage zur Frage der Peptonurie (Inaug.-Diss., Dorpat, 1892); and espe-

792 URINE.

monia; what has been designated as urine peptones seems to have been
chiefly deuteroproteoses.

In detecting the proteoses, the proteid-free urine, or urine boiled with addi-
tion of acetic acid, is saturated with ammonium sulphate, which precipitates the pro-
teoses. Several errors are here possible. The urobilin,which may give a reaction
similar to the biuret reaction, is also precipitated and may lead to mistakes (SAL-
KOWSKI, STOKVIS l). The following modification by BANG and DEVOTo's2 method
can be used to advantage: The urine is heated to boiling with ammonium sul-
phate (8 parts to 10 parts urine) and boiled for a few seconds. The hot liquid
is centrifuged for f to 1 minute and separated from the sediment. The urobilin
is removed from this by extraction with alcohol. The residue is suspended in
a little water, heated to boiling, filtered, whereby the coagulable proteid is retained
on the filter, and any urobilin still present in the nitrate is shaken out with chloro-
form. The watery solution, after removal of the chloroform, is used for the biuret
test. For clinical purposes this method is very serviceable.

According to SALKOWSKI the urine treated with 10-per cent hydrochloric
acid is precipitated with phosphotungstic acid, then warmed, the liquid decanted
from the resin-like precipitate, this washed with water, and then dissolved in a
little water with the aid of some caustic soda, warmed again until the blue color
disappears, cooled, and finally tested with copper sulphate. This method has
been somewhat modified by v. ALDOR and CERNY.S In regard to other more
complicated methods we refer to HUPPERT-NEUBAUER.

MORAWITZ and DIETSCHY 4 first remove the proteid from the urine made
faintly acid with acid potassium phosphate by the addition of double the volume
of 96-per cent alcohol and warming on the water-bath for several hours. [ From
the concentrated filtrate acidified with a little sulphuric acid the proteoses can
be precipitated by saturating with zinc sulphate. After the removal of the urobilin
by alcohol and extracting with water, the biuret test may be applied.

If the proteoses have been precipitated from a larger portion of urine by
ammonium sulphate, this precipitate is tested for the presence of different pro-
teoses for the reasons given in Chapter II. The following serves as a preliminary
determination of the character of the proteoses present in the urine. If the urine
contains only deuteroproteose it does not become cloudy on boiling, does not give
HELLER'S test, does not become cloudy on saturating with NaCl in neutral reaction,
but does become turbid on adding acetic acid saturated with this salt. In the
presence of only prptoproteose the urine gives HELLER'S test, is precipitated
even in neutral solution on saturating with NaCl, but does not coagulate on boil-
ing. The presence of heteroproteose is shown by the urine behaving like the
above with NaCl and nitric acid, but shows a difference on heating. It gradually
becomes cloudy on warming and separates at about 60° C. a sticky precipitate
which attaches itself to the sides of the vessel and which dissolves at boiling tem-
perature on acidifying the urine; the precipitate reappears on cooling.

In close relation to the proteoses stands the so-called BENCE-JONES
proteid, which occurs in the urine in rare cases in diseases with changes

cially Stadelmann, Untersuchungen iiber die Peptonurie, Wiesbaden, 1894; Ehrstrom,
Bidrag till kannedomen om Albumosurien, Helsingfors, 1900; Ito, Deutsch. Arch,
f. klin. Med., 71.

iSalkowski, Berlin, klin. Wochenschr., 1897; Stokvis, Zeitschr. f. Biologic, 34.

2Devoto, Zeitschr. f. physiol. Chem., 15; Bang, Deutsch. med. Wochenschr., 1898.

3 Salkowski, Centralbl. f. d. med. Wissensch., 1894; v. Aldor, Berl. klin. Wochenschr.,
36; Cerny, Zeitschr. f. analyt. Chem., 40.

4 Arch. f. exp. Path. u. Pharm., 54.

QUANTITATIVE ESTIMATION OF PROTEID IN URINE. 793

in the spinal marrow. It gives a precipitate on heating to 40-60° C.,
which on further heating to boiling dissolves again more or less completely,
depending upon the reaction and upon the amount of salt present. In
salt-free solution the precipitate is not dissolved, on heating to. boiling,
at least not always. It does not separate on dialysis, but can be pre-
cipitated from the urine by double the volume of a saturated ammonium-
sulphate solution or by alcohol. It has also been obtained as crystals
(GRUTTERINK and DE GRAAFF, MAGNUS-LEVY x). This body shows a
varying behavior in the different cases in which it has been found and its
nature has not been explained. From the investigations of the above-
mentioned and other experimenters (MOITESSIER, ABDERHALDEN and
ROSTOSKI) we can draw the conclusion that this proteid is similar to the
proteoses in several reactions, but that nevertheless it stands close to the
genuine protein bodies. It also yields primary as well as secondary
proteoses on peptic digestion (GRUTTERINK and DE GRAAFF), and yields
the same hydrolytic cleavage products as the other proteins (ABDERHALDEN
and ROSTOSKI).

Quantitative Estimation of Proteid in Urine. Of all the methods pro-
posed thus far, the COAGULATION METHOD (boiling with the addition of
acetic acid) when performed with sufficient care gives the best results.
The average error need never amount to more than 0.01 per cent, and it
is generallv smaller. With this method it is best to first find how much
acetic acid must be added to a small portion of the urine, which has been
previously heated on the water-bath, to completely separate the pro-
teid so that the filtrate will not respond to HELLER'S test. Then coagulate
20-50-100 cc. of the urine. Pour the urine into a beaker and heat on
the water-bath, add the required quantity of acetic acid slowly, stirring
constantly, and heat at the same time, Filter while warm, wash first
with water, then with alcohol and ether, dry and weigh, incinerate and
weigh again. In exact determinations the filtrate must not give HEL-
LER'S test.

The separate estimation of GLOBULINS and ALBUMINS is done by carefully
neutralizing the urine and precipitating with MgSO4 added to saturation (HAMMAR-
STEN), or simply by adding an equal volume of a saturated neutral solution of
ammonium sulphate (HOFMEISTER and POHL 2). The precipitate consisting of
globulin is thoroughly washed with a saturated magnesium-sulphate or half-
saturated ammonium-sulphate solution, dried continuously at 110° C., boiled
with water, extracted with alcohol and ether, then dried, weighed, incinerated,
and weighed again. The quantity of albumin is calculated as the difference
between the quantity of globulin and the total proteids.

Approximate Estimation of Proteid in Urine. Of the methods suggested for
this purpose none has been more extensively employed than ESBACH'S.

1 Magnus-Levy, Zeitschr. f. physiol. Chem., 30 (literature); Grutterink and de
Graaff, ibid., 34 and 36; Moitessier, Compt. rend. soc. biolog., 57; Ville and Derrien,
ibid., 62; Abderhalden and Rostoski, Zeitschr. f. physiol Chem., 46; see also Hopkins
and Savory, Journ. of Physiol., 42.

2 Hammarsten, Pfliiger's Arch., 17; Hofmeister and Pohl, Arch. f. exp. Path. u.
Pharm., 20.

794 URINE.

ESBACH'S * Method. The acidified urine (with acetic acid) is poured into a
specially graduated tube to a certain mark, and then the reagent (a 2-per cent
citric-acid and 1 per cent picric-acid solution in water) is added to a second mark,
the tube closed with a rubber stopper and carefully shaken, avoiding the pro-
duction of froth. The tube is allowed to stand twenty-four hours, and then the
height of the precipitate on the graduation is read off. The reading gives directly
the quantity of proteid in 1000 parts of the urine. Urines rich in proteid must
first be diluted with water. The results obtained by this method, are, however,
dependent upon the temperature; and a difference in temperature of 5° to 6.5°
C. may cause an error of 0.2-0.3 per cent deficiency or excess in urines containing
a medium quantity of proteid (CHRISTENSEN and MYGGE). The method sug-
gested by TSUSCHIJA 2 seems to be more reliable, and consists in precipitating the
proteid by an alcoholic solution of phosphotungstic acid containing hydrochloric
acid.

Other methods for the approximate estimation of proteid are the optical
methods of CHRISTENSEN and MYGGE, and of WALBUM,S of ROBERTS and STOLLNI-
KOW as modified by BRANDBERG, with HELLER'S test, which has been simplified
for practical purposes by MITTELBACH. The density methods of LANG, HUPPERT
and ZAHOR are also very good. In regard to these and other methods we refer to
HUPPERT-NEUBAUER'S Harn- Analyse, 10. Aufl.

There is at present no trustworthy method for the quantitative estimation
of proteoses and peptone in the urine.

Nucleoalbumin and Mucin. According to K. MORNER traces of urinary
mucoids may pass into solution in the urine; otherwise normal urine con-
tains no mucin. There is no doubt that there may be cases where true
mucin appears in the urine; in most cases mucin has probably been mis-
taken for so-called nucleoalbumin. The occurrence, under some circum-
stances, of nucleoalbumin in the urine is not to be denied, as such sub-
stances occur in the renal and urinary passages; still in most cases this
nucleoalbumin, as shown by K. MORNER;* is of an entirely different kind.

All urine, according to MORNER, contains a little proteid and in
addition substances which precipitate proteid. If the urine freed from
salts by dialysis is shaken with chloroform after the addition of 1-2 p. m.
acetic acid, a precipitate is obtained which acts like a nucleoalbumin.
If the acid filtrate is treated with seralbumin, a new and similar precipitate
is obtained, due to the presence of a residue of the substance which pre-
cipitates proteids. The most important of these proteid-precipitating
substances is chondroitin-sulphuric acid and nucleic acid, although the
latter appears to a much smaller extent. Taurocholic acid may in a few
instances^ especially in icteric urines, be precipitated. The substances
isolated by different investigators from urine by the addition of acetic
acid and called " dissolved mucin " or " nucleoalbumin " are considered

1 In regard to the literature on this method and the numerous experiments to
determine its value, see Huppert-Neubauer, 10 Aufl., 853 and Neuberg, Der Harn,
s. 765.

2 Christensen, Virchow's Arch., 115; Tsuschija, Centralbl. f. Med., 1908.

3 Deutsch. med. Wochenschrift, 1908.

4 Skand. Arch. f. Physiol., 6.

DETECTION OF NUCLEOALBUMINS. 795

by MORNER to be a combination of proteid chiefly with chondroitin-
sulphuric acid, and to a less extent with nucleic acid, and also perhaps
with taurocholic acid.

As normal urine habitually contains an excess of substances capable
of precipitating proteids, it is apparent that an increased elimination of
so-called nucleoalbumin may be caused simply by an augmented excretion
of proteid. This happens to a still greater extent in cases where the
proteid as well as the proteid-precipitating substance is eliminated to an
increased extent.

Detection of so-called Nucleoalbumins. When a urine becomes cloudy
or precipitates on the addition of acetic acid, and when it gives a more
typical reaction with HELLER'S test after the dilution of the urine than
before, one is justified in making tests for mucin and nucleoalbumin.
As the salts of the urine interfere considerably with the precipitation
of these substances by acetic acid, they must first be removed by dialysis.
As large a quantity of urine as possible is dialyzed (with the addition of
chloroform) until the salts are removed. The acetic acid is added until
it contains 2 p. m., and the mixture allowed to stand. The precipitate
is dissolved in water by the aid of the smallest possible quantity of alkali
and precipitated again. In testing for chrondroitin-sulphuric acid a
part is warmed on the water-bath with about 5 per cent hydrochloric
acid. If positive results are obtained on testing for sulphuric acid and
reducing substance, then chondroproteid was present. If a reducing
substance can be detected but no sulphuric acid, then mucin is probably
there. If it does not contain any sulphuric acid or reducing substance,
a part of the precipitate is exposed to pepsin digestion and another part
used for the determination of any organic phosphorus. If positive results
are obtained from these tests, then nucleoalbumin and nucleoproteid
must be differentiated by special tests for nuclein bases. No positive
conclusion can be drawn except by using very large quantities of urine.
The filtrate from the nucleoalbumin can be used for the ordinary proteid
tests.

Nudeohistone. In a case of pseudoleucsemia A. JOLLES found a phos-
phorized protein substance which he considers as identical with nucleohistone.
Histone is claimed to have been found in some cases by KREHL and MATTHES,
and by KOLISCH and BURIAN. l

The nitrogen contained in the substances precipitated by alcohol, called the
" colloidal nitrogen " by SALKOWSKI and whose quantity is doubled in carcinoma
as compared to the normal, consists in great part of oxyproteic acids. Accord-
ing to SALKOWSKI and KOJO 2 this can be precipitated by basic lead acetate and
the nitrogen determined therein.

Blood and Blood-coloring Matters. The urine may contain blood from
hemorrhage in the kidneys or other parts of the urinary passages (ILSJMA-

1 Jolles, Ber. d. deutsch. chem. Gesellsch., 30; Krehl and Matthes, Deutsch. Arch,
f. klin. Med., 54; Kolisch and Burian, Zeitschr. f. klin. Med., 29.

2Salkowski, Berl. klin. Wochenschr., 1905 and 1910; Kojo, Zeitschr. f. physiol.
Chem., 73.

796 URINE.

TURIA). In these cases, when the quantity of blood is not very small,
the urine is more or less cloudy and colored reddish, yellowish red, dirty
red, brownish red, or dark brown. In recent hemorrhages in which the
blood has not decomposed the color is nearer blood-red. Blood-corpuscles
may be found in the sediment, sometimes also blood-casts and smaller
or larger blood-clots.

In certain cases the urine contains no blood-corpuscles, but only dis-
solved blood-coloring matters, haemoglobin, or, and indeed quite often,
methsemoglobin (H^EMOGLOBINURIA). The blood-pigments appear in the
urine under different conditions, as in dissolution of blood in poisoning
with arseniuretted hydrogen, chlorates, etc., after serious burns, after
transfusion of blood, and also in the periodic appearance of hsemoglo-
binuria with fever. In haemoglobinuria the urine may also have an abun-
dant grayish-brown sediment rich in proteid which contains the remains
of the stromata of the red blood-corpuscles. In animals, haemoglobinuria
may be produced by many causes which force free haemoglobin into the
plasma.

To detect blood in the urine,* we make use of the microscope, the spec-
troscope, the guaiac test, and HELLER'S or HELLER-TEICHMANN'S test.

Microscopic Investigation. The blood-corpuscles may remain undis-
solved for a long time in acid urine; in alkaline urine, on the contrary,
they are easily changed and dissolved. They often appear entirely
unchanged in the sediment; in some cases they are distended and in
others unequally pointed or jagged like a thorn-apple. In hemorrhage of
the kidneys a cylindrical clot is sometimes found in the sediment which is
covered with numerous red blood-corpuscles, forming casts of the urinary
passages. These formations are called blood-casts.

The spectroscopic investigation is naturally of very great value; and if
it be necessary to determine not only the presence but also the kind of
coloring-matter, this method is indispensable. In regard to the optical
behavior of the various blood-pigments we must refer to Chapter V.

Guaiac Test. Mix in a test-tube equal volumes of tincture of
guaiac and old turpentine which has become strongly ozonized by the
action of air under the influence of light. To this mixture, which must
not have the slightest blue color, add the urine to be tested. In the
presence of blood or blood-pigments, first a bluish-green and then a beau-
tiful blue ring appears where the two liquids meet. On shaking the mixture
it becomes more or less blue. Normal urine or one containing proteid
does not give this reaction. According to LIEBERMANN l this reaction
is brought about by the blood pigments acting as catalyst upon the
organic peroxides existing in the turpentine, accelerating the decomposi-
tion of these and the active oxygen taken up by the guaiaconic acid
which is oxidized to guaiac blue (guaiaconic acid ozonide). Urine con-

1 Pfliiger's Arch., 104.

BLOOD PIGMENTS. H^MATOPORPHYRIN. 797

taining pus, even when no blood is present, gives a blue color with these
reagents; but in this case the tincture of guaiac alone, without tur-
pentine, is colored blue by the urine (VITALI 1). This is at least true
for a tincture that has been exposed for some time to the action of air
and sunlight. The blue color produced by pus differs from that pro-
duced by blood-coloring matters by disappearing on heating the urine
to boiling. A urine alkaline by decomposition must first be made faintly
acid before performing the reaction. The turpentine should be kept
exposed to sunlight, while the tincture of guaiac must be kept in a
dark glass bottle. These reagents to be of use must be controlled by a
liquid containing blood. With positive results, however, this test is
not absolutely decisive, because other bodies may give a similar reaction,
but when properly performed it is so extremely delicate that when it
gives negative results any other test for blood is superfluous.2

As the delicacy of the above-mentioned tests is sufficient for ordinary
purposes it is not necessary to give the new blood-tests suggested recently.

HELLER-TEICHMANN'S Test. If a neutral or faintly acid urine containing
blood is heated to boiling, one always obtains a mottled precipitate consist-
ing of proteid and haematin. If caustic soda is added to the boiling-hot test,
the liquid becomes clear and turns green when examined in thin layers (due to
haematin alkali), and a red precipitate, appearing green by reflected light, re-forms,
consisting of earthy phosphates and haematin. This reaction is called HELLER'S
blood-test. If this precipitate is after a time collected on a small filter, it may be
used for the hsemin test (see page 293). If the precipitate contains only a little
blood-coloring matter with a larger quantity of earthy phosphates, then wash
it with dilute acetic acid, which dissolves the earthy phosphates, and use the
residue for the preparation of TEICHMANN'S haemin crystals. If, on the contrary,
the amount of phosphates is very small, then first add a little MgCk solution
to the urine, heat to boiling, and add simultaneously with the caustic potash
some sodium-phosphate solution. In the presence of only very small quantities
of blood, first make the urine very faintly alkaline with ammonia, add tannic
acid, acidify with acetic acid, and use this precipitate in the preparation of the
haemin crystals (STURVE 3).

0. and R. ABLER 4 have recommended leucomalachite green or benzidine in
the presence of peroxide and acetic acid as especially sensitive reagents for blood.

Haematoporphyrin. Since the occurrence of hsematoporphyrin in the
urine in various diseases has been made very probable by several investi-
gators, such as NEUSSER, STOKVIS, MACMUNN, LE NOBEL, COPEMAN, and
others,5 SALKOWSKI has positively shown the presence of this pigment
in the urine after sulfonal intoxication. It was first isolated in a pure

1 See Maly's Jahresber., 18.

2 For more details in regard to the preparation of the reagents and the performance
of the reaction see O. Schumm., Zeitschr. f. physiol. Chem., 50.

3 Zeitschr. f. anal. Chem., 11.

4 Zeitschr. f . physiol. Chem., 41.

5 A very complete index of the literature on haematoporphyrin in the urine may be
found in R. Zoja, Su qualche pigmento di alcune urine, etc., in Arch. Ital. di. clin.
Med., 1893.

798 URINE.

crystalline state by HAMMARSTEN 1 from the urine of insane women after
sulfonal intoxication. According to GARROD and SAiLLET2 traces of
haematoporphyrin (SAILLET'S urospectrin) regularly occur in normal
urines. It is also found in the urine during different diseases. It was
found in great abundance in a case of typhoid fever (ARNOLD3) but
otherwise it generally occurs only in small amounts. It has been found in
considerable quantities in the urine after the lengthy use of sulfonal.

Urine containing haematoporphyrin is sometimes only slightly colored,
while in other cases, as for example, after the use of sulfonal, it is more
or less deep red. In these last-mentioned cases the color depends, in
greatest part, not upon the haematoporphyrin, but upon other red or
reddish-brown pigments which have not been sufficiently studied.

In the detection of small quantities of haematoporphyrin proceed as
suggested by GARROD. Precipitate the urine with a 10-per cent caustic-
soda solution (20 cc. for every 100 cc. of urine). The phosphate pre-
cipitate containing the pigment is dissolved in alcohol-hydrochloric acid
(15-20 cc.) and the solution investigated with the spectroscope. In more
exact investigations make the solution alkaline with ammonia, add enough
acetic acid to dissolve the phosphate precipitate, shake with chloroform,
which takes up the pigment, and test this solution with the spectroscope.

In the presence of larger quantities of hsematoporphyrin the urine
is first precipitated, according to SALKOWSKI, with an alkaline barium-
chloride solution (a mixture of equal volumes of barium-hydroxide solu-
tion, saturated in the cold, and a 10-per cent barium-chloride solution),
or, according to HAMMARSTEN,4 with a barium-acetate solution. The
washed precipitate, which contains the haematoporphyrin, is allowed
to stand some time at the temperature of the room, with alcohol contain-
ing hydrochloric or sulphuric acid, and then filtered. The filtrate shows
the characteristic spectrum of haematoporphyrin in acid solution and gives
the spectrum of alkaline haematoporphyrin after saturation with ammonia.
If the alcoholic solution is mixed with chloroform and a large quantity
of water added and carefully shaken, sometimes a lower layer of chloro-
form is obtained which contains very pure haematoporphyrin, while the
upper layer of alcohol and water contains the other pigments besides
some hiematoporphyrin.

Other methods which have no advantage over this one of GARROD have been
suggested by RIVA and ZOJA as well as SAILLET. 5

BAUMSTARK 6 found in a case of leprosy two characteristic coloring-matters
in the urine, " urorubrohaematin " and " urofuscohaematin," which, as their

1 Salkowski, Zeitschr. f. physiol. Chem., 15; Hammarsten, Skand. Arch. f. Physiol., 3.

2Garrod, Journ. of Physiol., 13 (contains review of literature) and 17; Saillet,.
Revue de Medecine, 16.

* Zeitschr. f . physiol. Chem., 82.

4 Salkowski, 1. c.; Hammarsten, 1. c.

6 Riva and Zoja, Maly's Jahresber., 24; Saillet, 1. c. See also Nebelthau, Zeitschr.
f. physiol. Chem., 27.

6 Pfliiger's Arch., 9.

PUS. BILE ACIDS. 799

names indicate, seem to stand in close relation to the blood-coloring matters.
Urorubrohcematin, C68H94N8Fe2026, contains iron and shows in acid solution an
absorption-band in front of D and a broader one back of D. In alkaline solution
it shows four bands — behind D, at E, beyond F, and behind G. It is not soluble
either in water, alcohol, ether, or chloroform. It gives a beautiful brownish-red
non-dichroic liquid with alkalies. Urofuscohcematin, CesHioeNgC^e, which is free
from iron, shows no characteristic spectrum; it dissolves in alkalies, producing
a brown color. It remains to be proven whether these two pigments are related
to (impure) hsematoporphyrin.

Melanin. In the presence of melanotic cancers dark pigments are some-
times eliminated with the urine. K. MORNER has isolated two pigments from
such a urine, of which one was soluble in warm 50-75 per cent acetic acid, while
the other, on the contrary, was insoluble. The .one seemed to be phymatorhusin
(see Chapter XV). Usually the urine does not contain any melanin, but a
chromogen of melanin, a melanogen. In such cases the urine gives EISLET'S
reaction, becoming dark-colored with oxidizing agents, such as concentrated
nitric acid, potassium bichromate, and sulphuric acid, as well as with free sulphuric
acid. They also give THORMAHLEN'S reaction namely a beautiful blue coloration
with sodium nitroprusside and then acetic acid. Urine containing melanin or
melanogen is colored black by a ferric-chloride solution (v. JAKSCH x).

In a case of melanotic sarcoma H. EPPINGER 2 has isolated from the urine
a crystalline melanogen of the composition CflHi2N2S04, and which was insoluble
in ether. It gave the ordinary melanogen reactions and, according to him is
probably an amidated ethereal sulphuric acid of methylpyrrolidinoxycarboxylic
acid, which is derived from tryptophane.

Pus occurs in the urine in various inflammatory affections, especially
in catarrh of the bladder and in inflammation of the pelvis of the
kidneys, or of the urethra.

Pus is best detected by means of the miscroscope. The pus-cells are
rather easily destroyed in alkaline urines. In detecting pus we make
use of DONNA'S pus test, which is performed in the following way: Pour
off the urine from the sediment as carefully as possible, place a small
piece of caustic alkali on the sediment, and stir. If the pus-cells have
not been previously changed, the sediment is converted by this means
into a slimy tough mass.

The pus-corpuscles swell up in alkaline urines, and dissolve, or at least
are so changed that they cannot be recognized under the microscope.
The urine in these cases is more or less slimy or fibrous, and the proteid
can be precipitated in large flakes by acetic acid, so that it might possibly
be mistaken for mucin. The closer investigation of the precipitate
produced by acetic acid, and especially the appearance or non-appearance
of a reducing substance after boiling it with a mineral acid, demonstrates
the nature of the precipitated substance. Urine containing pus always
contains proteid.

Bile-acids. The reports in regard to the occurrence of bile-acids in the
urine under physiological conditions do not agree. According to DRAGEN-
DORFF and HONE traces of bile-acids occur in the urine; according to MAO

^hormahlen, Virchow's Arch., 108; v. Jaksch, Zeitschr. f. physiol. Chem., 13.
2 Bioch. Zeitschr., 28.

800 URINE.

KAY and v. UDR£NSZKY and K. MORNER* they do not. Pathologically
they are present in the urine in hepatogenic icterus, although not invar-
iably.

Detection of Bile-acids in the Urine. PETTENKOFER'S test gives the most
decisive reaction; but as it gives similar color reactions with other bodies, it must
be supplemented by the spectroscopic investigation. The direct test for bile-
acids is easily performed after the addition of traces of bile to a normal urine.
But the direct detection in a colored icteric urine is more difficult and gives very
misleading results; the bile-acid must therefore always be isolated from the urine.
This may be done by the following method of HOPPE-SEYLER, which is slightly
modified in non-essential points.

HOPPE-SEYLER'S Method. Concentrate the urine and extract the residue
with strong alcohol. The filtrate is freed from alcohol by evaporation and then
precipitated by basic lead acetate and ammonia. The washed precipitate is
treated with boiling alcohol, filtered hot, the filtrate treated with a few drops
of soda solution, and evaporated to dryness. The dry residue is extracted with
absolute alcohol, filtered, and an excess of ether added. The amorphous or,
after a longer time, crystalline, precipitate consisting of the alkali salts of the
biliary acids is used in performing PETTENKOFER'S test.

Bile-pigments occur in the urine in different forms, of icterus. A
urine containing bile-pigments is always abnormally colored — yellow,
yellowish brown, deep brown, greenish yellow, greenish brown, or nearly
pure green. On shaking it froths, and the bubbles are yellow or yellowish
green in color. As a rule icteric urine is somewhat cloudy, and the sedi-
ment is frequently, especially when it contains epithelium-cells, rather
strongly colored by the bile-pigments.

Detection of Bile-coloring Matters in Urine. Many tests have been
proposed for the detection of these substances. Ordinarily we obtain
the best results with the following three tests :

* GMELIN'S test may be applied directly to the urine ; but it is better to
use ROSENBACH'S modification. Filter the urine through a very small
filter, which becomes deeply colored from the retained epithelium-cells
and bodies of that nature. After the liquid has entirely passed through
apply to the inside of the filter a drop of nitric acid which contains only
very little nitrous acid. A pale-yellow spot will be formed which is sur-
rounded by colored rings which appear yellowish red, violet, blue, and
green from within outward. This modification is very delicate, and it
is hardly possible to mistake indican and other coloring-matters for the
bile-pigments. Several other modifications of GMELIN'S direct test, e.g.,
with concentrated sulphuric acid and nitrate, etc., have been proposed,
but they are neither simpler nor more delicate than ROSENBACH'S modifica-
tion.

HUPPERT'S Reaction. In a dark-colored urine or one rich in indican
good results are not always obtained with GMELIN'S test. In such cases,
as also in urines containing blood-coloring matters at the same time,
the urine is treated with lime-water, or first with some CaCb solution,

1 Cited from Huppert-Neubauer, Ham- Analyse, 10. Aufl., 229.

BILE PIGMENTS. 801

and then with a solution of sodium or ammonium carbonate. The pre-
cipitate which contains the bile-coloring matter is filtered, washed, dis-
solved in alcohol which contains 5 cc. of concentrated hydrochloric acid
in 100 cc. (I. MUNK), and heated to boiling, when the solution becomes
green or bluish green. According to NAKAYAMA 1 this reaction is more
delicate on using a mixture of ferric chloride, acid, and alcohol.

HAMMARSTEN'S Reaction. For ordinary cases it is sufficient to add
a few drops of urine to about 2-3 cc. of the reagent (see page 432), when
the mixture immediately after shaking turns a beautiful green- or bluish
green, which color remains for several days. In the presence of only
very small quantities of bile-pigments, especially when blood or other
pigments are simultaneously present, pour about 10 cc. of the acid or
nearly neutral (not alkaline) urine into the tube of a small centrifugal
machine and add BaCb solution and centrifuge for about one minute.
The liquid is decanted and the sediment stirred with about 1 cc. of the
reagent and centrifuged again. A beautiful green solution is obtained
which may be changed, by the addition of increased quantities of the acid
mixture, to blue, violet, red, and reddish yellow. The green color may
be obtained in the presence of 1 part bile-pigment in 500,000-1,000,000
parts urine. In the presence of large amounts of other pigments calcium
chloride is better suited than barium chloride.

BouMA2 has suggested the use of alcohol containing ferric chloride
and hydrochloric acid instead of the above-mentioned acid mixture. He
has also worked out a colorimetric method of quantitative estimation
of bilirubin in urine by means of this reagent.

As above indicated, we have a great many other tests besides these
given above. A very complete summary of these tests and the literature
thereof can be found in the work of OBERMAYER and POPPER.

For ordinary purposes the above-mentioned tests are sufficiently
delicate, and according to HAMMARSTEN it is not advisable, as also in the
case of the detection of proteid, sugar, etc., to increase the delicacy of
a test so that it shows the presence of the traces of the questionable
substance in normal urine. If in certain cases a greater delicacy is
required than is obtained with the above tests, then we must recommend
the flotation test of OBERMEYER and POPPER 3 with iodine and salt.

MEDICINAL COLORING-MATTERS produced from santonin, rhubarb, senna, etc.,
may give an abnormal color to the urine and may be mistaken for bile-pigments,
or, in alkaline urines, perhaps for blood-coloring matters. If hydrochloric acid
is added to such a urine, it becomes yellow or pale yellow, while on the addition
of an excess of alkali it takes on a more or less beautiful red color.

1 Munk, Arch. f. (Anat. u.) Physiol., 1898; Nakayama, Zeitschr. f. physiol. Chem.,

2 Deutsch. med. Wochenschr., 1902 and 1904
» Wien. med. Wochenschr., 21.

802 URINE.

Sugar in Urine.

The occurrence of traces of glucose in the urine of perfectly healthy
persons has been, as above stated (page 749), quite positively proven. If
sugar appears in the urine in constant and especially in large quantities,
it must be considered as an abnormal constituent. In a previous chapter
several of the principal causes of glycosuria in man and animals were men-
tioned, and the reader is referred to Chapters VII and VIII for the essen-
tial facts in regard to the appearance of sugar in the urine.

In man the appearance of glucose in the urine has been observed
under various pathological conditions, such as lesions of the brain and
especially of the medulla oblongata, abnormal circulation in the abdomen,
diseases of the heart, lungs and liver, cholera, and many other diseases.
The continued presence of sugar in human urine, sometimes in very con-
siderable quantities, occurs in DIABETES MELLITUS. In this disease there
may be elimination of 1 kilogram or even more of glucose per day.
In the beginning of the disease, when the quantity of sugar is still very
small, the urine often does not appear abnormal. In the more developed,
typical cases the quantity of urine voided increases considerably, to
3-6-10 liters per day. The percentage of the physiological constituents
is as a rule very low, while their absolute daily quantity is increased.
The urine is pale, but of a high specific gravity, 1.030-1.040 or even higher.
The high specific gravity depends upon the quantity of sugar present,
which varies in different cases, but may reach 10 per cent. The urine is
therefore characterized in typical cases of diabetes by the very large
quantity voided, by the pale color and high specific gravity, and by its
containing sugar.

That the urine after the introduction into the system of certain medici-
nal agents or poisonous bodies contains reducing substances, conjugated
glucuronic acids, which may be mistaken for sugar, has already been men-
tioned.

Glucose in urine. The properties and reactions of this sugar have been
considered in a previous chapter, and it remains but to mention the methods
for the detection and quantitative determination of glucose in the urine.

The detection of sugar in the urine is ordinarily, in the presence of not
too small quantities, a very simple task. The presence of only very small
quantities may make its detection sometimes very difficult and laborious.
A urine containing proteid must first have the proteid removed by coagu-
lation with acetic acid and heat before it can be tested for sugar.

The tests which are most frequently employed and are especially
recommended are as follows:

TROMMER'S Test. In a typical diabetic urine or one rich in sugar this
test succeeds well, and it may be performed in the manner suggested on

SUGAR IN URINE. 803

page 214. This test may lead to very great mistakes in urine poor in
sugar, especially when they have at the same time normal or increased
amounts of physiological constituents, and therefore it cannot be recom-
mended to physicians or to persons inexperienced in such work. Normal
urine contains reducing substances, such as uric acid, creatinine, and others,
and therefore a reduction takes place in all urines on using this test. A
separation of copper suboxide does not generally occur, but still if one
varies the proportion of the alkali to the copper sulphate and boils, there
takes place an actual separation of suboxide in normal urines, or a peculiar
yellowish red liquid due to finely divided cuprous hydroxide. This occurs
especially on the addition of much alkali or too much copper sulphate,
and by careless manipulation the inexperienced worker may therefore
sometimes obtain apparently positive results in a normal urine. On the
other hand, as the urine contains substances such as creatinine and
ammonia (from the urea), which in the presence of only a little sugar
may keep the copper suboxide in solution, the investigator may easily
overlook small quantities of sugar that may be present.

The delicacy of TROMMER'S test can be increased by the suggestion made by
WoRM-MtJLLER.1 As by this rather complicated and tedious method small
amounts of sugar cannot be detected in certain urines, and also as special urines
from healthy persons readily give inconclusive results, and finally as SCHONDORFF
has shown in numerous cases that the physiological sugar content of the urine
responds to this test in perfectly healthy persons because of its extreme delicacy,
it does not seem advisable in HAMMARSTEN'S opinion to recommend this test to
the physician. BANG and BOHMANSSON 2 have recently also shown its unre-
liability.

ALMEN'S bismuth test, which has been incorrectly called NYLANDER'S
test, is performed with the alkaline-bismuth solution prepared as described
on page 214. For each test 10 cc. of urine are taken and treated with
1 cc. of the bismuth solution and boiled for a few minutes. In the
presence of sugar the urine becomes dark yellow or yellowish brown;
then it grows darker, cloudy, dark brown, or nearly black, and non-
transparent. After a longer or shorter time a black deposit appears,
the supernatant liquid gradually clears, but still remains colored. In
the presence of only very little sugar the test does not become black or
dark brown, but simply deeper colored, and not until after some time
is there seen on the upper layer of the phosphate precipitate a dark or
black layer (of bismuth?). In the presence of much sugar a larger
amount of the reagent may be used without disadvantage. In a urine
poor in sugar only 1 cc. of the reagent for every 10 cc. of the urine must
be employed.

1 In regard to this test see Pfliiger, Pfliiger's Arch., 105 and 106; Hammarsten,
ibid., 116, and Zeitschr. f. physiol. Chem., 50.

2 Schondorff, Pfluger's Arch., 121; Bohmansson, Bioch. Zeitschr., 19.

804 URINE.

Small amounts of proteid may retard this reaction and reduce the
delicacy of the test. Large quantities of proteid may, however, give
rise to an error by forming bismuth sulphide, and therefore it must
always be first removed. The assertion of BECHHOLD that mercury
compounds in the urine disturb the test has not been substantiated by
ZEIDLITZ on properly performing the test, and recently REHFUSS and
HAWK 1 came to the same conclusion. Those sources of error which
in TROMMER'S test are caused by the presence of uric acid and creatinine
are removed by using this test. The bismuth test is, moreover, readily
performed, and on this account is to be recommended to the physician.

The bumping and ejection of the fluid can be readily prevented by heating
over a very small flame after the test has been brought to a boil, and by gently
shaking the contents of the not too narrow test-tube. The recommendation
of heating for a longer time in the water-bath, fifteen minutes or more, is to be
discarded, as the delicacy of the test is thereby so much increased that it gives
a reaction with a physiological sugar content of 0.02 per cent.

When the amount of sugar in the urine is not less than 0.1 per cent
a positive reaction is obtained if the test is boiled for 2-3 minutes and
then allowed to stand quietly for 5 minutes. The phosphate precipitate
is then black or nearly black. In detecting smaller quantities of sugar
— 0.05 per cent, the test as a rule must be boiled longer — about 5 minutes.

The value of this test lies in the fact that it positively detects small
quantities of sugar — 0.1 per cent or somewhat less, and that when the
urine gives negative results we can consider it free from sugar in a clinical
sense. Like TROMMER'S test it is a reduction test, and shows also certain
other reducing bodies besides the sugar. These bodies are certain con-
jugated glucuronic acids which may appear in the urine. After the use
of certain therapeutic agents, such as rhubarb, senna, antipyrine, salol,
turpentine and others, the bismuth test gives positive results. From
this it follows that we should never be satisfied with this test alone, espe-
cially when the reduction is not very great.

According to BOHMANSSON and BANG this test is perfectly reliable
if about 20 cc. of the urine is treated with 5 cc. of 25 per cent HC1 and
2 grams blood-charcoal (a teaspoonful) added and shaken every once in
a while during five minutes and then filtered. The filtrate on neutraliza-
tion with caustic soda is used for the ALMEN test. The disturbing reduc-
ing substances are removed by the animal charcoal, but the sugar is not.

According to ANDERSEN 2 this procedure cannot be used in the quan-

1 Bechhold, Zeitschr. f. physiol. Chem., 46; Zeidlitz, Upsala Lakaref. Forh. (N. F.),
11 (Hammarsten Festschr.); Rehfuss and Hawk, Journ. of biol. Chem., 7.

2 Bohmansson and Bang, Bioch. Zeitschr., 19, and Zeitschr. f. physiol. Chem.,
63; Andersen, Bioch. Zeitschr., 37.

SUGAR IN URINE. 805

titative estimation of sugar as a part of the sugar is retained by the
use of hydrochloric acid and blood-charcoal. According to ANDERSEN
the pigments and the disturbing substances can be removed by per-
cipitation with mercuric nitrate. It can be more simply done by treating
40 cc. of the urine with 10 cc. acetic acid of 50 per cent strength and 4
grams blood-charcoal, shaking as above described and filtering. In the
presence of acetic acid no sugar is taken up by the charcoal and as this
simple method can be used for the quantitative estimation it can therefore
be used in the qualitative tests for sugar.

Fermentation Test. On using this test the process must vary accord-
ing as the bismuth test shows small or large quantities of sugar. If a
rather strong reduction is obtained, the urine may be treated with yeast
and the presence of sugar determined by the generation of carbon
dioxide. In this case the acid urine, or that faintly acidified with a little
tartaric acid is treated with compressed yeast, or yeast which has pre-
viously been washed by decantation with water. Pour this urine
to which the yeast has been added into a SCHROTTER'S gas burette or a
LOHNSTEIN'S saccharimeter (see below). As the fermentation proceeds,
the carbon dioxide collects in the upper part of the tube, while a correspond-
ing quantity of liquid is expelled below. As a control in this case two
similar tests must be made, one with normal urine and yeast to learn
the quantity of gas usually developed, and the other with a sugar solu-
tion and yeast to determine the activity of the yeast. According to
VICTOROW l the fermentation is complete after six hours at a tem-
perature of 34-36° C.

If, on the contrary, only a faint reduction with the bismuth test
is found, no positive conclusion can be drawn from the absence of any
carbon dioxide or the appearance of a very insignificant quantity. The
urine absorbs considerable amounts of carbon dioxide, and in the presence
of only small amounts of sugar the fermentation test as above performed
may lead to negative or inaccurate results. In this case proceed in the
following way: Treat the acid urine, or urine which has been faintly
acidified with tartaric acid, with yeast whose activity has been tested
by a special test on a sugar solution, and allow it to stand six to twelve
hours at about 34-36° C. Then test again with the bismuth test, and
if the reaction now gives negative results, then sugar was previously
present. But if the reaction continues to give positive results, then it
shows, if the yeast is active, the presence of other reducing, unfer-
mentable substances.

In performing the fermentation test care should be taken that the urine
be acid before as well as after fermentation. If the reaction becomes

1 Pfliiger's Arch., 118.

806 URINE.

alkaline during fermentation (alkaline fermentation), then the test
must be discarded. The vessel must be perfectly clean and strongly
heated before use. To make sure the urine may be boiled before fer-
mentation.1

If a good polariscope is at hand it must not be forgotten to control
the results of the fermentation by determining the rotation before and
after fermentation. The phenylhydrazine test also, in many otherwise
doubtful cases, gives good service in testing urines for sugar.

Phenylhydrazine Test. Can be performed in the following manner:
20-25 cc. urine in a test-tube or in a beaker covered with a watch-glass
are treated with 1 gram phenylhydrazine hydrochloride and 2 grams
sodium acetate, and after solution of the salts it is warmed on the water-
bath for three-quarters of an hour. In the presence of sugar even dur-
ing the warming, a precipitate occurs, or in the presence of only a little
sugar, at least after the gradual cooling, a yellow, crystalline precipitate
forms. If the precipitate is very slight, it can be collected to advantage
by means of a centrifuge and investigated by aid of the microscope.
One finds at least a few phenylglucosazone crystals in the sediment
while the appearance of smaller or larger yellow platelets or strongly
refractive, brown globules is not indicative of sugar. In the presence
of large amounts of sugar in the urine a large quantity of the yellow
needles of phenylglucosazone or a mass of them are obtained.

This reaction is very reliable, and by it the presence of 0.03 per cent
sugar can be detected (ROSENFELD, GEYER2). In doubtful cases it is
necessary to investigate the nature of the precipitate. For this purpose
dissolve a large quantity of the crystals in hot alcohol, treat the filtrate
with water, and boil off the alcohol. Still better, the precipitate is
dissolved, according to NEUBERG, in some pyridine, and again precipi-
tated as crystals by the addition of benzene, ligroin, or ether. If the
characteristic yellow crystalline needles, whose melting-point (204-
205° C.) may also be determined, are now obtained, then this test is
decisive for the presence of sugar. It must not be forgotten that fructose
gives the same osazone as glucose, and that a further investigation is
necessary in certain cases, and also that the impure crystals of phen-
ylglucosazone have a much lower melting-point than the pure ones.

The following modification by A. NEUMANN is simple, practical, and at the
same time sufficiently delicate. 5 cc. of the urine are treated with 2 cc. of acetic
acid (30-per cent) saturated with sodium acetate, 2 drops of pure phenylhydrazine

1 On the performance of the fermentation test and certain sources of error, see
Salkowski, Berlin, klin. Wochenschr., 1905 (Ewald-Festnummer), and Pfluger, Pfliiger's
Arch., 105 and 111.

2 Rosenfeld, Deutsch. med. Wochenschr., 1888; Geyer, cited from Roos, Zeitschr.
f. physiol. Chem., 15.

SUGAR IN URINE. 807

added, and the mixture boiled in a test-tube until it measures 3 cc. After quickly
cooling warm again and then allow it to cool slowly. After 5-10 minutes beautifully
formed crystals are obtained even in the presence of only 0.02 per cent sugar.
According to the experience of HAMMARSTEN this modification, even in the presence
of 0.1 per cent sugar in concentrated urines, does not always give a positive reac-
tion. SALKOWSKI x has suggested an even more simple method.

The value of the phenylhydrazine test has been considerably debated,
and the objection has been made that glucuronic acids also give a similar
precipitate. A confounding with glucuronic acid is, according to HIBSCHL,
not to be apprehended when the test is heated in the water-bath for a
long time (one hour). KTSTERMANN found this precaution insufficient,
and Roos states that the phenylhydrazine test always gives a positive
result with human urine, which coincides with E. HOLMGREN'S 2 and
HAMMERSTEN'S experience. This test only shows a non-physiological
quantity of sugar when a rather abundant crystallization is obtained from
a small quantity of urine (about 5-10 cc.) Too great a delicacy of this
test is not to be recommended.

RUBNER'S test is performed as follows: The urine is precipitated with an excess
of a concentrated lead-acetate solution and the filtrate carefully treated with
enough ammonia to produce a flocculent precipitate. It is then heated to boiling,
when the precipitate becomes flesh-colored or pink in the presence of sugar.

Polarization. This test is of great value, especially as in many cases
it quickly differentiates between glucose and other reducing, sometimes
levogyrate, substances, such as the conjugated glucuronic acids. In
the presence of only very little sugar the value of this test depends on
the delicacy of the instrument and the dexterity of the observer. As a
urine which shows no rotation or is actually faintly levorotatory, may
contain 0.2 per cent glucose or perhaps even more, this test must be
combined with the fermentation test if we are seeking very small amounts
of sugar. The sugar in these cases can be detected only by the use of a
very accurate and delicate instrument. This method is in many cases
not serviceable for the physician. If the urine is to be clarified and
partly decolorized by precipitation with lead acetate, it must be done in
acid solution with acetic acid.3

In the isolation of sugar and carbohydrates from the urine the benzoic-acid
esters may be prepared according to BAUMANN'S method. The urine is made
alkaline with caustic soda to precipitate the earthy phosphates, the filtrate treated
with 10 cc. of benzoyl chloride and 120 cc. of 10 per cent caustic soda solution
for every 100 cc. of the filtrate (REINBOLD 4), and shaken until the odor of benzoyl

1 Neumann, Arch. f. (Anat. u.) Physiol., 1899, Suppl. See also Margulies, Berlin,
klin. Wochenschr., 1900; Salkowski, Arbeiter aus dem pathol. Inst., Berlin, 1906.

* Hirschl, Zeitschr. f. physiol. Chem., 14; Kistermann, Deutsch. Arch. f. klin.
Med., 50; Roos, 1. c.; Holmgren, Maly's Jahresber., 27.

3 See Grossmann, Bioch. Zeitschr., 1.

4 Pfliiger's Arch., 91.

808 URINE.

chloride has disappeared. After standing sufficiently long the ester is collected,
finely divided, and saponified with an alcoholic solution of sodium ethylate in the
cold according to BAISCH'S method,1 and the various carbohydrates separated
according to his suggestion.

If small quantities of sugar are to be isolated from the urine, precipitate the
urine first with sugar of lead, filter, precipitate the filtrate with ammoniacal
basic lead acetate, wash this precipitate with water, decompose it with H2S when
suspended in water and use the filtrate for the special tests. SCHONDORFF 2
has suggested a method for the detection and estimation of very small amounts
of sugar based upon the work of PATEIN and DUFAU. This method depends
upon precipitating the nitrogenous substances with mercuric nitrate.

To the physician, who naturally wants simple and quick methods,
the bismuth test is especially to be recommended. If this test gives neg-
ative results, the urine is to be considered as free from sugar in a clinical
sense. If it gives positive results, the presence of sugar must be con-
trolled by other tests, especially by the fermentation test.

Other tests for sugar, as, for example, the reaction with orthonitrophenyl-
propiolic acid, picric acid, diazobenzene-sulphonic acid, are superfluous. The
reaction with a-naphthol, which is a reaction for carbohydrates in general, for
glucuronic acid and mucin, may, because of its extreme delicacy, give rise to
mistakes, and is therefore not to be recommenced to physicians. Normal urines
give this test, and if the strongly diluted urine gives the reaction the presence
of great quantities of carbohydrates may -be suspected. In these cases more
positive results are obtained by using other tests. This test requires great clean-
liness, and it has the inconvenience that sufficiently pure sulphuric acid is not
always readily procurable. Several investigators, such as v. UDRANSKY, LUTHER,
Roos and TREUPEL,3 have investigated this test in regard to its applicability
as an approximate test for carbohydrates in the urine.

Quantitative Determination of Sugar in the Urine. The quantity of
sugar can be determined by titration, by fermentation of the sugar, by
polarization, and also in other ways.

The titration methods are based upon the property of the sugar to
reduce metallic oxides in alkaline solutions. As the titration liquids
(cupric oxide solution in the FEHLING-SOXHLET, PAVY, BANG, BERTRAND
methods and mercuric oxide in KNAPP'S method) are also reduced by
other urinary constituents, these reduction methods always give too high
results. When large quantities of sugar are present, as in typical
diabetic urine, which generally contains a lower percentage of normal
reducing constituents, this is indeed of little account; but when small
quantities of sugar are present in an otherwise normal urine, the mistake
may, on the contrary, be important, as the reducing power of normal
urine may correspond to 5 p. m. glucose (see page 749). In such cases
the titr.ation procedure must be employed in connection with the fer-
mentation method, which will be described later.

1 Zeitschr. f. physiol. Chem., 19.

8 Pfliiger's Arch., 121, which cites the work of Patein and Dufau.

' See Roos and Treupel, Zeitschr. f. physiol. Chem., 15 and 16.

ESTIMATION OF SUGAR IN URINE. 809

Of the titration methods with copper solutions the method suggested
by BANG is the simplest, and at the same time seems to be more reliable
than any of the others. For this reason we will describe only this method
and refer to the original works and to HOPPE-SEYLER-THIERFELDER,
Handbuch der Chem. Analyses, 1909, for description of the titration of
FEHLING'S solution according to SOXHLET l and to the titration accord-
ing to PAVY and KuMAGAWA-SuTO.2

BANG'S First Method.3 The principle of this method is that when urine
is boiled with an excess of a solution of potassium carbonate, potassium
thiocyanate and copper sulphate, copper thiocyanide is formed, and this
remains in solution as a colorless compound. The excess of cupric
oxide remaining is determined by titration with a solution of hydroxyl-
amine until the blue color disappears. The quantity of sugar is calculated
from the quantity of hydroxylamine used.

The following solutions are necessary: (a) A copper salt solution
containing 25 grams cupric sulphate in 2 liters, and (6) a solution con-
taining 6.55 grams hydroxylamine sulphate in 2 liters.

The copper solution is prepared in the following manner: Dissolve 100 grams
potassium bicarbonate in 1300 cc. water in a 2-liter graduated flask, and if nec-
essary warm to 50-60° C. After complete solution of the bicarbonate, add
400 grams potassium thiocyanate and 500 grams potassium carbonate. To
this solution, which must have the temperature of the room, add very slowly
150 cc. of a copper sulphate solution, which contains 166.67 grams copper sul-
phate (CuS04+5HiiO) per liter, then add water up to 2 liters. This solution
unfortunately does not keep indefinitely, still, according to ANDERSEN, it can be
kept in the dark up to 3 months and its strength controlled by titration with the
hydroxylamine solution. The hydroxylamine solution is prepared by dissolving
200 grams potassium thiocyanate in about 1500 cc. water in a 2-liter graduated
flask and adding a solution of 6.55 grams hydroxylamine sulphate in water; then
add water to the 2-liter mark. This solution, on the contrary keeps, but it must
be kept in a dark-colored bottle. Equal volumes of each of these two solutions
should exactly correspond to each other, and this can be determined by titrating
at ordinary temperature 50 cc. of the copper solution (plus 10 cc. water) with the
hydroxylamine solution.

The presence of proteid does not interfere with the reaction, and it
is not necessary to remove the proteid. The urine for titration should
not contain more than 0.6 per cent sugar. If the amount is lower, then
10 cc. of urine is used directly; if it is higher, then the urine is corre-
spondingly diluted and of this diluted urine we also make use of 10 cc.
in the titration. The quantities of sugar given in the table below vary
between 0.9 and 60 milligrams in 10 cc.

Performance of the Determination. 10 cc. of the sugar fluid are placed
in a glass flask and treated with 50 cc. of the copper solution. This is
heated on a wire-gauze to boiling, boiled for three minutes, cooled
quickly with water to the temperature of the room and then the hydrox-

1 Journ. f. prakt. Chem., (N. F.), 21.

2 Pavy, The Physiology of the Carbohydrates, London, 1894; Kumagawa and Suto,
Salk'owski's Festschr., 1904; Sahli, Deutsch. med. Wochenschr., 1905.

3 Bang, Bioch. Zeitschr., 2, 11, 32, and 38. See also Funk, Zeitschr. f. physiol.
Chem., 56 and 69; Jessen-Hansen, Bioch. Zeitschr., 10 and Andersen, ibid., 15 and 26.

810

URINE.

ylamine solution allowed to flow in from a burette until the blue color
disappears and the solution is colorless, or, in urine poor in sugar, is yellow.
The sugar in milligrams is directly obtained from the amount of hydroxyl-
amine solution used by referring to the following reduction table : l

Hydroxyl-
amine
solution
used.

Milligrams
sugar.

Hydroxyl-
amine
solution
used.

Milligrams
sugar.

Hydroxyl-
amine
solution
used.

Milligrams
sugar.

Hydroxyl-
amine.
solution
used.

Milligrams
sugar.

0.75

60.0

13.00

39.0

25.50

23.5

38.00

10.4

1.00

59.4

13.50

38.3

26.00

22.9

38.50

9.9

1.50

58.4

14.00

37.7

26.50

22.3

39.00

9.4

2.00

57.3

14.50

37.1

27.00

21.8

39.50

9.0

2.50

56.2

15.00

36.4

27.50

21.2

40.00

8.5

3.00

55.0

15.50

35.8

28.00

20.7

40.50

8.1

3.50

54.3

16.00

35.1

28.50

20.1

41.00

7.6

4.00

53.4

16.50

34.5

29.00

19.6

41.50

7.2

4.50

52.6

17.00

33.9

29.50

19.1

42.00.

6.7

5.00

51.6

17.50

33.3

30.00

18.6

42.50

6.3

5.50

50.7

18.00

32.6

30.50

18.0

43.00

5.8

6.00

49.8

18.50

32.0

31.00

17.5

43.50

5.4

6.50

48.9

19.00

31.4

31.50

17.0

44.00

4.9

7.00

48.0

19.50

30.8

32.00

16.5

44.50

4.5

7.50

47.2

20.00

30.2

32.50

15.9

45.00

4.1

8.00

46.3

20.50

29.6

33.00

15.4

45.50

3.7

8.50

45.5

21.00

29.0

33.50

14.9

46.00

3.3

9.00

44.7

21.50

28.3

34.00

14.4

46.50

2.9

9.50

44.0

22.00

27.7

34.50

13.9

47.00

2.5

10.00

43.3

22.50

27.1

35.00

13.4

47.50

2.1

10.50

42.5

23.00

26.5

35.50

12.9

48.00

1.7

11.00

41.8

23.50

25.8

36.00

12.4

48.50

1.3

11.50

41.1

24.00

25.2

36.50

11.9

49.00

0.9

12.00

40.4

24.50

24.6

37.00

11.4

12.50

39.7

25.00

24.1

37.50

10.9

For every TO cc. hydroxylamine solution used more than given in the table
between 49.00-15.00, subtract 0.1 milligram from the corresponding sugar value
and 0.2 milligram between 15.00-1.0.

The yellow color of the urine may be somewhat disturbing for the
end reaction so that with little experience an error of 0.5 cc. hydroxylamine
solution ( = about 0.5 milligram sugar) may occur. In order to decolorize
the urine we can precipitate, according to ANDERSEN, with mercuric
nitrate, when the greatest part of the disturbing reducing substances are
removed, and then the excess of mercury removed by caustic soda and
shaking with zinc. Still simpler is the suggestion mentioned on page
805 with blood-charcoal after acidification with acetic acid.

BANG 2 decolorizes by the addition of 2 cc. alcohol of 95-97 per cent
and a teaspoonful of blood-charcoal to 18 cc. urine, shaking and filtering
immediately. By this means 50 per cent of the other reducing substances

1 This table is given with the permission of the publisher, Julius Springer, Berlin,
where it can be obtained at a low cost.

•Andersen, Bioch. Zeitschr., 15 and 37; Bang, ibid., 38.

ESTIMATION OF SUGAR IN URINE. 811

are removed. If an acidified urine is used for the titration then the urine
is added to the copper solution and not the reverse.

BANG'S Second Method. As the reagents necessary for the preceding titration
are expensive, and as the copper solution only keeps for three months, and the
preparation of the solutions requires great exactitude and is somewhat difficult,
and as the method gives somewhat higher results than other reduction methods
due to the high alkali and salt content of the solutions, BANG l has recently
modified his original method. Instead of potassium thiocyanate he uses potassium
chloride, which can also keep the cuprous oxide in solution as a colorless com-
pound. Also the non-reduced cupric oxide remaining, as in the early method, is not
determined, but the cuprous oxide formed in the reduction with the sugar is directly
determined by titration. This is done by means of a N/100 (or N/10 or N/25)
iodine solution, which in the alkaline liquid acts oxidizingly with the formation
of cupric oxide, according to the formula: CuCl+I+K2CO3=CuC03+KCl+KL
Starch solution is used as indicator. As the potassium chloride can only hold
small amounts of cuprous oxide in solution, and as the end-reaction with the blue
iodine-starch cannot be determined with ease in the presence of large amounts
of cupric oxide in solution, but can easily be done with the faintly blue coloration
due to cupric oxide, by this method a maximum of 10 milligrams sugar can only be
determined. On this account a urine rich in sugar must be diluted considerably
before titration. It must also be remarked that the iodine does not only react
with the cuprous oxide but also with other urinary constituents, and the importance
of this method on titration with rich urines, poor in sugar, has not been sufficiently
investigated. This method has given good results with pure sugar solutions and
with blood; but as its use for the determination of sugar in the urine has not
been sufficiently tested, we have only given the chief points of the method.

BERTRAND'S 2 Titration Method is more complicated than BANG'S method and
does not seem to have any special advantages over this latter, at least in regard
to the determination of sugar in the urine. A part of the cuprous oxide here also
remains in solution and like the titration, according to FEHLING, the cuprous
oxide sometimes settles only with difficulty. As this method seems to be used
extensively we will give the principles of the method.

The method consists in boiling the sugar solution (sugar urine) with an excess
of FEHLING'S solution. The cuprous oxide, freed from copper salt by decantation
and washing (under special precautions), is dissolved by ferric sulphate in sulphuric
acid, and the ferrous sulphate produced is determined by titration with potassium
permanganate, standardized by oxalic acid. The equations of the reactions are
as follows:

1. Cu20+Fe2(S04)3+H2S04 = H20+2CuS04+2FeS04

2. 10FeS04+2KMn04+8H2S04 = 8H20+5Fe2(S04)3+ 2MnS04+K2S04.

2 Cu are equivalent to 2 Fe, and as these are equivalent to 1 mol. oxalic acid,
then from the amount of oxalic acid (ammonium oxalate) used in the standardiza-
tion of the potassium permanganate solution the quantity of copper separated as
cuprous oxide can be readily calculated. The corresponding quantity of sugar
may be found in a special table.

For exact determinations of sugar the method as suggested 'by ALLIHN
and modified by PFLUGER 3 is the best suited.

1 Bioch. Zeitschr., 49.

a Bulletin de la Soc. chim., (3), 35, (1906).

« Pfluger's Arch., 66.

812 URINE.

The TITRATION ACCORDING TO KNAP? depends on the fact that mercuric
cyanide in alkaline solution is reduced to metallic mercury by glucose. The
titration liquid should contain 10 grams of chemically pure dry mercuric cyanide
and 100 cc. of caustic-soda solution of a specific gravity of 1.145 per liter. When
the titration is performed as described below (according to WORM-MULLER and
OTTO), 20 cc. of this solution should correspond to exactly 0.05 gram of glucose.
If the process is carried out in other ways, the value of the solution is different.

In this titration also, the quantity of sugar in the urine should be between
\ and 1 per cent, and the extent of dilution necessary be determined by a pre-
liminary test. To determine the end-reaction as described below, the test for the
excess of mercury is made with sulphuretted hydrogen.

In performing the titration allow 20 cc. of KNAPP'S solution to flow into a
flask and dilute with 80 cc. of water, or when the urine contains less than 0.5
per cent of sugar use only 40-60 cc. After this heat to boiling and allow the diluted
urine to flow gradually into the hot solution, at first 2 cc., then 1 cc., then 0.5 cc.,
then 0.2 cc., and lastly 0.1 cc. After each addition let it boil f minute. When
the end-reaction is approaching, the liquid begins to clarify and the mercury
separates with the phosphates. The end-reaction is determined by taking a
drop of the upper layer of the liquid into a capillary tube and then blowing it out
on pure white filter-paper. The moist spot is first held over a bottle containing
fuming hydrochloric acid and then over strong sulphuretted hydrogen. The
presence of a minimum quantity of mercury salt in the liquid is shown by the
spot becoming yellowish, which is best seen when it is compared with a second
spot that has not been exposed to the gas. The end-reaction is still clearer when
a small part of the liquid is filtered, acidified with acetic acid, and tested with
sulphuretted hydrogen (OiTo)1. As the added quantity of urine contains 0.050
gram sugar the calculation of the percentage content in sugar, bearing in mind
the extent of dilution, is very simple.

This titration, unlike the previous one, may be performed equally well by
daylight and by artificial light. It is applicable even when the quantity of sugar
r\ the urine is very small and that of the other urinary constituents is normal. It
is more easily performed, and the titration liquids may be kept without decom-
posing for a long time (WORM-MULLER and his pupils 2) . There is diversity of
opinion, nevertheless, among investigators on the value of this titration method.

ESTIMATION OF THE QUANTITY OF SUGAR BY FERMENTATION. This
may be done in various ways: the simplest method, and one at the same
time sufficiently exact for ordinary cases, is that of ROBERTS. This
consists in determining the specific gravity of the urine before and after
fermentation. In the fermentation of sugar, carbon dioxide and alcohol
are formed as chief products, and the specific gravity is lowered, partly
on account of the disappearance of the sugar and partly on account of
the production of alcohol. ROBERTS found that a decrease of 0.001 in
the specific gravity corresponded to 0.23 per cent sugar, and this has been
substantiated since by several other investigators (WORM-MULLER and
others). If the urine, for example, has a specific gravity of 1.030 before
fermentation and 1.008 after, then the quantity of sugar contained therein
was 22X0.23 = 5.06 per cent.

In performing this test the specific gravity must be taken at the same
temperature before and after the fermentation. The urine must be
faintly acid, and when necessary it should be acidified with a little hytlro-

1 Journal f. prakt. Chem., 26.

2 Pfluger's Arch., 16 and 23.

ESTIMATION OF SUGAR IN URINE. 813

chloric acid or sulphuric acid, The activity of the yeast must, when
necessary, be controlled by a special test. Place 200 cc. of the urine
in a 400 cc. flask, add a piece of compressed yeast the size of a pea, and
subdivide the yeast through the liquid by shaking; close the flask with
a stopper provided with a finely-drawn-out glass tube, and allow the test
to stand at the temperature of the room or, still better, at 30-35° C.
After twenty-four hours the fermentation is ordinarily ended, but this
must be verified by the bismuth test. After complete fermentation filter
through a dry filter, bring the filtrate, to the proper temperature, and
determine the specific gravity.

If the specific gravity be determined with a good pycnometer sup-
plied with a thermometer and an expansion-tube, this method, when the
quantity of sugar is not less than 0.4-0.5 per cent, gives, according to
WoRM-MtiLLER, very exact results, but this has been disputed by BuDDE.1
For the physician the method in this form is not serviceable. Even when
the specific gravity is determined by a delicate urinometer which can
give the density to the fourth decimal, exact results are not obtained,
because of the ordinary errors of the method (BUDDE); but the errors
are usually smaller than those which occur in titrations made by unskilled
hands.

When the quantity of sugar is less than 1.5 per cent, these methods
cannot be used. Such small amounts cannot, as already mentioned,
be determined by titration directly, because of the reducing power of nor-
mal urine. In such cases, it is better to first determine the reducing power
of the urine by titration according to BANG or KNAPP, then ferment the
urine with the addition of yeast and titrate again. The difference
found between the two titrations calculated as sugar gives the true
quantity of the latter.

The determination of the sugar by fermentation can be so performed
that the loss in weight due to the C02 can be estimated, or the volume
of the gas measured. For this last purpose LOHNSTEIN 2 has constructed
a special fermentation saccharometer, and his " precision saccharometer "
is to be recommended. Based upon LOHNSTEIN'S instrument, WAG-
NER3 has constructed a "fermentation saccharo-manometer," which
has certain advantages over LOHNSTEIN'S apparatus.

ESTIMATIONS OF SUGAR BY POLARIZATION. In this method the
urine must be clear, not too deeply colored, and, above all, must not
contain any other optically active substances besides glucose. The
urine may contain several levorotatory substances such as proteids,
/3-oxy butyric acid, conjugated glucuronic acids, the so-called LEO'S sugar
and less often cystine, all of which are unfermentable. The proteid is
removed by coagulation, and the others are detected by the polariscope
after complete fermentation. The fermentable fructose is detected in a
special manner (see below), and the dextrorotatory milk-sugar differs
from glucose in its not fermenting readily. By using a delicate instru-

1 Roberts, The Lancet, 1862; Worm-Muller, Pfliiger's Arch., 33 and 37; Budde,
ibid., 40, and Zeitschr. f. physiol. Chem., 13. See Lohnstein, Pfliiger's Arch., 62.

2 Berlin, klin. Wochenschr., 35, and Allg. med. Central-Ztg., 1899; Goldman, Chem.
€entralbl., 1907, 1, 1149.

3 Munch, rned. Wochensch., 1905.

814 URINE.

ment and with sufficient practice very exact results can be obtained by
this method. The value of this procedure consists in the rapidity with
which the determination can be made. In using instruments specially
constructed for clinical purposes the accuracy is less than with the less
expensive fermentation test. Under such circumstances, and as the
estimation by means of polarization can be performed with exactitude
only by specially trained chemists, it is hardly worth while to give this
method in detail, and the reader is referred to handbooks for hints in the
use of the apparatus.

HASSELBACH and LINDHARD : have recently suggested a method for the
quantitative estimation of sugar which is based on the decolorization of an alkaline
saf ranin solution in the presence of sugar.

Fructose (levulose). Levogyrate urines containing sugar have been
noted by several investigators, although the nature of the sugar was not
well known to the earlier observers. In recent years several positively
authentic cases of levulosuria have been described, and also cases of
diabetes have been found where fructose exists in the urine besides glucose.
Reports on this subject do not agree, however.2

Fructose may be. detected as follows : The urine is levorotatory, and
the levorotatory substance ferments with yeast. The urine gives the
ordinary reduction tests and the ordinary phenylglucosazone. With
methylphenylhydrazine it gives the characteristic fructose methyl-
phenylosazone, and it also gives SELIWANOFF'S reaction on heating
after the addition of an equal volume of hydrochloric acid and a little
resorcin. With this test it must be remarked that too lengthy or too
strong heating must not be applied, since other carbohydrates may also
give the reaction (see page 218 and the works of ROSIN and UMBER3).
In the presence of fructose a red coloration appears. After cooling it
can be neutralized with soda and shaken out with amyl alcohol, (RosiN)
or with acetic ether (BORCHARDT). The amyl alcohol removes a red
pigment which gives a band in the spectrum between E and b and on
stronger concentration also a band 'in the blue at F. The acetic ether
in the presence of fructose becomes yellow, and this is more characteristic
according to BORCHARDT than ROSIN'S method, which has certain
fallacies. The simultaneous presence of nitrites and indican disturbs
the test, and in this case first remove the nitrous acid by boiling the
urine, acidified with acetic acid or hydrochloric acid for one minute. In
order to remove other disturbing pigments, MALFATTI suggests the
oxidation of the urine with a little hydrochloric acid and potassium
permanganate. JoLLES4 has suggested a method for detecting fructose
besides glucose by means of a diphenylamine solution.

1 Bioch. Zeitschr., 27.

'See Borchardt, Zeitschr. f. physiol. Chem., 55 and 60; W. Voit, ibid., 58 and 61;
Adler, Pfliiger's Arch., 139.

1 Umber, Salkowski's Festschrift, Berlin, 1904; Rosin, ibid., and Zeitschr. f.
physiol. Chem., 38.

4 Rosin, 1. c.; Borchardt, 1. c.; Malfatti, Zeitschr. f. physiol. Chem., 58; Jolles
and Mauthner, Chem. Centralbl., 1910, 1, 483.

LACTOSE IN URINE. 815

Maltose sometimes occurs in the urine according to LEPINE and BOULUD.
GEELMUYDEN, l who also held this view, now states that maltose does not occur
in the urine.

Laiose is a substance named by HUPPERT and found by LEO 2 in diabetic, urines
in certain cases, and which he considers as a sugar. It is levogyrate, amorphous,
and does not taste sweet, but rather sharp and salty. Laiose has a reducing
action on metallic oxides, does not ferment, and gives a non-crystalline, yellowish-
brown oil with phenylhydrazine. There is no positive proof as yet that this
substance is a sugar.

L :ctose. The appearance of lactose in the urine of pregnant women
was nrst shown by the observations of DE SINETY and F. HOFMEISTER,
and this has been substantiated by other investigators. After the inges-
tion of large quantities of milk-sugar some lactose may be found in the
urine (see Chapter VIII on absorption). LANGSTEIN and STEINITZ have
observed the passage of lactose and also of galactose 3 into the urine of
nurslings with disease of the stomach. The passage of lactose into the
urine is called lactosuria.

The positive detection of this sugar in the urine is difficult, because
it is, like glucose, dextrogyrate, and also gives the usual reduction tests.
If urine contains a dextrogyrate, non-fermentable sugar which reduces
bismuth solutions, then it is very probable that it contains lactose. It
must be remarked that the fermentation test for lactose is, according to
the experience of LUSK and VoiT,4 best performed by using pure cultivated
yeast (saccharomyces apiculatus). This yeast only ferments the glucose
while it does not decompose the milk-sugar. VOIT claims that if RUB-
NER'S test is performed without heating to boiling, but only to 80° C.,
the color becomes yellow or brown in the presence of lactose, instead
of red. The most positive means for the detection of this sugar is to
isolate the sugar from the urine. This may be done by the method
suggested by F. HoFMEisTER.5

R. BAUER 6 detects galactose as well as lactose in the urine by oxidation with
concentrated nitric acid, producing mucic acid.

Cammidge's reaction, which is recommended in the diagnosis of acute diseases
of the pancreas, consists in that certain urines do not give the phenylhydrazine
reaction directly, but only after boiling with an acid. The reason of this is not
known and the reaction is partly due to cane-sugar, in part to pentoses or gluco-
ronic acid and in part to mixtures of bodies.

1 Lepine and Boulud, Compt. Rend., 132; Geelmuyden, Zeitschr. f. klin, Med., 70.

2 Virchow's Arch., 107.

3 Hofmeister, Zeitschr. f. physiol. Chem., 1, which also contains the pertinent
literature. See also Lemaire, ibid., 21; Langstein and Steinitz, Hofmeister's Beitrage, 7.

4 Carl Voit, Ueber Die Glycogenbildung nach Aufnahme verschiedener Zuckeraten,
Zeitschr. f . Biologie, 28.

5 Hofmeister, Zeitschr. f. physiol. Chem., 1, which also contains the pertinent
literature.

6 Zeitschr. f. Physiol. Chem., 51.

816 URINE.

Pentoses. SALKOWSKI and J ASTRO WITZ first found in the urine of
persons addicted to the morphine habit a variety of sugar which was a
pentose and yielded an osazone which melted at 159° C. Since this
several other cases of pentosuria have been observed, and according to
KULZ and VOGEL and others small amounts of pentose also occur in the
urine of diabetics, as also in the urine of dogs with pancreatic or phlorhizin
diabetes.1

The pentose isolated by NEUBERG from the urine in chronic pentosuria
was cW-arabinose. LUZZATTO and KLERCKER studied cases of pento-
suria and found Z-arabinose. In alimentary pentosuria the Z-arabinose
of the plant food may be found in the urine. The appearance of pentoses
in the urine after eating fruits and fruit-juices has been repeatedly
observed by BLUMENTHAL and also by v. JAKSCH. According to COMI-
NOTTI 2 pentoses habitually occur in human urine on a mixed diet.

A urine containing pentose reduces bismuth as well as copper solu-
tions, although the reduction is not so rapid, but appears gradually.
If only pentose is present, the urine does not ferment, but in the presence
of glucose small amounts of pentose may also undergo fermentation.
The preparation of the osazone serves in the detection of pentoses and
when obtained from the urine it melts at 156-160° C. The phloroglucin
or orcin tests can also be employed (see page 209). Of these the last is
most preferable, especially as it excludes a confusion with the conjugated
glucuronic acids.

The orcin test can be performed as follows: 5 cc. of the urine are mixed
with an equal volume of HC1 sp.gr. 1.19, a small amount of orcin added and
the whole heated to boiling. As soon as a greenish cloudiness appears
cool the mixture off and shake carefully with amyl alcohol. The amyl-
alcohol solution is used in the spectroscopic examination. The pre-
cipitation of a bluish-green pigment is in itself significant.

BIAL 3 uses as reagent 30 per cent hydrochloric acid, which contains 1 gram
of orcin and 25 drops of a ferric-chloride solution (62.9 per cent of the crystalline
salt) in 500 cc. of the acid. 4.5 cc. of the reagent are heated to boiling and then a
few drops (not more than 1 cc.) of the urine are added to the hot but not boiling
liquid. In the presence of pentose the liquid turns a beautiful green. The use-
fulness of BIAL'S reagent is questioned by several experimenters. The delicacy
is too great and the possibility of confounding with other carbohydrates is not
excluded. In regard to the numerous modifications of this test and to JOLLES'
reaction we refer to page 209. The same for the quantitative estimation of pen-

*In regard to the literature, see footnote 1, page 208. See also Blumenthal,
"Die Pentosurie," Deutsche Klinik, 1902.

2 Blumenthal, Deutsche Klinik, 1902; v. Jaksch, Centralbl. f. innere Medizin,
1906; Cominotti, Bioch. Zeitschr., 22.

3 Deutsch. med. Wochenschr., 1903; see also footnote 4, page 209.

CONJUGATED GLUCURONIC ACIDS. 817

toses. JoLLES1 considers the preparation of the osazone as a specially conclusive
test and the distillation of the osazone with hydrochloric acid and testing the
distillate with BIAL'S reagent.

ROSENBERGER 2 believes he has detected a heptose in the urine in a case of
diabetes. According to him and to GEELMUYDEN 3 probably different varieties
of sugar, which are not well known, can possibly occur in urine of diabetics.

Conjugated Glucuronic Acids. Certain conjugated glucuronic acids
such as menthol- and turpentine-glucuronic acid may spontaneously
decompose in the urine, and in this case they may readily lead to a con-
fusion with pentoses. The urine should always be fresh as possible
for these examinations.

A confusion of the glucuronic acids, which have a reducing power on
copper or bismuth solutions, with glucose and fructose, can be pre-
vented by the fermentation test. They may also be distinguished from
glucose by their optical behavior, as the conjugated glucuronic acids
are levogyrate. On boiling with an acid, dextrorotatory glucuronic acid
is produced and the levorotation is changed to dextrorotation.

The conjugated glucuronic acids, like the pentoses, give the phloro-
glucin-hydrochloric-acid test. On the contrary they do not give the
orcin test directly, but only after cleavage with the setting free of glucuronic
acid. On using BIAL'S reagent no mistaking for pentoses occurs, although
this statement requires further substantiation. The pentoses may also
be isolated and identified by their osazones. Certain readily decomposable
glucuronic acids can here give phenylhydrazine compounds. In order to
detect glucuronic acid in the osazone precipitate, we can, as suggested
by NEUBERG and SANEYOsm4 take a knife point (about 8 milligrams)
of the precipitate, dissolve in 4 cc. strong hydrochloric acid, dilute with
4-cc. water, heat to boiling, add at least 0.1 gram naphthoresorcin, warm
for | minute, allow to slowly cool to 50° and shake with benzene. In the
presence of glucuronic acid the benzene solution is violet with an absorp-
tion in the yellowish-green.

The occurrence of conjugated glucuronic acids in the urine is shown
when the urine does not give the orcin-hydrochloric-acid reaction directly,
but only after boiling with the acid. The naphthoresorcin reaction,
as suggested by TOLLENS, can also be used. To 5 cc. urine add 0.5 cc.
of a 1 per cent alcoholic solution of naphthoresorcin and 5 cc. hydro-
chloric acid (sp.gr. 1.19), boil for one minute, allow to stand four minutes,

1 Jolles, Bioch. Zeitschr., 2, Centralbl. f. inn. Med., 1907 and 1912, and Zeitschr.
f. anal. Chem., 46.

2 Zeitschr. f. physiol. Chem., 49.

3 Rosenberger, Centralbl. f. inn. Med., 28; Geelmuyden, Zeitschr. f. klin. Med.,
58, 63, and 70.

4 Bioch. Zeitschr., 36.

818 URINE.

cool and shake with ether. In the presence of glucuronic acid the ether
becomes violet or blue, and shows the absorption bands given on page
223. According to NEUBERG this test, which is not specific for glucuronic
acid, is best performed with the naphthoresorcin in substance. This
test is more conclusive, if, as suggested by NEUBERG and ScHEWKET,1
the residue from an ethereal extract of the acidified urine is used.

The surest method is that suggested by MAYER and NEUBERG, which
consists in precipitating the urine with basic lead acetate, decomposing
the precipitate with H^S, boiling with dilute sulphuric acid in order to
split the conjugated acid, and then after neutralizing with soda, prepar-
ing the characteristic bromphenylhydrazine compound of glucuronic
acid (see page 223) with ^-bromphenylhydrazine hydrochloride and
sodium acetate. HERVIEUX 2 has slightly modified this method. In
regard to the quantitative estimation of glucuronic acid we must refer
to the work of C. ToLLENS.3

Inosite seems to be a normal urinary constituent, although it occurs
only in very small quantities (HOPPE-SEYLER, STARKENSTEiN4). In
diabetes insipidus, as well as after excessive drinking of water, it occurs
in large quantities in the urine because of a more abundant washing-
out of the tissues.

For the detection of inosite we make use of the method given on page 581,
with the modifications suggested by MEILLERE and STARKENSTEIN.

Acetone Bodies (acetone, acetoacetic acid, /3-oxybutyric acid). These
bodies, whose occurrence in the urine and formation in the organism have
been the subject of numerous investigations, occur in the urine espe-
cially in diabetes mellitus, but also in many other diseases.5 According
to v. JAKSCH and others, acetone is a normal urinary constituent, though
it may occur only in very small amounts (0.01 gram in twenty-four hours).

In regard to the origin of these bodies it was formerly considered that
they were produced by an increased destruction of protein. One of the
various reasons for this was the increase in the elimination of acetone

1 B. Tollens, Ber. d. d. chem. Gesellsch., 41, 1788, and C. Tollens, Zeitschr. f. physiol.
Chem., 56; Neuberg, Bioch. Zeitschr., 24; Neuberg and Schewket, ibid., 44; see also
Mandel and Neuberg, ibid., 13.

2 Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Hervieux, Compt. rend,
eoc. biol., 63.

3 Zeitschr. f. physiol. Chem., 61.

4 Starkenstein, Zeitschr. f. exp. Path. u. Therap., 5, which contains the literature.

6 In regard to the extensive literature on acetone bodies the reader is referred to
Huppert-Neubauer, Harn-Analyse, 10. Aufl., and v. Noorden's Lehrb. d. Pathol. des
Stoffwechsels. Berlin, 1906, and for recent work, Magnus-Levy, Die Azetonkorper,
Ergbn. d. inn. Med. u. Kinderheilk., I.

ACETONE BODIES. 819

and acetoacetic acid during inanition (v. JAKSCH, FR. MuLLER1). This
also stands in accord with the observations that a considerable increase
in the quantity of acetone and acetoacetic acid eliminated is observed
in such diseases as fevers, diabetes, digestive disturbances, mental dis-
eases with abstinence and cachexia, where the body protein is largely
destroyed. The formation of acetone bodies from protein is also indi-
cated by the fact that acetone has been obtained as an oxidation prod-
uct from gelatin and protein (BLUMENTHAL and NEUBERG, ORGLER 2) .
The investigations of EMBDEN and collaborators are more conclusive.
After EMBDEN and KALBERLAH showed that the liver is an organ where
acetone is formed, EMBDEN, SALOMON and SCHMIDT 3 showed by exper-
iments on extirpated livers, that butyric acid, oxybutyric acid, leucine,
tyrosine and in fact those aromatic bodies which, like tyrosine, phenyl-
alanine, phenyl-a-lactic acid and homogentisic acid contain a combustible
benzene nucleus, are transformed, in the liver, into acetone. Research,
which has been continued further by EMBDEN and his collaborators
and substantiated by others, such as BAER, and BLUM, BORCHARDT
and LANGE, NEUBAUER and GROSS, SCHMITZ and FR. SACHS 4 has shown
that there can be no doubt that certain amino-acids, especially leucine,
are strong acetone formers, and consequently that acetone can be formed
from protein. Protamines and histones can also increase the acetone
elimination (BORCHARDT) or, as we say, may have a " ketoplastic "
action, and it is therefore possible that acetone can be formed from
arginine with a-amino-valerianic acid as intermediary step (BORCHARDT
and LANGE).

As we cannot deny the possibility of a formation of acetone from pro-
teins, on the other hand we have observations which are inconsistent with
the origin of the acetone bodies entirely from the proteins. Thus no par-
allelism exists between the acetone bodies and the nitrogen excretion
in diabetics, and the fact, that in man no certain relation exists between
the acetone elimination and the nitrogen and sulphur excretion, seems to
show that the acetone bodies are not entirely derived from the proteins.
In man the excretion of acetone does not increase with the rise in the

1 v. Jaksch, Ueber Acetonurie und Diaceturie. Berlin, 1885; Fr. Miiller, Bericht
liber die Ergebnisse des an Cetti ausgefuhrten Hungerversuches. Berlin, klin. Wochen-
schr., 1887.

2Blumenthal and Neuberg, Deutsch. med. Wochenschr., 1901; Orgler, Hofmeis-
ter's Beitrage, 1.

8 Hofmeister's Beitrage, 8.

4Embden, ibid., 11, with Marx, Engel, Lattes and Michaud, ibid., 11; Baer and
Blum, Arch. f. exp. Path. u. Pharm., 55, 56, and 62; Borchardt, ibid, 53, with Lange,
Hofmeister's Beitrage, 9; Neubauer and Gross, Zeitschr. f. physiol. Chem., 67; Schmitz,
Bioch. Zeitschr., 28; Fr. Sachs, ibid., 27.

820 URINE.

quantity of protein, and an increase in the latter above the average causes
a diminution in the elimination of acetone (ROSENFELD, HIRSCHFELD,
FR. VoiT1).

The carbohydrates cannot be considered as material for the forma-
tion of acetone bodies. It is. generally admitted that in man the
exclusion of carbohydrates from the food or the diminution in their
amount or their assimilation may lead to more or less increased elimina-
tion of acetone bodies. This behavior may occur in diabetes as well as
in starvation and in the above-mentioned diseased conditions. The
increased elimination of acetone with food lacking carbohydrates also
occurs in healthy persons with a fatty diet but with a sufficient supply of
calories in other ways (alimentary acetonuria). With an abundant
supply of carbohydrates the elimination of acetone bodies may be greatly
diminished or even stopped entirely. The carbohydrates therefore
act " antiketoplastic," and a similar retarding action can be produced
by certain other substances, such as glycerin (HIRSCHFELD), lactic acid
and glutaric acid (BAER and BLUM) alanine and asparagin (FORSSNER,
BORCHARDT and LANGE2). Certain bodies like glycerine, lactic acid,
alanine, asparagin, which cause a sugar formation or increased elimina-
tion of sugar, act in the same way.

It must not be overlooked that the conditions are different in man
and in other carnivora (GEELMUYDEN, FR. VOIT). In dogs the elimina-
tion of acetone bodies is not increased in starvation, but is reduced; it
is augmented with increased quantities of meat, runs parallel with the
nitrogen excretion, and is not diminished by carbohydrates (FR. VOIT 3) .
In spite of this divergent behavior an unmistakable relation also exists
in the dog between the elimination of acetone bodies and the carbo-
hydrate metabolism, because in phlorhizin diabetes the acidosis occurs
only after the glycogen has been consumed (MARUM 4) .

As the carbohydrates cannot be acetone-formers, then a second
source only remains, namely, the fats. As proof of this there are certain
cases of diabetes with strong elimination of acetone bodies (0-oxybutyric
acid) where the quantity of protein transformed was too small to account
for the acetone bodies (MAGNUS-LEVY). The free elimination of acetone
bodies in starvation may also depend upon the fact that a great part of

1 Hirschfeld, Zeitschr. f. klin. Med., 28; Geelmuyden, see Maly's Jahresber., 26,
and Zeitschr. f. physiol. Chem., 23 and 26; Rosenfeld, Centralbl. f. innere Med., 16;
Voit, Deutsch. Arch. f. klin. Med., 66.

2Borchardt and Lange, 1. c.; Hofmeister's Beitrage, 9; which also cites other
works; Baer and Blum, ibid., 10; Forssner, Skand. Arch. f. Physiol., 25.

3 See footnote 1.

4 Hofmeister's Beitrage, 10.

ACETONE BODIES. 821

the body fat is consumed, and in several cases a certain relation has
been found between the fat consumed and the acetone bodies eliminated.
Certain investigators (GEELMUYDEN, SCHWARZ, WALDVOGEL) have
also observed an increase in the acetonuria on partaking of fatty food,
and FORSSNER l has indeed found a certain parallelism between the
acetone elimination and the fat taken up. For the present the fats are
considered as the most important source of the acetone bodies.

The three acetone bodies occurring in the urine, as above stated, are
acetone, acetoacetic acid and /3-oxybutyric acid, and this last is considered
as the mother-substance of the other two. If /3-oxybutyric acid,
CHs.CHOH.CH2.COOH, is introduced into the animal body, it is burnt
if the quantity is not too great, while if in excess it passes into the urine
as acetoacetic acid, CHs.CO.CH2.COOHr This acid can also be burnt,
but if large quantities are introduced it appears in part in the urine and
readily splits into acetone, CHs.CO.CHs, and CO2. Acetone is in part
burnt in the animal body, but a part is eliminated by the kidneys and
especially by the lungs. We can imagine that the /3-oxybutyric acid is
a physiological metabolic product which normally is completely changed
into acetoacetic acid and acetone, and in diabetes and especially with
lack of carbohydrates is formed to an increased extent, or its combustion
made more difficult, so that in the first place acetone and acetoacetic
acid pass into the urine and in severe cases also /3-oxybutyric acid (acidosis) .
In this connection it must be borne in mind that, because of the previously-
mentioned (page 774) reversibility of the process, the direction may also
be reversed, that is acetoacetic acid can also be changed into /3-oxybutyric
acid in the animal body and this has been proven by perfusion of livers
(FRIEDMANN and MAASE) as well as in animals (DAKIN) and in diabetics

(0. NEUBAUER)2.

Since leucine in perfusion experiments with livers yields acetoacetic acid
(EMBDEN and ENGEL) and also, as BAER and BLUMS found, that leucine and iso-
valeric acid increased the /3-oxybutyric acid elimination in diabetics, it has been
accepted that a formation of /3-oxybutyric acid takes place from the leucine
with isovaleric acid as an intermediary product :

(leucine CH3)2CH.CH2.CH(NH2).COOH-^(CHS)2CH.CH2.COOH, isovaleric acid),
Valine («-amino-valeric acid (CH3)2CH.CH(NH2).COOH) is, on the contrary,
not an acetone former.

1 Magnus-Levy, Arch. f. exp. Path. u. Pharm., 42; Geelmuyden, 1. c., and Norsk,
Magasin for Laegevidenskaben, 1900; see also Zeitschr. f. physiol. Chem., 41; Schwarz,
Deutsch. Arch. f. klin. Med., 1903; Waldvogel, Centralbl. f. inn. Med., 20; Forssner,
Skand. Arch. f. Physiol., 22 and 23.

2 Friedmann and Maase, Bioch. Zeitschr., 27; Dakin, Journ. of biol. Chem., 8;
Neubauer, Maly's Jahresb., 40, 849.

3 Arch. f. exp. Path. u. Pharm., 55 and 56; Embden and Engel, Hofmeister's
Beitrage, 11.

822 URINE.

In regard to the formation of acetone bodies from fat it must be
remarked that glycerin has an antiketoplastic action, and that the
fatty acids can only be considered. As to the behavior of these in the
formation of acetone, EMBDEN and MARX* have shown that only those
normal fatty acids which contain an even number of carbon atoms are
acetone formers, while those with an uneven number of carbon atoms
are without action in this regard. This is true at least for the acids from
n-decanoic acid to n-butyric acid, which latter is a strong acetone former.
As in diabetics a greater number of oxybutyric acid molecules can be
eliminated than corresponds to the number of fatty acid molecules decom-
posed, it seems as if more than one molecule of j8-oxybutyric acid is pro-
duced from one molecule of fatty acid. We cannot therefore admit
of a simple demolition of the fatty acids to butyric acid (by consecutive
oxidation attacks in the ^-position), but rather a destruction of the fatty
acid molecules into several parts, and these take part in the formation of
/3-oxybutyric acid.

A synthetical formation of ^-oxybutyric acid has been accomplished by GEEL-
MUYDEN and others, but especially by MAGNUS-LEVY, starting* with acetaldehyde,
according to the hypothesis of SPIRO. It is also interesting that FRIEDMANN 2
has shown by perfusion experiments with livers that aldehyde ammonia, and to
a greater extent aldol, are acetone formers. It must therefore be admitted that
first a condensation of the aldehyde to aldol takes place, CH3.COH+CH3.COH
= CH3.CH(OH).CH2.COH, and that 0-oxybutryic acid, CH3.CH(OH).CH2.COOH,
is formed from this by oxidation.

According to the above-mentioned perfusion experiments it must
be admitted that the liver is important in the formation of acetone
bodies, and EMBDEN and LATTES have found that the ability of the liver
of the dog with pancreas diabetes or phloridzin diabetes to produce
acetone is much greater than the liver of the normal animal. On the
other hand, as shown by EMBDEN and MiCHAUD,3 in dogs and oxen the
liver also has a strong destructive action upon acetoacetic acid. A
similar action is also found in the kidneys, muscles and spleen of dogs
and pigs. The destructive action of fresh organs is much stronger upon
acetoacetic acid than upon acetone. They could not find any special
cleavage products, and the above-mentioned, so-called demolition may
therefore perhaps in part be a reformation of /3-oxybutyric acid from the
acetoacetic acid.

Acetone, CsHeO, dimethylketone, CHs.CO.CHs, is a thin, water-
clear liquid, boiling at 56.3° and possessing a pleasant odor of fruit,

1 Hofmeister's Beitrage, 11.

2 Geelmuyden, Zeitschr. f. physiol. Chem., 23 and 26; Magnus-Levy, Arch. f. exp.
Path. u. Pharm., 42; Friedmann, Hofmeister's Beitrage, 11.

8 Embden and Lattes, Hofmeister's Beitrage, 11; Embden and Michaud, ibid., 11.

ACETONE. 823

which in diabetes gives a pomaceous or fruit odor to the urine as well as
the expired air. It is lighter than water, with which it mixes in all
proportions, also with alcohol and ether. The most important, reactions
for acetone are the following:

LIEBEN'S lodoform Test. When a watery solution of acetone is treated
with alkali and then with some iodo-potassium-iodide solution and gently
warmed, a yellow precipitate of iodoform is produced, which is known
by its odor and by the appearance of the crystals (six-sided plates or stars)
under the microscope. This reaction is very delicate, but it is not char-
acteristic of acetone. GUNNING'S modification of the iodoform test con-
sists in using an alcoholic solution of iodine and ammonia instead of the
iodine dissolved in potassium iodide and alkali hydroxide. In this case,
besides iodoform, a black precipitate of nitrogen iodide is formed, but
this gradually disappears on standing, leaving the iodoform visible. This
modification has the advantage that it does not give any iodoform with
alcohol or aldehyde. On the other hand, it is not quite so delicate,
but still it detects 0.01 milligram of acetone in 1 cc.

FROMMER'S 1 Test. This reagent is a 10 per cent alcoholic solution
of salicylaldehyde. Add 1-2 cc. of this solution to 10 cc. of the solution
(urine) and add to this mixture 1 gram KOH in substance, when a
carmine-red color will be observed. If necessary warm to about 70° C.
This reaction is just as delicate as the above.

REYNOLD'S Mercuric-oxide Test is based on the power of acetone to
dissolve freshly precipitated HgO. A mercuric-chloride solution is pre-
cipitated by alcoholic caustic potash. To this add the liquid to be
tested, shake well, and filter. In the presence of acetone the filtrate
contains mercury, which may be detected by ammonium sulphide.
This test has about the same delicacy as GUNNING'S test. Aldehydes
also dissolve appreciable quantities of mercuric oxide.

LEGAL'S Sodium Nitroprusside Test. If an acetone solution is treated
with a few drops of a freshly prepared sodium-nitroprusside solution
and then with caustic-potash or soda solution, the liquid is colored ruby-
red. Creatinine gives the same color; but if the mixture is saturated
with acetic acid, the color becomes carmine or purplish red in the presence
of acetone, but yellow and then gradually green and blue in the presence
of creatinine. With this test paracresol responds with a reddish-yellow
color, which becomes light pink when acidified with acetic acid and can-
not be mistaken for acetone. ROTHERA 2 has suggested a modification
which is more delicate by using ammonium salts and ammonia.

PENZOLDT'S Indigo Test depends on the fact that orthonitrobenzaldehyde
in alkaline solution with acetone yields indigo. A warm saturated and

1 Berl. klin. Wochenschr., 1905. 2 Journ. of Physiol., 37.

824 URINE.

then cooled solution of the aldehyde is treated with the liquid to be tested
for acetone and next with caustic soda. In the presence of acetone the
liquid first becomes yellow, then green, and lastly indigo separates; and
this may be dissolved with a blue color by shaking with chloroform;
1.6 milligrams acetone can be detected by this test.

Acetoacetic Acid, C^eOs, acetylacetic acid, diacetic acid, CHs.CO.
CH2.COOH, is a colorless, strongly acid liquid which mixes with water,
alcohol, and ether in all proportions. On heating to boiling with water,
and especially with acids, it decomposes into carbon dioxide and ace-
tone, and therefore gives the above-mentioned reactions for acetone.
It differs from acetone in that it gives a violet-red or brownish-red
color with a dilute ferric-chloride solution. For the detection of this
acid we make use of the following reactions which may be applied directly
to the urine:

GERHARDT'S Reaction. Treat 10-15 cc. of the urine with ferric-
chloride solution until it fails to give a precipitate filter, and add some
more ferric chloride. In the presence of acetoacetic acid a wine-red
color is obtained. The color becomes paler at the room temperature
within twenty-four hours, but more quickly on boiling (differing from
salicylic acid, phenol, sulphocyanides). A portion of the urine slightly
acidified and boiled does not give this reaction on cooling, on account
of the decomposition of the acetoacetic acid.

ARNOLD and LIPLIAWSKY'S Reaction. 6 cc. of a solution contain-
ing 1 gram of p-aminoacetophenone and 2 cc. of concentrated hydro-
chloric acid in 100 cc. of water are mixed with 3 cc. of a 1 per cent potas-
sium-nitrite solution and then treated with an equal volume of urine.
A few drops of concentrated ammonia are now added and violently
shaken. A brick-red coloration is obtained. Then take 10 drops to
2 cc. of this mixture (according to the quantity of acetoacetic acid in
the urine), add 15-20 cc. H<C1 of sp.gr. 1.19, 3 cc. of chloroform, and
2-4 drops of ferric-chloride solution and mix without shaking. In the
presence of acetoacetic acid the chloroform is colored violet or blue
(otherwise only yellowish or faintly red). This reaction is more delicate
than the preceding test and reacts with 0.04 p.m. acetoacetic acid. Large
amounts of acetone (but not the quantity occurring in urines) give this
reaction according to ALLARD.*

BONDI and SCHWARZ'S 2 Reaction. 5 cc. of the urine is treated drop by drop
with iodine-potassium iodide solution until the color is orange-red. Then warm
gently and when the orange-red color has disappeared add the iodine solution
again until the color remains permanent on warming. Then boil, when the irritat-
ing vapors of iodo-acetone will attack the eyes. Acetone does not give this reaction.

1 Arnold, Wien. klin. Wochenschr., 1899, and Centralbl. f. innere Med., 1900;
Lipliawsky, Deutsch. med. Wochenschr., 1901; Allard, Berl. klin. Wochenschr., 1901.

2 Wien. klin. Wochenschr., 1906.

ACETOACETIC ACID. 0-OXYBUTYRIC ACID. 825

Detection' of Acetone and Acetoacetic Add in the Urine. Before test-
ing for acetone test for acetoacetic acid; as this acid gradually decom-
poses on allowing the urine to stand, the specimen must be as fresh as
possible. In the presence of acetoacetic acid the urine gives the above-
mentioned tests. In testing for acetone in the presence of acetoacetic
acid make the urine slightly alkaline and shake in a separatory funnel
with ether free from alcohol and acetone. Remove the ether and shake
it with water, which takes up the acetone, and test for acetone in the
watery solution.

In the absence of acetoacetic acid the acetone may be tested for
directly in the urine; this may be done by FROMMER'S test or LEGAL'S
test. These tests, which are only approximate, are of value only when
the urine contains a considerable amount of acetone.

For a more accurate test we distill at least 250 cc. of the urine faintly acidified
with sulphuric acid, care being taken to have a good condensation. Most of the
acetone is contained in the first 10-20 cc. of the distillate. A better result may
be obtained by distilling a large quantity of urine until about TO has been dis-
tilled off, acidify the distillate with hydrochloric acid, redistill and repeat this
.several times, collecting the first portion of each distillation. The final distillate
is used for the above reactions.1 SALKOWSKI and BORCHARDT have called attention
to the fact that in the distillation of an acidified urine containing sugar for the
detection or estimation of acetone, a substance giving iodoform can be formed
from the sugar if the distillation is carried too far. According to BORCHARDT 2
the urine must therefore first be diluted with water, or the concentration prevented
by the addition of water dropAvise during distillation.

The quantitative estimation of acetone (also that formed from the
acetoacetic acid) is done by distilling the urine after the addition of acetic
acid or a little sulphuric acid. The quantity of acetone in the distillate
can be determined, according to the HUPPERT-MESSINGER method, by
converting it into iodoform by means of potassium iodide and then titrat-
ing the quantity of iodine used in the formation of the iodoform. The
precipitation of the acetone as p-nitrophenylhydrazone-acetone by means
of p-nitrophenylhydrazine in acetic acid solution can also be used for
determining the acetone in the distillate (v. EKENSTEIN and BLANKSMA
and MOLLER). In regard to these methods we refer to.3 EMBDEN and
SCHLIEP and FoLiN4 have suggested methods for determining the quan-
tity of acetone and acetoacetic acid separately. In regard to these
estimations we must refer to the work of EMBDEN and ScHMiTz.5

/3-Oxybutyric Acid, C4H803, = CH3.CH(OH).CH2.COOH, ordinarily
forms an odorless syrup, but may also be obtained as crystals. It is readily
soluble in water, alcohol, and ether. It is levorotatory ; (a)^— —24.12°

1 See also Salkowski, Pfliiger's Arch., 56.

2 Hofmeister's Beitrage, 8.

3 Hoppe-Seyler, Thierfelder, 8. Aufl., 617 and 618.

4Embden and Schliep, Centralbl. f. d. ges. Phys. u. Path. d. Stoffwechsel, 1907;
Folin, Journ. of biol. Chem., 3. In regard to the quantitative estimation of acetone
see also O. Sammet, Zeitschr. f. physiol. Chem., 83.

6 Abderhalden's Handbuch der biochemischen Arbeitsmethoden, Bd. 3.

826 URINE.

for solutions of 1-11 per cent and has a disturbing action upon the deter-
mination of sugar by means of the polariscope. It is not precipitated
by basic lead acetate or by ammoniacal lead acetate, neither does it
ferment. On boiling with water; especially in the presence of a mineral
acid, this acid decomposes into a-crotonic acid, which melts at 71-72°
C., and water, CHH.CH(OH).CH2.COOH = H2O+CH3.CH:CH.COOH.
It yields acetone on oxidation with a chromic-acid mixture.

Detection of Q-Oxybutyric Acid in the Urine. If a urine is still levo-
gyrate after fermentation with yeast, the presence of oxybutyric acid
is probable. A further test may be made, according to KULZ, by evaporat-
ing the fermented urine to a syrup and, after the addition of an equal
volume of concentrated sulphuric acid, distilling directly without cool-
ing, a-crotonic acid is produced, which distills over, and, after collect-
ing in a test-tube, crystals which melt at +72° C. separate on cooling.
If no crystals are obtained, shake the distillate repeatedly with ether and
let this spontaneously evaporate. The crystals which separate out
can be purified according to EMBDEN and SCHMITZ by redissolving in ether,
evaporating the chief part of the ether and precipitating with petroleum-
ether, which removes the volatile fatty acids and benzoic acid.

The quantitative estimation is done by complete extraction of the
/3-oxy butyric acid by ether and determining the specific rotation. The
extraction can be done according to MAGNUS-LEVY l or according to
BERGELL.2 Other methods of estimating 0-oxybutyric acid have been
suggested by DARMSTADTER, BOEKELMAN and BOUMA.S In regard to
the quantitative estimation we refer to EMBDEN and SCHMITZ .4

EHRLICH'S 5 Urine Test. Mix 250 cc. of a solution which contains 50 cc. of
HC1 and 1 gram of sulphanilic acid in one liter, with 5 cc. of a \ per cent solution
of sodium nitrite (which produces very little of the active body, sulphodiazo-
benzene). In performing this test treat the urine with an equal volume of this
mixture and then supersaturate with ammonia. Normal urine will become
yellow or orange after the addition of ammonia (aromatic oxyacids may after a cer-
tain time give red azo bodies which color the upper layer of the phosphate sediment) .
In pathological urines there sometimes occurs (and this is the characteristic
diazo reaction) a primary yellow coloration, with very marked secondary red
coloration on the addition of ammonia, and the froth is also tinged with red.
The upper layer of the sediment becomes greenish. The body which gives this
reaction is unknown, but it especially occurs in the urine of typhoid patients
(EHRLICH). Opinions differ in regard to the significance of this reaction. If
the urine is made alkaline with sodium carbonate instead of ammonia and treated

1 See Hoppe-Seyler, Thierfelder's Handbuch, 8. Aufl., 619, and Geelmuyden, Ham-
marsten's Festschr., 1906.

2 Zeitschr. f. physiol. Chem., 33.

3 Darmstadter, ibid., 37; Boekelman and Bouma, see Maly's Jahresber, 31.

4 See footnote 5, page 825.

5 Ehrlich, Zeitschr. f. klin. Med., 5. See also Clemens, Deutsch. Arch. f. klin.
Med., 63 (literature). Kutscher and Engeland, footnote 1, page 758.

CYSTINE. 827

with a freshly prepared solution of diazobenzene sulphonic acid made alkaline
with sodium carbonate, normal urine also gives an orange or Bordeaux-red
coloration. The known normal urinary constituents which give the diazo reac-
tion are the aromatic oxy acids, antoxyproteic acid and the imidazole -derivative
found by ENGELAND (see page 758).

EHRLICH'S reaction with p-dimethylaminobenzaldehyde has already been
discussed in connection with urobilinogen.

ROSENBACH'S urine test, which consists in adding nitric acid drop by drop
to the boiling-hot urine and obtaining a claret-red coloration and a bluish-red
foam on shaking, depends upon the formation of indigo substances, especially
indigo-red.1

Fat in the Urine. The elimination of a urine which in appearance and rich-
ness in fat resembles chyle is called chyluria. It habitually contains a proteid
and often fibrin. Chyluria occurs mostly in the inhabitants of the tropics.
Lipuria, or the elimination of fat with the urine, may appear in apparently healthy
persons, sometimes with and sometimes without albuminuria, in pregnancy, and
also in certain diseases, as in diabetes, poisoning with phosphorus, and fatty
degeneration of the kidneys.

Fat is usually detected by the microscope. It may also be dissolved with
ether, and may invariably be detected by evaporating the urine to dryness and
extracting the residue with ether.

Cholesterin is also sometimes found in the urine in chyluria and in a few other
cases.

Ammo-acids. Leucine and tyrosine have been repeatedly found in urine by
the older methods, especially in acute yellow atrophy of the liver, in acute phos-
phorus poisoning, and in severe cases of typhoid and smallpox. Since ^-naphtha-
lene sulphochloride has been used in the detection of amino-acids these bodies have
not only been repeatedly found in normal urine (glycocoll, see page 756), but also
in pathological urines.2

Cystine. BAUMANN and GOLDMANN claim that a substance similar
to cystine occurs in very small amounts in normal urine. This substance
occurs in large quantities in the urine of dogs after poisoning with phos-
phorus. Cystine itself is only found with positiveness, and even then
very rarely, in urinary calculi and in pathological urines, from which
it may separate as a sediment. Cystinuria occurs oftener in men than
in women. BAUMANN and v. UDRANSZKY found in urine in cystinuria
the two diamines, cadaverine (pentamethylendiamine) and putrescine
(tetramethylendiamine) , which are produced in the putrefaction of
proteins. Cases of cystinuria may occur with or without the occurrence
of diamines in the urine, and only rarely are the diamines found in the
urine as well as in the feces, which perhaps depends upon the fact, as
found by CAMMIDGE and GARROD 3 in one case, that the diamines occur

1 See Rosin, Virchow's Arch., 123.

2Ignatowski, Zeitschr. f. physiol. Chem., 42; Abderhalden and Schittenhelm,
ibid., 45; Abderhalden and Barker, ibid., 42. See also footnote 5, page 756, and 2, 757.

3 Baumann, Zeitschr. f. physiol. Chem., 8. In regard to the literature on cystinuria
see Brenzinger, ibid., 16; Baumann and Goldmann, ibid., 12; Baumann and v.
Udranszky, ibid., 13; Stadthagen and Brieger, Berlin, klin. Wochenschr., 1889;
Cammidge and Garrod, Journ. of Path, and Bacteriol, 1900 (literature on diamines
in the urine and feces); Loewy and Neuberg, Bioch. Zeitschr., 2; Wolf and Schaffer,
Journ. of biol. Chem., 4; Williams and Wolf, ibid., 6.

828 URINE.

only from time to time in the feces. Cystinuria is generally admitted as
rather an anomaly in the protein metabolism where the cystine for
unknown reasons is not destroyed as ordinarily. It is remarkable
that the cystine of the food-proteins is eliminated by the urine, while in
cystinurics, at least sometimes, such cystine introduced is quantitatively
transformed.1 Certain observations, such as the appearance of lysine
in the urine of cystinurics (ACKERMANN and KUTSCHER 2), make it probable
that the demolition of other amino-acids is diminished in cystinuria.
The properties and reactions of cystine have been given on pages 148
and 149.

Cystine is easily prepared from cystine calculi by dissolving them
in alkali carbonate, precipitating the solution with acetic acid, and redis-
solving the precipitate in ammonia. The cystine crystallizes on the
spontaneous evaporation of the ammonia. The cystine dissolved in
the urine is detected, in the absence of proteid and sulphuretted hydrogen,
by boiling with alkali and testing with a lead salt or sodium nitroprusside.
To isolate cystine from the urine, acidify the urine strongly with acetic
acid. The precipitate containing cystine is collected after twenty-four
hours and digested with hydrochloric acid, which dissolves the cystine
and calcium oxalate, leaving the uric acid undissolved. Filter, supersat-
urate the filtrate with ammonium carbonate, and treat the precipitate
with ammonia, which dissolves the cystine and leaves the calcium oxalate.
Filter again and precipitate with acetic acid. The precipitated cystine
is identified by the microscope and the above-mentioned reactions.
Cystine as a sediment is identified by the microscope. It must be purified
by dissolving in ammonia and precipitating with acetic acid; it is then
further tested. Traces of dissolved cystine may be detected by the
production of benzoyl-cystine, according to BAUMANN and GOLDMANN.
For the detection and estimation of cystine we can proceed to advantage
in the following manner, suggested by GASKELL.S The urine freed from
oxalates and phosphates by means of ammonia and calcium chloride is
treated with an equal volume of acetone and with acetic acid. The
crystals which precipitate are dissolved in ammonia and then purified
by reprecipitation with acetone.

VH. URINARY SEDIMENTS AND CALCULI.

Urinary sediment is the more or less abundant deposit which is found
in the urine after standing. This deposit may consist partly of organized
and partly^ of non-organized constituents. The first, consisting of cells
of various kinds, yeast-fungi, bacteria, spermatozoa, casts, etc., must
be investigated by means of the microscope, and the following only
applies to the non-organized deposits.

1 See Wolf and Schaffer, Journ.'of biol. Chem., 4; and Hele, Journ. of Physiol.. 39.

2 Zeitschr. f. Biol., 57.

* Journ. of Physiol., 36.

URINARY SEDIMENTS. 829

As previously mentioned (page 674), the urine of healthy individuals
may sometimes, even on voiding, be cloudy on account of the phosphates
present, or become so after a little while because of the separation of
urates. As a rule, urine just voided is clear, and after cooling shows
only a faint cloud (nubecula) which consists of urine mucoid, a few epithe-
lium-cells, mucous corpuscles, and urate particles. If an acid urine is
allowed to stand, it will gradually change; it becomes darker and deposits
a sediment consisting of uric acid or urates, and sometimes also calcium-
oxalate crystals, in which yeast-fungi and bacteria are often to be seen.
This change, which the earlier investigators called "ACID FERMENTA-
TION OF THE URINE," is generally considered as an exchange of the dihy-
drogen alkali phosphates with the urates of the urine. Monohydrogen
phosphates besides acid urates, quadriurates (page 708) or free uric acid
or a mixture of both, according to conditions,1 are thus formed.

Sooner or later, sometimes only after several weeks, the reaction
of the original acid urine changes and becomes neutral or alkaline. The
urine has now passed into the " ALKALINE FERMENTATION," which con-
sists in the decomposition of the urea into carbon dioxide and ammonia
by means of lower organisms, micrococcus urese, bacterium ureae, and
other bacteria. MUSCULUS 2 has isolated an enzyme from the micro-
coccus ureae which decomposes urea, which is soluble in water and is called
mease. During the alkaline fermentation volatile fatty acids, especially
acetic acid, may be produced, chiefly by the fermentation of the car-
bohydrates of the urine (SALKOWSKI 3) . A fermentation by which
nitric acid is reduced to nitrous acid, and another where sulphuretted
hydrogen is produced, may sometimes occur.

When the alkaline fermentation has advanced only so far as to render
the reaction neutral, there often occur in the sediment fragments of uric-
acid crystals, sometimes covered with prismatic crystals of alkali urate;
dark-colored spheres of ammonium urate, crystals of calcium oxalate,
and sometimes crystallized calcium phosphate are also found. Crystals
of ammonium-magnesium phosphate (triple phosphate) and spherical
ammonium urate are specially characteristic of alkaline fermentation.
The urine in alkaline fermentation becomes paler and is often covered
with a fine membrane which contains amorphous calcium phosphate
and glistening crystals of triple phosphate and numerous micro-organisms.

1 See Huppert-Neubauer, 10. Aufl., and A. Ritter, Zeitschr. f. Biologie, 35.

2 Musculus, Pfliiger's Arch., 12.

8 Salkowski, Zeitschr. f. Physiol. Chem., 13.

830 URINE.

Non-Organized Sediments.

Uric Acid. This acid occurs in acid urines as colored crystals which
are identified partly by their form and partly by their property of giving
the murexid test. On warming the urine they are not dissolved. On
the addition of caustic alkali to the sediment the crystals dissolve, and
when a drop of this solution is placed on a microscope-slide and treated
with a drop of hydrochloric acid, small crystals of uric acid are obtained
which can be easily seen under the microscope.

Acid U rates. These occur only in the sediment of acid or neutral
urines. They are amorphous, clay-yellow, brick-red, rose-colored, or
brownish-red. They differ from other sediments in that they dissolve
on warming the urine. They give the murexid test, and small micro-
scopic crystals of uric acid separate on the addition of hydrochloric
acid. Crystalline alkali urates occur very rarely in the urine, and as a
rule only in such as have become neutral but not alkaline, by alkaline
fermentation. The crystals are somewhat similar to those of neutral
calcium phosphate; they are not dissolved by acetic acid, however,
but give a cloudiness therewith due to small crystals of uric acid.

Ammonium urate may indeed occur as a sediment in a neutral urine
which at first was strongly acid and has become neutralized by the alkaline
fermentation, but it is only characteristic of ammoniacal urines. This
sediment consists of yellow or brownish rounded spheres which are often
covered with thorny-shaped prisms and, because of this, are rather
large and resemble the thorn-apple. It reacts to the murexid test. It
is dissolved by alkalies with the development of ammonia, and crystals
of uric acid separate on the addition of hydrochloric acid to this solution.

Calcium oxalate occurs in the sediment generally as small, shining,
strongly refractive quadratic octahedra, which on microscopical examina-
tion remind one of a letter-envelope. The crystals can only be mistaken
for small, not fully developed crystals of ammonium-magnesium phos-
phate. They differ from these by their insolubility in acetic acid. The
oxalate may also occur as flat, oval, or nearly circular disks with central
cavities which from the side appear like an hour-glass. Calcium oxalate
may occur as a sediment in an acid as well as in a neutral or alkaline
urine. The quantity of calcium oxalate separated from the urine as
sediment depends not only upon the amount of this salt present, but
also upon the acidity of the urine. The solvent for the oxalate in the
urine seems to be the diacid alkali phosphate, and the greater the quan-
tity of this salt in the urine the greater the quantity of oxalate in solu-
tion. When, as previously mentioned (page 829), the simple-acid phos-
phate is formed from the diacid phosphate, on allowing the urine to
stand, a corresponding part of the oxalate may be separated as sediment

URINARY SEDIMENTS. 831

Calcium carbonate occurs in considerable quantities as sediment in
the urine of herbivora. It occurs in but small quantities as a sediment
in human urine, and in fact only in alkaline urines. It either has the
same appearance as amorphous calcium oxalate or it occurs as some-
what larger spheres with concentric bands. It dissolves in acetic acid
with the generation of gas, which differentiates it from calcium oxalate.
It is not yellow or brown like ammonium urate, and does not give the
murexid test.

Calcium sulphate occurs very rarely as a sediment in strongly acid urine. It
appears as long, thin, colorless needles, or generally as plates grouped together.

Calcium Phosphate. The CALCIUM TRIPHOSPHATE, CasCPO^, which
occurs only in alkaline urines, is always amorphous and occurs partly
as a colorless, very fine powder, and partly as a membrane consisting of
very fine granules. It differs from the amorphous urates in that it is
colorless, dissolves in acetic acid, but remains undissolved on warming
the urine. CALCIUM DIPHOSPHATE, CaHP04+2H20, occurs in neutral
or only in very faintly acid urine.1 It is found sometimes as a thin
film covering the urine and sometimes as a sediment. In crystallizing,
the crystals may be single, or they may cross one another, or they may
be arranged in groups of colorless, wedge-shaped crystals whose wide
end is sharply defined. These crystals differ from crystalline alkali
urates in that they dissolve without a residue in dilute acids and do not
give the murexid test.

Ammonium-magnesium phosphate, TRIPLE PHOSPHATE, may separate
from an amphoteric urine in the presence of a sufficient quantity of ammo-
nium salts, but it is generally characteristic of a urine which is ammo-
niacal through alkaline fermentation. The crystals are so large that they
may be seen with the unaided eye as colorless glistening particles in the
sediment, on the walls of the vessel, and in the film on the surface of the
urine. This salt forms large prismatic crystals of the rhombic system
(coffin-shaped) which are easily soluble in acetic acid. Amorphous
magnesium triphosphate, Mgs(PO4)2, occurs with calcium triphosphate
in urines rendered alkaline by a fixed alkali. Crystalline magnesium
phosphate, Mg3(PO4)2+22H2O, has been observed in a few cases in
human urine (also in horse's urine) as strongly refractive, long rhombic
plates.

As more rare sediments we fine! cystine, tyrosine, hippuric acid, xanthine, hcema-
toidine. In alkaline urine blue crystals of indigo may also occur, due to a decom-
position of indoxyl-glucuronic acid.

1 In regard to the conditions for the appearance of these sediments in urines see
C. Th. Morner, Zeitschr. f. physiol. Chem., 58.

832 URINE.

Urinary Calculi.

Besides certain pathological constituents of the urine, all those urinary
constituents which occur as sediments take part in the formation of
urinary calculi. EBSTEIN* considers the essential difference between an
amorphous and crystalline sediment in the urine on the one side and urinary
sand or large calculi on the other to be the occurrence of an organic
frame in the latter. As the sediments which appear in normal acid urine
and in a urine alkaline through fermentation are diverse, so also are the
urinary calculi which appear under corresponding conditions.

If the formation of the calculus and its further development take place
in an un decomposed urine, it is called a PRIMARY formation. If, on the
contrary, the urine has undergone alkaline fermentation and the ammonia
formed thereby has given rise to a calculus formation by precipitating
ammonium urate, triple phosphate, and earthy phosphates, then it is
called a SECONDARY formation. Such a formation takes place, for
instance, when a foreign body in the bladder produces catarrh accom-
panied by alkaline fermentation.

We discriminate between the nucleus or nuclei — if such can be seen —
and the different layers of the calculus. The nucleus may be essentially
different in different cases, for quite frequently it consists of a foreign
body introduced in the bladder. The calculus may have more than
one nucleus. In a tabulation made by ULTZMANN of 545 cases of vesic-
ular calculi, the nucleus in 80.9 per cent of the cases consisted of uric
acid (and urates); in 5.6 per cent, of calcium oxalate; in 8.6 per cent,
of earthy phosphates; in 1.4 per cent, of cy stein; and in 3.5 per cent,
of some foreign body.

During the growth of a calculus it often happens that, for some reason
or other, the original calculus-forming substance is covered with another
layer of a different substance. A new layer of the original substance may
deposit on the outside of this, and this process may be repeated. In
this way a calculus consisting originally of a simple stone may be con-
verted into a so-called compound stone with several layers of different
substances. Such calculi are always formed when a primary is changed
into a secondary formation. By the continued action of an alkaline
urine containing pus, the primary constituents of a primary calculus
may be partly dissolved and be replaced by phosphates. Metamor-
phosed urinary calculi are formed in this way.

Die Natur und Behandlung der Harnsteine. Wiesbaden, 1884.

URINARY CALCULI. 833

Uric-acid calculi are very abundant. They are variable in size and
form. The size of the bladder-stone varies from that of a pea or bean to
that of a goose-egg. Uric acid stones are always colored; generally
they are grayish-yellow, yellowish-brown, or pale red-brown. The upper
surface is sometimes entirely even or smooth, sometimes rough or uneven.
Next to the oxalate calculus the uric-acid calculus is the hardest. The
fractured surface shows regular concentric, unequally colored layers
which may often be removed as shells. These calculi are formed pri-
marily. Layers of uric acid sometimes alternate with other layers of
primary formation, most frequently with layers of calcium oxalate.
The simple uric-acid calculus leaves very little residue when burnt on
a platinum foil. It gives the murexid test, but there is no material
development of ammonia when acted on by caustic soda.

Ammonium urate calculi occur as primary calculi in new-born or nurs-
ing infants, rarely in grown persons. They often occur as a secondary
formation. The primary stones are small, with a pale yellow or dark-
yellowish surface. When moist they are almost like dough; in the
dry state they are earthy, easily crumbling into pale powder. They
give the murexid test and develop much ammonia with caustic soda.

Calcium-oxalale calculi are, next to uric-acid calculi, the most abundant.
They are either smooth and small (HEMP-SEED CALCULI) or larger, of the
size of a hen's egg, with rough, uneven surface, or their surface is cov-
ered with prongs (MULBERRY CALCULI). These calculi produce bleeding
easily, and therefore they often have a dark-brown surface due to decom-
posed blood-coloring matters. Among the calculi occurring in man
these are the hardest. They dissolve in hydrochloric acid without
developing gas, but are not soluble in acetic acid. After gently heating
the powder, it dissolves in acetic acid with frothing. With more intense
heat it becomes alkaline, due to the production of quicklime.

Phosphate Calculi. These, which consist mainly of a mixture of the
normal phosphate of the alkaline earths with triple phosphate, may be
very large. They are as a rule of secondary formation and contain
besides these phosphates also some ammonium urate and calcium oxalate.
These calculi ordinarily consist of a mixture of three constituents —
earthy phosphate, triple phosphate, and ammonium urate — surrounding
a foreign body as a nucleus. Their color is variable — white, dingy white,
pale yellow, sometimes violet or lilac-colored (from indigo red). The
surface is always rough. Calculi consisting of triple phosphate alone
are seldom found. They are ordinarily small, with granular or radiated
crystalline fracture. Stones of mono-acid calcium phosphate are also
seldom obtained. They are white and have beautiful crystalline texture.
The phosphatic calculi do not burn up, the powder dissolves in acid
without effervescence, and the solution gives the reactions for phos-

834 URINE.

phoric acid and the alkaline earths. The triple-phosphate calculi generate
ammonia on the addition of an alkali.

Calcium-carbonate ^calculi occur chiefly in herbivora. They are seldom found
in man. They have mostly chalky properties, and are ordinarily white. They
are completely or in great part dissolved by acids with effervescence.

Cystine calculi occur but seldom. They are of primary formation, of various
sizes, sometimes as large as a hen's egg. They have a smooth or rough surface,
are white or pale yellow, and have a crystalline fracture. They are not very
hard and are consumed almost entirely on the platinum foil, burning with a bluish
flame. They give the above-mentioned reactions for cystine.

Xanthine calculi are very rarely found. They are also of primary formation.
They vary from the size of a pea to that of a hen's egg. They are whitish, yel-
lowish-brown or cinnamon-brown in color, of medium hardness, with amorphous
fracture, and on rubbing appear like wax. They burn up completely when heated
on a platinum foil. They give the xanthine reaction with nitric acid and alkali,
but this must not be mistaken for the murexid test.

Urostealith calculi have been observed only a few times. In the moist state
they are soft and elastic at the temperature of the body, but in the dry state
they are brittle, with an amorphous fracture and waxy appearance. They burn
with a luminous flame when heated on platinum foil and generate an odor similar to
resin or shellac. Such a calculus, investigated by KRUKENBERG,1 consisted of
paraffin derived from a paraffin bougie used as a sound on the patient. Perhaps
the urostealith calculi observed in other cases had a similar origin, although the
substances of which they consisted have not been closely studied. HORBACZEW-
SKI has recently analyzed a case of urostealith which, to all appearances, was
formed in the bladder. This calculus contained 25 p. m. water, 8 p. m. inorganic
bodies, 117 p. m. bodies insoluble in ether, and 850 p. m. organic bodies soluble
in ether, among which were 515 p. m. free fatty acids, 335 p. m. fat, and traces of
cholesterin. The fatty acids consisted of a mixture of stearic, palmitic, and
probably myristic acids. -

HORBACZEWSKI 2 has also analyzed a bladder stone which contained 958.7
p. m. cholesterin.

Fibrin calculi sometimes occur. They consist of more or less changed fibrin-
coagulum. On burning they develop an odor of burnt horn.

The chemical investigation of urinary calculi is of great practical impor-
tance. To make such an examination actually instructive it is necessary
to investigate, separately, the different layers which constitute the cal-
culus. For this purpose saw the calculus, previously wrapped in paper,
with a fine saw so that the nucleus becomes accessible. Then peel off the
different layers, or, if the stone is to be kept, scrape off enough of the
powder from each layer for examination. This powder is then tested by
heating on the platinum foil. It must not be forgotten that a calculus
is never entirely burnt up, and also that it is never so free from organic
matter that on heating it does not carbonize. Do not, therefore, lay too
great stress on a very insignificant unburnt residue or on a very small

1Chem. Untersuch. z. wissensch. Med., 2. Cited from Maly's Jahresber., 19, 422.
2 Zeitschr. f . physiol. Chem., 18.

URINARY CALCULI. 835

amount of organic matter, but consider the calculus in the former case
as completely burnt and in the latter as unaffected.

When the powder is in great part burnt up, but a significant quantity
of unburnt residue remains, then the powder in question contains as a
rule urates mixed with inorganic bodies. In such cases remove the urate
with boiling water and then test the filtrate for uric acid and the sus-
pected bases. The residue is then tested according to the following
scheme of HELLER, which is well adapted to the investigation of urinary
calculi. In regard to the more detailed examination the reader is
referred to special works on the subject.

836

URINE.
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CHAPTER XV
THE SKIN AND ITS SECRETIONS

IN the structure of the skin of man and vertebrates many different
kinds of substances occur which have already been considered, such as
the constituents of the epidermal formation, the connective and fatty
tissues, the nerves, muscles, etc. Among these the different horn struc-
tures, the hair, nails, etc., whose chief constituent, keratin, has been
spoken of in another chapter (Chapter II), are of special interest.

The cells of the horny structure show, in proportion to their age, a
different resistance to chemical reagents, especially fixed alkalies. The
younger the horn-cell the less resistance it has to the action of alkalies;
with advancing age the resistance becomes greater, and the cell-mem-
branes of many horn-formations are nearly insoluble in caustic alkalies.
Keratin (or the keratins) occurs in the horn structure mixed with other
bodies, from which it is isolated with difficulty. These are detected by
microchemical investigations, and according to UNNA 1 three different
substances can be detected in the horn substance, designated by him A-,
B- and C-keratin.

The A-keratin, which forms the envelope of the horn and hair cells and the
outer layer of the hair, is the purest keratin. It is not dissolved by fuming nitric
acid at the ordinary temperature and does not give the xanthoproteic reaction,
and its keratin nature is doubtful. The £-keratin, which occurs as the contents
of the nail cells, gives the xanthoproteic reaction like the C-keratin occurring in
hair, but differs from the C-keratin by being soluble in fuming nitric acid.

Besides these substances, which have been called keratins, the horn
structure also contains other proteins which are soluble in pepsin-
hydrochloric acid. Among these we find residue of nuclei and the
so-called trichohyalin in the hair, which is a substance of unknown
constitution and characterized by great insolubility. From these
statements it is evident that we are here dealing with a mixture of dif-
ferent substances and for this reason it is unnecessary to give the older
elementary analyses of the various epidermoidal structures.

1 Monatsch. f . prakt. Dermat., 44.

837

838 THE SKIN AND ITS SECRETIONS.

The quantity of sulphur and of mineral bodies is of certain interest.
The sulphur and cystine content of these structures can be found on
pages 113, 114 and in this connection it must be mentioned that, accord-
ing to the investigations of RUTHERFORD and HAWK/ the sulphur content
of human hair is higher in men than in women, at least for the Caucasian
race, and ajso that red hair has the highest sulphur content irrespective
of race or gender. Hair on incineration leaves considerable ash, which
in human hair varies between 2.6 and 16 p. m., and in animal hair
is still greater, even up to 71 p. m. in the hair of the deer. The ash
consists of large amounts of alkali and calcium sulphate, and its sulphur
probably originates from the organic substance, which make the state-
ments as to the composition of the ash of hair of little value. Calcium
occurs in larger amounts, especially phosphate as well as carbonate, and is
most abundant in white hair. The amount of iron oxide in 1000 grams
of the ash of human hair varies between 42.2 grams in blond and 108.7
grams in brown hair, and silicic acid between 66.1 grams in black and
424.6 grams in red hair (BAUDRIMONT). The nails are rich in calcium
phosphate, and the feathers rich in silicic acid, especially the feathers
of grain-eating birds. According to v. GORUP-BESANEZ 2 the quantity
of silicic acid in grain-eating birds was 400 p. m., and in meat, berries
and insect-eating birds the amount was only 270 p. m. of the total ash.
DRECHSEL claims that at least a part of the silicic acid exists in the
feathers in organic combination as an ester while according to CERNY 3 it
exists only as an accidental contamination.

According to GATJTIER and BERTRAND4 arsenic also occurs in the
epidermal formations. GAUTIER says that arsenic is of importance in
the formation and growth of the formations, and on the other hand the
hair, nails, and epidermis-cells are of great importance in the excretion
of arsenic.

The ability of the skin to take up chlorides as observed by WAHLGREN
and by PADTBERG,5 is remarkable. According to them the skin is an
important chloride depot, which stores up chlorides when supplied in
excess and gives them up when necessary.

The skin of invertebrates has been the subject, in a few cases, of
chemical investigation, and in these animals various substances have
been found, of which a few, though little studied, are worth discussing.
Among them tunicin, which is found especially in the mantle of the

1 Journ. of Biol. Chem., 3.

2 Lehr. d. physiol. Chem., 4. Aufl., 660, 661; Baudrimont, ibid.

3 Drechsel, Centralbl. f. Physiol., 11, 361; Cerny, Zeitschr. f. physiol. Chem., 62.

4 Gautier, Compt. Rend., 129, 130, 131; Bertrand, ibid., 134.

5 Wahlgren, Arch. f. exp. Path. u. Pharm., 61; Padtberg, ibid., 63.

TUNICIN. CH1TIN. 839

tunicata, and the widely diffused chitin, found in the cuticle-formation of
invertebrates, are of interest.

Tunicin. Cellulose seems, from the investigations of AMBRONN, to occur
rather extensively in the animal kingdom in the arthropoda and the mollusks.
It has been known for a long time as the mantle of the tunicata, and this animal
cellulose was called tunicin by BERTHELOT. According to the investigations
of WINTERSTEIN there does not seem to exist any marked difference between
tunicin and ordinary vegetable cellulose. On boiling with dilute acid, tunicin
yields glucose, as shown first by FRANCHIMONT and later confirmed by WIN-
TERSTEIN. By the action of acetic acid anhydride and sulphuric acid, upon
tunicate-cellulose, ABDERHALDEN and ZEMPLEN 1 obtained octoacetyl-cellobiose,
which also indicates the relationship with the plant cellulose.

Chitin is not found in vertebrates. In invertebrates chitin is alleged
to occur in several classes of animals; it occurs chiefly in cephalopods
(sepia scales) and especially in the arthropods, in which it forms the
chief organic constituent of the shells, etc. It has been found in the
plant kingdom as in fungi (GILSON, WINTERSTEIN 2). The question
whether there are two or more chitins or whether there is only one
is still disputed (KRAWKOW, ZANDER, WESTER 3) . No formula can be
given for the same reasons (SUNDWIK, ARAKI, BRACK 4).

Chitin is decomposed on boiling with mineral acids and yields, as
shown by LEDDERHOSE, glucosamine and acetic acid. HOPPE-SEYLER
and ARAKI found, on heating with alkali and a little water to 180°, that
chitin was split into a new substance, chitosan, and acetic acid, and that
this chitosan contained acetyl groups as well as glucosamine. FRANKEL
and KELLY as well as OFFER 5 have obtained acetylglucosamine,
(C6Hi2NO5)COCH3 and acetyldiglucosamine (C^Ifea^OoJCOCHs as
cleavage products of chitin, and they consider chitin as a polymeric
monacetyldiglucosamine.

The chitosan which v. FURTH and Russo 6 have obtained as a crys-
talline hydrochloric acid combination and which E. LOEWY has obtained
as a crystalline sulphate is, according to the latter, a polymeric monacetyl-
diglucosamine with at least two monacetyldiglucosamine groups. Accord-

1 Ambronn, Maly's Jahresber., 20; Berthelot, Annal. de Chim. et Phys., 56, Compt.
Rend., 47; Winterstein, Zeitschr. f. physiol. Chem., 18; Franchimont, Ber. d. deutsch.
chem. Gesellsch., 12; Abderhalden and Zemplen, Zeitschr. f. physiol. Chem., 72.

2 Gilson, Compt. Rend., 120; Winterstein, Ber. d. d. chem. Gesellsch., 27 and 28.
3Krawkow, Zeitschr. f. Biol., 29; Zander, Pfluger's Arch., 66; Wester, Chem.

Centralbl., 1909, II.

4 Sundwik, Zeitschr. f. physiol. Chem., 5; Araki, ibid., 20; Brach, Bioch. Zeitschr.,
38.

5 Ledderhose, Zeitschr. f. physiol. Chem., 2 and 4; Araki, 1. c., Frankel and Kelly,
Monatshefte f. Chem., 23; Offer, Bioch. Zeitschr., 7.

6 v. Fiirth and Russo, Hofmeister's Beitrage, 8; Loewy, Bioch. Zeitschr., 23; Brach,
I.e.

840 THE SKIN AND ITS SECKETIONS.

ing to v. FURTH and Russo on cleavage it yields 25 per cent acetic acid
and 60 per cent glucosamine. The formula is (C28H<5oN40i9)z according
to v. FURTH and collaborators and splits according to the equation:
(C28H5oN4Oi9)4+5zH2O = 4s(C6Hi3N05)+2z(CH3COOH). According
to BRACK, who admits of at least four glucosamine groups in chitosan,
the formula for chitin is (C32H54N402i)x and contains 4 acetyl for every
4 nitrogen atoms. The transformation into chitosan consists in a
rupture of one-half of the acetic acid groups in the chitin.

In a dry state chitin forms a white, brittle mass retaining the form
of the original tissue. It is insoluble in boiling water, alcohol, ether,
acetic acid, dilute mineral acids, and dilute alkalies. It is soluble in
concentrated acids. It is dissolved without decomposing in cold con-
centrated hydrochloric acid, but is decomposed by boiling hydrochloric
acid. According to KRAWKOW the various chitins behave differently
with iodine or with sulphuric acid and iodine, in that some are colored
reddish brown, blue, or violet, while others are not colored at all. Accord-
ing to WESTER chitin free from chitosan is not colored by iodine.

Chitin may be easily prepared from the wings of insects or from the shells
of the lobster or the crab, the last-mentioned having first been extracted by an
acid so as to remove the lime salts. The wings or shells are boiled with caustic
alkali until they are white, afterward washed with water, then with dilute acid
and water. The pigments remaining can be destroyed by permanganate. The
excess of this last can be removed by a dilute solution of bisulphite, washed with
water and then extracted with alcohol and ether.

Hyalin is the chief organic constituent of the walls of hydatid cysts. From a
chemical point of view it stands close to chitin, or between it and protein. In
old and more transparent sacs it is tolerably free from mineral bodies, but in
younger sacs it contains a great quantity (16 per cent) of lime salts (carbonate,
phosphate, and sulphate).

According to LUCRE l its composition is:

C H N O

From old cysts 45.3 6.5 5.2 43.0

From young cysts 44.1 6.7 4.5 44.7

It differs from keratin on the one hand and from proteins on the other by the
absence of sulphur, also by its yielding, when boiled with dilute sulphuric acid,
a variety of sugar in large quantities (50 per cent), which is reducing, fermentable,
and dextrogyrate. It differs from chitin by the property of being gradually
dissolved by caustic potash or soda, or by dilute acids; also by its solubility on
heating with water to 150° C.

The coloring matters of the skin and horn-formations are of different
kinds, but have not been extensively studied. Those occurring in the
stratum Malpighii of the skin, especially of the negro, and the black
or brown pigment occurring in the hair, belong to the group of those
substances which have received the name melanins.

1 Virchow's Arch., 19.

MELANINS. 841

Melanins. This group includes several different varieties of amorphous
black or brown pigments which are insoluble in water, alcohol, ether,
chloroform, and dilute acids, and which occur in the skin, hair, chorioidia,
in sepia, in certain pathological, formations, and in the blood and urine
in disease. From the true native melanins we must differentiate the
humus-like products obtained on boiling proteins with mineral acids
and which have been called melanoidins or melanoidic acid (Schmiedeberg)
and whose relation to the true melanins is still unknown.

The melanoidins are readily soluble in dilute alkali while the melanins
show a different behavior in this regard. Of the melanins a few such as
SCHMIEDEBERG'S sarcomelanin, and that from the melanotic sarcomata
of horses, the hippomelanin (NENCKI, SIEBER, and BERDEZ), which are
soluble with difficulty in alkalis, while others, such as the coloring matter
of certain pathological swellings in man, the phymatorhusin (NENCKI and
BERDEZ) are readily soluble in alkalies. The melanins, as above stated,
are in general insoluble in dilute mineral acids; from black sheep-
wool GORTNER l has isolated a melanin which was soluble in acetic acid
and in dilute mineral acids (see below).

Among the melanins there are a few, for example the choroid pig-
ment, which are free from sulphur (LANDOLT and others) ; others, on the
contrary, as sarcomelanin and the pigment of the hair (SIEBER) are rather
rich in sulphur (2-4 per cent), while the phymatorhusin found in cer-
tain swellings and in the urine (NENCKI and BERDEZ, K. MORNER) is
very rich in sulphur (8-10 per cent). Whether any of these pigments,
especially the phymatorhusin, contains any iron or not is an important
though disputed point, for it leads to the question whether these pigments
are formed from the blood-coloring matters.

According to NENCKI and BERDEZ the pigment, phymatorhusin, isolated by
them from a melanotic sarcoma did not contain any iron, and according to them
is not a derivative of haemoglobin. K. MORNER and later also BRANDL and L.
PFEIFFER found, on the contrary, that this pigment did contain iron, and they
consider it as a derivative of the blood-pigments. The sarcomelanin (from a sar-
comatous liver) analyzed by SCHMIEDEBERG contained 2.7 per cent iron which
was partly in organic combination and could not be completely removed by
dilute hydrochloric acid. The sarcomelanic acid prepared by SCHMIEDEBERG
by the action of alkali on this melanin contained 1.07 per cent iron. The sar-
comelanin investigated by ZDAREK and v. ZEYNEK also contained 0.4 per cent iron.
Recently WOLFF 2 prepared two pigments from a melanotic liver, of which one
was no doubt modified. The other, which was soluble in a soda solution, con-

1 Gortner, Journ. of biol. Chem., 8, and Bioch. Bulletin, 1, 1911.

2 Zdarek and v. Zeynek, Zeitschr. f. physiol. Chem., 36; Wolff, Hofmeister's Beitrage,
5. The literature on the melanins may be found in Schmiedeberg, " Elementarformeln
einiger Eiweisskorper, etc." Arch. f. exp. Path. u. Pharm., 39; also in Robert, Wiener
Klinik, 27 (1901), and Spiegler, Hofmeister's Betrage, 4, and especially v. Fiirth,
Centralbl. f. allg. Path. u. Path. Anat., ,15, 1907, 617.

842 THE SKIN AND ITS SECRETIONS.

tained 2.51 per cent sulphur and 2.63 per cent iron, which was in great part
split off by 20 per cent hydrochloric acid. From another liver he, on the con-
trary, obtained melanin free from iron but with 1.67 per cent sulphur. From
this melanin he obtained, by treatment with bromine, a hydro-aromatic body
which was related to xyliton (a condensation product of acetone). A similar

S-oduct could not be obtained from the pigment of the hair (SPIEGLER) nor from
ppomelanin (v. FURTH and JERUSALEM 1).

The difficulties which attend the isolation and purification of the
melanins have not been overcome in certain cases, while in others it is
questionable whether the final product obtained has not another com-
position from the original coloring matter, owing to the energetic chemical
processes resorted to in its purification. The elementary composition
shows widely varying results in the different melanins, namely, 48-60
per cent carbon, and 8-14 per cent nitrogen. Under these circumstances,
and as no doubt we have a large number of melanins having different
composition, it seems that a tabulation of the analyses of the different
preparations can only be of secondary importance.

GORTNEB differentiates between two different groups of melanins.
The one, to which the melanin isolated by him from sheeps-wool belongs,
is soluble in very dilute acid, has a protein nature and is called melano-
protein. By the action of strong alkali the nitrogen and hydrogen con-
tent is much reduced and the quantity of carbon increased. The melanin
is now insoluble in dilute acids, like the second group of melanins. The
melanoprotein on hydrolysis with hydrochloric acid yields besides amino-
acids, a black pigment, rich in carbon and insoluble in acids. The
melanin isolated by PIETTRE 2 form sarcomatous horse tumors, on alkali
hydrolysis, yielded amino-acids and a melanin much richer in carbon
and poorer in nitrogen, a melamin. The sepia melanin and also the
artificially prepared melanin by means of tyrosinase, had a similar behavior.
The melanin is, therefore, according to PIETTRE, composed of a protein
group and a pigment residue, which is insoluble in acids.

So little is known about the structural products of the melanins or
melanoids that it is impossible to give the origin of these bodies. As
undoubtedly there are several distinct melanins, their origin must also be
distinct. The ferruginous melanins should be considered as originating
from the blood-pigments until further research proves otherwise. Others,
on the contrary, cannot have this origin; for example, the pigments
of the hair and choroid, which are free from iron and which do not yield
haemopyrrol according to SPIEGLER. Several melanins — and this is also

1 Wolff, Hofmeister's Beitrage, 5; Speigler, ibid., 10; v. Fiirth and Jerusalem,
ibid., 10.

2 Gortner, 1. c. and Bull. Soc. Chim. de France (4) 11; Piettre, Compt. Rend., 153,
and Congres, internal., de Path. Comparee, Paris, 1912.

MELANINS. 843

true of the melanoids produced from proteins en cleavage with acids
(SAMUEL Y J) — yield indol or skatol and a pyrrol substance on fusion
with alkali, while hippomelanin, according to v. FURTH and JERUSALEM,
gives a fecal odor on this treatment, but does not yield any indol or skatol.
More characteristic than the two last mentioned bodies is a phenol-like
substance, which occurs to a slight extent, and gives a bluish-black
color with ferric chloride (v. FURTH).

The cyclic complexes of the proteins are rightly considered as the
mother-substance of the melanins (SAMUELY and v. FURTH and others),
and this view has received support by the behavior of tyrosine with
oxidases. It has been found that by the action of a plant oxidase,
BERTRAND'S tyrosinase,2 upon tyrosine, colored products and then
melanin-like substances are formed, v. FURTH with SCHNEIDER and
PRIBRAM, GESSARD, NEUBERG, DEWITZ and others3 have shown that
similar-acting tyrosinases also occur in the animal kingdom, in insects
and sepia, in melanotic tumors and in pigmented skin, and v. FURTH
and JERUSALEM have prepared an artificial melanin from tyrosine which
shows great similarity to hippomelanin. Finally NEUBERG and JAGER*
have also prepared extracts from melanotic growths which formed a dark-
brown pigment from adrenalin. As indicated above, we tend more and
more to accept the view that the melanins are derived from the cyclic
components of the proteins.

In addition to the coloring matters of the human skin it is in place here to
treat of the pigments found in the skin or epidermal formation of animals.

The beautiful color of the feathers of many birds depends in certain cases on
purely physical causes (interference-phenomena), but in other cases on coloring
matters of various kinds. Such a coloring matter is the amorphous reddish-
violet turacin, which contains 7 per cent copper and whose spectrum is very similar
to that of oxyhsemoglobin. It must be remarked that according to LAIDLAW 5
turacin or at least a pigment with the same properties can be obtained on boiling
hsematoporphyrin in dilute ammonia with ammoniacal copper solution. KRUKEN-
BERG 6 found a large number of coloring matters in bird's feathers, namely zooery-
thrin, zoofulvin turacoverdin, zoorubin psittacofulvin, and others which cannot be
enumerated here.

Tetronerythrin, so named by WURM, is a red amorphous pigment which is
soluble in alcohol and ether, and which occurs in the red warty spots over the eyes
of the heathcock and the grouse, and which is very widely spread among the
invertebrates (HALLIBURTON, DE MEREJKOWSKI MACMUNN). Besides tetronery-

1 Hofmeister's Beitrage, 2.

2 Compt. Rend., 122.

8 The literature can be found in v. Furth and Jerusalem, Hofmeister's Beitrage,
10.

4 Neuberg, Virchow's Arch., 192; Jager, ibid., 198.

5 Journ. of Physiol., 31.

6 Vergleichende physiol. Studien, Abth. 5, and (2. Reihe) Abth. 1, 151, Abth. 2, 1,
and Abth. 3, 128.

844 THE SKIN AND ITS SECRETIONS.

thrin MACMUNN found in the shells of crabs and lobsters a blue coloring matter,
cyanocrystallin, which turns red with acids and by boiling water. Hcematoporphyrin,
according to MACMUNN, also occurs in the integuments of certain of the lower
animals. The blue pigment occurring in the fins of the fish, crenilabrus paw,
is according to v. ZEYNEK l a chromoprotein.

In certain butterflies (the pieridinse) the white pigment of the wings consists,
as shown by HOPKINS, 2 of uric acid, and the yellow pigment of a uric-acid deriva-
tive, lepidotic acid, which yields a purple substance, lepidoporphyrin, on warming
with dilute sulphuric acid. The yellow and red pigment of the Vanessa are,
according to LINDEN, 3 of an entirely different kind. In this case we are dealing
with a compound between protein and a pigment which is allied to bilirubin or
urobilin, i.e., a compound similar to haemoglobin.

In addition to the coloring matters thus far mentioned a few others found in
certain animals (though not in the skin) will be spoken of.

Carminic Acid, or the red pigment of the cochineal, gives on oxidation, accord-
ing to LIEBERMANN and VoswiNCKEL,4 cocheniUic acid, Ci0H807, and coccinic acid.
C*H8Ot, the first being the tri-carboxylic acid, and the other the di-carboxylic
acid, of ra-cresol. The beautiful purple solution of ammonium carminate has two
absorption-bands between D and E which are similar to those of oxyhaemoglobin.
These bands lie nearer to E and closer together and are less sharply defined. Pur-
ple is the evaporated residue from the purple-violet secretion, caused by the action
of the sunlight, upon the so-called " purple gland " of the mantle of certain species
of murex and purpura. According to FRIEDLANDER 5 the pigment is a bromine
derivative of indigo and indeed di-bromindigo.

Among the remaining coloring matters found in invertebrates may be men-
tioned blue stentorin, actiniochrom, bonellin, polyperythrin, pentacrinin, antedonin,
crustaceorubin, janthinin, and chlorophyll.

Sebum when freshly secreted is an oily semi-fluid mass which solidifies
on the upper surface of the skin, forming a greasy coating. ROHMANN
and LINSER hold that sebum is a mixture of the secretion of the sebaceous
glands and of the constituents of the epidermis. HOPPE-SEYLER found,
in the sebum, a body similar to casein besides albumin and fat, while
ROHMANN and LTNSER claim that true fat occurs only ta a very slight
extent. On saponification the sebum gives an oil, dermolein, which
combines readily with iodine, and another body, dermccerin, which
melts at 64-65° and which occurs to a considerable extent in dermoid
cysts, and which is perhaps identical with the constituent of cysts,
called cetyl alcohol by v. ZEYNEK. According to AMESEDER this der-
mocerin is not a pure substance, and the cetyl alcohol obtained from
the fat of dermoid cysts is an eicosyl alcohol, C2oH42O, corresponding to
arachinic acid. Cholesterin is found in especially large quantities in

1 Wurm, cited from Maly's Jahresber., 1; Halliburton, Journ. of Physiol., 6; Merej-
kowski, Compt. Rend., 93; MacMunn, Proc. Roy. Soc., 1883, and Journ. of Physiol., 7;
v. Zeynek, Zeitschr. f. physiol. Chem., 34 and 36, and Wien. Sitz.-Ber. 121, 1912.

' Phil. Trans., 186.

3 Pfliiger's Arch., 98.

4 Ber. d. deutsch. chem. Gesellsch., 30.

42.

SEBUM. CERUMEN. 845

the vernix caseosa. RUPPEL l found on an average in the vernix caseosa
348.52 p. m. water and 138.72 p. m. ether extractives, and also mentions
the presence of isocholesterin. These claims are disputed by UNNA.2
In his experience isocholesterin does not occur in the vernix fat nor in
the sebum of man, although all kinds of sebum contain cholesterin.

According to UNNA and GoLODETZ3 the fat secretion (of the skin),
as the fat of the ball of the foot, and sebum are rich in oxy cholesterin,
while the cell fats of the outer skin does not contain any oxycholesterin,
The nails, .which are rather rich in oxycholesterin, are an exception.

On account of the opinion generally held that the wax of the plant
epidermis serves as protection for the inner parts of the fruit and plant,
LiEBREiCH4 has suggested that these combinations of fatty acids with
monatomic alcohols are the cause of the waxes having a greater resistance
as compared with the glycerin fats. He also considers that the choles-
terin fats play the r61e of a protective fat in the animal kingdom, and he
has been able to detect cholesterin fat in human skin and hair, in vernix
caseosa, whalebone, tortoise-shell, cow's horn, the feathers and beaks
of several birds, the spines of the hedgehog and porcupine, the hoofs of
horses, etc. He draws the following conclusion from this, namely,
that the cholesterin fats always appear in combination with the keratinous
substance, and that the cholesterin fat, like the wax of plants, serves
as protection for the skin-surface of animals. Of the sebum fats inves-
tigated by UNNA all contained, with the exception of the epidermis fat,
besides cholesterin, greater or smaller amounts of cholesterin ester. The
epidermis fat, on the contrary, was almost free from esters and consisted
chiefly of free cholesterin.

In the fatty protective substance secreted by the Psylla alni, SUNDVIK 5
found psylla-alcohol, C33H680, which exists there as an ester in combination with
psyllic acid, CsaHesCOOH. This alcohol has also been found in the wax of the
humble-bee.

Cerumen is a mixture of the secretion of the sebaceous and sweat
glands of the cartilaginous part of the outer passages of the ear. It
chiefly contains soaps and fat, fatty acids, cholesterin and protein, and
besides these a red substance easily soluble in alcohol and with a bitter-
sweet taste.6

1 Hoppe-Seyler, Physiol. Chem., 760; Linser with Rohmann, Centralbl. f. Physiol.,
19, 317; see also reference in ibid., 18, from Deutsch. Arch. f. klin. Med., 1904; Rtippel,
Zeitschr. f. physiol. Chem., 21; Ameseder, ibid., 52; Zumbusch, ibid., 59.

2 Monatsch. f. prakt. Dermat., 45.

3 Bioch. Zeitschr., 20.

4 Virchow's Arch., 121.

5 Zeitschr. f. physiol. Chem., 17, 25, 32, 53, 54 and 72.

6 See Lamois and Martz, Maly's Jahresber., 27, 40.

846 THE SKIN AND ITS SECRETIONS.

The preputial secretion, smegma prceputii, contains chiefly fat, also
cholesterin and ammonium soaps, which probably are produced from
decomposed urine. The hippuric acid, benzoic acid, and calcium oxalate
found in the smegma of the horse probably have the same origin.

We may also consider as a preputial secretion the castoreum, which is secreted
by two peculiar glandular sacs, in the prepuce of the beaver. The castoreum is a
mixture of proteins, fats, resins, traces of phenol (volatile oil), and a non-nitrog-
enous body, castorin, crystallizing from alcohol in four-sided needles, insoluble
in cold water, but somewhat soluble in boiling water, and whose composition is
little known.

In the secretion from the anal glands of the skunk, butyl mercaptan and alkyl
sulphides have been found (ALDRICH, E. BECKMANN *)•

Wool-fat, or the so-called fat-sweat of sheep, is a mixture of the secretion of
the sudoriparous and sebaceous glands. There is found in the watery extract a
large quantity of potassium which is combined with organic acid, volatile and non-
volatile fatty acids, benzoic acid, phenol-sulphuric acid, lactic acid, malic acid,
succinic acid, and others. The fat contains, among other bodies, abundant quan-
tities of ethers of fatty acids with cholesterin and isocholesterin. DARMSTADTER
and LIFSCHUTZ have found other alcohols in wool-fat besides myristic acid, also
two oxyfatty acids, lanoceric acid, CjoHeoCX, and lanopalmitic acid, Ci6H3203.
Isocholesterin, oxycholesterin and carnaubyl alcohol, C^ILgOH, are besides the
two last-mentioned acids, substances that are characteristic of wool-fat. Accord-
ing to ROHMANN 2 wool-fat contains a body lanocerin, which is the internal anhy-
dride of the above-mentioned lanoceric acid.

The secretion of the coccygeal glands of ducks and geese contains a body similar
to casein, besides albumin, nuclein, lecithin, and fat, but no sugar (DE JONGE).
The chief constituent is octadecyl alcohol, CisHssO, which represents 40-45 per
cent of the ethereal extract (ROHMANN). The fatty acids are oleic acid, small
amounts of caprylic acid, palmitic acid, and stearic acid, and optical isomers of
lauric and myristic acid. The fatty acids are in great part combined with the
octadecylic alcohol, and this is probably formed by the reduction of stearic acid or
oelic acid. The secretion also contains a substance related to lanocerin which
ROHMANN calls pennacerin. Poisonous bodies have been found in the secretion
of the skin of the salamander and the toad, namely, samandarin (ZALESKI, FAUST)
and bufidin (JORNARA and CASALI), bufotalin and the disputed bodies bufonin
and bufotenin (FAUST, BERTRAND and PHISALIX 3). The active constituents in
the poison of the rattle-snake and cobra, the crotalotoxin and the ophiotoxin have
been isolated and studied by FAUST. 4 They are free from nitrogen and have a
similar composition, namely, CsJ^Oai and C34H5202o and are classified in the
pharmacological group of sapotoxins by FAUST. Thalassin is the crystalline body
discovered by RICHET 5 which is the poisonous constituent of the feelers of the
sea nettle.

1 Aldrich, Journ. of Exp. Med., 1; Beckmann, Maly's Jahresber., 26, 566.

2 Darmstader and Lifschiitz, Ber. d. d. Chem., Gesellsch., 29 and 31; Rohmann,
Hofmeister's Beitrage, 5, and Centralbl. f. Physiol., 19, 317. See also Unna, 1. c., 45;
and Lifschiitz and Unna, ibid., p. 234.

3 De Jonge, Zeitschr. f. physiol. Chem., 3; Rohmann, 1. c.; Zaleski, Hoppe-Seyler's
Med.-chem. Untersuch., p. 85; Faust, Arch. f. exp. Path. u. Pharm., 41; Jornara and
Casali, Maly's Jahresbr., 3; Faust, Arch. f. exp. Path. u. Pharm., 47 and 49; Bertrand,
Compt. Rend., 135; Bertrand and Phisalix, ibid.

4 Arch. f. exp. Path. u. Pharm., 56 and 64.
6 Pfluger's Arch., 108.

PERSPIRATION. 847

The Perspiration. Of the bodies secreted by the skin, whose quantity
amounts to about ^j of the weight of the body, a disproportionately
large part consists of water. Next to the kidneys, the skin, in man, is
the most important means for the elimination of water. As the glands
of the skin and the kidneys stand near to each other in regard to their
functions, they may to a certain extent act vicariously.

The circumstances which influence the secretion of perspiration are numerous,
and the quantity of sweat secreted must consequently vary considerably. The
secretion differs in different parts of the skin, and it has been stated that the per-
spiration of the cheek, that of the palm of the hand, and that under the arm stand
to each other as 100:90:45. From the unequal secretion on different parts of the
body it follows that no results as to the quantity of secretion for the entire surface
of the body can be calculated from the quantity secreted by a small part of the
skin in a given time. In determining the total quantity a stronger secretion is as
a rule produced, and as the glands can with difficulty work for a long time with
the same energy, it is hardly correct to estimate the quantity of secretion per day
from a strong secretion during only a short time.

The perspiration obtained for investigation is never quite pure, but
contains cast-off epidermis-cells, also cells and fat-globules from the
sebaceous glands. Filtered perspiration is a clear, colorless fluid with
a salty taste and of different odors from different parts of the body. The
physiological reaction is acid, according to most reports. Under certain
conditions an alkaline sweat may be secreted (TRUMPY and LUCHSINGER,
HEUSS). An alkaline reaction may also depend on a decomposition
with the formation of ammonia. According to a few investigators the
physiological reaction is alkaline, and an acid reaction depends upon
an admixture of fatty acids from the sebum. CAMERER found that
the reaction of human perspiration in certain cases was acid and in
others alkaline. MORIGGIA found that the sweat from herbivora was
ordinarily alkaline, while that from carnivora was generally acid.
SMITH l showed that horse's sweat is strongly alkaline.

KiTTSTEiNER,2 who has found that human perspiration is nearly always
acid, has also found that the perspiration from the vola manus, when
not contaminated with sebum, is acid in reaction and that an acid reac-
tion is not necessarily dependent upon an admixture with sebum.

The specific gravity of human perspiration varies between 1.001
and 1.010. It contains 977.4-995.6 p. m., average about 982 p. m.
water. The solids are 4.4-22.6 p. m. The molecular concentration
also varies widely and the freezing-point depression depends essentially

1 Triimpy and Luchsinger, Pfliiger's Arch., 18; Heuss, Maly's Jahresber., 22;
Camerer, Zeitschr. f. Biologie, 41; Moriggia, Moleschott's Untersuch. zur Naturlehre,
11; Smith, Journ. of Physiol., 11. In regard to the older literature on perspiration,
see Hermann's Handbuch, 5, Thl. 1, 421 and 543.

2 Arch. f. Hyg., 73 and 78.

848 THE SKIN AND ITS SECRETIONS.

upon the content of NaCl. ARDIN-DELTEIL found A= —0.08-0.46°,
average— 0.327°. BRIEGER and DISSELHORST found with perspiration
containing 2.9, 7.07 and 13.5 p. m. NaCl that the A was equal to-0.322°,
—0.608° and —1.002°, respectively. TARUGI and TOMASINELLI l found A
to be 0.52° as an average. KiTTSTEiNER2 found that perspiration had an
average specific gravity of 1.0046 and the average quantities of nitrogen
and sulphur were 0.5 and 0.08 p. m. respectively. The NaCl content
increased with the rapidity of secretion while the nitrogen content
diminished. The organic bodies are neutral fats, cholesterin, volatile fatty
acids, traces of protein (according to LECLERC and SMITH always in
horses, and according to GATJBE regularly in man, while LEUBE 3 claims
only occasionally after hot baths, in BRIGHT'S disease, and after the use
of pilocarpin), creatinine (CAPRANIC A), ar omatic oxy acids, ethereal-sulphuric
acids of phenol and skatoxyl (KAST4), sometimes also of indoxyl, serine
(page 145) and lastly urea. The quantity of urea has been determined by
ARGUTINSKY. In two steam-bath experiments, in which in the course of
^ and | hour respectively he obtained 225 and 330 cc. of perspiration, he
found 1.61 and 1.24 p. m. urea. Of the total nitrogen of the perspiration
in these two experiments 68.5 per cent and 74.9 per cent respectively
belong to the urea. From ARGUTINSKY'S experiments, and also from
those of CRAMER,5 it follows that of the total nitrogen a portion, not to
be disregarded, is eliminated by the perspiration. This portion was
indeed 12 per cent, in an experiment of CRAMER, at high temperature
and powerful muscular activity, and ZUNTZ and his collaborators find
indeed more than 13 per cent in high altitudes. CRAMER also found
ammonia in the perspiration. In uraemia and in anuria in cholera,
urea may be secreted in such quantities, by the sweat-glands, that crystals
deposit upon the skin. The mineral bodies consist chiefly of sodium
chloride with some potassium chloride, alkali sulphate and phosphate.
The relative quantities of these in perspiration differ materially from
the amount in the urine (FAVRE, KASTC). The relation, according to
KAST, is as follows:

Chlorine : Phosphate : Sulphate:

In perspiration 1 : 0.0015 : 0.009

In urine... 1 : 0.1320 : 0.397

1 Ardin-Delteil, Maly's Jahresber., 30; Brieger and Disselhorst, Deutsch. med.
Wochenschr., 29; Tarugi and Tomasinelli, cited in Physiol. Centralbl., 22, 748.

*Lc.

3 Leclerc, Compt. Rend., 107; Gaube, Maly's Jahresber., 22; Leube, Virchow's
Arch., 48 and 50, and Arch. f. klin. Med., 7.

.4Capranica, Maly's Jahresber., 12; Kast, Zeitschr. f. physiol. Chem., 11.

5 Argutinsky, Pfliiger's Arch., 46; Cramer, Arch. f. Hygiene, 10.

6 Compt. Rend., 35, and Arch. ge"ner. de Med. (5), 2; Kast, 1. c.

EXCHANGE OF GAS THROUGH THE SKIN. 849

KAST found that the proportion of ethereal-sulphuric acid to the
sulphate-sulphuric acid in perspiration was 1:12. After the administra-
tion of aromatic substances the ethereal-sulphuric acid does not increase
to the same extent in the perspiration as in the urine (see Chapter XIV) .
The quantity of mineral substances was on an average 7 p. m.

Sugar may pass into the perspiration in diabetes, but the passage of the bile-
coloring matters has not been positively shown in this secretion. Benzoic add,
succinic acid, tartaric acid, iodine, arsenic, mercuric chloride and quinine pass
into the perspiration. Uric acid has also been found in the perspiration in gout
and cystine in cystinuria.

Chromhidrosis is the name given to the secretion of colored perspiration.
Sometimes perspiration has been observed to be colored blue by indigo (Bizio),
by pyocyanin, or by ferro-phosphate (COLLMANN 1). True blood-sweat, in which
blood-corpuscles exude from the opening of the glands, has also been observed.

The exchange of gas through the skin is of great importance for non-
scaly amphibians; in mammalia, birds and human beings it is of little
importance compared with the exchange of gas by the lungs. The
absorption of oxygen by the skin, which was first shown by REGNATJLT
and REISET, is small, and according to ZUELZER amounts under the
most favorable circumstances to yiir of the oxygen absorbed by the
lungs. The quantity of carbon dioxide eliminated by the skin increases
with the rise of temperature (AUBERT, ROHRIG, FUBINI and RONCHI,
BARRATT and according to WILLEBRAND beginning at 33 °)2. It especially
increases with hypersemia of the skin and in particular after muscular
activity. It is also greater in light than in darkness. It is greater dur-
ing digestion than when fasting, and greater after a vegetable than after
an animal diet (FUBINI and RONCHI). The quantity calculated by differ-
ent investigators for the entire skin surface in twenty-four hours varies
between 2.23 and 32.8 grams. According to SCHIERBECK and WILLE-
BRAND 3 the average quantity is 7.5-9 grams, and it is ordinarily given as
about 1.5 per cent of the quantity eliminated by the lungs. In a horse,
ZUNTZ, with LEHMANN and HAGEMANN,* found for twenty-four hours
an elimination of carbon dioxide by the skin and intestine which amounted
to nearly 3 per cent of the total respiration. Less than four-fifths of
this carbon dioxide came from the skin respiration. The same investi-
gators found that the skin respiration equals 2J per cent of the simulta-
neous lung respiration.

1 Bizio, Wien. Sitzungsber., 39; Collmann, cited from v. Gorup-Besanez's Lehrbuch,
4. Aufl., 555.

2 Zuelzer, Zeitschr. f. klin. Med., 53; Aubert, Pfliiger's Arch., 6; Rohrig, Deutsch.
Klin., 1872, 209; Fubini and Ronchi, Moleschott's Untersuch. z. Naturlehre, 12;
Barratt, Journ. of Physiol., 21; Willebrand, Skand. Arch. f. PhysioL, 13.

3 See Hoppe-Seyler, Physiol. Chem., 580; Schierbeck, Arch. f. (Anat. u.) Physiol.,
1892; Willebrand, 1. c.

4 Arch, f . (Anat. u.) Physiol., 1894, and Maly's Jahresber., 24.

CHAPTER XVI.
RESPIRATION AND OXIDATION.

DURING life a constant exchange of gases takes place between the
animal body and the surrounding medium. Oxygen is inspired and
carbon dioxide expired. This exchange of gases, which is called respira-
tion, is brought about in man and vertebrates by the nutritive fluids,
blood and lymph, which circulate in the body and which are in constant
communication with the outer medium on one side and the tissue-elements
on the other. Such an exchange of gaseous constituents may take place
wherever the anatomical conditions offer no obstacle, and in man it may
go on in the intestinal tract, through the skin, and in the lungs. As
compared with the exchange of gas in the lungs, the exchange already
mentioned, which occurs in the intestine and through the skin, is very
insignificant. For this reason we will discuss in this chapter only the
exchange of gas between the blood and the air of the lungs on one side
and the blood and lymph and the tissues on the other. The first is often
designated as external respiration, and the other, internal respiration.
Besides this we will discuss the oxidation processes caused by the internal
respiration.

I. THE GASES OF THE BLOOD.

Since the pioneer investigations of MAGNUS and LOTHAR MEYER, the
gases of the blood have formed the subject of repeated careful investiga-
tions by prominent experimenters, among whom must be mentioned first
C.LuDWiGand his pupils, and E. PFLUGER and his school; and C. BOHR.
By these investigations not only has science been enriched by a mass of
facts, but also the methods themselves have been made more perfect
and accurate. In regard to these methods, as also in regard to the laws
of the absorption of gases by liquids, dissociation, and related questions,
the reader is referred to text-books on physiology, on physics, and on
gasometric analysis.

The gases occurring in blood under physiological conditions are
oxygen, carbon dioxide and nitrogen, and traces of argon, and perhaps
also carbon monoxide. Traces of hydrogen and marsh-gas also some-
times occur. The nitrogen is found only in very small quantities, on an
average 1.2 vols. per cent. The quantity is here, as in all following

850

GASES OF THE BLOOD. 851

experiments, calculated for 0° C. and 760 mm. mercury pressure. The
nitrogen seems to be simply absorbed by the blood, at least in great
part. It appears, like argon, to play no direct part in the processes of
life, and its quantity varies but slightly in the blood of different blood-
vessels.

The oxygen and carbon dioxide behave otherwise, as their quantities
have significant variations, not only in the blood from different blood-
vessels, but also because many factors, such as a difference in the rapidity
of circulation and the ventilation of the lungs, a different temperature,
alkalinity of the blood, rest and activity cause a change. In regard to the
gases they contain, the greatest difference is observable between the blood
of the arteries and that of the veins.

The quantity of oxygen in the arterial blood (of dogs) is on an average
22 vols. per cent (PFLUGER, BOHR and HENRIQUES). In human blood
SETSCHENOW found about the same quantity, namely, 21.6 vols. per
cent. LEOWY in another manner has determined the quantity of oxygen
which the blood can take up by first shaking human venous blood with
air and then calculating from this the quantity of oxygen in human
arterial blood. He calculates the average amount as 18 vols. per cent.
Lower figures have been found for the blood of herbivora (such as horse,
sheep, rabbits) and birds (hen and ducks) namely, 14-10.7 per cent (ZUNTZ
and HAGEMANN, SCZELKOW, WALTER, JOLYET). Venous blood in dif-
ferent vascular regions has variable quantities of oxygen. By sum-
marizing a great number of analyses by different experimenters, ZUNTZ
has calculated that the venous blood of the right side of the heart con-
tains on an average 7.15 per cent less oxygen than the arterial blood.

The quantity of carbon dioxide in the arterial blood (of dogs) is about
40 vols. per cent (LUDWIG, SETSCHENOW, PFLUGER, P. BERT, BOHR
and HENRIQUES and others), or a little above. In herbivora and the
above-mentioned birds the quantity of carbon dioxide in the arterial
blood is higher than in the carnivorous dog. SETSCHENOW found 40.3
vols. per cent in human arterial blood. The quantity of carbon dioxide
in venous blood varies still more (LUDWIG, PFLUGER, and their pupils,
P. BERT, MATHIEU and URBAIN, and others). According to the calcula-
tions of ZUNTZ, the venous blood of the right side of the heart contains
about 8.2 per cent more carbon dioxide than the arterial. The average
amount may be put down as 50 vols. per cent. HOLMGREN found in
blood after asphyxiation even 69.21 vols. per cent carbon dioxide.1

1 All the figures given above may be found in Zuntz's " Die Gase des Blutes " in
Hermann's Handbuch d. Physiol., 4, Thl. 2, 33-43, which also contains detailed state-
ments and the pertinent literature, and Bohr in Nagel's Handbuch der Physiologic des
Menschen, Bd. 1, Hefte 1, 1905, and in Loewy, Handb. d. Bioch. of C. Oppenheimer,
Bd. 4.

852 RESPIRATION AND OXIDATION.

Oxygen is dissolved only in a small extent by the plasma, whose
absorbability for oxygen is 97.5 per cent of that of water, according to
BOHR. The greater part or nearly all of the oxygen is loosely combined
with the haemoglobin. The quantity of the oxygen which is contained in
the blood of the dog corresponds closely to the quantity which, from the
activity of the haemoglobin, we should expect to combine with oxygen
and from the quantity of haemoglobin contained therein. It is difficult
to ascertain how far the circulating arterial blood is saturated with oxygen,
as immediately after bleeding a loss of oxygen always takes place. Still
it seems to be unquestionable that it is not quite completely saturated
with oxygen, in life. The laws which regulate the binding of the oxygen
in the blood will be found in the discussion of the gas exchanged between
the blood and the air of the lungs.

The carbon dioxide of the blood occurs in part, and indeed, accord-
ing to the investigations of ALEX. SCHMIDT/ Zu-NTz-,2 and L. FREDERICQ,S
to the extent of at least one-third in the blood-corpuscles, also in part,
and in fact the greatest part, in the plasma or serum. BoHR4 claims
that about 30 mm. may be considered as the average pressure of the
carbon dioxide in the organism, and with such a pressure the quantity of
physically dissolved CO2 in 100 cc. of the blood amounts to 2.01 cc. As
the blood with this tension takes up about 40 vols. per cent C02, there-
fore about 5 per cent of the total carbon dioxide is simply dissolved.
Under the assumption that the blood corpuscles make up about one-
third of the volume of the blood, of the physically dissolved C02, 0.59
cc. exists with the corpuscles and 1.42 cc. with the plasma.

As the blood corpuscles in 100 cc. blood as above stated take up at
the above pressure about 14 cc. CO2, only a small part of its CO2 is physi-
cally dissolved. The chief mass of the C02 is loosely combined and the
constituent of these cells which unites with the CCb seems to be the
alkali combined with phosphoric acid, oxyhaemoglobin or haemoglobin,
and globulin on one side and the haemoglobin itself on the other. That
in the red blood-corpuscles alkali phosphate occurs in such quantities
that it may be of importance in the combination with carbon dioxide
is not to be doubted; and it must be allowed that from the diphosphate,
by a greater partial pressure of the carbon dioxide, monophosphate and
alkali carbonate are formed, while by a lower partial pressure of the
carbon dioxide, the mass action of the phosphoric acid again comes into
play, so that, with the carbon dioxide becoming free, a reformation of

1 Ber. d. k. sachs. Gesellsch. d. Wissensch. math.-phys. Klasse, 1867.
2Centralbl. f. d. med. Wissensch., 1867, 529.
8 Recherches sur la constitution du Plasma sanguin, 1878, 50, 51.
4 In regard to the work of Bohr we will refer here and in future to Nagel's Handbuch
der Physiologie des Menschen, Bd. 1.

CARBON DIOXIDE IN THE BLOOD. 853

alkali diphosphate takes place. It is generally admitted that the blood-
coloring matters, especially the oxyhaemoglobin, which can expel carbon
dioxide from sodium carbonate in vacuo, acts like an acid, and as the
globulins also act similarly (see below), these bodies may also occur in
the blood-corpuscles as an alkali combination. The alkali of the blood-
corpuscles must, therefore, according to the law of mass action, be divided
between the carbon dioxide, phosphoric acid, and the other constituents
of the blood-corpuscles which possess acidic properties, and among these
especially the blood pigments, because the globulin can hardly be of
importance on account of its small quantity. By greater mass action
or greater partial pressure of the carbon dioxide, bicarbonate must be
formed at the expense of the diphosphates and the other alkali combina-
tions, while at a diminished partial pressure of the same gas, with the
escape of carbon dioxide, the alkali diphosphate and the other alkali
combinations must be reformed at the cost of the bicarbonate.

Haemoglobin must nevertheless, as the investigations of SETSCHENOW l
and ZUNTZ, and especially those of BOHR and ToRUP,2 have shown, be
able to hold the carbon dioxide loosely combined even in the absence
of alkali. BOHR has also found that the dissociation curve of the car-
bon dioxide haemoglobin corresponds essentially to the curve of the
absorption of carbon dioxide, on which ground he and TORUP consider
the haemoglobin itself as of importance in the binding of the carbon
dioxide of the blood, and not its alkali combinations. According to
BOHR the haemoglobin takes up the two gases, oxygen and carbon dioxide,
simultaneously by the oxygen uniting with the pigment nucleus and the
carbon dioxide with the protein component. But as according to the
researches of ZUNTZ 3 the combination of haemoglobin with the alkali is
first split to any great extent with a carbon dioxide tension of more than
70 mm., it must be admitted that with the ordinary CO2 pressure in
the organism, the combination of the carbon dioxide in the blood cor-
puscles does not essentially take place through the agency of the alkali
but chiefly by means of the haemoglobin.

The chief part of the carbon dioxide of the blood is found in the
blood-plasma or the blood-serum, which follows from the fact that the
serum is richer in carbon dioxide than the corresponding blood itself.
By experiments with the air-pump on blood-serum it has been found
that the chief part of the carbon dioxide contained in the serum is given
off in a vacuum, while a smaller part can be removed only after the

1 Centralbl. f. d. med. Wissench., 1877. See also Zuntz in Hermann's Handbuch,
76.

2 Zuntz, 1. c., 76; Bohr, Maly's Jahresber., 17; Torup, ibid.

3 Centralbl. f. d. med. Wissensch., 1867.

854 RESPIRATION AND OXIDATION.

addition of an acid. The red blood-corpuscles also act as an acid, and
therefore in blood all the carbon dioxide is expelled in vacuo. Hence
a part of the carbon dioxide is in firm chemical combination in the serum.

Absorption experiments with blood-serum have shown us further that
the carbon dioxide which can be pumped out is in greater part loosely
chemically combined, and from this loose combination of the carbon
dioxide it necessarily follows that the serum must also contain simply
absorbed carbon dioxide. For the form of binding of the carbon dioxide
contained in the serum or the plasma, there are the three following pos-
sibilities: 1. A part of the carbon dioxide is simply absorbed; 2. Another
part is in loose chemical combination; 3. A third part is in firm chemical
combination.

The quantity of physically dissolved carbon dioxide in the serum
cannot be higher than about 2 vols. per cent, as the quantity of carbon
dioxide in the plasma corresponding to 100 cc. of blood is given above
as 1.42 cc.

The quantity of carbon dioxide in the blood-serum which is combined
as a firm chemical union depends upon the quantity of simple alkali
carbonate in the serum. This amount is not known, and it cannot be
determined either by the alkalinity found by titration, nor can it be cal-
culated from the excess of alkali found in the ash, because the alkali is not
only combined with carbon dioxide, but also with other bodies, especially
with protein. The quantity of carbon dioxide in firm chemical combi-
nation cannot be ascertained after pumping out in vacuo without the
addition of acid, because to all appearances certain active constituents
of the serum, acting like acids, expel carbon dioxide from the simple
carbonate. The quantity of carbon dioxide not expelled from dog-
serum by vacuum alone without the addition of acid amounts to 4.9
to 9.3 vols. per cent, according to the determinations of PFLUGER.1

From the occurrence of simple alkali carbonates in the blood-serum
it naturally follows that a part of the loosely combined carbon dioxide
of the serum which can be pumped out must exist as bicarbonate. The
occurrence of this combination in the blood-serum has also been directly
shown. In experiments with the pump, as well as in absorption experi-
ments, the serum behaves in other ways differently from a solution of bicar-
bonate, or carbonate of a corresponding concentration; and the action
of the loosely combined carbon dioxide in the serum can be explained
only by the occurrence of bicarbonate in the serum. By means of a
vacuum, the serum always allows much more than one-half of the carbon
dioxide to be expelled and it follows from this that in the pumping out

XE. Pfliiger's Ueber die Kohlensaure des Blutes, Bonn, 1864, 11. Cited from
Zuntz in Hermann's Handbuch, 65.

CARBON DIOXIDE IN THE BLOOD. 855

not only may a dissociation of the bicarbonate take place, but also a
conversion of the double sodium carbonate into a simple salt. As we
know of no other carbon-dioxide combination, besides the bicarbonate,
in the serum, from which the carbon dioxide can be set free by simple
dissociation in vacuo, it must be assumed that the serum contains other
weak acids, in addition to the carbon dioxide, which contend with it for
the alkalies, and which expel the carbon dioxide from simple carbonates
in vacuo. The carbon dioxide which is expelled by means of the pump,
and which, without regard to the quantity merely absorbed, is generally
designated as " carbon dioxide in loose chemical combination," is thus
only obtained in part in dissociable loose combinations; in part it origi-
nates from the simple carbonates, from which it is expelled, in vacuo, by
other weak acids.

These weak acids are thought to be in part phosphoric acid and in
part globulins. The importance of the alkali phosphates in the car-
bon dioxide combination has been shown by the investigations of FERNET;
but the quantity of these salts in the serum is, at least in certain kinds
of blood, for example, in ox-serum, so small that it can hardly be of
importance. In regard to the globulins, SETSCHENOW is of the opinion
that they do not act as acids themselves, but form a combination with
carbon dioxide, producing carboglobulinic acid, which unites with the
alkali. According to SERTOLi,1 whose views have found a supporter
in TORUP, the globulins themselves are the acids which are combined
with the alkali of the blood-serum. In both cases the globulins would
form, directly or indirectly, that chief constituent of the plasma or of
the blood-serum which, according to the law of mass action, contends
with the carbon dioxide for the alkalies. By a greater partial pressure
of the carbon dioxide the latter deprives the globulin alkali of a part of
its alkali, and bicarbonate is formed; by low partial pressure carbon
dioxide is set free and it is abstracted from the bicarbonate by the
globulin alkali. It must also be added that the above-mentioned car-
boglobulinic acid can perhaps be considered as a dissociable combination
of carbonic acid and protein.

The assumption that the proteins of the blood are bodies active in
combining with the carbon dioxide has received some support from the
investigations of SIEGFRIED 2 on the combination of carbon dioxide with
amphoteric amino bodies. SIEGFRIED has found that amino-acids com-
bine with carbon dioxide, thereby being converted into carbamino-

1 Hoppe-Seyler, Med. chem. Untersuch., 350.

2 Zeitschr. f. physiol. Chem., 44 and 46.

856 KESPIRATION AND OXIDATION.

H
acids— glycocoll for example, into carbamino acetic acid, CH2 — N — COOH,

COOH

and that the carbon dioxide can be readily split off from these compounds.
The peptones and serum proteins in the presence of calcium hydroxide
may also act in the same manner as amino-acids. Protein carbamino-
acids are formed, and the possibility of such a binding of carbon dioxide
must also be considered.

In the foregoing it has been assumed that the alkali is the most essen-
tial and important constituent of the blood-serum, as well as of the blood
in general, in uniting with the carbon dioxide. The fact that the quan-
tity of carbon dioxide in the blood greatly diminishes with a decrease
in the quantity of alkali strengthens this assumption. Such a condi-
tion is found, for example, after poisoning with mineral acids. Thus
WALTER found only 2-3 vols. per cent carbon dioxide in the blood of
rabbits into whose stomachs hydrochloric acid had been introduced. In
the comatose state of diabetes mellitus the alkali of the blood seems to
be in great part saturated with acid combinations, /3-oxybutyric acid
(STADELMANN, MINKOWSKI), and MINKOWSKI i found only 3.3 vols.
per cent carbon dioxide in the blood in diabetic coma.

Gases of the Lymph and Secretions.

The gases of the lymph are the same as in the blood-serum, and the
lymph stands close to the blood-serum in regard to the quantity of the
various gases, as well as to the kind of carbon-dioxide combination. The
investigations of DAENHARDT and HENSEN 2 on the gases of human
lymph are at hand, but it still remains a question whether the lymph
investigated was quite normal. The gases of normal dog-lymph were
first investigated by HAMMARSTEN.S This gas contained traces of
oxygen and consisted of 37.4-53.1 per cent C02 and 1.6 per cent N at 0°
C. and 760 mm. Sg pressure. About one-half of the carbon dioxide was
in firm chemical combination. The quantity was greater than in the
serum from arterial blood, but smaller than from venous blood.

The remarkable observation of BUCHNER, that the lymph collected
after asphyxiation is poorer in carbon dioxide than that of the breathing

1 Walter, Arch. f. exp. Path. u. Pharm., 7; Stadelmann, ibid., 17; Minkowski,
Mittheil a. d. med. Klinik in Konigsberg, 1888.

2 Virchow's Arch., 37.

8 Ber. d. k. sachs. Gesellsch. d. Wissensch., math.-phys. Klasse, 23.

GASES OF THE LYMPH AND SECKETIONS. 857

animal, is explained by ZUNTZ l by the formation of acid in the tissues,
and especially in the lymphatic glands, immediately after death, and
this acid in part decomposes the alkali carbonates of the lymph. <j

The secretions, with the exception of the saliva, in which PFLUGER
and KULZ found respectively 0.6 per cent and 1 per cent oxygen, are
almost free from oxygen. The quantity of nitrogen is the same as in blood,
and the chief mass of the gases consists of carbon dioxide. The quantity
of this gas is chiefly dependent upon the reaction, i.e., upon the quan-
tity of alkali. This follows from the analyses of PFLUGER. He found
19 per cent carbon dioxide removable by the air-pump and 54 per cent
firmly combined carbon dioxide in a strongly alkaline bile, but, on the
contrary, 6.6 per cent carbon dioxide removable by the air-pump and
0.8 per cent firmly combined carbon dioxide in a neutral bile. Alkaline
saliva is also very rich in carbon dioxide. As average for two analyses'
made by PFLUGER of the submaxillary saliva of a dog we have 27.5 per
cent carbon dioxide removable by the air-pump and 47.4 per cent chem-
ically combined carbon dioxide, making a total of 74.9 per cent. KULZ 2
found a maximum of 65.78 per cent carbon dioxide for the parotid saliva,
of which 3.31 per cent was removable by the air-pump and 62.7 per
cent was firmly combined. From these and other reports as to the
quantity of carbon dioxide removable by the air-pump and chemically
combined in the alkaline secretions it follows that bodies occur in them,
although not in appreciable quantities, which are analogous to the pro-
tein bodies of the blood-serum and which act like weak acids.

The acid or at any rate non-alkaline secretions, urine and milk, con-
tain, on the contrary, considerably less carbon dioxide, which is almost
all removable by the air-pump, and a part seems to be loosely combined
with the sodium phosphate. The figures found by PFLUGER for the
total quantity of carbon dioxide in milk and urine are 10 and 18.1-19.7
per cent respectively.

EwALD3 made investigations on the quantity of gas in pathological
transudates. He found only traces, or at least only very insignificant
quantities of oxygen in these fluids. The quantity of nitrogen was about
the same as in blood; that of carbon dioxide was greater than in the
lymph (of dogs), and in certain cases even greater than in the blood after
asphyxiation (dog's blood). The tension of the carbon dioxide was
greater than in venous blood. In exudates the quantity of carbon
dioxide, especially that firmly combined, increases with the age of the

1Buchner, Arbeiten aus der physiol. Anstalt zu Leipzig, 1876; Zuntz, I. c., 85.
2 Pfluger, Pfliiger's Arch., 1 and 2; Kiilz, Zeitschr. f. Biologie, 23. It seems as if
Ruiz's results were not calculated at 760 millimeters Hg. but rather at 1 meter.
» C. A. Ewald, Arch. f. (Anat. u.) Physiol., 1873 and 1876.

858 RESPIRATION AND OXIDATION.

fluid, while, on the contrary, the total quantity of carbon dioxide, and
especially the quantity firmly combined, decreases with the quantity
of pus-corpuscles.

II. THE EXCHANGE OF GAS BETWEEN THE BLOOD, ON THE ONE HAND,
AND PULMONARY AIR AND THE TISSUES, ON THE OTHER.

In Chapter I (page 42) it was stated that we are to-day of the
opinion, derived especially from the researches of PFLUGER and his
pupils, that the oxidations of the animal body do not take place in the
fluids and juices, but are connected with the form-elements and tissues.
It is nevertheless true that oxidations take place in the blood itself, al-
though, only to a slight extent; but these oxidations depend, it seems,
upon the form-elements of the blood, hence it does not contradict the
above statement that the oxidations exclusively occur in the cells and
chiefly in the tissues.

The gaseous exchange in the tissues, which has been designated
internal respiration, consists chiefly in that the oxygen passes from the
blood in the capillaries to the tissues, while the great bulk of the carbon
dioxide of the tissues originates therein and passes into the blood of the
capillaries. The exchange of gas in the lungs, which is called external
respiration, consists, as is seen by a comparison of the inspired and
expired air, in the blood taking oxygen from the air in the lungs and giving
off carbon dioxide. This does not exclude the fact that in the lungs, as in
every other tissue, an internal respiration takes place, namely, a com-
bustion with a consumption of oxygen and formation of carbon dioxide.
According to BOHR and HENRIQUES 1 the lungs take a variable but
sometimes a very important part in the total metabolism. This part,
which on an average is 33 per cent, but may even rise above 60 per
cent of the total ' metabolism, depends, these experimenters say, upon
the fact that the intermediary metabolic products formed in the tissues
are burnt in the lungs. It is also in part represented by the specific
work of the lungs.

What kind of processes take part in this double exchange of gas?
Is the gaseous exchange simply the result of an unequal tension of the
blood on one side and the air in the lungs or tissues on the other? Do
the gases pass from a place of higher pressure to one of a lower, according
to the laws of diffusion, or are other forces and processes active?

These questions are closely related to that of the tension of the
oxygen and carbon dioxide in the blood and in the air of the lungs and
tissues.

1 Centralbl. f. Physiol., 6, and Maly's Jahresber., 27.

GAS EXCHANGE. OXYGEN TENSION. 859

Oxygen occurs in the blood in a disproportionately large part as
oxy haemoglobin, and the law of the dissociation of oxy hemoglobin is
of fundamental importance in the study of the tension of the oxygen
in the blood.

Attempts have been made to prove this law by investigations on
pure solutions of haemoglobin, and HUFNER l has made very careful and
important determinations on such solutions. Recent investigations
of BOHR 2 and his pupils, as well as of LOEWY and ZuNTZ,3 have shown
that the conditions in the blood are different from a pure haemoglobin
solution, which, in part, may be due to a change in the haemoglobin
brought about in its preparation. A haemoglobin solution in which
alcohol is used in preparing it, combines more firmly with oxygen than
the blood, and the dissociation tension of the oxygen is greater in blood
than in such a haemoglobin solution.

The oxygen tension may be variable, as LOEWY 4 has shown, with
different individuals, and it is not the same in the blood of different
animals with the same oxygen pressure; for example, it is less in cat's
blood than in the dog, horse and human blood. The temperature is also
of great importance, as the dissociation tension increases with a rise in
temperature, and with the same pressure the blood combines with less
oxygen at a high temperature than at a low temperature. The influence
of the concentration of the haemoglobin manifests itself in that in dilute
solutions the oxygen is more firmly combined (H.UFNER, LOEWY and ZUNTZ,
BOHR) and that consequently blood made laky with water has a lower
dissociation tension and a firmer binding of the oxygen than undiluted
blood.

Of especial interest is the finding of BOHR, HASSELBALCH and KROGH 5
that the C02 present also influences the oxygen taken up, in that as the
carbon dioxide tension (also within physiological limits) increases the
oxygen taken up diminishes. The laws of oxygen absorption must
be determined by determinations upon blood itself, at the same time
observing the temperature and the carbon dioxide tension. A series of
determinations made by KROGH 6 upon horse's blood at 38°, and a con-
stant carbon dioxide tension, is given below. In calculating the results
in column 4 the quantity of oxygen chemically combined at 150 mm.
oxygen pressure is equal to 100.

1 Arch. f. (Anat. u.) Physiol., 1890 and 1894.

2 See Nagel's Handbuch, and Krogh, Skand. Arch. f. Physiol., 16.

3 Arch. f. (Anat. u.) Physiol., 1904.

4 Ibid.

5 Centralbl. f. Physiol., 17, and Skand. Arch. f. Physiol., 16.
e Skand. Arch, f . Physiol., 16.

860 RESPIRATION AND OXIDATION.

In 100 cc. blood Oxygen taken up

Oxygen
tension in mm.

Chemically
combined
oxygen

Oxygen
dissolved
in plasma

Per cent
chemically
combined

Dissolved in
100 cc. plasma

10

6.0

0.020

30.0

0.030

20

12.9

0.041

64.7

0.061

30

16.3

0.061

81.6

0.091

40

18.1

0.081

90.4

0.121

50

19.1

0.101

95.4

0.152

60

19.5

0.121

97.6

0.182

70

19.8

0.141

98.8

0.212

80

19.9

0.162

99.5

0.243

90

19.95

0.182

99.8

0.273

150

20.00

0.303

100.0

0.455

From the above table we see that even with an oxygen tension which
amounts to only one-half of the oxygen pressure in the air, the haemoglobin
is saturated in greatest part with oxygen. The dissociation is hence at
70-80 mm. pressure only slightly more than with a pressure of 150 mm.
and indeed even with as low a pressure as 40-30 mm., still 90-80 per cent
of the entire quantity of oxygen taken up chemically at 150 mm. is com-
bined with the haemoglobin.

From these and other observations it follows that the oxygen partial
pressure may sink to one-half of that existing in the atmospheric air
without markedly influencing the oxygen content of the blood. This
also coincides with the experience of FRANKEL and GEPPERT 1 on the
action of low air pressures upon the oxygen content of the blood of dogs.
With an air pressure of 410 mm. Hg, they found that the oxygen content
of arterial blood was normal. With an air pressure of 378-365 mm.
it was slightly diminished, and only on reducing the pressure to 300
mm. was a noticeable decrease observed. A. LoEWY2 found that
the lowest oxygen pressure of the alveolar air wherein the exchange
of material can go on normally both qualitatively and quantitatively,
is equal to 30 mm. Hg.

In regard to the above-mentioned action of low air pressure it must
be remarked that the alveolar oxygen tension is regulated by changes
in the respiration, so that with great diminution in the quantity of oxygen
of the inspired air, the alveolar air contains the same quantity of oxygen
as with a higher oxygen partial pressure of the inspired air (LOEWY).
For example, LOEWY found the same quantity of oxygen, namely, 6.1
per cent, in the alveolar air with 16 and with 10.5 per cent oxygen in
the inspired air, because the respiration in the latter case was 8.5 liters
per minute against only 4.9 liters in the first case.

It may be concluded from the large quantity of oxygen or oxyhsemo-

1 Ueber die Wirkungen der verdiinnten Luft auf den Organismus, Berlin, 1883.

2 A. Loewy, Untersuch. iiber die Respiration und Zirculation, etc., Berlin, 1895;
Centralbl. f. Physiol., 13, 449, and Arch. f. (Anat. u.) Physiol., 1900.

OXYGEN TENSION IN BLOOD. 861

globin in the arterial blood that the tension of the oxygen in the arterial
blood must be relatively higher. This is substantiated' by the earlier
observations of BERT and HUFNER, as well as by the determinations
of HERTER, FREDERICQ and others,1 using aerotonometric methods,
which will be mentioned below in connection with the carbon dioxide
tension. HERTER found the oxygen tension in the arterial blood of
dogs to be equal, on an average, to a pressure of 78.7 mm. Hg and FRE-
DERICQ, by a better method, found that it was equal to 91-99 mm. Hg.

The oxygen tension of the venous blood of dogs has been found by
aerotonometric means to be equal to 20.6-27.7 mm. (STRASSBURG,
FALLOISE), and by means of the lung-catheter (see below) equal to 25.5-27
mm. (WOLFBERG, NUSSBAUM). For human venous blood LOEWY and v.
ScHROTTER2 found an average of 37.68 mm. Concerning the question
as to the mechanism of taking up oxygen in the lungs these figures are of
less interest than the oxygen tension in the arterial blood, that is, that
which has left the lungs, whose tension is estimated as 90 to about 100
mm. Hg as given above.

These results do not coincide with the investigations of BOHR,S who
in many cases obtained essentially higher figures for the oxygen tension
in arterial blood.

fle experimented on dogs, allowing the blood, whose coagulation had been
prevented by the injection of peptone solution or infusion of the leech, to flow
from one bisected carotid to the other, or from the femoral artery to the femoral
vein, through an apparatus called by him an hsemataerometer. The apparatus,
which is a modification of LUDWIG'S rheometer (stromuhr}, allowed, according
to BOHR, of a complete interchange between the gases of the blood circulating
through the apparatus and a quantity of gas whose composition was known at
the beginning of the experiment and inclosed in the apparatus. The mixture
of gases was analyzed after an equalization of the gases by diffusion. In this
way the tension of the oxygen and carbon dioxide in the circulating arterial blood
was determined. During the experiment the composition of the inspired and
expired air was also determined, the number of inspirations noted, and the extent
of respiratory exchange of gas measured. To be able to make a comparison
between the gas tension in the blood and in an expired air whose composition was
closer to the unknown composition of the alveolar air than the ordinary expired
air, the composition of the expired air at the moment it passed the bifurcation of
the trachea was ascertained by special calculation. The tension of the gases in
this " bifurcated air " could be compared with the tension of the gases of the blood,
and in such a way that the comparison took place simultaneously. Recently

1 Bert, La pression barometrique, Paris, 1878; Herter, Zeitschr. f. physiol. Chem.
3; Hiifner, 1. c.; Fredericq, Centralbl. f. Physiol., 7, and Travaux der laborat. de
1'inst. de physiol. de Liege, 5, 1896.

2 Strassburg, Pfliiger's Arch., 6; Falloiso, Bull. Acad. Roy. Belg., 1902. Wolfberg,
Pfliiger's Arch., 4 and 6; Nussbaum, ibid., '/; Loewy and v. Schrotter, cited by Loewy
in Oppenheimer's Handb., 4, 76.

3 Skand. Arch. f. Physiol., 2, and Nagel's Handbuch der Physiologie.

862 RESPIRATION AND OXIDATION.

KROGH l constructed an apparatus, called by him microtonometer, to be used
for the same purpose.

BOHR found remarkably high results for the oxygen tension in arterial
blood in this series of experiments. They varied between 101 and 144
mm. Hg pressure. In eight out of nine experiments on the breathing
of atmospheric air, and in four out of five experiments on breathing air
containing carbon dioxide, the oxygen tension in the arterial blood was
higher than the " bifurcated air." The greatest difference, where the
oxygen tension was higher in the blood than in the air of the lungs, was
38 mm. Hg.

HUFNER and FREDERICQ 2 have made the objection to BOHR'S experi-
ments and views that a perfect equilibrium had probably not been
attained between the air in the apparatus and the gases of the blood.
FREDERICQ, by new experiments, presents strong objections to the
acceptance of BOHR'S findings, while on the other hand BOHR not only
defends his experiments, but also finds errors in the experiments of his
opponents, while HALDANE and SMITH'S3 experiments, making use of
an entirely different principle, tend to corroborate the high results attained
by BOHR.

HALDANE'S method is as follows : The individual experimented upon is allowed
to inspire air containing an exactly known but small quantity of carbon monoxide
(0.045-0.06 per cent), until no further absorption of carbon monoxide takes place
and the percentage saturation of the hemoglobin in the arterial blood with carbon
monoxide has become constant, as shown by a special titration method. This
percentage saturation is dependent upon the relation between the tension of the
oxygen in the blood and the tension of the carbon monoxide, as known from the
composition of the inspired air. When this last and the percentage saturation
with carbon monoxide and oxygen are known the oxygen tension in the blood
can be easily calculated.

According to this method HALDANE and SMITH found still higher
figures than BOHR for the oxygen tension in the blood, and they calculated
the average tension of the oxygen in human arterial blood to be equal
to 293 mm. Hg.

Based upon the experiments of A. and M. KROGH, which will be dis-
cussed below (page 864) A. KROGH 4 has presented objections to the
experiments of HALDANE and SMITH.

Let us now compare the figures for the oxygen tension of the arterial
blood as found by various investigators with the tension of the oxygen
in the air of the lungs.

1 Skand. Arch. f. Physiol., 20.

2Hiifner, Arch. f. (Anat. u.) Physiol., 1890; Fr6dericq, Centralbl. f. Physiol., 7,
and Traveaux du laboratorie de 1'institute de physiologie de Liege, 5, 1896.
8 Haldane, Journ. of Physiol., 18; Haldane and Smith, ibid., 20.
* Skand. Arch. f. Physiol., 23, 217, 253.

THE ALVEOLAR AIR. 863

Numerous investigations as to the composition of the inspired atmos-
pheric air as well as the expired air are at hand, and it can be said that
these two kinds of air at 0° C. and a pressure of 760 mm. Hg have the
following average composition in volume per cent.

f)_v Nitrogen Carbon

Oxygen (and argon) Dioxide

Atmospheric air 20.96 79.02 0.03

Expired air 16.03 79.59 4.38

The partial pressure of the oxygen of the atmospheric air corresponds
at a normal barometric pressure of 760 mm. to a pressure of 150 mm.
Hg. The loss of oxygen which the inspired air suffers in respiration
amounts to about 4.93 per cent, while the expired air contains about
one hundred times as much carbon dioxide as the inspired air.

The expired air is therefore a mixture of alveolar air with the residue
of inspired air remaining in the air-passages; hence in the study of
the gaseous exchange in the lungs the alveolar air must first be con-
sidered. There exists no direct determination of the composition of the
alveolar air in man, but only approximate calculations. From the
average results found by VIERORDT in normal respiration for the carbon
dioxide in the expired air, 4.63 per cent, ZUNTZ 1 has calculated the
probable quantity of carbon dioxide in the alveolar air as equal to 5.44
per cent. If we start from this value, with the assumption that the
quantity of nitrogen in the alveolar air does not essentially differ from
the expired air, and admit that the quantity of oxygen in the alveolar
air is 6 per cent less than the inspired air, it will be seen that the alveolar
air contains 15 per cent oxygen. As the total pressure of the air of the
lungs after deducting the aqueous tension of about 50 mm. can be cal-
culated as about 710 mm. the partial pressure of the oxgyen in man
can be put at about 106 mm. and that of the carbon dioxide as about
45 mm.

Based upon several respiration experiments upon different persons,
LOEWY has been able to calculate the composition of the alveolar air
of human beings almost at the atmospheric pressure, from the com-
position of the expired air and the depth of inspiration and expiration,
taking into consideration the air in the upper air-passages. He obtained
results which varied between 101 and 105 mm. Hg for the oxygen tension
and between 32-42 mm. for the carbon dioxide tension.

The alveolar oxygen tension in dogs can be calculated from the car-
bon dioxide content of the alveolar air and is also found to be above
100 mm. Hg.

If the oxygen partial pressure in the alveoli is put at about 105

1 See Zuntz, 1. c., Hermann's Handbuch, 105 and 106.

864 RESPIRATION AND OXIDATION.

mm. Hg and we compare this with the highest results obtained for the
oxygen tension of the arterial blood as determined by tonometric means,
we find that the taking up of oxygen in the lungs can be simply explained
according to physical laws as a diffusion process. The conditions are
quite different if we start with the high-tension results of BOHR, 101-144
mm. Hg, or the still higher results of HALDANE and SMITH. The oxygen
tension in the blood is, in many cases, according to these latter authors,
always higher than the tension in the lungs, as average for various races
of animals. In these cases the passage of oxygen from the lungs to the
blood cannot be explained simply by a diffusion. We must therefore
with BOHR, accept a special activity cf the lungs, and according to,
him a secretory activity of the lungs also exists besides diffusion. In his
most recent work BOHR 1 presents the view that the specific action of the
lungs essentially consists in maintaining a necessary difference in pressure
for the diffusion. Nevertheless besides this a secretory process is nec-
essary, especially for the taking up of oxygen. Based upon newer
measurements DOUGLAS and HALDANE 2 also advocate the view that the
taking up of oxygen can be brought about by diffusion alone, but that
with the existing lack of oxygen in the tissues an active secretion of
oxygen takes place in the lungs.

By means of a tonometer described by A. KROGH, he and M. KROGH
have compared the oxygen tension in the arterial blood with that in the
alveolar air. In these experiments the tension in the blood was always
found lower than in the alveolar air. From this A. KROGH 3 concludes
that the exchange of gas in the lungs is chiefly brought about by diffusion.
FREDERicQ4 has recently arrived at the same view by his experiments
on the respiratory exchange of gas in aquatic animals.

As reports on the taking up of oxygen are conflicting so also are those
on the giving up of carbon dioxide.

The tension of the carbon dioxide in the blood has been determined
in different ways by PFLUGER and his pupils WOLFFBERG, STRASSBURG,
and NussBAUM.5

According to the aerotonometric method the blood is allowed to flow directly
from the artery or vein through a glass tube which contains a gas mixture of a
known composition. If the tension of the carbon dioxide in the blood is greater
than the gas mixture, then the blood gives up carbon dioxide, while in the reverse
case it takes up carbon dioxide from the gas mixture. The analysis of the gas

iCentralbl. f. Physiol. 23, 274; Skand. Arch. f. Physiol., 22, 221 (1909).
2Skand. Arch. f. Physiol., 25, 169 (1911); Proc. Roy. Soc., 1911; see also Journ.
of Physiol., 44, 305 (1912).

3 Skand. Arch. f. Physiol., 20, 263; 23, 179, 200, 213, (1910).

4 Arch, intern, de Physiol., 10, 391 (1911).

5 Wolffberg, Pfliiger's Arch., 6; Strassburg, ibid.', Nussbaum, ibid., 7.

CARBON DIOXIDE TENSION. 865

mixture after passing the blood through it will also decide if the tension of the
carbon dioxide in the blood is greater or less than in the gas mixture; and by a
sufficiently great number of determinations, especially when the quantity of car-
bon dioxide of the gas mixture corresponds as closely as possible, in the beginning,
to the probable tension of this gas in the blood, we may learn the tension of the
carbon dioxide in the blood. As above mentioned the oxygen tension can be
determined by the same method.

According to this method the carbon-dioxide tension of the arterial
blood is on an average 2.8 per cent of an atmosphere,1 corresponding to
a pressure of 20 mm. mercury (STRASSBURG). In the blood from the
pulmonary aveoli NUSSBAUM found a carbon-dioxide tension of 3.81
per cent of an atmosphere, corresponding to a pressure of 27 mm. mer-
cury. STRASSBURG, who experimented in non-tracheotomized dogs
in which the ventilation of the lungs was less active and therefore the
carbon dioxide was removed from the blood with less readiness, found
in the venous blood of the heart, a carbon-dioxide tension of 5.4 per
cent of an atmosphere, corresponding to a partial pressure of 38.3 mm.
mercury.

Another method, which was first used by PFLUGER and his pupils
WOLFFBERG and NUSSBAUM, depends upon excluding a part of the lungs
be means of the lung catheter

The principle of this method is as follows: By the introduction of a catheter,
of a special construction, into a branch of a bronchus the corresponding lobe of
the lung may be hermetically sealed, while in the other lobes of the same lung,
and in the other lung, the ventilation remains unchanged, so that no accumulation
of carbon dioxide takes place in the blood. When the cutting off lasts so long that
a complete equalization between the gases of the blood and the retained air of
the lungs is assumed, a sample of this, air of the lungs is removed by means of
the catheter and analyzed.

When a complete exchange between the gases of the inclosed part of
the lungs and the gases of the circulating venous blood has taken place,
the tension of the gases in this part of the lungs can be considered as a
.measure for the gas tension in the venous blood, if we admit that the
gas exchange is due only to physical forces. In their experiments
WOLFFBERG and NUSSBAUM found only 3.6 per cent C(>2 in the air taken
out with the catheter. NUSSBAUM also determined the carbon-dioxide
tension in the blood from the right heart in a case simultaneous with
the catheterization of the lungs. He found almost identical results,
namely, a carbon-dioxide tension of 3.84 per cent and 3.81 per cent
of an atmosphere, which also shows that complete equalization between
the gases of the blood and lungs in the inclosed parts of the lungs had taken

1 Here and in the following discussion we mean by atmospheric pressure the pressure
in the lungs after subtracting the aqueous vapor tension (about 50 mm.), namely,
760—50 = 710 mm. mercury pressure.

866 RESPIRATION AND OXIDATION.

place. The method of catheter! zing the lungs is, as shown by LOEWT
and v. ScHROTTER,1 also applicable to man, and they found that the
carbon dioxide tension of human venous blood was equal to 6 per cent
of the atmospheric pressure in the lungs = 42.6 mm. Hg, while according
to LOEWY'S calculations the carbon dioxide tension in the respired lung
aveoli varied between 31.8 and 41.8 mm. Hg with an average of 37.3
mm. Hg for eleven cases.

According to these investigations the giving up of carbon dioxide
may also be explained by physical laws; but BOHR, in his experiments
above mentioned (page 861), has arrived at other results in regard to
the carbon-dioxide tension. In eleven experiments with inhalation of
atmospheric air the carbon-dioxide tension in the arterial blood varied
from 0 to 38 mm. Hg, and in five experiments with inhalation of air con-
taining carbon dioxide, from 0.9 to 57.8 mm. Hg. A comparison of the
carbon-dioxide tension in the blood with the bifurcated air gave in several
cases a greater carbon-dioxide pressure in the air of the lungs than in
the blood, and as maximum this difference amounted to 17.2 mm. in
favor of the air of the lungs in the experiments with inhalation of atmos-
pheric air. As the aveolar air is richer in carbon dioxide than the bifur-
cated air this experiment unquestionably proves, according to BOHR,
that the carbon dioxide has migrated against the high pressure.

In opposition to these investigations, FREDERicQ,2 in his above-men-
tioned experiments, obtained the same figures for the carbon-dioxide
tension in arterial peptone blood as PFLUGER and his pupils found for
normal blood. WEISGERBER,S in FREDERICQ'S laboratory, has made
experiments with animals which respired air rich in carbon dioxide, and
these experiments confirm PFLUGER'S theory of respiration. Recently
FALLOISE has made determinations of the carbon-dioxide tension of
venous blood by means of FREDERICQ'S aerotonometer. The carbon-
dioxide tension was found to equal 6 per cent of an atmosphere, hence
somewhat higher than the results found by PFLUGER'S pupils. To these
investigations BOHR has presented strong objections; he has demon-
strated the principles for the construction of the tonometer, and claims that
the earlier experiments with the tonometer are not conclusive, as a
complete equilibrium of the gas tension was not attained.

A certain importance has been ascribed to oxygen in regard to the
elimination of carbon dioxide in the lungs, in that it has an expelling
action on the carbon dioxide from its combinations in the blood. This
theory, first advanced by HOLMGREN, has recently found an advocate

11. c., footnote 2, page 861.

2 See footnote 1, page 861.

•Centralbl. f. Physiol., 10, 482; Falloise, see Maly's Jahresber., 32.

INTERNAL RESPIRATION. 867

in WERIGO. Still ZUNTZ has presented weighty objections to WERIGO'S
experiments, and BOHR 1 has later also shown that we have no positive
basis for the above assumption.

The conditions as to the elimination of carbon dioxide in the lungs
are not quite clear. On the one hand we have advocates of the view
that the gas exchange takes place simply according to physical laws
and is chiefly considered as a diffusion process. According to the former
views of BoHR,2 which he has supported by recent experiments, a diffusion
does take place, but the lung is a gland which has the power of secreting
gases, and the gas exchange in the lungs is essentially a secretory proc-
ess. In his most recent work BOHR,S after a thorough criticism of the
methods used in the measurement of the lung diffusion and based upon a
new calculation of the extent of diffusion, has come to the conclusion
that the specific activity of the lungs consists in that a difference in
pressure necessary for the diffusion is brought about.

That a true secretion of gases occurs in animals follows from the composition
and behavior of the gases in the swimming-bladder of fishes. These gases con-
sist of oxygen and nitrogen with only small quantities of carbon dioxide. In
fishes which do not live at any great depth the quantity of oxygen is ordinarily
as high as in the atmosphere, while fishes which live at great depths may, accord-
ing to BIOT and others, contain considerably more oxygen and even above 80 per
cent. MOREAU has also found that after emptying the swimming-bladder by
means of a trocar, new air collected after a time, and this air was richer in oxygen
than the atmospheric air, and contained even 85 per cent oxygen. BOHR, who
has proven and confirmed these statements, also found that this accumulation
is under the influence of the nervous system, because on the section of certain
branches of the pneumogastric nerve it is discontinued. It is beyond dispute that
there is here a secretion and not a diffusion of oxygen. Recently JAEGER 4 has
given a further explanation as to the secretory activity of the swimming-bladder.

From what has been said above (page 858) in regard to the internal
respiration, one can conclude that it consists chiefly that in the capil-
laries the oxygen passes from the blood into the tissues, while the car-
bon dioxide passes from the tissues into the blood.

The assertion of ESTOR and SAINT PIERRE that the quantity of oxygen
in the blood of the arteries decreases with the remoteness from the heart
has been shown to be incorrect by PFLUGER,S and the oxygen tension in
blood on entering the capillaries must be higher. The oxygen tension

1 Holmgren, Wien. Sitzungsber., 48; Werigo, Pfliiger's Arch., 51 and 52; Zuntz,
ibid., 52; Bohr, see Nagel's Handbuch der Physiologic.

2 Centralbl. f . Physiol., 21, 367.

3 Ibid., 23, 374; Skand. Arch. f. Physiol., 22, 221 (1909).

4Biot, see Hermann's Handbuch d. Physiol., 4, Thl. 2, 151; Moreau, Compt.
Rend., 57; Bohr, Journ. of Physiol., 15. See also Hiifner, Arch. f. (Anat. u.) Physiol.,
1892; Jaeger, Pfliiger's Arch., 94.

5 Estor and Saint Pierre with Pfltiger in Pfliiger's Arch., 1.

868 KESPIRATION AND OXIDATION.

of the plasma is of importance in the giving up of oxygen to the tissues,
as the blood corpuscles contain a supply of oxygen only sufficient to
replace that removed from the plasma by the tissue. This quantity
of oxygen, which is dissolved in the plasma and at the disposal of the
tissues, is dependent upon the oxygen tension in the blood and only
indirectly dependent upon the total quantity of oxygen in the blood.
As this tissue is almost or entirely free from oxygen, a considerable dif-
ference in regard to the oxygen pressure must exist between the blood
and the tissues. The possibility that this difference in pressure is suf-
ficient to supply the tissues with the necessary quantity of oxygen is
hardly to be doubted.

The animal body, it seems, also has the command over means of regu-
lating and varying the oxygen tension, and such a means is the carbon
dioxide produced in the tissue which, according to BOHR, HASSELBALCH,
and KROGH,1 raises the oxygen tension. This is of special importance
when the tension of the oxygen in the blood of the capillaries is very low;
then the ability of the carbon dioxide to raise the dissociation tension
of the oxyhaemoglobin comes into play, especially with low oxygen tension.
Another regulating moment is, BOHR claims, the specific oxygen capacity
of the blood, which means the relation of the maximum oxygen combina-
tion to the quantity of iron of the blood or the haemoglobin solution.

In regard to the carbon-dioxide tension in the tissue it must be
assumed a priori that it is higher than in the blood. This is found to
be true. STRASSBURG 2 found in the urine of dogs and in the bile a car-
bon-dioxide tension of 9 per cent and 7 per cent of an atmosphere,
respectively. The same experimenter has, further, injected atmospheric
air into a ligatured portion of the intestine of a living dog and analyzed
the air taken out after some time. He found a carbon-dioxide tension
of 7.7 per cent of an atmosphere. The carbon-dioxide tension in the
tissues is considerably greater than in the venous blood, and there is
no opposition to the view that the carbon dioxide simply diffuses from
the tissues into the blood according to the law of diffusion.

Several methods have been suggested for the study of the quantitative
relation of the respiratory exchange of gas. The reader must be referred
to other text-books for details as to these methods, and we will here
mention only the chief features of the most important methods. It must
also be remarked, in regard to these methods, that those of REGNAULT
and REISET and of PETTENKOFER, determine the total gas exchange,
and indeed for a long time, while the other three methods determine the
respiratory gas exchange alone, and this only for a short time.

1 Centralbl. f. Physiol., 17, and Skand. Arch. f. Physiol., 16.

2 Pfliiger's Arch., 6.

METHODS FOR DETERMINING RESPIRATORY EXCHANGE. 869

REGNAULT and REISET'S Method. According to this method the animal
or person experimented upon is allowed to respire in an inclosed space. The
carbon dioxide is removed from the air, as it forms, by strong caustic alkali, from
which the quantity may be determined, while the oxygen is replaced continually
in exactly measured quantities. This method, which also makes possible a direct
determination of the oxygen used as well as the carbon dioxide produced, has
since been modified by other investigators, such as PFLUGER and his pupils, SEEGEN
and NOWAK, and HOPPE-SEYLER, ROSENTHAL and OPPENHEIMER and especially
by ATWATER and BENEDICT.1

PETTENKOFER'S Method. According to this method the individual to be
experimented upon breathes in a room through which a current of atmospheric
air is passed. The quantity of air passed through is carefully measured. As it
is impossible to analyze all the air made to pass through the chamber, a small
fraction of this air is diverted into a branch line during the entire experiment,
carefully measured, and the quantity of carbon dioxide and water determined.
From the composition of this air the quantity of water and carbon dioxide con-
tained in the large quantity of air made to pass through the chamber can be
calculated. The consumption of oxygen cannot be directly determined in this
method, but may be calculated indirectly by difference, which is a defect in this
method. The large respiration apparatus of SONDEN and TIGERSTEDT as well
as of ATWATER and ROSA 2 are based upon this principle.

SPECK'S Method.3 For briefer experiments on man SPECK used the follow-
ing: He breathes through a mouthpiece with two valves, closing the nose with a
clamp, into two spirometer-receivers, where the gas-volume can be read off very
accurately. The air from one of the spirometers is inhaled through one valve
and the expired air passes through the other into the other spirometer. By means
of a rubber tube connected with the expiration-tube an accurately measured part
of the expired air may be passed into an absorption-tube and analyzed.

ZUNTZ and GEPPERT'S Method.* This method, which has been improved by
ZUNTZ and his pupils from time to time, consists in the following: The individual
being experimented upon inspires pure atmospheric air through a very wide feed-
pipe leading from the open air, the inspired and the expired air being separated by
two valves (human subjects breathe with closed nose by means of a soft-rubber
mouthpiece, animals through an air-tight tracheal canula). The volume of the
expired air is measured by a gas-meter and an aliquot part of this air collected and
the quantity of carbon dioxide and oxygen determined. As the composition of
the atmospheric air can be considered as constant within a certain limit, the
production of carbon dioxide as well as the consumption of oxygen may be readily
calculated (see the works of ZUNTZ and his pupils).

HANRIOT and RICHET'S Method 5 is characterized by its simplicity. These
investigators allow the total air to pass through three gasometers, one after the
other. The first measures the inspired air, whose composition is known. The
second gasometer measures the expired air, and the third the quantity of the

1 See Zuntz in Hermann 's Handbuch, 4, Thl. 2, and Hoppe-Seyler, Zeitschr. f .
physiol. Chem., 19; Rosenthal, Arch. f. (Anat. u.) Physiol., 1902; Zuntz and Oppen-
heimer, Arch. f. (Anat. u.) Physiol., 1905, and Bioch. Zeitschr., 14; Atwater and
Benedict, Bull. Dept. of Agriculture, Washington, 69, 109, and 136. See also Krogh,
Wien. Sitz. Ber., 115, III., and Skand. Arch. f. Physiol., 18.

2 Pettenkofer's method; see Zuntz, 1. c.; Sond4n and Tigerstedt, Skand. Arch,
f. Physiol., 6; Atwater and Rosa, Bull, of Dept. of Agriculture, 63. Washington.

8 Speck, Physiologic des menschlichen Atmens. Leipzig, 1892.

4 Pfliiger's Arch., 42. See also Magnus-Levy in Pfliiger's Arch., 55, 10, in which
the work of Zuntz and his pupils is cited.

5 Compt. Rend., 104.

870 KESPIRATION AND OXIDATION.

expired air after the carbon dioxide has been removed by a suitable apparatus.
The quantity of carbon dioxide produced and the oxygen consumed can be ^readily
calculated from these data.

Appendix

THE LUNGS AND THEIR EXPECTORATIONS

Besides proteins and the albuminoids of the connective-substance
group, lecithin, taurine (especially in ox-lungs), uric acid, and inosite
have been found in the lungs. POULET l claims to have found a special
acid in the lung-tissue, which he has called pulmotartaric acid. Glyco-
gen occurs abundantly in the embryonic lung, but is absent in the adult
organ. The proteolytic enzymes also belong to the physiological con-
stituents of the. lungs. They are active in the autolysis of the
lungs (JACOBY) as well as in the solution of pneumonic infiltrations (Fn.

MtJLLER).2

The lungs have a strong reducing property, which BOHR explains by
the extensive oxidation processes in the lungs. According to N. SIEBER
they also have the ability to decompose neutral fats, while RIEHLS
says they do not have the ability to invert milk sugar.

The black or dark-brown pigment in the lungs of human beings and domestic
animals consists chiefly of carbon, which originates from the soot in the air. The
pigment may in part also consist of melanin. Besides carbon, other bodies, such
as iron oxide, silicic acid, and clay, may be deposited in the lungs, being inhaled
as dust.

Among the bodies found in the lungs under pathological conditions
must be specially mentioned, proteoses (and peptones?) in pneumonia
and suppuration, glycogen, & slightly dextrorotatory carbohydrate
differing from glycogen, found by POUCHET in consumptives, and finally
also cellulose, which, according to FREUND,4 occurs in the lungs, blood,
and pus of persons with tuberculosis.

C. W. SCHMIDT found in 1000 grams of mineral bodies from the normal
human lung the following: NaCl 130, K2O 13, Na2O 195, CaO 19, MgO
19, Fe2O3 32, P205 485, SO3 8, and sand 134 grams. According to
OiDTMANN,5 the lungs of a 14-day old child contained 796.05 p. m. water,
198.19 p. m. organic bodies, and 5.76 p. m. inorganic bodies.

1 Cited from Maly's Jahresber., 18, 248.

2 Jacoby, Zeitschr. f. physiol. Chem., 33; Miiller, Verhandl. d. Kongress. f. inn.
Medizin, 1902.

3 N. Sieber, Zeitschr. f. physiol. Chem., 55; Riehl, Zeitschr. f. Biol, 48.

4 Pouchet, Compt. Rend., 96; Freund, cited from Maly's Jahresber., 16, 471.

8 Schmidt, cited from v. Gorup-Besanez, Lehrbuch, 4. Aufl., 727; Oidtmann,
ibid., 732.

PHYSIOLOGICAL OXIDATION PROCESSES. 871

The sputum is a mixture of the mucous secretion of the respiratory
passages, of saliva and buccal mucus. Because of this its composition
is variable, especially under pathological conditions when various prod-
ucts mix with it. The chemical constituents are, besides the mineral
substances, chiefly mucin with a little proteid and nuclein substance.
Under pathological conditions proteoses and peptones (?), which are
probably produced by bacterial action or by autolysis (WANNER, SiMON1),
volatile fatty acids, glycogen, CHARCOT'S crystals, and also crystals of
cholesterin, hsematoidin, tyrosine, fat and fatty acids, triple phosphates,
etc., have been found.

The form constituents are, under physiological circumstances, epithe-
lium-cells of various kinds, leucocytes, sometimes also red blood-cor-
puscles and various kinds of fungi. In pathological conditions elastic
fibers, spiral formations consisting of a mucin-like substance, fibrin
coagulum, pus, pathogenic microbes of various kinds and the above-
mentioned crystals occur.

The lung concretions contain chiefly calcium and phosphoric acid as inorganic
constituents. Silicic acid is, in ZICKGRAF'S opinion, an essential and constant
constituent, but according to GERHARTZ and STRIGEL 2 is not always constant.

III. HOW ARE THE PHYSIOLOGICAL OXIDATION PROCESSES BROUGHT

ABOUT?

After the oxygen passes from the blood to the tissues a very extensive
oxidation is there carried out, which in conjunction with cleavage processes
yields finally the products carbon dioxide, water, urea and other bodies.
Little is known as to the manner in which the organism carries out such
complete oxidations. Attempts have been made for a long time to
explain the mechanism of the oxidation processes. Thus ScHONBEiN3
believed in the presence in the organism of oxygen in a peculiar form,
suited for the oxidation. HoppE-SEYLER4 connects the oxidation with a
simultaneous reduction; reducible or readily oxidizable substances first rup-
ture the oxygen molecule ( = 02) into atoms and take one up ; the other
at the moment it is set free is especially able to oxidize. M. TRAUBE 5
believes that in the case that a readily oxidizable (auto-oxidizable) substance
is present, the oxidation is produced by means of the entire oxygen
molecule, and indeed in the manner that water is transformed at the same
time to hydrogen peroxide, for example

1 Wanner, Deutsch. Arch. f. klin. Med., 75; Simon, Arch. f. exp. Path. u. Pharm., 49.

2 Gerhartz and Strigel, Beitr. z. klin. d. Tuberkulose, 10, which also cites Zickgraf.
'Baseler, Verh., Bd. 1, 339 (1853); Sitzungsber. Bayer. Akad. Wiss., 1863, Bd.

1, 274.

* Zeitschr f. physiol. Chem., 2, 1 (1878).

5 Ber. d. d. chem. Gesellsch., Bd. 15 to 26 (1882 to 1893).

872 RESPIRATION AND OXIDATION.

With these views as basis and at the same time although independently
of each other ENGLER 1 and BACH 2 have developed a theory which for
the present is the one generally accepted. According to this theory,
peroxides of the hydrogen peroxide type are always formed as primary
oxidation products. The peroxides are either formed by a direct attach-
ment of oxygen with readily oxidizable substances or in consequence of a
simultaneous oxidation with other substances — in the last way, for
example, the formation of H2O2 in the oxidation of indigo-white to indigo
according to the formula:

/H
Indigo^ + O2 = Indigo -f- H^Cb

H

Indigo-white

Only certain substances have the ability either directly or indirectly of
forming peroxides. Certain protein-like substances occurring especially
in the plants which have this ability, have been called oxygenases by
BACH and CnoDAT.3 Most of the substances which are oxidized within
the organism lose their ability to be directly oxidized. The oxidation
of such substances can, according to BACH,4 be accomplished in that the
oxygen is transported to the substance to be oxidized from the peroxide
simultaneously present by means of special enzymes, the peroxidases.
These latter were first prepared from pumpkins and from horse-radish
roots. In the absence of peroxides or oxygenases the peroxidases are
without action. CHODAT and BACH 5 have also found that certain
preparations, which have previously been called oxidases, can be decom-
posed into oxygenases and peroxidases. According to BACH'S theory the
formation of peroxides is a constant process going on in the organism,
to which the organism accommodates itself, in that the cells by means of
the peroxidases can make use of the peroxides for the oxidation processes.
Besides this the organism also forms other enzymes, the so-called
catalases, which have the ability of decomposing the peroxides with the
formation of molecular oxygen (02) and in this way making a possible
excess of peroxides harmless.6 In reference to the behavior of the perox-

1 Verb, naturw. Verein, Karlsruhe, 20, XI (1896), Bd. 13, 72; see also Zeitschr.
f. physiol. Chem., 59, 327 (1909).

2Compt. Rend., 124, 951 (1897); see also Bioch. Centralbl., 1, 417, and 457 (1903);
9, 1 (1909).

3 Ber. d. d. chem. Gesellsch., 36, 600 (1903).

4 Ibid., 36, 600 (1903).

5 Ibid., 36, 606 (1903).

6 Bioch. Centralbl., 1, 460.

PHYSIOLOGICAL OXIDATION PROCESSES. 873

idases to heat the views are contradictory. CZYHLARZ and v. FURTH
found that the peroxidases from animal tissues were remarkably resistant
to high temperatures, while BATELLI and STERN 1 find that animal peroxi-
dases are destroyed even at 66° C.

According to BACH'S theory on the one hand, substances are nec-
essary for the oxidation, which take up oxygen with the formation of
peroxides (oxygenases) and on the other hand, substances which are
able to transport the oxygen from the peroxides to the oxidizable bodies
(peroxidases). In certain oxidations, for example in the phenols, the
peroxidases can be replaced by certain metallic combinations.2 The
iron, of the blood pigments, acts in this way in the guaiacum reaction
(see Chapter XIV). The oil of turpentine here represents the peroxide
and can be replaced by hydrogen peroxide. The oxidizable substance,
which becomes blue in the reaction, is the guaiaconic acid in the guaiac
gum.3

Irrespective of whether the division of the oxidation enzymes into
oxygenases and peroxidases can be carried out in all cases, there are
various oxidation processes, whose occurrence by a combination of oxy-
genase (or peroxide) with peroxidase (or metallic salt) can be explained only
with difficulty. According to BERTRAND'S* view the action of plant
oxidation enzymes is connected with their manganese content. Never-
theless BACH 5 has been able to prepare enzymes from plants which were
entirely free from iron as well as manganese salts. Starting from BER-
TRAND'S view, TRILLAT 6 has prepared solutions of manganese salts, alkali
and colloidal substances, which acted like oxidizing enzymes. DONY-
HENAULT 7 has prepared artificial " oxidases " from a faintly alkaline
solution of gum treated with a solution of manganese salt. According to
EULER and BOLIN 8 the salts of certain organic acids have the ability of
setting the oxidation power of manganese salts free. Similar observations
have been made by WOLFF.Q In the oxidation of auto-oxidizable substances
the presence of extremely small amounts of iron salts may be of advantage,

!Czyhlarz and v. Fiirth, Hofmeister's Beitrage, 10, 358 (1907); Batelli and Stern,
Bioch. Zeitschr., 13, 44 (1908).

2Ber. d. d. chem. Gesellsch., 43, 366 (1910).

3C. E. Carlson, Zeitschr. f. physiol. Chem., 48, 69 (1906). P. Richter, Arch.
d. Pharm., 244, 90 (1906).

«Compt. Rend., 124, 1032, 1355 (1897).

6 Ber. d. d. ehem. Gesellsch., 43, 364 (1910).
6Compt. Rend., 138, 274 (1904).

7 Bull. acad. roy. de Belgique, 1908, 105.

8 Zeitschr. f. physiol. Chem., 57, 80 (1908).

9 Ann. inst. Past., 24 (1910).

874 RESPIRATION AND OXIDATION.

for example with the lecithins (THUNBERG, WARBERG and MEYERHOF l
as well as in the oxidation of certain thio-compounds.2

BATELLI and STERN 3 have made careful investigations as to the
occurrence of peroxidases in the animal organism. In order to eliminate
the action of catalases which are present in the tissues and which,
as shown by earlier investigators, decompose the hydrogen peroxide,
these experimenters used ethyl hydrogen peroxide, C2Hs.O.O.H, on
which the catalases do not act. With ethyl hydrogen peroxide and
hydroidic acid nearly all animal tissues gave the peroxidase reaction,
wherein free iodine was formed. SCHEUNERT, GRIMMER and ANDRYEWSKI 4
make use of the following solution as a reagent for peroxidases: 100 cc.
fresh tincture of guaiacum and 0.1 to 0.2 cc. 3 per cent H2O2 solution.
Blood does not give any blue coloration with this reagent, but in the pres-
ence of large quantities of H2O2 or other superoxide solutions (ethylhy-
drogen peroxide, oil of turpentine) it does give a blue coloration. With
this active tincture of guaiacum these experimenters were able to detect
peroxidases in the salivary glands, as well as the mucous membrane
of the stomach and intestine of certain varieties of animals. The liver
was always free from peroxidases. On the other hand, BATELLI and STERN5
also tested the ability of various tissues of acting upon formic acid in the
presence of H202 with the evolution of carbon dioxide. In later works
these experimenters claim that in all animal tissues there exists a substance
of an unknown nature, the pnein, which has the ability of bringing about
the respiration in all animal tissues. Pnein, which is soluble in water,
dializable and resistant to temperature, increases the so-called chief respira-
tion, which is connected with the life of the cells and which stops more
or less rapidly after the death of the animal. The so-called accessory
respiration continues quite a long time after death, and this can continue
in the absence of cell elements and is of an enzymotic character. THUN-
BERG 6, who has constructed an apparatus for measuring the respiratory
exchange of gas in small organs and organisms (microrespirometer) finds
that the salts of certain organic acids (succinic acid, citric acid, malic
acid, fumaric acid) accelerate more or less the gas exchange in surviv-
ing frog's muscles. In their last communication BATELLI and STERN 7
differentiate between two kinds of oxidation catalysts: the oxidases
and the oxidones. The first to which, among others the tyrosinase, alcohol-

ASkand. Arch. f. Physiol., 24, 90 (1911); Zeitschr. f. physiol. Chem., 85, 412 (1913).

1 Thunberg, Lunds Univ. Arsskr., N. F., 2, Bd. 9 (1913).

« Bioch. Zeitschr., 13, 44 (1908).

4/Wd., 53,300(1913).

*Ibid., 21, 487 (1910); 30, 172 (1910); 33, 315 (1911).

6 Skand. Arch. f. Physiol., 17, 23, 24, 25 (1911).

T Bioch. Zeitschr., 46, 317, 343 (1912); Compt. rend., soc. biol., 74, 212 (1913).

PHYSIOLOCAL OXIDATION PKOCESSES. 875

oxidase, xanthin oxidase (see below) belong, are soluble in water, re-
sistant to alcohol and acetone and can be heated to 55-60° C. The
oxodones, which for example oxidize succinic acid to malic acid and
act upon p-phenyldiamine, cannot be extracted from the tissues by water;
they are injured or destroyed by alcohol, acetone or by being heated
to 55-60° C.

WARBURG J has carried on extensive experiments on the influence of
foreign bodies upon the respiration in the cells and has conformed the
results to OVERTON'S theory of lipoid membrane.

No deep oxidation processes have been produced under the influence
of the mentioned oxidizing substances outside of the organism. The
various divergent views on the nature of the oxidizing substances strik-
ingly indicates how little exact knowledge we have of this subject.
Perhaps the oxidation within the body takes place step by step, and it
seems possible that the consecutive stages of the reaction can be brought
about by different agents. A positive division of the so-called oxidizing
enzymes cannot be made at the present time. For in many cases it
is undecided whether we are dealing with enzymotic processes or with
non-enzymotic catalytic action of metallic salts, especially as the
reports on the heat-resistance of the active substances are contra-
dictory. In the enzymotic oxidations we are in many cases in doubt
whether the oxygen is transported directly to the oxidizing substance or
whether the oxidation is brought about by the system peroxide plus
peroxidase. When the oxidation cannot be shown as due to the just-
mentioned system, then the active enzyme is called simply oxidase.

In consideration of the substances upon which the oxidation enzymes act,
we can divide them for the present into the following groups, according to

OPPENHEIMER:2

1. Alcoholases, which transform alcohols into acids, for example the acetic-
acid-forming enzyme of certain varieties of bacteria.

2. Aldehydases, which oxidize aldehydes into acids, for example, salicylase.

3. Purine-oxidases, which oxidize hypoxanthine and xanthine into uric acid and
which act upon uric acid with the formation of allantoin. This last reaction is
produced by the action of the so-called uricase.

4. Phenol-oxidases, which oxidize various phenols and related bodies with the
formation of pigments. The guaiac reaction is of this kind.

5. Tyrosinases, which oxidize tyrosine and closely related bodies into dark
pigments.

The system peroxide -f peroxidase has been shown only in connection with
enzymes of group 4.

Besides the above-mentioned bodies, upon which the different classes of oxidiz-
ing enzymes act more specifically, we can mention the following as oxidase reagents.

^eitschr. f. physiol. Chem., 67, 69, 70, (1910); 71, 76 (1911).

8 Die Fermente und ihre Wirkungen, 3. Aufl., Spec. Teil, 351 (1909).

876 RESPIRATION AND OXIDATION.

1. Iodides in acid solution in the presence of H202. According to BACH and
CHODAT l this reaction is completely parallel with the guaiac reaction.

2. Formic acid with H202 (see above).

3. Amines (especially p-phenyldiamine) which forms colored products on
taking up oxygen.2

4. Leucobases or mixtures of their formers, which by oxidation are converted
into pigments. A solution of a mixture of a-naphthol and p-phenylen-diamine
made alkaline with soda gives indophenol on taking up oxygen (ROHMANN and

SPITZER3).

5. Certain benzene derivatives which on oxidation and loss of water are
transformed into diphenol derivatives, for example vanillin into dehydrodi vanillin.4

For quantitative estimation of the extent of oxidation BACH and CHODAT 5
use the transformation of pyrogallol into purpurogallin, which latter can be weighed.
BACH 6 determines' the amount of iodine set free in the reaction between hydrogen
peroxide and hydriodic acid and BATELLI and STERN 7 determine the quantity of
C02 formed in the oxidation of formic acid.

There is no doubt that reductions occur to a great extent in the animal
body and often go hand in hand with oxidations. The question as to
the extent in which special reduction enzymes are concerned, is still
undecided. As the oxidations are explained by the action of special
enzymes, so also we can admit of special reduction enzymes, so-called
reduclases or hydrogenases. To this group belongs the so-called " philo-
thion" (De REY-PAILHADE), which in the presence of sulphur and
water develops sulphuretted hydrogen, while others on the contrary do
not accept this view, and deny the enzyme nature of philothion.8 The
investigations of NASSE and ROSING 9 on the oxidation of protein in the
presence of sulphur contradict the enzymotic nature of this formation
of sulphuretted hydrogen, and the recent investigations of HEFFTER 10
have shown that certain reductions occurring in the tissues are not pro-
duced by enzymes. He has also shown that those reductions, which are
not influenced fcy HCN, like the reduction of pigments (methylene blue),

1 Ber. d. d. chem. Gesellsch., 35, 2466 (1902).

2 Bioch. Zeitsqhr., 46, 317 (1912).

« Ber. d. d. phem. Gesellsch., 28, 567 (1894).

4 In regard .to this and other reagents, see Zeitschr. f. physiol. Chem., 59, 359
(Engler and Herzog).

5 Ber. d. d. chem. Gesellsch., 37, 1342 (1904).
• Ibid., 37, 3785 (1904).

^ Bioch. Zeitschr., 31/443; 33, 282 (1911).

8 De Rey-Pailhade, Recherches expeY. sur le Philothion, etc., Paris (G. Masson),
1891, and Nouvelles recherches sur le Philothion, Paris (Masson), 1892; Bull. soc.
chim. (4), 1; Pozzi-Escot, Bull. soc. chim. (3), 27, and Chem. Centralbl., 1904, 1,
1645; Chodat and Bach, Ber. d. d. chem. Gesellsch., 36; Abelous and Ribaut, Compt.
Rend., 137, and Bull. soc. chim., (3), 31.

9E. Rosing, Unters. iiber die Oxydation von Eiweiss in Gegenwart von Schwefel,
Inorg.-Dissert, Rostock, 1891.

10 Med.-naturw. Arch., 1, 81-104; Marburg, cited in Chem. Centralbl., 2, 1907,
822; Thunberg, Ergebn. d. Physiol., 11.

REDUCTION PROCESSES. 877

of sulphur to H2S and others, can be brought about by the labile H of the
sulphydryl-compounds. In this manner for example the cysteine
(see Chapter II) acts and quickly reacts with sulphur with the formation
of H2S and similarly acting substances have been detected by HEFFTER
in various organs and organ extracts. We have here a group of reductions
which are not of an enzyme nature.

From the investigations of ABELOUS and ALOY 1 it follows that
plants as well as animal organs have the ability at the same time of oxidiz-
ing salicylaldehyde and of reducing nitrates to nitrites. On the other
hand SCHARDINGER, TROMMSDORFF and BACH2 have shown that fresh
cow's milk, which alone is without action upon methylene blue and on
nitrates, reduces these bodies in the presence of aldehydes into leucobases
or nitrites. Boiled milk does not have this power and the action is
explained by the presence of a reductase, the so-called SCHARDINGER
enzyme. The optimum of action lies at about 70° C. BACH found the same
action in various animal organs. The process may to be just as well
considered as an oxidation under the influence of an aldehydase whereby
the methylene blue or the nitrate gives up the oxygen for the oxidation
of the aldehyde. On the other hand STRASSNER 3 ascribes the reduction
of the methylene blue, to the above-mentioned reducing action of the
sulphydryl groups.

1 Compt. Rend., 138, 382 (1904); see also Pozzi-Escot, ibid., 138, 511.

2 Schardinger, Zeitschr. f. Unters. d. Nahrungs- und Genussmittel, '5, 22 (1902);
Trommsdorff, Centralbl. f. Bakter., 49, 291 (1909); Bach, Ber. d. d. chem. Gesellsch.,
42, 4463 (1909); Bioch. Zeitschr., 31, 443; 33, 282; 38, 154 (1911),

*Ibid., 29, 295 (1910).

CHAPTER XVII.

METABOLISM WITH VARIOUS FOODS, AND THEIR NECESSITY

TO MAN.

I. GENERAL DISCUSSION AND METHODS USED IN THE STUDY OF MATTER
AND FORCE METABOLISM.

THE conversion of chemical energy into heat and mechanical work
which characterizes animal life, leads to the formation of relatively
simple compounds — carbon dioxide, urea, etc. — which leave the organism,
and which, moreover, being very poor in energy, are for this reason of
little or po value to the body. It is therefore absolutely necessary for
the continuance of life and the normal course of the functions of the body
that the organism and its different tissues should be supplied with new
material to replace tjiat which has been exhausted. This is accom-
plished by means of food. Those bodies are designated as food which
have no injurious action upon the organism and which serve as a source
of energy and can replace those constituents of the body that have been
consumed in metabolism or that can prevent or diminish the consumption
of such constituents.

Among the numerous dissimilar substances which man and animals
take with the food all cannot be equally necessary or have the same value.
Some perhaps are unnecessary, while others may be indispensable. We
have learned by direct observation and a wide experience that besides the
oxygen, which is necessary for oxidation, the essential foods for animals
in general, and for man especially, are water, mineral bodies, proteins,
carbohydrates, and fats.

It is also apparent that the various groups of food-stuffs necessary for
the tissues and organs must be of varying importance; thus, for instance,
water and the mineral bodies have another value than the organic foods,
and these again must differ in importance among themselves. The
knowledge of the action of various nutritive bodies on the exchange of
material from a qualitative as well as a quantitative point of view must
be of fundamental importance in determining the value of different
nutritive substances relative to the demands of the body for food under
various conditions, and also in deciding many other questions — for instance,
the proper nutrition for an individual in health and in disease.

878

EXCRETA OF THE ORGANISM. 879

Such knowledge can be attained only by a series of systematic and
thorough observations, in which the quantity of nutritive material, rela-
tive to the weight of the body, taken and absorbed in a given time is
compared with the quantity of final metabolic products which leave the
organism at the same time. Researches of this kind have been made by
investigators, but above all should be mentioned those made by BISCHOFF
and VOIT, by PETTENKOFER and VOIT, and by VOIT and his pupils, by
RUBNER, ZUNTZ and by ATWATER.

It is absolutely necessary in researches on the exchange of material
to be able to collect, analyze, and quantitatively estimate the excreta
of the organism, so that they may be compared with the quantity and
composition of the nutritive bodies ingested. In the first place, one must
know what the habitual excreta of the body are and in what way these
bodies leave the organism. One must also have trustworthy methods
for their quantitative estimation.

The organism may, under physiological conditions, be exposed to
accidental or periodic losses of valuable material — such losses as occur
only in certain individuals, or in the same individual only at a certain
period; for instance, the secretion of milk, the production of eggs, the
ejection of semen or menstrual blood. It is therefore apparent that these
losses can be the subject of investigation and estimation only in special
cases.

The regular and constant excreta of the organism are of the very
greatest importance in the study of metabolism. To these belong, in
the first place, the true final metabolic products — carbon dioxide, urea
(uric acid, hippuric acid, creatinine, and other urinary constituents),
and a part of the water. The remainder of the water, the mineral bodies,
and those secretions or tissue constituents — mucus, digestive fluids, sebum,
perspiration, and epidermal formations — which are either poured into
the intestinal tract, or secreted from the surface of the body, or broken
off and thereby lost to the body, also belong to the constant excreta.

The remains of food, sometimes indigestible, sometimes digestible but not
acted upon, which are contained in the feces, and which vary considerably in
quantity and composition with the nature of the food, also belong to the excreta
of the organism. Even though these remains, which are never absorbed and
therefore are never constitutents of the animal fluids or tissues, cannot be con-
sidered as excreta of the body in a strict sense, still their quantitative estimation
is absolutely necessary in certain experiments on the exchange of material.

The determination of the constant loss is in some cases accompanied with the
greatest difficulties. The loss from the detached epidermis, from the secretion
of the sebaceous glands, etc., cannot be determined with exactness without dif-
ficulty, and therefore — as they do not occasion any appreciable loss because of
their small quantity — they need not be considered in quantitative experiments
on metabolism. This also applies to the constituents of the mucus, bile, pancreatic
and intestinal juices, etc., occurring in the contents of the intestine, and which,
leaving the body with the feces, cannot be separated from the other contents of

880 METABOLISM.

the intestine and therefore cannot be quantitatively determined separately. The
uncertainty which, because of the intimated difficulties, attaches itself to the
results of the experiment, is very small as compared to the variation which is caused
by different individualities, different modes of living, different foods, etc. Only
approximate values can therefore be given for the constant excreta of the human
body.

The following figures represent the quantity of excreta for twenty-
lour hours from a grown man, weighing 60-70 kilos, on a mixed diet.
The figures are compiled from the results of different investigators:

Grams.

< Water 2500-3500

Salts (with the urine) 20-30

Carbon dioxide 750-&00

Urea. . 20^40

Other nitrogenous urinary constituents 2-5

Solids in the excrement 20-50

These total excreta are approximately divided among the various
excretions in the following way; but still it must not be forgotten that
this division may vary to a great extent under different external circum-
stances: By respiration about 32 per cent, by the evaporation from the
skin 17 per cent, with the urine 46-47 per cent, and with the excrement
5-9 per cent. The elimination by the skin and lungs, which is sometimes
differentiated by the name " perspiratio insensiblis " from the visible
elimination by the kidneys and intestine, is on an average about 50 per
cent of the total elimination. This proportion, quoted only relatively,
is subject to considerable variation, because of the great difference in
the loss of water through the skin and kidneys under varying circum-
stances.

The nitrogenous constituents of the excretions consist chiefly of urea,
or uric acid in certain animals, and the other nitrogenous urinary con-
stituents. A disproportionately large part of the nitrogen leaves the body
with the urine, and, as the nitrogenous constituents of this excretion are
final products of the metabolism of proteins in the organism, the quantity
of proteins catabolized in the body may be easily calculated by multiply-
ing the quantity of nitrogen in the urine by the coefficient 6.25 (YBa = 6.25),
if it is admitted that the proteins contain in round numbers 16 per cent
of nitrogen.

Still another question is whether the nitrogen leaves the body only
with the urine or by other channels. The latter is habitually the case.
The discharges from the intestine always contain some nitrogen, which
consists in part of non-absorbed remnants of the food, but in chief part
and sometimes entirely of constituents of the epithelium and the secre-
tions. Under these circumstances it is apparent that one cannot give
any exact figures which are valid for all cases for that part of the nitrogen
of the excrement which originates in the digestive tract and in the digestive

NITROGEN ELIMINATION. NITROGEN DEFICIT. 881

fluids. It may not vary in different individuals only, but also in the same
individual after more or less active secretion and absorption. In the
attempts made to determine this part of the nitrogen of the excrement
it has been found that in man, on non-nitrogenous or nearly nitrogen-
free food, it amounts in round numbers to somewhat less than 1 gram
per twenty-four hours (RIEDER, RUBNER). Even with such food the
absolute quantity of nitrogen eliminated by the feces increases with
the quantity of food because of the accelerated digestion (TsuBOi 1),
and is greater than in starvation. MtiLLER2 found in his observations
on the faster CETTI that only 0.2 gram nitrogen was derived from the
intestinal canal.

The quantity of nitrogen which leaves the body under normal circum-
stances by means of the hair and nails, with the scaling off of the skin,
and with the perspiration cannot be accurately determined. It is
nevertheless so small that it may be ignored. Only in profuse sweating
need the elimination by this channel be taken into consideration.

The view was formerly held that in man and carnivora an elimination
of gaseous nitrogen took place through the skin and lungs, and because of
this, on comparing the nitrogen of the food with that of the urine and
feces, a nitrogen deficit occurred in the visible elimination.

This question has been the subject of much discussion and of numerous
investigations, the most recent by KROGH and OppENHEiMER.3 These
researches have shown that the above assumption is unfounded, and
moreover several authorities, especially PETTENKOFER and VOIT, and
GRUBER,4 have shown by experiments on man and animals that with
the proper quantity and quality of food the body can be brought into
nitrogenous equilibrium, in which the quantity of nitrogen voided with
the urine and feces is equal or nearly equal to the quantity contained in
the food. Undoubtedly we must admit, with VOIT, that a deficit of nitro-
gen does not exist, or it is so insignificant that in experiments upon
metabolism it need not be considered. Ordinarily, in investigations on
the catabolism of proteins in the body, it is only necessary to consider the
nitrogen of the urine and feces, but it must be remarked that the nitrogen
of the urine is a measure of the extent of the catabolism of the proteins

1 Rieder, Zeitschr. f. Biologie, 20; Rubner, ibid., 15; Tsuboi, ibid., 35.

2 Berlin, klin. Wochenschr., 1887.

3 See Regnault and Reiset, Annal. d. chem. et phys. (3), 26, and Annal. d. Chem.
u. Pharm., 73; Seegen and Nowak, Wien. Sitzungsber., 71, and Pfliiger's Arch., 25;
Pettenkofer and Voit, Zeitschr. f. Biologie, 16; Leo, Pfliiger's Arch., 26; Krogh,
Skand. Arch. f. Physiol., 18, and Wien. Sitz. Ber., 115, III; Oppenheimer, Bioch.
Zeitschr., 4.

4 Pettenkofer and Voit, hi Herrman's Handbuch, 6, Thl. 1; Griiber, Zeitschr. f.
Biologie, 16 and 19.

882 METABOLISM.

in the body, while the nitrogen of the feces (after deducting about 1 gram
on a mixed diet) is a measure of the non-absorbed part of the nitrogen of
the food. The nitrogen of the food, as well as of the excreta, is generally
determined by KJELDAHL'S method.

In the oxidation of the proteins in the organism, their sulphur is oxidized
into sulphuric acid, and on this depends the fact that the elimination of
sulphuric acid by the urine, which in man is but to a small extent derived
from the sulphates of the food, nearly makes equal variations with the
elimination of nitrogen by the urine. If the amount of nitrogen and sul-
phur in the proteins is considered as 16 per cent and 1 per cent respectively,
then the proportion between the nitrogen of the proteins and the sulphuric
acid, H2SO4, produced by their combustion is in the ratio 5.2 : 1, or about
the same as in the urine (see page 765). The determination of the quantity
of sulphuric acid eliminated in the urine gives us an important means of
controlling the extent of the transformation of proteins, and such a con-
trol is especially important in cases in which it is expected to study the
action of certain nitrogenous non-albuminous bodies on the metabolism
of proteins, or to decide the question whether a true protein combustion
and not only a washing out of the nitrogenous products of metabolism
from the tissues is taking place. A determination of the nitrogen alone
is naturally not sufficient in such cases. A perfectly positive measure
of the protein catabolism cannot be made from the sulphuric acid of the
urine, as the various protein substances have a rather variable sulphur
content, and on the other hand also a variable quantity of the sulphur
in the urine exists as so-called neutral sulphur.

In metabolism experiments the total sulphur of the urine as well as the
feces must be determined, and it may also be of importance to determine
the relation between the sulphuric-acid sulphur and the neutral sulphur
of the urine. The elimination of the sulphur originating from the proteins
does not, according to v. WENDT, HAMALAINEN and HELME and CH.
WOLFF l always run parallel with the protein nitrogen, and for the
white of egg the maxima of the elimination curves may indeed be
separated during a period of twenty-four hours (WOLFF). The sulphur
is more quickly eliminated than the nitrogen, and this behavior of sul-
phur gives in certain cases a more positive picture of the temporal
catabolism of protein than the nitrogen. This is of importance, as the
elimination of the nitrogen corresponding to a certain amount of protein
requires several days for completion. FALTA has also observed that the
chief amount of nitrogen in man on taking different proteins is secreted
with varying rapidity, and the same is true, according to HAMALAINEN

1Wendt, Skand. Arch. f. Physiol., 17; Hamalainen and Helme, ibid., 19; Falta,
Deutsch. Arch. f. klin. Med., 86; Ch. G. Wolff, Bioch. Zeitschr., 40.

CALCULATION OF METABOLISM. 883

and HELME, for the elimination of sulphur, as in their experiments the
sulphur elimination from white of egg required about six days and from
casein only two days. These conditions must be considered in metab-
olism experiments.

Besides lecithins and other phosphatides the body takes with its food
pseudonucleins as well as true nucleins, and these are absorbed more or
less completely from the intestinal tract and then assimilated. On the
other hand, the phosphorized protein substances, lecithins and phos-
phatides, are also decomposed within the body, and their phosphorus is
chiefly eliminated as phosphoric acid and also in part as organic phos-
phorus (see page 757). For these reasons the phosphorus is of great
importance in certain investigations on metabolism.

It is found, on comparing the nitrogen of the food with that of the
urine and feces, that there is an excess of the first; this means that the
body has increased its stock of nitrogenous substances — proteins. If,
on the contrary, the urine and feces contain more nitrogen than the food
taken at the same time, this denotes that the body is giving up part of its
nitrogen — that is, part of its own proteins has been decomposed.

We can, from the quantity of nitrogen, as above stated, calculate the corre-
sponding quantity of proteins by multiplying by 6.25. x Usually, according to
VOIT'S proposition, the nitrogen of the urine is not calculated as decomposed
proteins, but as decomposed muscle-substance or flesh. Lean meat contains on
an average about 3.4 per cent nitrogen; hence each gram of nitrogen of the urine
corresponds in round numbers to about 30 grams of flesh. The assumption that
lean meat contains 3.4 per cent nitrogen is arbitrary, and the relation of N : C
in the proteins of dried meat, which is of great importance in certain experiments
on metabolism, is given differently by various experimenters, namely, 1 : 3.22-
1 : 3.68. ARGUTINSKY found in beef, after complete removal of fat and subtrac-
tion of glycogen, that the relation was 1 : 3.24 (see Chapter X).

The carbon leaves the body chiefly as carbon dioxide, which is elimi-
nated by the lungs and skin. The remainder of the carbon is excreted in
the urine and feces in the form of organic compounds, in which the quan-
tity of carbon can be determined by elementary analysis. It was for-
merly considered sufficient to calculate the quantity of carbon in the urine
from the quantity of nitrogen according to the relation N : C = 1 : 0.67 to
0.72. This does not seem to be trustworthy, as this relation varies and
depends, according to TANGL, PFLUGER, LANGSTEIN, and STEiNiTZ,2 upon
the kind of food. TANGL has shown that the richer the food is in car-
bohydrates the more carbon and hence the more heat of combustion per

1 In calculating the protein catabolism from the nitrogen of the urine it must not
be forgotten that the food often contains nitrogenous extractives whose nitrogen cannot
be calculated as protein and for which a special correction must be made, if necessary.

2Tangl, Arch. f. (Anat. u.) Physiol., 1899, Suppl. Bd.; Pfluger in Pfliiger's Arch.,
79; Langstein and Steinitz, Centralbl. f. Physiol., 19.

884 METABOLISM.

gram of nitrogen does the urine contain. He found the following for 1
gram of nitrogen in the urine: With diet rich in fat 0.747 gram C and 9.22
calories; for carbohydrate-rich diet he found 0.936 gram C and 11.67
calories. The quantity of carbon in the feces can be calculated from the

. Q

quantity of nitrogen in the feces by using the quotient — = 9.2 (average

with mixed diet, according to ATWATER and BENEDICT.1)

The extent of the gas exchange can be determined by any of the
methods given on page 869. By multiplying the quantity of carbon dioxide
found by 0.273 one obtains the quantity of carbon eliminated as CCb.
If the total quantity of carbon eliminated in various ways is compared
with the carbon contained in the food, some idea can be obtained as to
the transformation of the carbon compounds. If the quantity of carbon
in the food is greater than in the excreta, then the excess is deposited;
while if the reverse be the case, it shows a corresponding loss of body
substance.

The nature of the substances here deposited or lost, whether they consist
of proteins, fats, or carbohydrates, is learned from the total quantity of the
nitrogen of the excretions. The corresponding quantity of proteins may be cal-
culated from the quantity of nitrogen, and, as the average quantity of carbon
in the proteins is known, the quantity of carbon which corresponds to the decom-
posed proteins may be easily ascertained. If the quantity of carbon thus found
is smaller than the quantity of the total carbon in the excreta, it is then obvious
that some other nitrogen-free substance has been consumed besides the proteins.
If the quantity of carbon in the proteins is considered in round numbers as 52.5
per cent, then the relation between carbon (52.5) and nitrogen (16) is 3.28, or in
round numbers 3.3 : 1. If the total quantity of nitrogen eliminated is multiplied
by 3.3, the excess of carbon in the eliminations over the product found represents
the carbon of the decomposed non-nitrogenous compounds. For instance, in
the case of a person experimented upon, 10 grams of nitrogen and 200 grams of
carbon were eliminated in the course of twenty-four hours; then these 62.5 grams of
protein correspond to 33 grams of carbon, and the difference, 200 — (3.3X10) =167,
represents the quantity of carbon in the decomposed non-nitrogenous compounds.
If we start from the simplest case, starvation, where the body lives at the expense
of its own substance, then, since the quantity of carbohydrates as compared with
the fats of the body is extremely small, in such cases in order to avoid mistakes
the assumption must be made that the person experimented upon has used only
fat and proteins. As animal fat contains on an average 76.5 per cent carbon,
the quantity of transformed fat may be calculated by multiplying the carbon by

100

=-rg = 1.3. In the case of the above example, the person experimented upon

would have used 62.5 grams of proteins and 167X1.3=217 grams of fat, of his
own body, in the course of the twenty-four hours.

Starting from the nitrogen balance, it can be calculated in the same way
whether an excess of carbon in the food as compared with the quantity of carbon
in the excreta is retained by the body as proteins or fat or as both. On the other
hand, with an excess of carbon in the excreta one can determine how much of the
loss of the substance of the body is due to a consumption of the proteins on the
one side and of non-nitrogenous bodies on the other side. How to especially

1 Bull, of Dept. of Agric., U. S., Washington, No. 136.

CALORIC VALUES OF FOOD-STUFFS. 885

calculate the part taken by the fats and carbohydrate will be shown in connection
with the calculation of the energy metabolism.

The quantity of water and mineral bodies voided with the urine and
feces can easily be determined. The quantity of water eliminated by
the skin and lungs may be directly estimated by means of the large
respiration apparatus.

The organic constituents of the body as well as the foodstuffs intro-
duced, represent a sum of chemical' energy which the body can use
for force. The exchange of material is also an exchange of force,
and the first stands in such close relation to the second that the study
of one cannot be separated from the other. The energy theory of
metabolism has exercised an extraordinarily fruitful influence upon
the entire study of metabolism and nutrition, and this is due in great
measure to the work of RUBNER.

This energy of the various foods may be represented by the amount
of heat which is set free in their combustion. This quantity of heat is
expressed as calories, and a small calorie is the quantity of heat necessary
to warm 1 gram of water from 0° to 1° C. A large calorie is the quantity
of heat necessary to warm 1 kilo of water 1° C. Here and in the follow-
ing pages large calories are to be understood. There are numerous
investigations by different experimenters, such as FRANKLAND, DAN-
ILEWSKI, RUBNER, BERTHELOT, STOHMANN, BENEDICT and OSBORNE,
and others, on the calorific value of different foodstuffs. The following
results, which represent the calorific value of a few nutritive bodies on
complete combustion outside of the body to the highest oxidation prod-
ucts, are taken from STOHMANN'S l work.

Calories.

Casein 5 . 86

Ovalbumin 5 . 74

Conglutin 5 . 48

Protein (average) 5.71

Animal tissue-fat 9 . 50

Butter-fat 9 . 23

Cane-sugar 3 . 96

Milk-sugar 3 . 95

Glucose 3. 74

Starch 4. 19

Fats and carbohydrates are completely burnt in the body, and one can
therefore consider their combustion equivalent as a measure of the living
force developed by them within the organism. We generally designate
9.3 and 4.1 calories for each gram of substance as the average for the
physiological calorific value of fats and carbohydrates respectively.

xSee Rubner, Zeitschr. f. Biologic, 21, which also cites the works of Frankland
and Danilewski; see also Berthelot, Compt. Rend., 102, 104, and 110; Stohmann,
Zcitschr. f. Biologie, 31; Benedict and Osborne (vegetable proteins), Journ. of biol.
Chem., 3.

886 METABOLISM.

The proteins act differently from the fats and carbohydrates. They
are only incompletely burnt, and they yield certain decomposition prod-
ucts, which, leaving the body with the excreta, still represent a certain
quantity of energy which is lost to the body. The heat of combustion
of the proteins is smaller within the organism than outside of it, and they
must therefore be specially determined. For this purpose RuBNER1
fed a dog on washed meat, and he subtracted from the heat of combustion
of the food the heat of combustion of the urine and feces, which cor-
responded to the food taken plus the quantity of heat necessary for the
swelling up of the proteins and the solution of the urea. RUBNER has
also tried to determine the heat of combustion of the proteins (muscle-
proteins) decomposed in the body of rabbits in starvation. According
to these investigations, the physiological heat of combustion in calories
for each gram of substance is as follows:

1 gram of the dry substance Calories.

Protein from meat 4.4

Muscle 4.0

Protein in starvation 3.8

Fat (average for various fats) 9.3

Carbohydrates (calculated average) 4.1

The physiological combustion value of the various foods belonging to
the same group is not quite the same. It is, for instance, 3.97 calories
for a vegetable protein, conglutin, and 4.42 calories for an animal protein
body, syntonin. According to RUBNER the normal heat value per 1
gram of animal protein may be considered as 4.23 calories, and of vegetable
protein as 3.96 calories. When a person on a mixed diet takes about
60 per cent of the proteins from animal foods and about 40 per cent from
vegetable foods, the value of 1 gram of the protein of the food is equivalent
to about 4.1 calories. The physiological value of each of the three
chief groups of organic foods, by their decomposition in the body, is in
round numbers as follows:

Calories.

1 gram protein 4.1

1 gram fat 9.3

1 gram carbohydrate 4.1

1 gram alcohol 7.1

These figures are generally used in the calculation of the energy con-
tent of various foodstuffs and diets.

The extent of gas exchange and the so-called respiratory quotient
is, besides the extent of nitrogen elimination, of the greatest importance
in the calculation of the extent of energy metabolism and the division
of the energy between the protein, fat and carbohydrate.

On comparing the inspired and expired air we learn, on measuring
them when dry and at the same temperature and pressure, that the volume

1 Zeitschr. f . Biologic, 21.

RESPIRATORY QUOTIENT. 887

of the expired air is less than that of the inspired air. This depends
upon the fact that not all of the oxygen appears again in the expired
air as carbon dioxide, because it is not only used in the oxidation of car-
bon, but also in part in the formation of water, sulphuric acid, and other
bodies. The volume of expired carbon dioxide is regularly less than the

volume of the inspired oxygen, and the relation — ^p, which is called the

respiratory quotient, is generally less than 1.

The magnitude of the respiratory quotient is dependent upon the kind
of substances destroyed in the body. In the combustion of pure carbon
one volume of oxygen yields one volume of carbon dioxide, and the
quotient is therefore equal to 1. The same is true in the burning of
carbohydrates, and in the exclusive decomposition of carbohydrates in
the animal body the respiratory quotient must be approximately 1. In
the exclusive metabolism of proteins it is close to 0.80, and with the decom-
position of fat it is 0.7. In starvation, as the animal draws on its own
flesh and fat, the respiratory quotient must be a close approach to the
latter figure. The respiratory quotient, which is calculated with exclusive
combustion of carbohydrate, fat and protein, as respectively, 1, 0.707 and
0.809 and with alcohol is 0.667, also gives important information as to
the quality of material decomposed in the body, especially with the
supposition that the carbon dioxide elimination is not influenced by some
special condition such as a change in the respiratory mechanism. Another
supposition is that no incomplete oxidation step in combustion is elimi-
nated.

The respiratory quotient can also be strongly influenced by inter-
mediary processes in the animal body, as by the formation of glycogen
from protein, or from fat or by the formation of fat from carbohydrates.
In the first case the quotient may be lower than 0.7 and in the last case
it can be higher than 1.

Knowledge as to the extent of oxygen consumption is of special
importance in the calculation of the energy metabolism from the extent
of gas exchange, and one can under some circumstances approximately
calculate the energy exchange from the calorific value of the oxygen
alone — with regard to the respiratory quotient (ZUNTZ and co-workers).
The calorific value of oxygen must be different for each of the three men-
tioned foodstuffs, as they require different quantities of oxygen for their
combustion. For fat and carbohydrate this calorific value can be readily
calculated, as these bodies are completely burnt into carbon dioxide and
water. One gram of starch uses 828.8 cc. oxygen in its combustion
and produces 828.8 cc. carbon dioxide, and 4183 calories of heat are
developed. For one liter ( = 1.43 gram) oxygen, 5047 calories are pro-
duced, therefore for every liter ( = 1.966 gram) carbon dioxide formed,

888 METABOLISM.

the same number, 5047 calories, are produced. In an analogous manner
the average calorific value of fat for 1 liter of oxygen, 4686 calories, and
for 1 liter carbon dioxide, 6629 calories, can be calculated.

These figures, which represent the physiological combustion values
per 1 gram of food-stuffs, derived from the carbon dioxide output or the
oxygen in-take in (grams or) liters which are represented by the quotients

' or , ' have been called the calorific coefficients.

With proteins, because of the unequal composition of the different
proteins, the results are uncertain and variable, and the calculation is
much more complicated. As example we will give the following calcula-
tion of ZUNTZ l for the fat-free dry substance of meat.

This substance consisted in 100 parts

52.38g.C.; 7.27g.H.; 22.68g.O.; 16.65g.N.; 1.02g.S.

Of which were found in the urine. 9.406 2.663 14.099 16.28 1.02

Of which were found in the feces. 1.471 0.212 0.889 0.37

Retained ............... 41.50C; 4.40H; 7.69O; O.ON; O.OS.

From this residue, with the taking up of 96.63 liters of oxygen, besides 39.6
grams water, 77.39 liters carbon dioxide were formed and the respiratory quotient
is therefore 0.801. Now 100 grams of such dry meat substance on complete
combustion yields 563.09 calories, and if we subtract the calorific value of the
corresponding urine (=113.70 calories) and feces (=17.76 calories), the sum,
131.46 calories, then 431.63 calories were set free in the body. For every gram

A Q 1 AQ

of nitrogen eliminated in the urine (16.28 gram) there is produced -1g 00 =26.51

iD.Zo
QA AQ

calories; the corresponding quantity of oxygen is '00 =5.91 liter 0 and the

77 39
corresponding quantity of C02 produced is j^og =4<^ ^ters C02. The calorific

value for 1 liter of oxygen consumed is therefore - ' = 4.485 calories, and for

1 liter of carbon dioxide produced . '„. =5.579 calories.

For milk protein ZUNTZ has calculated for 1 gram urea nitrogen 5.8 liters
oxygen, 4.6 liters carbon dioxide and 27 calories. The calorific value can be cal-
culated from this for 1 liter 0=4.66 and for 1 liter C02 = 5.87 calories. If we

take the average of these calculations we obtain the calorific coefficients T ' =4.57

Ij.Uz

Cal
and rrvT = 6.73 for protein.

For the three foodstuffs we have the following calorific values:

Per 1 liter Relative Per 1 liter Relative

Oxygen. value. Carbon dioxide. value.

Protein ............ 4.57 100 5.73 113.4

Fat ............... 4.69 102.6 6.63 131.3

Carbohydrate ...... 5.05 110.5 5.05 100.0

1 Zuntz, Loewy, Miiller and Caspar!, Hohenklima und Bergwanderungen, Berlin,
1906, pages 102, 103.

CALCULATION OF THE CALORIC VALUE. 889

The figures for the oxygen vary less than those for the carbon dioxide,
and this is a reason why the oxygen values are better suited than the
C02 values for calculating the energy production from the extent of gas
exchange. Other investigators have obtained results which correspond
more or less with the above values for the heat value of oxygen, and E.
VOIT and KuMMACHER,1 who have made calculations in another way,
have obtained still smaller differences for the relative oxygen value.

From what was said above we can calculate the extent of protein
metabolism, the corresponding development of energy and the correspond-
ing absorption of oxygen and carbon dioxide formation, from the quantity
of nitrogen in the urine. If we subtract the oxygen and carbon dioxide
values from the total, directly determined gas exchange, the result repre-
sents the fats and carbohydrates used. According to ZUNTZ from this
residue we can calculate the heat value of the oxygen used as well as the
division of the decomposition of the fat and carbohydrate by consider-
ing the respiratory quotient. For this purpose ZUNTZ and SCHUMBURG
have constructed a table, an abstract of which we give below, taken
from the work of MAGNUS-LEVY.2

R. Q. Calories value Division in per cent.

per 1 liter O. Carbohydrate. Fat.

1.000 5.047 100 0

0.950 4.986 83 17

0.900 4.924 66 34

0.850 4.863 49 51

0.800 4.801 32 68

0.750 4.740 15 85

0.707 4.686 0 100

As the calorific oxygen values in the combustion of protein, fat
and carbohydrate show no great differences among themselves, in those
cases where, as in starvation, the part taken by the proteins in the total
metabolism is relatively small, one can calculate the total energy exchange,
without any striking error, from the respiratory quotient and the oxygen
used. This is especially important in experiments of short duration
where the protein metabolism cannot be directly determined, but is
calculated from the nitrogen elimination occurring during a longer time.
The method of ZUNTZ and GEPPERT, mentioned on page 869, has shown
itself especially useful in the study of the material and force exchange
in these experiments of short duration, while the respiration apparatus
constructed on PETTENKOFER'S or the REGNAULT-REISET principle are
only useful in experiments over a longer period.

KAUFMANN 3 incloses the individual to be experimented upon in a capacious
sheet-iron room, which serves both as a respiration-chamber and a calorimeter,

1 Voit, Zeitschr. f. Biol., 44; Kummacher, ibid.

2 A. Magnus-Levy in v. Noorden's Handb. d. Pathol. des Stoffwecheels, Bd. 1. (1906).
* Arch. d. Physiologic (5), 8.

890 METABOLISM.

and which permits the estimation of the nitrogen of the urine and the carbon
dioxide expired, as well as the inspired oxygen and the quantity of heat produced.
If we start from the theoretically calculated formulae for the various possible
transformations of the proteins, fats, and carbohydrates in the body, it is clear
that other values must be obtained for the heat, carbon dioxide, oxygen, and
nitrogen of the urine, when one, for example, admits of a complete combustion
of proteins to urea, carbon dioxide, and water, or of a partial splitting off of fat.
Another relation between heat, carbon dioxide, and oxygen is also to be expected
when the fat is completely burnt or when it is decomposed into sugar, carbon
dioxide, and water. In this way, by a comparison of the values found in special
cases with the figures calculated for the various transformations, KAUFMANN
attempts to explain the various decomposition processes in the body under dif-
ferent nutritive conditions.

The organic foodstuffs serve in part to replace the necessary losses
of the organs and in part as sources of energy. Under all circumstances
a restitution of the protein-like constituents of the organs is necessary.
This replacement is, according to RUBNER, represented by the so-called
wear-and-tear quota (see below) which amounts to about 4-6 per cent of
the total energy transformed and which can be supplied by proteins only.
For the supply of the remaining exchange, which according to RUBNER
serves as source of energy, all three groups of organic foodstuffs can be
used, and investigations carried out by RUBNER have taught that these
foodstuffs can act as sources of energy in the animal body in a proportion
which corresponds with the respective figures of their heat value. This
is apparent from the following table. In this is found the weight of
the various foods equal to 100 grams of fat, a part determined from
experiments on animals and a part calculated from figures of the heat
values:

From Experiments From the Difference,

on Animals. Heat Value. per cent.

Syntonin 225 213 +5.6

Muscle-flesh (dried) .... 243 235 +4 . 3

Starch 232 229 +1.3

Cane-sugar 234 235 -0

Glucose 256 255 -0

From the given isodynamic value of the various foods it follows that
these substances replace one another in the body almost in exact ratio
to the energy contained in them. Thus in round numbers 227 grame of
protein and carbohydrate are equal to or isodynamic with 100 grams of
fat in regard to source of energy, because each yields 930 calories on com-
bustion in the oody.

By means of recent very important calori metric investigations, RUB-
NER l has shown that the heat produced in an animal in several series of
experiments extending over forty-five days corresponded to within 0.47
per cent of the physiological heat of combustion calculated from the decom-

Zeitschr. f. Biologic, 30.

ISODYNAMICS OF FOOD-STUFFS. 891

posed body and foods. ATWATER and his collaborators 1 have made some
very thorough investigations on this subject on men. In their experi-
ments they made use of a large respiration calorimeter, which not only
exactly determined the excreta, but also made a calorimetric determina-
tion of the heat given out by the person experimented upon, i.e., the work
performed. From the results of these experiments they found an almost
absolutely complete agreement between the calories found directly and
those calculated.

This isodynamic law is of fundamental value in the study of metabo-
lism and nutrition. The quantity of energy in the transformed foods
or the constituents of the body may be used as a measure for the total
consumption of energy, and the knowledge of the quantity of energy
in the foods must also be the basis for the calculation of dietaries for
human beings under various conditions.

The isodynamic theory has been accepted by a large number of inves-
tigators, but not by all. Certain of them, especially the French, accept
an isoglucosic instead of the isodynamic. According to this theory the
organism for its physiological functions can use glucose only, and as a
formation of glucose is possible from proteins as well as fats, those quan-
tities of food-stuffs are to be considered as equivalent which yield an
equal amount of glucose.

The heat value of a foodstuff can be directly determined in a calorim-
eter, but may also be calculated from its composition. If one subtracts
from the gross heat value of the food obtained in one way or another
the combustion heat of the feces and urine with the same diet, there is
obtained the net calorific value of the diet. This value, calculated in
percentage of the total energy content of the food, is called the physio-
logical availability by RuBNER.2 In order to elucidate this we will give
a few of RUBNER'S values. The loss in calories, as well as the physio-
logical availability, is calculated in percentages of the total energy
content of the food.

Food.
Cow's milk

Loss
In Urine.
5 13

in per cent.
In the Fecea.
5 07

Total loss
in per cent.
10 20

Availability
in per cent.
89 8

Mixed diet (rich in fat) . .
Mixed diet (poor in fat) .
Potatoes

.... 3.87
. ... 4.70
2 00

5.73
6.00
5 60

9.60
10.70

7 60

90.4
89.3
92 4

Graham bread.

. . . . 2 40

15.50

17.90

82 1

Rye bread

2.20

24.30

26.50

73 5

Meat . .

. 16.30

6.90

23.20

76.8

In order to simplify the calculation of the energy exchange there exist other
standard factors, besides the above-mentioned standard figures for the physiological

1 Bull, of Dept. of Agric., Washington, 44, 63, 69, and 109, and Ergebnisse der
Physiologic, 3.

2 Zeitschr. f . Biologic, 42.

892 METABOLISM.

calorific value of the organic foodstuffs, also for the carbon of the carbon dioxide,
and for the oxygen. Thus for 1 gram of meat (dry substance) free from fat
and extractives we have the calculated value of 5.44-5.77 calories. KOHLER l
found 5.678 calories for 1 gram of ash and fat-free dried-meat substance of the
ox and 5.599 calories for horse meat. According to FRENTZEL and SCHREUER 2
45.4 calories is calculated for 1 gram of nitrogen in fat and ash-free dried-meat
feces (dog), while 6.97 to 7.45 calories is calculated for 1 gram of nitrogen in meat-
urine. The calorific urine quotient — ^ — seems still, as above given, not to be
constant for human beings, but is dependent upon the variety of food.

II. METABOLISM IN STARVATION AND WITH INSUFFICIENT NUTRITION.

In starvation the decomposition in the body continues uninterruptedly,
though with decreased intensity; but, as it takes place at the expense of
the substance of the body, it can continue for a limited time only. When
an animal has lost a certain fraction of the mass of the body, death is the
result. This fraction varies with the condition of the body at the begin-
ning of the starvation period. Fat animals succumb when the weight of
the body has sunk to one-half of the original weight. Otherwise, accord-
ing, to CnossAT,3 animals die as a rule when the weight of the body has
sunk to two-fifths of the original weight. The period when death occurs
from starvation not only varies with the varied nutritive condition at the
beginning of starvation, but also with the more or less active exchange
of material. This is more active in small and young animals than in large
and older ones, but different classes of animals show an unequal activity.
Children succumb in starvation in 3-5 days after having lost one-fourth
of their body mass. Grown persons may, as observed upon Succi,4 and
other professional fasters, starve for twenty days or more without lasting
injury; and there are reports of cases of starvation extending over a
period of even more than forty to fifty days. Dogs may starve, accord-
ing to several observers, 50-60 days. HAWKS and co-workers have
recorded a case where a dog was starved for 117 days and lost about
63 per cent of its original weight. Snakes and frogs can starve for one-
half a year or even a whole year.

In starvation the weight of the body decreases. The loss of weight is
greatest in the first few days, and then decreases rather uniformly. In
small animals the absolute loss of weight per day is naturally less than
in larger animals. The relative loss of weight — that is, the loss of weight

1 Zeitschr. f. physiol. Chem., 31.

2 The works of Frentzel and Schreuer may be found in Arch. f. (Anat. u.) Physiol.,
1901, 1902, and 1903.

3 Cited from Voit in Hermann's Handbuch, 6, Thl. 1, 100.

4 See Luciani, Das Hungern. Hamburg u. Leipzig, 1890.

6 P. B. Hawk, P. E. Howe, and H. A. Mattil, Journ. of biol. Chem., 11.

METABOLISM IN STARVATION. 893

of the unit of the weight of the body, namely, 1 kilo — is, on the contrary,
greater in small animals than in larger ones. The reason for this is that
the smaller animals have a greater surface of body in proportion to their
mass than larger animals, and the greater loss of heat caused thereby
must be replaced by a more active consumption of material.

It follows from the decrease in the weight of the body that the absolute
extent of metabolism must diminish in starvation. If, on the contrary,
the extent of metabolism is referred to the unit of weight of the body,
namely, 1 kilo, it appears that this quantity remains almost un-
changed during starvation. The investigations of ZUNTZ, LEHMANN,
and others,1 on the professional faster CETTI, showed on the third and
sixth days of starvation an average consumption of 4.65 cc. oxygen per
kilo in one minute, and on the ninth to eleventh day an average of 4.73
cc. The calories, as a measure of the metabolism, fell on the first to
fifth day of starvation from 1850 to 1600 calories, or from 32.4 to 30 per
kilo, and it remained nearly unchanged, if referred to the unit of body
weight.2 In man the average daily energy consumption in starvation
amounts to about 30-32 calories per kilo.

The extent of the metabolism of proteins, or the elimination of nitrogen
by the urine, which is a measure of the same, diminishes as the weight
of the body diminishes. This decrease is not regular or the same during
the entire period of starvation, and the extent depends, as the experi-
ments made upon carnivora have shown, upon several circumstances.
During the first few days of starvation the excretion of nitrogen is greatest,
and the richer the body is in protein, due to the food previously taken,
the greater is the protein catabolism or the nitrogen elimination, accord-
ing to VOIT. The nitrogen elimination diminishes the more rapidly —
that is, the curve of the decrease is more sudden — the richer in proteins
the food was which was taken before starvation. This condition is
apparent from the following table of data of three different starvation
experiments made by VoiT3 on the same dog. This dog received 2500
grams of meat daily before the first series of experiments, 1500 grams of
meat daily before the second series, and a mixed diet relatively poor in
nitrogen before the third series.

Day of Starvation.
First

Grams of Urei
Ser. I.

. . . 60.1

Et Eliminated in i wentj
Ser. II.
26.5

r-iour flours.
Ser. III.
13.8

Second

24 9

18.6

11.5

Third

19 1

15.7

10.2

Fourth

. .. 17.3

14.9

12.2

Fifth

. . . 12.3

14.8

12.1

Sixth

. .. 13.3

12.8

12.6

Seventh

12.5

12.9

11.3

Eighth

... 10.1

12.1

10.7

1 Berlin, klin. Wochenschr., 1887.

2 See also Tigerstedt and collaborators in Skand. Arch. f. Physiol., 7.

3 See Hermann's Handbuch, 6, Thl. 1, 89.

894 METABOLISM.

In man and also in animals sometimes a rise in the nitrogen excretion
is observed about the second or third starvation day, which is then fol-
lowed by a regular diminution. This rise is explained by PRAUSNITZ,
TIGEESTEDT, LiANDERGREN,1 as follows: At the commencement of star-
vation the protein metabolism is reduced by the glycogen still present
in the body. After the consumption of the glycogen, which takes place
in great part during the first days of starvation, the destruction of pro-
teins increases as the glycogen action decreases, and then decreases again
when the body has become poorer in available proteins.

Other conditions, such as varying quantities of fat in the body, have
an influence on the rapidity with which the nitrogen is eliminated during
the first days of starvation. After the first few days of starvation the
elimination of nitrogen is more uniform. It may diminish gradually
and regularly until the death of the animals or there may be a rise in the
last days, a so-called premortal increase. Whether the one or the other
occurs depends upon the relation between the protein and fat content
of the body.

Like the destruction of proteins during starvation, the decomposi-
tion of {at proceeds uninterruptedly, and the greatest part of the* calories
needed during starvation are supplied by the fats. According to RUBNER
and VOIT the protein catabolism varies only slightly in starving animals
at rest and at an average temperature, and forms a constant portion
of the total exchange of energy; of the total calories in dogs 10-16 per
cent comes from the protein decomposition and 84-90 per cent from the
fats. This is at least true for starving animals which had a sufficiently
great original fat content. If on account of starvation the animal has
become relatively poorer in fat and the fat content of the body has fallen
below a certain limit, then in order to supply the calories necessary, a
larger quantity of protein is destroyed and the premortal increase now
occurs (E. VOIT). The reason for this premortal rise in protein catabol-
ism is still not completely understood (SCHULZ and collaborators2).

Since the fat has a diminishing influence on the destruction of pro-
teins corresponding to what was said above, the elimination of nitrogen
in starvation is less in fat than in lean individuals. For instance, only
9 grams of urea were voided in twenty-four hours during the later stages
of starvation by a well-nourished and fat person suffering from disease
of the brain, while I. MUNK found that 20-29 grams urea were voided
daily by CETTI,B who had been poorly nourished.

^rausnitz, Zeitschr. f. Biologie, 29; Tigerstedt and collaborators, 1. c.; Landergren,
Undersokningar ofver menniskans agghviteomsattning, Inaug.-Diss. Stockholm,
1902.

2Voit, Zeitschr. f. Biologie, 41; 167 and 502. See also Kaufmann, ibid., and N.
Schulz, ibid., and Pfliiger's Arch., 76, with Mangold, Stiibel and Hempel, ibid., 114.

8 Berl. klin. Wochenschr., 1887.

METABOLISM IN STARVATION. 895

The investigations on the exchange of gas in starvation have shown,
as previously mentioned, that its absolute extent is diminished, but
that when the consumption of oxygen and elimination of carbon dioxide
are calculated on the unit weight of the body, 1 kilo, this quantity
quickly sinks to a minimum and then remains unchanged, or, on the
continuation of the starvation, may actually rise. It is a well-known
fact that the body temperature of starving animals remains almost con-
stant, without showing any appreciable decrease, during the greater
part of the starvation period. The temperature of the animal first sinks
a few days before death, which occurs at about 33-30° C.

From what has been said about the respiratory quotient it follows
that in starvation it is about the same as with fat and meat exclusively
as food, i.e., approximately 0.7. This is often the case, but it may occa-
sionally be lower, 0.65-0.50, as observed in the cases of CETTI and Succi.
This can be explained by an elimination of acetone bodies by the urine;
a part can be accounted for perhaps by a formation and deposition of
glycogen from protein.

Water passes uninterruptedly from the body in starvation even when
none is taken. If the quantity of water in the tissues rich in proteins
is considered as 70-80 per cent, and the quantity of proteins in them
20 per cent, then for each gram of protein destroyed about 4 grams of
water are set free. This liberation of water from the tissues is generally
sufficient to supply the loss of water, and starvation is ordinarily not
accompanied with thirst.

The loss of water calculated on the percentage of the total organism must
naturally be essentially dependent upon the previous amount of fatty tissue in the
body. In certain cases the starving animal body has indeed been found richer
in water; but if we bear these conditions in mind, then, it seems, according to
BoHTLiNGK,1 that, from experiments upon white mice, the animal body is poorer
in water during inanition. The body loses more water than is set free by the
destruction of the tissues.

The mineral substances leave the body uninterruptedly in starvation
until death, and the influence of the destruction of tissues is plainly
perceptible by their elimination. Because of the destruction of tissues
rich in potassium the proportion between potassium and sodium in
the urine changes in starvation, so that, contrary to the normal condi-
tions, the potassium is eliminated in proportionately greater quantities.

* Contrary to the above BOHTLINGK with starving white mice, and KATSUYAMA 2
with starving rabbits found a greater excretion of sodium than potassium.

1 Arch, des sciences biol. de St. P£tersbourg, 5.

2B6htlingk, 1. c.; Katsuyama, Zeitschr. f. physiol. Chem., 26.

896

METABOLISM.

MUNK observed, in CEirf s case, an increase in the elimination of
phosphoric acid in relation to the ^-elimination, which indicates an
increased decomposition of bone-substance, and this explanation is
supported by the fact that a simultaneous increase in the elimination of
lime and magnesia occurs. Recently WELLMANN 1 showed that in rabbits,
the increase in the elimination of phosphorus, calcium and magnesium
in starvation corresponds to the loss in the bones of these constituents.

The question as to the participation of the different organs in the loss
of weight of the body during starvation is of special interest. In elucida-
tion of this point we give the following results of CHOSSAT'S experiments
on pigeons, and those of VOIT 2 on a male cat. The results are percentages
of weight lost from the original weight of the organ.

Pigeon (CHOSSAT). Male Cat (Vorr).

Adipose tissue 93 per cent. 97 per cent.

Spleen 71 67

Pancreas 64 17

Liver 52 54

Heart 45 3

Intestine 42 18

Muscles 42 31

Testicles — 40

Skin 33 21

Kidneys 32 26

Lungs 22 18

Bones 17 4

Nervous system 2 3

The total quantity of blood, as well as the quantity of solids contained
therein, decreases, as PANUM and others3 have shown, in the same pro-
portion as the weight of the body. Concerning the loss of water by
different organs authorities disagree, LuKjANOw4 claiming that the
various organs differ from each other in this respect.

The above-tabulated results cannot serve as a measure of the metabol-
ism in the various organs during starvation. For instance, the nervous
system shows only a small loss of weight as compared with the other
organs, but from this it must not be concluded that the exchange of
material in this system of organs is least active. The conditions may be
quite different; for one organ may derive its nutriment during starva-
tion from some other organ and exist at its expense. A positive con-
clusion cannot be drawn in regard to the activity of the metabolism in
an organ from the loss of weight of that organ in starvation. Death
by starvation is not the result of the death of all the organs of the body,

1 Munk, Berl. klin. Wochenschr., 1887; Wellmann, Pfluger's Arch., 121.

2 Cited from Voit in Hermann's Handbuch, 6, Part I, 96 and 97.

3 Panum, Virchow's Arch., 29; London, Arch. d. scienc. biol. de St. P^tersbouig, 4.

4 Zeitschr. f. physiol. Chem., 13.

METABOLISM IN STARVATION. 897

but it depends more likely upon the disturbance in the nutrition of a few
less vitally important organs (E. VOIT 1).

In calculating or determining the loss of weight of the . organs in
starvation the original fat content of the organs must be considered.
With the consideration of the fat content of the organs, determined or
estimated in a special way before the starvation period and at the end,
E. VoiT2 found the following loss of weight in the supposed fat-free
organs in starvation, namely, muscles 41 per cent, viscera 42 per cent,
skin 28 per cent, and skeleton 5 per cent.

The quantity of urine nitrogen sinks in starvation corresponding to
the protein catabolism, but to a varying degree in different individuals.
The lowest value observed thus far in man was 2.82 grams per diem as
found by E. and O. FREUND on the twenty-first day in the faster Succi.
Calculated on 1 kilogram of body weight, the urine nitrogen, as is to be
expected, shows striking differences in different persons; in CETTI and
Succi it was 0.150-0.200 gram on the fifth to tenth day of starvation.
The division of the nitrogen in the urine in starvation is unlike that in
the normal condition. The relative amount of urea diminishes, as
shown by E. and O. FREUND, BRUGSCH and CATHCART,S so that instead
of being about 85 per cent of the total nitrogen under normal conditions
it can sink to 54 per cent (BRUGSCH). At the same time because of the
abundant formation of acetone bodies (starvation acidosis) the quantity
of ammonia increases considerably (BRUGSCH, CATHCART). A relative
increase in the neutral sulphur of the urine also takes place (BENEDICT,
CATHCART4). Creatine also occurs in starvation urine and according
to HAWK 5 and co-workers the elimination of creatine is much greater than
the creatinine a few days before the premortal nitrogen elimination.

One must differentiate between the real starvation metabolism and the
metabolism in the inanition condition, the basal requirement (MAGNUS-
LEVY) or the maintenance value (LOEWY 6) . With this we understand the
metabolism in uniform, medium temperature, with absolute bodily rest
and inactivity of the intestinal canal. As a measure of this we deter-
mine the gas exchange in a person lying down with as perfect com-
plete muscular rest as possible, or sleeping in the early morning and
at least twelve hours after a light meal not rich in carbohydrates. This

1 Zeitschr. f . Biologie, 41.

2 Ibid., 46.

3 E. and O. Freund, Wien. klin. Rundschau, 1901, Nos. 5 and 6; Brugsch, Zeitschr.
f. exp. Path. u. Therap., 1 and 3; Cathcart, Bioch. Zeitschr., 6.

4 Zeitschr. f. klin. Med., 36; Cathcart, 1. c.
6 Journ. of biol. Chem., 11.

6 Magnus-Levy in v. Noorden's Handbuch, and Loewy in Oppenheimer's Handbuch
d. Biochemie, Bd. 4.

898 METABOLISM.

basal requirement is the measure of the energy necessary for the per-
formance of all the functions necessary to maintain life during rest; and
all work above this minimum activity is called productive increase by
MAGNUS-LEVY. The basal requirement is almost constant for the same
individual and serves as the starting point in the study of the action of
different influences such as work, food, diseased conditions, etc., upon
metabolism. The extent of this basal requirement, as determined by
the gas exchange according to the ZUNTZ-GEPPERT method, and by
JOHANSSON l and collaborators amounts in men of 60-70 kilos body
weight to about 220-250 cc. oxygen and 160-200 cc. carbon dioxide per
minute, which equals 20-24 grams carbon dioxide per hour. JOHANSSON
found in forced complete muscular rest 20.7 grams CO2 per hour and 24.8
grams C(>2 in the ordinary resting. GIGON 2 found about 23.4 grams
C02 and 21 grams oxygen for the basal requirement. According to MAG-
NUS-LEVY the total daily metabolism can be calculated for the basal
requirement as 1625 calories, or including the rise due to the partaking
of food as 1800 calories. According to GIGON the basal requirement
consists of 15.22 per cent protein, 15-35.2 per cent carbohydrates and
44.5-70 per cent fat.

The food may be quantitatively insufficient, and the final result of
this is absolute inanition. The food may also be qualitatively insufficient
or, as we say, inadequate. This occurs when any of the necessary
nutritive bodies are absent in the food, while the others occur in sufficient
or perhaps even in excessive amounts.

Lack of Water in the Food. The quantity of water in the organism is
greatest during foetal life and then decreases with increasing age. Nat-
urally, the quantity differs essentially in different organs. The enamel,
with only 2 p. m. water, is the tissue poorest in water, while the teeth
contain about 100 p. m. and the fatty tissue 60-120 p. m. water. The
bones, with 140-440 p. m., and the cartilage with 540-740 p. m. are
somewhat richer in water, while the muscles, blood and glands, with 750
to more than 800 p. m., are still richer. The quantity of water is even
greater in the animal fluids (see preceding chapter), and the adult body
contains in all about 630 p. m. water.3 It follows from what has been
given in Chapter I in regard to the great importance of water for living
processes, that if the loss of water is not replaced by fresh supply, the
organism must succumb sooner or later. Death occurs indeed sooner
f romHack of water than from complete inanition (LANDAUER, NOTHWANG) .

1 The literature can be found in the works of Magnus-Levy and Loewy.

2 Johansson, Skand. Arch. f. Physiol., 7, 8, 21, and Nord. Med. Arch. Festband,
1897; see also Magnus-Levy; Gigon, Pfliiger's Arch., 140.

8 See Voit, in Hermann's Handbuch, 6, part 1, 345.

LACK OF MINERAL SUBSTANCES. 899

If water is withdrawn for a certain time, as LANDAUER and espe-
cially STRAUB have shown, it has an accelerating influence upon the
decomposition of protein. This increased destruction has, according to
LANDAUER, the purpose of replacing a part of the water removed, by the
production of water by means of the increased metabolism. The depriva-
tion of water for a short time may, according to SriEGLER,1 especially in
man, cause a diminution in the protein metabolism by means of a reduced
protein absorption.

Lack of Mineral Substances in the Food. In the previous chapters
attention has repeatedly been called to the importance of the mineral
bodies and also to the occurrence of certain mineral substances in certain
amounts in the various organs. The mineral content of the tissues and
fluids is not very great as a rule. With the exception of the skeleton,
which contains as average about 220 p. m. mineral bodies (VOLKMANN 2),
the animal fluids or tissues are poor in inorganic constituents, and the
quantity of these amounts as a rule, only to about 10 p. m. Of the
total quantity of mineral substances in the organism, the greatest part
occurs in the skeleton, 830 p. m., and the next greatest in the muscles,
about 100 p. m. (VOLKMANN).

The mineral bodies seem to be partly dissolved in the fluids and partly
combined with organic substances, but nothing definite can be given as
to the kind of combination, or whether they occur in stoichiometric
proportions, or whether they are simply adsorption combinations. In
accordance with this the organism persistently retains, with food poor
in salts, a part of the mineral substances, also such as are soluble, as the
chlorides. On the burning of the organic substances the mineral bodies
combined therewith are set free and may be eliminated. It is also
admitted that they in part combine with the new products of the com-
bustion, and in part with organic nutritive bodies poor in salts or nearly
salt-free, which are absorbed from the intestinal canal and are thus retained
( VOIT, FORSTER 3) .

If this statement is correct, it is possible that a constant supply of
mineral substances with the food is not absolutely necessary, and that the
amount of inorganic bodies which must be administered is insignificant.
The question whether this is so or not has not, especially in man, been
sufficiently investigated; but generally we consider the need of mineral

1 Landauer, Maly's Jahresber., 24; Nothwang, Arch. f. Hyg., 1892; Straub, Zeitschr.
f. Biol., 37 and 38; Spiegler, ibid., 41.

2 See Hermann's Handbuch., 6, pt. 1, 353.

3 Forster, Zeitschr. f . Biologic, 9. See also Voit, in Hermann's Handbuch, 6,
Part 1, 354. In 'regard to the occurrence and the behavior of the various mineral
constituents of the animal body see the work of Albu and Neuberg, Physiologic und
Pathologic des Mineralstoffwechsel, Berlin, 1906.

900 METABOLISM.

substances by man as very small. It may, however, be assumed that
man usually takes with his food a considerable excess of mineral sub-
stances.

Experiments to determine the results of an insufficient supply of
mineral substances with the food in animals have been made by several
investigators, especially FCRSTER. He observed, on experimenting with
dogs and pigeons with food as poor as possible in mineral substances,
that a very suggestive disturbance of the functions of the organs, par-
ticularly the muscles and the nervous system, appeared, and that death
resulted in a short time, earlier in fact than in complete starvation. On
observations made upon himself, TAYLOR l found on partaking less than
0.1 gram salts per diem that the chief disturbance occurred in the mus-
cular system.

BUNGE in opposition to these observations of FORSTER'S has suggested
that the early death of these cases was not caused by the lack of mineral
salts, but more likely by the lack of bases necessary to neutralize the sul-
phuric acid formed in the combustion of the proteins in the organism;
these bases must then be taken from the tissues. In accordance with
this view, BUNGE and LuNiN2 also found, in experimenting with mice,
that animals which received nearly ash-free food with the addition of
sodium carbonate were kept alive twice as long as those which had the
same food without the sodium carbonate. Special experiments also
show that the carbonate cannot be replaced by an equivalent amount of
sodium chloride, and that to all appearances it acts by combining with
the acids formed in the body. The addition of alkali carbonate to the
otherwise nearly ash-free food may indeed delay death, but cannot pre-
vent it, and even in the presence of the necessary amount of bases death
results from lack of mineral substances in the food.

With an insufficient supply of chlorides with the food the elimination
of chlorine by the urine decreases constantly, and at last it may stop
entirely, while the tissues still persistently retain the chlorides. It has
already been stated (Chapter VIII) how chloride starvation influences
other functions, especially the secretion of gastric juice. If there be a
lack of sodium as compared with potassium, or if there be an excess of
potassium compounds in any other form than KC1, the potassium com-
binations are replaced in the organism by NaCl, so that new potassium
and sodium compounds are produced which are voided with the urine.
The organism becomes poorer in NaCl, which therefore must be taken
in greater amounts from the outside (BUNGE). This occurs continuously

1 University of California Publications, Pathol., 1. •

2 Bunge, Lehrbuch der physiol. Chem., 4. Aufl., 97; Lunin, Zeitschr. f. physiol.
Chem., 5.

ALKALI CARBONATES. PHOSPHATES AND EARTHS. 901

in herbivora, and in man with vegetable food rich in potash. For human
beings, and especially for the poorer classes of people who live chiefly
on potatoes and foods rich in potash, common salt is not only a condi-
ment, but a necessary addition to the food (BUNGE *). On the behavior
of chlorides, especially sodium chloride, in the animal body as well as the
elimination or the retention of NaCl in diseases, we have an abundance
of investigations, which may be found in ALBU and NEUBERG's2 work,
previously cited.

Lack of Alkali Carbonates or Bases in the Food. The chemical processes
in the organism are dependent upon the presence in the tissues and tissue-
fluids of a certain reaction, and this reaction, which is habitually alkaline
toward litmus and neutral toward phenolphthalein, is chiefly due to the
presence of alkali carbonates and carbon dioxide and in a lesser degree to
alkali phosphates. The alkali carbonates are also cf great importance,
not only as a solvent for certain protein bodies and as constituents of
certain secretions, such as the pancreatic and intestinal juices, but they
are also a means of transportation of the carbon dioxide in the blood.
It is therefore easy to understand that a decrease below a certain point
in the quantity of alkali carbonate must endanger life. Such a decrease
not only occurs with lack of bases in the food which brings about various
disturbances and death by a relatively great production of acids through
the burning of the proteins, but it also occurs when an animal is given
dilute mineral acids for a period. The importance of ammonia as a
means of neutralizing the acids produced or introduced into the body
as well as the unequal resistance of man and other animals toward this
action of acids has already been discussed in Chapter XIV.

Lack of Phosphates and Earths. With the exception of the value of
the alkaline earths as carbonates and more especially as phosphates in
the physical composition of certain structures, such as the bones and
teeth, their physiological importance is almost unknown. The importance
of calcium for certain enzymotic processes and of calcium ions for the
functions of the muscles, and especially for cell life, gives an indication of
the necessity of the alkaline earths to the animal organism. Little is
known of the need of these earth in adults, and no average results can
be given. According to KOCHMANN and PETZSCH 3 we cannot conceive
of a certain calcium minimum (in dogs) as the Ca needs vary with dif-
ferent foods. With a Ca equilibrium we can cause an increased elimi-
nation of calcium by increasing the quantity of protein, of fat, or of
carbohydrate in the food and this probably depends upon a giving up

1 Zeitschr. f . Biologie, 9.

2 See footnote 3, page 899.

3 Kochmann, Bioch. Zeitschr., 31, with Petzsch, ibid., 32.

902 METABOLISM.

of calcium phosphate by the skeleton. It is impossible to give
positive figures for the need of phosphates or phosphoric acid, whose
value is recognized not only in the construction of the bones, but also
in the functions of the muscles, the nervous system, the glands, the
organs of generation, etc. The extent of this need is most difficult to
determine, as the body shows a strong tendency, when increased amounts
of phosphorus are introduced, to retain more than is necessary. The
need of phosphates, which, according to EnRSTROM,1 corresponds in adults
to a minimum of 1 to 2 grams phosphorus, is relatively smaller in adults
than in young, developing animals, and in these latter the question of
the result of an insufficient supply of earthy phosphates and alkaline
earths upon the bone tissue is of special interest. For details we refer
to Chapter IX and to the cited work of ALBU-NEUBERG.

Another important question is, How far do the phosphates take part
in the construction of the phosphorized constituents of the body or to
what extent are they necessary? The experiments of ROHMANN and his
pupils 2 with phosphorized (casein, vitellin) and non-phosphorized pro-
teins (edestin) and phosphates show that with the introduction of casein
and vitellin a deposition of nitrogen and phosphorus takes place, while
with non-phosphorized protein and phosphates this does not seem to
occur. The body apparently does not have the power of building up
the phosphorized cell constituents necessary for cell life from non-phos-
phorized proteins and phosphates. On the contrary, according to the
observations of several investigators, the lecithins seem to possess this
power. As known from the investigations of MEISCHER, the develop-
ment of the generative organs of the salmon, which are very rich in nuclein
substances and phosphatides, from the muscles which are relatively poor
in organic-combined phosphorus, seem to indicate a synthesis of phos-
phorized organic substance from the phosphates. The investigations
of HART, McCoLLUM and FULLER,S who found that pigs with food
poor in phosphorus develop just as well with inorganic phosphates as
with organic phosphorus compounds, also indicate such a formation.
The recent investigations of MCCOLLUM 4 on rats show that these animals
can take up the entire need of phosphorus for the skeleton as well as for
the reformation of nucleins and phosphatides in the form of inorganic

1 Skand. Arch, f . Physiol., 14.

2 The literature on feeding experiments with phosphorized and non-phosphorized
food can be found in McCollum, Amer. Journ. of Physiol., 25.

3 Hart, McCollum and Fuller, Amer. Journ., of Physiol., 23. See also Lipschiitz,
Pfliiger's Arch., 143. The literature on the phosphorus metabolism can also be
found in Albu and Neuberg, Physiologic und Pathologic des Mineralstoffwechsel,
Berlin, 1906.

4 Amer. Journ. of Physiol., 25.

LACK OF IKON. 903

phosphorus. Also the investigations of v. WENDT and HOLSTI 1 show
that a synthesis of organic phosphorized substances from phosphates is
very probable. The feeding experiments of OSBORNE and collabora-
tors, which we will soon discuss, and wilich extend over a long period
where the animals were fed with proteins, fat, carbohydrates and min-
eral substances free from phosphorus, give especially strong proof of
the ability of the animal to construct phosphatides and nucleins from only
inorganic phosphorus.

Lack of Iron. As iron is an integral constituent of haemoglobin,
absolutely necessary for the supply of oxygen, it is an indispensable
constituent of food. Iron is a never-failing constituent of the nucleins
and nucleoproteins, and herein lies another reason for the necessity
of the introduction of iron. Iron is also of great importance in the
action of certain enzymes, the oxidases. In iron starvation, iron is
continually eliminated, even though in diminished amounts; and with
an insufficient supply of iron with the food the formation of haemoglobin
decreases. The formation of haemoglobin is not only enhanced by the
supply of organic iron, but also, according to the general view, by inor-
ganic iron preparations. The various divergent reports of this question
have already been given in a previous chapter (on the blood).

In the absence of protein bodies in the food the organism must nourish
itself by its own protein substances, and with such nutrition it must sooner
or later succumb. By the exclusive administration of fat and carbohy-
drates the consumption of proteins in these cases is very considerably
reduced. For a long time we believed in the view suggested by C. and E.
VOIT 2 that with a nitrogen- free diet the protein metabolism could never
be reduced to as small a value as in starvation, but now, due to the investi-
gations of HlRSCHFELD, KUMAGAWA, KLEMPERER, SlV^N, LiANDERGREN"

and recently those of THOMAS,S we learn that the protein metabolism
with such a diet can be smaller than in complete starvation. With exclusive
feeding of sugar, according to THOMAS, the nitrogen elimination can be
reduced in a few days to the wear and tear quota, and he has observed
an elimination of only 30 milligrams nitrogen per day and per kilo of
body weight.

The absence of fats and carbohydrates in the food affects carnivora and
herbivora somewhat differently. It is not known whether carnivora

1 v. Wendt, Skand. Arch. f. Physiol., 17; Holsti; ibid., 23. See also Gregersen,
Zeitschr. f. physiol. Chem., 71.

2 Zeitschr. f. Biologic, 32.

'Hirschfeld, Virchow's Arch., 114; Kumagawa, ibid., 116; Klemperer, Zeitschr.
f. klin. Med., 16; Sive"n, Skand. Arch. f. Physiol., 10 and 11; Landergren, 1. c., 11;
footnote 1, page 894, also Maly's Jahresber., 32; Karl Thomas, Arch. f. (Anat. u.)
Physiol., 1909 and 1910, Suppl. Bd.

904 METABOLISM.

can be kept alive for any length of time by food entirely free from fat
and carbohydrates.1 But it has been positively demonstrated that they
can be kept alive a long time by feeding exclusively with meat freed as
much as possible from visible fat (PFLUGER2). Human beings and
herbivora, on the contrary, cannot live for any length of time on such
food. On the one hand they lose the property of digesting and assimilating
the necessarily large amounts of meat, and on the other a distaste for
large quantities of meat or proteins soon appears. The elimination of
acetone bodies with an exclusion of carbohydrates from the food of man
is of interest (see Chapter XIV).

A question of greater importance is whether it is possible to maintain
life in an animal for any length of time with a mixture of simple organic
and inorganic foodstuffs. The earlier experiments carried out by many
investigators to decide this question have not yielded satisfactory results.
and ROHMANN 3 was first able, by feeding a mixture of several proteins
with fat, starch, glucose and salts, to keep mice alive for a long time, and
was also able to raise young mice by artificial feeding of the mother and
then the small animals. ROHMANN concludes from his experiments
that for the continuous maintenance or for development of the animal
a mixture of different proteins is necessary, but more recently he4 has
found that this can be accomplished by a single protein, and the results
of his experiment coincide well in this regard with the investigations of
OSBORNE and MENDEL (and E. FERRYS).

In experiments with white mice these investigators have found that
on feeding with a mixture of only one protein with cane-sugar, starch,
fat, agar-agar and mineral substances, adult mice could be kept for 169-
259 days without changing their body weight. The reason why the
adult mice could not be maintained for a still longer time and why young
mice did not grow was that certain substances of unknown kind were
lacking. Such substances occur in milk, and by adding to the food, milk
from which the proteins have been removed, although the food contained
only one protein, the animals can be kept alive for a longer time — 500-600
days, and the normal growth accomplished as well. These proteins were,
especially, casein, lactalbumin, ovalbumin, hemp-seed edestin, wheat
giutenin and excelsin, while on the contrary they were not able to pro-

1 See Horbaczewski, Maly's Jahresber., 31, 715.

2 Pfluger's Arch., 50.

3F. Rohmann, Klin, therap. Wochenschr., No. 40, 1902, and Allg. med. Centralbl.
Zeitung, 1908, No. 9.

4 Rohmann, Bioch. Zeitschr., 39.

6Th. B. Osborne and L. B. Mendell, Science, 34. The Carnegie Institution, Wash-
ington, Parts 1 and 2, 1911; with Edna Ferry, Journ. of biol. Chem., 12 and 13, and
Zeitschr. f. physiol. Chem., 80.

FEEDING WITH FOOD-STUFFS AND LIPOIDS. 905

duce a sufficient growth with pea-legumin, zein, gliadin and hordein when
added to the other foodstuffs and protein-free milk. These experiments
showed that animals fed with gliadin as the only protein had the normal
ability to produce offspring and had the ability to produce milk necessary
for their food.

In another series of experiments it was shown that the protein-free
milk could be replaced by a proper mixture of salts and that the organic
constituents of such milk were not necessary. On feeding with fat, car-
bohydrates, casein and such a salt mixture they were able to attain
normal growth in a series of experiments of more than 80 or 100 days.
Growth was produced in the animals also in the absence of substances
soluble in ether (lipoids). This is remarkable, as according to the
observations and experiments of STEPP, lipoids are necessary for the
normal nutrition.

According to STEPP 1 a food which is adequate but not quite genuine
for mice can be made genuine by the addition thereto of certain substances
soluble in alcohol-ether from milk, egg-yolk, brain, etc. These substances,
which are neither fat nor cholesterin, and which he calls lipoids, are partly
heat-labile and correspondingly lose their action by continuously boiling
with alcohol or by a lengthy boiling of the natural food-stuffs with alcohol
or water. A proper food for mice can be so changed by continuous
boiling with alcohol so that all animals fed with it die, while the changes
in the food brought about in this way can be counteracted by the lipoids
obtained under conditions where the lengthy action of heat is prevented.
Mice, which die with an otherwise sufficient food but free from lipoids
may be kept alive by the addition of the undestroyed lipoids to the
same food.

Recently it has been suggested that beside the foodstuffs in the ordinary
sense, other constituents of our food exist which are of the very greatest
importance for life. The investigations of FUNK as well as those of
SUZUKI, SHIMAMURA and ODAKE on the constituents of rice-bran give a
specially striking proof of this. According to C. FUNK 2 rice-bran contains
a substance called vitamine, CiyH^o^O?, which belongs to the pyrimidine
group and which also occurs in yeast, milk residue and beef-brains. This
substance, which is absent in polished rice, causes the disease Beri-Beri
in man and polyneuritis in birds. SUZUKI, SHIMAMURA and ODAKE have
also isolated from rice-bran a substance which they call oryzanine, which
is soluble in alcohol and necessary for animal life. With mixtures of
protein, carbohydrates, fat and salts without oryzanine these investiga-
tors could not keep hens, pigeons and mice alive and dogs could not be

1 Bioch. Zeitschr., 22, and Zeitschr. f. Biol., 57 and 59.

2 C. Funk, Journ. of Physiol., 43 and 45; Suzuki, Shimamura and Odake, Bioch.
Zeitschr., 43.

906 METABOLISM.

kept alive with boiled meat and polished rice. They emaciate quickly
and rapidly recover again if they receive oryzanine.

It follows from the above that there exists a certain unexplainable
contradiction between the important observations of STEPP and those
of the other investigators on the one side and the very interesting, prolonged
experiments of OSBOKNE and MENDEL with pure foodstuffs on the
other side.

IH. METABOLISM WITH VARIOUS FOODS.

For carnivora, as above stated,, meat as poor as possible in fat may
be a complete and sufficient food. As the proteins moreover take a special
place among the organic nutritive bodies by the quantity of nitrogen they
contain, it is proper that we first describe the metabolism with an exclu-
sively meat diet.

Metabolism with food rich in proteins, i.e., feeding only with meat as
poor in fat as possible.

By an increased supply of proteins the catabolism and the elimination
of nitrogen is increased, and this in proportion to the supply of proteins.

If a certain quantity of meat has daily been given to carnivora as
food and the quantity is suddenly increased, an augmented catabolism
of proteins, or an increase in the quantity of nitrogen eliminated, is the
result. If the animal is daily fed for a certain time with larger quantities
of the same meat, a part of the proteins accumulates in the body, but
this part decreases from day to day, while there is a corresponding daily
increase in the elimination of nitrogen. In this way a nitrogenous
equilibrium is established; that is, the total quantity of nitrogen eliminated
is equal to the quantity of nitrogen in the absorbed proteins or meat.
If, on the contrary, an animal in nitrogenous equilibrium, having been
fed on large quantities of meat, is suddenly given a small quantity
of meat per day, it uses up its own body proteins, the amount de-
creasing from day to day. The elimination of nitrogen and the catab-
olism of proteins decrease constantly, and the animal may in this case,
also pass into nitrogenous equilibrium, or almost into this condition
These relations are illustrated by the following table (VOIT) : l

Grama of Meat in the Food per Day.

Before the Test.

During the Teat.

1. .

500

1500

2

, . . . •. 1500

1000

Grams of Flesh Metabolized in Body per Day.

1

2345

6 7

1222

1310 1390 1410 1440

1450 1500

1153

1086 1088 1080 1027

1 Hermann's Handbuch, 6, Part I, 110.

METABOLISM WITH FOOD RICH IN PROTEINS. 907

In the first case (1) the metabolism of meat before the beginning of
the actual experiment on feeding with 500 grams of meat was 447 grams,
and it increased considerably on the first day of the experiment, after
feeding with 1500 grams of meat. In the second case (2), in which the
animal was previously in nitrogenous equilibrium with 1500 grams of
meat, the metabolism of flesh on the first day of the experiment, with
only 1000 grams meat, decreased considerably, and on the fifth day an
almost nitrogenous equilibrium was obtained. During this time the
animal gave up daily some of its own proteins. Between that point below
which the animal loses from its own weight and the maximum, which
seems to be dependent upon the digestive and assimilative capacity of
the intestinal canal, a carnivore may be kept in nitrogenous equilibrium
with varying quantities of proteins in the food.

The supply of proteins, as well as the protein condition of the body,
affects the extent of the protein metabolism. A body which has become
rich in proteins by a previous abundant meat diet must, to prevent a loss
of proteins, take up more protein with the food than a body poor in pro-
teins.

In regard to the rapidity with which the protein catabolism takes
place FALTA l found in man but not, or at least not to the same extent,
in dogs, that quite great differences exist between the different proteins.
Thus on feeding pure proteins the chief amount of the nitrogen is more
quickly eliminated after feeding casein than after genuine ovalbumin.
This latter is more easily demolished after a previous modification by
coagulation than in the native state, which indicates that an unequal
resistance of the different proteins toward the digestive juices plays a
part. HAMALAINEN and HELME2 have also obtained similar results.
Even on feeding with easily decomposable proteins it always takes several
days before the total nitrogen corresponding thereto is eliminated, which
depends, according to FALTA, upon a progressive demolition of the pro-
tein. From the unequal rate at which the different proteins are decom-
posed it follows that in the passage from a diet poor in protein to one
rich in protein the time within which nitrogenous equilibrium occurs
depends chiefly upon the kind of protein contained in the food.

PETTENKOFER and VOIT have made investigations on the metabolism
of fat with an exclusively protein diet. These investigations have shown
that by increasing the quantity of proteins in the food the daily metab-
olism of fat decreases, and they have drawn the conclusion from these
experiments, that there may even take place a formation of fat under
these circumstances. The objections presented by PFLUGER to these

1 Deutsch. Arch. f. klin. Med., 86.

2 Skand. Arch. f. Physiol., 19.

908 METABOLISM.

experiments, as well as the proofs of the formation of fat from proteins,
are also given in Chapter IX.

According to PFLUGER'S doctrine, the protein can influence the formation of
fat only in an indirect way, namely, in that it is consumed instead of the non-
nitrogenous bodies and hence the fat and fat-forming carbohydrates are
spared. If sufficient protein is introduced with the food to satisfy the total nu-
tritive requirements, then the decomposition of fat stops; and if non-nitrogenous
food is taken at the same time, this is not consumed, but is stored up in the animal
body, the fats as such, and the carbohydrates at least in great part as fat.
ir PFLUGER defines the " nutritive requirement " as the smallest quantity of
lean meat which produces nitrogenous equilibrium without causing any decom-
position of fat or carbohydrates. At rest and at an average temperature it is
found in dogs to be 2.073 to 2.099 grams of nitrogen l (in meat fed) per kilo of
flesh weight (not body weight, as the fat, which often forms a considerable fraction
of the weight of the body, cannot as it were be used as dead measure). Even
when the supply of protein is in excess of the nutritive requirements, PFLUGER
found that the protein metabolism increases with an increased supply until
the limit of digestive power is reached, which limit is about 2600 grams of meat
with a dog weighing 30 kilos. In these experiments of PFLUGER'S not all of the
excess of protein introduced was completely decomposed, but a part was retained
by the body. PFLUGER therefore defends the proposition " that a supply of
proteins only, without fat or carbohydrate does not exclude a protein fattening."

• From what has been said on protein metabolism in starvation and
with exclusive protein food, it follows that the protein catabolism in the
animal body never stops, that the extent is dependent in the first place
upon the extent of protein supply, and that the animal body has the prop-
erty, within wide limits, of accommodating the protein catabolism to the
protein supply.

These and certain other peculiarities of protein catabolism have led
VOIT to the view that not all proteins in the body are decomposed with
the same ease. VOIT differentiates between the proteins fixed in the
tissue-elements, so-called organized proteins, tissue-proteins, from those
proteins which circulate with the fluids in the body and its tissues and
which are taken up by the living cells of the tissues, from the interstitial
fluids washing them, and destroyed. These circulating proteins or supply
proteins are, he claims, more easily and quickly destroyed than the tissue-
proteins. When, therefore, in a fasting animal which has been previously
fed with meat, an abundant and quickly decreasing decomposition of
proteins takes place, while in the further course of starvation this protein
catabolism becomes less in quantity and more uniform, this depends upon
the fact that the supply of circulating proteins is destroyed chiefly in the
first days of starvation and the tissue-proteins in the last days.

The tissue-elements constitute an apparatus of a relatively stable
nature, which has the power of taking proteins from the fluids washing
the tissues and appropriating them, while their own proteins, the tissue-

1 See Schondorflt, Pfluger's Arch., 71.

METABOLISM WITH FOOD RICH IN PROTEINS. 909

proteins, are ordinarily catabolized to only a small extent, about 1 per
cent daily (Vorr). By an increased supply of proteins the activity of
the cells and their ability to decompose nutritive proteins is also increased
to a certain degree. When nitrogenous equilibrium is obtained after
an increased supply of proteins, it indicates that the decomposing power
of the cells for proteins has increased so that the same quantity of proteins
is metabolized as is supplied to the body. If the protein metabolism is
decreased by the simultaneous administration of other non-nitrogenous
foods (see below), a part of the circulating proteins may have time to
become fixed and organized by the tissues, and in this way the mass of
the flesh of the body increases. During starvation or with a lack of pro-
teins in the food the reverse takes place, for a part of the tissue proteins
is converted into circulating proteins which are metabolized, and in this
case the flesh of the body decreases.

VOIT'S theory has been criticised by several investigators and espe-
cially by PFLUGER. PFLUGER'S belief, based on an investigation made
by one of his pupils, SCHONDORFF/ is that the extent of protein destruc-
tion is not dependent upon the quantity of circulating proteins, but
upon the nutritive condition of the cells for the time being — a view
which does not widely differ from VOIT if the author does not misunder-
stand PFLUGER. VoiT2 has, as is known, stated that the conditions for
the destruction of substances in the body exist in the cells, and also that
the circulating protein is first catabolized after having bean taken up
by the cells from the fluids washing them. Besides this, certain inves-
tigations conclusively show that the extent of protein catabolism is depend-
ent upon the concentration of the decomposable proteins at the place
where the decomposition is taking place. Thus in confirmation with
the earlier investigations of v. GEBHARDT and KRUMMACHER, THOMAS,
v. HOESSLIN and LESSER 3 have recently shown that on feeding with
a certain quantity of protein, less protein was catabolized when the pro-
tein was supplied piecemeal, i.e., in several small portions during the day
instead of at one time. That the peculiarity of the nitrogen elimination
in starvation arid after sufficient protein supply depends essentially
upon the concentration of the decomposable proteins (or more correctly the
decomposable nitrogenous substances) is no doubt also generally admitted.4

1 Pfliiger, Pfluger's Arch., 54; Schondorff, ibid., 54.

2 Zeitschr. f. Biologic, 11.

3 K. Thomas. Arch. f. (Anat. u.) Physiol., 1909; H. v. Hoesslin and E. J. Lesser,
Zeitschr. f. physiol. Chem., 73, when also the works of v. Gebhardt and Kummacher are
cited.

4 See also E. Voit and A. Korkunoff, Zeitschr. f. Biol., 32, and O. Frank and R.
Trommsdorff, ibid., 43.

910 METABOLISM.

Recent investigations, especially those of FoLiN,1 which show that the
amount of certain nitrogenous urinary constituents, such as creatinine,
uric acid and the combinations containing neutral sulphur, are almost
independent of the quantity of protein taken as food, while the quantity
of urea is determined by the protein partaken of, tend to substantiate
VOIT'S view that we must differentiate between the real cell protein
and the food protein. This has also led FOLIN to differentiate between
endogenous and exogenous protein metabolism. The chief point in
VOIT'S theory that all the proteins in the body do not behave alike
and that the organized proteins which have been fixed in the cells and
have been introduced into the cell structure are less readily catabolized
than the proteins occurring in the nutritive fluids or temporarily taken
up from these, must also be considered as not disputed. RuBNER2
differentiates also between the deposited protein (growth protein, and
deposited by the activity of the cells melioration protein) in the body on
the one hand and the protein temporarily incorporated with the body
(supply protein and catabolized in passing to a protein-poor diet, transitory
protein) on the other hand.

This question is intimately connected with another, namely, whether the
food proteins taken up by the cells are metabolized as such or whether they are
first organized, i.e., are converted into specific cell protein. The observations
of PANUM, FALCK, ASHER and HAAS and others 3 on dogs have shown that the
nitrogen elimination increases almost immediately after a meal and in the fifth or
sixth hour according to these experimenters, when according to SCHMIDT-MULHEIM 4
about 59 per cent of the eaten protein is absorbed, do not indicate that a trans-
formation of the food protein into organized protein occurs before it is catab-
olized. The recent investigations upon the deep cleavage of proteins in digestion
and the generally accepted protein syntheses from amino-acids have made this
question lose its special interest.

On account of the above-stated action of the concentration of the
catabolizable nitrogenous material upon the protein decomposition or
nitrogen elimination, it is not possible to replace the quantity of protein
catabolized in starvation by the exclusive feeding of protein administered
at one time and in quantities corresponding to the food proteins. This
always requires large amounts of protein. Even on the fractional intro-
duction of natural protein v. HOESSLIN and LESSER were unable to pro-
duce a nitrogen equilibrium with quantities of protein equal to the starva-

1 Amer. Journ. of Physiol., 13.

2 Arch. f. (Anat. u.) Physiol., 1911.

3 Panum, Nord. Med. Arkiv., 6; Falck, see Hermann's Handbuch, 6, Part I, 107;
Asher and Haas, Bioch. Zeitschr., 12. For further information in regard to the curve
of nitrogen elimination in man, see Tschenloff, Korrespond. Blatt Schweiz. Aerzte,
1896; Rosemann, Pfluger's Arch., 65, and Veraguth, Journ. of Physiol., 21; Schlosse,
Maly's Jahresber., 31.

4 Arch. f. (Anat, u.) Physiol., 1879.

NUTRITIVE VALUE OF GELATIN. 911

tion protein; the elimination of nitrogen was always somewhat increased.
On the fractional introduction of protein, THOMAS l was nevertheless able
in dogs to produce nitrogenous equilibrium without essentially raising
the protein metabolism (in comparison with the starvation value). In
experiments upon himself he was not able to produce this.

It has been stated above that other foods may decrease the catab-
olism of proteins. Gelatin is such a food. Gelatin and the gelatin-formers
do not seem to be converted into protein in the body, and this last cannot
be entirely replaced by gelatin in the food. For example, if a dog is fed
on gelatin and fat, its body sustains a loss of proteins even when the
quantity of gelatin is great enough so that the animal with an amount of
fat and meat containing just the same quantity of nitrogen as the gelatin
in question, remains in nitrogenous equilibrium. On the other hand,
gelatin, as VOIT, PANUM and OERUM2 have shown, has great value as
a means of sparing the proteins, and it may decrease the catabolism of
proteins to a still greater extent than fats and carbohydrates. This is
apparent from the following summary of VOIT'S experiments upon a dog:

Food per Day. Flesh.

Meat. Gelatin. Fat. Sugar. Catabolized. On the Body.

400 0 200 0 450 -50

400 0 0 250 439 -39

400 200 0 0 356 +44

I. MUNK 3 has later arrived at similar results by means of more deci-
sive experiments, and the recent investigations of KRUMMACHEB and
KIRCHMANN 4 show the extent of the sparing action of gelatin upon pro-
teins. The extent of protein destruction during gelatin feeding was com-
pared with the extent of protein catabolism in starvation, and it was
found that 35-37.5 per cent of the quantity of protein decomposed in
starvation could be spared by gelatin. The physiological availability
of gelatin was found by KRUMMACHER to be equal to 3.88 calories for 1
gram, which corresponds to about 72.4 per cent of the energy-content of
the gelatin.

The value of gelatin has been found by MURLIN 5 to be dependent to
a high degree upon the protein condition of the body, on the calorific
value of the food and the quantity of carbohydrates in the latter. If
in a man weighing 70 kilos, 51 calories per kilo were partaken, the quan-
tity of nitrogen eliminated was 10 per cent more than the starvation

1 v. Hoesslin and Lesser, 1. c.; Thomas, Arch. f. (Anat. u.) Physiol., Suppl. Bd., 1910.

2 Volt, 1. c., 123; Panum and Oerum, Nord. Med. Arkiv., 11.

3 Pfliiger's Arch., 58.

4 Krummacher, Zeitschr. f. Biologic, 42; Kirchmann, ibid., 40.
6 Amer. Journ. of Physiol., 19.

912 METABOLISM.

value, and when two-thirds of the total calories partaken of were sup-
plied by carbohydrates, 63 per cent of the total nitrogen could be replaced
by gelatin nitrogen.

The reason why gelatin cannot entirely replace protein has been sought
for in the fact that gelatin does not contain all the amino-acids of the
proteins (such as tyrosine and tryptophane), or does not contain a suf-
ficient amount of the various amino-acids. The correctness of this
explanation was first shown by KAUFMANN by an experiment on himself,
where he showed that gelatin after addition of tyrosine, tryptophane
and cystine could be made equivalent to protein. The conclusive proof
was given later by ABDERHALDEN l when he showed that completely
decomposed gelatin on the addition of a mixture of amino-acids, among
them also tyrosine and tryptophane, could be made equivalent to proteins.

As it has been possible to replace the proteins in the food by their
cleavage products or mixtures of amino-acids,2 it is easily understandable
that also proteoses or peptones can completely or partly replace the
protein. Their ability in this regard is essentially dependent upon their
constitution, i.e., their content of the different amino-acids. As the
proteoses and peptones are produced by cleavage and as therefore in
one proteose we hae certain atomic comp loies and in others again
these may be absent or only exist to a slight extent, it is conceivable that
different investigators 3 have obtained contradictory results because of
the use of different proteoses and peptones.

We have a number of investigations on the action of amides upon
metabolism, which are mostly connected by the use of asparagin. These
investigations have in part led to conflicting results; but they indicate
that carnivora 'and herbivora act differently, that the results are depen-
dent upon the rapidity with which the asparagin is absorbed and also
upon the bacterial action in the intestine, and that in herbivora a protein-
sparing action can be brought about by asparagin.4 If, as is generally

1 Martin Kaufmann, Pfliiger's Arch., 109; Abderhalden, Zeitschr. f. physiol. Chem.,
77.

2 See Abderhalden and collaborators, Chapter VIII; also Abderhalden, Zeitschr.
f. physiol. Chem., 77, and especially 83.

3 In regard to the literature on the nutritive value of the proteoses and peptonea
see Maly, Pfltiger's Arch., 9; P16sz and Gyergyay, ibid., 10; Adamkiewicz, 'Die Natur
und der Nahrwerth des Peptones" (Berlin, 1877); Pollitzer, Pfliiger's Arch., 37, 301;
Zuntz, ibid., 37, 313; Munk, Centralbl. f. d. med. Wissensch., 1889, 20, and Deutsch.
med. Wochenschr., 1889; Ellinger, Zeitschr. f. Biologic, 33 (literature). Blum, Zeitschr.
f. physiol. Chem., 30; Henriques and Hansen, Zeitschr. f. physiol. Chem., 48.

4 Weiske, Zeitschr. f. Biologic, 15 and 17, and Centralbl. f. d. med. Wissensch., 1890,
945; Munk, Virchow's Arch., 94 and 98; Politis, Zeitschr. f. Biologic, 28. See also
Mauthner, ibid., 28; Gabriel, ibid., 29; and Voit, ibid., 29, 125; Kellner, Maly's Jahres-

METABOLISM WITH A MIXED DIET. 913

admitted, the amino-acids can serve in the building up of the proteins,
then there is no use denying that their amides can also be used by the
animal body.

Recently GRAFE, ABDERHALDEN 1 and their collaborators have carried
on investigations on the value of ammonia and of urea as protein sparers
and protein formers. These investigations have shown that ammonia
or urea under special conditions of experimentation may cause a nitrogen
retention, but we are not justified in believing that a synthesis of protein
from ammonia takes place.

Metabolism on a Diet Consisting of Protein, with Fat or Carbohydrates.
As the various foodstuffs can replace each other as sources of energy in
the food it follows that the non-nitrogenous foodstuffs can be used
instead of the proteins and can reduce the catabolism of these. Thus
the fat cannot completely arrest or prevent the catabolism of proteins,
but it can decrease it and so spare the proteins. This is apparent from
the following table by VoiT.2 A is the average for three days, and B
for six days.

Food. Flesh.

Meat. Fat. Metabolized. On the Body.

A 1500 0 1512 -12

B 1500 150 1474 +26

According to VOIT the adipose tissue of the body acts like the food-
fat, and the protein-sparing effect of the former may be added to that of
the latter, so that a body rich in fat may not only remain in nitrogenous
equilibrium, but may even add to the store of body proteins, while in a
lean body with the same food containing the same amount of proteins
and fat there would be a loss of proteins. In a body rich in fat a greater
quantity of proteins is protected from metabolism by a certain quantity
of fat than in a lean body.

Like the fats the carbohydrates have a sparing action on the proteins.
By the addition of carbohydrates to the food, carnivora not only remain
in nitrogenous equilibrium, but the same quantity of meat which in
itself is insufficient and which without carbohydrates would cause a loss

her., 27, and Zeitschr. f. Biologie, 39; Pfluger's Arch., 113; Kellner and Kohler, Chem.
Centralbl., 1, 1906; Voltz, Pfluger's Arch., 107, 117, with Yakuwa, ibid., 121; v.
Strusiewicz, Zeitschr. f. Biol., 47; Rosenfeld and Lehmann, Pfluger's Arch., 112;
Lehmann, ibid., 115; M. Miiller, ibid., 117; Henriques and Hansen, Zeitschr. f. physiol.
Chem., 54.

1 Grafe, Zeitschr. f. physiol. Chem., 78, 82, 84, with Schlapfer, ibid., 77, with
Turban, ibid., 83; Voltz, ibid., 74; Abderhalden with Hirsch or Laupe, ibid., 80,
82-84; Peschek, Bioch. Zeitschr. 45.

2 Voit, in Hermann's Handb., 6, 130.

914 METABOLISM.

of weight in the body may with the addition of carbohydrates produce
a deposit of proteins. This is apparent from the following table:1

Food. Flesh.

Meat. Fat. Sugar. Starch. Metabolized. On the Body.

500 250 ... ... 558 - 58

500 ... 300 ... 466 +34

500 ... 200 ... 505 - 5

800 ... ... 250 745 + 55

800 200 ... ... 773 +27

2000 ... ... 200-300 1792 +208

2000 250 ... ... 1883 +117

The sparing of protein by carbohydrates is greater, as shown by the
table, than by fats. According to VOIT the first is on an average 9 per
cent and the other 7 per cent of the administration protein without a
previous addition of non-nitrogenous bodies. Increasing quantities of
carbohydrates in the food decrease the protein metabolism more regularly
and constantly than increasing quantities of fat. ATWATER and BENE-
DICT 2 also found that the carbohydrates had a somewhat greater sparing
action upon proteins than fats.

Because of this great protein-sparing action of carbohydrates the her-
bivora, which as a rule partake of considerable quantities of carbohydrates,
assimilate proteins readily (Voii).

The greater protein-sparing action of carbohydrates as compared with
that of the fats occurs, as shown by LANDERGREN,3 to a still higher degree
with food poor in nitrogen or in nitrogen starvation, in which cases the
carbohydrates have double the protein-sparing action as compared with
an isodynamic quantity of fat. This different behavior of the fats
and the carbohydrates is also shown in the experiments of RUBNER and
THOMAS 4 that on the exclusive feeding of sugar the nitrogen elimination
is reduced to the wear and tear quota while on the exclusive feeding of
fats the nitrogen requirement was about two to three times as great as
the wear and tear quota.

The protein-sparing action of the carbohydrates and fats has generally
been studied through the one-sided feeding with one or the other of these
two groups of foodstuffs. The question may be raised whether the differ-
ence observed between the fats and carbohydrates could not also be
brought about by the simultaneous supply of carbohydrates and fat in
varying proportions. TALLQUISTS made a series of experiments on this

1 Voit, in Hermann's Handb., 6, page 143.

2 See Ergebnisse der Physiologie, 3.

3 1. c., Inaug.-Diss., and Skand. Arch. f. Physiol., 14. Wimmer, Zeitschr. f. Biol.,
57, has given further proofs of the strong protein-sparing action of carbohydrates in
nitrogen starvation.

4 See Thomas, Arch. f. (Anat. u.) Physiol. Suppl. Bd., 1910.

5 Finska Lakaresallskapets Handl., 1901. See also Arch. f. Hygiene, 41.

LIMIT OF PROTEIN REQUIREMENT. 915

subject. In one of the periods 16.27 grams N, 44 grams fat, and 466
grams carbohydrate were given; in a second, 16.08 grams N, 140 grams
fat, and 250 grams carbohydrate, containing almost the same number
of calories, namely, 2867 and 2873. In both cases an almost complete
nitrogenous equilibrium was reached and the carbohydrate did not
spare more protein than the fat. It is therefore possible that the fat has
about the same protein-sparing action as an isodynamic amount of car-
bohydrate when the quantity of carbohydrates does not sink below a
certain minimum, which is not known for the present.

This condition as well as the extent of protein-sparing action of the
carbohydrates stands, according to LANDERGREN, in close relation to
the formation of sugar in the body. The animal body always needs
sugar, and a lack of carbohydrates in the food leads to a part of the pro-
teins being used in the sugar formation. This part can be spared by
carbohydrates but not by fats, from which, according to LANDERGREN,
the carbohydrates cannot be formed. In this also lies the probable
reason why the fats, on being fed exclusively but not with a sufficient
supply of carbohydrates, have a much lower protein-sparing action than
the carbohydrates. The fats cannot prevent the protein catabolism
necessary for the formation of sugar on a diet lacking in carbohydrates.

The law as to the increased protein catabolism with increased pro-
tein supply also applies to food consisting of protein with fat and car-
bohydrates. In these cases the body tries to adapt its protein catabolism
to the supply; and when the daily calorie-supply is completely covered
by the food, the organism can, within wide limits, be in nitrogenous
equilibrium with different quantities of protein.

The upper limit to the possible protein catabolism per kilo and per
day has been determined only for herbivora. For human beings it is
not known, and its determination is from a practical standpoint of second-
ary importance. What is more important is to ascertain the lower limit,
and on this subject we have several older experiments upon man as well
as upon dogs by HIRSCHFELD, KUMAGAWA, KLEMPERER, MUNK, ROSEN-
HEiM,1 and others. It follows from these experiments that the lower
limit of protein requirement for human beings, for a week or less, is about
30-40 grams or 0.4-0.6 gram per kilo with a body of average weight,
v. NooRDEN2 considers 0.6 gram protein (absorbed protein) per kilo
and per day as the lower limit (threshold of protein requirement). The
above-mentioned figures are valid only for short series of experiments;
still there exists the observation of E. VOIT and CONSTANTINIDI 3 on

1 See footnote 3, page 903; also Munk, Arch. f. (Anat. u.) Physiol., 1891 and 1896
Rosenheim, ibid., 1891; Pfluger's Arch., 54.

2 Grundriss einer Methodik der Stoffwechseluntersuchungen. Berlin, 1892.
» Zeitschr. f. Biologic, 25.

916 METABOLISM.

the diet of a vegetarian when the protein condition was kept almost
but not completely normal for a long time with about 0.6 gram of pro-
tein per kilo. CASPARI J has also made observations upon a vegetarian
for a period of 14 days with an average of 0.1 gram nitrogen (recalculated
as equal to 0.62 gram protein) per kilo, where a nearly complete nitrog-
enous equilibrium was observed as the average result.

According to VOIT'S normal figures, which will be spoken of below,
for the nutritive need of man, an average workingman of about 70 kilos
weight, requires on a mixed diet about 40 calories per kilo (true calories
or net calories). In the above experiments with food very poor in pro-
tein the demand for calories was considerably greater; as, for instance,
in certain cases it was 51 (KUMAGAWA) or even 78.5 calories (KLEMPERER).
It therefore seems as if the above very low supply of protein was pos-
sible only with great waste of non-nitrogenous food; but in opposition
to this it must be recalled that in VOIT and CONSTANTINIDI'S experiments
upon the vegetarian, who for years was accustomed to a food poor in
protein and rich in carbohydrate, the calories amounted to only 43.7
per kilo. In the case studied by CASPARI a supply of 41 calories per
kilo was entirely sufficient.

SIVEN has shown by experiments upon himself that the adult human
organism, at least for a short time, can be maintained in nitrogenous
equilibrium with a specially low supply of nitrogen without increasing
thje calories in the food above the normal. With a supply of 41-43
calories per kilo he remained in nitrogenous equilibrium for four days
with a supply of nitrogen of 0.08 gram per kilo of body weight. Of the
nitrogen taken, a part was of a non-protein nature and the quantity of
true protein nitrogen was only 0.045 gram, corresponding to about 0.3
gram of protein per kilo of body weight. That this low limit, which
by the way holds only for a short time, has no general validity follows
from other observations. Thus CASPARI 2 also, in an experiment on him-
self, could not attain complete nitrogenous equilibrium on a much greater
nitrogen supply. The protein minimum also seems to vary in different
individuals.

The protein minimum can also be different for other reasons. It
varies, as mentioned by RUBNER, not only with the kind of foodstuffs,
but also with the nutritive condition of the body. The needs of the
cells for protein varies with the nutritive condition of the body. Where
the protein is eagerly demanded, less supply of protein suffices, and where
the demand is low more protein must be offered (RUBNER). The more the

1 Physiologische Studien iiber Vegetarismus, Bonn, 1905.

2 Siven, Skand. Arch. f. Physiol., 10 and 11; Caspar!, Arch. f. (Anat. u.) Physiol.,
1901.

WEAR AND TEAR QUOTA. 917

body has become reduced the lower is the protein minimum, according to

RUBNER.1

As mentioned in the early part of this chapter, the body always suffers
a certain loss of nitrogen through the falling out of the hair and other
epidermis formations, by the secretions, etc.; but to this also belongs the
constant loss of nitrogenous substance which every cell sustains because
of its activity. This unpreventable loss of nitrogen has been included
by RUBNER under the name " wear and tear " quota, and this quota,
which corresponds to the nitrogen elimination with a perfectly nitrogen-
free diet, and hence is a protein minimum, may rise to 4 to 6 per cent of
the total calorific needs. The energy supply of the food is under these
conditions entirely assumed by the non-nitrogenous foodstuffs, and when
this quota is replaced by protein the body is in a condition of lowest
nitrogenous equilibrium.

All proteins do not have the same value in replacing the protein
minimum. MICHAUD 2 determined the protein minimum in dogs by
feeding entirely with nitrogen-free food, and he found that this min-
imum can be covered by the corresponding quantity of protein specific
of the animal, but not by the same quantity of an alien protein, like
gliadin and edestin. v. HOESSLIN and LESSER have found on the con-
trary in experiments with dogs that proteins specific to the animal were
only unessentially superior to the proteins of horse flesh, and E. VOIT
and LISTERER found for the three kinds of protein, beef-muscle, aleuronat
and casein, that the relation was 100 : 106 : 121. THOMAS 3 has carried
out experiments on man with different foods and has found that the
nitrogen of various kinds of proteins has an unequal value in replacing
the wear and tear quota. By the expression " biological equivalence "
of the nitrogenous foodstuffs he denotes the number of parts of body
nitrogen which can be replaced by 100 parts of the food-nitrogen and
he fourffe the following equivalence: for beef = 104. 7, milk = 99. 7, casein =
70.14, wheat flour = 39. 6, potatoes = 78. 9, peas = 55.7, and corn = 29.5.
Also in consideration of the different content of nitrogenous extractives
in the food these figures therefore show that different proteins have essen-
tially different values for the replacement of the nitrogen minimum.

The purposes of the protein as foodstuff are, according to RUBNER, as
follows: (1) To compensate for the wear and tear quota; (2) betterment
of the condition of the cells; and (3) dynamogenic purpose. In the
accomplishment of this third purpose the protein splits into a nitrogenous

1 Rubner, Theorie d. Ernahrung nach Vollendung des Wachstums, Arch, f . Hyg.,
66, 1-80, and Ernahmngsorgange beim Wachstum des Kindes, ibid., 66, 81-126.

2 Zeitschr. f. physiol. Chem., 59.

3 v. Hoesslin and Lesser, 1. c., E. Voit and Listerer, Zeitschr. f. Biol., 53; Thomas,
Arch. f. (Anat. u.) Physiol., 1909.

918

METABOLISM.

and a non-nitrogenous part. The potential energy set free immediately
as heat in the combustion of the nitrogenous part, which is quantitatively
used within the region of the chemical heat regulation but is otherwise
lost, has been called the specific dynamic action by RuBNER.1 The
remainder of the energy which is represented by the non-nitrogenous
part of the proteins, serves, like all other foodstuffs, in satisfying the
energy requirement of the cells. According to RUBNER only non-nitrog-
enous groups (of the proteins, fats and carbohydrates) come almost
entirely, if not completely, in consideration for purposes of energy.

In close relation to the second purpose, the betterment of the condi-
tion of the cells, stands the question as to the conditions favoring the deposi-
tion of flesh in the body, which is closely associated with the question as
to the conditions of fattening the body. In this connection it must be
remembered in the first place that all fattening presupposes an overfeed-
ing, i.e., a supply of foodstuffs which is greater than that catabolized in
the same time.

In carnivora a flesh deposition may take place on the exclusive feeding
with meat. This is not generally large in proportion to the quantity of
protein catabolized. In man and herbivora, who cannot cover their
calorific needs by protein alone, this is not possible, and the question as
to the conditions of fattening with a mixed diet is of importance.

These conditions have also been studied in carnivora, and here, as
VOIT has shown, the relation between protein and fat (and carbo-
hydrates) is of great importance. If much fat is given in proportion
to the protein of the food, as with average quantities of meat with con-
siderable addition of fat, then nitrogenous equilibrium is but slowly
attained and the daily deposit of flesh, though not large, is quite constant,
and may become greater in the course of time. If, on the contrary,
much meat besides proportionately little fat is given, then the deposit
of protein with increased catabolism is smaller day by day, and nitrog-
enous equilibrium is attained in a few days. In spite of the somewhat
larger deposit per diem, the total flesh deposit is not considerable in
these cases. The following experiment of VOIT may serve as example:

Number of
Days of Ex-
perimentation.

Food.

Total
Deposit of
Flesh.

Daily
Deposit of
Flesh.

Nitrogenous
Equilibrium.

Meat, Gram3.

Fat, Grama.

32

500

250

1792

56

Not attained

7

1800

250

854

122

Attained

The greatest absolute deposition of flesh in the body was obtained in
these cases with only 500 grams of meat and 250 grams of fat, and even

1 Rubner, 1. c., and Gesetze des Energieverbrauches, 70.

PROTEIN FATTENING. 919

after 32 days nitrogenous equilibrium had not occurred. On feeding
with 1800 grams of meat and 250 grams of fat nitrogenous equilibrium
was established after seven days; and though the deposition of flesh
per day was greater, still the absolute deposit was not one-half as great
as in the former case.

The possibility of a protein fattening in man and animals (dogs, sheep)
is shown by the series of experiments of KRUG, BORNSTEIN, SCHREUER,
HENNEBERG, PFEIFFER and KALB and others l and there is no doubt
that such a fattening is possible. That we are here not dealing with an
increase in the number of cells, but rather an enlargement of the volume
of the same is the generally accepted view. Theories as to the value and
nature of this protein-fattening are still divergent, as we must differentiate
between flesh accumulation or actual organ formation and protein
accumulation or deposition of dead protein, and opinions vary in
regard to the question how far the one or the other of these occur.
By determining the relation between P2Os and N in muscles, kidneys
and liver in dogs and hens in starvation and in fattening, GRUND 2 has
tested this possibility experimentally. If we are dealing with the deposi-
tion of dead protein then the relationship of the P2O5 to the N would
change in favor of the nitrogen; GRUND found only a very slight change
of this kind, which was not conclusive, and according to him the various
organs have correspondingly a certain tendency of maintaining the rela-
tion between phosphorus and nitrogen unchanged in starvation as well
as in fattening.

It is difficult to produce a permanent flesh deposit in adult man by
overfeeding alone. It is to a much greater degree a function of the specific
growth energy of the cells and the cell-work than the excess of food.
Therefore there is observed, according to v. NOORDEN, abundant flesh
deposition (1) in each growing body; (2) in those no longer growing, but
whose body is accustomed to increased work; (3) whenever, by previous
insufficient food or by disease, the flesh condition of the body has been
diminished and therefore requires abundant food to replace it. The
deposition of flesh is in this case an expression of the regenerative energy
of the cells.3

The experiences of graziers show that in food-animals a flesh deposit
does not occur, or at least is only inconsiderable, on overfeeding. The

1 Krug, Cited by v. Noorden, Lehrb. der Path, des Stoffwechsel, 1. Aufl., p. 120;
Bornstein, Berl. klin. Wochenschr., 1898, and Pfliiger's Arch., 83 and 106; Bornstein
and Schreuer, Pfliiger's Arch., 110; Henneberg and Pfeiffer, see Maly's Jahresb.,
20; Pfeiffer and Kalb, ibid., 22.

2 G. Grund, Zeitschr. f. Biol., 54.

8 See also Svenson, Zeitschr. f. klin. Med., 43.

920 METABOLISM.

individuality and the race of the animal are of importance for flesh deposi-
tion.

The conditions in young, growing individuals differ from those in
adults. In the first the protein is necessary for the building up of the
growing tissue and in them an abundant true flesh deposition takes place.
For this protein fattening the amount of supply does not take first place,
but rather the energy of development.

As above stated (Chapter IX), in regard to the formation of fat in the
animal body, the most essential condition for a fat deposition is an over-
feeding with non-nitrogenous foods. The extent of fat deposition is
determined by the excess of calories administered over those actually
needed. But as protein and fat are expensive nutritive bodies as com-
pared with carbohydrates, the supply of greater quantities of carbo-
hydrates is important for fat deposition. The body decomposes less
substances at rest than during activity. Bodily rest, besides a proper
combination of the three chief groups of organic foods, is therefore
also an essential requisite for an abundant fat deposit.

E. GRAFE and D. GRAHAM 1 report an experiment on a dog in which they were
able to keep the body weight nearly constant for about two months by excessive
food with about 210 per cent of the minimum need of calories and with a diet
very rich in non-nitrogenous food-stuffs. No fattening occurred in this case;
the calories produced were considerably increased and the author considers this
case as an accommodation to the food and a luxus-consumption of non-nitrogenous
food-stuffs.

Action of Certain Other Bodies on Metabolism. Water. If a quan-
tity in excess of that which is necessary, is introduced into the organism,
the excess is quickly and principally eliminated with the urine. This
increased elimination of urine causes in fasting animals (Vorr, FORSTER),
but not to any appreciable degree in animals taking food (SEEGEN, SAL-
KOWSKI and MUNK, MAYER, DuBELiR2), an increased elimination of
nitrogen. The reason for this increased nitrogen excretion is to be found
in the fact that the drinking of much water causes a complete washing
out of the urea from the tissues. Another view, which is defended by
VOIT, is that because of the more active current of fluids, after taking
large quantities of water, an increased metabolism of proteins takes place.
VOIT considers this explanation the correct one, although he does not
deny that by the liberal administration of water a more complete washing
out of the urea from the tissues takes place. Opinions on this subject
are not yet in accord, and recently HEILNER has advocated VOIT'S

1 Zeitschr. f . physiol. Chem., 73.

2 Voit, Untersuch. iiber den Einfluss des Kochsalzes, etc. (Miinchen, 1860); Forster,
cited from Voit in Hermann's Handbuch, 6, 153; Seegen, Wien. Sitzungsber., 63;
Salkowski and Munk, Virchow's Arch., 71; Mayer, Zeitschr. f. klin. Med., 2; Dubelir,
Zeitschr. f . Biologic, 28.

ACTION OF SALTS AND ALCOHOL UPON METABOLISM. 921

view. The recent investigations of ABDEEHALDEN 1 show a washing
out of the retained nitrogen by the partaking of water.

We have the thorough investigations of HAWK2 and his co-workers
on the action of drinking of water upon the digestion and absorption of
foods as well as upon the putrefaction processes in the intestine and the
elimination of allantoin and purine bodies in the urine.

When the body has lost a certain amount of water, then the abstinence
from water (in animals) is accompanied by a rise in the protein metabo-
lism (LANDAUER, STRAUB 3). In regard to the action of water on the
formation of fat and its metabolism, the theory that the free drinking
of water is favorable for the deposition of fat seems to be generally
admitted, while the drinking of only very little water acts against its
formation. For the present we have no conclusive proofs of the correct-
ness of this view. i

Salts. In regard to the action of salts — for example sodium chloride
and the neutral salts — which partly depends upon the use of large and
varying amounts of salt in the experiments, the authors disagree. Inves-
tigations of STRAFB and RosT4 show that the action of salts stands in
close relation to their power of abstracting water. Small amounts of
salt which do not produce diuresis have no action on metabolism. On
the contrary, larger amounts, which bring about a diuresis, which is
not compensated by the ingestion of water, produce a rise in the pro-
tein metabolism. If the diuresis is compensated by drinking water,
then the protein metabolism is not increased by salts, but is diminished
to a slight degree. An increased nitrogen excretion caused by taking
salts can be increased by the ingestion of water, thus increasing the
diuresis, and the action of salts seems to bear a close relation to the
demand and supply of water.

Alcohol. The question as to how far the alcohol absorbed in the
intestinal canal is burnt in the body, or whether it leaves the body
unchanged by various channels, has been the subject of much discussion.
To all appearances the greatest part of the alcohol introduced (95 per
cent or more) is burnt in the body (STUBBOTIN, THUDICHUM, BODLANDER, !
BENEDICENTI 5) . As the alcohol has a high calorific value (1 gram = 7.1

1 See R. Neumann, Arch. f. Hygiene, 36; Heilner, Zeitschr. f. Biologic, 47 and 49;
Hawk, University of Pennsylvania Med. Bull., xviii; Abderhalden, Zeitschr. f. physiol.
Chem., 59.

2 See Journ. of biol. Chem., 10 and 11, Arch, of internat. Med., 1911, Journ. of
Amer. Chem. Soc., 33 and 34.

3 Landauer, Maly's Jahresber., 24; Straub, Zeitschr. f. Biologic, 37.

4W. Straub, Zeitschr. f. Biologic, 37 and 38; Rost, Arbeiten aus d. Kaiserliche
Gesundheitsamte, 18 (literature). See also Griiber, Maly's Jahresber., 30, 612.
6 Arch. f. (Anat. u.) Physiol., 1896, which contains the literature.

922 METABOLISM.

calories), then the question arises whether it acts sparingly on other
bodies, and whether it is to be considered as a nutritive substance. The
earlier investigations made to decide these questions have led to no
decisive result. The thorough investigations of ATWATER and BENEDICT,
ZUNTZ and GEPPERT, BJERRE, CLOPATT, NEUMANN, OFFER, ROSEMANN/
and others, seem to show positively that, in man, alcohol can diminish the
consumption not only of fat and carbohydrates, but also the proteins,
although at first, due to its poisonous properties, it may increase the pro-
tein metabolism for a short time. The nutritive value of alcohol can
be of special importance in certain cases only, as large amounts of alcohol
taken at one time, or the continued use of smaller quantities, has an injur-
ious action on the organism. Alcohol may therefore be regarded as a
foodstuff only in exceptional cases, and in other respects must be con-
sidered as an article of luxury.

Coffee and tea have no action on the exchange of material which can
be positively proven, and their importance lies chiefly in their action
upon the nervous system. It is impossible to enter into the effect of
various therapeutic agents upon metabolism.

IV. THE DEPENDENCE OF METABOLISM ON OTHER CONDITIONS.

The so-called basal requirement which was previously mentioned,
i.e., the extent of metabolism with absolute rest of body and inactivity
of the intestinal tract, serves best as a starting-point for the study of
metabolism under various external circumstances. The metabolism
going on under these conditions leads in the first place to the production
of heat, and it is only to a subordinate degree dependent upon the work
of the circulatory and respiratory apparatus and the activity of the glands.
According to a calculation by ZuNTz,2 only 10-20 per cent of the total
calories of the basal requirement belongs to the circulation and respira-
tion work.

The magnitude of the basal requirement depends in the first place
upon the heat production necessary to cover the loss of heat, and this
heat production is in turn dependent upon the relation between the weight
and the surface of the body.

Weight of Body and Age. The greater the mass of the body the greater
the absolute consumption of material; while, on the contrary, other

1 In regard to the literature on this subject, see the works of O. Neumann, Arch,
f. Hygiene, 36 and 41, and Rosemann, Pfliiger's Arch., 86 and 94. A summary of
the entire literature upon alcohol can be found in Abderhalden, " Bibliographic der
gesamten wissenschaftlichen Literatur tiber den Alcohol und den Alcoholismus,"
Berlin and Wien, 1904. See also Rosemann in Oppenheimer's Handb. d. Bioch., Bd.
4,1.

2 Cited from v. Noorden's Handbuch, 1. Aufl., page 97.

DEPENDENCE OF WEIGHT OF BODY AND AGE. 923

things being equal, a small individual of the same species of animal metab-
olizes absolutely less, but relatively more as compared with the unit of
the weight of the body. With increasing bodily weight the total metab-
olism per kilo of animal diminishes, which is true first for individuals of
the same species of animals, but also seems to have a certain correctness
on the comparison of different species of animals. It must be remarked
that the relation between flesh and fat in the body exerts an important
influence. The extent of the metabolism is dependent upon the quantity
of active cells, and a very fat individual therefore decomposes less sub-
stance per kilo than a lean person of the same weight. According to
RUBNER 1 the importance of the size of the flesh or cell-mass in the body
is overestimated. In his investigations on two boys, one of whom was
corpulent and the other normally developed, and on comparing the food-
need with that found by CAMERER for boys of the same weight, RUBNER
came to theVesult that the exchange of force in the corpulent boy almost
completely corresponded with that in the non-corpulent boy of the same
weight. By approximately estimating the quantity of fat in the body
RUBNER was also able, from the protein condition, to compare the cal-
culated exchange of energy with that actually found. The exchange
per kilo amounted to 52 calories in the lean and 43.6 calories in the fat
boy, while, if the protein condition was a measure, one would expect
an exchange of calories of only 35 calories for the fat person. We can-
not therefore admit of a diminished activity of the cell-mass in the fat boy,
but rather an increased activity. According to RUBNER it is not the
flesh -mass (protein mass) alone, but its variable functional changes,
which determine the extent of decomposition. In women, who generally
have less body weight and a greater quantity of fat than men, the metab-
olism in general is smaller, and the latter is ^ordinarily about four-fifths
that of men.

The essential reason why small animals catabolize relatively more
substance than large ones, when calculated per kilo body weight, is that
the bodies of smaller animals have greater surface in proportion to their
mass. On this account the loss of heat is greater, which causes increased
heat production, i.e., a more active metabolism. This is also the reason
why young individuals of the same kind show a relatively greater metab-
olism than older ones. If the heat production and carbon-dioxide elim-
ination is calculated on the unit of surface of the body, we find, on the
contrary, as the experiments of RUBNER, RicHET,2 and others show,
that they vary only slightly from a certain average in individuals of
different weights.

1 Beitrage zur Ernahrung im Knabenalter, etc. Berlin, 1902.

2 Rubner, Zeitschr. f. Biologic, 19 and 21; Richet, Arch, de Physiol., 5 (2).

924 METABOLISM.

According to RUBNER'S rule as to the influence of the surface, which
has been recently formulated by E. VOIT, the need of energy in homceo-
thermic animals is influenced by the development of their surface when
their body is given rest, medium surrounding temperature, and relatively
equal protein condition. This rule applies not only to adult human beings,
but also to children and growing individuals (RUBNER, OPPENHEIMER,
SCHLOSSMANN and MURSCHHAUSER). The surface is the essential factor
in determining the extent of exchange of energy. In order to show
this we will give here, from a work of RUBNER/ the figures representing
the quantity of heat in calories for 1 square meter of surface for twenty-
four hours:

Adult, medium diet, rest 1189 calories.

Adult, medium diex, work 1399 ' '

Suckling 1221 "

Child with medium diet 1447

Aged men and women 1099 ' '

Women 1004 "

The variation in the calorific values2 found by many investigators,
which is sometimes not very small, suggests the fact that the surface
rule is not alone decisive for the exchange of material in resting animals.
Still it is generally considered that it is of the greatest importance in
metabolism.

The more active metabolism in young individuals is apparent when
we measure the gaseous exchange as well as the excretion of nitrogen.
As example of the elimination of urea in children the following results of
CAMERER 3 are of value :

Age. Weight of Body Urea in Grams.

in Kilos. Per Day. Per Kilo.

liyears 10.80 12.10 1.35

3

5

7

9

12

15

13.30 11.10 0.90

16.20 12.37 0.76

18.80 14.05 0.75

25.10 17.27 0.69

32.60 17.79 0.54

35.70 17.78 0.50

In adults weighing about 70 kilos, from 30 to 35 grams of urea per day
are eliminated, or 0.5 gram per kilo. At about fifteen years of age the
destruction of proteins per kilo is about the same as in adults. The
relatively greater metabolism of proteins in young individuals is explained
partly by the fact that the metabolism of material in general is more
active in young animals, and partly by the fact that young animals are,
as a rule, poorer in fat than those full grown.

1 Rubner, Ernahrung im Knabenalter, page 45; E. Voit, Zeitschr. f. Biologic, 41;
Oppenheimer, ibid., 42; Schlossmann and Murschhauser, Bioch. Zeitschr., 18 and 26.
J See Magnus-Levy, Pfluger's Arch., 55; Slowtzoff (u. Zuntz), ibid., 95.
1 Zeitschr. f . Biologic, 16 and 20.

SEX. REST AND WORK. 925

That young individuals show a more active metabolism than adults,
follows, as above stated, principally from the relatively greater body
surface in the first as compared to the latter. According to TIGERSTEDT
and SONDEN, the greater metabolism in young animals depends neverthe-
less, also in part, on the fact that in these individuals the decomposition
in itself is more active than in older ones. The period of growth has a
considerable influence on the extent of metabolism (in man), and indeed
the metabolism, even when calculated on the unit of surface of body, is
greater in youth than in old age. This view is strongly disputed by
RUBNER. He does not deny that differences exist between young and
adult individuals which may be considered as a deviation from the above
rule; still these differences may, he claims, be dependent upon the work
performed, the food, and the nutritive condition. MAGNUS-LEVY and
FALK1 have reported observations which support the conclusions of
SONDEN and TIGERSTEDT.

Nurslings have a behavior different from older children, as with them
during the first months of life, and especially the first three days, the
metabolism, calculated on the unit of surface, is strikingly low, and
lower than with adults. After about two weeks it attains about the same
height as adults (SCHERER, FoRSTER2).

In old age the metabolism is very much reduced; and even when calcu-
lated upon the square meter of surface of body it is lower than in an*
individual of medium age.

The question as to what extent sex specially influences metabolism
remains to be investigated. TIGERSTEDT and SONDEN found that in 'the
young the carbon-dioxide elimination, per kilo of body weight, as well as
per square meter of body surface, was considerably greater in males
than in females of the same age and the same weight of body. This
difference between the sexes seems to disappear gradually, and at old
age it is entirely absent. The investigations of .MAGNUS-LEVY and
FALK oppose these observations. They investigated by means of the
ZUNTZ-GEPPERT method, not only children, but also adults and old
persons of both sexes, but could not observe any positive influence of
the sex upon metabolism.3

irFigerstedt and Sonden, Skand. Arch. f. Physiol. 6; Rubner, 1. c.; and Arch. f.
Hygiene, 66; Magnus-Levy, Arch. f. (Anat. u.) Physiol., 1899, Suppl.

2 Cited by A. Loewy in Oppenheimer's Handb., Bd. 4, 189.

•Tigerstedt and Sonden, Skand. Arch. f. Physiol., 6; Magnus-Levy and Falk,
Arch. f. (Anat. u.) Physiol., 1899, Suppl. In regard to metabolism and its relation
to the phases of sexual life and especially under the influence of menstruation and
pregnancy, see the investigations of A. Ver Eecke (Bull. acad. roy. de me"d. de Bel-
gique, 1897 and 1901, and Maly's Jahresber., 30 and 31). See also Magnus-Levy
in-v. Noorden's Handb. d. Pathol. d. Stoffwechsels.

926 METABOLISM.

As the metabolism may be kept at its lowest point by absolute rest
of body and inactivity of the intestinal tract, it is manifest that work
and the ingestion of food have an important bearing on the extent of
metabolism.

Rest and Work. During work a greater quantity of chemical energy
is converted into kinetic energy, i.e., the metabolism is increased more or
less on account of work.

As explained in a previous chapter (X), work, according to the gen-
erally accepted view, has no material influence on the excretion of nitro-
gen. It is nevertheless true that several investigators have observed,
in certain cases, an increased elimination of nitrogen; this increase does
not seem to be directly related to the work, but to be caused by secondary
circumstances. These observations have been explained in other ways.
For instance, work may, when it is connected with violent movements
of the body, easily cause dyspnoea, and this last, as FRANKEL l has shown,
may occasion an increase in the elimination of nitrogen, since diminution
of the oxygen supply increases the protein metabolism. In other series
of experiments the quantity of carbohydrates and fats in the food was
not sufficient; the supply of fat in the body was decreased thereby, and
the destruction of proteins was correspondingly increased. Other condi-
tions, such as the external temperature and the weather,2 thirst, and
drinking of water, can also influence the excretion of nitrogen. The
prevailing sentiment is that muscular activity has hardly any influence
on the metabolism of proteins.

•On the contrary, work has a very considerable influence on the elimi-
nation of carbon dioxide and the consumption of oxygen. This action,
which was first observed by LAVOISIER, has later been confirmed by
many investigators. PETTENKOFER and VOIT 3 have made investigations
tions on a full-grown man as to the metabolism of the nitrogenous as well
as of the non-nitrogenous bodies during rest and work, partly while
fasting and partly on a mixed diet. The experiments were made on a
full-grown man weighing 70 kilos. The results are contained in the
following table :

Consumption of

Proteins.

Fat. Carbohydrates.

C02 Eliminated.

O Consumed.

Fasting . . .

/ Rest . . .
1 Work...

79
. 75

209
380

716

1187

761
1071

Mixed diet .

/Rest
\ Work . . .

.137
.137

72
173

352
352

912
1209

831
980

1 Virchow's Arch., 67 and 71. In regard to disputed views see C. Voit, Zeitschr.
f. Biol., 49, and Frankel, ibid., SO.

8 See Zuntz and Schumburg, Arch. f. (Anat. u.) Physiol., 1895.
» Zeitschr. f . Biologic, 2.

WORK AND GAS EXCHANGE. 927

In these cases work did not seem to have any influence on the destruc-
tion of proteins, while the gas exchange was considerably increased.

ZUNTZ and his pupils l have made important investigations on the
extent of the exchange of gas as a measure of metabolism during work
and caused by work. These investigations not only show the impor-
tant influence of muscular work on the catabolism of material, but they
also indicate, in a very instructive way, the relation between the extent
of metabolism of material and its utilization for work of various kinds.
We can refer only to those which are of special physiological interest.

The action of muscular work on the gas exchange does not alone appear
with hard work. From the researches of SPECK and others we learn that
even very small, apparently quite unessential movements may increase
the production of carbon dioxide to such an extent that by not observing
these, as in numerous older experiments, very considerable errors may
creep in. JOHANSSON 2 has also made experiments upon himself, and
finds that on the production of as complete a muscular inactivity as
possible the ordinary amount of carbon dioxide (31.2 grams per hour
at rest in the ordinary sense) may be reduced nearly one-third, or to an
average of 22 grams per hour.

The quantity of carbon dioxide eliminated during a working period
is uniformly greater than the quantity of oxygen taken up at the same
time, and hence a raising of the respiratory quotient was usually con-
sidered as caused by work. This rise does not seem to be based upon the
character of the chemical processes going on during work, as we have a
series of experiments made by ZUNTZ and his collaborators, LEHMANN, KA'T-
ZENSTEIN and HAGEMANN,S in which the respiratory quotient remained
almost wholly unchanged in spite of work. According to LoEWY4 the
combustion processes in the animal body go on in the same way in work
as in rest, and a raising of the respiratory quotient (irrespective of the
transient change in the respiratory mechanism) takes place only with
insufficient supply of oxygen to the muscles, as in continuous fatiguing
work or excessive muscular activity for a brief period, also with local
lack of oxygen caused by excessive work of certain groups of muscles.
This varying condition of the respiratory quotient has been explained by

1 See the works of Zuntz and Lehmann, Maly's Jahresber., 19; Katzenstein, Pfliiger's
Arch., 49; Loewy, ibid.; Zuntz, ibid., 68; Zuntz and Slowtzoff, ibid., 95; and especially
the large work "Untersuch. iiber den Stoffwechsel des Pferdes bei Ruhe und Arbeit,"
Zuntz and Hagemann, Berlin, 1898; Hohenklima und Bergwanderungen by Zuntz,
Loewy, Miiller and Caspari, which also contains a bibliography.

2Nord. Med. Arkiv. Festband, 1897; also Maly's Jahresber., 27; Speck, "Physiol.
des menschl. Atmens," Leipzig, 1892.

3 See footnote 1.

4 Pfliiger's Arch., 49.

928 METABOLISM.

KATZENSTEIN by the statement that during work two kinds of chemical
processes act side by side. The one depends upon the work which is
connected with the production of carbon dioxide, also in the absence
of free oxygen, while the other brings about the regeneration which takes
place by the taking up of oxygen. When these two chief kinds of chemical
processes make the same progress the respiratory quotient remains
unchanged during work; if by hard work the decomposition is increased
as compared with the regeneration, then a raising of the respiratory
quotient takes place. If, on the contrary, moderate work is continued
and performed in a way so that irregularities and occasional changes
in the circulation and respiration are excluded or are without importance,
then the respiratory quotient may correspondingly remain the same
during work as in rest. Its extent is thus determined in the first place
by the nutritive material at its disposal (ZUNTZ and his pupils) .

The4 theory of -LOEWY and ZUNTZ, that the raising of the respiratory quotient
during work is to be explained by an insufficient supply of oxygen, is opposed
by LAULANIE.! He has observed the reverse, namely, a diminution in the
respiratory quotient during continuous excessive work, and this is not reconcilable
with the above statements. He considers that sugar is the source of muscular
energy, and that the rise in the respiratory quotient is due to an increased combus-
tion of sugar. Its diminution, he explains, is caused by a re-formation of sugar
from fat which takes place at the same time and is accompanied by an increased
consumption of oxygen.

* In sleep metabolism decreases as compared with that during waking
hours, and the most essential reason for this is the muscular inactivity
during sleep. The investigations of RUBNER upon a dog, and of JOHANS-
SON 2 upon human beings, teach us that if the muscular work is elim-
inated the metabolism during waMng hours is not greater than in sleep.
The action of light also stands in close connection with the question
of the action of muscular work. It seems positively proven that metabo-
lism is increased under the influence of light. Most investigators, such as
SPECK, LOEB, and EWALD,S consider that this increase is due to the move-
ments caused by the light or an increased muscle tonus, and in man an
increase in metabolism under the influence of light with complete rest
has not been observed. Divergent results have been obtained in animals,
and our knowledge of the truth is not yet complete.4

Mental activity does not seem to have any influence on metabolism
according to the means at hand for studying this influence.

i Arch, de Physiol. (5), 8, 572.

'Rubner, Ludwig-Festschr., 1887; Loewy, Berl. klin. Wochenschr., 1891, 434;
Johansson, Skand. Arch. f. Physiol., 8.

'Speck, 1. c.; Loeb, Pfliiger's Arch., 42; Ewald, Journ. of Physiol., 13.
4 See larger handbooks for the literature on this question.

HEAT REGULATION IN ANIMALS. 929

The Action of the External Temperature also stands in close relation
to muscular work, namely to the question as to whether the chemical
heat regulation is independent of the muscular activity. .The heat
regulation, as is well known, is of two kinds, namely the chemical heat
regulation, which consists in a change in the metabolism and which man-
ifests itself as an increased heat production due to the increased metabo-
lism at low temperatures, and the physical heat regulation, which occurs
generally at higher temperatures and is caused by changes in the con-
ditions in the heat elimination of the thermal equilibrium.

In regard to the chemical heat regulation, which will only be discussed
here, we must differentiate between cold-blooded and warm-blooded
animals. In the first the metabolism rises with an increase in the surround-
ing temperature, while in the second group the conditions are different.
The experiments of SPECK, LOEWY and JOHANSSON 1 on human beings
have shown that the lowering of the external temperature is without
influence upon the extent of metabolism (measured by the gas exchange)
only as long as all natural and non- voluntary movements of the muscles
are excluded; otherwise the metabolism is raised. A chemical heat regu-
lation, i.e., a rise in metabolism without noticeable movements of the
muscles, is not accepted in man, or at least it has not been proven. The
heat regulation, in man, at lower temperatures seems to be brought about
by the natural or reflex production of muscle action, nor has a chemical
heat regulation in the reverse sense, namely, a fall in the catabolism by
raising the external temperature, been shown in man. The investigations
of EYKMAN 2 upon inhabitants of the tropics also show the same result,
namely, that in human beings no appreciable chemical heat regulation
occurs.

In animals the conditions are different so far as that a chemical heat
regulation in the true sense has been positively shown. The investiga-
tions of RuBNER3 on various animals have shown that the reduction
of the external temperature with these, causes a considerable chemical
heat regulation by increasing the metabolism without any chill or shiver
movements. On sufficient cooling the temperature of the body may fall
irrespective of the increased metabolism, and at a certain limit of body
temperature the exchange of material becomes still lower with decreasing
temperature. According to RUBNER many animals can bear a temperature
of 0° C. for days in absolute rest. If the natural muscular activity is
eliminated by poisoning with curare or by section of the spinal cord, then,
as shown by PFLUGER 4 and his pupils, the warm-blooded animal behaves

1 Speck, 1. c.; Loewy, Pfluger's Arch., 46; Johansson, Skand. Arch. f. Physiol., 7.

2 Virchow's Arch., 133, and Pfluger's Arch., 64.

8 Arch. f. Hyg., 37, and Handbuch d. Hyg., Bd. 1, Leipzig, 1911.
4 See footnote 2, page 591.

930 METABOLISM.

like a cold-blooded animal, and the metabolism decreases parallel with
the body temperature. In normal animals, on the contrary, the body
temperature can be kept constant, on lowering the external temperature,
by an increased metabolism; but also in such animals because of a rise
in the external temperature a rise in the metabolism above a certain
limit can also take place.

A very interesting and important question is the action of high altitude
upon the oxidation processes, the economy of temperature, the protein
exchange and the general metabolism. The results of the laborious
and important investigations on this subject may be found in the large
work of N. ZUNTZ, A. LOEWY, F. MULLER and W. CASPAR!.1

That the ingestion of food raises the metabolism has been known for
a rather long time, and this has been studied by ZUNTZ, v. MERING, MAG-
NUS-LEVY, VOIT, RUBNER, JOHANSSON and collaborators, also by HEILNER
and by GiGON.2 It follows from these investigations that this rise in
metabolism, which in man, on sufficient supply of food, amounts to a rise
of 10-15 per cent of the basal requirement and with abundant supply of
food may be still larger (35 per cent in the researches of JOHANSSON,
TIGERSTEDT and collaborators), has a double cause, namely, partly a
digestion work (ZUNTZ) and partly a chemical decomposition (specific
dynamic action of RUBNER) which takes place at the same time.

The sum of all the work which is necessary for the chemical trans-
formation of the foods, as well as for the mechanical division and trans-
portation of the food in the intestinal canal, is called the digestion work by
ZUNTZ. That such work exists has been shown by ZUNTZ and v. MERING
by comparative tests of the different action upon metabolism by
foods introduced per os and intravenously, and recently CoHNHEiM3
has shown that in sham feeding an increased catabolism of non-
nitrogenous body constituents took place. The influence of digestion
work in ZUNTZ 's sense is especially apparent in herbivora, in which this
work, according to ZUNTZ and collaborators, may amount to the consump-
tion of more than 50 per cent of the total energy content of the raw
fodder.

1 Hohenklima und Bergwanderungen in ihrer Wirkung auf den Menschen, Berlin,
1906.

2 Zuntz and v. Mering, Pfluger's Arch., 15; Zuntz, Naturw. Rundschau, 21 (1906),
with Hagemann, 1. c., with Magnus-Levy, Pfluger's Arch., 49; Magnus-Levy, ibid.,
55, and v. Noorden's Handbuch; Voit, Hermann's Handbuch, 6; Rubner, Zietschr.
f. Biol., 19 and 21; and Arch. f. Hyg., 66; Johansson, Skand. Arch. f. Physiol., 21,
with Koraen, ibid., 13; Heilner, Zeitschr. f. Biol., 48 and 50; Gigon, Pfluger's Arch.,
140.

3 Arch. f. Hyg., 57.

SPECIFIC DYNAMIC ACTION. 931

On partaking of large amounts of food, especially proteins, by car-
nivora, the digestion work in the above sense is not sufficient to account
for the increase in metabolism, and in these cases, besides this, we must
accept an increase in the chemical transformation process in the animal
body brought on by the foodstuffs in an unknown manner (specific
dynamic action of foodstuffs, according to RUBNER). The only real
difference in opinion between the various experimenters consists, so far
as HAMMARSTEN can see, in that according to the ZUNTZ school, normally
on supplying sufficient food it is the digestion work in the above sense
which chiefly causes the rise in metabolism after taking food, while
according to the views of VOIT-RUBNER, with which HEILNER agrees,
it is on the contrary the specific dynamic action.

That the proteins or their cleavage products, without regard to the
digestion work, cause a rise in the metabolism seems to be generally
accepted. This rise, according to GIGON, is not proportional to the
protein supply, as on supplying quantities of protein represented by
1: 2: 4: 3 the oxygen absorption was in the proportion 1:3:6:9 and the
carbon dioxide elimination was in the proportion 1:4:8:12. On the
introduction of glucose GIGON found, as first shown by JOHANSSON/
that the introduction of carbohydrate caused a proportional rise in the
carbon dioxide elimination to a maximal limit of 150 grams. The
conditions on supplying fat are harder to judge, but GIGON found no rise
in metabolism on introducing oil.

The rise in the gas exchange occurring after feeding protein and sugar
is added, according to GIGON, entirely to the basal metabolism. A
substitution in the basal metabolism of the catabolized body constituents
by the food taken does not take place according to GIGON and, as example,
the protein is not replaced from catabolism by the sugar introduced.
The isodynamic law does not apply to the metabolism occurring the
first few hours after supplying food, as shown by JOHANSSON and HELL-
GREN, and GIGON 2 believes that the foodstuffs first pass into the various
depots of the body to be later used for purposes of energy. Proteins
serve only to a slight degree to replace the catabolized body protein;
the remainder is stored up in part as glycogen and in part as fat. The
fat is deposited as such and the carbohydrates are deposited as glycogen
and fat.

As the three foodstuffs influence the metabolism in very different ways we
can, according to GIGON, speak of a specific action of the foodstuffs. This
action, according to him, is more of a material than of a dynamic kind, and the
expression, specific dynamic action, may lead to an erroneous conception.

1 Skand. Arch, f . Physiol., 21.

2 Johansson with Hellgren, Hammarsten's Festschrift, 1906; Gigon, Skand. Arch. f.
Physiol., 21.

932 METABOLISM.

V. THE NECESSITY OF FOOD BY MAN UNDER VARIOUS CONDITIONS.

Various attempts have been made to determine the daily quantity of
organic food needed by man. Certain investigators have calculated
from the total consumption of food by a large number of similarly fed
individuals — soldiers, sailors, laborers, etc. — the average quantity of
foodstuffs required per head. Others have calculated the daily demand
for food from the quantity of carbon and nitrogen in the excreta, or cal-
culated it from the exchange of force of the persons experimented upon.
Others, again, have calculated the quantity of nutritive material in a
diet by which an equilibrium was maintained in the individual for one or
several days between the consumption and the elimination of carbon
and nitrogen. Lastly, still others have quantitatively determined, dur-
ing a period of several days, the organic foodstuffs daily consumed by
persons of various occupations who chose their own food, by which they
were well nourished and rendered fully capable of work.

Among these methods a few are not quite free from objection, and
others have not as yet been tried on a sufficiently large scale. Neverthe-
less the experiments collected thus far serve, partly because of their
number and partly because the methods correct and control one another,
as a good starting-point in determining the diet of various classes and
similar questions.

If the quantity of foodstuffs taken daily be converted into calories
produced during physiological combustion, we then obtain some idea of
the sum of the chemical energy which under varying conditions is intro-
duced into the body. It must not be forgotten that the food is never
completely absorbed, and that undigested or unabsorbed residues are
always expelled from the body with the feces. The gross results of calo-
ries calculated from the food taken must therefore, according to RUBNER,
be diminished by at least 8 per cent. This figure is true at least when
the human being partakes of, a mixed diet of about 60 per cent of the
proteins as animal, and about 40 per cent of the proteins as vegetable
foodstuffs. With more one-sided vegetable food, especially when this
is rich in undigestible cellulose, a much larger quantity must be sub-
tracted.

The following summary contains a few examples of the quantity of
food which is consumed by individuals of various classes of people under
different conditions. In the last column we also find the quantity of
living force which corresponds to the quantity of food in question, calcu-
lated as calories, with the above-stated correction. The calories are
therefore net results, while the figures for the nutritive bodies are gross
results.

FOOD REQUIREMENT IN MAN.

933

Soldier during peace 119

Soldier light service 117

Soldier in field 146

Laborer 130

Laborer at rest 137

Cabinetmaker (40 years). 131

Young physician 127

Young physician 134

Laborer (36 years) 133

English smith 176

English pugilist 288

Bavarian wood-chopper. . 135

Laborer in Silesia 80

Seamstress in London ... 54

Swedish laborer 134

Japanese student 83

Japanese shopman 55

Proteins. Fat. "dJgJJ; Calories. Authority.

40

529

35

447

46

504

40

550

72

352

68

494

89

362

102

292

95

422

71

666

88

93

208

876

16

552

29

292

79

485

14

622

2784 PLAYFAIR.1

2424 HlLDESHEIM.

2852 HlLDESHEIM.

2903 MOLESCHOTT.

2458 PETTENKOFER and VOIT.

2835 FORSTER.*

2602 FORSTER.

2476 FORSTER.

2902 FORSTER.

3780 PLAYFAIR.

2189 PLAYFAIR.

5589 LIEBIG.

2518 MEINERT.3

1688 PLAYFAIR.

3019 HlTLTGREN and LANDERGREN.4

2779 EuKMAN.6

394 1744 TAWARA.*

We have a very large number of complete investigations upon the
diet of people of different vocations in America, but they are too exten-
sive to enter into, hence we must refer to the original publications of

ATWATER.6

It is evident that persons of essentially different weight of body
who live under unequal external conditions must need essentially dif-
ferent food. It is also to be expected (and this is confirmed by the table)
that not only the absolute quantity of food consumed by various persons,
but also the relative proportion of the various organic nutritive substances,
shows considerable variation. Results for the daily need x>f human
beings in general cannot be given. For certain classes, such as soldiers,
laborers, etc., results may be given which are valuable for the calculation
of the daily rations.

Based on extensive investigations and a very wide experience, VOIT
has proposed the following average quantities for the daily diet of adults:

For men .

Proteins.

118 grams

Fat.

56 grams

Carbohydrates.

500 grams

Calories.

2810

But it should be remarked that these data relate to a man weighing
70 to 75 kilos and who was engaged daily for ten hours in not too fatiguing
labor.

The quantity of food required by a woman engaged in moderate work

1 In regard to the earlier researches cited in this table, we refer the reader to Voit,
in Hermann's Handbuch, 6, 519.

2 Ibid,, and Zeitschr. f . Biologic, 9.

3 Armee- und Volksernahrung, Berlin, 1880.

4 Untersuching iiber die Ernahrung schwedischer Arbeiter bei frei gewahlter Kost,
Stockholm, 1891. Maly's Jahresber., 21.

5 Cited from Kellner and Mori in Zeitschr. f . Biologic, 25.

6 Report of the Storrs Agric. Expt. Station, Conn., 1891-1895, and 1896, and U. S.
Depart, of Agriculture, Bull. 53, 1898.

934 METABOLISM.

is about four-fifths that of a laboring man, and we may consider the
following as a daily diet with moderate work :

Proteins. Fat. Carbohydrates. Calories.

For women 94 grams 45 grams 400 grams 2240

The proportion of fat to carbohydrates is here as 1:8-9. Such a
proportion often occurs in the. food of the poorer classes who chiefly live
upon the cheap and voluminous vegetable food, while this ratio in the
food of wealthier persons is 1 :3-4. It would be desirable if in the above
rations the fat were increased at the expense of the carbohydrates, but
unfortunately on account of the high price of fat such a modification
cannot always be made.

In examining the above figures for the daily rations it must not
be forgotten that those for the various foodstuffs are gross results.
They consequently represent the quantity of those which must be taken
in, and not those which are really absorbed. The figures for the calories
are, on the contrary, net results.

The various foods are, as is well known, not equally digested and
absorbed, and in general the vegetable foods are less completely consumed
than animal foods. This is especially true of the proteins. When,
therefore, VOIT, as above stated, calculates the daily quantity of pro-
teins needed by a laborer as 118 grams, he starts with the supposition
that the diet is a mixed animal and vegetable one, and also that of the
above 118 grams about 105 grams are absorbed. The results obtained
by PFLUGER and his pupils BOHLAND and BLEIBTREU 1 on the extent of
the metabolism of proteins in man with an optional and sufficient diet
correspond well with the above figures, when the unequal weight of body
of the various persons experimented upon is sufficiently considered.

As a rule, the more exclusively a vegetable food is employed, the
smaller is the quantity of proteins in it. The strictly vegetable diet
of certain people, as that of the Japanese and of the so-called vegeta-
rians, is therefore a proof that, if the quantity of food be sufficient, a
person may exist on considerably smaller quantities of proteins than
VOIT suggests. It follows from the investigations of HIRSCHFELD, KUMA-
GAWA and KLEMPERER, SIVEN, and others (see pages 903, 915) that an
almost complete or indeed a complete nitrogenous equilibrium may be
attained by the sufficient administration of non-nitrogeneous nutritive
bodies with relatively very small quantities of proteins.

If we bear in mind that the food of people of different countries
varies greatly, and that the individual also takes essentially different
nourishment according to the external conditions of living and the influence
of climate, it is not remarkable that a person accustomed to a mixed

1 Bohland, Pfluger's Arch., 36; Bleibtreu, ibid., 38.

PROTEIN REQUIREMENT. 935

diet can exist for some time on a strictly vegetable diet deficient in pro-
teins. No one doubts the ability of man to adapt himself to a heteroge-
neously composed diet when this is not too difficult of digestion and is
sufficient in quantity; nor can we deny that it is possible for a man
to exist for a long time with smaller amounts of protein than VOIT
suggests, namely 118 grams. Thus O. NEUMANN 1 experimented on him-
self during 746 days in three series of experiments, and his diet consisted
of 74.2 grams protein, 117 grams fat, and 213 grams carbohydrates = 2367
gross calories, with a weight of 70 kilos and with ordinary laboratory
work. These figures cannot be compared with those obtained by VOIT'S
worker, weighing 70 kilos, whose work was harder than a tailor's and
easier than a blacksmith's; for example, the work of a mason, carpenter,
or cabinet-maker. The very extensive investigations recently performed
by CHITTENDEN 2 on the determination of the extent of protein necessary
are of great interest. These investigations, upon a total of twenty-
six persons, extended over a period of five to twenty months, and con-
sisted of careful observations upon the manner of living, food taken,
nitrogen elimination, and the ability of performing work. The individuals
were divided into three groups. The first consisted of five professional
men (four assistants and one professor) . The second group was composed
of thirteen soldiers (of the sanitary corps of the United States army) who
besides their daily work were given gymnastic exercises for six months.
The third group consisted of eight athletic students who were trained in
different kinds of sport.

In all the persons experimented upon the original nitrogen content
of the food, which corresponded to VOIT'S value or were somewhat higher,
was gradually reduced more or less. The total calories supplied were
not increased above the original value, but rather diminished to a reason-
able extent. The bodily as well as the mental ability was repeatedly
tested. As it is not possible to enter into the details of the investiga-
tion the following will be sufficient to show the results. With a diet
corresponding to VOIT'S values the amount of urine nitrogen per day is
16 grams, corresponding to a total protein catabolism in the body of 100
grams, or 1.43 grams per kilo. The corresponding results for the above
three groups may be found in the following table, where for comparison
H AMMARSTEN also includes the figures for VOIT'S diet :

Urine

Nitrogen.

Catabolized Protein.

Protein per Kilo.

Min.

Max.

Min.

Max.

Min.

Max.

Group 1

5

.69

8.99

35.

6

56.

19

0.

61

0.

86

Group 2

7

.03

8.39

43

.9

52

.44

0

.74

0

,87

Group 3

7

.47

11.06

46,

,7

69,

10

0,

75

0,

92

Volt's figures

16

100

1.43

1 Arch. f. Hygiene, 45.

8 R. H. Chittenden, Physiological Economy in Nutrition, New York, 1904.

936 METABOLISM.

The chief results from these investigations are that on partaking of
amounts of protein much smaller than VOIT'S figures, without changing
the original supply of calories and indeed diminishing the same, the
persons experimented upon remained not only in nitrogenous equilibrium,
but in perfect health, and were not only able to perform ordinary work,
but were indeed regularly able to perform much greater work.

From these investigations, which extended over a long period and
were carried on with special care in exactitude, it cannot be denied that
man can for a long time exist with much smaller quantities of protein
than VOIT'S figures call for, which is also derived from the experience of
vegetarians, and from people living almost entirely upon vegetable food.
On the other hand it must not be forgotten that VOIT'S figures represent
average results not theoretically necessary, but which have been shown
to be the actual diet developed from habit, custom, conditions of life and
climate, with sufficient nourishment and free selection for centuries in
Middle and North Europe. A rational change in this food requirement
based upon scientific facts is just as difficult to determine as it is to carry
out practically. Certain standard figures for the general needs of nutri-
tion cannot be established because the conditions in various countries
are different and must necessarily be so. The numerous compilations
(of ATWATER and others J) on the diet of different families in America
have given the figures 97-113 grams protein for a man, and the very
careful investigations of HULTGREN and LANDERGREN have also shown
that the laborer in Sweden with moderate work and an average body
weight of 70.3 kilos, with optional diet, partakes 134 grams protein, 79
grams fat, and 522 grams carbohydrates. The quantity of protein
is here greater than is necessary, according to VOIT. On the other hand
LAPicQUE2 found 67 grams protein for Abyssinians and 81 grams for
Malaysians (per body weight of 70 kilos), materially lower figures.

If we compare the figures on page 933 with the average figures pro-
posed by VOIT for the daily diet of a laborer, it would seem at the first
glance as if the food consumed in certain cases was considerably in excess
of the need, while in other cases, as, for instance, that of a seamstress
in London, it was entirely insufficient. A positive conclusion cannot,
therefore, be drawn if we do not know the weight of the body, as well
as the labor performed by the person, and also the conditions of living.

1 Atwater, Report of the Storrs Agric. Expt. Station, Conn., 1891-1895 and 1896;
also Nutrition investigations at the University of Tennessee, 1896 and 1897; U.S
Dept. of Agriculture, Bull. 53, 1898. See also Atwater and Bryant, ibid., Bull. 75.
Jaffa, ibid., 84; Grindley, Sammis, and others, ibid., 91.

2 Hultgren and Landergren, 1. c.; Lapicque, Arch, de Physiol. (5), 6.

WORK AND FOOD REQUIREMENT. 937

It is certainly true that the amount of nutriment required by the body
is not directly proportional to the body weight, for a small body consumes
relatively more substance than a larger one, and varying quantities of
fat may also cause a difference; but a large body, which must maintain
a greater quantity, consumes an absolutely greater amount of substance
than a small one, and in estimating the nutritive need one must also
always consider the weight of the body. According to VOIT, the diet
for a laborer with 70 kilos body weight requires 40 calories for each kilo.
EKHOLM 1 calculates, basing it upon his experiments, that for a man
weighing 70 kilos, busied with reading and writing, the net calories are
2450 and the gross calories 2700, or 35 and 38.6 calories per kilo. In the
ordinary sense for a resting man, the general food requirement is calculated
in round numbers as 30 calories for every kilo. The minimum figure
for metabolism during sleep and in as complete rest as possible has been
found by SONDEN, TIGERSTEDT and JOHANSSON 2 to be 24-25 calories.

As several times stated above, the demands of the body for nourish-
ment vary with different conditions of the body. Among these condi-
tions two are especially important, namely, work and rest.

In a previous chapter, in which muscular labor was spoken of, it was
seen that all foodstuffs have almost the same power of serving as a source
of muscular work, and that the muscles, it seems, select that foodstuff
which is supplied to them in the greatest quantity. As a natural sequence
it is to be expected that muscular activity requires indeed an increased
supply of foodstuffs, but no essential change in their relation as compared
to rest.

Still this does not seem to hold true in daily experience. It is a well-
known fact that hard-working individuals — men and animals — require
a greater quantity of proteins in the food than less active ones. This,
contradiction, is however, only apparent, and it depends, as VOIT has
shown, upon the fact that individuals used to violent work are more
muscular. For this reason a person performing severe muscular labor
requires food containing a larger proportion of proteins than an individual
whose occupation demands less violent exertion. Another fact is that
the diet rich in proteins is often concentrated and less bulky, and also
that in many cases of training, a diet yielding as little fat as possible is
selected.

If we compare the results for the needs of food in work and rest which
are obtained under conditions which can be readily controlled, it is found
that the above statements are in general confirmed. As example of this

1 Skand. Arch. f. Physiol., 11.

* Sonden and Tigerstedt, Skand. Arch. f. Physiol., 6; Johansson, ibid., 7; Tigerstedt,
Nord. Med. Arkiv. Festband., 1897.

938 METABOLISM.

the following tables give the rations of soldiers in peace and in the field
and the average figures from the detailed data of various countries : l

A. Peace Ration. B. War Ration.

/ Proteins. Fat. Carbohydrates. Proteins. Fat. Carbohydrates.

Minimum. . . 108 22 504 126 38 484

Maximum. . . 165 97 731 197 95 688

Mean 130 40 551 146 59 557

The following figures for the daily ration are obtained from the above
averages:

Proteins. Fat. Carbohydrates. Calories.

In peace. 130 40 551 2900

In war 146 59 557 3250

If we calculate the fat in its equivalent quantity of starch, then the
relation of the proteins to the non-nitrogenous foods is:

In peace 1 : 4 97

In war 1 : 4 . 79

The relation in both cases is nearly the same. Similar results are
obtained when we start with Vorr's figures for a soldier in manoeuver A
(hard work) and B (strenuous work) in war.

Proteins. Fat. Carbohydrates. Calories.

A 135 80 500 3013

B... 145 100 500 3218

The relation here, when the fat is recalculated as starch, in both cases is
the same, or equal to 1:5.

If we calculate that portion of the total calories supplied which falls
to each group of the foodstuffs, it is found that 16-19 per cent comes
from the protein in rest as well as with medium and strenuous work.
For the fat and the carbohydrates the variations are greater; the chief
quantity of calories comes from the carbohydrates. Of the total calories
16-30 per cent comes from the fat and 50-60 per cent from the carbo-
hydrates.

The importance of the food-demand for working individuals is shown
by the figures given on page 933 for a wood-chopper in Bavaria. A
need of more than 4000 calories occurs but seldom, and with very hard
work the demand may rise even to 7000 calories (ATWATER and BRYANT,

JAFFA2).

1 Germany, Austria, Switzerland, France, Italy, Russia, and the United States.
It is not known by the author whether these figures have been changed in the last
few years in the various countries, and hence whether they must be modified or not.

8 See footnote 1, page 936.

WORK AND FOOD REQUIREMENT. 939

As more work requires an increase in the absolute quantity of food,
so the quantity of food must be diminished when little work is performed.
The question as to how far this can be done is of importance in regard
to the diet in prisons and poorhouses. We give below the following as
example of such diets:

Proteins. Fat. Carbohydrates. Calories.

Prisoner (not working).. 87 22 305 1667 SCHUSTER.!

Prisoner (not working).. 85 30 300 1709 VOIT.

Man in poorhouse 92 45 332 1985 FoRSTER.2

Woman in poorhouse. .. 80 49 266 1724 FORSTER.

The figures given by VOIT are, he says, the lowest reported for a non-
working prionser. He considers the following as the lowest diet for old
non-working people:

Proteins. Fat. . Carbohydrates. Calories.

Men 90 40 350 2200

Women 80 35 300 1723

In calculating the daily diet it is in most cases sufficient to ascertain
how much of the various foodstuffs must be administered to the body
in order to keep it in the proper condition to perform the work required
of it. In other cases it may be a question of improving the nutritive
condition of the body by properly selected food; and there are also cases
in which it is desired to diminish the mass or weight of the body by an
insufficient nutrition. This is especially the case in obesity, and all the
dietaries proposed for this purpose are chiefly starvation cures, which
is readily apparent if we study such dietaries.

iSee Voit, Unters. der Host, Miinchen, 1877, page 142. See also Hirschfeld,
Maly's Jahresber., 30.
8 Ibid., page 186.

940

FOOD TABLES.

TABLE I.— FOODS.1

1. Animal Foodstuffs.

1000 Parts contain

Relation of
1:2:3.

a. MEAT WITHOUT BONES.

Fat beef2 183 166

Beef (average fat ») 196

Beef2 190 120

Corned beef (average fat) 218 115

Veal 190 80

Horse, salted and smoked 318 65

•Smoked ham 255 365

Pork, salted and smoked 3 100 660

Meat from hare 233 11

" chicken 195 93

" partridge 253 14

" wild duck 246 31

b. MEAT WITH BONES.

Fat beef2.. 156 141

Beef (average fat1) 167 83

Beef, slightly corned 175 93

Beef, thoroughly corned 190 100

Mutton, very fat 135 332

1 ' average fat 160 160

Pork, fresh, fat 100 460

" corned, fat 120 540

Smoked ham 200 300

c. FISHES.

Hiver eel, fresh, entire 89 220

Salmon, " " 121 67

Anchovy, " " 128 39

Flounder, " " 145 14

River perch, fresh, entire 100 2

Torsk " " 86 1

Pike, " " 82 1

Herring, salted, entire 140 140

Anchovy, " " 116 43

Salmon (side), salted 200 108

Kabeljau (salted haddock) 246 4

Codfish (dried ling) 532 5

(dried torsk) 665 10

Fish-meal from variety of GADUS 736 7

11

18

18

117

13

125

100

40

12

11

14

12

15

85

100

8

10

5

60
70

6
10
11
11

8
8
6

100

107

132

178

106

59

87

640
688
672
550
717
492
280
130
744
701
719
711

544
585
480
430
437
520
365
200
340

352
469
489
580
440
455
461
280
334
460
472
257
116
170

150

150

167

180

88

150

70

80

90

333
333
333
250
450
450
450
340
400
100
100
100
150

100
100
100
100
100
100
100
100
100
100
100
100

100
100
100
100
100
100
100
100
100

100
100
100
100
100
100
100
100
100
100
100
100
100
100

1 The results in the following tables are chiefly compiled from the summary of ALM^N and of
KONIG. We here designate as " waste " that part of the foods which is lost in the preparation
or that which is not used by the body; for instance, bones, skin, egg-shells, and the cellulose
vegetable foods.

1 Meat such as is ordinarily sold in the markets in Sweden.

•Pork, chiefly from the breast and belly, such as occurs in the rations of Swedish soldiers.

ANIMAL FOODS.
TABLE I.— FOODS— (Continued).

941

1. Animal Foodstuffs.

1000 Parts contain

Relation of
11 : 2 : 3.

Proteids and ,_,
Extractive.

2

1

3

11
3*

4

i

5

1

6

1

1

:2

= 3 '

d. INNER ORGANS (FRESH).
Brain

116
196
184
163
221
150

182

190
220
7
3
304
35
35
41
37
230
334
89
106
122
160
103

123
110
92
150
88
90
115
115
114
77
80
111
110
117
140
101
70
232
220
270

103
56
92
106
38
170

2

150
160
850
990

35
7
9
257
270
66
70
93
107
307
7

17
10
11
39
10
3
17
15
20
10
14
21
10
60
60
58
7
21
15
15

11

7

50
50
38
35
40
50
456
4
5

7

676
740
768
439
550
768
688
720
725
480
514
654
720
563
660
656
770
537
530
520

11
17
10
10
13
10

9

50

55
15

175

7
7
7
6
60
50
56
8
10
13
8

18
8
3
50
17
8
18
20
15
16
11
26
7
30
20
17
2
36
25
25

770
720
714
721

728
670

807

610
565
119

7
217
873
901
905
665
400
500
329
654
756
520
875

140
120
120
130
330
131
140
110
110
400
370
140
146
130
100
140
146
137
150
125

135

26
12
6
192
5

22
20
16
17
11
48
7
100
20
28
5
37
60
45

100
100
100
100
100
100

100

100
100
100
100

100
100
100
100
100
100
100
100
100
100
100

100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

89
28
50
65
17
113

1

79
73
12100
33000

100
20
22
695
117
19
79
88
88
192
7

14
11
12
26
11
3
15
13
18
14
18
19
9
51
43
57
19
9
7
6

6
0
0
0
0
0

0

0
0
100
0

143
143
93
95
17
15
512
4
4
0
7

549
654
835
292
625
853
600
626
634
623
634
589
654
481
471
662
1100
231
240
192

Beef-liver

Beef -heart

Heart and lungs of mutton ....
Veal-kidney.

Ox tongue (fresh)

Blood from various animals
(average results)

e. OTHER ANIMAL FOODS.
Variety of pork-sausage (Mett-
wurst)

Butter

Lard

JVleat extract •

Cow's milk (full) . ...

" " (skimmed)

Buttermilk

Cream

Cheese (fat)

11 (noor).

TTpn'ff ecrcr entirG

' ' ' ' without shell.

Volk of esrff

White of egg

2. Vegetable Foodstuffs.

Wheat (grains)

Wheat-flour (fine)

'" " (very fine)

Wheat-bran

Wheat-bread (fresh)

Ivlacaroni . . .

Rve (crrains}

Rye-flour

Rye-bread (dry).

(fresh, coarse)

' ' (fresh, fine)

Barley (grains)

Scotch barley . .

Oat (grains)

' ' (peeled)

Corn

Rice (peeled for boiling)

French beans

Peas (yellow or green, dry) ....
Flour from peas

942

FOOD TABLES.
TABLE I.— FOODS— (Continued).

2. Vegetable Foodstuffs.

1000 Parts contain

Relation of
1:2:3.

1

1|

|]

JP

2

1

3

!i

4

i

5

3

i

6

£

1

• 2

= 3

Potatoes

20
14
10
25
19
27
31
14
10
12
32
219
4
5
242
140

2
2
2

4
2
1
5
3
1
1
4
25

537

480

200
74
90
50
49
66
33
22
23
38
60
412
130
90
72
180

10
7
10
8
12
6
19
10
4
7
9
61
3
6
29
50

760
893
873
904
900
888
908
944
956
934
877
160
832
849
54
55

8
10
15
9
18
12
8
7
6
8
18
123
31
50
66
95

100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100

10
14
20
16

11

4
16
21
10

8
12
12

222
343

1030
529
900
200
258
244
106
157
230
317
188
188
3250
1800
30
129

Turnips

Carrot (yellow)

Cauliflower

Cabbage

Beans

Spinach

Lettuce

Cucumbers

Radishes

Edible mushrooms (average). . .
Same dried in the air (average).
Apples and pears

Various berries (average)
Almonds

Cocoa

TABLE II.— MALT LIQUORS

1000 Parts by Weight contain

*

1 Carbon
Dioxide.

Alcohol.

Extract.

£

05

a

1

<j

Glycerine.

4

Porter.

871

2

54

76

7

13

3.0

4

Beer (Swedish)

887

28

15

*--' •

f

>5

5

' ' (Swedish export)

885

32

7

f

r3

3

Draught-beer

911

2

35

55

8

10

31

2.0

?,

?,

Lager-beer

903

2

40

58

4

7

47

1.5

?,

?,

881

2

47

72

6

13

1.7

3

916

3

25

59

5

,

4.0

.

2

Swedish " Svagdricka "

945

?,?,

7

^

?

v '

3

3

WINES AND OTHER ALCOHOLIC LIQUORS.

943

TABLE III.— WINES AND OTHER ALCOHOLIC LIQUORS

1000 Parts by Weight contain

Water.

Alcohol, Vol.
Per Cent.

Extract.

i

£

e®

•a-12

sjS

-n°£
wPn W
<

Glycerine.

1

Carbon Di-
oxide, Vol.
Per Cent.

Bordeaux wine

883

94

23

6

5.9

2 0

White wine (Rheingau)
Champagne

863
776

115
90

23
134

4
115

5.0
6.0

1 0

2.0
1 0

,

Rhine wine (sparkling)

801

94

105

87

6 0

1 0

2 0

> 60-70

Tokay

808

120

72

51

7 0

9 0

3 0

Sherry

795

170

35

15

5 0

6 0

5 0

Port wine

774

164

62

40

4.0

2 0

3 0

Madeira

791

156

53

33

5.0

3 0

3 0

Marsala

790

164

46

35

5 0

4 0

4 o

Swedish punch

479

263

332

Brandy

460

.

.

French cognac ....

, .

550

_ _

_

r

Liqueurs

442-590

__

260-475

944 INDEX TO SPECTRUM PLATE.

SPECTRUM PLATE

1. Absorption spectrum of a solution of oxy haemoglobin.

2. Absorption spectrum of a solution of hcemoglobin, obtained by the action of an

ammoniacal ferro-tartrate solution on an oxy haemoglobin solution.

3. Absorption spectrum of a faintly alkaline solution of methcemoglobin.

4. Absorption spectrum of a solution of hcematin in ether containing oxalic acid.

5. Absorption spectrum of an alkaline solution of hcematin.

6. Absorption spectrum of an alkaline solution of hcemochromooen, obtained by the

action of an ammoniacal ferro-tartrate solution on an alkaline-hasmatin solution.

7. Absorption spectrum of an acid solution of hcemaioporphyrin.

8. Absorption spectrum of an ammoniacal solution of urobilin after the addition of a

zinc-chloride solution.

8.

INDEX OF AUTHORS

Abderhalden, E., enzymes, 50, 53, 54, 62,
65, 525; protein hydrolysis, 84, 106,
107, 109, 114, 121-125, 132, 133, 142,
146, 154, 262, 288, 507, 605, 634, 662;
polypep tides, 86-90, 132, 133, 509;
protein reaction, 101; ichthylepidin,
121; proteoses, 127; cystine and cys-
tinuria, 148, 776; tryptophane, 157,
158; histidine, 160, 161; diamino-
trioxydodecanoic acid, 165; glyco-
proteid, 167; proteoses in the blood,
263, 264; blood serum, 266, 269, 270;
blood corpuscles, 275, 277, 304; blood
analyses, 328, 334, 338; iron per-
parations, 340; blood and high altitudes,
341; adrenalin, 376, .380; melanin, 380;
cholesterin, 445, 448; gastric contents,
481; digestion, 484, 513; duodenal
secretion, 489, 490; absorption and
synthesis of proteins, 529, 530; fat,
559; milk, 646, 659, 662, 667; urea
formation, 682; nuclein metabolism,
702, 705; amino-acid storage, 721, 722;
alcaptonuria, 735-737; urinary sul-
phur, 752; polypetides in the urine,
763; amino acids in the urine, 756, 827;
pyridine, 787; Bence-Jones proteid,
792, 793; tunicin, 839; nutritive value
of gelatin, 912; ammonia as protein
sparer, 912; water and metabolism
920; alcohol, 921; nin-hydrin, 101;
caprine, 144; tyrosine reagent, 154

Abel, J., 379, 621, 683

Abeles, M., 266, 749

Abelmann, M., 532, 539, 540

Abelous, J., 757, 877

Abelsdorf, G., 616

Ach, L., 698

Achalme, 307

Achard, Ch., 265

Acheles, W., 698, 757

Ackermann, D., putrefaction bases, 47;
histidine, 82, 159, 160; proline, 154;
aporrhegmen, 166; blood corpuscles,
274, 275; meat bases, 578; lysine in
the urine, 828

Ackroyd, H., 717

Adam, H., 311

Adamkiewicz, A., 100, 912

Adamson, 17

v. Anrep, R., 287, 723

Adler, O., proteoses, 209; fructose, 217,

814; ochronosis, 550; blood, 797
Adler, R., proteoses, 134; pentoses, 209;

fructose, 217; blood, 797
Adrian, C., 730
Adriance, J., 662, 665
Adriance, V., 662, 665
Aducco, V., 675, 757
Agulhon, 50
Albanese, M., 711
Albert, R., 41
Albertoni, P., 415, 502, 533
Albrecht, E., 273
Albro, A., 506
Albu, A., gastric juice, 466; urine poison,

758; mineral metabolism, 758, 769,

899, 901, 902
v. Alder, L., 792
Aldrich, J. B., 846
Alexander, F., 133, 652
v. Alfthan, K., 749
Allard, E., 825
Allaria, G. B., 659
Allen, S., 471
Alleis, R. A., 157, 380
Allihn, F., 656, 811
Allison, F. G., 689
Almagia, M., 410, 583, 706
Alms' n, A., xanthine, 188; sugar test,

214; meat, 599, 600; food-stuffs, 940
Aloy, J., 877
Alsberg, C., 104, 184
Altmann, R., 179
Amberg, S., 71, 642, 643
Ambronn, H., 839
Amerman, G., 470
Ameseder, Fr., 239, 627, 844
Amiradzibi, S., 575
Amthor, K., 237, 559
Andersen, A. C., 154, 804, 809, 810
Anderson, R. J., 580
Anderson, N., 332
Andersson, J., 376
Andryewskij, P., 874
Anselm, R., 435
Ansiaux, G., 97, 253
Authon, 213
Appleyard, J. R., 28
Araki, T., blood pigments, 285; nucleic

945

946

INDEX OF AUTHORS

acids, 508; lactic acid, 582-585, 748,

749; chitin, 839
Ardin-Delteil, P., 848
Argiris, A., 112, 114, 606
Argutinsky, P., 600, 848, 883
Arinkin, M., 43
Armstrong, E. T., enzyme, 55, 56, 58,

59, 65; glucoside, 200; osone, 203
Armstrong, H. E., 59, 65
Arnheim, J., 408
Arnold, J., 377
Arnold, V., protein reaction, 101; cystein,

149; urine reaction, 696; nephrosein,

734; hsematoporphyrin, 797; aceto-

acetic acid, 824, 825
Arnold, W., 288
Arnschink, L., 537
Arnstein, R., 715
Aron, H., 281, 552, 554, 555
Arrhenius, S., dissociation theory, 4;

catalysis, 33, 34; enzymes, 53; Schutz's

rule, 57; toxin-antitoxin combination,

67,68 '
Arronet, H., 328
Ateaga, T. F., 400
Arthus, M., blood coagulation, 250, 251,

313, 314, 316-318, 320; fibrinolysis,

255; serum lipase, 265, glycolysis, 332;

casein,* 648, 650
Artmann, P., 23
Asahina, Y., 297
Ascher, E., 145
Ascherson, 646
Ascoli, A., antolysis, 43; nucleic acids,

172, 185; uracil 194; glutinase, 505;

protein absorption, 526;) placenta, 642;

urea formation, 682; uric acid, 706
Asher, L., blood sugar, 264; lymph, 364,

349-352; spleen, 372; thyroids, 374;

liver, 381, absorption, 528; nitrogen

elimination, 910
Aso, K., 97
Astaschewsky, 593
Athanasiu, J., 384, 561
Atwater, W. O., metabolism, 595, 597,

879, 891, 914; respiration apparatus,

868, 869; urine quotient C:N, 884;

alcohol, 921; diets, 932, 936, 939
Aubert, H., 849
Auche, A., 267, 431
Austin, A. E., 392
Austrian, C. R., 702
Autenrieth, W., 766 -
Aynand, M., 308, 314, 315
Ayres, W. C., 615
Akerman, J., 477

Baas, H., 501, 720

Babcock, 653

Babkin, B., 107, 499, 682

Bach, A., oxygenases, peroxides and
peroxidases, 871, 873; catalases, 873;
oxidation processes, 875; philothion,
877; reduction processes, 877

Bacmeister, 439

Baer, J., 43; cystin, 148; thiolactic acid,
151; lactic acid, 583; ammonia elimina-
tion, 675, 676; acetone bodies, 818,
819, 822

v. Baeyer, A., 37, 38, 212

Baginsky, A., 438, 555

Baglioni, S., 333

Bailie, A., 387

Bainbridge, F. A., 53, 351

Baisch, C., 749, 808

Baker, J. L., 226

Balch, A., 414

Baldi, D., 385

Baldoni, A., 509, 783

Balean, H., 301

Balke, P., purine bases, 188, 193, 714;
phosphocarmic acid, 578

Bang, B., 671

Bang, 1., histone, 108; guanylic acid,
183, 185; nucleohistone, 307; blood
sugar, 329; blood, 329; estimation of
sugar, 329, 808-811; lymph glands
and thyrnus, 365-368; "glycogen, 402;
saliva, 457; rennin enzyme, 473; chlor-
ine estimation in the urine, 759; pro-
teoses in the urine, 792; sugar tests, 803,
804; sugar formation, 399

Barbera, A. G., 356, 349, 415

Barbieri, J., 152, 448

Barbieri, N. A., 615/630

Barcroft, J., 1277-279

Bardach, Fr., 726

Bardach, K., 684 .

Bardier, E., 757

Barendrecht, H. P., 65

Barker, B., 307

Barker, L. F., 827

Barral, 332

Barratt, W., 849

Barezczewski, C., 210

Bartholomans, E., 297, 295, 429

Bartoschewitsch, S. F., 724

Basch, K., 667-669

Baserin, O., 443 *.

Bashford, E., 722

Baskoff, 385, 386

Bass, 727

Bassow, 461

Bastianelli, G., 492

Batelli, F., fermentation, 407; uricolysis,
706; peroxidases, 873, 874; oxidation
processes, 874, 875

Baudrirnont, 838

Bauer, Fr., 182

Bauer, H., 646

Bauer, J., 525, 561

Bauer, K., 757

Bauer, M., 160

Bauer, R., 113, 815

Baum, Fr., 159

Baumann, E., diamines, 47; cystine, and
cystinuria, 149, 827, 828; thiolactic
acid, 149, 150; carbohydrates,. 215;

INDEX OF AUTHORS

947

iodothyrin, 376; deamidation, 410;
intestinal putrefaction, 514, 720; ethereal
sulphuric acids, 515, 724-729, 784;
hippuric acid, 720; oxyacids, 734,
homogentisic acid, 735, 736, 738, 739;
carbohydrates in the urine, 749, 808;
urinary sulphuric acids, 764; sarcosine,
776; behavior of aromatic bodies, 778,
780, 783; mercapturic acids, 786

Baumann, L., 157, 288, 484, 513

Baumgarten, A., 360

Baumgarten, O., 403, 758

Baumstark, F., brain constituents, 605,
606, 612, 613; urinary pigments, 799

Baumstark, Rob., 512, 524

Bayer, H., 136

Bayer, R., 372

Bayliss, W. M., enzyme, 54, enterokinase,
492, 496; secretin, 492, 498; intestinal
enzymes, 492, 493, 503; trypsinogen
and trypsin, 496, 497, 503, 506; casein
digestion, 652

Beatty, W. A., 89

Beaumont, W., 461, 481

Bebeschin, K., 673

Beccari, L., 435

Bechamp, A., 633, 655, 661

Bechhold, H., colloids, 17, 18, 30, 32, 51;
uric acid, 708; sugar determination, 804

Becht, F. C., 349

Beck, A., 744

Beck, C., 595

Beckmann, E., 4

Beckmann, Ernst, 846

Beckmann, W., 768

Becquerel, A., 338, 665

Beger, C., 667

Behrend, R., 698, 699

v. Behring, E., 66

Beier, Karl, 441

Beitler, C., 152

Bellamy, H., 496

Bellori, E.r 653

van Bemmelen, J. M., 29, 31

Bence, J., 311

Bence Jones, H., 792

Bendix, E., 208, 209, 394

Benedicenti, A., 126, 729, 921

Benedict, F. G., metabolism in work,
595, 597; respiration, 868; calorimetry,
885; sparing of proteins, 914; alcohol,
921

Benedict, H., 595, 752, 897

Benedict, S. R., 689, 693, 752

Benedikt, R., 236

Benedikt, St., 214

Benrath, A., 477

Berard, E., 97, 633

Berdez, J., 841

Berenstein, M., 521

Bergell, P., carbohydrates in proteins,
84, 262; lecithin, 245; polypeptides,
508; placenta, 642; casein, 662;
oxybutyric acid, 826

Berger, W. M., 456

Bergh, E., 116

Bergholz, R., 509

Bergin, T. J., 519

v. Bergmann, G., 266, 267, 442

Bergmann, P., 376, 466, 542-543

Bergmann, Wolfg, 761

Berlioz, A., 758

Berlinerblau, M., 334

Bernard, Claude, blood sugar, 332; gly-
colysis, 332; glycogen, 389; 390, 398,
sugar puncture, 402; diabetes, 405;
pancreas, 495, 501, 502, fat absorption,
538; muscle glycogen, 592

Bernert, R., 83, 233, 358

Bernheim, A., 357, 359

Bernheim, R., 766

Bernstein, J., 602

Bernstein, N. O., 495

Bert, P., mammary glands, 643, 669;
gases of blood, 851, 861

Bertagnini, C., 783

Bertarelli, E., 64

Berthelot, M. P. E., division law, 27;
fat cleavage, 501; tunicin, 839, calor-
imetry, 885

Bertin-Sans H., 285, 287

Bertrand, G., arsenic, 72, 373, 838;
xylonic acid, 210; sugar determina-
tion, 808, 811; tyrosinase, 842; reptile
poison, 846; oxidases, 873

Bertz, F., 558

Berzelius, J. J., catalytic reactions, 33;
saliva, 458 1

Besbokaia, M., 496

Best, Fr., 481

Bezzola, C., 706

Bial, M., pentoses, 208, 209, 816; glucur-
onic acids, 221; diastase, 265, 346,
398; glycogen, 396

Bialobrzeski, M., 292

Bialocour, F., 485 .

v. Bibra, E., 389

Bickel, A., 464, 465

Bidder, F., buccal mucus, 453, 554;
saliva, 458; gastric juice, 465; pan-
creatic juice, 499; bile, 518; fat
absorption, 538

Biedert, Ph., 660

Biehler, A., 124

Biel, J., 658, 662

Bielfeld, P., 387

Bienenfeld. B., 660

Bienstock,'B., 519, 520

Biernacki, E., blood, 309, 326; pepsin,
467; trypsin, 503; intestinal putrefac-
tion, 517, 519, 724

Bierry, H., cataphoresis, 50; filtration, 51;
enzymes, 53, 71, 231; pancreatic juice,
500

Biffi, U., 267, 652, 744

Billard, G., 45

Billitzer, J., 26, 27

Biltz, W., glycogen, 19, 20; colloids, 22,

948

INDEX OF AUTHORS

23; adsorption, 28, 29, 69; dextrin
B, 230

Bin£t, P., 541

Bing, H. J., 245, 331, 385, 398

Bingel, A., 266

Bing, C., 777

Biondi, C., 42

Biot, J. B., 867

Birchard, Fr., 134, 144

Biscaro, G., 653

Bischoff) Th., 879

Bizio, G., 849

Bizio, J., 390

Bizzozero, J., 307, 314

Bjerre, P., 921

Bjorn-Andersen, H., 769

Blachstein, A., 582

Blanck, F. C., 696

Blankenhorn, E., 606

Blanksma, J., ammo-sugar, 167; 219,
oxymethylfurfurol, 211, 215, 217; ace-
tone, 825

Bleibtreu, L., 326, 690, 934

Bleibtreu, M., 326, 563, 636

Bleile, A. M., 332

Blendermann, H., 410, 734, 780

Bliss, C. L., 126

Blix, M. G., 326

Bloch, Br., 735, 736

Blondlot, N., 518

Blood, A., 52

Blum, F., haloginized protein, 82; Mil-
Ions reaction, 99; adrenalin glycosuria,
380

Blum, L., autolysis, 45; protein nitrogen,
77; protogon, 126; alcoptonuria, 735,
cystin, 776; tyrosin cleavage, 779, ace-
tone bodies, 818, 819, 822; food value
of albumoses, 912

Blumenthal, F., gelatin, 83; indol and
skatol, 159; pentoses, 208, 815, 816;
nucleoproteins, 383; glycogen, 394;
assimilation limit, 534; urine indican,
728; acetone, 818

Boas, J., 488

Bocarius, N., 621

Bocchi, O., 741

Bock, C., 398, 400

Bock, J., 285

Bode, A., 658

Boden, E., 707

Bodlander, G., 921

Bodon, K., 356

Bodong, A., 318, 323

Boedeker, C., 727

Bodtker, E., 680, 759

Boehm, P., 381

Boehm, R., 448, 581, 590, 591

Boehner, R., 154

Boehtlingk, R., 895, 896

Boekelmann, W. A., 826

Boeri, G., 716, 752

Boettger, 214

Bogdanow, E., 586, 596

Bogdanow-Beresowski, 455

Bogen, H., 464

Bogomoloff, Th., 743

Bohland, K., urea nitrogen, 680; urea,
690; uric acid, 701; ammonia, 768;
protein need, 934

Bohm, V., 402

Bohmansson, G., 803, 804

Bohr, Chr., blood pigments, 276, 278,
279, 282, 287; egg hatching, 637; gases
of blood, 850-853; metabolism in
lungs, 858, 869; oxygen tension, 859,
861-863, 867; carbon dioxide tension,
865, 866; specific action of lungs, 864,
867; air-bladder, 867; oxygen capacity,
868

Du Bois-Reymond, E., 592, 602

Du Bois, Reymond, R., 312

Bokorny, T., 212

Bolaffio, C., 630

Boland, G. W., 365

Boldyreff, W., gastric digestion, 481, 511;
intestinal juice, 490-493

Boljarski, N., 694

Bolin, J., 873

Boll, F., 615

Bonamartini, G., 96

Bonanni, A., 437, 438, 785

Bondi, J., 642, 643

Bondi, S., lipoproteids, 87; sericin, 122,
123; bile acids, 419, 421, 423; acetoace-
tic acid, 825

Bondzinski, St., oxyproteic acid, 83;
koprosterin, 448; ovalbumin, 633;
urine purines, 711; urochrom, 741;
oxyproteic acids in urine, 753, 754

Bonnema, A., 646

Bookman, S., 721, 722

Boos, P., 454

Borchardt, L., elastin albumose, 263,
527; sugar formation, 398; fructose
uria, 814; acetone, 818, 819, 825

Bordet, J., antienzymes, 64; sensibiliza-
tors, 69; blood coagulation, 314, 318,
320

Borissow, P., 462, 717

Borkel, C., 136

Bornstein, K., 595, 918

Boruttau, H., 581

Bosshard, E., 149

Bostock, G., 676

Bottazzi, Ph., freezing point, lowering of
blood, 12; blood corpuscles, 304; gly-
cogen, 384, 390, 391; heart muscle, 569;
smooth muscle, 602; placenta, 640

Bouchard, Ch., 393, 757, 758

Bouchet, A., 680

Bouchez, 767, 772

Boudet, 264

Boulud, glucuronic acids, 221, 331; pen-
toses, 264; sugar in blood, 264, 329-
332; glycolysis,333; maltose in urine, 814

Bouma, J., indican, 730; bile pigments,
801, oxybutyric acid, 826

INDEX OF AUTHORS

949

Bourcet, P., 269, 336

Bourquelot, E., 395

Bouveault, L., 144

Brach, H., 839

Bradley, H. C., 500

Brahm, C., 559, 785

Brahm, B., 179, 182, 183, 211

Brand, J., 414, 437, 438

Brandberg, J., 793

Brandenburg, K., 310

Brandl, J., 841

Brasch, W., 144, 534

Brat, H., 209

Brauer, L., 440

Braun, K., 64

Braunstein, A., 408

Brau tlecht, C., 78

Bredig, G., colloidal metals 14; surface
tension, 26; catalysis, 33-37; assym-
etric synthesis, 59

Brienl, F., 103

Brenzinger, K., 827

Bretschneider, A., 329, 330

Brewster, J. F., 155

Brieger, L., putrefaction products, 47;
neurine, 246; intestinal putrefaction,
514; skatol, 521; neuridine, 606, 612,
628; urine indican, 727, 729; skatoxyl-
sulphuric acid, 732 ;] cystinuria, 827;
perspiration, 848

Briggs, C. E., 4Q2

Brigl, P., 179, 183

Brion, A., 774

Brodie, T. G.,k256, 400, 509

Brodley, H. C., 72

Brook, F. W., 82

Brooks, Cl., 399

Browinski, J., 266, 741, 754

Brown, A. J., 55, 56, 65

Brown, E. W., 705, 717

Brown, H. T., inverting enzymes, 55;
starch hydrolysis, 226, 229, 456, sacha-
rase, 492'

Brown. R., 19

Brown, T. Graham, 594

Browne, C. A. (jr.), 647

Brubacher, H., 554, 557

v. Briicke, E., blood coagulation, 316;
glycogen, 392; pepsin 467-469, fat
emulsion, 511; protein absorption,
525; carbohydrates in urine, 749

Brugsch, Th., bile pigments, 443; pan-
creas/532; uric acid, 700, 701; hip-
puric acid, 721; amino-acids in urine,
757; urine in hunger, 897

Bruhns, G., 193

Brunner, E., 36

Brunner, Th., 665

Bruno, G., 501, 502, 506, 510, 758

Brunton-Blaikie, 572 .

de Bruyn, Lobry, 19, 201, 220

Bryant, A. P., 936, 938

Buchanan, A., 256

Buchner, E., alcohol fermentation, 41,

205; lactic acid fermentation, 207;
sugar test, 213

Buchner, H., 41, 856

Buchtala, H., 113-115, 147, 426

Budde, V., 812

Biilow, K., 95, 227

Biinz, R., 612, 613

Burger, L., 545

Biirker, K., 314, 315

Bufalini, 339

Bugarszky, St., 271, 326

Buglia, G., 510, 572, 602, 603

Bull, H., 237

v. Bunge, G., serum, 270; blood cor-
puscles, 304; blood coagulation, 313;
blood analysis, 328; iron preparations,
340; blood and high altitude, 341;
iron in liver, 387; gastric juice, 485;
cartilage, 550; hamatogen, 629, 637;
milk, 657, 664, 666-668; .hippuric acid,
722, 723; mineral requirement, - 900,
901

Bunsen, R., 690

Buntzen, J., 339

Buraczewski, J., 83

Burchard, H., 447

Burckhardt, A. E., 270

Burian, R., purine bases and their
enzymes, 193, 195, 371, 373, 572, 594,
702, 703, 713; uric acid formation, 700,
702, 703, 705; uric acid cleavage, 706;
histone in urine, 795

Burow, R., 371, 663

Busch, P. W., 349, 350

Butlerow, A., 211, 212

Butterfield, E., 277, 303

Bywaters, H. W., 260, 263

Cade, A., 464

Cahn, A., 476, 615

Camerer, W., milk, 656, 657, 662, 664-
666; urine nitrogen, 680; uric acid elim-
ination, 700; purine bases, 715; meta-
bolism, 922, 924

Camerer, W., jr., 847

Cameron, A. T., 78, 109

Cam is, M., 279, 592

Cammidge, P. J., 815, 827

Campani, A., 763

Campbell, G., 628, 629, 633, 634

Campbell, J. F., 432-434

Camps, R., 739

Camus, L., pancreatic juice, 496; secretin,
498; vesiculase, 622

Cannon, W. B., 479-484, 524

Cappelli, J., 304, 602

Cappezzuoli, C., 369, 557

Capranica. St.. 190, 848

Carbone, D., 613

Carlier, E, W., 313, 347

Carlinfanti, E., 235, 236

Carlini, C., 381

Carlson, A. J., 348, 349, 454

Carlson, C. E., 873

950

INDEX OF AUTHORS

Carnot, Ad., 552, 558

Carvallo, J., 485, 486

Casali, A., 847

Caspari, W., high altitude 341, 888;

protein metabolism, 595, 916, milk fat;

669; vegetable diet, 915-916
Castoro, N., 161
Cathcart, E. P., autolysis, 44; glycogen,

396; stomach, 479; gastric digestion,

481; protein absorption, 527; creatine

and creatinine, 594, 693; milk sugar,

670; starvation urine, 897
Cavazzani, E., cerebrospinal fluid, 361;

glycogen cleavage, 399; absorption,

535, phosphocarnic acid, 578; muscular

work, 592; semen, 620
Cernj, C., 838
Cerny, T., 583, 792
Chabbas, J., 617
Chabrie, C., 557
Chan'delon, Th., 592
Chaniewski, 563
Charnas, D., 746, 747
Chassevant, A., 706
Chauveau, A.,~ sugar formation, 412;

fat formation, 562; muscular work;

592, 597
Cherry, Th., 68
Chigin, P., 462
Chistoni, A., 314, 315
Chittenden, R. H., keratin, 119; elastin,

116, 117; gelatin, 118, 120; albumoses

and peptones, 128-131, 137; saliva,

455-458; pepsin, 470, 471; trypsin, 506;

tendon mucoid, 545, 551; myosin,

567, 568; neurokeratin, 605, 613, 614;

protein requirement, 934
Chodat, R., oxidases, peroxides and

peroxidases, 871; oxidation processes,

875; |" philothion," 877
Chossat, Th., 892, 896
Christenn, G., 662
Christensen, A., 793
Ciamician, G., 159
Cingolani, M., 699
Citron, H., 766
Clapp, S. H., 89, 107
Clar, C., 701
Clarke, T. W., 287
Glaus, R., 408

Clausmann, P., 693, 552, 558 i
Clemens, Paul, 785, 827
Clemm, C. G., 665
Clerk, B., 265
Cleve, P. T., 422, 423
Cloetta, M., 290, 292, 293, 753
Cloez, 441
Clopatt, A., 921
Closson, O. E., 400
Cobliner, 332
Cohn, Felix, 485]
Cohn, Max, 500
Cohn, Michael, 455
Cohn, R., leucinimid, 145; carbohydrate

formation, 410, 411; fate of aromatic
substances in the animal organism, 778,
783, 784; furfural, 784, pyridin, 787

Cohn, Th., 621, 717

Cohnheim, J., 456

Cohnheim, O., lipoid action, 10; proteins,
76, 95; blood and high altitudes, 341,
340; glycolysis; 407, 408; gastric
juice, 465; peptic digestion, 481, 484;
absorption, 484, 528, 542-543; erepsin,
491, 493; pancreatic juice, 497; con-
nective tissue, 512; peristalsis, 524;
digestion work, 930; accommodation
of digestive enzymes, 53

Cohnstein, J., 336

Cohnstein, W., 309, 352

Colasanti, G, 591, 593, (598, 748, 749

Cole, S. W., protein reaction, 100; trypto-
phane, 155-158

Collmann, 849

Comaille, A., 653, 668

Comesatti, G., 380

Comiotti, L., 816

Connstein, W., 265

Conradi, H., 45, 324, 520

Constantinidi, A., 535, 915, 916

Constantino, A., 572

Contejean, Ch., phlorhizin diabetes,
400; gastric juice, 465, 476; pyloric
secretion, 477

Copemann, M., 797

Coranda, G., 682

Cordua, H., 301

Corin, G., protein coagulation, 97; fibrino-
gen, 253; proteins of egg-white, 633

Coronedi, G., 560

Corper, H. J., 372, 449-450, 701, 706

Corvisart, L., 502

Costantiho, A., 586, 602, 603, 748

Le Count, E. R., 114

Courant, G., milk, 644, 649, 650, 659

Cousin, H., 242, 248

Couvreur, E., 393

Cramer, C. D., 253

Cramer, E., 122, 848

Cramer, Tr., 531

Cramer, W., absorption, 525, 528, 530;
protagon, 606-608; placenta, 640;
creatine, 692; hippuric acid, 722;
blood coagulation, 320

Cremer, M., glycogen, 58; 332, 390,
393, 395; pentoses, 208; glycolysis,
332; phlorizin-diabetes, 400; sugar
formation, 412; fat formation, 562

Cristea, G., 336

Crittenden, A. L., 454

Croftan, A., 334

Croft-Hill, A., 58, 225

Croner, W., 488, 536

Cronheim, W., 488-

Croockewitt, J. H., 122

Csokas, J., 648, 658

v. Csonka, F., 657

Cummis, G. W., 506, 567, 568

INDEX OF AUTHORS

951

Cunningham, R. H., 540
Curtius, Th., 85, 86, 422 <
Cutler, W. D., 168, 544, 545
Cybulski, N., 379
Czernecki, W., 266, 358
Czerny, Adalb, 307
Czerny, F., 407 j
Czerny, V., 485, 525
v. Czyhlarz, E., 873

Daddi, L., 339, 669

Daenhardt, C., 856

Dakin, H. D., autolysis, 44; mandelic
acid ester, 62; arginase, 89, 161, 574,
681, 682; protamine, 109, 110; valine,
141; serine, 145; proline, 154; color
reactions, 157; lysin, 163; hexon bases,
164; oxalic acid, 716, 773; alcap-
tonuria, 737; cleavage of fatty acids
774, 781; uramino acids, 786; acetone
formers, 822; sugar formation, 412;
lactic acid, 583; a-amino acids, 775

Daland, J., 329

van Dam, W., 474, 650

Danilewski, A., plasteines, 58, 135; pro-
tein sulphur, 79; retarding substances,
487, 492; muscle protein, 566-569;
milk globules, 646

Danilewsky, B., 885

Danilewsky, W., 241

Dareste, C., 620, 628

Darmstadter, E., 826

Darmstadter, J., 846

Darmstadter, L., 448

Dastre, A., fibrinogen, 252; fibrinolysis,
255; glycogen, 307, 346; blood coagula-
tion, 314; liver, 383, 384, 388; gly-
cogen, 395, 397, 399; bile, 414, 434,
435, 511; enterokinase, 496, 497; fat
absorption, 538

Dantzenberg, P. J. W., 783

Danwe, F., 49

Davidoff, W., 75

Day, H., 480

Dean, A. L., 228, 529

Decaisne, E., 667

Deetjen, H., 308, 314, 315

Dehn, W. M., 760

Dekhuysen, C., 13

Delezenne, C., enzyme retardation, 65;
papain, 65; blood coagulation, 250,
313, 318, 320, 324, 325, 351; intestinal
juice, 490-492, enterokinase, and pan-
creatic juice, 493, 495-497, 509; secre-
tin, 498

Delfino, A., 640

Demant, B., 491

Demoor, J., 453

Denigds, G., tyrosin, 154; indol and
skatol reactions, 158, 159; inosite,
580; homogentisic acid, 739

Denis, P. S., 257

Denis, W., tyrosin, 154; urea in blood,
333, 336; hypophysis, 592; creatine,

692, 696; uric acid, 708, 711, 334;
urine sulphur, 752

Denk, W., 336

Dennemark, L., 658

Derrien, E., glucose, 215; blood pig-
ments, 281, 285, oxymethylfurfurol,
419; Bence- Jones, protein, 792

Desgrez, A., 393

Dencher, P., 532, 539, 540

Devilard, P., 360

Devoto, L., 792

Dewitz, J., 843

Diakanow, C., 243-245

Diamare, V., 494

Dick, M., 702

Diels, O., 445, 446, 448

Diesselhorst, G., 848

Dietrich, M., 104, 652

Dietschy, R., 792

Dietz, W., 56, 60

Dietze, Alb., 506

Dillner, H., 633

Dimitz, L., 248, 614

Disque, L., 740, 742, 744

Ditthorn, Fr., 167, 173, 219

Dittrich, P., 285

Ditz, H., 726

Doblin, A., 329, 333

Dorpinghaus, Th., carbohydrates in pro-
tein substances, 84;] protein hydrolysis,
113, 141, 142, 252

Dohm, M., 406

Dombrowsky, St., urinary pigments,
740, 741; pxyproteic acids in urine,
753, 754; urinary bases,l757

de Domenicis, A., 287

de Dominicis, N., 404

Donath, J., 360

Donne, A., 799

Dony-Henault, O., 873

Donze, G., 680

Doree, Ch., 448

Dormann, E., 298

Dorner, G., 574

Douglas, C. G., 286

Douglas, Gordon C., 864

Doyon, M., fibrinogen, 252, 253, 335;
serum lipase, 265; blood coagulation,
324, 325; glycolysis, 332; bile, 415, 416,
434, 439

Dragendorff, D., 800

Drechsel, E., proteins, 76, 79, 94, 123;
diamino-acetic acid, 163; lysin, 163,
164; purin bases, 188; thyroid gland,
375; jecorin, 385; urea formation,
681, 683; carbamic acid, 683; silicic
acid ester, 838

Dreser, H., 13, 71

Dreyfus, G. L., 465

Droop-Richmond, H., 646

Drosdoff, W., 335

Dubelir, D., 920

Ducceschi, V., 475, 569

Duclaux, E., 97, 647

952

INDEX OF AUTHORS

Ducleau, E., 55
Dudley, H. W., 583, 775
During, Fr., 554
Dufau, E., 808
Dufourt, 415, 416, 439, 592
Duggan, C. W., 97
Dull, G., 229
Dumas, J. A., 684
Dunham, E., 180, 239, 673
Dunlop, J. C., 595, 715

Ebbeke, U., 757

Ebstein, E., 114, 208, 209

Ebstein, W., 457, 727, 831

Eckhard, C., 452

Edelstein, E., 43

Edelstein, F., 657, 662

Edic, E., 246, 402

Edkins, J. S., 463, 477, 509

verEecke, A., 925

Ehrenfeld, R., proteins, 83; leucine, 143;
tyrosin, 153

Ehenreich, M., 49, 505

Ehrenthal, W., 521

Ehrlich, F., amino-acids, 143, 144, 153,
206; fermentation, 157; fusel oil, 206

Ehrlich, P., side chain theory, 67,
68; amboceptors, 69; dimethylamino-
benzaldehyde, 159, 219, 746, 827;
bilirubin, 431; urine test, 826

Ehrmann, R., 405

Ehrstrom, R., histone, 108; phosphate,
761,762,796; albumoseuria, 791

Eichholz, A., 260, 633, 743

Eichhorst, H., 525

Einhorn, M., 159

Ekbom, A., 426

Ekehorn, G., 760

van Ekenstein, A., amino-sugar, 167,
219; carbohydrates, 201, oxymethyl-
furfurol, 211, 215, 217; acetone, 825

Ekholm, K., 936

Elias, H., 609

Ellenberger, W., 479, 480, 658, 659

Ellinger, A., isoserin, 145; tryptophan,
155, 156; arginin, 163; blood coagula-
tion, 324; lymph formation, 351, 352;
pancreatic secretion, 500; urine in-
dican, 728, 730; tri-indylmethane pig-
ments, 734; kynurenic acid, 739;
oxyphenyllactic acid, 780; food value
of albumoses, 912

Ellmer, A., 237

Ely, J., 455, 457

Embden, G., cystein and cystine, 79,
147, 149; serine, 145; glycolysis, 333,
408; carbohydrate formation. 410;
liver blood perfusion, 529, 530, 775,
786; lactic acid, 583, 584, 333; gly-
cocoll in urine, 756; acetone bodies, 819
822, 825, 826

Embden, H., 735

Emerson, R. L., 507

Emich, Fr., 517

Emmerling, A., 113
Emmerling, O., 58, 59, 225
Emsmann, Otto, 463
Engel, 662

Engel, H., lipases, 57, 476; lactic acid
formation, 585; acetone bodies, 818,

Engel, St., 658, 663

Engeland, R., agmantin, 162; aporrheg-
men, 166; carnitin, 577; methvl-guani-

din, 698; urine bases, 757, 826^ 827
Engelmann, G. J., 595
Engler, C., 871, 875
Eppinger, H., 375, 406, 676, 799
Eppinger, P., 290, 292, 293, 354
Epstein, A., 721, 722

Erben, Fr., oxystearic acid, 233; leuco-
cytes, 307, 342; blood analysis, 342;

chyle fat, 346; urine nitrogen, 681,

urein, 691
Erdelyi, A., 482
Erikson, A., 50, 63
Erlandsen, A., phosphatides, 242, 243

245, 246, 586; cuorin, 248, 249; phlor-

hizindiabetes, 400
Erlanger, J., 541
Erlenmeyer, E., 142, 152
Erlenmeyer, E. (jr.) 145, 148, 152
d'Errico, G., 391
Esbach, G., 793
Escher, Heins, H., 623, 631
Estor, A., 867
Etard, A., 578
Etti, C., 642
Ettinger, J., 43
Eulenburg, 398
v. Euler, H., activators, 52; enzymes, 53,

56, 70; phosphoric acid ester, 205;

erepsin, 493; oxidation processes, 873
Eves, F., 457
Ewald, Aug., proteins, 112, 119; hsema-

toidin, 301; digestion, 508 Jj visual

purple, 615; corpora lutea, 623
Ewald, C. A., 857, 928
Ewins, A. I., 380
Eykman, C., 326, 929, 932
Eymonnet, 757

Fabian, E., 396

Fahr, G., 587

Fajans, K., 35

Falck, 910

Falk, Edm, 642

Falk, Ernst, 924, 925

Falk, Fr., 248, 613

Falloise, A., bile, 416; gastric lipase,

476; intestinal enzyme; 492, 493;

chloralsecretin, 499; blood gas tension.

861, 866
Falta, W., thyroid gland, 375; diabetes

405, 406, 409, 412; alcaptonuria, 735,

736; protein metabolism, 882, 883, 907
Fano, G., 251
Farkas, K., 638

INDEX OF AUTHORS

953

Farmer, Ch., 688

Farwik, B., 599

Fasal, H., 106, 107, 153, 652

Faust, E., sepsine, 47; gelatin, 118;
poisons in the secretion of the skin and
salivary glands, 847

Favre, P. A., 848

Fawitzki, A., 685

Fedeli, C., 686

Feder, L., 682

Fehling, H., 214, 808

Fehrsen, A., 338

Feigin, P., 721

Feinshmidt, J., 408

v. Fenyvessy, Bela, 751, 785

Fermi, CL, 255, 486, 505

Fernet, E., 855

Ferry, E., 904

Fick, A., 596

Fiessinger, N., 307, 364

Filchne, W., 440

Filhol, 670

De Filippi, F., 397, 534, 757

Fine, M. S., 692

Fingerling, G., 667

Fink, H., 295

Finkler, D., 41

Fischer, Ch., 140

Fischer, hmil, enzymes, 58, 59; specif-
icity of enzymes action, 62, 65; amino-
acids, 85, 140-145, 148, 152-155;
polypeptides, 86-89, 124, 132, 133,
136; protein hydrolysis, 113, 119, 120,
132, 133, 136, 507; ornithin, 162;
lysin, 163; diaminotrioxydodecanoic
acid, 165; purin bases, 186, 188-191;
pyrimidine bases, 194, 195; carbo-
hydrates, 197-202, 211, 212, 215, 216;
glucosamine, 218; glucuronic acid, 220;
isomaltose, 225; lactose fermentation,
654; uric acid, 698; urine purines, 711;
conjugated glucuronic acids, 751

Fischer, Hans, blood pigment derivatives,
295-297, 295; bile acids, 425, 427;
bilirubin, hydrobilirubin, urobilinogen
and urobilin, 429, 744, 746, 747, 429;
kpprosterin, 448; urobilinoids, 743;
bilirubic acid, 429

Fischer, H. W, 587

Fischer, Martin, 400

Fischler, E., 686

Fischler, M., 434, 744

Fisher, H. L., 694

Fiske, P. S., 36, 38

Fitzgerald, 476

Flack, M., 374

Flacher, Fr., 379

Flamand, CL, 152, 153, 734

Flanders, Fr., 723

Flatow, L., 738, 739, 779, 780

Flatow, R., 711

Fleckseder, R., 455

Fleig, C., 416, 498, 499

Fleischer, R., 402 •>

Fleischl, E., 303, 441

Fleischmann, W., 647

Fleischer, M. S., 320

Fleitmann, 79

Fletcher, W. M., 459, 585, 593

Flint, A., 448, 488

Floresco, N., 384, 388, 434

Fliickiger, M., 749, 750

Foa C., 455, 645, 659

Folin, O., cystine, 149; tyrosine test, 154;
blood alkalinity, 309; urea, 333, 335;
urine acidity, 677; nitrogen determina-
tion, 688; urea determination, 688-
690; urein, 691; methylurea, 691;
creatinine and creatine, 691-693, 696,
697; uric acid, 700, 708, 711/334;
hippuric acid, 723; urinary sulphur,
752, 766; ammonia, 768, 769; acetone,
825; protein metabolism, 910

Fordos, M., 365

LaForre.F., 179, 180, 185

Forrest, J. R., 553

Forschbach, J., 396

Forssner, G., 756, 819, 820

Forster, J., mineral metabolism, 72, 899;
transfusion, 344; water and metabol-
ism, 920; metabolism of sucklings, 925;
diets, 932, 939

Fosse, R., 681

Foster, M. L., 159

Foster, N. B., 694

Franckel, P., 465

Frankel, A., 685, 860

Frankel, Sigm., proteins, 79, 102; pep-
tones, 131; thiolactic acid, 151; his-
tidin, 160; albumin, 219; cephalin,
248; thyroids, 375; adrenalin, 380;
glycogen, 396; gastric juice, 476;
chondrosin, 548; brain phosphatides,
605, 609; brain analyses, 613, 614;
neottin, 630, kidney phosphatides,
673; hompgentisic acid, 738; chitin,
839; protein cleavage, 925

Frankel, W., 34

Framm, F., 120, 213, 332

Franchimont, A. P., 839

Frank, E., 265, 329-332

Frank, Fr., 702, 706

Frank, O., 535-537, 591. 910

Frankland, E., 885

Franz, Fr., 251

Frauenberger, Fr., 547

Frazer, J. C. W., 3

Fredericq. L., proteincoagulation 97;
serumglobulin, 260; hsemocyanin, 303,
304; blood coagulation, 313' blood
gases, 852, 861, 862, 864, 866

Frehn, A., 663

Fremy, E., 603, 636

Frenkel-Heiden, 360

Frenzel, J., glycogen, 391, 393; work and
fat destruction, 596, 598; meat, 600;
calories and nitrogen, 892

Frerichs, F. Th., synovia, 362, human

954

INDEX OF AUTHORS

bile, 437; saliva, 458; uric acid de-
composition, 705

Freudberg, A., 309

Freund, E., serumglobulins, 259; albu-
moses in blood, 263; hsematinogen, 299;
blood coagulation, 313, 320; glycogen,
395; digestion blood, 530; chlorine
determination, 759; lungs, 870; starva-
tion metabolism, 987

Freund, O., 897

Freundlich, H., 22; surface tension, 28;
adsorption, 29

Frey, W., 756

Freytag, Fr., cerebrosides, 364, 607, 610,
protagon, 606, 607, 610

Fridericia, L. S., 597

FriedenthaL H., 467, 525

Friedjung, J. K., 664

Friedlander, G., 525

Frienlander, P., 843

Friedmann, E., protein sulphur, 79;
thiolactic acid, 113, 151; albumoses,
134; isoleucine, 144; cystine, 148;
cysteinic acid and taurine, 148, 151,
i 442; adrenalin, 379; demolition of
fatty acids, 774, 776, 781; of aromatic
substances, 779-781, 786; furfurol,
784; acetone former, 820, 822

Friend, W. M., 274, 356

Fries, H., 593

Frohlich, 379

Fromherz, K., 735, 736, 780

Fromholdt, G., 743, 744

Fromm, E., 785

Fromme, A., 57, 476

Frommer, V., 823

Frpuin, A., thyroid gland, 377; gastric
juice, 463, 464; intestinal juice, 490-
492; pancreatic juice, 496

Frugoni, C., 405 •

Fubini, S., 849

Fuchs, D., 114, 154

Fiirbringer, P., 715, 791

v. Fiirth, O., peroxyproteic acids, 83;
xanthoprotein, 83; nucleic acids, 184;
cholin, 247; iodothyrin, 376; supra-
renin, 379; adrenalinglucosuria, 405;
cholic acid, 424; secretin, 498; bile
and fat splitting, 502; muscles, 566-
572, 589, 590; camosin, 575; smooth
muscles, 602; chitosin, 839; melanins,
841-843; tyrosinase, 843, peroxydase,
873

Fuld, E., rennin action, 57, 474, 650;
fibrin formation, 256, 318, 319, 324;
pepsin determination, 470; womans'
milk, 660, 662

Fuller, J. G., 902

Funk, C., amino-acid combination, 87,
88; glutamic acid, 146; urinary sul-
phur, 752; sugar determination, 809;
vitamine, 905

v. Funke, 595

Gabriel, S., cystine, 148; bones, 552;
teeth, 558; ovalbumin, 633; food-
value of aspargin, 912
Gache, J., 496
Gajrlio, G., 334, 582, 773
Galdi, F., 354
Galeotti, G., 96, 706
Galimard, J., 113
Galli, P., 267
Gallois, 580

Gamgee, A., nucleoproteins, 175; nucleic
acids, 182; blood pigments, 280;
intestinal juice, 490; protagon, 606,
607; pseudocerebrin, 611
Gammeltoft, S. A., 689, 690, 767

Ganassini, D., 455, 456, 709

Gansser, E., 278

Gardner, J. A., 448

Garrod, A. E., hsematoporphyrin, 295,
299, 797; alcaptonuria, 738; homo-
gentisic acid, 738, 739; urochrome, 740,
741; urobilin, 743, 745; uroerythrin,
748; cystinuria, 827

Gaskell, J. F.. 828

Gassmann, Th., 552, 557, 558

Gatin-Gruzewska, Z., glycogen, 19, 20,
390, 391; seralbumin, 261

Gaube, T., 848

Gaule, J., 340

Gaunt, 41

Gautier, A., ptomaines, 47; arsenic, 72,
269, 336, 368, 373, 838;" glycogen, 392;
fat formation, 562; muscles, 578;
protein of hen egg, 633, 634; xantho-
creatininine, 698; fluorine, 552, 558

Gautier, CL, 252, 253, 654, 744

Gawinski, W., 756

v. Gebhardt, F., 591, 909

Geelmuyden, H. C., sugar in urine, 814,
816; acetone bodies, 819, 820, 822, 826

Geiger, W., 145

Geissler, 791

Generali, F., 374

Gengou, O., 64, 314, 318, 320

v. Genser, 665

Gentzen, M., 728

Geoghegan, E. G., 609, 610, 614

Gephart, F., 245, 680, 689, 769

Geppert, J., blood alkalinity, 309; respira-
tion, 860, 869, 890; alcohol, 921

Gerard, E., 446, 699

Gerber, N., 662

Gerhardt, C., 824

Gerhardt, D., 744, 745

Gerhartz, H., 870

Gerngross, O., 195

Gertner, W., 416

Gessard, C., 843

Gewin, J. W. A., 475

Geyer, J., 806

Geyger, A., 735

Giacosa, P., mucins, 167; blood pigments,
302; frog egg, 636; iron in urine, 770;
aromatic substances, 779

INDEX OF AUTHOKS

955

Giaja, J., 71, 231

Gibson, R., 103

Giertz, H., 104, 177

Gies, W. J., elastin, 116-118; hexone
bases, 164; mucin substances, 168,
169, 472, 544, 545, 551; lymph, 349-
351; pancreatic juice, 500; ligaments
and tendons, 545; bones, 551; pro-
tagon, 606, 608; phrenosin, 611;
urein, 691

Gigon, A., polypep tides, 65; diabetes,
405, 409; amino-acid supply, 721,
722; amino-acids in urine, 756; basal-
requirement, 898, 931; metabolism,
929-931

Gilbert, 531, 563

Gill, F. W., 689, 752

Gilson, E., 242, 245, 839

Ginsberg, S., 534

Ginsberg, W., 754, 756

Githens, Th., St., 270

Giunti, L., 773

Gizelt, A., 498

Gjaldbak, I. K., 58, 135, 165

Glaessner, K., pseudopepsin, 466, 489,
490; erepsin, 493; pancreatic juice,
500, 509

Gleiss, W., 593

Glendinning, T. A., 55

Gley, E., iodine in blood, 269; blood
coagulation, 324; lymphagogues, 351;
thyroidea, 374; pancreatic juice, 496,
498; heart muscle; 599, vesiculase,
622

Glikin, W., fat, 233; lecithin, 241, 244,
245, 663; liver nitrogen, 387; choles-
terin, 445; milk, 663

Glund, W., 87

Gmelin, L., 432, 458

Gmelin, W., 465

Gogitidse, S., 669

Goldmann, E., cystine and cystinuria,
149, 827, 828; iodothyrin, 376

Goldmann, F., 813

Goldschmidt, C., 687

Goldschmidt, F., 133

Goldschmidt, H., 36, 456

Golodetz, L., 844

Gompel, M., 65

Gonnermann, M., 140, 508

Goodbody, W., 515

Goodwin,' R., 130

Gorodecki, 443

Gortner, R. A., 841, 842

v. Gorup-Besanez, E. F., 356, 437, 838

Gossmann, H., 495

Gosio, B., 582

Goto, M., 110, 111, 709

Gottlieb, R., urea, 333; bile, 435; creatine
and creatinine, 573, 692, 698; urine
purines, 711; oxyproteic acid, 754;
iron in urine, 770

Gouban, F., 366, 367

Gourlay, F., 373

deGraaf, C. J. H., 792, 793

Grabowski, J., 297

Graebe, C., 524

Graffenberger, L., 554

Grafe, E., 54, 912, 919

Graham, D., 919

Graham, Th., 13, 14, 18, 31, 96

Granstrom, E., 718, 769

Grawitz, E., 340

Grebe, F., 396

Green, E. H., 121

Green, J. R., 255, 316

Greer, James Richard, 348, 349

Gregersen, J. P., 903

Gregor, A., 698

Gregor, G., 766

Grehant, N., 333, 335, 684, 773

Griesbach, W., 333

Griffiths, A. B., 614, 758

Grimaux, E., 85, 86, 323

Grimbert, L., 677, 746, 747

Grimm, F., 744

Grimmer, W., 482, 490, 874

Grindley, H. S., 689, 752, 936

Griswold, W., 457

Grober, J., 467

Grober, A., 732

Grohe, B., 485

Groll, S., 339

Grosjean, A., 324

Gross, A., 358, 629

Gross, O., pepsin determination, 469;

trypsin determination, 506; lens, 619;

alkaline earths in urine, 769
Gross, W., 463, 780, 818
Grossenbacher, H., 372
Grosser, P., 733
Grossmann, H., 808
Grouven, H., 599
Grube, K., 396, 400
Gruber, D., 229
Gruber, M., 881, 920
Griibler, G., 94
Griinbaum, D., 642
Griindler, J., 777
Griinhagen, A., 361
Griitzner, B., 790
Griitzner, P., pepsin determination, 469;

gastnc contents, 479, 480; Brunner's

glands, 489, 490; pancreatic diastase.

501

Grund, G., 20S, 209, 389, 919
Grutterink, A., 792, 793
Gryns, G., 7
Gscheidlen, R., rhodan, 455, 456, 751;

lactic acid, 582; urea, 684
Gubler, A., 348, 349
Gudzent, F., 707, 708
Gumbel, Th., 77
Giinther, G., 622
Gtirber, A., ion permeability, 8, 310;

serumalbumin, 261, 263; serum, 267;

bile, 440; amniotic fluid, 642
Guerin, G., 758

956

INDEX OF AUTHORS

Guest, H. H., 106, 107 .

Guggenheim, M., 50, 380

Guigan, H., Me, 399

Guillemonat, A., 371, 387

Guinochet, E., 358

Guldberg, C. M., 32

Gulewitsch, W., arginine, 161, 162;
thymin, 195; choline, 247; trypsin,
508; meat extractive, bases, 575-578

Gullbring, A., 421

Gumilewski, 491

Gumlich, E., 680, 685, 761

Gundermann, K., 82

Gunning, J. W., 823

Gusserow, A., 716, 717

Guth, F., 233

Guyenot, E., 457

Gyergyai, A., 912

de Haan, J., 306

Haas, E., 528, 910

Haberlandt, L., 265

Habermann, J., proteins, 83; amino-
acids, 143, 146, 153

Hamalainen, J., glucuronic acid conjuga-
tion, 785; sulphur elimination, 882,
883; "protein catabolism, 907

Handel, M., 549

Hansel, E., 487, 663

Baser, 771

Hausermann, E., 340

Hafner, A., 233

Hagemann, O., skin breathing, 849;
blood gases, 851; metabolism, 926,
927, 929

Hagen, W., 193

Hahn, M., fermentation, 41; casein
digestion, 651, urea formation, 683,
684

Haig, A., 701

Haiser, F., inosinic acid, 178-180; 182,
183; carnine, 577

Haldane, J., blood pigments, 285, 286,
302; amount of blood, 343; oxygen
tension, 862, 863

Hall, W., purine bases, 193, 520, 572;
iron absorption, 340; amino-acids in

urine, 757

Hallauer, B., 440

Halle, W. L., 380

v. Hallervorden, E., 682, 685, 767

Halliburton, W. D., protein coagula-
tion, 97; dextrins, 234, choline; 246;
fibrin ferment, 256; serum albumin,
261; blood serum, 270; stroma of
blood corpuscles, 274; tetronerythrin,
304, 843; leucocytes, 306; blood
coagulation, 323; pericardial fluid,
356; cerebro-spinal fluid, 360; liver,
382; glycogen, 391; 'pancreatic renin,
509; myxcedema, 545; bone marrow,
553; muscles, 566-570; brain proteins,
604, 605; diseases of the nervous sys-
tem, 614; kidneys, 673

Halpern, H., 372

Halsey, J. T., 152

Ham, C. E., 301

Hamburger, C., 265, 456

Hamburger, E. W., 435

Hamburger, H. J., blood corpuscles, 6-8.
273, 304, 305, 311; blood alkalinity,
269; blood serum, 271; phagocytoses,
306; blood alkalinity, 309, 310; lymph
formation, 350, 351; ascitic fluid^ 359;
intestinal juice, 490, 491; enterokinase,
496; absorption", 542-543

Hammarsten, O., rennin action, 54, 63,
473, 474, 475, 650; nucleoalbumin,
105; mucin substances, 168-169;
helicoproteid, 173; nucleoproteins,
208, 383, 494, 495 ; fibrinogen,
fibrin and blood coagulation, 253,
254, 257, 263, 316; fibrin globulin,
258; globulins, 259-261, 263, serum-
albumin, 262; blood plasma and serum,
267-270, haematoporphyrin, 299, 797,
798; gases of lymph, 346, 856; transu-
dates, 354, 356, 357, 359; synovia, 362;
human bile, 414, 437, 438; bile of
various animals, 417, 427, 439; bile acids
417, 420-427, 438; bile pigments, 267,
432, 434, 801; phosphatides, 435,
439; saliva, 457; pepsin, 466, 467;
trypsin, 503; pseudomucin, 625, 626;
perch eggs, 630, 636; casein, 649;
lactoprotein, 653; urea in bile, 679;
protein in urine, 793; sugar determina-
tion in urine, 806

Hammerbacker, F., 458

Hammerl, H., 521

Hammerschlag, A., 308, 309

Hammerschlag, G., 637

Handovsky, H., 18

Henriot, M., lipases, 59, 265; d]iabetes,
404; respiration quotient, 563; respira-
tion, 869

Hansen, C., lecithin, 242; protein syn-
thesis, 529; fatty tissue, 559; fat of
yolk, 630; fat of milk, 669; food value
of albumoses, 912; asparagin, 912

Hansen, W., 233

Hanssen, O., amyloid, 171, 172, 173,
547

Harden, A., co-enzyme, 52, 205; phos-
phoric acid ester, 60, 205; dioxyacetone,
205; glycolysis, 407, 583

v. Hardt-Stremayr, 77

Hardy, W. B., colloids, 15, 20, 21, 25, 26,
30; ovalbumin, 634

Hari, P., 54

Harkink, L, 594, 693

Harley, V., sugar of blood, 398; intestinal
putrefaction, 515; pancreas, 532, 539,
540; large intestines, 541, urinary
pigments, 740, 744

Harms, H., 552

Harnack, E., ash-free protein, 95; iodo-
spongin, 122; blood pigments, 286,

INDEX OF AUTHORS

957

287; hydramnion, 642; oxalic acid
poisoning, 728, 729; sulphur in urine,
752; demolition of halogen substi-
tuted methane, 777; galic and tannic
acid, 785

Harries, C., 83

Harris, J. F., 77, 179, 185

Hart, A. S., 116, 117

Hart, E., protein nitrogen, 77; albumoses,
134; histidine, 160; hexone bases, 164;

Hart, E. B., 902

Hartley, P., 384, 385

Hartogh, 412

Hartung, C., 636

Hartwell, J. A., 130

Hasebroek, K., lecithin, 245, 515; peri-
cardial fluid, 356; digestion products,
472

Haskins, H. D., 680, 683, 691

Haslam, H. C., salting out of proteins,
95, 250; albumoses, 139

Hasselbalch, K. A., reaction of blood, 75,
310; methsemoglobin, 283, 284; egg-
hatching, 637; sugar determination,
814; oxygen intake, 859; oxygen
tension, 867

Hatcher, R. A., 397

Hauff, 665

Hausmann, W., nitrogen in proteins, 77,
119; haBmatoporphyrin, 295; kopros-
terin, 448; cholesterin, 449-450

Hawk, P. B., keratin, 112; bones, 551;
Eck's fistula, 683; sugar determina-
tion, 804; hair, 838; metabolism, 892,
897, 920

Hay, M., 358

Haycroft, J. B., protein coagulation, 97;
blood coagulation, 251, 313; diabetes,
403; biliverdin, 433

Hayem, G., 307, 342

Heckel, F., 141

Hedenius, 116, 434

Hedin, S. G., blood corpuscles, 7, 8;
autolysis, 42-44; adsorption, 49, 50;
enzymes, 54-57; retardation of enzyme
action, 63-65; rennin-antirennin bind-
ing, 68; elastin, 116; histidine, 159,
160; arginine, 161; lysine, 163; blood.
326; haBmatocrit, 326; lienases, 371;
rennin zymogeiv. 473; rennin enzyme,
474; muscle pro tease, 571

Hedon, E., pancreas diabetes, 406;
absorption of sugar, 533; of fat, 539,
540

Heffter, A., liver, 385; muscle, 565, 589;
lactic acid, 593; hyposulphite in urine,
753; foreign substances in urine, 773;
hippuric acid synthesis, 783; reduc-
tions, 877

Heger, P., 381

Heidenhain, M., 101

Heidenhain, R., lymphagogues and lymph,
11, 345, 349-351; transudates, 353;
bile, 414, 415; saliva, 453, 459; stom-

ach, 461, 476, 477; pyloric secretion,
477; pancreas and its secretion, 494,
495, 497, 503; absorption, 528, 534;
smooth muscles, 602

Heilner, E., protein assimilation, 525;
metabolism, 920, 929; specific dynamic
action, 930

Heinemann, H. N., 597

Heinsheimer, Fr., 476

Heintz, W., 235, 582

Heiss, E., 555

Heitzmann, G., 555

Heckma, E., intestinal juice, 490-493,
enterokinase, 496, 497

Hele, T. Sh., 738, 828

Heller, Fl., protein test, 99, 789; uroxan-
thin, 727; urinary pigments, 740;
blood test, 796; urinary calculi, 834-
835

Hellgren, W., 931

Hellwig, 602

Helm, 461

Helme, W., 882, 883, 907

Hetper, J., 292

Hemmeter, J. C., 463, 464, 519

Hempel, E., 894

Henderson, L. J., 309, 675, 677

Henderson, Y., 77, 402, 529

Henkel, Th., 647

Henneberg, W., 510, 918

Henninger, A., 137

Henogque, A., 303

Henri, V., cataphoresis, 50, 51; enzymes,
56; white of egg, 65; saccharase, 65;
diastase, 71

Henriques, R., 239

Henriques, v., plastein, 58, 135; formol
titration, 165; lecithin, 242; lecithin
sugar, 331, 332; protein synthesis,
529; fat tissue, 559; fat of yolk, 630;
fat of milk, 669; urea determination,
689, 690; hippuric acid, 723; urine nitro-
gen, 756; ammonia, 768; gases of blood,
851; metabolism in lungs, 858; food
value, albumoses, 912; asparagin, 912

Hensel, Marie, 726

Hensen, V., 349, 856

Henze, M., gorgonin and iodogorgonic
acid, 123, 146; protein of octopodes,
175; ha?mocyanin, 303, 304; blood
corpuscles in ascitic fluid, 304; liver,
388; spongosterin, 448; muscles, 603

Heptner, F. K., 438

1'Heritier, 665

Herlaut, L., 185

Herlitzka, A., 455

Hermann, E., 72

Hermann, L., blood in starvation, 339;
formation of feces, 521; muscular
work, 592; allantoin, 716, 717

Heron, J., 229, 456, 492

Herrmann, A., 507, 700, 701

Hermann,. Edm., 336

Kerry, A., 325

958

INDEX OF AUTHORS

Herter, C. A., indol and skatol, 159, 732;

urorosein, 732, 733
Herter, E., saliva, 458; ethereal sulphuric

acids, 724, 726, 784; oxybenzoic acids,

783; oxygen tension, 861
Herth, Rj 137
Hervierux, Ch., indol and indican, 266,

728-732; skatol red and urorosein,

733; uroerythrin, 748; glucuronic acids,

817

v. Herwerden, M., 651
Herzen, A., spleen and digestion, 372;

gastric juice secretion, 462; pancreatic

juice, 496, 498
Herzfeld, A., 203, 224
Herzfeld, E., 267, 433
Herzog, R. O., enzymes, 41; histidin,

160; lactic acid, 582, 616; oxydase

reactions, 875
Hess, K., 297
Hess, L., 43
Heubner, O., 258, 569
Heuss, E., 847
Hewlett, A. W., 541
Hewlett, R. T., 97
Hewson, W., 6, 313
Heyl, F. W., 185
Heymann, F., 626
Heynsius, A., 128, 432-434
Hiestand, O., 245
Hilbert, P., 779
Hildebrandt, H., oxalic acid, 716, 773;

amino-benzoic acids, 783; toluols, 783;

glucuronic acid conjugation, 785, 786
Hildebrandt, P., antiemulsin, 64; mam-
mary glands, 644, 668; glucuronic

acids, conjugation, 750
Hildebrandt, W., 267, 744
Hildesheim, 932
Hildesheimer, A., 726
Hilger, 171
Hilger, A., 579
Hill, A. V., 277, 278
Hiller, E., 554
Hirsch, Rahel., glycolysis, 408; hippuric

acid, 721; amino-acids in urine, 756
Hirsch, Paul, 722, 912
Hirschberg, A., 307
Hirschfeld, E., 485

Hirschfeld, F., work and nitrogen elimina-
tion, 595; uric acid, 700; acetone

bodies, 819; protein catabolism, 903,

915, 934; daily ration, 939
Herschfeld, H., 791
Hirschl, J. A., 806
Hirschler, A., 517. 518, 519, 724
Hirschstein, L., 700, 756
His, W., 550

His, W. (jr), 193, 707, 787
Hlasiwetz, H., 146
Hochhaus, H., 340
Hober, R., osmotic pressure, 13, 15;

precipitation properties of ions, 25;

alkalinity of blood, 310, 311; absorp-

tion, 542; permeability, 588; urine
acidity, 677

Hone, J., 800

Hoernes, Ph., 78

Horth, F., 582

v. Hoesslin, H., 909-911, 917

Hoyrup, M., 161

Hofbauer, L., 456

van't Hoff, J., osmotic pressure, 2, 3;
catalysis, 33, 35; glucosides, 59

Hoffman, Ch., 152

Hoffmann, A., 722

Hoffmann, F. A., transudates, 354, 357,
361, 362; sugar in blood, 398; glucos-
curia, 400

Hoffmann, J., 675

Hoffmann, P., 770

Hofmann, tyrosin test, 153

Hofmann, Fr., 559-561

v. Hofmann, Karl, 621

Hofmann, K. B., 116, 124

Hofmeister, F., gelatin, 31; cell enzymes,
44; protein nitrogen, 77, 82; amino-
acids, binding of 85; removal of pro-
teins, 102; collagen and gelatin, 118,
120; albumoses and peptones, 133, 136,
139; grouping of proteins, 159; serum-
globulins, 259, 261; pus, 364; actions
of stomach, 479, 480; protein absorp-
tion, 527, 528; assimilation limit, 533;
blood serum and earthy phosphates,
557; ovalbumin and protein crystalliza-
tion, 633, 634; urea formation, 684;
creatinine, 694; protein in urine, 793;
lactose in urine, 815

Hofmeister, V., 480, 510

Hohlweg, H., 267, 740, 741

Holde, D., 233

Hotlinger, A., 328

Holmgren, E. S., 452, 806

Holmgren, Fr., 852, 866

Holmgren, L, 489, 566, 569

Holobut, Th., 696

v. Hoist, G., 169, 354, 362

Holsti, O., 903

Honore, Ch., 526

v. Hoogenhuyze, C. J., creatine and
creatinine, 573, 594, 691-694

Hooker, D., 351

Hooper, C. W., 442

Hopkins, F. G., halogen proteins, 82;
protein reaction, 100; tryptophan, 155-
158; protein crystallization, 263, 633,
634; lactic acid formation, 533; uric
acid, 699, 710, 711, 843; urobilin, 743,
746; Bence-Jones protein, 792; butter-
flies, 843

Hoppe-Seyler, F., oxydation, 41; ovovi-
tellin, 105; collagen, 118; proteins,
167; nucieins, 175; xanthin, 189;
lecithin, 243-245; blood plasma, 269;
blood corpuscles, 274, 304, blood pig-
ments, 275-278, 281, 283, 286, 287-
290, 293-302; urobilinoids, 295, 743;

INDEX OF AUTHORS

959

tlycogen, 307, 390; blood analysis,
28; chyle, 347; pericardial fluid, 356;
Eus, 363-365; strumacystica, 374;
ilc, 435, 436, 440; excretin, 523;
cartilage, 549, 550; bones and teeth,
553, 558; lactic acids, 583, 585, 593,
748; retina, 615; ovovitellin, 628;
milk, 647, 648, 656; bile acids in urine,
800; inosite, 817; chitin, 839; sebum,
844; skin breathing, 849; respiration
apparatus, 868; oxydation, 871

Hoppe-Seyler, G., blood pigment deter-
mination, 302; phenol elimination,
725; indoxyl, 728-732, urobilin, 744,
745, 747

Hopwood, A., 87

Horbaczewski, J., keratin, 112; elastin,
116, 117; purine bases, 188, 189;
uric acid, 373, 698, 700-702; urostealith,
834, 835; metabolism, 904

Hornborg, A. J., 464, 465

Honre, R, M., 251

Horodynski, W., 334, 336

Horowitz, L. M., 520

Horton, E., 59

Hoshiai, Z., 787

Hougardy, A., 261

Howe, P. E., 892

Hovvel, W. H., 257, 266, 319, 322

Hryntschak, Th., 723

Huber, A., 251, 255

Hudson, C. S., 55, 57, 199

v. Hiibl, 236, 238

Huebner, R., 159

Hiifner, G., leucin, 142; blood pigments,
277-279, 283-286, 289; spectrophoto-
metry, 302, 303; bile, 420; gases
of bird egg, 636; urea determination,
690; oxygen tension, 859, 861, 862;
air bladder, 867

Hiirthle, K., 264

Hugounenq, L., biliverdin, 434; haemato-
gen, 629; clupervin, 636; ash of milk,
and of child, 666

Huiskamp, W., fibrinogen and fibrin for-
mation, 253, 254, 256, 258, 320; nucleo-
protein in blood, 258; thymus histone,
366, 367

Hulett, G. A., 29

Hultgren, E. O., utilization of nutr tives,
531,535,542, 543; diete,9c2, 936

Hummelberger, F., 126

Humnicki, V., 448

Hundeshagen, F., 122, 242

Hunter, A., 106, 111, 134, 758

Hupfer, Fr., 72l

Huppert, H., Schiitze's rule, 57; diges-
tion products, 133; glycogen, 307, 391;
bile pigment reactions, 432; pepsin de-
termination, 469; flesh, 601; urea, 686;
uroleucinic acid, 739; urine albumin,
794; laiose, 814; acetone, 825

Hurtley, W. H., 287, 739

Hurwitz, S. H., 253

Husson, 671

Hutch inson, Rob., 553

Hybbinette, S., 748

Ibrahim, J., 497

Ide, 578

Ignatowski, A., 827

Inagaki, C., serum albumin, 262; serum-
globulin, 270; blood corpuscles, 341;
protein absorption, 528; muscle rigor,
590

Inoko, Y., 277

Inouye, K., 179, 574, 748, 749

Inoye, Z., 476

Irisawa, T., 334, 749

Ishihara, H., 423

Ito, M., 791

Iversen, A., 621, 623

Iwanoff, 763

Iwanoff, L., 182, 206, 508

Izer, G., autolysis, 43; uric acid, 704-706

Jaarswelde, G. J., 723

Jablonsky, J., 499

Jackson, H. C., 739

Jacobs, W. A., serine, 145; nucleic acid,
178-185; ribose, 211, glucothionic
acid, 369; sphingosin, 611; cerebronic
acid, 611

Jacobsen, A., 331

Jacobsen, C. A., 673

Jacobsen, O., 437, 438, 779

Jacoby, M., autolysis, 42, 45, 869; phos-
phorous poisoning, blood, 253, 255,
256; pepsin determination, 470; tryp-
sin determination, 506; fertilization,
640; uric acid demolition, 706

Jacubowitsch, 458

Jaeckle, H., 233, 235, 559

Jaderholm, A., 285, 289

Jaeger, A., 867, 843

Jaffa, M. E., 936, 939

Jaffe, M., ornithine and ornithuric acid,
162, 783; bile pigments, 432, 433;
urobilin, 521; creatine and creatinine,
574, 575, 693, 695; phenylsemicar-
bazide, 687; urethane, 692; uric
acid, 703, 704, 708; indican in urine,
727-730; kyenuric acid, 739; urobilin,
740, 741, 744, 746; conjugated glu-
curonic acids, 751, 786; behavior of
organic substances, 778, 779, 783, 786;
furfurol, 784; thiophene, 784; guani-
dine acetic acid, 787

de Jager, L., 675, 677, 734, 756, 769

Jahnson-Blohm, G., 63

Jakowsky, M., 513

v. Jaksch, R.. blood alkalinity, 309;
urea, 333; brain, 605; volatile fatty
acids, 748; melanin, 799; pentoseuria,
816; acetone, 818

Jalowetz, E., 226

Jamieson, G. S., 123

Janney, N., 768

960

INDEX OF AUTHORS

Jansen, B. C. P., 491, 581

Jappelli, G., 453

Jaquet, A., 277

Jastrowitz, H., 469, 716, 757

Jastrowitz, M., 208, 815

Jelinek, J., 407, 583

Jensen, P., 581, 587

Jerome, W., Smith, 753

Jerusalem, E., nucleic acids, 184; lactic
acid, 488; melanins, 841-843

Jesner, S., 617

Jess, A., 619

Jessen-Hansen, H., 165, 809

Joachim, Jul., 259, 353, 357, 359

Jochmann, G., 307

Jodlbauer, A., 50, 552

Johansson, F., 757

Johansson, J., E., serumalbumin, 262;
tissue and gas metabolism, 597, 898,
927-931, 936; digestion work and
specific dynamic action, 930

Johnson, St., 691, 694

Johnson, T. B., protein sulphur, 79;
thiopplypeptides, 87; nucleic acid, 185;
cytosine, 194

Jolin, S., 376

Jolles, A., pentoses, 209, 210, 816; bile
pigments, 431; milk, 664; urine
acidity, 677; urinometer, 679; uric
acid, 711; urobilin, 743; albumin in
urine, 787; nucleohistone, 795; fructose
determination, 814

Joly, 670

Jolyet, 851

Jones, D. B., 84, 106, 107

Jones, W., autolysis, 44- nucleopro-
teids, 175; nucleic acids, 182, 183;
thy mine, 195; purin substances and
their enzymes, 368, 371, 373, 701-703,
706

Jonescu, D., 65

de Jonge, D., 846, 847

Jornara, D., 847

Josephsohn, A., 739

Jlinger, E., 427

Jiirgensen, E., 20, 97

Jungfleisch, E., 27, 585

Junkersdorf, P., 413

Juschtschenko, A., 373

Just, I., 339

Justus, J., 71

Juvalta, N., 778

Kaas, K., 78, 634
Kahn, R., 402
Kalb, G., 918
Kalberlah, Fr., 585, 818
Kalmus, E., 289
Kanitz, A., 506
Kapfberger, G., 53
Kareff, N., 253
Kashiwabara, M., 710
Kast, A., intestinal putrefaction, 519;
urinary sulphur, 752; chlorine excre-

tion, 758; halogen substituted methane,
777; perspiration, 848

Kastle, J. H., 56, 59

Kato, Kan, 636

Katsuyama, K., 161, 334, 582, 896

Katz, A., 744

Katz, J., 598, 599

Katzenstein, A., 926, 927

Kauder, G., 261

Kaufmann, M., glycogen, 397; sugar of
blood, 398; sugar formation, 412;
fat formation, 562; urea, 572; sugar
utilization, 592; lactose, 670; urea
formation, 684; metabolism - experi-
ments, 890

Kauffmann, M. (Frankfurt), 247, 360, 713

Kaufmann, Martin, 894, 912

Kaup, J., 595

Kausch, W., 395

Kautzsch, K., polypeptides, 87; glu-
tamic acid, 147; proline, 154; adrenalin
379; digestion, 484, 513

Kaznelson, H., 464

Keller, A., 757

Keller, Fr., 525

Keller, W., 783

Kellner, O., utilization of nutriments,
531; protein catabolism in work, 595:
asparagin, nutrition value, 912; diets,
932

Kelly, A., 839

Kempe, M., 157, 158

Kennaway, E. L., 83, 109, 700, 715

Kendall, A. J 520

Kermauner, F., 521

Kerner, G., 694

Kiermayer, 211

Kiesel, A., 247

Kikkoji, T., 640

Kiliani, H., 197

Kirchmann, J., 911

Kirk, R., 739

Kirschbaum, P., 613

Kistermann, C., 806

Kitagawa, F., 608, 611

Kittsteiner, C., 847

Kjeldahl, J., methods for nitrogen deter-
mination, 687, 688

Klages, A., 427

Klatte, F., 205

v. Klaveren, H. K. L., 288

Klecki, K., 521

Klein, W., 675

Kleine, F., 752, 767

Kleiner, 1. S., 717

Klemensiewicz, R., 476, 477

Klemperer, G., urochrom, 740, 741;
oxalic acid, 773; protein metabolism,
903, 915, 934

af Klercker, O., 691, 692, 694, 815

Klingemann, F., 670

Klingemann, W., 484

Klingenberg, K., 779

INDEX OF AUTHORS

961

Klug, F., tryptophan, 155; pepsin, 466,
472; trypsin, 502, 503; phosphoric
acid excretion, 762

v. Knaffl-Lenz, E.,, 124, 390

Knapp, K., sugar test, 214; sugar deter-
mination, 808, 811

v. Knieriem, W., cellulose, 510; urea

formation, 682; uric acid formation, 704

Knopfelmacher, W., 233, 559

Knoop, F., histidin, 159, 160; methyl-
imidazol, 201; demolition of fatty
acids, 774, 781; synthesis of amino
acids, 775, 786; demolition of aromatic
substances, 779, 781

Knop, W., 690

Knorr, L., 297

Kobert, H. .W., 276

Kobert, R., cyanmethsemoglobin, 285;
iron in urine, 770; melanins, 841

Kobrak, E., 662

Koch, W., lecithins, 241-245; cephalin,
248; brain analyses, 605, 607, 609,
612-614; milk, 663

Kocher, Th., 375

Kochs, W., 722

Kochmann, M., 902

Kobner, 440, 441

Koefoed, E., 647

Kohler, A., 600, 892, 912

Koelichen, K., 35

Koelker, A. H., 455

Konig, J., 599, 657-659

Koppe, H., blood corpuscles, 7, 8, 273,
305, 326; hydrochloric acid of stomach,
477

v. Korosy, K., digestion, 484, 513;
parenternally introduced protein, 525;
digestion blood, 527, 530

Koster, H., 650

Koettgen, E., 616

Kohler, R., 708

Kohlrauch, A., 776

Kojo, K., 795

Kolisch, R., 698, 795

Kondo, K., indol and skatol, 159; lactic
acid, 333, 585; cholesterin, 385; chon-
droitin sulphuric acid, 548, 549; urinary
phosphorous, 757

Koraen, G., 598, 929

Koranyi, A., 13, 311, 351

Korkunoff, A., 910

Korn, A., 469

Korndofer, G., 572

Korowin, 500

Kossel, A., protein nitrogen, 77, 78;
nitration of protamines, 83; arginase,
89, 161, 574, 681, 682; histones, 107-
109; protamines, 109-111, 137; pro-
tein hydrolysis, 122, 141, 145, 154;
histidin, 159, 160; hexone bases, 161,
164; arginine, 161, 162; agmatine, 162;
ornithin, 163; lysin, 163; nucleopro-
teins, 174; nucleic acids, 179, 185;
purin bases, 186, 187, 190-193, 367,

386, 495; pyrimidine bases, 194, 195;

primary and secondary cell constituents,

240; haemoglobin, 277; nucleohistone,

307, 366; blood plates, 308; pus, 364;

protagon, 606, 607, 610; cerebrosides,

607, 610; ichthulin, 630
Kossler, A., 725
Kostin, S., 287
Kostytschew, S., 184, 185
Kotake, Y., 179, 393, 394, 711, 780
Kowalewski, K., 132, 185, 704, 767
Kowarski, A., 572
Kraft, F., 17
Kramm, W., 695
Kranenburg, W. R. H., 476
Kraske, B., 333
Krasnosselsky, T., 369
Kratter, Jul., 560
Kraus, Fr., 384, 410, 411, 583
Krause, R. A., 692
Krauss, E., 730
Krauze, L., 83
Krawkow, N. P., amyloid, 171, 172;

chitin, 839, 840
Kreglinger, G., 341
Krehl, L., 795
Kreis, H., 233
Kresteff, S., 477
Kreuzhage, C., 595
Krieger, H., 261, 263, 763
Krimberg, R., 575-578
Kristeller, L., 692
Krober, E., 209
Kronig, B., 71
Krogh, A., oxygen in blood, 859, 862, 864,

867; microtonometer, 861, 864; car-
bon dioxide tension, 864; metabolism

experiments, 868, 881
Krogh, M., 690, 862, 864, 866
Kronecker, F., 723
Krshyschkowsky, K., 463
Kriiger, A., 79, 120, 230
Kriiger, Fr., modification of proteins, 97;

haemoglobin, 280; leucocytes, 305;

blood, 335, 336; spleen, 371; liver,

388; sulphocyanides in saliva, 455;

milk, 654
Kriiger, M., purin bases, 186, 188; in

feces, 520; in urine, 711-715; ammonia,

768

Kriiger, Th. R., 136, 578, 598
Krug, B., 918
Krukenberg, F. C. W., keratinalbumoses,

113; skeletins, 121; cornein, 122;

cornicrystallin, 123; hyalogens, 170;

171; lipochrome, 267; hsemoerythrin,

303, 304; muscle extractives, 572, 577;

bird egg, 636; uroostealith, 834-5;

bird feathers, 843
Krumbholz, C. J., 536
Krummacher, O., sugar formation, 412;

work, 595; calorific power, 889; protein

metabolism, 909; nutritive value of

gelatin, 911

962

INDEX OF AUTHORS

Krzemecki, A., 82

Kudo, T., 476, 479, 506

Kiibel, F., 457

Kiihling, O., 784, 785

Kuhne, W., enzymes, 40; neurokeratin,
112, 605, 614; gelatin, 119; albumoses,
and peptones, 128, 129, 132, 135-139;
paraglobulin, 258; haematoidin, 301;
glycogen, 390; gastric digestion, 472,
485; pancreas and its enzymes, 500,
502, 503, 508, 509; fat emulsion, 536;
muscles, 566-568, 589; smooth mucles,
602; pigments of the eye, 615, 616;
corpora lutea, 623

Kulz, C., 355

Kiilz, E., cystine, 147; pentoses, 208, 815;
isomaltose, 225; glycogen, 390-394,
397, 592; diabetes, 400, 403; saliva,
454, 456; gastric juice, 476, 477;
pancreatic diastase, 500; gases of milk,
664; conjugated glucuronic acids, 777,
785; oxybutyric acid, 826

Kulz, R., 283, 857

Kueny, L., 215

Kiister, F. W., 28

Kiister, W., blood pigments, 283, 289-
298, 430; bile pigments, 425, 428-431,
433, 434, 429

Kiittner, S., 652

Kuhn, 617

Kuliabko, A., 494, 691

Kullberg, S., 205

Kumagai, T., 737

Kumagawa, M., fat determination, 238;
fat formation, 562; sugar determination,
809; protein metabolism, 903, 915, 934

Kunkel, A. J., arsenic, 72; carbon
monoxide blood test, 287; iron prep-
arations, 340; bile, 435, 443; iron in
urine, 770

Kuprianow, J., 582

Kurajeff, D., coagulose, 58, 135; halogen
protein, 83; protamine, 109; trypto-
phan, 155

Kurbatoff, D., 237

Kurpjuweit, O., 520

Kusmine, K., 351

Kusumoto, Ch., 448, 449

Kutscher, Fr., protein nitrogen, 77;
gelatin, 83; proteolysis, 106, 107, 122,
146; histone, 107, 108; protamine, 109;
digestion products, 132; histidine, 159,
160; arginine, 161, 162; agmatin, 162,
lysine, 163, 164; hexone bases, 164;
aporrhegmen, 166; cytosin, 194;
thymin, 368; erepsin, 493; guanidine,
507; choline, 507; intestinal digestion,
513; absorption, 527, 528; bases of
meat extract, 575, 576; methylguani-
dine, 698; urinary bases, 757, 826, 828

Kuwschinski, P., 499

Kyes, P., 241

Laache, S., 342

Ladenburg, A., 621
Laidlaw, P. P., 301, 380, 843
Laitinen, T., 309
Lambling, E., 680
Lampe, A., 912
Lampel, H., 78, 126
Lanceraux, 406
Landau, A., 361
Landauer, A., 518, 899, 920
Landergren, E., utilization of nutriments,
531, 535; metabolism, 894, 903, 914;
diets, 932, 935
Landois, L., 275, 343
Landolt, H., 841
Landsteiner, K., 69
Landwehr, H., 749
Lane-Claypton, J. E., 43
Lang, G.,' 484, 793
Lang, J., 151, 349
Lang, S., 703, 704, 776
de Lange, C., 242, 664
Lange, F., 818, 819
Langer, L., 235
Langgaard, A., 660
Langhaus, A., 230
Langhaus, Th., 301
Langbeld, K., 81, 426
Langley, J. N., 457, 459, 477
Langstein, L., carbohydrates in proteins,
84, 168, 262; digestion products, 132;
skatosin, 159; fibrin and leucocytes,
253; blood globulin, 260; rest nitro-
gen in blood, 267; serumprotein, 270;
deamidation, 410, 411, 775; lactic acid,
583; ovoglobulin 633; ovalbumin, 633,
634; ovomucoid, 634; casein, 662;
alcaptonuria, 735, 736, 739; C:N
quotient, 772, 884; lactose in urine, 815
Lankester, E. R., 303, 304
Lannois, E., 533, 846
Lapicque, L., 371, 387, 388, 936
Lappe, J., 492
Laptschinsky, M., 619
Laqueur, E., autolysis, 43; lipase, 47o;
casein and rennin coagulation, 648-651
Laqueur, ,T., 340
Larin, A. M., 469
Larsson, K. O., 759
Lassaigne, J. L., 717
Lassar, O., 309

Lassar-Cohn, 422-425, 427, 435
Latarjet, A., 464

Latschenberger, J., bile pigments, 441,
442; iron in bile and liver, 443; pro-
tein absorption, 525
Latschinoff, P., 422-426
Lattes, L., 822
Laulanie, F., 563, 597, 927
Lauritzen, M., 769
de Laval, G., 656
Laves, E., 660
Laves, M., 397, 403, 404
Laveson, H., 479
Lavoisier, M., 926

INDEX OF AUTHORS

963

Lawes, 563

Lawrow, D., coagulose, 58, 135; histone,
107, 108; digestion products, 132; his-
tidine, 160; blood pigments, 288;
conjugated glucuronic acids, 785

Laxa, O., 649, 651

Lazarus-Barlow, W. S., 351

Lea, Sh., 65

Leathes, J. B., lymph, 13; autolysis, 44;
liver fat, 384, 385; protein absorption,
527; ovarial fluid, 626; uric acid, 700

Leavenworth, Ch., 78, 84, 163

Lebedeff, A./384, 560, 561, 669

v. Lebedew, A., 205

Leclerc, A., 848

Leconte, P., 463

Ledderhose, G., 218, 839

Ledoux, A., 324

Leers, O., 285

van Leersum, E. C., 209, 221

Lefevre, K. U., 222

Lefmann, G., 692

Legal, E., 158, 823

Lehmann, C., fat formation, 563; meta-
bolism in hunger, 893; in work, 926,
927; asparagin, food value, 912

Lehmann, C. G., 458, 632

Lehmann, Fr., 849

Lehmann, K. B., 288, 560

Lehndorff, H., 360

Leichtenstern, O., 338, 339

Leick, 559

Lemaire, F., 749, 815

Lenk, E., 423, 589, 590

Leo, H., liver fat, 384, 561; diabetes,
404; acidity in gastric juice, 489;
laiose, 814; nitrogen deficit, 881

Lepage, L., 498, 499

Lepine", R., glucuronic acids, 221, 331;
pentoses, 264; sugar in blood, 264, 329,
330; glycolysis, 332, 333, 346, 407,
408; glycogen, 393, phlorhizin-diabetes,
400; absorption, 533; urinary sulphur,
752, 753; urinary phosphorous, 757;
urinary poisons, 758; urine maltose, 814

Leponois, E., 758

Lerch, 619

Lesein, W., 606, 607

Lesnik, M., 778, 779

Lesser, E. J., 909-911, 917

Lesser, K. A., 350

Letsche, E., blood serum, 264, 266;
methsemoglobin, 283; bile acids, 419,
420, 423

Leube, W., 787, 848

Leuchs, H., serine, 145; arabinose, 201;
glucosamine, 218; oxyprolin, 155

Levene, P. A., autolysis, 44, 620; poly-
peptides, 89 ; nucleoalbumin, 104;
albumin hydrolysis, 106, 107, 119, 134,
141, 143, 144, 146; plasteins, 135;
tendon-mucin, 168, 544; nucleic acids,
178-185, 643; guanine, 190; pyrimi-
dinc bases, 194, 195, 507, ribose; 211;

glucothionic acids, 307, 369, 387, 547,
643; spleen, 369; liver, 383; phlor-
hizin diabetes, 400; glycolysis, 408,
333; trypsin, 502, 503; brain protein,
604; cerebroside, 609, 611; sphingosin,
611; cerebronic acid, 611; ichthulin,
630; urea, 682, 689 ; creatin and
creatinine, 692; lactic acid, 583

Levi-Malvano, M., 235, 236

Levison, L., 470

Levites, S., 78, 449-450, 476, 514

Levy, A. G., 309

Levy, H., 784

Levy, Ludw., 571

Levy, M., 557

Lewandowsky, M., 525

Lewin, Karl, 721, 728

Lewin, L., blood pigments, 281, 285, 286,
288, 289; hydroquinone, 727; urobilin,
745

Lewinsky, J., 269, 270, 721

Lewis, D. H., 53

Lewis, Th., 602

Lewy, B., 621

v. d. Leyen, E., 728

Lichtwitz, L., 66, 749

Liddle, L. M., 107

Lieb, Ch., 702

Lieben, A., 301, 306, 823

Liebermann, C., 447, 63.6, 843

Liebermann, H., 754

Liebermann, L., protein reaction, 100;
lecithalbumins, 105; nucleins, 175,
176; hen's egg, 628, 630, 632, 637,
638; kidneys, 673; Guaiac test, 796

Liebermeister, G., 258

v. Liebig, J., mineral substances, 72;
fat formation, 563; work and meta-
bolism, 594, 596; urea, 686; diets, 932

Lieblein, V., 361, 684

Liebrecht, A., 82

Liebreich, O., 606, 844

Liechti, P., 726

Liepmann, W., 642

van Lier, E. H. B., 545

van Lier, G. A., 8

Lifschiitz, J., oleic acid, 237; cholesterin,
447, 449; isocholesterin, 446; wool fat,
846

Likhatscheff, A., 785

Lilienfeld, L., nucleo histone, 108, 307;
fibrin-ferment and blood coagulation, i
256, 314, 316; blood plates, 308;
thymus, 366, 368

Lillie, R. S., colloids, 16, 17, 31; salt |
action, 73

v. Limbeck, R., 309, 310

Limpricht, H., 572

Lindberger, W., 506, 517

Lindemann, L., 208

v. Linder, M., 843

Linden, S. E., 23, 26

Lindhard, J., 814

Lindvall, V., 112

964

INDEX OF AUTHORS

Ling, A. R., 226

Linn, K., 445, 446

Linnert, K., 612, 613

Linossier, G., 675

Linser, P., 844

Lintner, C. J., 229

Lipliawsky, A., 824, 825

Lipp, A., 152

Lippich, Fr., metallic albuminates, 96;
leucine, 143; excrements, 521; urein,
691 ; uramino acids, 786

v. Lippmann, E. O., 153

Lipschiitz, A., 902

Lister, J., 313

Ljubarsky, E., 239

Ljungdahl, M., 402

Lloyd-Jones, E., 308

Lochhead, A. C., 606

Lochhead, J., 640

Loche, F. S., 72, 592

Lockemann, G., 307

Locquin, R., 144

Loeb, A., 415, 767, 333

Loeb, J., muscles, 9; Overton's theory, 10;
enzymes, 70; antagonistic salt action,
72, 73; artificial fertilization, 639, 640;
metabolism, 928; fundulustrials, 70,
73,74

Loeb, L., 256, 319

Loeb, W., 37, 212, 597

Loebisch, Wilh., 643, 668

Loebisch, W. F., 434, 544

Lohlein, W., 469, 505, 506

Loning, H., 611

Lonnberg, J., 549, 673

Lonnqvist, B., 462

Loeper, M., 265

Lorcher, G., 474

Loeschcke, K., 392

Lotsch, E., 484

Loevenhart, A. S., enzymes, 56, 59, 71.
501,502, 563

Loew, O., protein, 78, 82, 97, 126; sugar
synthesis, 212

Lowe, S., 10

Lowenthal, S., 43

Loewenthal, W., 511

Loewi, O., phlorhizin-diabetes, 400; sugar
formation, 412; protein synthesis, 529;
urea formation, 682; allantoin, 718;
conjugated glucuronic acids, 750; phos-
phorous metabolism, 761

Loewit, M., 314

Loewy, A., diamines, 163; blood alkalin-
ity, 310; high altitudes, 341, 929;
liver nitrogen, 387; work and meta-
bolism, 595; acid action, 676; amino-
acids in urine, 757; cystinuria, 827;
gases of blood, 851, 852, 859, 861, 865;
alveolar air, 860, 863, 865; metabol-
ism, 888, 925-929; maintenance value,
897, 898

Loewy, E., 839

Lohmann, A., lysine, 164; choline, 246,

247, 379, 507; methylguanidme, 698;
urinary bases, 757

Lohnstein, Th., 679, 804, 813

Lohrisch, H., 510

Lombardi, M., 96

Lombroso, U., 532, 535, 536, 539, 540

London, E. S., enzymes, 53; nucleic
acids, 472; gastric lipase, 476; diges-
tion, 482-485, 513, 514; pancreatic
juice, 498; Eck's fistula, 529, 702;
small intestines, 541; creatinine, 694;
nuclein metabolism, 702, 705; starva-
tion blood, 896

Long, J. H., lecithin, 244, 245; casein,
648, 649; urine nitrogen, 680; urinary
coefficient, 771; elimination of alkali
earths, 769

Longcope, W. T., 43

L6pez-Suarez, J., 476

Lorrain-Smith, J., 343

Losev, G., 29

Lossen, F., 83

Lessen, J., 325

Lottermoser, A., 19, 23

Luchsinger, B., 394, 847

Luciani, L., 339, 892

Luckhardt, A. B., 348

Ludwig, C., bile, 441; gastric digestion,
485; pancreatic juice, 495; absorption
of proteins, 526, 527; of sugar, 534;
gases of blood, 850, 851

Ludwig, E., 239, 627, 709, 710

Liicke, A., hyalin, 171, 840; pus, 365;
benzoic acid reaction. 722

Liidecke, T., 243

Liidecke, K., 247

Liithje, H., sugar formation, 408, 410,
412; nitrogen retention, 530; oxalic
•acid, 715

Lukjanow, S., 415, 896

Lukomnik, L, 136

Lummert, W., 384, 563

Lundsgaard, Chr., 75, 310

Lunin, N., 900

Lusk, Gr., phlorhizin-diabetes, 399, 400,
407, 409, 412, 413; lactose in intestines,
532; in urine, 815

Lussana, F., 398

Luther, E., 808

Luzzatto, A., 815

Lyman, I. F., 702

Lyon, E. P., 73

Lyttkens, H., 265, 329, 332

Maas, O.. 126

Maase, C., 774, 779, 780, 822

Macadam, J., 595

Macallum, A. B., 271, 340, 586, 769

Maccallum, A., (jr.), 711

MacCallum, J. B., 490

McClenden, J. F., 630

McClendon, 73

MacCollum, E. V., 902, 903

McCrudden, F. H., 557

INDEX OF AUTHORS

965

Macfadyen, A., 41, 512

v. Mach, W., 703

Mackay, J. C. H., 800

Mackie, W. C.,»573, 574

MacLean, H., 243, 247

Maclean, H., 407, 583

Macleod, J., glycoscuria, 402; bone mar-
row, 553; phosphocarnic acid, 578,
593; carbamic acid, 683, 691

MacMunn, Ch., A., hsematoporphyrin,
295, 797, 843; echinochrom, 304;
cholohaematin, 435; myohaematin,
571; urobilinoid, 743; tetronerythrin,
843

Madsen, Th., 50, 57, 449-450

Magnanini, G., 159

Magne, H., 670

Magmer, 770

Magnus-Alsleben, E., 772

Magnus, G., 850

Magnus, R., 52, 524

Magnus Levy, A., spleen, 372; thyroid
gland, 374, 376; liver, 382, 388, 389;
diabetes, 413; salivary glands, 452;
pancreas, 495 ; analyses of muscles, 599,
600; of brain tissue, 614; kidneys, 673;
hippuric acid, 721, 722; fatty acids
in urine, 748; benzoicglucuronic acid,
751; Bence-Jones' protein, 792; ace-
tone bodies, 818, 820, 821, 826; respira-
tion, 869; metabolism, 889, 923-925,
929, 897^898

Maignon, F., 581

Maillard, L. C., creatinine, 693; indoxyl
sulphuric acid, 727, 730; urinary sul-
phur, 752; urinary phosphorous, 762;
ammonia, 766

Maillard, M. L., 71

Majert, W., 621

Makris, C., 660

Malcolm, J., 761

Malengreau, F., 242, 366, 367

Maleniick, W. D., 109, 110

Malfatti, H., urine, purin bases, 715,
ammo-acids, 756; ammonia, 768, 769;
phosphorous elimination, 788; fruc-
tose determination, 814

Mall, F., 121

Mallevre, A., 510

Maly, R., oxyproteic acids, 82, 83;
peptones, 137; bile pigments, 429,
431-433, 743; saliva, 453; hydro-
chloric acid secretion, 477; putrefac-
tion, 517; luteins, 631

Manasse, P., 385

Manche, M., 592

Manchot, W., 279, 286, 298

Mancini, St., 208, 267, 740, 741

Mandel, J. A., glutamic acid, 147; nucleic
acids, 179, 643; adenine-hexose com-
pound, 180; guanylic acid, 183; naph-
thoresorcin reaction, 223; glucothionic
acids, 307, 369, 387, 643, 673; spleen,
369; liver, 383; mammary glands, 668;

reno-sulphuric acid, 673; urinary phos-
phorous, 757
Mandel, R., 407
Mandelstamm, E., 415
Mangold, E., 390, 894
Manicardi, C., 578
Mann, S., 612, 613
Manning, T. D., 491
Mansfeld, G., 265, 360, 377
Maquenne, L., starch, 227, 229, 230;

cellose, 231; protein assimilation, 525;

sugar absorption, 534; inosite, 579;

sarcosin, 776; digestion work, 929,

930

Marcet, 188, 523
Marchetti, G., 560
Marchlewski, L., leaf and blood pigments,

276, 277, 296, 297; hasnin, 292;

cholohaematin and bilipurpurin,435
Marcus, E., 259
Marcuse, W., 592, 593
Mares, F., 700, 702
Marfori, P., 773
Margulies, 806
Maiie, P. L., 307, 364
Marino-Zucco, 246
Mark, H., 385
Markewicz, M., 767
Marquardsen, E., 519
Marshall, J., 735
Martin, C. J., fibrin ferment, 57, 256;

toxin-antitoxin combinations, 68; blood

coagulation, 323
Martin, S. H., 510
Martz, 846
Marum, A., 820
Marx, A., 756, 818, 822
Marxer, A., 406
Maschke, O., 94, 695
Masius, J. B., 429, 521
Massen, V., 683
Masuyama, M., 628
Mathews, A., arbacin, 108; protamine,

109, 623; lysine, 163; nucleic acids,

184; fibrinogen, 252, 253
Mathieu, E., 851
Matthes, M., 519, 795
Mattili, H. A., 892
Mauthner, J., 149, 445, 814, 912
Maw as, L, 253
Maximowitsch, S., 261, 262
May, R., 208

Mayeda, M., 172, 173, 462
Mayer, Arthur, 770
Mayer, A., 105
Mayer, E. W., 445, 448
Mayer, J., 920
Mayer, L., 532, 540
Mayer, Mart., 253, 270
Mayer, P., isoserin, 145; cystin, 147-149;

mannoses, 201, 203; glucuronic acids,

221, 222; lecithin, 244; conjugated

glucuronic acids, 331, 749, 750, 817;

phosphatides, 385; deamidation, 410;

966

INDEX OF AUTHORS

inosite, 579; oxalic acid, 716; indican,

728; skatoxyl-glucuroriic acid, 732
Mayo-Robson, A. W., 414
Mays, K., 502, 503, 616
Mazurkiewicz W.. 499
Meara, F. S., 131
Medigreceanu, FL, 182, 277, 481
Meek, W. I., 253
Mehu, C., 351, 745, 746
Meigs, E. B., 589, 590, 603
Meillere, G., inosite, 579, 580, 818;

urinary chlorine compounds, 758
Meinert, C. A., 531, 932
Meinertz, J., 385

Meisenheimer, J., 41, 205, 207, 213
Meissl, E., 563
Meissl, Th., 642
Meissner, F., digestion products, 471,

472; urea formation!, 684; allantoin,

716, 717; hippuric acid, 720
Meister, V., 684
Mellanby, E., 573, 574
Mellanby, J., serum proteins, 259, 322;

peptone-blood, 325; creatine and crea-

tinine, 692, 694
Mendel, Lafayette B., enzymes, 52, 53;

lymph formation, 351; saliva, 454;

trypsinogen, 496; protein absorption,

525; creatine, 693; uric acid, 702, 705;

allantoin, 717; kynurenic acid, 740;

artificial feeding, 904, 906
Mendes de Leon, M. A., 662
Menozzi, A., 449-450
Menschutkins, 35
Menzies, J. A., 285
de Merejkowski, C., 843
v. Mering, J., urochloralic acid, 221, 777;

sugar in blood, 264; blood from portal

vein, 336, 532; glycogen, 394; phlorizin

diabetes, 399, 400; pancreatic diabetes,

404; amylolysis, 456, 500
Merunowicz, J., 293
Mesermitzki, 628
Messinger, J., 825
Messner, E., 529
Mester, Br., 519, 732, 752
Mett, By 469
Meyer-Betz, Fr., hsematoporphyrin, 295;

bilirubin, 429, 744; urobilinoids, 743;

urobilinogen, 744, 746, 747
Meyer, C., 388
v. Meyer, E., 23
Meyer, E., 440
Meyer, Erich, 735, 739, 783 .
Meyer, G., 531
Meyer, G. M., amino-acids in blood, 266;

glucose, 408, 333; urea, 682, 689;

lactic acid, 583
Meyer, H., 221, 703, 704
Meyer, Hans, 267
de Meyer, J., 408
Meyer, K., 64
Meyer, Kurt, 32, 396
Meyer, Lothar, 850

Meyer, P., 425, 429

Meyer-Wedell, L., 385

Michaelis, H., 265

Michaelis, L., cataphoresis, 20; colloid
envelopment, 23; adsorption, 49-51,
97, 102; reaction of blood, 75; coagula-
tion of proteins by heat, 20, 97; albu-
moses, 134; sugar-of blood, 264, 235,
328, 329; butyrinases, 265; protein
absorption, 525; milk, 657

Michaud, L., 401, 818, 822, 917

Michel, A., 261, 262, 263

Micheli, F., 139

Micko, K., 521, 578

v. Middendorff, M., 336

Mieg, W., 631

Miescher, F., protamines, 109-111; nu-
clein, 175; nucleic acid, 179; pus, 364,
365; spermatozoa, 622, 623; salmon
metabolism, 902

Miethe, A., 281, 285, 286, 289

Migay, Th., 481

Miller, I. R., 701-703, 706

Millon, M. E., 99. 653

Mills, W., 715

Milroy, J. A., 290

Milroy, T. H., 175, 761

Minami, D., 501

Minkpwski, O., blood alkalinity, 309;
ascitic fluid, 358; glycogen, 397;
sugar of blood, 298; phlorhizin-
diabetes, 399, 400; pancreatic diabetes,
493-406; duodenal diabetes, 405; bile
pigment, 442, 443; pancreas and
absorption, 532; fat absorption, 535,
539, 540; lactic acid, 582, 749; uric
acid, 703-705; allantoin, 717; his-
tozym, 723; blood in diabetes. 856

Mitchell, P. H., 702

Mitjukoff, K., 626

Mittelbach, F., 254, 739, 793

v. Mituch, A., 637

Miura, K., 264, 393, 492

Miyamota, S., 467

Moeckel, K., 265

Mollenberg, R., 338

Moeller, J., 521

Moller, S., 825

Morner, C., Th., albumoid, 114, 546, 549,
617; gelatin, 118, 120, 546; ichthy-
lepidin, 121; gorgqnin and pennatulin,
122; cornicrystalline, 123; proteins
of anthozoa, 123; tyrosin test, 154;
membranin, 171, 550, 617; fructose,
217; vitreous humor, 545, 616; car-
tilage tissue, 546-550; cornea, 550;
mucoid, 545, 550, 551; bones, 555;
crystalline lense, 617, 618; sugar
in egg-white, 632; ovomucoid, 634,
635; perca globulin, 636; homogentisic
acid, 738, chlorine determination,
760; gallic and tannic acid, 785; cal-
cium diphosphate in urine sediment,
831

INDEX OF AUTHORS

967

Morner, K. A. HM sulphur of proteins,
79; cystine and cystein, 106, 113, 114,
147-149; thiolactic acid, 79, 113, 151;
albuminate, 126, 569; pyro-racemic
acid, 150; proteins of serum, 259, 260,
262; hsemin, 292, 293; blister fluid,
361; hydrochloric acid determination,
489; chondroitin sulphuric acid, 547,
673; muscle pigments, 571; urinary
nitrogen, 680, 685; urea determina-
tion, 688, 689; fatty acids in urine,
748; urinary nubecula, 757; acetanilid,
779; albumin in urine, 787; nucleo-
albumin of urine, 794; melanins, 799,
841 ; bile acids in urine, 800

Mohr, Fr., 488, 759

Mohr, L., diabetes and sugar formation,
406, 412, 413; purines in urine, 713

Mohr, P., 112

Moitessier, J., 287, 758, 792, 793

Moleschott, J., 932

Molisch, H., 215

Moll, L., 103

Monari, A., 593, 594, 698

Monod, O., 654

Moor, Ovid., 690, 691

Moore, J., 213

Moore, B., theory of Overton, 10; adsor-
pates, 12, 29; colloids, 16; glycoseuria,
402; bile and fatty acids, 511, 536;
intestinal contents, 519; fat synthesis,
535; fat emulsion, 536

Mooser, W., 726

Moraczewski, W., excrements, 521; heart
muscle, 599; pseudonuclein, 651; in-
dican of urine, 728, 729

Morat, J., 592

Morawitz, P., fibrin and leucocytes, 147;
serum proteins, 270; blood coagula-
tion, 314, 315, 318-321, 324, 325;
detection of albumoses in urine, 792

Morax, V., 724

Moreau, A., 867

Moreau, J., 229, 230

Morel, A., fibrinogen, 252, 253; serum
lipase, 265; blood coagulation, 234;
glycolysis, 332; haBmatogen, 629; milk,
654

Morel, I., 677

Morel, L., 501

Moreschi, A., 449

Morgen, A., 667

Morgenroth, J., 64, 68

Mori, Y., 531, 932

Moriggia, A., 847

Morishima, K., 382

Moritz, 479

Moritz, F., 354, 400, 401, 675

Moriya, G., 582, 606

Morkowin, N., 109

Morochowetz, L., 546

Morris, G. H., 41, 226, 229

Morse, H. N., 3

Moruzzi, G., 247

Moscatelli, R., allantoin, 355, 359; lactic
acid, 593, 748, 749

Moscati, G., starch assimilation, 532;
glycogen, 581, 591; placenta, 640

Mosen, R., 308

Mosse, M., , pseudochylous appearance,
358; sugar in blood, 398; hydrochloric
acid in stomach, 476; ethereal sulphuric
acids, 724

Mott, F. W., choline in blood, 246; in
cerebrospinal fluid, 360; diseases of the
nervous system, 614

Mottram, W. H., 384, 385

Mouton, H., 65

Miihle, P., 136

Muhsam, J., 335

Miiller, A., 15, 23

Miiller, Alb., 489

Muller, Eduard, 307

Muller, Erich, 510

Muller, Ernst, 419, 421, 423

Muller, Fr., 136

Muller, Franz, 377, 378, 888, 926, 929

Miiller, Friedrich, autolysis of pneu-
monic infiltrations, 45, 364, 869; glu-
cosamine from proteins, 83, 84, 168,
169, 170, 626; high altitudes,. 341;
starvation (indican) 516; fat absorp-
tion, 538, 539; ethereal sulphuric
acids, 724; urobilin, 744; sulphur of
urine, 752, 753; aniline, 779; acetone
bodies, 818; feces nitrogen, 881

Muller, Joh., 580, 592, 628

Muller, Jul., 727

Muller, Max, 600, 721

Muller, Martin, 578

Muller, Paul, 448, 521

Muller, Paul, Th., 252, 253, 270, 553

Muller, W., 609, 610

Miintz, A., 669

Miinzer, E., 682, 685

Muther, A., 210

Muirhead, A., 683

Mulder, G. J., Ill

Munk, I., chyle and lymph, 346-349;
sulphocyanides, 455, 456, 751; in-
testinal contents, 519; absorption of
protein, 525, 526; of sugar, 534; of fat,
535, 537, 538; fat synthesis and fat
formation, 560, 563; work and meta-
bolism, 595; smooth muscles, 602;
milk, 655, 656; urea, 682; phenol
elimination, 725; phosphoric acid elim-
ination, 762; bile pigment reaction, 801 ;
starvation metabolism, 895, 896;
nutritive value of gelatin, 911; of
albumoses, 912; of asparagin, 912;
protein requirement. 915; water and
metabolism, 920

Murlin, J. R., 911

Murray, Fr. W., 500

Murschhauser, H., 923

Musculus, F., amylolysis, 229, 456, 500;
urochloralic acid, 777; urease, 829

968

INDEX OF AUTHORS

Myers, V., 195, 692, 693
Mygge, J., 793

Mylius, F., starch iodide, 228; bile acids,
419, 422-425

v. NageirC., 227

Nageli, K., 5

Nageli, O., 676, 677

Nagano, J., 491, 532, 533

Nakaseko, R., 393

Nakayama, M., 182, 493, 801

van Name, W. G., 118, 121

Nasse, H., muscle experiments, 9; blood,
336,337; lymph, 349; spleen, 371

Nasse, O., proteins, 78, 99, glutin, 120;
dextrins, 230; glycolysis, 332; gly-
cogen, 390, 391, 590-592; saliva, 456;
457; musculin, 568, 570; oxidates, 877

Naunyn, B., glycogen, 394; bile pig-
ments and liver, 442, 443; demolition
of aromatic substances, 778, 779

Nawratzki, 360

Nebelthau, E., 393, 799

Necker, F., 791

Nef, J. U., 213, 214

Neilson, C. H., 37, 53, 454

Neimann, W., glucuronic acids, 221, 222,
750, 751

Nelson, L., 109, 110 .

Nencki, L., 779

Nencki, M., protein sulphur, 78; trypto-
phan, 155; skatol acetic acid, 155;
indol, 157; blood pigments, 276, 277,
280, 291-295; hsBmatoporphyrin, 295;
diabetes, 403; gastric juice, 465-467,
476, 477, 485; enzymes of the stomach,
475;cleavage of esters, 501; intestinal
digestion, 511, 512; intestinal put-
refaction, 514; reaction in intestine,
519; ammonia, 528, 683, 768; urea,
572, 682-684; carbamic acid, 683;
urosein, 733, 740; urobilinoids, 743;
demolition of aromatic substances,
778, 780, 785, melanins, 841

Neppi, B., 505

Nerking, J., lecithin, 241, 244; glycogen,
392; bone marrow, 553; milk, 663

Nernst, W., permeability of membranes,
9; division rule, 27; diffusion, 36;
toxin-antotixin reaction, 69; gas chains,
74, 272

NersessorT, N., 744

Neubauer, C., creatin, 575; creatinine,
691; ammonia, 766

Neubauer, E., 245, 404

Neubauer, O., protein reaction, 100;
acids of alcaptonurics, 736, 738, 739;
demolition of amino-acids, 775, 779,
780, 786; conjugation of glucuronic
acid, 777; acetone formation, 780, 818,
822

Neuberg, C., autolysis, 43; putrefactive
products of proteins, 82; gelatin, 83;
artificial phosphoproteins, 105; iso-

leucine, 144; isoserine, 145; oxyamino-
succinic acid, 147; cystine, 147-149;
proiine, 154; tryptophan, 157, 158;
diamine formation, 163; amyloid, 171,
172; nucleic acids, 178, 179, 182, 183;
sugar demolition, 200; mannoses, 201,
203; pentoses, 203, 208-211, 815;
sugar-free fermentation, 206; glucose,
215; galactose, 216; laevulose, 217, 218,
355; glucoseamine, 219, 626, 629;
aminoaldehyde, 219, 220; glucuronic
acids, 221, 223, 749-751, 817; amino-
acids in blood, 267; glucothionic acid,
369, 673; melanin, 380; softening of
liver, 386, 387; glycogen, 393; deamdia-
tion, 410, 411; cholesterin, 445, 447;
chondrosin, 548; inosite, 579; lactic
acid, 583; lactose, 655; renosulphuric
acid, 673; urine, 678; heteroxanthine,
713; phenol determination, 725, 726;
skatoxyl-glucuronic acid, 732; amino-
acids in urine, 756; mineral meta-
bolism, 758, 769; tataric acid, 774;
phenylhydrazine test, 806; acetone 818;
cystinuria, 827; tyrosinase, 843; min-
eral metabolism, 899, 901, 902

Neumann, Alb., nucleic acids, 179, 184,
185; pyrimidine bases, 194, 195; orcin
test, 209; iron in urine, 770; phenyl-
hydrazine test, 806

Neumann, E., 443

Neumann, Jul., 338

Neumann, O., 921, 934

Neumann, R., 920

Neumann, Walt, 136

Neumeister, R., keratins, 112; albumoses
and peptones, 128-131; tryptophan,
155; dextrins, 230; glycogen, 391;
protein assimilation, 525; ovomucoid,

Neusser, E., 797

Nickles, J., 635

Nicloux, M., 56, 264

Nicolaier, A., 710

Niemann, A., 532, 539

Nierenstein, E., 469

Nilson, G., 228

Nilson, L. F., 658

Nishi, M., 404

Njegovan, VI., 245

Le Nobel, C., 295, 743, 797

Noel-Paton, D., lymph, 349; liver, 384,
385; glycogen, sugar formation, 399;
bile, 414, 438; creatine metabolism,
573-575; lactose. 670

Noguchi, H., 449-450

Nogueira, A., 673

Nolf, P., osmotic pressure, 13; fibnnogen
252, 253; fibrinolysis, 255, 256; albu-
moses in blood, 263; blood coagulation,
318, 319-322, 324, 325, 320; saliva,
452; absorption, 526, 527; carbamic
acid, 683, 691

Noll, A. ,184, 614, 535

INDEX OF AUTHORS

969

v. Norden, C., spectrophotometry, 302;
diabetes, 401, 404, 405, 412; liver
and urinary nitrogen, 685; ethereal
sulphuric acids, 724; albumin in urine,
787; acetone bodies, 818; metabolism,
915, 919

v. Noorden, K., 333

Nothwang, Fr., 899

Noskin, J., 375

Novi, J., 435, 459

Novy, F., 126

Nowak, J., 600, 868, 881

Niirenberg, A., 376

Nuremberg, A., 56

Nussbaum, M., 860, 864, 865 '

Nuttal, G., 516

Nylander, E., 214

Nylen, S., 457,

Obermayer, Fr., protein precipitation,
102; globulins, 103; bile pigments,
434, 801, 802; indican detection, 729,
730

Obermiiller, K., 445, 446

Odake, S., 905, 906

Oddi, R., 511, 675

Odenius, R., 643

Oertel, H., 757

Oertmann, E., 41

Oerum, H. P., (sr.), 627, 911

Oerum, H. P. T. (jr.), 342, 437, 730

Oesterberg, E., 680, 693

Offer, Th. R., pentose-amine, 219; gly-
cogen, 396; chitin, 839; alcohol, 921

Offringa, J., 282

Ofner, R., 217, 218

Ogata, M., 485

Ohta, K., 774

Oidtmann, H., 366, 368, 870

Oker-Blom, M., 8, 377

Okunew, W., 135

Olinger, J., 529

Oliver, G., 379

Ollendorff, G., 203

Olsavszky, V., 762

Omeliansky, V., 510

Omi, K., 492, 526

van Oondt, 401

Opie, E. L., 307

Oppenheimer, C., serumalbumin, 261;
parenteral protein assimilation, 525;
if, respiration, 868; 'oxydation enzymes,
875; nitrogen elimination, 881; sur-
face rule, 923

Oppenheimer, Max, 583

Oppenheimer, S., 583

Oppler, B., 329, 513

Orban, R., 492

Orgler, A., acetone, 83, 818; chondrosin,
548; uric acid, 638

Orndorff, W. R., 428, 430, 431

Orton, K., 738, 739

Oeborne, Th. B., proteins, 77-79, 84,
104, 106-108, 163; polypeptides, 89;

nucleic acids, 179, 185; ovovitellin,
628, 629; protein of white of egg, 633,
634; calorimetry, 885; phosphorous
metabolism, 903; artificial nutrition,
904, 906

Osborne, W. A., 581

Ostertag, 659

Ostwald, Wilh., 28, 29; catalysis, 33, 35,
36

Ostwald, Wo., 15, 31, 279

Ostwald, A., halogen protejn, 82; thy-
roid glands, 373, 375, 376; di-iodoty rosin,
508; urinary globulin, 791

Otori, J., mucin, 169; transudates, 355;
guanidine, 507, 574; pseudomucin,
626

Ott, A., 769

Otte, P., 486

Otto, J. G., sugar of blood, 265; blood
pigments, 277, 283, 284, 302; blood,
328, 329, 335, 338, 339, 532; skatoxyl-
sulphuric acid, 732; sugar determina-
tion, 811

Overton, E., plasmolysis, 6, 8; muscle
experiments, 9, 588; amphibians, 9;
theory of permeability, 9-11

Owen, Rees, 347

Paal, C., proteins, 78; gelatin peptones,

120; alkali albuminate, 126; peptone,

130, 131, 137
Pacchioni, D., 381
Pachon, V., blood coagulation, 324;

stomach extirpation, 485, 486; trypsino-

gen, 496
Paderi, 332
Padtberg, J. H., 838
Pages, C., blood coagulation, 251, 316,

317; rennin action, 650; milk, 667
Pagniez, Ph., 314, 315
Paigkull, L., exudates, 353, 354, 357, 358,
"~ bile, 417

Painter, H. M., 130, 457
Panek, K., 753, 754, 763
Panella, A., 266, 578, 602
Panormoff, A., 581, 633, 635
Panum, P., serum casein, 258; starvation

blood, 338, 339, 896; transfusion, 340,

344; amount of blood, 343; nitrogen

elimination, 910, nutritive value of

gelatin, 911
Panzer, Th., proteins, 83; chylus, 347,

cerebrospinal fluid, 360; bile, 423;

colloid, 625, 626
Pappenhusen, Th., 484
Paraschtschuk, S., 669
Parastschuk, S. W., 474
Parcus, E., 610
Parke, J. L., 632, 635
Parker, W. H., 16, 536
Parmentier, E., 660
Parnas, J., 248, 583
Partridge, C. L., 371, 702
Paschutin, V., 350, 492

970

INDEX OF AUTHORS

Pascucci, O., 273, 274

Pascheles, W., 776

Pasqualis, G., 749

Pasteur, L., 40, 41, 516

Patein, G., 808

Patten, A. J., 79, 149, 160

Patten, J. B., 457

Paul, Th., 71, 707

Pauli, W., colloids, 17, 18, 20, 25, 30;-
proteins, 95, 97; gelatin, 31, 119

Pauly, H., histidin, 82, 159-161, adrenalin,
379

Pautz, W., 361, 492, 617

Pavy, F. W., carbohydrate groups in
proteins, 84, 396; isomaltose, 264;
glycogen, 392, 398; sugar in blood and
diabetes, 400; self-digestion of stomach,
486; work and metabolism, 595; sugar
determination, 808, 809

Pawlow, J. P., secretion of enzymes, 52,
53; Schiitze's rule, 57; bile fistula, 414;
saliva, 454; stomach and gastric juice,
461-463, 465, 468; stomach enzymes,
474; pyloric reflex, 481; intestinal
juice, 490; pancreatic juice and enzymes
of pancreas, 495-498, 499, 501; diges-
tion in intestines, 513; ammonia in
blood, 683; Eck's fistula, 684

Payer, A., 338

Pearce, R. G., 399

Peiper, G., 309

Pierce, G., 71

Peju, P., 253

Pekelharing, C. A., cataphoresis, 50, 51;
fibrin ferment and blood coagulation,
256, 257, 316, 317, 319, 320; nucleo-
proteins, 258, 569; stomach enzymes,
466-468; creatin and creatinine, 594,
692, 693

Penny, E., 725

Penzoldt, F., 402, 824

Pernou, M., 371

Perrin, J., 15, 20

Peschek, E., 912

Petersen, P., 599

Petit, A., 758

Petrowa, M., 416

Petry, E., 651

v. Pettenkofer, M., bile acid test, 418;
fat formation, 560, 561; work and
metabolism, 594, 596, 597; respira-
tion apparatus, 868, 869; metabolism
experiments, 879, 881, 908, 926; diets,
932

Pittibone, C., 690

Petrone, A., 315

Petzsch, E., 902

Pfaff, F., 414

Pfannenstiel, J., 620

Pfaundler, M., 132, 133, 681

Pfeffer, W., 2, 3

Pfeiffer, E., 662, 665

Pfeiffer, L., 841

Pfeiffer, Th., 918

Pfeiffer, Wilh., 705

Pfleidener, 470, 471

Pfliiger, E., oxydations, 41; life of cells,
45; carbonic acid of lymph, 347; gly-
C9gen, 390, 392, 393, 394, 413, 549;
diabetes and sugar formation, 405, 408,
412, 413; duodenum and diabetes, 405;
gases of saliva, 453, 857; bile and fatty
acids, 511, 536; fat formation, 561,
562; muscle metabolism, 591, 595,
597; gases of milk, 657, 857; urinary
nitrogen, 680; urea determination, G90;
sugar tests, 803, 805; sugar determina-
tion, 811; gases of blood and respira-
tion, 850, 851, 854, 858, 866-868;
N:C quotient in urine, 884; protein
metabolism, 904, 008, 909; external
temperature and metabolism, 929;
protein allowance, 934

Phisalix, C., 847

Picard, J., 616, 617

Piccard, 109

Piccolo, G., 301, 623

Pick, A., 470

Pick, E. P., albumoses and peptones, 131,
133-135, 139; serumglobulins, 259;
peptozym, 325; thyreoidea and adren-
alin-glycosuria, 376

Pick, F., 399

Pickardt, M., 355, 549, 550

Pickering, J. W., 85, 86, 323

Picton, H., 22, 26

Pierallini, G., 773

Piettre, M., stroma of blood corpuscles,
275; blood pigment, 281, 285; hsemin,
292; hyoglycocholic acid, 421; melanin,
842

Pigeand, J., 354

Pighini, G., 613

Piloty, O., blood and blood pigments,
220, 277, 290, 292-298, 295, 297;
bile pigments, 429, conjugated glu-
curonic acids, 751

Pilz, O., 137

Pilzecker, A., 440

Pimenow, P., 462

Pincussohn, L., 23

Pineles, Fr., 376

Pinkus, S. N., proteins and their crystal-
lization, 82, 95, 263, 633, 634

Piontkowski, L. F., 463

Piria, 153

Planer, J., 486

Plattner, E., 418

Plaut, M., 756

Playfair, 932

Pletnew, D., 493, 532

Plimmer, R. H., Aders, enzymes, 53;
gelatin hydrolysis, 119; nucleopro-
teins, 174, 630; livetin, 629; ichthulin,
630; hatching of the egg, 637; casein
digestion, 652; uric acid, 702

P16sz, P., blood corpuscles, 274; liver
382, 383; urinary pigments, 740;

INDEX OF AUTHORS

971

proteid in urine, 787, albumoses,
nutrition value, 912

Pod a, H., 521

Poduschka, P., 717

Poehl, A., 517, 621

Pohl, J., dextrin, 230; globulin determina-
tion, 261, 793; liver, 383; acid poison-
ing, 676; urea, 682; allantoin, 716, 717;
oxalic acid, 773; demolition of fatty
acids, 774; phthalic acid, 778

Poleck, 631

Policard, A., 324

Polimanti, O., 561

Politis, G., 912

Pollak, H., 105

Pollak, L., 505, 776

Pollitzer, S., 912

Polwzowa, W., 482, 514

Ponfick, E., 343, 344

Ponamarew, 489, 490 .

Pons, Ch., 364, 548, 757

Popel, W., 339

Popielska, Helene, 463

Popielski, L., enzymes, 53, 324; saliva,
454; pancreatic juice, 496, 498

Popowsky, N., 158

Popper, H., glycogen, 395; bile pigments,
434, 801, 802; pancreatic juice, 500,
509

Porcher, Ch., lactose, 670; indican of
urine, 728-732; skatpl red and uroro-
sein, 733; uroerythrin, 748; phthalic
acid, 778

Porges, O., 245, 259

Porteret, E., 393

Portier, P., 407, 532

Posner, C., 620, 621, 787

Posner, E. R., 169, 472, 606

Posselt, L., 122

Posternak, S., 578, 579

Pottevin, H., 59, 226

Pouchet, A. G., carnine, 577, 711;
urinary poisons, 757; lungs, 870

Poulet, V., 869

Poulsen, W., 550

Poulsson, E., 572

Pozerski, E., 65, 498

Pozzi-Escot, E., 877

Prausnitz, W., 400, 521, 894

Pray on, I., 693

Pregyl, Fr., keratins, 112, 114; koilin,
115, 124; carbon monoxy haemoglobin,
290; dehydrocholan, 418; bile acids,
423, 426; intestinal juice, 490, 491;
colloid, 626; ovalbumin, 634; oxypro-
teic acid, 753; polypeptides in urine,
756; C:N quotient, 772

Presch, W., 752, 753

Preti, L., 64, 71, 706

Preusse, C., phenols in urine, 725-727;
behavior of aromatic substances in
animal body, 778, 779, 786

Prevost, J. L., 541, 684

Preyer, W., 288, 642

Preysz, K., 762

Pribram, E., 778

Pribram, H., 106

Pribram, R., 766

Pridgent, G., 242

Pringle, H., histopeptone ,109; prota-

mines, 109, 110; absorption, 525, 528,

530; blood coagulation, 320
Pringsheim, H., 230, 510
Pristley, J. H., 37
Prochownik, L., 642, 643
Proscher, F., 431, 659, 667
Profitlich, W., 389
Prutz, W., 728
Prym, O., 372, 496
Przibram, H., 571
Pugliese, A., 319, 476
Pulvermacher, G., 212
Pupkin, Z., 309
Pyman, F. L., 160

Quagliarello, G., 675
Quevenne, Th., 348, 349
Quincke, G., 23, 646
Quincke, H.,-301, 340
Quinquand, Ch., urea, 333, 335; muscle
work, 592; fatty acids in urine, 773
Quinton, R., 13

Raaschou, C. A., 183

Rabinowitsch, A. G., 484

Rachford, B. K., 501, 506, 511

Radenhausen, P., 646

Radziejewski, S., 559, 560

Radzikowski, C., 462

Raehlmann, E., 616, 617

Raineri, G., 642

Rakoczy, A., 475

Ramsden, W., 30, 97

Ranc, A., 267

Ranke, H., 700

Ranke, J., 344

Ransom, H., 449

Raper, H. S., 136

Rapp, R., 41

Raske, K., 140, 148

Rauchwerger, D., 445, 447

Raudnitz, R. W., 644, 649

Reach, F., 381, 466, 597

Reale, E., 716, 752

Reemlin, E. B., 513

Reese, H., 756

Regnault, H. V., 849, 868, 881

Reh, A., 104, 366, 652

Rehfuss, M., 804

Rehn, E., 253

Reich, M., 770

Reich, O., 771

Reichel, H., 650

Reich-Herzberge, F., 508

Reid, E. W., 16

v. Reinbold, B., 283, 507, 808, 283

Reinecke, 558

Reiset, J., 849, 868, 881

972

INDEX OF AUTHORS

Reiss, E., 260

Reiss, W., 619

Reitzenstein, A., 711

Rekowski, L^ 786

Rennie, J., 494

Ren vail, G., 769

Rettger, I., 257, 322

Rettger, L. F., 496

Reuss, A., 355, 357

Rewald, B., 179, 180, 210, 211

Reye, W., 254

Reynolds, J. E., 823

de Rey-Pailhade, J., 877

Rhodin, N. J., 43

v. Rhorer, L., 677

Ribaut, H., 877

Richards, A. N., albumoids, 116, 117, 118;

hexone bases, 164; saliva, 455, 456
Richaud, A., 599
Richet, Ch., gastric juice, 465; fat forma-

tion, 458; urea, 682; uric acid, 706;

thalassin, 846; respiration, 869; sur-

face rule, 923; spleen, 372
Richter, Max, 621
Richter, P., 873
Richter, P. F., osmotic pressure, 13;

amino-acids in blood, 267; softening

of the liver, 386, 387, 685
Rieder, H., 881
Riegel, 464
Riegel, M., 647
Riegler, E., 290
Riehl, M., 869
Riess, L., 748, 749, 780
Riesser, O., 162, 611
Rmaldi, U., 572
Ringer, A. L, 721
Ringer, A. J., 412
Ringer, L., 316
Ringer, S., 72

Ringer, W. E., 50, 51, 675, 677, 708
Ringstedt, O. T., 675
Ritter, A., sugar in blood, 398; phlor-

hizin-diabetes, 400; fat absorption,

535 ; urinary fermentation, 829
Ritter, F., 439, 440, 445, 449
Rittausen, H., proteins, 94; leucinimide,

143; milk, 655
Riva, A., 740, 748, 799
Rivalta, F., 354
Roaf, H. E., lipoid theory, 10; adsorpate

12, 29; osmotic pressure of protein, 17,

17; lecithin, 246; glycoseuria, 402
Roberts, F., 279
Roberts, W., 509, 793, 812, 813
Robertson, T. B., lipoid theory, 10;

protein salts, 95; crystallized albumin,

262; globulins, 263, 270; casein, 648,

649

Roch, G., 790
Rockwood, C. W., 757
Rockwood, D., bile and fatty acids, 511,

536; intestinal contents, 519
Rockwood, E. W., 525

Rodhe, E., 100

Rodier, A., 338

Roden, H., 63

Roeder, G., 194, 195

Rohmann, F., amylolysis and diastase,
225, 265, 346, 456"; glycolysis, 333;
blood, 335; glycogen 393, 398; in-
testinal juice, 491, 492; bile and
putrefaction, 518; absorption, 532-
534, 538; muscles, 565, 589; casein,
649; phosphorous metabolism, 761,
902; sebum, 844; wool fat, 846;
coccygeal glands secretion, 846, 847;
oxydase action, 875; artificial feeding,
904

Rohrig, A., 591, 849

Rose, B., 650

Rose, C., 558

Rose, Heinrich, 429, 295

Rosing E., 877 .

Roger, G. H., 381

Roger, H., 457, 510

Rogozinski, F., 136

Rohde, A., 703

Rokitansky, P., 748

Rona, P., cataphoresis, 20; enzymes, 56;
adsorption, 97, 102; thymus histone;
109; gelatin, 119; albumoses, 134;
sugar in blood, 264, 265; butyrinases
265; calcium in serum, 269; sugar
in blood, 328, 329; glucose, 333,
duodenal, secretion, 489, 490; protein
absorption, 525, 529; milk, 657; alcap-
tonuria, 735, 736

Ronchese, A., 768, 769

Ronchi, J., 849

Roos, E., 376, 761, 806, 807

Roosen, O., 698

Rorive, F., 217

Rosa, E. B., 869

Rose, W. C., 692, 693

Rosemann, R., gastric juice, 465, 479;
milk, 670; nitrogen elimination, 910;
, alcohol, 921

Rosenbach, O., 749, 800, 827

Rosenbaum, A., 408

Rosenberg, Br., 401

Rosenberg, S., duodenal diabetes, 405;
* bile, 415; pancreatic juice, 497; putre-
faction, 518; pancreas and absorp-
tion, 532, 535, 539

Rosenberger, F., 579, 816

Rosenfeld, B., 484

Rosenfeld, F., 728, 748, 912

Rosenfeld, G., fat and fat formation, 384,
560-562, 669; uric acid, 700; phenyl-
hydrazine test, 806; acetone bodies, 819

Rosenfeld, L., 136

Rosenfeld, M., 290, 293

Rosenfeld, R., 264

Rosenheim, O., tryptophane reaction, 157;
cerebrospinal fluid, 360; pancreatic
lipase, 502; muscle-work, 592; pro-
tagon, 606-609

INDEX OF AUTHORS

973

Rosenheim, Th., 915

Rosenqvist, E., 412

Rosenstein, A., chyle and lymph, 347,
348, 349; absorption, 526, 534, 537

Rosenstein, W., 403

Rosenthal, J., 868

Rosenthaler, L., 59, 66

Rosin, H., fructose, 217, 814; indican,
730; skatol pigments, 733; Rosen-
bach's urine test, 827

Rossi, O., 360

Host, E., 920

Rost, Fr., 265

Rostoski, O., 792, 793

Roth, O., 267

Roth, W., 351

Rothberger, C. J., 683

Rothera, C. H., 77, 148, 824

Rothmann, A., 573, 692

Rotmann, F., 355

Rotschy, A., 295

Roux, E., 227

Rovida, C. L., hyaline substance, 274,
364

Rovighi, A., 517, 724

Rowland, S., 41-43, 371, 571

Rowntree, L. G., 183, 642, 643

Rozenblat, H., 462

Rubbrecht, R., 270

Rubner, M., protein sulphur, 79; sugar
test, 215, 806, 815; utilization of
nutriments, 531, 534, 535, 538; fat
formation, 562, 563; milk, 664; meta-
bolism experiments, 879, 885, 890,
894, 922, 923, 928, 929; nitrogen
of excrements, 881; heat of combus-
tion, 885, 886, 891, 932: utilization
quota, 890, 916; protein catabolism,
910, 914, 916, 918; specific dynamic
action, 917, 930; surface rule, 923, 924

Rubow, W., 586

Rudinger, C., 375, 406

Riidel, G., 707

Ruff, O., 200, 203

Ruge, E., 515

Rulot, H., 255, 256

Rumpf, Th., 412, 767

Runeberg, J. W., 357

Ruppel, W. G., 606, 660, 844

Russel, 653

Russo, M., 839

Russo, Ph., 744

Rutherford, A., 838

Rutherford, Th. A., 112

Ryan, L. A., 603

Rywosch, D., 273

Sabanejew, A., 137

Sabbatani, S., 316

Sachaijin, 328

Sacharow, N., 135

Sachs, Fritz, papain, 65; pentoses, 210;
nuclease, 182, 493, 508; hydrochloric
acid secretion, 477; acetone formers, 818

Sachs, H., 64

Sachsse, R., 215, 228

Sackur, O., 648, 649

Saaikoff, W., 118

Sagelmann, A., 482

Sahli, H., 303, 325, 809

Saiki, T., 603, 748, 749

Saillet, haematoporphyrin, 295, 797, 798;
urobilin and urobilinogen, 740, 743,
744, 746, 747

Sainsbury, 316

de Saint-Martin, L., 286

Saint Pierre, C., 867

Saito, S., 334, 582

Salaskin, S., digestion products, 132;
plastein, 135; leucinimid, 143: blood
alkalinity, 309; ammonia, 334, 336,
528, 683; erepsin, 393; urea, 682, 684;
liver and acid formation, 685; uric
acid formation, 703, 704

Salkowski, E., autolysis, 42; denaturing
of proteins, 97; pseudpnuclein, 104,
651; albumoses, 131; in urine, 792;
putrefaction products, 141, 514; skatol
carbonic acid, 155, 732, 733; indol, 158;
pentoses, 208, 209, 815; glucuronic
acid, 221; cerebrospinal fluid, 360;
synovin, 362; liver-proteins, 383, 384;
glycogen, 393; cholesteiin, 447; saliva,
459; trypsin, 503; putrefaction, 517;
corpse wax, 560; flesh, 600; dermoid
cyst, 627; dextrose in white of egg,
632; ovomucoid, 634; casein, 651,
652; urea, 682, 684, 690; creatinine,
696; uric acid, 705, 709, 710; purme
bases, 714; oxalic acid, 716, 773;
allantoin, 716, 717; hippuric acid, 720;
phenaceturic acid, 723; ethereal-sul-
phuric acids, 724; indican, 729, 730;
urobilin, 745; urine, fatty acids, 748,
829; carbohydrates, 749, 815; sulphur
compounds, 752, 753; adializable
urinary constituents, 757, 795; urinary
sulphur acids, 764; alkalies, 766;
demolition of various substances, 776,
777, 783; hapmatoporphyrin, 797;
sugar tests, 805, 806; acetone deter-
mination, 825; water and metabolism,
920

Salkowski, H., 141, 514, 720

Salomon, Georg, purin bases, 187; glyco-
gen, 207; lactic acid, 334; urinary purines,
711-713

Salomon, H., 410, 818

Salomon, W., 682

Salomone, G., 78

Salomonsen, K. E., 740, 741

Samec, M., 141

Sammet, O., 825

Sammis, J. L., 936

Samuely, F., proteolysis, 107; amino-
acids in urine, 756; cystine demoli-
tion, 776; melanoids, 843

Sandgren J., 265, 329, 332

974

INDEX OF AUTHORS

Sandmeyer, W., pancreatic diabetes, 405;

absorption, 532, 539, 540
Sandstrom, J., 374
Saneyoshi, S., 774, 777, 817
Sasaki, K., 757

Sasaki, T., 146, 147, 781, 784
Sato, T., 369
Satta, G., 681
Sauer, K., 402
Sauerbeck, E., 494
Savare, M., 640, 641, 757
Savory, H., 792
Sawitsch, W., 496, 499
Sawjalow, W., 58, 135, 475
Saxl, P., 43, 387, 569
Scaffidi, V., ferratin, 384; iron in liver,

387; purine bases, in muscles, 572,

594, 603; uric acid, 706
Schade, H., 707
Schafer, E., 379
Schaeffer, 50, 51
Schaffer, F., 78

Schaffer, Ph., 692, 711, 768, 827, 828
Schalfejeff, M., 292, 293
Schardinger, F., 581, 877, 230
Scheele, M. H., 454
Scheermesser, W., 136
Scheibe, A., 659, 664
Schemiakine, A. J., 477, 478
Schenck, Fr., 83, 398
Schenck, M., 422, 423
Schepowalnikoff, N. P., 495
Scherer, Fr., 925
v. Scherer, J., lymph, 348; inosite, 579,

580; meta and para-albumin, 625
Scheuer, M., 464
Scheunert, A., gastric digestion, 479, 482;

duodenal secretion, 490; pancreatic

stones, 509; cellulose, 510; peroxydase,

874

Schewket, O., 817
Schierbeck, N. P., saliva, 457; gases of

stomach, 486; trypsin action, 506;

excrements, 520; skin breathing, 849
Schiff, A., 469
Schiff, H., protein, 78; biuret test, 101;

cholesterin, 448; urea, 686; uric acid, 709
Schiff, M., spleen, 372; liver, 381, sugar

of liver, 398; bile, 416, 541; charging

theory, 477, 496
Schindler, S., 368
Schittenhelm, A., nucleic acid, 182, 514;

blood coagulation, 318, 323; deamida-

tion enzymes, 371; purine bases, 520;

urea formation, 682; uric acid and its

formation, 373, 699-703, 705, 706, 709;

amino-acids in urine, 756, 757, 827;

ammonia, 767, 768
Schlapfer, V., 912
Schlatter, K., 485, 486
Schlesinger, A., 457
Schlesinger, W., 747
Schliep, L., 825
Schlosing,,Th., 768

Schloessing, C., 22

Schlossberger, J. E., 665, 670

Schlossmann, A., 656, 658, 659, 663, 923

Schmey, M., 387, 599

Schmid, Jul., 702, 715

Schmidt, Ad., 513, 523

Schmidt, Albr., 621

Schmidt, Alex., blood coagulation, 256,
257, 305, 314-317, 319, 321-323;
fibrin oplastic substances, 258, 319,
320; blood corpuscles, of frogs, 375;
leucocytes, 305, 306; cell protein, 306,
307, 367; saliva, 453, 454; gases of
blood, 852

Schmidt, C., serum, 270; blood, 328;
lymph, 348; transudates, 353; saliva,
458; mucus of the mouth, 453, 454;
gastric juice, 465; pancreatic juice,
499, 500; bile, 518; fat absorption,
538; osteomolacia, 555

Schmidt, C. H. L., 82

Schmidt, C. W., 870

Schmidt, E., 572

Schmidt, Ernst., 695

Schmidt, Fr., 818

Schmidt, Hub.. 101

Schmidt, P., 7il

Schmidt-Muhlheim, 251, 526, 910

Schmidt-Nielsen, Signe, 50

Schmidt-Nielsen, Sigvae, rennin, 50, 651;
casein, 649, 650

Schmiedeberg, O., albumin crystals, 94;
salmine, 110, 110; alkali albuminate,
126; onuphin, 171; nucleic acids, 179,
184, 185; nucleosin, 195; glucuronic
acids, 221, 725, 730, 731, 785; ferra-
tin, 383; chondroitin sulphuric acid,
547-549; urea, 682; hippuric acid,
722, 723; histozym, 723; melanin
substances, 840, 841

Schmitz, E., 775, 780, 786, 818, 826

Schmitz, H., 136

Schmitz, K., 517, 519, 724

Schmutzer, I., 708

Schneider, A., 328

Schneider, E., 455, 739

Schoffer, A., 142

Schonbein, C. F., 766, 871

Schondorff, B., urea, 333, 572, 664, 679
690; thyroidea, 376; glycogen, 390,
394, 396, 581; phlorhizin action, 400;
uric acid, 701; sugar in urine, 803, 808;
protein metabolism, 908, 909

Schone, A., 208

Scholz, H., 728, 729

Schottelius, M., 516

Schotten, C., fellic acid, 427; intestinal
putrefaction, 720; fatty acids in urine,
748, 773; damaluric and damolic
acid, 758; behavior of aromatic sub-
stances in animal body, 780

Schoubenko, G., 79

Schoumow-Simanowski, E. O., 467, 477,
485

INDEX OF AUTHORS

975

Schreiber, E., 701

Schreiner, Ph., 621

Schrener, M., flesh nitrogen, 600; calorific

value of nitrogen, 892; protein feeding,

918

Schrodt, M., 553
v. Schroder, W., urea, 12, 333, 679, 682,

684; uric acid, 334, 703, 704
Schroter, F., 182

v. Schrotter, H., 130, 131, 137, 861, 865
Schryver, S. B., 43, 427
Schiile, 464
Schiile, A., 456
Schiitz, E., Schiitz's rule, 57; digestion

products, 133; pepsin determination,

469; stomach movement, 479; lactic

acid in urine, 749
Schiitz, J., pepsin determination, 469;

action, 471; hydrochloric acid, 489;

bile and fat splitting, 502, 511
Schiitze, A., 64

Schiitzenberger, P., 85, 86, 130
Scbultze, B., 563
Schultze, E., 680
Schultze, F. E., 72
Schultzen, 0., diabetes, 403; urea, 682,

683; lactic acid in urine, 748, 749;

sarcosin, 776; behavior of aromatic

substances in the animal body, 778-

780

Schulz, Art., 299
Schulz, Fr. N., proteins, 79, 95, 98; oxy-

proteins, 83; histone, 108; galactos-

amine, 167, 173, 219; serumalbumin,

261, globin, 288; starvation blood, 339;
- premortal protein metabolism, 894
Schulz, H., 376, 546, 770
Schulz, C., 457
Schulz, E., products of hydrolysis of

proteins, 141, 153; phenylalanine, 152;

histidine, 160; arginine, 161; lysine

163; hexone bases, 164; vernine, 178-

180; hemicelluloses, 231; phosphatides,

245; isocholesterin, 448
Schulze, E., 650
Schulze, F., 22
Schulze, H., 14, 21
Schumburg, W., 474, 889, 926
Schumm, O., blood, 342, 796; chyle fat,

347; sugar formation, 412; pancreatic

cyst, 500

Schunck, C. A., 276, 277, 296, 631
Schunck, E., 740
Schur, H., uric acid, 700, 702, 706;

urinary purines, 702, 713
Schurig, 387
Schuster, A., 531, 938
Senuurmanns-Stekhoven, 282
Schwalbe, E., 315, 384, 561
Schwann, Th., 414, 528
Schwarz, Carl, choline, 247; iodothyrin,

376; glycoseuria, 405; digestion, 483;

secretin, 498
Schwarz, H., 216

Schwarz, Hugo, 116, 117, 118

Schwarz, Karl, 572, 575

Schwarz, L., 126, 477, 820

Schwarz, O., 402, 487, 825

Schwarzschild, M ., 502, 508

Schweissenger, O., 790

Schwiening, 42, 43

Schwinge, W., 338

Scofield, H., 433

Scott, F. H., 174, 630, 637

Scott, L., 122

Sczelkow, 851

Sebauer, R., 555

Sebelien, J., peptones, 130; milk, 644,
651, 652, 655, 656, 658; casein diges-
tion, 651; carbohydrates in milk, 655

Seegen, J., sugar in blood, 264, 398, 592
597; sugar formation, 405; amylolysis,
456; respiration, 868; nitrogen deficit,
881 ; water and metabolism, 920

Seelig, P., 517

Seeman, J., protein substances, oxyda-
tion, 83; galactoseamine, 167; erepsin,
493; intestinal contents, 513; absorp-
tion, 527, 528; creatine and creatimne,
573; ovomucoid, 635

Segale, M., 72

Seisser, Ph., 702, 705, 709

Seitz, W., 381, 389

Seliwanoff, Th., 217

Selmi, 47

Semmer, G., 275

Senator, H., 729

Senter, G., 56

Sera, Y., 393, 394

Sertoli, E., 855

Sestini, F. and L., 699

Setschenow, J., 851, 853, 855

Shackell, L. F., 73

Shepard, C. W., 720

Shibata, N., 384, 561

Shimamura, T., 905, 906

Shinidzu, Y., 238

Shaw-Mackenzie, J. A., 502

Siau, R. L., 264, 400

Sieber, N., protein sulphur, 79; blood
pigments, 280, 292, 293; haematopor-
phyrin, 295; glycolysis, 333; diabetes,
403; gastric juice, 361, 362, 485;
stomach enzymes, 475; intestinal diges-
tion, 512; UmikofPs reaction, 664;
urosein,7 33, 740; urobilinoids, 743;
nitrobenzaldehyde, 784; melanins, 841;
lungs, 869

Siedentopf, H., 19

Siegert, F., 559

Siegfried, M.; peptone substances and
kyrines, 121, 132, 133, 136-138, 146:
reticulin, 121, 544; lysine, 163; car-
bamino reaction, 166, 855; jecorin, 385;
phosphocarnic acid, 578, 585, 593, 598;
orylic acid, 653; milk-nucleon 653, 663;
phenol excretion, 725, 726

v. Siewert, A., 292

976

INDEX OF AUTHORS

Signorelli, E., 757

Sikes, A. W., 663

Silbermann, M., 145, 147

Simacek, E., 407, 583

Simon, Fr., 606

Simon, G., 656, 658

Simon, L., G., 510

Simon, O., 45, 364, 870

de Sinety, L., 815

Sittig, O., 355

Siven, V. O., uric acid, 700; urinary
purines, 713; protein metabolism, 903;
916, 934

Sivre, A., 482

Siwertzow, D., 241, 244

Sjoqvist, J., enzymes, 57; peptones 137;
hydrochloric acid determination, 488,
489; intestinal concrements, 524;
urinary nitrogen, 680, 685; urea
determination, 688, 689

Skita, A., 145

v. Skramlik, E., 675

Skraup, Zd., protein nitrogen, 77, 78;
hydrolysis of proteins, 119, 124, 146,
147; alkali albuminate, 126; oxyamino-
acids, 165; carbohydrate, 215

Skworzow, W., 575, 576

Slansky, P., 585

Slavw, 379, 380

Slosse, A., 333, 684, 910

Slowtzoff, B., pentosan, 208; liver, 389 ;
semen, 620; milk coagulation, 651;
metabolism, 923, 926

v. Slyke. D. D., deamidation, 77, 78, 88;
hy'drolysis of albumin, 106, 107, 134,
141, 143, 144; plastein, 135; amino-
acids in blood, 266; casein, 648, 649

van Slyke, L. L., 648, 649

Small, Fr., 707

Smetanka, F., 700, 702

Smirnow, A., 784

Smith, F., 847, 848

Smith, Herbert, 457, 551

Smith, Lorrain, 862, 863

Smith, W. J., 752

Socin, C. A., 395

Socoloff, N., 437

Soldner, F., milk, 647-650, 655-657, 662,
664-668

Sorensen, S. P. L., isoelectric point, 20;
determination of the reaction, 74, 75;
coagulation of proteins by heat, 20,
97; glycocoll, 140; phenylalanine, 152;
proline, 154; arginine, 161; ornithin,
162, 163; formol titration, 165; hip- I
puric acid, 723; urinary nitrogen, 756;
ammonia, 768

Soetber, F., 763

Solera, L., 455, 456

Solley, Fr., 118, 120

Sollmann, T., chyle, 347; bile, 440; mus-
cles, 566, 567, 570; uterine fibroma, 627

Sommer, A., 384

Sommerfeld, 438

Sommerfeld, P., 465

Sonden, K., respiration apparatus, 869;

metabolism, 924, 925, 936
Sorby, H. C., 636
Soret, J., 281
Sourder, C., 502
Le Sourd, L., 314
Soudat, 665
Southgate, 506
Soxhlet, glucose, 216; galactose, 216;

maltose, 225; fat formation, 563;

milk, 647, 650, 656, 667. 669; sugar

titration, 808, 809
Spack, Wl., 124
Spanpani, G., 669
Spangaro, S., 250
Spanjer-Herford, R., 455
Speck, C., 869, 926, 928
Spiegler, A., 899
Spiegler, E., 790, 841, 842
Spiro, K., colloids, precipitation, 25, 102;

swelling, 31; diffusion, 32; gelatin, 119;

glycocoll, 140; pyrazinedicarbonic,

acid, 220; serumglobulms, 259; blood

coagulation, 318, 319, 324; rennin

action, 650; urine acidity, 675; oxy-

butvric acid formation, 822
Spiro," P., 593
Spitzer, W., glycolysis, 333, 407; liver,

383; uric acid formation, 702, 875
Spriggs, E. J., 469
Spring, 14

Staal, J. Ph., 676, 733
Stade, W., 57, 476
Stadelmann, E., tryptophan, 155; icterus,

301; adrenal bodies, 377; bile, 414-

416, 438, 441-444, 541; intestinal

putrefaction, 519; nitrogen excretion,

685; ammonia, 768; pentoseuria, 791;

diabetes, blood, 856
Stadthagen, M., diamines, 47; adenine,

191; xanthocreatinine, 698; urinary

sulphur, 752, cystinuria, 827
Stadler, G., 434
Staehlin, R., 354
Stanek, V., 247

Stangassinger, R., 573, 692, 698
Stange, M., 233
Starke, J., 102
Starke, K., 262, 263

Starkenstein, E., 398,402, 579, 817, 818
Starling, E. H., colloids, 16; enzymes, 54;

lymph formation, 351; hormones, 375;

enterokinase, 492, 496; secretin, 492,

498; intestinal enzymes, 492, 493;

pancreatic erepsin, 493, 503; trypsino-

gen and trysin, 496, 497
Stassano, H., 496, 497
Stassow, B. D., 541
Stauber, A.. 493
Stavenhagen, A., 41
Steel, M., 768
Steenbock, H., 723
Steensma, F. A., 158, 488, 732

INDEX OF AUTHORS

977

Steiff, R., 724

Steiger, E., 161

Steil, H., 599

Stein, G., 445

Steinitz, Fr., phosphorous metabolism,

761; C:N quotient, 772, 884; lactose

in urine, 815
Stenger, E., blood pigments, 281, 285,

286,289; urobilin, 745
Stepanek, J. O., 628
Stepp, W., 905, 906
Stern, E., 35
Stem, Fr., 750
Stern, H., 441, 442
Stern, Heinrich, 621
Stern, L., uricolysis, 706; peroxidase,

873, 874; oxydation processes, 874, 875
Stern, M., 248, 630
Stern, R., 440, 724
Steudel, H., arginine, 162; hexone bases,

164; mucin, 169; nucleic acids, 179,

181, 183, 184; pyrimidine bases, 194,

195; glucoseamine, 219
Stewart, C. W., 471
Stewart, G. N., 326, 566, 567, 570
Steyrer, A., 571
Sticker, G., 455, 458
Stiles, P. G., 400, 457
Stock, J., 297, 297
Stockmann, R., 595
Stoltzner, H., 554
Stoffregen, A., 791
Stohmann, F., 510, 885
Stokes, 282, 289
Stoklasa, J., lecithin, 241; glycolysis,

407, 408, 509; lactic acid formation,

583; fermentation enzyme of milk, 653
Stokvis, B. J., bile pigments, 432, 433,

743; benzoic acid, 723; urobilin, 745,

792 ; hsematoporphyrin, 797
Stolnikow, J., 793
Stolte, K., 220, 396, 682
Stoltzner, W., 554
Stolz, Fr., 379
Stomberg, H., 256, 322
Stone, W., 208
Stookey, L. B., 136, 394
Stoop, F., 145, 148
Storch, V., 646, 671
Strada, Fr., 364
Stradomsky, N., 773
Strashesko, N. D., 462
Strassburg, G., gases of lymph, 347;

tension of the gases of blood, 861, 864,

868

Strassburger, J., 523
Strassuer, W., 877
Straub, W., 403, 899, 920
Strauch, F. W., 124
Strauss, Edw., 122
Strauss, H., fructose, 217, 218, 264;

blood, 264; transudates, 264, 355, 358;

bile, 437; lactic acid fermentation, 485;

amino acid storage, 721, 722

Strecker, A., 242, 245, 422

Strickler, E., 658

Strigel, A., 870

Strohmer, F., 563

Strusiewicz, B., 912

Struve, H., 797

Strzyzowski, C., 293

Stubel, H., 894

Sttttz, 791

Subbotin, V., 339, 921

Suckrow, Fr., 400

Sugg, E., 653

Suida, W., 445

Suleima, Th., 513

O'Sullivan, C., 54, 55, 57

Sundberg, C., 467, 468

Sunde, E., 655

Sundvik, E., purin bases, 188, 190;

glucoseamine, 218; uric acid, 699;

conjugated glucuronic, acids, 751, 777;

chitin, 839; psylla alcohol, 846
Suter, F., 79, 113, 151
Suto, K., 238, 809
Suwa, A., 572, 780
Suzuki, W., cystine, 148; muscles, 572;

crab meat, 578; phytase, 579; oryza-

ninc, 905, 906
Svedberg, The., 14, 20
Svenson, N., 919
Swain, R. E., 159
Symmers, D., 757
Syniewski, V., 227, 229
v.Szontagh, F., 653, 658, 660, 662
Szydlowski, Z., 474
Szymonowicz, L., 379

Tachan, H., 145

Takahashi, D., 269, 329

Takaishi, M., 579

Takamine, J., 379

Takada, K., 787

Tallqvist, T. W., 914

Tammann, G., 60, 66

Tanaka, T., 371

Tangl, Fr., blood serum, 264, 271; blood

analysis, 326; sugar in blood, 398;

fat, 482; egg, development, 637, 638;

casein, 648, 658; milk, 668; C:N

quotient, 772, 884
Tanret, C., 200
v. Tappeimer, H., enzymes, 50; cellulose,

510, 515; bile acids, 541
v. Tarchanoff, J., 441, 632
Tarugi, B., 848
Tarulli, L., 675
Tawara, 932
Taylor, A. E., enzymes, 56; liver fat,

384; fat formation, 561; mineral

starvation, 900
Tebb, Chr., reticulin, 121; glycogen, 392;

amylolysis, 456; saccharate, 492; mal-
tose, 500; protogon, 606-609; choles-

terin in brain, 613
Tedesko, Fr., 676

978

INDEX OF AUTHORS

Teeple, J., 428, 430, 431

Teichmann, L., 292

Tengstrom, B. St., 418

Terrat, P., 758

v. Terray, P., bile and putrefaction, 518,

520; oxalic acid, 716; lactic acid in

urine, 748, 749
Terroine, E. F., 105, 500-503
Terry, O. P.. 454
Terunchi, Y., 682
Tezner, E., 455, 456
Thannhauser, S. J., 429
Theissier, 247
Thesen, J., 572, 729
Thevenot, 247
Thiele, O., 754
Thiemich, M., 384
Thierf elder, H., barium, 72; galactose,

216, 610, 611; glucuronic acid, 223;

cephalin, 248; digestion and micro-
organism, 516; protagon, 607, 608;

cerebron and cerebroside, 608-611;

yolk phosphatides, 630; mammary

glands, 643, 669; sphingosin, 611;

cerebronic acid, 611
Thies, Fr., 379, 380
Thiroloix, J., 406
Thiry, L., 490, 491
Thorner, W., 644 .
Thomas, K, 595, 611
Thomas, Karl, 903
Thomas, P., 492

Thomas, W., 488, 909, 911, 914, 917
Thompson, W. H., 574, 682
Thorns, H., 687
Thonnahlen, J., 799
Thudichum, L. W., phosphatides, 239,

240, 242, 245, 248; bilirubin, 431;

brain phosphatides, 605, 607-609; cere-

brosides, 609-611; sphingosin, 611;

lutein,631; paraxanthine, 714; urinary

pigments, 740; alcohol in animal

organism, 921

Thunberg, T., 873, 874, 876
Tichomirow, N. P., 468
Tidemann, F., 458
Tiemann, H., 652, 658
Tigerstedt, K., 762
Tigerstedt, R., respiration apparatus,

869; metabolism, 893, 894, 924, 925,

936; digestion work, 930
Tissot, J., 592
Tobler, L., 341, 484, 763
Toepfer, G., 530, 759
Tollens, B., carbohydrates, 197, 208-211,

216, 217; glucuronic acids, 223, 817;

urea, 687; naphthoresorcin-reaction,

223, 817

Tollens, C., 724, 725, 750, 817
Tolmatscheff, 656, 662, 665
Tomasinelli, G., 848
Tomaszewski, Zd., 716
Tompson, E., 54, 55, 57
Torup, S., carbon monoxyhaemoglobin,

287, 288; globulins and carbonic

acids, 853, 855
Totani, G., 161, 787
Tower, R. W., 121
Towles, C., 692, 693
Toyonaga, M., 388
Traube, J., 10, 11; absorption, 542;

oxydation, 871
Traube, M., 1, 9
Traxl, W., 78
Treupel, G., 749, 808
Treves, Z., 78

Trier, G., 178-180, 240, 243
Trifanowski, D., 437
Trillat, A., 873
Tritschler, F., 773
Troisier, J., 744
Troller, J., 464
Trommer, C., 214
Trommsdorff, R., 877, 910
Triimpy, D., 847
Trunkel, H., 119, 120
Truthe, W., 230
Tschenloff, R., 910
Tschernoruzki, M., 307
Tscherwinsky, N., 563
Tschirjew, S., 344
Tsuboi, J., 339, 881
Tsuschija, J., 744, 747, 793
Tuczek, F., 459 J

Tiillner, H., 698
Turk, W., 146
Turkel, R., 585
Turban, K., 912
Turby, H., 491

Udranszky, L., diamines, 47; bile acids,
419, 800; urinary pigments and humus
substances, 740; carbohydrates in
urine, 749, 808; cystine, 827

Uffelmann, J., 488

Uhlik, M., 280, 282, 283

Ulrich, Chr., 226

Ultzmann, R., 832

Umber, F., albumoses, 133; nucleins,
175; transudates, 354; gastric juice,
464, 465; proteins of pancreas, 494;
Isevuloseuria, 814

Umikoff, N., 664

Underbill, F. P., protozym, 324; glyco-
seuria, 400, 402; saliva, 454; allantoin,
717; lactic acid in urine, 748, 749

Unna. P. G., 837, 844, 845

Ulpiani, C., 244, 699

Urano, F., 573, 587

Urbain, V., 851

Ure, A., 783

Usher, Fr., 37

Ussow, 506

Ustjanzew, W., 542

Vahlen, E., 425
Valenciennes, A., 603, 636
Valenti, A., 663

INDEX OF AUTHORS

979

de Vamossy, Z., 379

Vandegrift, G. W., 545

Vandevelde, A. J. J., 653,

Vanlair, C., 429, 521

Vasilin, H., 720, 723

Vassale, G., 374

Vaubel, W., 82, 99

Vauquelin, L. N., 717

Vay, Fr., 383, 592

v. d. Velde, A., 723

v. d. Velden, R., 724

Velichi, J., 602

Vella, L., 490

Veraguth, O., 910

Verhaegen, A., 465

Verneuil, 461

Vernois, M., 665

Vernon, H. M., erepsin, 57, 492, 493;
white of egg, 65; pancreatic enzymes,
i 497, 500, 501, 503, 507, 509; muscle
rigor, 590

Verploegh, H., creatin and creatinine,
573, 691-694, 698

Verzar, Fr., 404

Viault, P., 340

Victorow, C., 804

Vierordt, K., 302, 863

Vigno, L., 526

Vignon, L., 122

Vila, A., blood corpuscles, 275, 281;
blood pigments, 285, 292; musculamine,
578

Ville, J., oxymethyl furfurol, 215; blood
pigments, 281, 285; bile acids, 419;
fat absorbtion, 539, 540; urinary
chlorine compounds, 758; Bence-Jones
protein, 792

Villiers, A., 758

Vincent, Sw., 376-602

Vines, S. H., 493, 502

Vinci, S., 314, 315

Virchow, R., 171, 301

Vitali, A., 796

Vitali, D., 758

Vitek, 407

Voegtlin, C., 507, 692, 694, 703

Voltz, W., 646, 912

Vogel, H., 372

Vogel, J., pentoses, 208, 815; isomaltose,

i 225; lactase, 492; amylolysis, 456, 500

Vogelius, 391

Voges, O., 680

Vohl, H., 579

Voit, C., glycogen, 390, 393, 395, 534;
bile and putrefaction, 518; excrements,
520; absorption, 525, 537, 538; fat
formation, 560, 561, 663; work and
metabolism, 594, 597, 925; nitrogen
in meat, 600, 883; urea formation,
684; phosphoric acid excretion, 762;
standard numbers, 772, 915; lactose
detection, 815; metabolism experiments,
879, 881, 920, 926, 929; starvation
metabolism, 898, 903; water content

of body, 898; mineral metabolism, 899;
protein catabolism, 903, 906, 908-910,
913, 914; nutritive value of gelatin
diets, 932-936, 938

Voit, E., glycogen, 395; "bones, 554, 555;
fat formation, 562, 563; calorific value
of oxygen, 889; of nutritive substances,
892; starvation metabolism, 894, 896
897; nitrogen excretion, 910; vege-
table diet, 915; protein minimum, 917;
surface rule, 923

Voit, Fr., galactose fermentation, 216;
thyroidea and metabolism, 376; glyco-
gen formation, 395; sugar elimina-
tion, 395, 534; feces formation, 521;
lactose, 532; curare poisoning, 591;
acetone bodies, 819

Voit, W., 814

Voitinovici, A., 106, 114, 121

Volhard, F., 469, 476, 506

Volhard, J., 759, 760

Volkmann, A. W., 899

Vorlander, D., 247

v. V9rnveld, J. A., 341

Vossius, A., 441

Voswinckel, H., 843

Vozdrik, A., 675, 677

de Vries, H., 2, 5, 6

Vulpian,jL, 377, 441

Waage, P., 32

Wachsmann, M., 501

Wachsmuth, L., 356

Walchli, G., 117

de Waele, H., 653

Wagner, B., 813

Wagner, H., 572, 577

Wahlgren, V., 417, 420, 440, 838

Wait, Ch., 595

Wakemann, A. J., 386, 387, 737, 774

Walbum, L. E., 50, 793

Waldvogel, R., 820

Walker, J., 28

Wallace, G. B., 89

Walter, Fr., 682, 851, 856

Walter, G., 173, 630

v. Walther, P., 535, 537

Walton, J. H., 35

Wanach, R., 304

Wang, E., 730, 731

Wanklyn, J. A., 647

Wauner, Fr., 870

Warburg, O., leucine, 142, 143; fertiliza-
tion, 640; phenylamino acetic acid, 786;
oxydation processes, 873, 874

Warfield, L. M., 455

Warmbold, H., 647

Warren, J., 593

Wasbutzki, M., 519

Wassiliew, W., 499

Wasteneys, H., 73

Waterman, N., 379

Waymouth, Reid, E., 533

Weber, 635

080

INDEX OF AUTHORS

Weber, O. H., 341, 555, 340

Weber, S., 594, 692, 693

Wechselmann, Ad., 733

Wechsler, E., 83, 117

Wedenski, N., 749

Wegrzynowski, L., 716

Wehrle, E., 404

Weidel, H., 189

Weigert, Fr., 163

Weigert, R., 265

Weil, Arth., 161, 605, 144

Weil, F. J., 423

Weiland, W., 786

Weinland, E., lactase, 53, 492, 532;

glycogen, 390, 395; sugar formation,

412; retarding substances, 466, 487,

892; fat formation, 561 ;
Weintraud, W., 403, 404, 685, 761
Weis, Fr., 506
Weisbach, 613
Weiser, St., 264
Weisberger, G., 866
Weiske H., cellulose 510; bones, 554,

555; asparagin, nutritive value, 912
Weiss, F., 78, 83, 109, 111, 162
Weiss, H. R., 505, 506
Weiss, J., 721
Weiss, Sigm., 395, 592
Weisz, Mor., 741, 752
Weizmann, Ch., 87
Wellman, O., 896
Wells, H. G., keratin, 114; hypophysis,

380; liver nitrogen, 387; uric acid,

701-703, 706
Wendel, A., 143
v. Wendt, G., 882, 903
Wenz, R., 128
Wenzel, F., 178-180, 577
Werenskjold, F., 659
Werigo, B., 95, 866
Werncken, G., 651
Werner, A., 552
Wertheimer, E., 440, 498, 499
Werther, M., 459, 590, 591, 593
v. Westenrijk, N., 309
Wester, D. H., 839, 840
WetzelJG., 122, 160, 178
Weyl, Th., albumin crystals, 94; car-

bonmonoxy methaemoglobin, 287; amni-

otic, fluid, 642, 643; creatinine, 695;

benzoic acid, 723; nitrate, 766
Wheeler, H., L., iodogorgonic acid, 123;

phenylalanine, 152; nucleic acid, 185;

cytosine, 194
Whipple, G. H., 253, 442
White, B., 705
Withney, J. L., 406 '
Wichmann, A., 261, 652
Widdicombe, J. H., 492
Wichowski, W., urea determination, 688;

uric acid, and allantoin, 705, 706,

716-718; hippuric acid, 721
Wicland, H., 423
Wiener, H., autolysis, 43; serumglobulins,

259, 261, 335, nucleic acids, 514; uric
acid, 699, 700, 702, 704-706; oxalic
acid, 716

Wiener, K., 182

Wihelmy, 33

Wilke, K., 298

Willcock, Ed., 634

Willdenow, C., 164

v. Willebrand, E. A., 849

Willheim, R., 103

Williams, D., 510

Williams, H. B., 827

Willstatter, R., proline 154; lecithin, 263;
glycerinphosphoric acid, 247; chloro-
phyll and blood pigments, 277, 296,
297; cholesterin, 445, 448; carotein
volk lutein, xanthophyll, 631

Wilson, R. A., 606, 608

Wimmer, M., 914

Windaus, A., histidin, 159, methylimi-
dazol, 201; cholesterin, 445, 448, 449

Windrath, H., 529

Winkler, 559

Winogradow, A., 416

Winteler, L., 416

Winter, J., 660

Winterberg, A., 334, 675, 683

Winternitz, Hugo, blood pigment deter-
mination, 302; amounts of haemoglobin,
338; bile, 440; putrefaction, 517,
724; iodized fat, 560, 668, 669

Winternitz, M. C., 371, 702

Winterstein, E., amino-acids, 141, 153;
arginine, 161; lysine, 163; hexone
bases, 164; phosphatides, 245; phytin,
579; colostrum, 658; tunicin, 839;
chitin, 839

Wislicenus, J., 596

Wittmaack, K., 663

Woeber, A., 126

Wohler, Fr., hippuric acid synthesis, 39,
783; urea, 689; demolition of uric acid,
705, allantoin, 716, 717

Worner, E., 607, 611, 711

Wohl, A., 200

Wohlgemuth, J., autolysis, 43; enzymes,
53, 71; oxyaminosuberic acid, 147;
oxydiamino-sebacic acid, 165; pen-
toses, 203, 208, 210; diastase in blood,
298; ferratin, 383; glycogen, 393, 394;
cystine demolition, 442; gastric juice,
476; pancreatic juice, 500; pancreatic
diastase, 501; pancreatic rennin, 509;
enzyme of egg-yolk, 628; woman's
milk, 660, 661; conjugaged glucuronic
acids, 751; amino acids in urine, 756

Wolf, C. G., glycogen, 397; urinary
nitrogen, 680; creatinine, 693; cystin-
uria, 827, 828; urinary sulphur, 882

Wolfenstein, R., 737

v. Wolff, E., 595

Wolff, H., 219, 358, 841

Wolff, J., 873

Wolff, L. W., 19

INDEX OF AUTHORS

981

Wolffberg, S., 395, 861, 864, 865

Woikcw, M., 735, 736

Woll, F. W., 645

Wolter, O., 770

Weltering, H., 340, 383

Woods, H. S., 246

Wooldridge, L. C., strorna, cf blood cor-
puscles, 274; tissue fibrinogen, 306,
307; blood coagulation, 315, 321, 323,
324

Worm-Miiller, J., blood, 339, 340, 343,
344; sugar test, 803; sugar determina-
tion, 811-813

Worms, W., 635

Woronzow, W. N., 381

Wright, A., fibrin ferment and coagula-
tion, 256, 313, 323; blood alkalinity,
309; diabetes, 400

Wr6blewski, A., fermentation, 41 ; pseudo-
nuclein, 104; starch, 227; pepsin, 466,
470; enzyme action, 510; enzymes
of brain, 606; milk, 660, 661

Wulff, C.. 160, 714

Wurm, W. A., 843

Wurster, C., 768

Wurtz, A., 346, 625

Yagi, S., 448

Yakuwa, G., 912

Yoshimoto, 443

Yoshimura, K., 579

Young, P. A., 435

Young, R. A., 230, 391

Young, W. J., co-enzymes, 52, 205; car-
bohydrate-phosphoric acid ester, 60,
205 ; dioxyacetone, 205

Yvon, 752

Zangerle, M., 626

Zah6r, H., 794

Zaitschek, A., milk, 651, 653, 658-662,

668

Zak, E., 354, 769, 791
Zaleski, J., leaf and blood pigments, 277,

296; blood pigments, 291-295, 301;

ammonia, 334, 336, 528, 683, 768;

urea, 684; liver and acid formation,

685, 703, 704
Zaleski, St., iron of liver, 383, 388; milk,

666, 667; reaction of intestinal contents

519

Zalesky, N., 552, 847
Zander, E., 839
Zanetti, C. II., ,seromucoid, 260, 263;

bile, 417; ovomucoid, 635

Zangermeister, W., 302, 642

Zaribnicky, Fr., 348

Zaudy, 701

Zdarek, E., 841

v. Zebrowski, E., 453

Zeehuisen, H., 777

Zeidlitz, P. V., 804

Zegla, P., 398

Zeller, A., 758, 777

Zemplen, G., 839

v. Zeynek, R.,, dermoid-cyst-fat, 239, 627,
844; blood pigments, 283, 285, 289,
290; liver, 602; bile, 438; sarcomelanin
841; chromoproteid, 843

Zickgraf, G., 83, 870

Ziegler, E., 779

Ziegler, J., 32, 708, 716, 717

Zillessen, H., 593

de Zilwa, L., 500

Zimmermann, R., 372, 725, 726

Zimnitzki, S., 517

Zink, J., 237, 559

Zinnowsky, O., 277

Zinsser, A., 476

Zisterer, ,!., 917

Zobel, S., 581

Zoja, L., oxyproteic acids, 83; elastin, 122;
ovalbumin, 633; urobilin, 744; uroery-
thrin, 748; hsematoporphyrin, 797, 798

Zsigmondy, R., colloids, 15, 19, 21, 23

Zuelzer, G., lecithin, 245; diabetes,
405, 406; skin breathing, 849

v. Zumbusch, L., 434, 844

Zuntz, N., blood, 310, 338, 340, 341;
glycogen, 391; sugar in blood, 398;
phlorhizin diabetes, 400; digestion,
506, 542; protein assimilation, 525;
muscle fat, 586, 596; muscle metabol-
ism, 591, 595, 597; pig milk, 659;
high altitudes, 341, 848; skin breath-
ing, 849; gases of blood, 851-853, 859,
866, carbonic acid of lymph, 856;
alveolar air, 863; respiration, 868,
869; metabolism, 879, 884, 890, 893^
922, 923, 926, 927, 929; calculation
of calorific power, 888; nutritive value
of albumoses, 912; alcohol, 921; diges-
tion work, 930

Zuntz, L., 309

Zuntz, E., digestion products, 132-134,
139; gastric digestion, 482, 484; absorp-
tion, 484, 526, 532, 540; trypsinogen
and its activation, 496, 497; intestinal
digestion, 513; muscles ,572

Zweifel, P., 456, 500, 748, 749

GENERAL INDEX

Abderhalden and Schmidt's reaction for

proteins, 101

Abiuret products, 132, 513, 526
Absorption, 524-543

chemical processes during,

524-525

effect of extirpation of the
pancreas upon,
531, 532

removing portions
of intestine upon,
541, 542

of amino acids from intes-
tine, 526-528

of bile constituents, 540, 541
carbohydrates, degrees of

rapidity of, 532-534
of fat, 535-540
of nonbiuret giving products

from intestine, 526-528
of mineral substances, 540
peptones from intestine, 526-

528
of proteose from intestine,

526-528
of undigested protein, 525,

526

theory of, 542, 543
Acetanilide, fate of in organism, 779
Acethaemin, 292
Acetone bodies, 776

in urine, 818-828
origin of, 818, 819
fate in organism, 821
formation from fat, 820-822
formation from protein, 819
former, 781
in urine, 822-824
quantitative estimation of, in

urine, 825

Acetophenone, fate of in organism, 785
Acetylation in organism, 786
Acetyldiglucosamine, 839
Acetyl equivalent of fats, 238
Acetylglucosamine, 784
Acetylene haemoglobin, 288
Acetylpara-amidophenol, 780
Achilles tendon, 545
Acholia, pigmentary, 439

Achroodextrin, 229, 458
Acid abietinic, 447

acetic, in intestines, 513
in stomach, 465
in urine, 748, 773
acetoacetic in urine, 821, 824, 825

fate in organism, 821
acetylaminobenzoic, 784
albuminates, 125-127

absorption of, 533
in peptic digestion, 472
alloxyproteic.'in urine, 752, 754, 755
amides, behavior in organism, 775
aminoacetic. See Glycocoll.
aminocaproic. See Leucine, 141
a-diamino-/3-dithiolactic, 148
a-y-diamino valeric,, 163
a-e-diaminocaproic, 163
d-a-amino-n-caproic, 144
aminoethylsulphonic, 150
a-aminoglutaric, 146, 117
a-aminoisobutylacetic. See Leucine.
a-amino-valeric, 140. SeeValine.
aminobenzoic, behavior in organ-
ism, 783

aminocinnamic, 778, 780
a-amino-/3-oxypropionic, 145. See

Serum.
a-amino-/3-thiolactic, 148, 150. See

Cystine,

a-aminopropionic, 140. See Alanine,
aminoglucuronic, 548
aminohippuric, 783
aminophenylacetic, behavior in or-
ganism, 781
aminosuccinic. See Aspartic acid,

146

antoxyproteic in urine, 752
arachidic, 232, 627,645,845
aspartic, 85, 146

quantity in proteins, 106,

107, 115, 125
relation to formation of

urea, 682, 776
relation to formation of

uric acid, 702
/3-amino-a-oxypropionic. See Iso-

serine, 146
/3-irnidazol-a-aminopropionic, 159

984

GENERAL INDEX

Acid benzole, conjugation of, in organism,

782

in urine, 723
glucuronic, 751

benzoyl-aminoacetic. See Hippuric
acid, 719

bilinic, 429

bilianic, 423

bilirubinic, 430

bromphenylmercapturic, 780

butyric, fermentation, 214, 516

camphoglucuronic, 222, 751, 786

capric, 232

caproic, 232

caprylic, 232

carbamic, 267

conjugation of, 786
fate of in organism, 786
in blood, 267, 683
in urine, 691, 692

carbamino-acetic, 164, 856

carboglobulinic, 855

carbolic. See Phenol.

carminic, 844

carnaubic, 239, 673

carnic, 653

caseanic, 85, 165

caseinic, 85, 165

cephalic, 248

cepholinic, 248

cerebrinic, 612

cerebrinic-phosphoric, 612

cerebronic, 611

cerotic, 239

cheno-taurocolic, 422, 427

chitaminic, 219

chitaric, 219

chlorhodinic, 366

chlorphenylmercapturic, 786

cholalic, 423-425

constitution of, 423

cholanic, 425

choleic, 425

cholest2rinic, 423

cholic, 423, 424

properties of, 424
tests for, 424

choloidanic, 423

choloidic, 427

cholylic, 423

chondroitic. See Chondroitinsul-
phuric acid, 547

chondroitin-sulphuric, 171, 173, 547

chrysophanic, elimination in urine,
787

cilianic, 423

cinnamic, behavior in organism,
719

citric, in milk, 647, 658, 663

coccinic, 844

cochenillic, 844

combined hydrochloric, 488

cresol-sulphuric, 724-727

crotonic, 825

Acid cumic, conjugation of in organism,

783
cuminuric, synthesis of in organism,

783

cyanuric, 687, 698
cysteinic, 149
damaluric, 758
damolic, 758
dehydrobillinic, 429
dehydrocholeic, 425
desaminoalbuminic, 127
desoxycholic, 425, 426

properties of, 426
diacetic. See Ace to-acetic
dialinic, relation to formation of

urine, 704
diamino acetic, 85
diaminotrioxydodecanoic, 85, 165
dimethylaminobenzoglucuronic in

urine, 751, 786
p-dimethylaminobenzoic, 786
dioxydiaminosuberic, 165
dioxyphenylacetic in urine, 735
dioxyphenyl-lactic, 738
dioxystearic, 236, 647
doeglic, 237
elaic, 236
elaidic, 236
ellagic, 524
equivalent, 238
erucic, 232
ethylidenelactic, 582
euxanthic, 222, 223

in urine, 785

euxanthonglucuronic, 222
excretolic, 523
fellic, 426
fermentation lactic, 582

in blood, 335
in coagulation of
milk, 585, 645
in muscle, 604
in stomach, 487,

488

formic, fate in organism, 748, 773
furfuracrylic in urine, 784
furfuracryluric in urine, 784
gadoleic, 237
galactonic, 217
gallic, fate of in organism, 785

in urine, 734
gentisic, 336, 737

behavior in organism, 784
gluconic, 198

in diabetes, 403
glucosaminic, 201, 219, 221
in milk, 643
glucothionic, 387, 673

in milk, 643
glucuronic, 221-223

conjugation with, 785
elimination, effect of for-
eign substances on,
750

GENERAL INDEX

985

Acid glucuronic, formation of in organism,

751

in bile, 417, 433
in blood, 264
in urine, 750
isolation of, 222
preparation of, 223
properties of, 222
quantitative estimation

of, 223
glutamic, 82, 86, 106, 107, 115, 125,

147

glutinic, 119
glyceric, 145
glycerophosphoric, 248

in urine, 749, 757

glychollic, fate of in organism, 713
glycocholic, 417, 419, 420

absorption of, 548
in feces, 517
preparation of, 421
properties of, 420
glycocholeic, 420-421

preparation of, 421
properties of, 420
glycosuric in urine, 735
glycuronic, 221
glyoxylic, 719

as reagent, 100
guanidineacetic, fate of in organism.

See Glycosamine, 787
6-guanido-a-amino valeric. See Arg-

inine, 161
guano bile, 421
guanylic, 178, 181, 183, 184

of liver, 383
haematinic, 296, 430
hsematopyrrolidinic, 291
haemoglobin, 285
hippuric, 267, 719-723

formation of, 139

in organism,

719
in urine of herbivora, 720,

721

occurrence of, 719 720
preparation of, 722-723
properties of, 722
quantitative estimation of,

723

reactions for, 722
synthesis of in organism,

782
synthetical preparation of,

719
theories of formation in

organism, 721
homogentisic, 778

formation of in organ-
ism, 735-737
in urine, 727, 734-740
mother substances of,

735-736
preparation of, 739

Acid homogentisic, properties of , 738-739
quantitative estima-
tion of, 739
quantity eliminated,

735

test for, 739

homoglytisic, origin of, 736
hydrochloric action upon ptyalin, 455
secretion of

bile, 416
secretion of
pancrea-
tic juice,
498

pylorus,
480, 482
antifermentive action,

485

hydrochinon -sulphuric, 724
hydrocinnamic, behavior in animal

body, 720

hydrocyanic, effect on blood pig-
ments, 285 1
pepsin diges-
tion, 471
trypsin di-
gestion, 506

hydroparacoumaric, in intestinal pu-
trefaction,!)^
in urine, 734

hydroquinone carboxylic, 738
hydroquinonesulphuric, 691, 724, 728
hyoglycocholic, 421, 427
hypogaeic, 239

imidazolaminoacetic in urine, 758
imidazol propionic, 82
indolacetic, 82

in urine, 733

indolaminopropionic, 82, 155
indol-carboxylic, 728
indol-propionic, 82, 155
indoxyl-carboxylic, 728
indoxylglucuronic, 728
indoxyl-sulphuric, 724

formation of in

organism, 728
in urine, 728-731
inosinic, 178, 181-183, 185
iodogprgpnic, 123
isobilianic, 423
. isocholanic, 425
isocholic, 427

isophonppyrrol carboxylic, 429, 430
iso valeric, 774
jecoleic, 237
kynurenic, 740, 758

in urine, 734
kyroproteic, 83
lactic, 145

formation of in active
muscles, 593

in blood, 333
in blood, 334
in cerebrospinal fluid, 360

986

GENERAL INDEX

Acid lactic, in relation to diabetes, 408
origin of, 583, 584
quantitative determination in

gastric contents, 488
test for in gastric contents, 488
transformation into uric acid,

704

lactophosphocarnic, 645, 653
lanoceric, 846
lanopalmitic, 846
lauric, 232, 647, 846
lepidotic, 844
leucinic, 142
levulinic, 211, 654
linolenic, 232
linolic, 232
lithobilic, 524
lithocholic, 427
lithofellic, 427, 524
lithuric, 758
lysalbinic, 127
lysuric, 164
maleic, 290

malic, fate in organism, 668, 676
mandelic, 780
mannonic, 212
margaric, 235
melanoidic, 841
menthol-glucuronic, 817
mesitylenic, conjugation of in organ-
ism, 783

mesitylenuric synthesis of in organ-
ism, 783

metaphosphoric as precipitant of
proteids, 98, 789

in nucleins, 176
in pseudonucleins,

methylethyl-«-aminopropionic, 143.

See Isoleucine.
methylguanidine-acetic. See Crea-

tine, 573

methylhydantoic, 776
monoxystearic, 233, 236
mucic, 217, 394, 654
mucpnic, 778
myristic, 232
naphtholglucuronic, 787
neurostearic, 611
nitrobenzoic, 784
nitrohippuric, 783

nitrophenolpropiolic, fate in organ-
ism, 728, 731
test for dex-
trose, 216,808
norisosaccharic, 219, 630
nucleic, 622

of milk, 643
yeast, 178, 185
nucleotinic, 179
nucleotinphosphoric, 179
oleic, 232

properties of, 236
for, 237

Acid ornithuric, 163

synthesis of in organism,

783

orotic, 654
orylic, 653

oxalic in urine, 716, 717
oxaluric, 699, 715
oxaminic, 83
oxonic, 699

oxyaminosuberic, 85, 148
oxyaminosuccinic, 85, 147
oxy-a-pyrolidinecarboxylic. See oxy-

proline.

oxybenzoic, fate in organism, 783
0-oxybutyric, 821, 825-827

detection of in urine,

826

fate in organism, 821 ]
formation from amino

acids, 821

properties of, 825, 826
quantitative estima-
tion of, in urine 826
oxydiaminosebacic, 85, 165
oxydiaminosuberic, 85, 164
oxyhydroparacoumaric, in urine, 734
oxyisovaleric, 774
oxymandelic, 739

oxymethylpyrazine-carboxylic, 221
p-oxyphenylacetic, 515

in urine, 734, 784
p-oxyphenyl-a-aminopropionic. See

Tyrosine, 152
p-oxyphenylpropionic, 515, 734

in urine, 784

oxyphenylpyroracemic, 737
oxyproteic in urine, 752, 754
oxyprotosulphonic acid, 83
oxyquinoline carboxylic. See Kynu-

renic acid,
palmitic, 232, 235

properties of, 236
parabanic, 699
paralactic, 582

origin of, 583, 584
in urine, 749
paranucleic, 652
paraoxyphenyl-acetic in urine, 734,

735
paraoxyphenylpropionic, 82

in urine,

734, 735

pepsin-hydrochloric, 473
peroxyproteic, 83
phenaceturic in urine, 723
phenol glucuronic, 222, 725, 749, 751
phenol-sulphuric, 724, 725-727
phenylacetic, 82

conjugation of in or-
ganism, 783
phenylaminoacetic, 780
phenyl-a-aminopropionic. See Phe-

nylalanine, 151
phenylbutyric, 781

GENEKAL INDEX

987

Acid phenylcaproic, 781

phenylketopropionic, 781
phenyllactic, 736, 778, 780
phenyl oxyprop ionic, 781
phenylpropionic, 82
phenyl pyroracemic, 737
phenyl valeric, 781

in perspiration, 848
in urine, 724

phonopyrrolcarboxylic, 298
phosphocarnic, 267, 572, 577-579

in active muscles, 594

in urine, 757

source of muscular

energy, 598
as nuclein amounts
in blood of differ-
ent animals, 326
in urine, quantitative
estimation of, 763-
764

phthalic, fate in organism, 779
physetoleic, 239
plasminic, 186
polypeptide phosphoric, 652
protalbinic, 127
protic, 572
protocatechuic, fate in organism,

726

pseudonucleic. See Paranucleic acid
psyllic, 845
pulmotartaric, 870
pyinic, 366

pyridine-carboxylic, fate of in or-
ganism, 784
pyridinuric, 784
pyrocatechin-sulphuric, 724

in urine, 727
pyrocholoidanic, 424
a-pyrolidine carboxylic. See Pro-
line, 154

pyromucic in urine, 784
pyromucinornithuric in urine, 784
pyromucuric, 784
pyroracemic, 150

effect of yeast upon,

206
pyrrolidone-carboxylic. See a-Pro-

line, 113

pyruvic, effect of yeast upon, 206
quinic, 719

racemic, fate in organism, 773
renosulphuric, 673
rhodizonic, 580
saccharic, 198, 222

behavior in diabetics, 403
relation to glycogen for-
mation, 394
salicylic, conjugation of in organism-

783

salmo-nucleic, 178
sarcolactic, 582

in brain, 606

in lymphatic glands, 366

Acid sarcolactic, in muscular work and

rigor, 591-595
in the bones, 556
passage into urine, 748
sarcomelanic, 841
scymnol, 417
scymnol-sulphuric, 417
sebacic, 236
silicic, in blood, 268
in bones, 555
in connective tissue, 549
in feathers and hair, 839
in hen's eggs, 632, 637, 639
in urine, 769
skatol acetic, 82, 156
skatolaminoacetic, 156
skatol-carboxylic in urine, 733
skatoxylglucuronic, 222, 732, 750
skatoxyl-sulphuric, 724, 732, 733
origin of, 732
stearic, 232

properties of, 235
succinic, 83

in fermentation of milk, 645
in intestines, 513
in perspiration, 848
in spleen, 370
in thyroid, 373
in transudates, 355, 359, 361
in urine, 749, 773
sulphosalicylic as a reagent, 790
sulphuric, action on peptic digestion,
469. See also Ethereal sulphates
and Mineral substances,
tannic, fate of in organism, 785
tartaric, relation to glycogen for-
mation, 394
in organism, 773
in perspiration, 848
tartronic, 704
taurocarbamic, 776
taurocholic, 417, 421-422

preparation of, 421, 422
properties of, 422
taurocholeic, 422, 423
tetraoxyaminocaproic, 548
therapinic, 237
thiolactic, 150
thiophenuric in urine, 784
thymic, 179

thymonucleic, groups, 181
thiolactic, 85
toluic, conjugation of in organism,

783

toluric, synthesis of in organism, 783
trichlorethylglucuronic. See Uro-

chloralic acid,
triticonucleic, 178, 185
2-, 4-, 5-trimethylpyrrol-3-propionic,

429

trioxybenzoic. See Gallic,
turpentine glucuronic, 785, 817
tyrosinesulphuric, 153
uraminobenzoic, 783

GENERAL INDEX

Acid uramin-salicylic, 783
urea glucuronic, 750
uric, 187, 267

amounts formed in organism.

707

as a pigment, 844
effect of food on elimination of,

700

endogenous origin, 702
exogenous origin, 702
fate of in organism, 705, 706
formation of from purines, 701,

702

in birds, 703, 704
of in the organism,

701-707

from lactic acid, 704
in blood, 334
in muscles, 572
in urinary sediment, 830
in urine, 698-712
occurrence of, 699, 700
preparation from urine, 710
preparation of, 698
properties of, 698-699, 707, 708
quantitative estimation of in

urine, 710
relation to urea
elimination, 701
quantity in various urine, 699,

700

synthetic formation of, in or-
ganism, 705
tests for, 709

various factors effecting elim-
ination, 700-702
urocanic, 758
urochloralic, 777
uroferric in urine^ 752, 755
uroleucic in urine, 736, 739
uronitrotoluolic in urine, 786
uroproteic in urine, 754
uroxanic, 699
ursalicylic in urine, 783
ursocholeic, 427
valeric, 80, 142
vanillinic, 780
whey, 645

xanthobilirubinic, 429
xanthopyrrolcarboxylic, 299
yeast-nucleic, 179, 185
Acids, amino, 92

aromatic, fate of in organ-
ism, 780-783
investigations on,
of demolition in
organism, 780-
783

as acetone formers, 819
as carbon dioxide binders,

855, 856

conjugation with, 786
deamidation, 682
fate of, in organism, 774-776

Acids, amino, formation in tryptic diges-
tion, 507
of, in liver, 530
how absorbed, 528
in globin of blood pig-
ments, 289

in homogentissic acid for-
mation, 736-738
in liver tissue, 383
in lymphatic glands, 366
in muscles, 572
in transudates and exu-

dates, 355
in serum, 267
in urine, 756, 827
investigation on the demo-
lition of, in organism,
774-776

of pseudomucin, 626
Sorensen's, formol titration

for, 139-167
synthesis of, in organism,

786
sugar formation from, in

liver, 412

transformation of, into
urea, in the organism,
683, 684
aromatic, fate of, in organism, 784

-oxy, in urine, 733, 734
bile, 267

detection of, 418, 419, 427, 428
biliary, properties of alkali salts,

418

caseonphosphoric, 652
cholic, preparations of, 426
conjugated glucuronic in urine,

817-818

desaminoproteic, 83
diamino, 84, 159-163
ethereal sulphuric, 723-733

amounts in

urine, 724
in urine, 765
quantitative es-
timation of,
726
synthesis in

liver, 381

excitants for bile secretion, 416
fatty, 232-239, 265

amounts in blood of differ-
ent animals, 328
in brain, 605
in blood, 334
in perspiration, 848
in pus, 365, 366
in urine, 748, 773, 829
investigation on the demol-
ition of, in organism, 773,
774
series, fate of, in organism,

773, 774
glucuronic, conjugated, 222

GENERAL INDEX

989

Acids, glycocholic, preparation of, 421

in large intestine, from putrefac-
tion, 515
in spleen, 370
in thymus, 368
in thyroid gland, 373
in transudates and exudates, 355
kyroproteic, 83
lactic, 582-586

detection of, 585
in bones, 556
in urine, 703, 704, 748
mercaptic, elimination of, 786

properties of, 585
melanoidic, 841

mineral, alkali-removing action of,
and action on the elimination of
ammonia, 675, 676, 762, 768, 856
893

monoamino. See Acids, Ammo,
neutralization of, in the organism,

675, 676

nucleic, 175, 177-186
complex, 181
effect of gastric juice on,

473

enzymotic, cleavage of, 508
plant, 185-186
simple, 181
organic, behavior in animal body,

767, 772, 773, 776, 777
oxyamino, 79, 80, 84, 148, 219
oxyfatty, in animal fat, 232, 238
oxy, in urine, detection of, 735
oxypropionic, 582
oxyproteic in urine, quantitative

estimation of, 755, 756
phthalic, fate of, in the organism.

778

proteic in blood, 267
in serum, 267
in urine, 752, 754
thymonucleic, 178
thymus-nucleic, 178
uramino, 786
ureido glucuronic, 750
volatile fatty, in urine, 748
Acidosis, 820, 821
Acrite, 212
Acrolein, 234, 237
a-acrose, 212
/3-acrose, 212
Actiniochrom, 844
Activators, 60
Adamkiewicz-Hopkin's reaction for tryp-

tophane, 157

Adamkiewicz's reaction for protein, 100
Adaptation of the glands, 454, 462, 463,

494-496

Addison's disease, 377, 379
Adelomorphic cells, 489
Adenase, 48, 188

Adenine, 178, 181, 185, 187, 188, 192, 193,
712

Adenine-hexose compound, 180

Adenosine, 180

Adialyzable bodies in urine, 757

Adipocere, 560

Admissibility, theories of,, 9

Adrenalin, 378-380

bodies, 377, 378
constitution of, 378
function of, 379, 380
properties of, 379
tests for, 379
" Adsorpates," 12
Adsorption, 49, 62, 63, 69

in relation to permeability,

11, 12

-(Egagrophilae, 524
^Erotonometric method, 864
Age, effect upon metabolism, 922, 924
Agglutenins, 47, 69
Agmatine, 162

Air bladder, of fishes, gases of, 867
Alanine, 85, 106, 107, 109, 111, 113, 125,

140, 145

Alanylalanine, 88
Alanylalanineglycin, 86
Alanylglycine, 87, 508
Alanylleucine,' 86, 508
Albamine, 220
Albumin, 49

detection of, in urine, 791
Albuminates, 104, 125-127
acid, 92, 126

alkali, 91, 92, 125, 126, 533
Albuminoids, 92, 112
Albuminose, 623
Albumins, 91, 92, 93, 102, 103, 106

quantitative estimation of, in

urine, 793

properties of, 102, 103
Albumoids, preparation of, 549
Albuminuria, 771, 787
Albuminoids, 92

Albuminous bodies, in general, 94-97
Albumoid, properties of, 618
Albumose, alkali, 127
Albumoses, 130
Alcapton in urine, 727
Alcaptonuria, 735-740
Alcohol, aminoethyl, 240
cetyl, 239
melissyl, 239
myricyl, 239
Alcoholase, 47, 48
Alcoholases, 875
Alcoholic fermentation, 41
Aldehydases, 875
Aldehydes, fate of, in organism, 775, 780,

781

Aldoses, 197
Alexines, 266

Alimentary acetonuria, 820
glycosuria, 533

Alizarin, 'elimination in urine, 556, 787
Alkali earths. See Mineral substances.

990

GENERAL INDEX

Alkaloids, fate of, in organism, 787
Alkyl sulphides, 846
Allantoin, 699, 717-719

elimination of during poison-
ing, 717, 718

formation of, in organism, 717
occurrence of, 717
preparation of, 718
properties of, 718
tests for, 718
Alloisoleucine, 145
Alloxan, 86
Alloxuric bases, 186, 712

preparation of, 714. See

Purines.

Alveolar air, 853
Ambergris, 524
Amboceptors, 69
Ambrain, 524
Amicrons, 19
Amidases, 702
Amidoguanine, 718
Amidomyelin, 609
Amidulin, 227

Amino acids. See Acids, Amino.
Amino aldehyde, 219

butyrqbetaine in urine, 787
cerebrinic acid, glucoside, 612
oxypurine. See Guanine.
oxypyrimidine. See Cytosine.
sugars, 219-221
Aminophenol, 779
Aminopurine, 191
Aminosuccinic-acid amide. See Aspara-

gine
Ammonia, amounts in urine, 766

detection of, in urine, 768
elimination in relation to acid

formation, 766, 767
importance of to organism, 767
in blood, 334
in urine, 766-768
in venous blood, 336
quantitative estimation of, in

urine, 768
transformation of. in the liver,

767
Ammonium magnesium phosphate, in

intestinal calculi, 232
in urinary calculi, 834
salts, relation to glycogen

formation, 394
relation to urea for-
mation, 683, 767
relation to uric acid

formation, 702
urate, in urinary calculi, 834
in urinary sediment,

829

Amniqtic fluid, 642
Amphicreatine, 578
Amygdalase, 48
Amygdalin, cleavage of, 59
Amylase, 47, 48

Amylodextrin, 227, 229

Amyloid, 172, 231

preparation of, 174

Amylopectin, 227

Amylopsin, 501

Amylose, 227

soluble, 227

Amylum. See Starch, 226

Anaphylaxis, 70

Aniline, fate of in organism, 779

Animal oxidation, 42

gum in urine, 749

Anions, 5

Anisotropous muscle substance, 565

Antedonin, 844

Antialbumate, 472

Antialbumid, 472

Antibodies, 66, 68

Antidonin, 844

Antienzyme, 63

Antigens, 66, 68, 69

Antiketoplastic action, 820

Antimony, action on N-elimination, 679

passage into milk, 671. See
Mineral substances.

Antipepsin. See Enzymes, 464

Antipeptone, 130

Antipyrine, fate of, in organism, 785

Antirennin, 69, 474

Antithrombin, 322

Antitoxins, 47, 66

Antitrypsins, 503

Anuria in cholera, 848

Aorta elastin, 116

Apatite in bone ash, 553

Amphopeptone, 129

Aporrhegmas, 167

Aqueous humor, 361

Arabinoses, 198, 210. Structural formu-
lae for, 198

d-arabinosinine, 201

Arabite, 198

Arachnoidal fluid, 355

Arbacin, 108

Arbutin, 394, 727

Arginase, 48, 91, 161

Arginine, 85, 106, 107, 108, 109, 111, 113,

115, 117, 119, 125, 161, 162
in various proteins, 165
cleavage into creatine, 575

Arginine-histone peptone, 139

Argon in blood, 850. See also Gases

Arnold and Lipliawsky's reaction for
acetoacetic acid in urine, 824

Arnold's reaction for proteins, 100
urine reaction, 696

Aromatic combinations, fate in organism,
7767784

Arsenic poisoning, 439-441

on nitrogen elimination, 679

Arterin, 276

Ascitic fluid, 357

Asparagine, 146

Asparagus, effect on odor of urine, 764

GENERAL INDEX

991

Assimilation limit, 533

Atheromatous cysts, 142

Atmidalbumin, 130

Atmidalbumose, 130

Atmidkeratin, 113

Atmidkeratose, 113

Atropine, effect on uric acid elimination,

700

Auto digestion, 43
Auto-oxidation,
Autolysis, 43

as a protective agent, 46
effect of arsenic upon, 44
CO2 on, 44
in organic colloids, on

44

oxygen upon, 44
radium on, 44
reaction upon, 43
importance of enzymes on, 45
various processes in, 44
Autolytic processes in life, 45
Autotoxines, influence on putrefaction, 529

Bacteria, influence on putrefaction, 520

Bacterial action in intestinal canal, 518

Bacteriolysins, 69

Bacterium ureae, 829

Balsam of copaiba, fate of, in organism,

787

Bang's method of estimating sugar, 809
Barium, 22

Basal requirement, 897, 922
Basedow's disease, 375
Baumann's test for dextrose, 216
Beer vinegar bacteria, enzymes of, 10
Beeswax, 239

Bela v. Bitto's reaction for acetone, 823
Bence- Jones' protein, 792
Benzaldehyde, 36
Benzene homologues, fate of, in organism,

779
ring, theory for splitting of, in

organism, 778
Benzidine blood test, 798
Benzoylation of carbohydrates, 215, 216,

749, 808

Benzoylchloride test for dextrose, 215
Benzoyl cystine, 150
Betaine, 246, 572
Bezoar stone, oriental, 426, 532
Bifurcated air, 853
Bile-acids, 419-428

detection of, 418, 419

in animal fluids,

427, 428

formation of, 442
in blood, 267, 333
in urine, 799-800
origin of, 440

relation to bile pigments, 444
Bile, 413, 442

absorption of, from the liver, 444
amounts secreted, 414-445

Bile, chemical formation of, 440-444

coloring matters, detection of, in

blood, 435

composition of, in disease, 439
concretions, cholesterin stones, 445

pigment stones, 445
constituents, absorption of, 540, 541
constituents of, 417, 435
effect of in absorption of fats, 536-

540

effect on putrefaction, 518, 519
gases of, 857
Hiifner's, 419
human, 437

composition of, 438
pigments, in, 438
properties of, 437

importance in absorption of fat, 511
mucus, 415, 437
passage into blood, 444
urine, 444

phosphorized constituents of, 439
properties of, 416, 417
putrefaction of, in intestine, 516
quantitative composition of, 436, 437
pigments, 427

formation of, 440-442
in feces, 522
in urine, 800, 801
origin of, 440, 441
relation to bile acids, 444
relation to blood pig-
ments, 430, 442, 443
relation to urinary pig-
ments, 743. 744
test for, 432, 433
salts, 417-419

properties of, 418
tests for, 418, 419
secretion, cholagogues, 415

effect of therapeutic agents

on, 415

Biliary fistula, 413, 421
Bilicyanin, 435
Bilifulvin, 427
Bilifuscin, 434
Bilihumin, 435
BiliphaBin, 427
Biliprasin, 434
Bilipurpurin, 435
Bilirubin, 427, 431, 516

derivatives of, 428-430
detection of, in blood, 435
Ehrlich's test for, 429
Hedenin's test for, 433
hemi-, 429

Huppert's test for, 429
hydro-, 428
of blood serum, 268
preparation of, 433
properties of, 430-432
quantitative estimation of, 433
Biliverdin, 433, 434

in excrements, 530

992

GENERAL INDEX

Biliverdin in feces, 530
properties, 434
preparation of, 434
Biological equivalence, 917

protein reactions, 266, 533
Bismuth, passage into milk, 670
Bitter substances, effect upon secretion,

461, 482
Biuret, 86

base, 86

reaction for proteins, 101
Blister-fluid, 362
Blood, 308-344

acid lactic, in, 334

alkalies in, 310, 311

ammonia in, 334

analyses of blood from various

animals, 328
arterial, 335, 336

quantity of carbon di-
oxide in, 851

quantity of oxygen in, 851
at different periods of life,,
" buffy coat," 312
casts in urine, 796
clot, 251

coagulation, accelerating sub-
stances, in cell, 315,
316
calcium salts in, 316,

317

lime salts in, 316
methods of retarding,

312

prevention of, 251
retarding substances

in, 312-326
theories for, 317, 321-

3231

changes in viscosity, 311
coloring matters, in urine. See
also Blood
p i g m e n ts,
795-799
relation to bile
pigments,
442, 443

corpuscles, 250, 272-305-308
constituents of, 274
determination of vol-
ume in blood, 326,
327
effect of water on, 6

salts on, 6
experiments with, 6
haemagglutination, 275
haemoglobin, 274, 276
haemolysis of, 273
in lymph, 346
isolation of, 274
mineral bodies in, 304
number of, 272
size of, 272
non-nucleated, 250

Blood-corpuscles, nucleated, 250

protection of salts upon,

6

quantitative constitu-
tion of, 304

quantitative determi-
nation of, 326, 327,
328

red, osmotic phenom-
ena with, 8
stromata, 274
stroma-fibrin, 275
stroma, 272, 273, 274
white, 250, 305-308

number of, 305
constituents of,

306-308

defibrinated, 252
degree of dissociation in, 271
determination of reaction, 271, 272
form elements of, 272-276
detection of, in urine, 796
determination of H ions in, 310
during pregnancy, 337
effect of alkalinity on carbon-
dioxide content, 856
electrical conductivit y,cleavages in,
enzymes in, 53, 332
fat in, 334
fatty acids in, 334
from muscular veins, 337
from veins of glands, 337
gas exchange in, 858-870
gases of, 857
gas tension in, 859-868
hepatic vein, from the, 336
human, analysis of, 328
importance of haemoglobin in oxy-
gen carbon dioxide exchange in,
853

influence of food on, 338
injection of, 343, 344
in urine, 795-799
laky, 312
leucaemic, properties of, 342

constituents of, 342
leucocytes, increase in number of,

342
manner of binding of carbon

dioxide in, 853, 854
menstrual, 337
mineral substances in, 335
non-coagubility of circulating, the-
ories for, 319, 320
of various animals, analysis of,

328

of the two sexes, 337
of woman, analysis of, 328
oxidation in, 858
pigment arterin, 276
pigments, 276-305

acid hsematinic, 296
acid haemoglobin, 286
carbohaemoglobin, 288

GENERAL INDEX

993

Blood pigments, carbon dioxido - haemo-
globin, 288

carbon monoxide-haemo-
globin, 286, 287
carbon-monoxide methae-
moglobin, 287
chlorocruorin, 303
cryptopyrrol, 297, 299
cyanhsemoglobin, 285
cyanmethaemoglobin, 285
decomposition, products

of, 288, 289
detection of, 286, 287
formation of, 286
globin, 289

properties of, 289
haematin, 277, 290-292

formula for, 291
preparation of,

291
properties of,

291
spectroscopic

action of,292
haematin, reduced, 289
haematinogen, 300
haematocrystallin, 278
haematoglobulin, 278
haematoidin, 301

relation to
bile pig-
ments, 301

haematoporphyrin, 277
haematoporphyrin, spec-
troscopic examination
of, 300

haematoporphyrin, prep-
aration of, 300
haematoporphyrin, pro-
duction of, 294-301
haematoporphyrin, for-
mula for, 294
haematoporphyrin, rela-
tion to haematin, 294
haematoporphyrin, rela-
tion to bile pigments,
295

haematoporphyrin, be-
havior in animal body,
295

haematoporphryin, rela-
tion to plant pigments,
296
haemin, 292-294

formula for, 292
properties of, 293
preparation of,

293

haemachromogen,289, 290
haemerythrin, 303
haemin crystals, 292

prepara-
tion, of,
293

Blood pigments, haemochrom, 276
haemocyanin, 303
haemoglobin, 274, 276
haemoglobin, composi-
tion of, 277
haemoglobin, molecular

weight of, 278
haemoglobin, gas combin-
ing ability, 280
hemoglobin, prepara-
tion from oxyhaemo-
globin, 283

haemoglobin, quantita-
tive determination of,
301

haemoglobin, Hoppe-Sey-
ler's colorimetric meth-
od, 301
haemoglobin, reduced,

282

haemoglobin, spectro-
scopic quantitative
method for, 302
haemopyrrol, 297,

299

haemorrhodin, 288
haemochromogen, 276
haemoverdin, 288
in urine, 795-799
isohaemopyrrql, 297
kathaemoglobin, 288
mesoporphyrin, 294,

300
methaemoglobin, 283-

285

methaemoglobin, proper-
ty of, 284, 285
methaemoglobin, prepa-

aration of, 285
nitric oxide-hsemoglobin.

288

neutral haematin, 288
phlebin, 276
phonoporphyrin, 295
phyllohsemin, 296
phyllopyrrol, 298, 299
phylloporphyrin, 296
porphyrinogen, 295
oxyhaernatin, 290
oxyhaemocyanin, 303
oxyhaemoglobin, prop-
erties of, 280, 281
parahaemoglobin, 281
parahasmoglobin, prop-
erties of, 281
photomethaemoglobin,

285

purple cruorin, 282
quantitative estimation

of, 301-304
sulphhaemoglobin, 287
sulphur methaemoglobin,

287
tetronerythrin, 303

994

GENERAL INDEX

Blood plasma, 250, 252-264

analysis of, 269
plates, 250, 308

in coagulation, 314
poikillocytosis, 342
portal vein, from the, 336
quantitative composition of, 326-

335
estimation of urea in,

691
quantity of, 343

bleeding of, 343
in organs, 344
reaction of, 76, 309-311
red corpuscles, decrease in number

of, 341
effect of hemorrhage

on, 341
effect of transfusion,

340
effect of transuda-

tion from, 340
effect of pressure on,

340

increase of, 340
increase of, theories

for, 341

refraction coefficient of serum, 311
rennin acting enzyme in, 474
serum, acids in, 267

action of, on starch, 226
analyses of, 269
constituents of, 264-269
properties of, 264
rest in nitrogen, 267
pigments of, 268
quantitative analysis of

mineral bodies, 270, 271
specific gravity, determina-
tion of, 309

spleenic vein, from the, 336, 337
" sucre virtual," 331
" sucre immediat," 331

urea in, 333, 334
uric acid in, 334
vascular regions,

335-344

venous, 335, 336
quantity

composition of,

of carbon diox-
ide in, 851
quantity of oxygen in, 851
Blueberry, pigments of, in urine, 787
Blue milk, 671
Blue stentorin, 844
Boar sperms, 622
Boas' test, for lactic acid, in gastric juice,

487

for HCL, 486, 487

Body, relation of weight and age of, to
absolute consumption of mate-
rial, 913
weight decrease during starvation,

885
Boiling-point, elevation of by colloids, 17

Bombicesterin, 449

Bondi-Schwarz's test for acetoacetic acid.

824
Bone, 551-558

ash, analysis of, 553

at different ages, 555

catabolism in starvation, 896

components of, 551

earth, analysis of, 552

effect of food upon, 556

marrow, 554

rachitic, 557

rachitis of, 555

softening of, 555
Bonellin, 844

Bony structure, matrix of, 551
Borneol, fate of, in organism, 785
Bottcher's spermine crystals, 621
Bottger-Almen's test, 215, 802
" Bowman's disks," 566
Boyle-Marriot's law, 28
Bradoxidizable substances, 4
Brain, constituents of, 604-606

epileptic, analysis of, 613
gray and white substance, com-
pared, 605

human, analysis of, 612-613

paralytics, analysis of, 613

phosphatides of, 606T609
quantitative composition of, 612
British gum, 229
Bromhaemin, 294
Bromides, in saliva, 459

relation to formation of gas-
tric juice, 477
Bromine, 22, 123

Bromoform, behavior in animal body, 775
Bromthymine, 195
Bromtoluene, behavior in animal body,

775

Brownian molecular motion, 20
Briicke's quantitative method for pep-
sin, 469

Buccal mucus, 453
" Buffy coat," 312
Bufidin, 846
Bufonin, 846
Bufotalin, 846
Bufotenin, 846
Bufotin, 846
Bunge and Schmeideberg, quantitative

method for hippuric acid, 723
Burbot, sperms of, 181
Bursae mucosae, 361
Butalanine, 495
Butter, 647

Butterflies, pigments of, 844
Butylmercaptan, 844
Butyrinase in blood, 266
Butyrine, mono enzymotic synthesis of, 60
Byssus, 92, 122, 123

Cadaver alkaloids, 82
Cadaverine, 47, 82, 164

GENERAL INDEX

995

Cadaverine in urine, 827

Caecum, 514

Caffeine, 187

Calcium carbonate in urinary sediment,

831

importance to enzymotic proc-
esses, 255, 256, 307, 312, 313,
498, 649, 650
in urine, 768(769
manner of excretion, 769
oxalate in urinary sediment, 830
phosphate in urinary sediment,

831
salts in blood coagulation, 308,

309

sulphate in urinary sediments,
831. See also Mineral sub-
stances.

Calculi-ammonium urate, 833
-calcium carbonate, 834

oxalate, 833
-cystin, 834
-fibrin, 834
Heller's scheme for investigating,

833

hemp seed, 833
intestinal, 531
-mulberry, 833
pancreatic, 509
-phosphate, 833
salivary, 459
-uric acid, 833
urinary, 828-829, 832-836
urinary, scheme for chemical anal-
ysis of, 836
urinary, chemical investigation of,

834-836

-urostealith, 834
-xanthine, 834

Caliphora larvae, fat formation, 561
Calomel, effect upon excrement, 529
Calorific coefficients, 888
value of fat, 888
value of proteins, 888

milk protein, 888
starch, 887
urine quotient, 884
Cammidge's reaction for sugar, 815
Camphors, fate of, in organism, 000
Cane-sugar, 49, 224. See Sucrose.
Canirine, 578
d-Caprine, 144

Capronica's test for guanine, 191
Capsule of crystalline lens, 170, 617
Caramel, 214, 225

Carbamino reaction, Siegfried's, 1 66
Carbazole, fate of, in organism, 779
Carbohaemoglobin, 287
Carbohydrate (phosphatized), 244?
Carbohydrates, absorption of, 532-534

as a source of muscular

energy, 598
benzoyl esters of, 202
classification of, 197

Carbohydrates, cyanhydrin synthesis for,

200
effect of gastric juice on,

473
ether-like combinations,

202

fate of, in organism, 885
fermentation of, 203-207
hydrazones, 202-203
in urine, 749-752
in venous blood, 336
osazones, 202-203
phosphoric acid esters of,

204-205
relation to histidine and

purines, 202
See Various sugars.
Carbolic urines, 728
Carbon dioxide, acids, amino as binders

of, 855, 856
effect of alkalinity on

content in blood, 856
formation, calculation of,

889
haemoglobin, 287, 853

as a binder

of, 853

influencing oxygen ab-
sorption, 859
manner of binding in

blood, 853-854
mechanism of elimina-
tion, 852-853
physical explanation of

the giving up of, 866
proteins as binders of, 855
quantity in arterial blood,
851

venous blood

851

tension, 865
tension in tissue, 868
Carbon, elimination in organism, 883-884
monoxide blood, test for, 286

haemochromogen, 288
haemoglobin, 279, 285,

288, 300

methaemoglobin, 286
poisoning, * 285, 402,

582, 679

Carboxylase, 206
Carnaubon, 240, 673
Carniferrine, 578
Carnine, 572, 577, 712
Carnitine, 572, 577
Carnomuscarine, 578
Carnosine, 572, 576-577
Carotin, 631
Cartilage, 546-550

components of, 540
constituents of, 549-550
gelatin, 549

hyalin, action "of trypsin upon,
508

996

GENERAL INDEX

Caseid, 649

Casein, 49, 84, 91, 106, 647-652
cleavage products, of, 106
coagulation of, 649

a two-faced proc-
ess, 650

theory, of, 650, 651
composition of, 647
properties of, 647, 648
hexone bases in, 165
human, preparation of, 652
of woman's milk, 661
peptic digestion of, 651, 652
preparation of, 652
solutions of, properties of, 649
Casemates, 648, 649
Caseinokyrin, 136
Caseoses, 130
Castoreum, 846
Castor lipase, 234
Castorin, 846

Catabolism of protein, 907, 908, 909, 911
Catalases, 43, 872

definition of, 33
equilibrium constant of, 33
in heterogeneous systems, 36
mass action on, 32
measurement of, 32
of benzaldehyde, 36
of diaceton alcohol, 35
of diazoacetic ether, 34
of esters, 32

of hydrogen peroxide, 35
reaction velocity of, 32
velocity coefficient, 34
function of, 7
Catalysts, 35
Catalytic processes, compared (organic

and inorganic), 37
Cataphoresis, 20, 50
Cataract of lens, 619
Cations, 5
Cell-globulin, 274

-membrane, animal effect of gastric

juice on, 473
of plant, effect of gastric

juice on, 473
Cells, adelomorphic, 461

boundary layer of, 21
cover, 461
delomorphic, 461
lymphoid, 300
mineral constituents of, 22
pepsin, 461
permeability of, 34
rennin, 461
Cellobiose, 231
Cellose, 231
Cellulose, 231

fermentation of, in the intes-
tine, 511
Celluloses, 226
Cement, of teeth, 557
Cephalin, 248-249, 605

Cephalopoda, flesh of, 572, 604
Cerebrin, 607, 609-610

in pus-corpuscles, 364, 365
Cerebron, 605, 609, 611-612

cleavage products of, 611
preparation of, 611
properties of, 611
Cerebrosides, 605, 607, 609
Cerebrospinal fluid, 360-361
Cerolein, 239
Cerumen of skin, 845
Cetin, 238, 239
Cetyl alcohol, 239, 627, 845
Chalaza, 634
Charcoal bone, ability to absorb trypsin,

703

Charcot's crystals, 871
Charcpt-Leyden crystals, 621
Chemical processes, plants and animals,

37

" Chemical tonus," 591
Chief cells. See Adelomorphic cells.
Chitin, 122, 839, 839-840
Chitosamine, 219, 626
Chitosan, 168, 840
Chitose, 221
Chloral hydrate, fate of, in organism, 777

secretin, 415, 500
Chloramine, 40
Chlorbenzene, behavior in animal body,

786

Chlorides. See Mineral substances.
of urine, 758-761
in urine, quantitative estima-
tion of, 759
quantity of, 759
Chlorine, in teeth, 558
in blood, 333
Chlorochrome, 384
Chlorocruorin, 299
Chloroform, behavior in animal body, 760,

775
influence upon elimination of

chlorine, 760

influence upon muscles, 591
influence upon protein, 96
Chlorometer, 762
Chlorophan, 617
Chlorophyl, 38, 844
Chlorosis, 340
Chlortoluene, behavior in animal body,

783

Cholagogues, 415
Cholecyanin, 430
Choleprasein, 434
Cholepyrrhin, 427
Cholera bacilli, behavior toward gastric

juice, 485
blood, 334
perspiration, 848
Cholestanol, a and 0, 446
Cholestenon, 443
Cholesteriline, 445
Cholesterin, 10, 265, 613

GENERAL INDEX

997

Cholesterin, amounts in blood of different

animals, 328
constitution of, 445
derivatives of, 446
ester, 264, 360, 444, 445, 613.

844

importance of, 449
in ascitic fluid, 359
in chyle, 347
in lymphatic glands, 366
in lymph, 348
in pus corpuscles, 364
in pus serum, 363, 365
in spleen, 370
occurrence of, 446
of sebum of skin, 845
preparation of, 450
properties of, 446
tests for, 447-448
Cholesterone, 445
Choline, 240, 246-248, 607

in cerebrospinal fluid, 360
in thyroid, 373
occurrence of, 247
preparation of, 247
properties of, 247
Cholohsematin, 435
Chondrigen, 546
Chondrin, 121
Chondrin-balls, 549

Chondroalbuminoid, elementary composi-
tion of, 551

Chondroproteins, 168 ,172-174
Chondromucoid, 172, 546-547

elementary composition

of, 551

preparation of, 548
Chondroitin, 547
Chondrosin, 171, 547
Chorda saliva, 448
Choroid coat, 619
Chromaffine tissue, 378
Chromhidrosis, 849
Chromogens. See Urinary pigments.
Chromoproteins, 92, 93, 167
Chyle, quantitative composition of, 346-

347

Chyluria, 827
Chylous ascites, 358
Chyme, 478
Chymosin, 474
Ciamician and Magnanini's reaction for

indol, 158

Circulating proteins, 908-910
Cleavage, hydrolytic, 16

, processes, 15
Clupeine, 110
Coagulation, intravascular, 324

method for quantitative pro-
tein in urine, 793
Coagulins of blood, 320, 321
Coagulose, 59
Coaguloses, 135
Coapeptides, 136

Coaproteoses, 136
Cobra-poison, 310, 320
Cochineal, 844
Cocosite, 581
Codfish, eggs, 630

sperms, 107
Co-enzymes, 60

Coffee, action on metabolism, 912
Coilin, 92
Collagen, 92, 549, 551

analysis of, 118

in lymphatic glands, 366

preparation of, 118, 121

properties of, 119
Colloid, 171, 624, 625

cysts, 624

effect of charge upon, 23

effect of various ions upon, 22

envelope, 45

from uterine fibroma, 627

substances, non-permeability of,
9

suspension, electrolytic precipita-
tion, 21
" Colloidal nitrogen," 795

substances, preparation of. 14
Colloids, 49

adsorption, 27-30

boiling-point, elevation of, 17

character of, 14

classification of, 15

diffusion of, 18

disperse phase, 27

dispersion means, 27

effects of the different ions, 25

electrical transportation of sus-
pended particles, 20

electrolyte precipitation of, 24

emulsion, 15

examples of, 14

filterability of, 17

freezing-point, depression of, 17

hydrophile, 15

in relation to surface tension and
adsorption, 24

irreversible, 21

migration of to poles, 21

molecular movement of, 20

optical properties of, 19

precipitation of, 21

precipitation phenomena, the-
ories of, 25

protective. 23

relationship to crystalloids, 14

relative size of, 18

reversible, 21

suspension, 15

suspension, precipitation of, 70

Tyndal's phenomenon, 19

ultra microscope, use of in, 19
Colon, effect of extirpation of, 549
Coloring matter. See Pigments.
Colostrum, 658

composition of, 658

998

GENERAL INDEX

Colostrum of woman's milk, 665

Combustion, physiological heat of, 879

Complements, 69

Conalbumin, 633

Conchiolin, 122, 123

Concrements, intestinal, 523, 524. See

also Calculi,
prostatic, 623
Concretions of lungs, 871
Conglutin, 878, 879
Conjugated glucuronic acids, fate of in

organism, 777
sulphuric acids, fate of in

organism, 777
Connective tissue, analysis of, 545

components of, 544
fibrils of, 545

Copper, occurrence in blood, 268, 333
in bile, 416, 433, 440
in liver, 387
in pigments, 299
Cornea, 550
Corneal mucoid, elementary composition

of, 551
tissue, 619

Cornein, 122, 123-124
Cornicrystallin, 123
Corpora lutea, 623
Corpse-wax, 560
Corpus callosum, 613, 614
Corpuscles, blood. See Blood corpuscles,
colloid, 624
colostrum, 645
Gluge's, 624

Corpuscula amylacea, 612
Cover cells, 456, 476, 478
Crab extract, 576
Crangitine, 578
Crangonine, 578
Creatine, 267

detection of, 576

formation of, in active muscles,

594

in organism, 787
in ascitic fluids, 350
in urine, 692

mother substance of, 574
origin of, 574
preparation of, 573, 576
production from arginine, 575
properties, 575-576
relation of to creatinine, 692-693
relation to catabolism of pro-
tein, 574
Creatinine, 572, 573-576

elimination, effect of disease

upon, 694
effect of muscular
activity upon.
694

effect of starva-
tion upon, 694
Folin's colorimetric method
for, 697

Creatinine, formation of, in active mus-
cles, 594

mother substances of, 693
preparation of, 697
properties of, 695, 696
quantitative estimation of, 697
quantity of, in urine, 692
relation of, to creatine, 692-

693

-zinc chloride, 695
Crenilabrine, 110
Crenilabrus payo, 844
Croners-Conheim's test for lactic acid in

gastric juice, 488
Crude fibre, digestion of, 549

silk, 121, 122
Cruor, 252
Cruorin, purple, 282
Crusocreatinine, 578
Crustaceorubin, 844
Crusta inflammatoria, 306

phlogistica, 306
Cryptopyrrol, 430
Crystalbumin, 618
Crystalfibrin, 618
Crystallin, alpha and beta, properties of,

618

Crystalline lens, 617-619
Crystalloids, 13
Crystals, Charcot-Leyden, 621
Cuorin, 249

Curare, 348, 398, 588, 920
CyanhaBmoglobin, 284
Cyanhydrins, formation of, 200
Cyanmethaemoglobin, 284
Cyanocrystallin, 637, 844
Cyanurin, 741
Cyclopterine, 110
Cymene, fate of, in organism, 779
Cyprinine, 110

hexone bases in, 165
Cysteine, 80, 85, 148. 150, 619
Cystic fluid, protein bodies in, 626
Cystine, 80, 85, 100, 107, 113, 114, 115,

116, 125, 148-150
in urine, 827-828
preparation of, 828
protein-, 148
stone-, 148
Cystinuria, 827, 828

Cysts, characteristic constituents of. 624
colloid, 624
dermoid, 627
intraligamentary, 627
myxoid, 624
papillary, 627
parqvarial, 627
proliferous, 624
serous, 624
tubo-ovarial, 627
Cytidine, 180, 185
Cytin, 368

Cytoglobin, 307, 315, 366
Cytosine, 178, 181, 185, 193-194

GENERAL INDEX

999

Cytosine, detection of, 194
Cytotoxin, 69
Cytozym, 319

Deamidation, 411, 536, 582, 702, 774
Dehydrochloride hsBmin, 293
Dehydrocholon, 418, 423
Deniges's test for tyrosine, 154
Dentin, 553
Dermocerin, 844
Dermolein, 844
Desamidoprotein, 78
Descemet's membrane, 171, 551, 618
Desoxyhsematoporphyrin, 291
Deutercaseoses, 129
Deuteroelastose, 117
Deuteromyosinose, 130
Deuteroproteose, 129
Deuterospongenose, 122
Deuterovitellose, 130
Development, work of, 642
Dextrin, 229

hydrolytic cleavage products of,

229

Dextrins, 226

Dextrose, 212. See Glucose.
Diabetes, duodenal, 406
mellitus, 403

acetone bodies in, 818
pancreas, artificial, 405

relation to adrenals

and thyroids, 406
phlorhizin, 400
sugar eliminated in, origin of,

409-411

Diabetic sugar, 212
Diaceton alcohol, 35
Diamine, 24, 131, 163, 757, 928
Dialysis, 14
Diarginylalanine, 111
Diarginylproline, 111
Diarginylserine, 111
Diarginylvaline, 111
Diastase. See Enzymes, 48, 64.

action of, on starch paste, 226
Diazoacetic ether, 34

Diazobenzenesulphonic-acid test for dex-
trose, 216
Diazo-reaction, Ehrlich's, 755

.for histidine, 161
Dibenzoylornithine, 161
Diet, average daily adult, 923

for people in different vocations, 924,

927
Diffusion, 1

of colloids, 18
streams, 5

Digestion, effect of extirpation of pan-
creas upon, 531-532
gastric, effect of fats on, 481
in the stomach, 478-489

time of, 481

movement of food in stomach
during, 479-480

Digestion, movements of stomach during,

479

peptic, 132

Digestion- work, 930-931
Diglucosamine, 220
Diglycyl-glycine, 85
Dihydrocholesterin, 443, 446
Di-isobutyldiacylpiperazine, 142
Di-iodotyrosin, 123
Di-leucyl-cystine, 85
Di-leucyl-glycyl-glycine, 85
Dimethylaminobenzaldehyde, fate of, in

organism, 786
Dimethylfulvene, 7
Dimethylguanidine in urine, 76SC_
Dimethylindol, 729
Dimethylketone, 822
Dimethyltoluidine, fate of, in organism,

786

Dimethylxanthine (1, 7), 713
Dioxyacetone, 205
Dioxybenzene, 778
o-Dioxybenzene, 727
p-Dioxybenzene, 728
Dioxymethylene creatinine, 574
Dioxynapththaline, 778
Dioxypurine, 188
Dioxypyrimidine, 220
Dipalmitylolein, 233
Dipentosamine, 220
Dipeptides, 85-89
Diphosphatides, 239
Disaccharides, 223-226
Disperse phase, 47
Dispersion means, 47
Dissociation, degree of, 5
Di-stearyl lecithin, 148, 241, 242 •-
Di-stearylolein, 233
Di-stearyl-palmatin, 233
Di-tetraoxybutylpyrazine, 220
Dithiopiperidine, 88
Donne" s pus test, 798
Dotterplattchen, 93, 628, 638
Dulcite, 198
Dye stuffs, behavior of living cells

toward, 10
Dyslysins, 427
Dysoxidizable substances, 4
Dyspeptone, 472
Dysproteoses, 129

Ear, fluids of the inner, 619

Earthy phosphates. See Phosphates.

earthy.

Echinochrom, 299
Echinococcus cysts, fluid of, 362
Eck's fistula operation, 398, 537, 542, 680
Edestan, 108, 468
Edestin, 84

cleavage products of, 107
hexone bases in, 165
Eel-meat, 601

serum, 250, 327
Egg shell, 636-641

1000

GENERAL INDEX

Egg, white of the, 632-636

yolk of hen's, 628
Eggs, chemical energy in, 639
development of, 637
fertilization of, 639-640
incubation, 637-638

change in solid con-
tent during, 638
exchange of gases, 637-

638
Loeb's experiments on fertilization,

639

Ehrlich's diazo reaction, 753, 847
glucosamine test, 221
side chain theory, 67, 71
test for bilirubin, 429
urine test, $26
Eicosyl alcohol, 844
Elaiidin, 236
Elastic substance, action of trypsin on,

508

tissue, analysis of, 545
Elastin, 92

analysis of, 116
effect of gastric juice on, 473
hexone bases in, 165
in lymphatic glands, 366
peptone, 117
preparation of, 117
properties of, 117
Elastoses, 130

Electrolytes, amphoteric, 90, 93
Elephant, bones of, 553
Emulsin, 48, 58, 59, 62, 64
Emulsoids, properties of, 15
Enamel of teeth, 557, 891
Encephalin, 607, 609, 610
Endocrinic glands, 374, 375
Endoenzymes, 52
Endolymph, 619

Energy content of various food stuffs, 886
development, calculation of, 889
exchange, 891

calculation of, 889
metabolism calculation of, 886,

887

Enterokinase, 496
Enzymes, 37-70

action on glucpsides, 62
action, specificity of, 61
action, retardation of, 62
activators, 52
adenase, 703
alcoholases, 875
aldehydases, 875
amylopsin, 501
anti, 63-64, 266
antipepsin, 467
arginase, 682
butyrinases, 266
catalases, 266, 872
classification of. 47
co-,52
deamidizing, 703

Enzymes, deviation, 64
diastases, 266

effect of bile upon, 511-512
endo-, 52
esterases, 266
extra cellular, 52
fat splitting, 501-503
fermentation, 653
formation of, 52
gastric lipase, 476-478
general properties, 48
glutinase, 505
glycolytic, 332
glyoxylase, 584
guanase, 703
heat production of, 54
histozyn, 723
hydrogenases, 876
in amniotic fluid, 642
in ascitic fluids, 359
in blood, 52
in blood serum, 266
in bile, 435
in brain, 606
in fatty tissue, 558
in gastric juice, 466
in intestinal juice, 491-492
in leucocytes, 307
in liver, 386
in lungs, 870
in lymph, 346
in mammary glands, 643
in milk, 653
in muscle, 572

in pancreatic gland, 495, 500
in pancreatic juice, 496
in placenta. 641
in prostate, 621
in pus cells, 365
in pyloric secretion, 478
in saliva, 455
in spleen, 370, 371
in thymus, 369
in thyroid gland, 373
intracellular, 52
in urine, 757
in yolk, 628
lipases, 260
maltase, 266, 458
modes of action of, 54
myosin ferment, 570
nuclease, 504
nucleases, 703
oxidases, 266
oxidones, 875
oxygenase, 872
pancreas press juice, action of,

62

pancreatic rennin, 509
pepsin, 466-476
peroxidases, 872
phenol-oxidases, 875
phytase, 579
polypeptide-splitting, 266

GENERAL INDEX

1001

Enzymes, proteolytic, 82, 266, 703
pseudopepsin, 466
ptyalin, 456-460
purine-oxidases, 875
quantitative determination of,

58

reactivation, 52, 64
reductases, 876, 877
rennin, 266, 474
retardation by charcoal, 62
retarding substances, 65
reversibility of enzyme action,

58

salivary diastase, 456
Schutz's rule for, 58
secretion of, 52
eteapsin, 501-503
syntheses, 38, 39
synthesis of hippuric acid, 39
trypsin, 503-509
tyrosinases, 875
urease, 829
uricase, 706
uricolase, 706
urocolytic, 706
xanthin oxidase, 703
Enzymotic processes, 40

fermentation processes, 41
hydrolytic cleavages, 40, 58
synthetic processes, 58
reactions, 55

laws of, 55, 56, 57
Epidermis, 112, 834, 835, 845
Epidermoidal structures, 837-838
Epiguanine, 187
Epiguanine in urine, 712, 714
Epinephrin, 378
Episarkine, 187

in urine, 712, 714
Epitpxiod, 71
Equilibrium constant, 33

nitrogenous, 906
Erepsin. See Enzymes, 48, 593-494

action of, 493
Ereptases, 503
Erythrite, relation to glycogen formation,

393

Erythrocytes, 275
Erythrodextrin, 229
Erythropsin. See Visual purple, 615
Esbach's quantitative method for pro-
tein in urine, 794
Ethal, 239

Ether, action on blood. 273
Ethyl alcohol, action on metabolism, 912
behavior in animal body,

775, 912
benzene, behavior in animal body,

778

butyrate, enzymotic synthesis of, 60
mercaptan, behavior in animal

body, 776
secretin, 823
sulphide, from protein, 79

Ethyl sulphide, behavior in animal body,

776
sulphuric acid, behavior in animal

body, 776

Ethylene glycol, glycogen formation, 394
Euglobulin, 259

Euxanthon, fate of, in organism, 785
Excelsin, 108, 160
Exchange of force, 871, 877, 879
Excrements, 521, 549, 873, 874

in biliary fistulas, 519
Excreta, regular and constant, 879
analysis of, 880

nitrogenous constituents of, 880
Excretin, 523
Exostosis, 556

Expectorations of lungs, 870-871
Extirpation of large intestine, effect of, 548
Extra cellular enzymes, 52
Extractive Jbodies of brain, 606

of kidneys, 673

of mammary glands,

643

of milk plasma, 647
non-nitrogenous, 579
of pancreatic gland, 495
substances of liver, 386

of muscles, 572-588

Extractives nitrogenous, of muscle, 572
of bone marrow, 554
of bodies of cystic fluids, 627
of testes, 620
of yolk, 628
Exudates, gases of. 857
Eye, fluids of, 615-619

lens of, insoluble protein of, 618
soluble protein of, 618
pigments of, 615-617
tissues of, 615-619

Fat-cells, membrane of, 558

action of trypsin

on, 508
-globule, 646
Fats, 38, 232-249

absorption of, 535-540

effect of bile upon,

536-540

acetone formers, 820, 822
amounts in blood of different animals,

328

catabolism in starvation, 894
chemical methods for investigating,

238

deposition of, 920
destruction of, during work, 596
detection of, 237
effect of extirpation of pancreas on

absorption of, 539, 540
effect of gastric juice on, 473
effect on glycogen content of liver,

394

emulsion of, 511
fate of, in organism, 885

1002

GENERAL INDEX

Fats, formation from carbohydrates, 563
from glycogen in liver,

397

from protein, 560-562
of, in organism, 559
hydrolysis of, 234
human, 559
in blood, 334

serum, 265
in chyle, 346, 347
in kidney, 673
in liver, 384
in lymph, 348
in lymphatic glands, 366
in muscle, 586
in pus corpuscles, 364
in pus-serum, 363
in spleen, 370
in synovial fluid, 362
in thymus, 368
in urine, 827
in woman's milk, 660
in yolk, 630

manner of absorption of; 535-536
metabolism of, in starvation, 894

with an exclusive pro-
tein diet, 907-908
muscular energy, source of, 598
of different animals, 559
pancreatic splitting of, 502
properties of, 233
saponification of, 234
storing up of, 563
syntheses of, 60
Fatty degeneration, 385, 560

series, fate of, in organism, 773-774
tissue, 558-564

analysis of, 558
constituents of., 558
Feathers, mineral substances of, 838

pigments of, 843-844
Feces, appearance of, 521, 522
constituents of, 521, 879
pigments in, 522
reaction of, 522
Feeding experiments to show value of

different foodstuffs, 904-906
Fermentation, 8, 9, 203, 207

lactic acid, in stomach, 485
processes, 41

test in urine, 803, 804, 809,
- 810, 812
for sugar, 803
Ferments, 41
Ferratin, of liver, 383
Ferrine, 384
Fertilization membrane, bringing about,

640

Fever elimination of ammonia. 768
Fibrin, -106, 251, 254-261
action of, 257
cleavage products of, 106
coagulation, 256, 257
elementary composition of, 263

Fibrin, ferment, 256

globulin, 258, 259
Henle's, 620

in blood coagulation, 317
in blood during pregnancy, 337
in venous blood, 336
manner of formation, 322
peptic digestion of, 472
plastic substance, 258
preparation of, 254-255
production, manner of, 315
properties of, 255
quantitative estimation of, 255
Fibrinogen, 91, 252-261

amounts in blood, in poison-
ing, 253

detection of, 254
elementary composition, 263
formation, seat of, 252
in coagulation of blood, 317
in venous blood, 336
occurrence of, 252
preparation of, 254
properties of, 253
purification of, 254
quantitative estimation, 254
relation to fibrin. 257, 258
transformation into fibrin, 256
Fibrinolysis, 255, 322
Fibroin, 92, 122, 124

analysis of, 125
properties of, 124
Filterability of colloids, 17

preparation of the filter, 18
Fischer-Weidel's reaction, 189
Fleischl's hsemometer, 299
Florence's sperm reaction, 621
Fluorine content in teeth, 558

content in organs and tissues,

553

Folin and Dennis' test for tyrosine, 154
Folin's method for urea, 689
Folin-Schaffer's quantitative method for

uric acid, 711-712
Food, amount of, for an average daily

diet, 933, 934
chemical energy introduced with,

932

definition of, 878
influence on blood, 338
necessity under various conditions,

932-939

needs of, in work and rest, 937-939
requirements for men in various

vocations, 933
stuff, determination of heat value,

891

energy content of, 886
organic, uses for in organism,

890
physiological availability of,

891
Foods, energy of, 885

essential to life, 878

GENERAL INDEX

1003

Foods, importance of various, 878
Formaldehyde, 38

formation of, from carbon-
ic acid, 2

relation to glycogen for-
mation, 397
transformation into sugar,

1

Formol titrable nitrogen in urine, 755, 756
titration, Sorensen's, for amino

acids, 166

Freezing-point depression, for mamma-
lians, 12
in the frog, 12
in the inverte-
brates, 12
in the lower

fishes, 13
in the higher

fishes, 13
in the eel, 13
in urine, 13
of colloids, 17
molecular lowering of, 4
Fructosazine, 221
Fructose, 217-218

in blood serum, 265
in urine, 814-815
structural formulae for, 197, 199
tests for, 217-218
Fruit-sugar, 217. See Levulose.
Fuld and Levison's method for testing

pepsin, 470

Furbringer's test for proteid, 790
Furfurol, 208-209

fate of, in organism, 784
Fuscin, 617

Gadushistone, 108
Galactosamine, 220
Galactose,..654, 816

structural formula for, 199
tests for, 217
Galactoside, 202

Gall bladder, secretion of, 416, 437
Gall-stones, 444
Gallois'.inosite test, 578
Ganassini's reaction for uric acid, 707
Gas exchange, a measure of metabolism,

927-928

between blood and pul-
monary air, 858-870
between blood and tissues,

858-870

in muscle activity, 592
in starvation, 895
methods for the quantita-
tive determination of,
868-870

through skin. 849
rise of, 931
tension in blood, 859-868

methods of determining, 864-
866

Gases, in bile, 857

in birds' eggs, 636
in blood, 857, 850-856
in exudates, 857
in blood serum, 269
in gastric digestion, 485-486
in lymph, 346, 856-858,
in milk, 657, 658
in muscles, 588
in saliva, 857
in stomach, 485-486
in transudates, 355
in urine, 770, 857
in woman's milk, 664
produced in putrefaction, 515-516
Gastric contents, indicators for determin-
ing acids in. 487
nature of acids in, 487
quantitative determina-
tion of lactic acid in,
488
test for lactic acid in,

488

digestion, absorption of cleavage
products in stomach,
484

degree of, 483, 484
effect of fats on, 481
gases in, 485, 486
time of passage through
the stomach of dif-
ferent foods, 482, 483
juice, 461-466

action of foreign substances

on secretion of, 462
action of saliva on secre-
tion of, 463
composition of, 465
•constituents in, 466
degree of acidity in, 487
obtainment, free of saliva

461-462
origin of hydrochloric acid

in, 477
secretion of, 461, 462-463

in man, 464
lipase, 476-478

Geissler's albumin-test papers, 790
Gel, 15
Gels, 30-32

Gelatin, 31, 78, 80, 118, 119-121, 152
analysis of, 118
as a foodstuff, 912
forming substances of bones, pep-
tic digestion of, 472
forming substances of cartilage,

peptic digestion of, 472
forming substance of connecting
tissue, action of trypsin on, 508
forming substances of connective
tissue, peptic digestion of, 472
from peptic digestion, 473
hexone bases in, 165
in egg development, 639

1004

GENERAL INDEX

Gelatin, in protein catabolism, 911
oxidation of, 83
pancreatic digestion of, 508
peptic digestion of, 473
peptones, 120, 121
preparation of, 118
properties of, 119
protein-sparer, 911, 912
Gelatose, pro to-, 120

deutero-, 120
Gelatoses, 120, 473
Generation, organs of, 620-642
Generative organs, female, 623-642
secretions, male, 620-623
Gerhardt's test for acetoacetic acid, 824
Glands, albuminous, 541

Brunner's, secretion of, 489
fundus, 460, 461
Lieberkuhn's, secretion of, 490
mammary, 643

constituents of, 643
mixed, 451
mucous, 451, 460

membrane, in the intes-
tine, 489-493
membrane of stomach,

460

pancreatic, 494-495
pyloric, 460, 461
salivary, 451-460

analysis of, 451

Gliadin, cleavage products of, 107
Globan, 104, 276
Globulins, 78, 91, 92, 93, 106

detection of, in urine, 791
properties of, 104
quantitative estimation of, in

urine, 791, 793
Globuloses. 130
Glucocyanhydrin, 200
Glucomaines in urine, 757, 758
Glucoproteins, 167, 168-174
Gluconose, 206
Glucopeptose, 200
Glucoproteins, phosphorized, 105
Glucoproteose, 133
Glucosamine, 84, 219

from blood globulin, 260
from seralbumin, 262
preparation of, 220
tests for, 219
d-glucosamine, 201
Glucosan, 213
Glucose, 59, 212-217
in blood, 330
in urine, 749, 802-814
from blood globulin, 260
osimine formation, 201
preparation of, 216
properties of, 213
structural formula for, 197, 198,

199

tests for, 213-216
Glucosides, 202

Glucosides, action of enzymes on, 62
Glucoside-splitting enzymes, 16, 202, 491
Glucosoxime, 200
Glucuronates, conjugated, properties of,

751, 752

conjugated in urine,750-752
.Glucurone, 222
Gluge's corpuscles, 623
Gluteins, 119
Glutelins, 92
Gluten casein, hexone bases in, 165

proteins, hexone bases in, 165
Glutin, 92
Glutinase, 505
Glutokyrin, 138
Glycerides, tri-, 232
Glycerine, 265

relation to glycogen formation,

394, 397, 412

Glycine. See Glycocoll, 139
Glycocoll, 85, 106, 107, 109, 113, 115, 119,

124, 125, 139
amounts in proteins, 106, 107,

113, 115, 125

conjugation with, 782-785
formation of, in organism, 721
importance of, in uric acid

formation, 720, 721
Glycocyamine, 693
Glycogen, 229, 390^14, 581-582, 637
amount in liver, 390, 391
amount in muscles, 390, 391
consumption of, in muscles, 592
content increased by, 393, 394,

396, 397

fat formation from, 397
formation, a cell function, 398
formation from sugar, 395
in lymph, 346
in lymphatic glands, 366
in placenta, 641
origin of, 393
origin in muscles, 397
preparation of, 392
properties of, 391, 392
pseudoglycogen-formers, 395,

396

quantitative estimation of, 393
synthesis of, 58
synthesis of, in liver, 381
true glycogen formers, 395, 396
transformation into sugar, 398,

399

Glycolaldehyde, 38
Glycolysis, 265, 332, 407-409, 570
Glycolytic enzyme, 265
Glycoproteins, 92, 167, 168-174

in blood, 264
Glycosuria adrenalin, 402, 403

relation of pancreas
and adrenals, 406
alimentary, 401
diabetic, relation of pancreas
to, 405

GENERAL INDEX

1005

Glycosuria piqure, 402
salt, 401

sugar-puncture, 402
Glycylalanine, 86
Glycylasparaginyl leucine, 86
Glycylglycin, 86
Glycyl leucine, 86

proline anhydride, 86
tyrosine, 86
valanine anhydride, 86
Glyoxal-methyl, 202
Glyoxyldiureide. See Allantoin, 717
Gmelin's test, for bile pigments, 429

in urine, 800
Goitre, 376
Gorgonin, 123
Graafian follicles, 623, 624
Grape sugar, 212. See Glucose.
Guaiac test for blood, 281, 795
Guanadine, 162
Guanase, 48, 188, 187, 382, 368, 702. See

Enzymes.

Guanidobutylamine, 162
Guanine, 178, 181, 183, 185, 187, 188, 190,

191, 712

Capronica's test for, 191
epi-, 187
-hexoside, 180
Weidel's reaction for, 191
Guano, 188, 699
Guanosine, 180 .
Guanovulit, 637
Gulose, 211
Gums, 229, 230
plant, 226
vegetable, 230

Gunning's modified Lieben's test, 822
Gunzburg's test for HC1 in gastric juice,

487
Gynesin in urine, 758

Hsemaphilia, 329
Haemase, 50
Haemataerometer, 861
Hsematin, 289-291

neutral, 215
reduced, 288
Hsematinogen, 296
Hsematinometer, 297
Haematocrit, 326
Haematocrystallin, 278
Haematogen, 629, 637
Ha3matoidin, 871
Haematoblasts, 308
Hsematoporphyrin, 294-296, 844

relation to bilirubin,

295, 428, 440
relation to chlorophyl,

276, 295
relation to urobilin,

295, 440, 742
in urine, 740, 797-798
Hsematoscope, 303

Heematuria, 795, 796
Hsemerythrin, 299
Hsemin; 292-294, 297, 800

crystals, 292, 293, 800 .
Hsemochrom, 275, 270
Hsemochromogen, 276, 288, 289
Haemocyanin, 92
Haemoglobin, 92, 108

amounts in blood, 328
carbon dioxide binder, 853
exudation of, 6
in blood during pregnancy,

337
in CO2— O2 exchange in

blood, 853
in venous blood; 326
transformation into bile pig-
ments, 442
Haemoglobinuria, 796
Haemoglutination, 275
Hsemolysins, 69
Haemolysis, 273
Haemometer, 303

Hasmopyrrol, 276,^291, 295, 428, 843
Haemorrhodin, 287
Haemoverdin, 287
Hair, 837

-balls, 524

human, sulphur content of, 838
lanugo, 642
Hammarsten's test for bile pigments, 430,

800

Haptogen-membrane, 646
Haptophore, 67

Hanriot and Richet's method for deter-
mining respiratory exchange, 869
Haser's coefficient, 771
Heat regulation, chemical, 929
physical, 929

Hedenius' bilirubin test, 433
Helicoproteid, 174
Heller's blood test, 797

scheme for investigating calculi,

837
Heller-Teichmann's test, 797

in urine, 788
for proteid, 98
Hemicelluloses, 231
Hemicollin, 120
Hemielastin, 117

in blood, 264
Hemipeptone, 130
Hemolysins, 70
Hemp seed calculi, 835
Henle's fibrin, 620
H£nocque's haematoscope, 299
Henriques and Gammeltoft's method for

urea, 690

Heparphosphatide, 386
Hepatopancreas, 494
Heptapeptides, 85, 87
Heptose in urine, 817
Heptoses, 197
Heterocaseoses, 129

1006

GENERAL INDEX

Heterocyclic compounds, fate of, in

organism, 778-787
Heterolysis, 18
Heterosponginose, 122
Heterosyntonose hexone bases in, 165
Heteroxanthine, 187

in urine, 712, 713
Hexapeptides, 85
Hexobioses, 224
Hexone bases, 161

in various proteins, 165
Hexoses, 197, 211-218

syntheses of, 212
Hippokoprosterin, 449
Hippomelanin, 841
Hirudin, 251
Histidine, 85, 106, 107, 109, 110, 111, 115,

117, 119, 125, 159-161
diazo reaction for, 161
in various proteins, 165
Weidel's reaction for, 160
Histidyl-histidine, 85
Histone in urine, 795
Histone-peptone, 109
Histones, 78, 91, 92, 93
Gadus-, 108
hexone bases in, 165
Lota-, 108

properties of, 108-109
Histozym, 48

Hoffmann's test for tyrosine, 153
Holozym, 319

Homocerebrin, 607, 609, 610
Homocyclic compounds, fate of, in organ-
ism, 778-787
Hopkin's quantitative method for uric

acid, 711

Hoppe-Seyler's reaction for xanthine, 190
CO blood test, 286
colorimetric method, 297
test for bile acid, 179
Hordein, 107, 156
Hormone, 375
Hormones, 407
Horn structures, 837

mineral content of, 838
sulphur content of, 838

See also Keratin.
Hiifner's bile, 419
Humor, aqueous, 352, 359, 617
Huppert-Messinger method of estimating

acetone, 825
Huppert-Schiiltz's method of testing

pepsin, 468
Huppert's test for* bile pigments, 429,

430, 799
Hyalin, 840
Hyalines, 171
Hyaline substance, 364
Hyalogens, 171
Hyalomucoid, 617
Hydantoins, 786
Hydraemia, 340, 352
Hydramnion, 642

Hydrazine poisoning, 718

Hydrazones, 202-203

Hydrobilirubin, 743

Hydrocele and spermatoccle fluids, 359,

360

Hydrochinon in urine, 727
Hydrogel, 14
Hydrogen, colorimetric method, 75

determination of, 75

determination of, in fluids con-
taining CO2, 76

electromotive method, 75

ion content, 75

peroxide, 35
Hydrogenases, 876
Hydrolytic cleavage processes, 40
Hydroquinone, 727
Hydrosol, 14

Hydroxylamine poisoning, 718
Hyperacidity, 486
Hyperglycaemia, 401, 402
Hyperthyreoidismus, 378
Hypertonic solution, 6
Hypnotics and glycogen formers, 394
Hypophysis, 380
Hypotonic solution, 6
Hypoxanthine, 178, 187, 188, 191, 192,

572, 712
detection of, 191

Ichthidin, 630, 636

Ichthin, 636

Ichthulin, 92, 174, 630, 636

Ichthylepidin, 122

Icterne, 413, 428, 431, 440, 441

urine, 799
Ignotine, 576
Ileum, extirpation of, 549
Imbibition, 30
Imidazol derivatives in urine, 757, 826

structural formula of, 186
Immune bodies, 66, 69
Immunity, 63-70

active, 70
passive, 70

theory of, Arrhenius', 67, 68
theory of Erlich, 67, 68
Immunization, 66
Indican, 517, 728

elimination of, 728

effect of putrefaction

on, 729

excretion of, 729, 730
quantitative method for, 731
tests for, 730, 731
Indigo-blue of urine, 741
Indigo-red formation, source of, 731
Indigotin in urine, 728, 731
Indirubin in urine, 731
Indol, 46, 82, 117, 157-159, 267, 515, 522,

843

fate of, in organism, 786
formation of, in organism, 728, 729

GENERAL INDEX

1007

Indol, methyl-, 157
tests for, 158
Indoxyl, 724
Infraproteins, 93
Inosine, 180, 577
Inosite, 579-581

in adrenal bodies, 377
preparation of, 580
properties of, 580
Inositogen, 577

" Integral factor," in uric acid elimina-
tion, 705
Internal friction, 18

effect of acids and alka-
lies upon, 19
effect of NaCl upon, 19
of hydrophile colloids, 19
of suspension colloids, 18
Intestinal fermentation, 512, 513

juice, constituents of, 491, 492

properties of, 491
putrefaction, 512, 513
Intestine, chemical processes in, 510-524
decomposition by microbes in,

513

large, putrefaction in, 515
small, digestion and absorption
of various foods in, 513-514
walls of, chemical processes in,

527-529

Intracellular enzymes, 52
Intraligamentary cysts, 627
Inulin, 228

Inversion of sugars, 224
Invert sugar, 224
Invertase. See Enzymes, 48
Invertin, 225. See Enzymes.
Iodine combinations, passage into milk,

667

passage into per-
spiration, 849
passage into saliva,

457

in blood, 268, 333, 337
in glands, 378, 379
in perspiration, 848
in proteins, 81, 125
lodoform, fate in organism, 795
lodospongin, 124
lodothyrin, 373, 376

of thymus, 368
lodothyrioglobulin, 373, 376
Ions, 5, 70-76

action of, on enzymes, 70
Iron, absorption, 338-339
and blood, 339, 639
elimination, 433, 440, 770
importance of, in metabolism, 903
in spleen, 371
in urine, 769
results in lack of, 895
Islands of Langerhans, 407
Iso-casein, 649
Isocholesterin, 449

Isodynamic law in metabolism, 890-892
Isoelectric point, 21, 26
Isolactose svnthesis of, 58
Isoleucine, 85, 106, 107, 143-145
Isomaltose, 223, 224, 225-226

in urine, 749

synthesis of, 58

splitting of, 58
Isoserine, 146
Isosmqtic solutions, 3
Isotonic solution, 5
Isotropous muscle substance, 564

Jacoby's quantitative method for pepsin,

470

Jaffe's creatinine reaction, 695
Jaffe-Obermayer's indican test, 729
Janthinin, 844
"Jaune indien," 222
Jecorin, 605

in liver, 385
in spleen, 370

Jejunum, effect of extirpation of. 549
Jerusalem's test for lactic acid in gastric

juice, 488

Kaolin, 16
Karyogen, 623
Kephir, 518, 654, 659

effect on putrefaction, 518
-lactase. 57. 58

Kerasin, 607, 609, 610-611, 613
Keratin. A, B, C of horn and hair, prop-
erties of, 837

action of gastric juice on, 473
Keratins, 92, 112-116

composition of, 113

Ketone, behavior in animal body, 776, 777
Ketoplastic action, 819
Ketoses, 197
Kidneys, constituents of, 672-674

formation of hippuric acid in,

722

quantitative analysis of, 673
relation to formation of urea, 686
Kinase, in thrombin formation, 323
occurrence in intestine, 492
Kinases, 51
Kjeldahl's method for total nitrogen,

688

Klupeovin, 698
Knapp's method of estimating sugar,

812

reaction for dextrose, 215
Knpp-Hiipner's method for urea, 696
Koilin, 115
Koprosterin, 448

Kossel's reaction for hypoxanthine, 192
Kossler and Penny's method for phenol-
sulphuric acids, 726
Kramm's creatinine reaction, 696
Kumyss, 654, 659

1008

GENERAL INDEX

Kyrin, 137

caseino- 137

gluto-, 137

proto-, 137
Kyrines, 167

carbarn ino reaction with, 167

Lactacidase, 207
Lactalbumin, 91, 652-654

cleavage products of, 106
composition of, 652
properties of, 653
Lactase, 48, 64
Lactimide, 156
Lactocrit, 657
Lactoglobulin, 652
Lactones, 198
Lactoprotein, 653
Lactose, 223, 224, 647, 654-655

in milk, conversion into lactic

acid, 644
in urine, 815
preparation of, 655
properties of, 654
tests for, 655
theory of formation in mammary

gland, 643
Lactosuria, 811
Laiose in urine, 815
Langerhans, islands of, 494
Lanocerin, 844
Lanolin, 446
Lanugq hair, 642
Large intestine, effect of extirpation of,

548

Latebra, 624
Lead in blood, 333

in the liver, 389
passage into milk, 670
Lecithin, 105, 586

amounts in blood of different

animals, 328
from blood serum, 261
in chyle, 347
in lymph, 348
in pus corpuscles, 364, 365
in pus-serum, 363
quantitative determination of,

246
Lecithins, 10, 242-249

hydrolytic products of, 342
in metabolism, importance of,

902

preparation of, 246
properties of, 244
structure of, 242
Lecithoproteins, 92
Legal's reaction for indol, 158
Legumin, cleavage products of, 107
Lense, of oxen, analysis of, 619
Leo's sugar, 810
Lepidoporphyrin, 844
Lethal, 238, 239
Leucaemia, blood, 187, 341, 698

Leuca3mia, spleen, 375

urine, 710, 764
Leucine, 85, 106, 107, 109, 111, 113, 115,

123, 124, 125, 141-143, 267
conversion into isoamyl alcohol,

206

in brain, 607
in lymphatic glands, 366
in spleen, 370
in thyroid gland, 373
in urine, 755, 827
Leucinimide, 143
Leucocytes, 305

constituents affecting coagu-
lation, 315

in venous blood, 336
osmotic phenomena with, 8
See also white blood cor-
puscles

Leucomaines, 25
Leuconuclein, 367

in blood, 316
Leucopoliin, 609
Leucyl peptides, 85-88
Levulins, 228
Levulose, 217

in urine, 814-815
Lichenin, 228

Lieberkiihns solid alkali albuminate, 126
Lieben's iodoform test for acetone, 822
Liebermann-Burchart's. for cholesterin,

447

Liebermann's reaction for protein, 100
Liebig's method for urea, 688
Lienases, 371

Lifschiitz's reaction for cholesterin, 445
Ligamentum nuchse, 115, 116, 546
Lignin, 231

Lime salts, manner of excretion, 769
Lipanin, absorption of, 545
Lipase, 47, 48

pancreatic, 57

Li pases, plants, 64. See also Enzymes.
Lipochromes, 268
Lipopeptides, 88
Lipoids, 241

as a special limiting layer, 10
in brain, 605

in relation to osmosis in cells, 10
sulphurized, 605
Lipuria, 827
Lithium, 22, 333
Liver, 381-450

acetone formation in, 819, 822

amino acids, synthesis of, 382

autolysis of, 387

assimilation in, 381

bile pigments, formation of, 381

blood pigments, destruction of.

388

-cells, reaction of, 382
conjugation of glucuronic acid in,

382
constituents of, 382

GENERAL INDEX

1009

Liver, creatin-creatine metabolism, 694,

695

deamidation in, 382
destroying of uric acid in, 707
ethereal sulphuric acids, formation

of, 382

formation of amino acids in, 530
formation of bile in, 382
glycogen formation, 381, 390
glycogen content in, extirpation of

the adrenals, 402
glycolysis in, theories for, 408
iron in, 387, 388
" organ plasma " of, 382
pigments of, 384
processes going on in, 381, 382
protein synthesis in, 529, 530
t storehouse for protein, 389
synthetical formation of uric acid

in, 705
urea formation, 382

of, from ammonia,

'767

uric acid formation in, 702, 703
Livetin, 629
Lotahistone, 108
Lungs, concretions of, 871
constituents of, 870
expectorations of, 870, 871
gas, exchange in, 867
Lutein, 631

Luteins, in blood serum, 268
Lymph, 345-380

amount secreted, 349
composition of, 345-348
coagulation of, 346
formation of, 350-352
gases of, 856-858
origin of, 345
osmotic pressure in, 348
secretion of, circumstances in-
fluencing, 349, 350
Lymphagogues, 350
Lymphatic glands, 366

quantitative composi-
tion of, 366
Lymphocytes, 300
Lymphoid cells, 305
Lysatine, 160
Lysatinine, 160

Lysine, 85, 106, 107, 109, 111, 113, 115,
117, 119, 123, 124, 125, 163,
164, 267

in various proteins, 165
peptone, 138
Lysines, 47

Lysylglycylpeptide, 89
Lysyl lysine, 85

Magnesium in urine, 768, 769

triphosphate in urinary sedi-
ment, 831

Maintenance value, 897
Maltase, 48, 458. See also Enzymes.

Malt dextrin, 229
Malt diastase, 225

action of, upon starch, 226
Maltose, 223, 224, 225
in urine, 815 ?
synthesis of, 58
Malt sugar, 225
Mammary gland, theory of formation of

lactose in, 643
Mandelic acid ester, racemic, 62

nitrile glucoside, 59
Manganese, 268, 333, 442
Mannite, 198
Mannose, 198. 216
Manonpse, 206
Marcitine, 47
Margarin, 235

Maschke's reaction for creatinine, 695
Mass action, 32, 74
Mastic, 22
Meconium, 523

Medicinal coloring matters in urine, 801
Medulla, 607

Medullary fibers, composition of, 614
Melanin, 617, 842

in urine, 799
Melanins, of skin, 841

mother substances of, 843
Melanogen in urine, 799
Melanoidin nitrogen, 78
Melanoidins, 841
Melano-protein, 842
Melanotic cancers, 802, 842, 843
Membrane, semi-permeable, 26
Membranin, 171, 550

properties of, 617
Menthol, fate of, in organism, 785
Mercuric salts poisoning, 23
Mesitylene, fate of, in organism, 779
Mesoinosite, 582
Mesoporphyrin, 295
Mesoxalyl urea, 698
Metabolism, 878-939

affected by sleep, 928
as affected by external tem-
perature, 929

as affected by high alti-
tude, 930
as affected by ingestion of

food, 930

as affected by light, 928
as affected by mental activ-
ity, 928

basal requirement, 897, 898
calculation of, 889
conditions affecting, 922-931
effect of age upon, 922-925
effect of alcohol on, 921, 922
effect of coffee and tea upon,

922
effect of rest and work upon,

926-928

effect of salts on, 921
effect of sex upon, 925, 926

1010

GENERAL INDEX

Metabolism, effect of weight of body

upon, 922-925
effect of water on, 920, 921
importance of lecithins in,

902
importance of phosphates

in, 902

in active and inactive mus-
cles, 591-598

inanition condition, 897-898
in starvation, 892-906
lack of mineral substances in

food, 899, 900
water in food, 898
maintenance value, 897-898
measured by gas exchange,

927, 928

with absence of carbohy-
drates in food, 903, 904
with absence of fats in food,

903-904
with absence of proteins

in food, 903

with a mixed diet, 913-920
of fat with an exclusive

protein diet, 907, 908
with foods rich in protein,

906T913
with insufficient supply of

chlorides, 900, 901
with lack of bases in foods,

901
with lack of earths in food,

901, 902
with lack of iron in foods,

903
with lack of phosphates in

food, 901, 902

with various foods, 906-922
Metacasein, 651
Metalbumin, 624, 625
Metanitrobenzaldehyde, fate of, in the

body, 784
Metaproteins, 93
Metazym, 319
Methaemoglobin of thyroid gland, 374.

See also Blood pigments.
Methal, 239
Methose, 212

Methylation in organism, 787
Methylenitan, 212

Methylethylmaleic acid anhydride, 290
Methylglycocoll. See Sarcosin.
Methylglyoxal in relation to lactic acid

fermentation, 584
Methylguanidine, 578, 698

in urine, 758
Methyl guanine (7), 714
Methylindol, 157 See Skatol.
Methylindolin, 729

Methylimidazol formation of, 201-202
Methyl pentoses, 199, 208
Methylphenylhydrazine, test for levulose,
218

Methyl pyridine, behavior in animal body,

778, 781
chloride in urine. 756,

758

Methylpyridylammonium hydroxide, 787
Methylthiophene, 784
Methyluracil, 194
Methyluramine, 574, 694
Methyl-urea, 691

in urine, 712, 713
Methylxanthine, 187
Mett's quantitative method for pepsin,

469
Micro-organisms in intestines, 511, 514,

517, 520

Microrespirometer, 874
Microtonometer, 861
Milk, 643-671

albumin, quantitative determina-
tion of, 656
ash, quantitative composition of,

666
casein, quantitative determination

of, 656
coagulation, a two-faced process,

650

coagulation of, 645
cows, 644, 645
cows, quantitative composition of,

657

effect on putrefaction, 518
fat, quantitative determination of,

657

constituents of, 647
theory for origin of, 669
foodstuffs in, 666
gases of, 857
globules. 645-647
goat's, 659
human, 660-671
human, compared to cow's milk,

660, 661, 663
influence of food on composition of,

667, 668
mare's, 659
lactose quantitative determination.

of, 657

mineral bodies, quantitative de-
termination of, 656, 657, 658
of carnivora, 659
of various animals, composition of,

660
passage of foreign substances into,

670

plasma, 647

preparation, composition of, 659
proteins, quantitative determina-
tion of, 656

quantitative analysis of, 656, 657
secretion, chemistry of, 668-671
solids, quantitative determination

of, 656

souring of, 645
sugar, 226. See Lactose.

GENERAL INDEX

1011

Milk, sugar, theory for origin of, 670

quantitative determination

of, 657
theory for origin of casein, 668

permanent emulsion, 646
uterine, 641

various fermentations of, 645
woman's, quantitative composition,

652, 653

quantity of mineral sub-
stances in, 654
Millon's reaction for proteins, 99

in hydrocele and
spermatocele fluids,
359

Mineral bodies in milk, 656
Mineral substances, absorption of, 540

amounts in blood of
different animals,
328

distribution of, 72
effect on metabolism,

921
importance to life of

cells, 72
in bile, 436
, in blood, 335

in blood serum, 268,

269

in bone structure, 552
in brain, 615
in cartilage, 550
in cells, 72
in cerebrospinal fluid,

361

in chyle, 347
in connective tissue,

546

in cystic fluid, 627
in egg shell, 636
in feathers, 838
in gastric juice, 466
in hair, 838
in intestinal juice, 491
in kidneys, 673
in liver, 387-389
in lungs, 870
in lymph, 346, 348
in muscles, 586-588,

599

in nails, 838
in pancreatic gland,

495
in pancreatic juice,

500
in pericardial fluid,

356

in pus corpuscles, 365
in pus-serum, 363
in retina, 615
in semen, 621
in smooth muscles,

602
in spermatozoa, 622

Mineral substances, in starvation, 895,

896

in synovial fluid, 362
in urine, 758-770
in white of egg, 635
in woman's milk, 654
in yolk, 632
lack of, in food, 899,

900

of spleen, 361
toxicity of, 73
Mingen in urine, 756, 758
Mohr's quantitative method for chlorides,

759

Molisch's test for sugar, 216
Monaminophosphatides, 241
Monomethylxanthine, 712
Monosaccharides, 197-218

cyanhydrin formation,

200

oxime formation, 200
transformation of, 201
Moore's test for sugar, 214, 224
Morner-Sjoqvist and Folin's method for

urea, 689
Morphine, elimination in urine, 749, 787

in milk, 670
Moss-starch, 228
Mucin, 619

action of trypsin on, 508
" dissolved," 794
effect of gastric juice on, 473
in urine, 757, 794
pseudo, 171
substances, 168-172
Mucilages, 226

vegetable, 230
Mucinoids, 171
Mucins, analysis of, 169

amino acids in. 170
cleavage products of, 170
true, 168, 169
Mucoid, osseo-, 172
Mucoids, 169, 171

effect of gastric juice on, 473
Mucous of urine, 674
Mulberry calculi, 833
Murexide test, 707
Muscarine, 246
Muscle-pigments, 571
Muscle-plasma, 566

preparation and proper-
ties of 566. 567
proteins of, 569 570
Muscle-serum, 566

smooth, 601-603

constituents of, 602
extractives in, 602
" snow," 566
"-stroma," 569
syntonin, 569
Muscles, 565-603

acid rigor of. 590

active, acid reaction of, 593

1012

GENERAL INDEX

Muscles, active, characteristics of, 596

amount of fat in, 600

an isotropous substance of, 555,
556

chemical rigor of, 590

dead, proteins of, 567

elementary analysis of, 601

extractive bodies of, 572-588

"flesh quotient," 601

haemoglobin, 571

heat rigor of; 590

imbibition rigor of, 590

isotropous substance of, 555, 556

metabolism in, 591-598

of different animals, ash anal-
yses of, 599

of different animals, analyses of,
599

proteins of, 566-572

quantitative composition of, 598-
601

reaction of, 565

rigor mortis of, 588-591

striated, 565-601

water rigor of, 590
Musculamine, 578
Muscular action, source of, 597
Musculin, 568, 570
Myelin, 608
" Myeline forms," 605
Myoalbumin, 569
Myocynine, 578
Myogen, 570, 571
Myogen-fibrin, 570, 571

soluble, 570

preparation of, 570, 571
Myoglobulin, 568, 571
Myohaematin, 571
Myoproteid, 571
Myosin, 84, 567, 568, 570

as related to fibrinogen, 254

coagulation of, 570

ferment, 570

fibrin, 568, 570

in blood corpuscles, 306

preparation of, 568
Myosinogen, 569, 570, 571
Myosinoses, 130
Myricin, 239
Mytilite, 581
Mytolin, 569
Myxcedema, 374
Myxoid cysts, 624

Nails, 837

mineral substances of, 838
Naphthalene, fate of, in organism, 779,
. 785

Napthoresorcin, reaction for conjugated
glucuroric acid, 218, 223,

818

reaction for levulose, 218
Naphthylisocyanate compounds of amino-
acids. See various Amino-acids.

Narcotics, relation to glycogen formation.

394

Neosine, 578
Neossin, 171
Neottin, 631
Neozym, 320

Nephrorosein in urine, 734
Nerve fibres, analysis of, 614
Nerves, 604-615
Neubauer's and Rolide's reaction for

proteins, 100

Neuberg's test for levulose, 218
Neuberg-Rauchwerger's reaction for cho-

lesterin, 444

Neuridine, 606, 612, 628
Neurine, 246
Neurochitin, 614, 615
Neuroglia, 601

Neuroglobulin, a and ft, 604, 605
Neurokeratin, 605, 614, in various nerves,

amounts in, 614
Nicotine, effect upon gases of stomach,

485

Ninhydrin reaction, 101
Nitrates in urine, 766
Nitriles, fate in organism, 775
Nitrobenzaldehydes, fate of, in organism,

783, 784

Nitrobenzene, fate of, in organism, 782
Nitrogen colloidal, 795

elimination in starvation, 893,

894

through various
channels, 880, 881
gaseous elimination of, 881.
See also Gases in various tis-
sues and fluids.

Nitrogenous equilibrium, 918, 919, 934
Nitrotoluol, behavior in animal body,

786
Novaine, 577

in urine, 758
Nuclease, 504, 508

Nucleuses, 48, 182. See also Enzymes.
Nucleinases, 182
Nuclein bases, 186 i

in lymphatic glands, 366
plates, 308
in pus-cells, 365
true, 176
Nucleins, 176, 177

action of trypsin on, 508
effect of gastric juice on, 473
pseudo-, 177
Nucleoalbumins, 91, 92, 93, 177

detection of in urine, 795
properties of, 104
in urine, 794
Nucleohistone, 307

in blood, 316
in urine, 795
of thyrnus, 367, 368
Nucleon, 578, 620, 666
Nucleo proteins, 92, 167, 174-177, 252

GENERAL INDEX

1013

Nucleo proteins, action of trypsin on, 508
cleavage products of, 177-

195

of blood, 258
of milk, 653
of the spleen, 369
peptic digestion of, 472
Nucleosidases, 182
Nucleosides, 180, 211
Nucleosin, 195
Nucleotides, 179

mono-, 179
poly-, 179
Nucleotin, 179, 182
Nucleotoprotamines, 174, 623
Nutritive need of man, 916
requirement, 908
Nylander AlmeVs test, 215

sugar test, 802

Obermayer's indican test, 729

Obermuller's cholesterin reaction, 445

Oblitine, 578

Ochronose, 550

Octodecapetide, 85, 87

Oedematous fluid, and its constituents, 363

Oesophageal fistula, 459

Oil-turpentine, behavior in animal body,

749, 784
Olein, 236

mono and tri enzymotic synthesis

of, 60

Oligaemia, 340
Oligocythsemia, 340
Oliguria, 771
Olive oil, absorption, 544

effect upon bile secretion, 415
Onuphin, 171
Oocyan, 636
Oorodein, 636
Opalisin, 652

Opium, passage of, into milk, 670
Organic phosphorous compounds in urine,

757

in urine, 762
sulphur compounds in urine, 752,

765

Orcin-hydrochloric acid test, 209, 223
Orcin, test for pentoses in urine, 817
Oriental bezoar-stone, 524
Ornithine, 85, 162, 163
Orthonitrobenzaldehyde, fate of in the

body, 784
Orthonitrophenylpropiolic acid, test for

dextrose, etc. See Acid nitrophenyl-

propiolic.

Orthonitrotoluene, fate of, in organism, 785
Osamines, 204
Osazones, 202, 203, 215
Osimines, 201
Osmometer, 16
Osmosis, 5
Osmotic pressure, 1-13

determination of, 16

Osmotic pressure, effective, 12

effect of various sub-
stances upon, 17
electrolytes, abnormal,

due to ionization,
in higher animals, 13
in lower sea animals, 13
in lymph, 13
in milk and bile, 13
non-electrolytes, 4
of animal fluids, 12
of colloids, 16-18
of the blood, in selachii,

in saliva, 13
in urine, 13
Osones, 203
Ossein, 551

Osseoalbuminoid, elementary composi-
tion of, 551
Osseomucoid, 172, 551

elementary composition of,

551

Osteomalacia, 556, 557
Osteosclefosis, 556
Otoliths, 619
Ovalbumin, 633-635

cleavage products of, 106
preparation of, 634
Ovaries, 623

cysts in, 624
Ovin, 631

Ovoglobulin, 84, 633
Ovokeratin, 114
Ovomucin, 634

Ovomucoid, 171. 633, 635, 636
properties, 635
preparation, 635
Ovovitellin, 84, 91

composition of, 628, 629
preparation of, 630
Ovum, 623, 628
Oxamide, 83
Oxidases, 188

artificial, 873. See also En-
zymes
Oxidation in animal tissues, theories of, 42

in the organism, 850-877
Oxidones, 874, 875
Oxydase, 47, 48

Oxygen absorption, calculation of, 889
activation, 4-8
tension in blood, 859-861
tension, regulation of in organism,

868

Oxygenases, 872
Oxyha3matin, 289-291
Oxyhsemocyanin, 299
Oxyhsemoglobin, 276, 278-282

absorption bands of 281,

282

action of trypsin on, 508
effect of gastric juice on,
473

1014

GENERAL INDEX

Oxyhsemoglobin, law of dissociation of,

859

preparation of, 282
See also Blood pig-
ments.

Oxyphenylethylamine, 82

Oxyproline, 85,^106, 107, 125, 155

Oxyprotein, 83

Oxypurine, 190

Oxypyrimidine, 195

Oxyquinolines, fate of, in organism, 785

Ozone, occurrence in the organism, 4

Palmitin, 235
Pancreas, 494-510

importance of, to absorption of

carbohydrates, 535
Pancreatic diastase, 501

gland, constituents of, 495
juice, 494-501

action on fats, 502
action of, on starch, 226
amount secreted, 500
constituents of, .500
excitants for the secre-
tion of, 498
properties of, 500
lipase, 57. See also En-
zymes.

secretion of, 496
rennin, 509. See also En-
zymes.

secretion, human, 500, 501
Papa in, 51
Paracasein, 650
Parachymosin, 474, 567
Paracresol, 515

formation in putrefaction, 515,

723, 724

Paraglobulin, 258
Parahaemoglobin, 280
Parahistone, 109
Paralbumin, 624, 625
Paraminophenol, 779
Paramucin. 626
Paramyosinogen, 568, 570, 571
Paranuclein. See Pseudonuclein
Paraovarial cysts, 627
Paraoxypropiophenone, 785
Parapeptone, 472
Parathyroids, 377

Paraxanthine in urine, 712, 713, 714
Parenchymatous organs, action of trypsin

on, 508

Parenteral canal, introduction of pro-
tein by way of, 533-541
Parotid, 448

saliva, 450
Peaglobulin, 84
Pectin bodies, 229, 231
Pemphigus chronicus, 359
Pennacerin, 846
Pennatulin, 123
Pentacrinin, 844

Pentamethylenediamine, 47. See Cada-

verin

Pentapeptides, 85
Pentosamines, 220
Pentosans, 207

Pentoses, 197, 198, 199, 207-211
in urine, 816

quantitative estimation, 209
test, orcin-hydrochloric, 209
tests for, 209
Pentosides, 180
Pentosuria, 816

Penzoldt's test for acetone, 823
Pepsin, 57, 64, 466-476

action on proteins, 468-473
characteristic property of, 468
digestion of proteins, products of,

472

digestion, effect of foreign sub-
stances upon rapidity of, 470
digestion, rapidity of, 471
digestion of various bodies, 472,

473

mother substances of, 477
nature of, 467
occurrence of, 466
preparation of, 468
properties of, 467, 468
quantitative methods for, 469, 470
relation to rennin, 475
test for, 469

testing for, in gastric juice, 487
Pepsinogen, 477

Peptases, 503. See also Enzymes.
Peptic-glutin peptone, 135
peptones, 135

zymolysis, effect of bile upon, 511
Peptidases, 48
Peptides, 68, 85-89, 93, 126, 127, 156

behavior toward enzymes, 8,
266, 492, 510, 511. See Poly-

peptides.
in urine, 755

relation to alcaptonuria, 735
relation to urea formation, 777
Peptinuria, 791
Peptochondrin, 549
Peptoid, 507
Peptone-plasma, 251
Peptones, 92, 127-132
anti-, 132
as foodstuff, 912
carbamino reaction with, 167
from peptic digestion 472
in urine, 791
properties of, 131
quantitative estimation, 138
separation from proteoses, 136,

138

Percaglobulin, 637
Pericardial fluid, 356, 357

analysis of, 356
Perilymph, 619
Peritoneal fluid, 357

GENERAL INDEX

1015

Permeability, causes for,

of blood corpuscles, 7
Overtoil's solubility theory,

10

shortcomings of, 10, 11
Traube's solution tenacity

theory, 11

Peroxidases, 872. See also Enzymes.
Peroxydase, 50
Perspiration, 847-849

circumstances influencing,

847

constituents of, 848
properties of, 847
Pettenkofer's method for determining

respiratory change, 869
Pit test for bile-acid, 798
test for bile, 418
Pfaundler's method of precipitating urine,

681

Phaseomannite, 579
Phenol, 515

as precipitant of proteids, 98
in urine, 515, 723, 726, 785, 787
Phenol-oxidases, 875
Phenols, 117, 723

fate of, in organism, 775, 784
Phenylalanine, 85, 109, 114, 151, 152

amounts in proteins, 106,

107, 113; 115, 125
Phenylethylamine, 82
Phenylisocyanate compounds of amino
acids. See various
Acids, amino.
peptone, 138
Phenylmethylketone, 785
Philothion, 876
Phlebiri, 276
Phosphates, importance of, in metabolism,

902

in urine, 761-764
in urine, quantity of, 762
See also Mineral substances.
Phosphatides, 239-249, 265, 586

in adrenal bodies, 377
in bile, 435, 439
in kidney, 673
in liver, 385
in yolk, 630
Phosphaturia, 763
Phosphoglycoproteins, 174, 177
Phosphoproteins, 91, 92, 93

properties of, 104
Phosphorus compounds, action on bile,

437, 441
action on blood,

252, 254

action on urea
elimination,
679, 685
organic, in urine,

756

metabolism, 875,
876, 894

Phosphorus-containing urinary constitu-
ents, 756, 875

elimination in organism, 883
elimination in relation to ni-
trogen elimination, 762, 763
elimination in relation to pur-

ine metabolism, 763
Phosphatides, properties of, 241
Photomethaemoglobin, 284
Phrenin, 612
Phrenosin, 609, 610, 611
Phyllocyanin, 295
Phylloerythrin, 435
Phylloporphyrin, 276, 295
Phymatorhusin, 841

in urine, 799

Physical chemistry in biology, 1
Physiological oxidation processes, 871-877

salt solution, 7
Phytase, 579
Phytin, 579
Phytosterines, 446

Picolin, behavior of, in animal body, 784
Pigments, fate of, in organism, 787
medicinal in urine, 801
of bile, 427, 433, 435, 436
of birds' eggs, 636
of blood, 275, 298
of blood serum, 268
of butterflies, 844
of corpora lutea, 297, 623
of eye, 614, 616
of feathers, 843, 844
of human skin, 840-843
of liver, 384
of lobster, 638, 844
of lower animals, 299, 844
of lungs, 870
of muscle, 571
of placenta, 641
of pus, 366
of skin, 840-844
of stones, 441
of urine, 733, 734, 740, 748, 798,

799

of yolk, 631
Pilocarpine, effect upon elimination of

CO2 in stomach, 485
effect upon elimination of

uric acid, 700

effect upon secretion of in-
testinal juice, 490
effect upon secretion of sali-
va, 457

Piperdine-glycosuria, 403
Piquie, 402

Piria's test for tyrosine, 153
Placenta, 641
Plant cells, 5

osmotic experiments with, 5
gums, 226

Plasmolysing power of differently con-
structed salts, 6
Plasmolysis, 5

L016

GENERAL INDEX

Plasmolysis, substances bringing about, 5
Plasmolytic experiments with animal

cells, 7

Plasmoschisis, 314
Plasmozym, 319
Plastein, 59
Plasteines, 135
Plastin, 178

Plattner's crystallized bile, 396
Pleural fluid, 357
Pnein, 874
Pneumonic infiltrations, solutions of, 18,

368, 869

Poikilocytosis, 342
Polycythaemia, 340
Polynucleotides, 179
Polypeptide-like bodies in urine, 756
Polypeptides, 86-91, 92. See Peptides.
action of trypsin on, 509
cleavage of, by enzymes, 62
E. Fischer's synthetical

preparation of, 88
methylated, 88
syntheses of, 86
synthetically produced, 87
sulphur containing, 88
synthetic, comparison of
properties with natural pro-
teins, 90. See also Pep-
tides.

Polyperythrin, 844
Polysaccharides, colloid, 226-231
Polyuria, 771

Potassium in urine, 766. See also Min-
eral substances.
Praglobin, 307

Precipitation, Traube's membrane, 1
Precipitins, 66
Preglobulin, 315, 368
Preputial, secretion of skin, 846
Proenzymes, 51
Prolamine, 78
Prolamins, 106
Proline, 85, 109, 111, 154, 155

amounts in proteins, 106, 107,

113, 115, 125
Propepsin, 477
Propeptone, 126
Propylalanine, 85
Propyl benzene, behavior in animal body,

778
Propylene glycol, relation to glycogen

formation, 394
Prosecretin, 463, 499
Prostate, secretion of, 621
Prostatic concrements, 172, 611, 623
Prosthetic group, 174
Protagon, 605, 606-608, 609

cleavage products of, 607
elementary composition of, 606
nature of, 606, 607
preparation of, 608
properties of, 608
Protamine nucleate, 623

Protamines, 91, 92, 93, 108

hexone bases in, 165
preparation of, 112
properties of, 109-112
Proteans, 93
Protease, 47, 48

alpha retardation of, 63
Proteases, 48

Protective colloids, 37, 44, 131
Protein, absorption, effect of cellulose on,

531
catabolism, 907, 908, 909, 911

importance of sulphur

in, 882, 883
in active muscle, 594
in starvation, 894
destruction of, during work, 595
ochrome, 155
requirement, 933-936

lower limit, 915
sparers, 913-915

substances, diarginylalanine, 111
diarginylproline, 111
diarginylserine, 111
diarginylvaline, 111
Proteins, 38, 77-195

absorption of, 525-531
acid, 125

action of neutral salts on, 98
action of nitrous acid upon, 79
action of pepsin on, 468-473
action of trypsin on, 505, 507
adsorption compounds, 95
adsorption of, 97
albuminates, 104
albuminoids, 112
albuminous, 102-138
albumins, 106
albumose, alkali, 127
alcohol soluble, 92
alkali, 126

amino acids in, linking of, 90
amounts in blood of different

animals, 328
analysis, 118
atmidkeratin, 113
atmidkeratose, 113
Bence-Jones, 792
byssus, 122, 123
carbon dioxide binders in blood

855

casein, 106
chitin, 122
chondrin, 121
chondro-, 168, 172-174
chromo-, 167
circulating, 908-910
classification of, 91-93
cleavage products, 93, 106
clupeine, 110
coagulated, 93
coagulation of, 96
collagen, 118, 119
color, reaction for, 99

GENERAL INDEX

1017

Proteins, composition of, 77, 94
compound, 92, 167-177
conchiolin, 122, 123
conjugated, 92, 93
cornein, 122, 123, 124
cornicrystalline, 123
crystalline, 94
cyclopterine, 110
cyprinine, 110
derived, 93
deutero, 120
deuteroelastose, 117
effect on glycogen content of

liver, 394, 397
elastin, 116-118
fate of, in organism, 886
fattening, 919
fibrin, 106
fibroin, 122, 124
foodstuff purposes of, 917, 918
forms of nitrogen in, 77, 78
gelatin, 118, 119-121
gelatin-peptones, 120
gelatose, 120
gelatoses, 120
globan, 104
globulins, 103-104
gluco-, 167, 168-174
glucoproteins, phosphorized, 105
gluteins, 119
glyco-, 92, 167, 168-174
gorgonin, 123
helicp-, 174
hemicollin, 120
hemielastin, 117
hordein, cleavage of, 107
hydrolysis of, 81
ichthylepidin, 122
in amniotic fluid, 642
in aqueous humor, 361
in ascitic fluids, 359
in bone, 551
in bone marrow, 554
in brain, 604
in cartilage, 546
in cerebrospinal fluid, 360
in chyle, 347
in connective tissue, 544
in crystalline lens? 617, 618
in cystic fluids, 625-627
in dead muscles, 567
in fat globules, 647
in horn substance, 837
in hydrocele and spermatocele

fluids, 359
in kidneys, 672
in liver, 382, 383
in lungs, 870
in lymph, 346, 348
in lymphatic glands, 366
in mammary glands, 643
in milk, 647-654
in milk plasma, 647
in muscles, 566-572

Proteins, in muscle-plasma, 569, 570

in pancreatic gland, 495

in pericardial fluid, 356

in placenta, 641

in prostate secretion, 621

in pus corpuscles, 364, 365

in pus-serum, 363

in retina, 615

in salivary glands, 451

in sebum of skin, 844

in semen, 620

in smooth muscle, 602

in spermatozoa, 623

in sputum, 871

in synovial fluid, 362

in testes, 620

in thymus, 366, 367

in transudates and exudates,
353, 354

in urine, 787-795

in urine, detection of, 788

in urine, quantitative estima-
tion of, in urine, 793

in veins, 336

in venous blood, 336

in white of egg, 633-636

in yolk, 628-630

iodo, 123

keratins, 112-116

koilin, 115

lecithalbumins, 105

lecitho, 92

Lieberkiihn's solid alkali albu-
minate, 126

metabolism calculation of, 889

metabolism of, in starvation,
893,894 '

metabolism with food rich in
proteins, 90(5, 913

method of synthesis, 86

membranins, 617

modified, 96

molecular weight, 98

mucins, true, analysis of, 168-
172

mucinoids, 171

mucoids, 171

native, 96

Neubauer and Rohde's test,
100

nuclei of aliphatic series, 85

nuclei of carbocyclic series, 85

nuclei of heterocyclic series, 85

nucleo, 92, 105, 167, 174-177

nucleo albumins, 104, 105

ovokeratin, 114

ovovitellin, 105, 108

parenterally introduced, absorp-
tion of, 525

pennatulin, 123

phospho, 92, 104

phosphoglycoproteins, 174, 177

phosphorized importance of, in
metabolism. 902

1018

GENERAL INDEX

Proteins, precipitation of, 95, 96, 98, 99
preparation, 118, 121
prolamine, 106
properties of, 94-96, 107, 108,

protamine, constitution of, 111

protamines, 109-112

proto-, 120

protoelastin, 117

protones, 110

pseudomucins. 171

pseudonucleins, 104

putrefaction of,

putrefactive products of, 82

quantitative estimation, 101

reaction of, 91

reactions, precipitation, 98

reticulin, 121, 122

salmine, 110

salting put of, 95

scombrine, 110

semiglutin, 120

eeralbumin, cleavage products

of. 106

[serglobulins, cleavage of, 106
sericin, 122, 124
simple, 91, 92, 93, 94

cleavage products of,

125-195
skeletins, 122

constitution, 122
source of muscular energy, 595-

597

spongin, 122, 123
sturine, 110
sulphur content,
syntheses "from amino acids,

529, 530

synthesis of, in organism, 530
synthetically formed, 59
tests for, 99, 100, 101
tissue, 908-910
xanthoprotein reaction, 99
zein, cleavage products of, 107
Prpteose-like substances in blood serum,

264
Proteoses, 92, 127-132

absorption, 534, 538

as foodstuff, 912

carbamino reaction with, 167

detection of, in urine, 792

deutero, 130

dys, 130

from peptic digestion, 472

gluco-, 134

hetero, 130, 134

in blood, 262, 263, 534, 535

in intravascular coagulation,

325

in lungs, 870
in stomach, 483, 484
in urine, 791
primary, 130
properties of, 131

Proteoses, proto-, 130, 134

quantitative estimation, 138

secondary, 130

separation from peptones, 136,

138

syn-, 134
Prothrombin, 255, 256, 257, 312, 314, 315,

«317

in circulating plasma, 318
Protocaseoses, 129
Protoelastose, 117
Protogelatose, 119
Protogen, 126
Protokyrins, 138
Protones, 110
Protoplasm, 5

Protoproteose, 131, 133, 135
Protosyntonose hexone bases in, 165
Proto-toxoids, 71
Pseudocerebrin, 611
Pseudoglobulin, 259
Pseudohaemoglobin, 283
Pseudomucin, 171

beta, 625, 626
detection of, 626
hydrolytic cleavage prod-
ucts of, 626

Pseudonuclein/629, 651
Pseudonucleins, 104, 177
Pseudopepsin, 464, 489
Pseudoxanthine, 578
Psittacofulvin, 843
Psylla-alcohol, 845
Ptomaines, 46, 82

1 in urine, 757, 758

Ptyalin, 48, 456-460

action of, 456, 457
best reaction for, 457
condition of, in the intestine, 510
effects of foreign substances upon,

457, 458

preparation of, 456
Purine bases, 186-193, 267, 715
detection of, 193
in active muscles, 594
in adrenal bodies, 377
in spleen, 370
preparation of, 193
quantitative estimation of,

715
transformation into uric

acid, 702, 703
bodies, in pus-corpuscles, 364

composition of, 187
oxidases, 875. See Enzymes,
skeleton, 186

structural formula of, 186
tri-oxy-, 2, 6, 8, 187
Purines, in lymphatic glands, 366
in thymus, 368
in thyroid gland, 373
Pus, 363

cells, analysis of, 365
corpuscles, 364-366

GENERAL INDEX

1019

Pus, constituents of, 364
in urine, 799
serum, 363

analyses of, 363, 364
analyses of ash of, 364
Putrefaction, 43, 46

factors influencing, 516-520
in intestines, importance of,

517
preventive substances for,

518

Putrefactive products, absorption of, 515
conjugation of, 515
elimination of, in

urine, 515
in large intestine,

515
Putrescine, 47, 82, 162

in urine, 827
Putrine, 47
Pyin, 363, 366
Pyloric secretion, 478
Pyocyaneus protease, 57
Pyocyanin, 366
Pyogenin, 364
Pyosin, 364
Pyoxanthose, 366

Pyrazine, formation from glycocoll, 220
Pyridine, fate of in organism, 787

structural formula of, 186
Pyrimidine, 193

bases, 177, 178, 193-195
Pyrin, 357

Pyrocatechin in urine, 727
Pyrrol derivatives, 154, 158, 276, 290,

740
Pyrrolidone-carboxylic acid, 113

Quercinite, 581
Quercite, 394
Quinidine, as catalyst, 60
Quinine, as catalyst, 60

effect upon spleen, 375

Cal
Quotient, -, 888

^,892

C to N, 771

flesh, 602

nitrogen to homogentistic acid,

735
respiratory quotient, 412, 563,

593, 879, 887, 918
urea to nitrogen, 777, 877
calorific urine, 892

^,887

Q

TV , in f eces, o84

C
Quotient, =^, in urine, 771

N

^r, in urine, 883

N

•s-, in urine, 882

calculation of, 889
in starvation, 895
respiratory, 887, 927, 928

Rachitis, 556, 557

Reaction, of a solution, determination of

74

velocity, '32

Reactivation enzymes, 52
Receptors, 67

Reducing substances in urine, 749-752
Reductase, 47, 48
Reductases, 876, 877
Reductions in animal body, 876
Reductonovaine in urine, 758
Regnault-Reiset's method for determining

respiratory exchange, 869
Reichert-Meissl's equivalent for fats, 238
Rennet, 649
Rennin, 48, 49, 57, 474-476

action of on charcoal, 62

anti, 64, 69

occurrence of, 474

pancreatic, 509

preparation of, 475

properties of, 475

relation to pepsin, 475

retardation of, 63

retarding substances for, 474

testing for in gastric juice, 487

See also Enzymes,
zymogen, 474
Resacetophenone, 785
Resins, fate of in organism, 787
Respiration, 850-877

apparatus, experiments with,

889, 890
external, 858

gas tension in blood, 859-868

importance of haemoglobin

in oxygen-carbon dioxide

exchange in blood, 853

internal, 858, 867

mechanism of carbon dioxide

elimination, 852, 853
processes taking part in the

gas exchange, 858

Respiratory exchange, methods for deter-
mining, 868T870
quotient in active muscle, 596

in diabetes 412-413
Rest carbon, 268
Rest nitrogen, in serum, 267

in stomach, 483, 484
and work, effect on metabolism,
926-928

1020

GENERAL INDEX

Reticulin, 92, 121, 544

composition of, 121
in lymphatic glands, 336
properties of, 121
preparation of, 122
Retina, constituents of, 615

pigments of, 615-617
Reynold's test for acetone, 823
Rhamnose, 208

relation to glycogen formation,

394

Rhodophan, 617
Rhodopsin, 615

Ribose, structural formula for, 199, 211
d-Ribose, 178
Ricin, 470, 505

in testing pepsin, 470
Rigor mortis, 601

of muscles, 588-591
Ringer-Locke's solution, 70 «3
Ritthausen's method of determining pro-
teins in milk, 656
Roch's test for proteid, 995
Rosenbach's bile pigment test, 799

urine test, 827
Rotation, specific, 199
Rovida's hyaline substance, 274, 302, 367
Rubner's reaction for dextrose, 215
sugar test in urine, 809

Saccharase, 48, 49, 57. See also Enzymes,
action of charcoal on, 63
retardation of, 63
Saccharides, mono, 197-218
di, 197, 223-226
poly, crystalline, 197
poly, colloid, 197, 226
tri, 197-226

Saccharose, 223, 224, 225. See Sucrose.
Sachsse's reaction for dextrose, 295
Sahidin, 609
Sahli's haemometer, 299
Saliva, the, 451-460

action of, in stomach, 480

on starch, 226
chorda, 452
constituents of, 453
diastatic power of, 71
gases of, 857
human, quantitative composition

of, 458

quantity secreted, 459
mixed buccal, constituents of, 455
paralytic, 452
parotid, 453, 454
physiological importance of, 459,

460

properties of, 453, 454, 455
sublingual, 453
submaxillary, 452
sympathetic, 452
Salivary concrements, 460
Salkowski's creatinine reaction, 696

reaction for chloesterin, 444

Salmine, 110

Salt action on enzymes, 70-76
glycosuria, 402
plasma, 251
Samandarin, 846
Sandmeyer's method of inducing pancreas

diabetes, 405

Santonin, elimination of in urine, 787, 800
Sapokrinin, 499
Saponin, 273, 446, 447
Sarcolemma, relation to permeability, 9
Sarcomelanin, 841
Sarcosine, fate in organism, 786
Sarkine. See Hypoxanthine, 190
Sarkosine, 776
Schalfejeff's, haemin, 292
Scherer's inosite test, 578
Schiff's reaction, 686

reaction for cholesterin, 445
Schreiner's bases, 621
Schutz-Borrissow's rule of ferment action,

54, 471, 476, 503, 506
Schutz's rule, 58
Schweitzer's reagent, 231
Scleroproteins, 93 j
Sclerotic, 619
Scombrine, 110
Scombron, 108
Scyllite, 370, 581
Scymnol, 418
Sebelien's method of determining proteins

in milk, 656
Sebum of skin, 844
Secretin, 463, 492, 498, 499
Secretion enzymes, 52

of prostate, 621
Sedimentum lateritium, 708
Segregation, 52
Seliwanoff's reaction for levulose, 218,

815

Semen, 620, 621

Semicarbazide, poisoning therewith, 718
Semiglutin, 119, 120
Seminose, 216, 217
Semi-permeable, membrane, 1
Senna, elimination of coloring matter of,

in urine, 787, 802, 827
Sensibilizators, 69
Sepia, 381, 841, 843
Sepsine, 47

Seralbumin, 84, 91, 252, 261-264
crystalline, 262
composition of, 262
elementary composition of,

263

preparation of. 262, 263
properties of, '262, 263. See

also Proteins,
quantitative estimation of,

263

Serglobulin, 84, 91, 252, 258-261
detection of, 261
elementary composition of,
263

GENERAL INDEX

1021

Serglobulin, para-, 258

preparation, 259, 261

properties, 260

quantitative estimation, 261.

See also Proteins.
Sericin, 92, 122, 124
Serine, 106, 107, 111, 113, 115, 125, 145

iso-, 146
Serolin, 265
Seromucoid, 261, 264
Serosamucin, 354

Serum, casein, 258. See Serum glo-
bulin.

normal. See Blood serum.
Sham feeding, 459, 464, 821
Shark, bile, 416, 433, 681
urea, 334, 433, 681

Shell, membrane of hen's egg, 636-641
Side-chain theory, 71
Siegfried and Zimmermann's method for

phenol-sulphuric acids, 726
Siegfried's carbamino reaction, 166
Silk gelatin, 123, 124
Sinistrin. animal, 174
Skatol, 46, 82, 117, 157-159, 515

522, 723, 732, 843
fate of in organism, 786
Skatosine, 159
Skatoxyl, 724

tests for, 732
Skeletins, 122

composition of, 122
Skin, 837-849

exchange of gas through, 849
horn formations of, 840
human, pigments of, 840-843
melanins of, 841
perspiration of, 847-849
pigments of 840-844
sebum of, 844
secretion, preputial, 846
secretions of, 837-849

of various animals, 846
Smegma prseputii, 844
Snake poison, effect upon blood, 255, 310,

325
Soaps, 265

in chyle, 347
Sodium alcoholate as a saponification

agent, 234, 237
compounds, division among form

elements and fluids, 22, 23
in urine, 766. See Mineral sub-
stances.
Solanin, 273
Solution tenacity, 10

in relation to Osmosis Relation-
ship to surface tension, 11
Sorbin, 218
Sorbinose, 218
Sorbite, 198
Sorensen's formol titration for amino-

acids, 166
Specific dynamic action, 918

Speck's method for determining respira-
tory exchange, 869
Spermaceti, 238

oil, 238, 239 \
Spermatin, 623
Spermatocele fluids, 359, 360
Spermatozoa, 620, 622, 623

properties of, 622
constituents of, 622
Florence's test for, 621
osmotic phenomena with, 8
Spermme, 621, 622

crystals, Bottcher's, 621
Sphygmogenin, 378
Sphyngomyelin, 608, 609
Sphyngosin, 611
Spiegler's test for proteids, 789
Spinal marrow, 614
Spirographin, 171
Spleen, 369-373

analyses of, 372
constituents of, 370
pathological processes in, 372
physiological functions of, 372
uric acid formation in, 702, 703
Spongin, 92, 122, 123

iodo-, 123
Sponginoses, 122
Spongosterin, 449
Sputum, 871

form constituents of, 871
Stachyose, 226
Starch, 226-228

artificial, 227
cellulose, 227
granulose, hydrolysis, 228
gum, 229
soluble, 227

Starvation, bone, catabolism in, 896
catabolism of fat in, 894
gas exchange in, 895
loss in weight of different

organs in, 896
metabolism, 892-906
metabolism of fats in, 894
mineral substances in, 895.

896
nitrogen content of urine in.

897
nitrogen elimination in, 893,

894
protein in, 894

metabolism in, 893.

894

time interval to death, 892
urea content in, 897
urinary constituents in starva-
tion, 899
water in, 895
Steapsin, 476, 501-503

effect upon, of bile, 511
Steapsinogen, 478

activation of, by bile, 511
Stearine, 234, 235

1022

GENERAL INDEX

Steensma's modification of Gunzburg's

test, 487

Stenson's test, 590
Stentorin blue, 844
Stercobilin, 428, 429, 529, 743

in feces, 532
Stercorin, 448 '
Stethal, 239

Stoke's reduction fluid, 282
Stomach, absorption of cleavage products

in, 484

action of salivary diastase in, 480
contents. See Chymus.
digestion and absorption of

various |food in, 513, 514
fistulas, 365, 460
gases in, 485, 486
glands, 458

lactic acid fermentation in, 485
movement of food in, during

digestion, 479, 480
movement within, during diges-
tion, 479

self-digestion of, 486
Stone-cystine, 148, 150
Stroma-fibrin, 275

of blood corpuscles, 712,

273

Stromata, 273, 274
Strvchnine, and glycogen transformation,

391, 394

Sturgeon, spermataozoa of, 110, 181
Sturine, 110

hexone bases in, 165
Stutz's test for proteid, 794
Subcutaneous oedema, fluid of, 362
Sublingual glands, 448
saliva, 450

Submaxillary glands, 446
Submicrons, 19
Substrate, 47
"Sucre immediat," 331
" Sucre virtuel," 331
Sugar, amounts in blood of different

animals, 328
amounts in blood, 331
detection of in urine, 802-808
fats, in liver, 412, 413
gelatin, 139
glycogen, 393, 395
in aqueous humor, 361
in ascitic fluids, 359
in blood, 329, 330
in blood serum, 265
in cerebrospinal fluid, 360
in lymph, 346

in transudates and exudates, 355
in urine, 802-818
in venous blood, 336
proteins, in liver, 409-411
Sulphaemoglobin, 286
Sulphate quantitative estimation of, in

urine, 765
Sulphates, amounts in urine, 765

Sulphates in urine, 764-766. See also

Mineral substance,
ethereal in urine, 765
quantitative estimation of, 765
Sulphatide, 609
Sulphocyanides, occurrence in urine, 752,

775

Sulphonal intoxication, urine, 294, 797
Sulphur, compounds in urine, 752, 753

elimination, importance of in

protein metabolism, 882, 883
in proteins, 78, 79. See also

various proteins.
in urine, 752, 874, 875, 890
methsemoglobin, 286
Sulphuretted hydrogen in urine, 753
Suprarenin, 387

Surface tension, in relation to osmosis. 10
Suspension colloids, precipitation of, 70
Suspensoids, properties of, 15
Sympathetic saliva, 449
Synovia, 360
Synovial cavities around joints, fluid in,

360

analysis of, 362
fluid, 362

Synoviamucin, 362
Synovin, 362
Synproteose, 135
Syntoniri, hexone bases in ,165 ]
Syntoxoids, 71

Tamia, 389
Talose, 211
Tartar, 460
Tatalbumin, 632
Taurine, 150, 151

fate of in organism, 786
Tears, 619

Teichmann's crystals, 292, 796
Tendon mucoid, 544

elementary composition of, 551
Terpenes, fate of in organism, 785
Testes, 620
Tetanolysin, 72

Tetanotoxine and gastric juice, 485
Tetraglyclyglycine, 85, 510
Tetramethylene diamine, 47. See Putras-

cine.

Tetrapeptides, 85, 87, 89, 510
Tetronerythrin, 843, 844
Tetroses, 197
Theobromine, 187
Theophylline, 187

Thiophene, behavior in animal body, 784
Thiotolene, 784
Thrombin, 48, 256, 315, 317, 324

action of, 256

formation of, 318

preparation of, 257

properties of, 256

pro-, 256
Thrombogen, 318, 321

formation of, 322

GENERAL INDEX

1023

Thrombokinase, 318, 319
Thromboplastic substances, 326
Thrombosin, 320

-lime, 320
Thrombozym, 321

formation of, 322
Thymine, 178, 181, 185, 195
Thymus, 366-369

analysis of, 369
constituents of, 368
functions of, 369
Thyreoglobulin, 375, 376
Thyreoproteid, 375
Thyroid, gland, 373-376

constituents of, 373
effect of extirpation of, 374
Tissue, connective, analysis of, 545
elastic analysis of, 545
fatty, 558-564
fibrinogen, 307
proteins, 908-910

Tissues, end products of oxidation in, 871
gas exchange in, 858-870
gelatinous, 545
mucous, 545
oxidation in, 858

physiological oxidation in, 871
theories for oxidation processes

in, 871, 872
Tollen's reaction for pentoses, 209

Rorive test for levulose, 218
test for glucuronic acid, 223
Toluene, fate of in organism, 779
Toluylenediamine, poisoning with, 441
Tonus, chemical, 590
Tooth, cement, 557
dentin, 557
enamel, 557
structure, 557
Toxin, cysts, 69
Toxins, 46, 66

a method of measuring, 67
Toxoid, 67

proto, syn, epi, 67
Toxons, 67
Toxophore, group, 67
Tradescantiadiscolor, 6
Transfusion, fluid for mamallian heart, 72

composition of, 72, 73
Transudates and exudates, constituents

found in, 353-355
distinguishing feature in, 356
formation of, 352, 353
osmotic pressure in, 356
pathological, gases of, 857
reaction of, 356
Trichohyalin, 837
Triglycerides, 232
Triglyclyglycin, 85, 510

ethyl ester, 86

Trimethylamine in urine, 757
Trimethylvinylammonium hydroxide, 246.

See Neurine.
Triolein, 233, 236

Trioses, 197

Trioxypurine, 2, 6, 8, 187. See Uric acid.

Tripalmitin, 233, 235

Tripeptides, 85, 509, 510

Triple phosphates, in urinary concrements

831-833
in urinary sediments,

831

Trisaccharides, 200
Tristearin, 233, 234
Trommer's test, 214, 793, 803
Trypsin 49, 57, 503-509

action of, 504, 505
action of charcoal on, 62
digestion action of foreign bodies

on, 506

formation of, 497
preparation of, 504
properties of, 503
quantitative estimation of, 505
rapidity of, under various con-
ditions, 506
retardation of, 63
tests for, 505. See also Enzymes,
upon other bodies, 508
Trypsinogen, 504

activation of, 496, 497, 498
activators of, 497
Tryptic digestion, 505

products, of 507
Tryptophane, 85, 111, 119, 155-157

Adamkiewicz - Hopkin's

reaction, 157
amounts in proteins, 106,

107

effect of yeast upon, 206
quantitative, colorimetric

method for, 157
Tryptophol, 157, 206
Tubo-ovaria, 628
Tunicin, 838, 839
Turacin, 843
Turacoverdin, 844
Turpentine, fate of in organism, 787
Tyrosinases, 875
Tyndall's phenomenon, 40
Tyrosine, 85, 109, 111, 119, 123, 124,

152-154, 267
amounts in proteins, 106, 107,

113, 115, 125
effect of yeast upon, 206
importance of in homogentisic

acid formations, 737
tests for, 153
Tyrosol, 153, 206

Uffelmann's test for free lactic acid in

gastric juice, 487
Umikoff's reaction, 663
Uracil, 178, 185, 194, 195

Weidel's reaction for, 194
Wheeler and Johnson reaction for,

194
Uranium salts, and glycosuria, 402

1024

GENERAL INDEX

Urate, ammonium, in urinary sediment, 830
Urates, acid, in urinary sediment, 830

of urine, 674
Ultra violet rays, 50
Umbilical cord, 545
Urea, 267, 679, 691

compounds of, 687
creatinine, 682

formation, anhydride theory, 684
from ammonium salts, 683
from arginine, 682
in ascitic fluids, 359
in blood, 333, 334
in cerebrospinal fluid, 360
in lymph, 346
in muscles. 572
in the organism, 682
in transudates and exudates, 355
in veins, 336
mesoxalyl, 699

mother substances of, 682, 683
nitrate, 686
occurrence of, 679
other organs besides liver, 685
oxalate, 687
oxidation theory, 684
physiological significance of, 680
preparation of, 679, 687, 688
properties of, 686

quantitative methods for 689-691
quantity voided, 68
tests for, 686
Urease, 48, 829
Urein, 691

Ureotheobromine, 713
Urethane, 692
Uric acid stones, 832
Uricolysis, 704
Uridine, 180, 185
Urinary calculi, 828, 829, 832-836

calcium carbonate, 834
calcium oxalate? 833
chemical investigation of,

834-836
cystine, 834
fibrin, 834
phosphate, 833
scheme for chemical,

analysis of, 836
uric acid, 833
urostealith, 834
xanthine, 834
pigments, 740-748
sediments, 674, 828, 829

acid hippuric in, 831
ammonium magnesium

phosphate in, 831
ammonium urate in.

830
calcium carbonate in,

831

calcium oxalate in, 830
calcium phosphate in,
831

Urinary sediments, calcium sulphate in,

831

cystin in, 831
haematoidine in, 831
magnesium triphos-

phate in, 831
non-organized, 830,

831
triple phosphates in,

831

tyrosine in, 831
urates, acid in, 830
uric acid in, 830
xanthine in,
Urine, 670-836

abnormal color of, due to foreign

substances, 787
acetoacetic acid in, 824, 825
acetone bodies in, 818-828
acetone in, 822-824
acid fermentation of, 829
alcaptonuric, test for, 739
alkaline due to ammonium car-
bonate, 676
fermentation of, 829
Almen's bismuth test for sugar,

803

amino-acids in, 827
ammonium urate calculi, 833
average composition of, 772
Bang's quantitative methods for

sugar, 809-811
Bertrand's quantitative method

for sugar, 811

Bial's test for pentoses, 816
bile-acids in, 799, 800
bile-pigments in, 800-801
casual constituents of, 772-787
cholesterin in, 827
cloudy, reasons for, 674
color of, 674
conjugated glucuronic acids in,

817,818

cystine in, 827, 828
degree of acidity, 675
detection of acetone and aceto-
acetic acid in, 825
Bence-Jones protein in,

792
betaoxybutyric acid in,

826'

bile-acids in, 800
blood in, 796
conjugated glucuronic

acids in, 817,818
fructose in, 814
hsematoporphyrin in,

798

lactose in, 815
peritose in, 816
pigments in, 800, 801
proteins in, 787-781
proteoses in 791, 792
sugar in, 802-808

GENERAL INDEX

1025

Urine, determination of acidity, 676,677,
ion acidity, 677,

678
specific gravity

678, 679

division of the nitrogen of, 681

Ehrlich's urine test, 826

end products of acids amino

aromatic, 780-783

amino-acids, 774-

776

benzene ring and
homologues, 778,
779

fatty series, 773,774
heterocyclic com-
pounds, 778-787
homocyclic com-
pounds, 778-787
fat in, 827

fermentation test for sugar, 805
fructose in, 814, 815
gases of, 857
glucose in, 802-814
guaiac test for blood in, 796
haematoporphyrin in, 797, 798
Heller-Teichmann's test for blood

in, 797

heptose in, 817
indican, 728

inorganic constituents of, 758-770
inosite in, 818
isolation of sugar from, 807
Kjeldahl's method for total nitro-
gen, 688
Knapp's quantitative method for

sugar, 812
lactose in, 815
laiose in, 815
levulose in, 814, 815
maltose in, 815

medicinal coloring matter in, 801
melanin in, 799
microscopic investigation of blood

corpuscles, 796
nitrogen content during starvation,

897

Nylander's test for sugar, 803
odor of, 674
organic physiological constituents

of, 679-758

osmotic pressure of, 678
0-oxybutyric acid in, 825-827
passage of sugar into, 534
pathological constituents of, 787-

828

pentoses in, 816
percentage division of nitrogenous

substances, 681

phenylhydrazine test for sugar, 806
physical properties of, 674-679
physico-chemical methods in, 772
physiological constituents of, 679-
787

Urine, pigments in, 798, 799

polarization of, for sugar, 807

pus in, 799

quantitative composition of, 770-

277

determination of su-
gar, 808-814
determination of pro-

teid, 793
determination of total

nitrogen, 688
estimation of acetone

in, 825
estimation of /3-oxy-

butyric acid, 826
estimation of sugar
by fermentation,
812

estimation of sugar
by polarization,
813-814

method for nitrogen
in very small quan-
tities of urine, 689
quantity of, 770

conditions affecting,

770
of solids excreted, 770,

771

reaction of, 674, 675
Rubner's test for sugar, 807
sedimentatum lateritium, 674
specific gravity of, 678
spectroscopic investigation for

blood, 796
sugar in, 802-818
taste of, 674
transparency of, 674
Trommer's test for sugar, 802
urea content in starvation
Urinometer, 678
Urobilin, 429, 516, 740, 743-746
detection of, 747
formation of in organism, 744
in blood serum, 268
preparation of, 746
properties of, 745
quantitative estimation of, 747
tests for, 745, 746
Urobilinogen, 740

detection of in urine, 746
formation of in organism.

744, 746, 747
in blood serum, 268
preparation of, 746, 747
properties of, 746
quantitative estimation of,

747

Urobilinoids, 743
Urochrome, preparation of, 742

quantitative estimation of, 742
Urochrome, 740, 741, 742
in urine, 755
Ilrocyanin, 741

1026

GENERAL INDEX

Uroerythrin, 740, 748

tests for, 748
Urofuscohsematin, 798
Uroglaucin, 741
Urohaematin, 741
Urohodin, 741
Urohypertensin, 758
Urohypotensin, 758
Uromelanins, 741
Urophain, 740
Uropyrryl, 741
Urorosein, 741

in urine, 733
Urorubin, 741
Urospectrin, Saillet's, 797
Urostealith calculi, 834
Urotheobromine, 713
Urotoxic coefficient, 758
Urorubrohsematin, 798
Uroxanthine, 728
Uterine milk, 642
Uterus, colloid, 627

Valine, 111, 140,141

amounts in proteins, 106, 107, 113,
115, 125

Vanillin, behavior in animal body, 779

Van Slyke's method for amino acids, 79

Van't Hoff's rule, 201

Velocity coefficient, 34

Vernine, 178

Vernix caseosa, 845

Vesiculase, 621, 622

Viridinine, 47

Visual purple, 615-617

preparation of, 617
properties of, 616
regeneration of, 616

Visual red, 615

Vitali's pus-blood test, 796

Vitamine, 905

Vitellin, 628

cleavage products of, 106

Vitellolutein, 632

Vitellorubin, 632

Vitelloses, 130

Vitiatine, 578

in urine, 758

Vitreous humor, 617

Voit's normal average diet, 933

Volhard and Lohlein's quantitative
method for pepsin, 470

Volhard's quantitative method for
chlorides, 760

Walden's reversion, 89
Water, importance to life, 71, 72

lack of in food, 899
Wear and tear quota, 890, 917
Weidel's reaction for histidine, 160
xanthine, 190

Weiss' sparing theory, 396

Weyl's creatinine reaction,695

Wheeler and Johnson reaction for Uracil

194
Whey, 645

acid, 645

protein, 650

sweet, 645
Witch's milk, 665
Wool-fat constituents of, 846
Work affecting metabolism, 926-928
Worm, Miiller's sugar test, 803

Xanthine, 86, 178, 187, 188, 189, 190, 712
in ascitic fluids, 359
in urinary calculi, 833, 834
in urinary sediments, 831
in urine, 710
para, 187

Seyler's reaction for, 190
Weidel's reaction for, 190

Xantho creatinine, 578

in urine, 698

Xantho melanin, 84 t

Xanthoproteic reaction for proteins, 99,
173

Xanthoprotein, 84

Xanthophan, 617

Xanthosine, 180

Xylene, fate of in organism, 779

Xyliton, 842

Xyloses, structural formula for, 198, 210

Yeast, 59,62

action of, on glucose, 226
maltase, 266
Yoghurt, 654, 659
Yolk, constituents of, 628

fat of, 630

membrane, 628

mineral bodies of, 632

of hen's egg, 628

pigments of, 631

phosphatides of, 630

proteins of, 628-630

spherules, 628, 636

Zein, 77, 105, 163
Zinc, in the bile, 433

in the liver, 389

passage into milk, 670
Zooerythrin, 843
Zoofulvin, 843
Zoorubin, 843

Zuntz and Geppert's method for deter-
mining respiratory exchange, 869
Zymase, 41
Zymogen, rennin, 474
Zymogens, 51, 461
Zymoplastic substances in blood, 315

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