1

WORKS OF
PROFESSOR J. A. MANDEL

PUBLISHED BY

JOHN WILEY & SONS.

Handbook for Biochemical Laboratory.

12mo, cloth, $1.6U.

TRANSLATIONS,

A Text-book of Physiological Chemistry.

By Olof Hammarsten, Emeritus Professor of Medi-
cal and Physiological Chemistry in the University
of Upsala. Authorized translation, from the
author's enlarged and revised 7th German edition,
by John A. Mandel, Professor of Chemistry in the
New York University and Bellevue Hospital Med-
ical College. 8vo, viii 4-964 pages, cloth, 4.00 net.

A Compendium of Chemistry, Including: General, In-
organic, and Organic Chemistry.

By Dr. Carl Arnold Professor of Chemistry in the
Royal Veterinary School of H jnriover. Authorized
translation from the eleventh enlarged and revised
German edition, by John A. Mandel. Small Svo,
xti + 627 pages, cloth, $3.50.

A TEXT-BOOK

OF

PHYSIOLOGICAL CHEMISTEY

BY

OLOF IjtAMMARSTEN

EMERITUS PROFESSOR OF MEDICAL. AND PHYSIOLOGICAL, CHEMISTRY IN THE
UNIVERSITY OF UPSALA

FROM THE AUTHOR'S ENLARGED AND REVISED
SEVENTH GERMAN EDITION

BY

JOHN A. MANDEL, Sc.D.

PROFESSOR OF CHEMISTRY IN THE NEW YORK UNIVERSITY AND
BELLEVUE HOSPITAL MEDICAL, COLLEGE

SIXTH EDITION
TOTAL ISSUE, TEN THOUSAND

NEW YORK
JOHN WILEY & SONS

LONDON: CHAPMAN & HALL, LIMITED
1912

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

BY

JOHN A. MANDEL

THE SCIENTIFIC PRESS

B08ERT ORUMMONO AND COMPANY

BROOKLYN, N. V.

PREFACE TO THE SEVENTH GERMAN EDITION.

THIS edition differs from the earlier ones by containing a new
chapter on " Physical Chemistry in Biology." This chapter has been
written by Professor S. G. HEDIN, of Upsala. In order to give space
for this new, second chapter, without changing the number of chapters
of the earlier editions, the fifth chapter of these earlier editions on " the
animal cell " has been dropped and its contents incorporated in the
other chapters. For example, in this edition the nucleic acids, as well
as the purine and pyrimidine bases, are treated of in connection with
the nucleoproteins, and the phosphatides are discussed with the fats in
Chapter V, a change which also seems necessary for other reasons. In
general, the plan of the book remains unchanged. The numerous publi-
cations in physiological chemistry which have appeared since the publica-
tion of the sixth edition have necessitated a thorough revision and
rewriting of all the chapters. Unfortunately a considerable increase in
the size of the book could not be prevented.

OLOF HAMMARSTEN.
UPSALA, November 16, 1909.

in

TRANSLATOR'S PREFACE TO THE SIXTH AMERICAN

EDITION.

ALTHOUGH a number of excellent works on Physiological Chemistry
have appeared during the past few years, HAMMARSTEN'S " Physiologischen
Chemie " maintains its early popularity; and I am confident that all
workers in biochemical research are thankful to Professor HAMMARSTEN
for the labor and care that he has exercised in the prepartion of the present
edition.

The work of translating and editing has been a labor of love, inas-
much as I feel that it will be of aid in the advance of this department
of chemical science.

I am indebted to my assistant, Mr. A. O. GETTLER, for help given
in revising the proof.

JOHN A. MANDEL.

NEW YORK, April, 1911

CONTENTS.

CHAPTER I.

PAGE

INTRODUCTION 1

CHAPTER II.
PHYSICAL CHEMISTRY IN BIOLOGY 26

CHAPTER III.
THE PROTEIN SUBSTANCES 76

CHAPTER IV.
THE CARBOHYDRATES 192

CHAPTER V.
ANIMAL FATS AND PHOSPHATIDES 225

CHAPTER VI.
THE BLOOD : 242

CHAPTER VII.
CHYLE, LYMPH, TRANSUDATES, AND EXUDATES 327

CHAPTER VIII.
THE LIVZR 360

CHAPTER IX.

DIGESTION 426

CHAPTER X.

TISSUES OF THE CONNECTIVE SUBSTANCE 519

vii

viii CONTENTS.

CHAPTER XI.

PAGE

MUSCLES 533

CHAPTER XII.
BRAIN AND NERVES 574

CHAPTER XIII.
ORGANS OF GENERATION 589

CHAPTER XIV.
THE MILK 610

CHAPTER XV.
THE URINE 638

CHAPTER XVI.
THE SKIN AND ITS SECRETIONS 789

CHAPTER XVII.
CHEMISTRY OF RESPIRATION 801

CHAPTER XVIII.

METABOLISM 822

INDEX TO AUTHORS 887

GENERAL INDEX. . 929

PHYSIOLOGICAL CHEMISTRY.

CHAPTER I.
INTRODUCTION.

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 are only called upon to appropriate and assimilate
material already existing 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, carbohy-
drates, fats, resins, organic acids, etc. The chemical work which is per-
formed 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 + H2O = 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 PRIESTLEY 3 are of greater interest

1 Ber. d. d. chem. Gesellsch., 3.
2Zeitschr. f. Electrochem., 12.
3 Proc. Roy. Soc. London, 78, Series B.

2 I INTRODUCTION.

in thai, they show the formation of formaldehyde in the photolytic
decomposition of moist carbonic acid in the presence of chlorophyll.
These investigations also do not seem to be entirely free from exception.
The conception as to the formation of sugar from formaldehyde is also
often different from that explained by v. BAEYER'S idea, and his view as to
the assimilation of carbonic acid constitutes a hypothesis 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 dependent
either directly, as the herbivora, or indirectly, as the carnivora, upon
plant-life, from which they drive 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 animal
body, undergo within the animal organism a cleavage and oxidation,
#nd 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 chem-
ical 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 syn-
theses, 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 oxidation 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 intermediate
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 car-
bon dioxide, while in the light the oxidation processes going on in the
green parts are overshadowed or hidden beneath the more intense reduc-
tion processes. As in the animal, we also find a heat production in fer-
mentation produced by plant organisms ; and even in a few of the higher

SYNTHESIS AND OXIDATIONS. 3

plants — as the aroidece when bearing fruit — a considerable development
of heat has been observed. On the other hand, in the animal organism,
besides oxidation and splitting, reduction processes and syntheses 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.

WoHLER1 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 (amirioacetic acid). Since
the discovery of this synthesis, which may be expressed by the follow-
ing equation:

C6H5.COOH + NH2.CH2.COOH - NH (C6H5.CO) .CH2.COOH + H2O,

Benzoic acid Glycocoll Hippuric acid

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
ha3moglobin), 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.

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 oxida-
tion in the form-elements is induced and how it proceeds cannot be
answered with certainty.

When a substance is oxidized by neutral oxygen at the ordinary
temperature or at the temperature of the body, the substance is said

1 Berzelius, Lehrb. d. Chemie, iibersetzt von Wohler, 4, p. 356, Abt. 1, Dresden, 1831.

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

4 INTRODUCTION

to be easily oxidized or autooxidized, and the process is considered as a
direct oxidation or autooxidation. As the oxygen of the inspired air;
and that of the blood, is neutral molecular oxygen, the old assumption
that ozone occurs in the organism has now been discarded for several
reasons. On the other hand, the chief groups of organic nutritives,
carbohydrates, fats, and proteins, the last two forming the chief mass
of the animal body, are not autooxidizable substances. They are on
the contrary bradoxidizable (TRAUBE) or dysoxidizable bodies. They
are nearly indifferent to neutral oxygen, and it is therefore a question
how an oxidation of these and other dysoxidizable bodies is possible
in the animal body.

In explanation it is very generally admitted that the oxygen is made
active and this causes a secondary oxidation. It is generally con-
ceded that in autooxidation a cleavage of neutral oxygen takes place.
The autooxidizable substance splits the oxygen molecule and combines
with one of the oxygen atoms, while the other free atom as active oxygen
may oxidize the dysoxidizable substances simultaneously present. Such
a subordinate oxidation is called an indirect or secondary oxidation.
The explanation of animal oxidations has been attempted in different
ways by the supposition that the oxygen is made active and thus pro-
duces secondary oxidation.

The cause of the animal oxidation is considered, by PFLUGER and
several other investigators, to be dependent upon the special constitu-
tion of the protoplasmic proteins or the living protoplasmic substance.
This investigator calls the proteins outside of the organism, or those
which occur in the animal fluids, " non-living proteins," and considers
them to be somewhat different from those occurring in living protoplasm.
The latter are called " living proteins " (PFLUGER), " active proteins "
(LoEw), or " biogens " (VERWORN). The living protoplasmic molecule
differs from the ordinary non-living protein by being more unstable and
therefore having a greater inclination toward intramolecular changes
of the atoms.

The reason for these greater intramolecular movements PFLUGER ascribes to
the presence of cyanogen, and LATHAM attributes it to the presence of a chain
of cyanalcohols in the protein molecule. VERWORN, 1 on the contrary, claims
an intramolecular introduction of oxygen into a large hypothetical protoplasmic
molecule, the " biogen molecule," which is supposed to contain a nitrogen or an
iron complex as an oxygen receptor or carrier, and a side-chain of aldehydic
character like that of the carbohydrates, as an oxidizable group.

According to LoEW,2 who bases his claim upon special investigations and

1 Pfliiger, Pfliiger's Archiv, 10; Latham, Brit. Med. Journal, 1886; Verworn,
Die Biogenhypothese, Jena, 1903.

2Loew and Bokorny, Pfliiger's Archiv, 25; O. Loew, ibid., 30; and specially O.
Loew, Die chemische Energie der lebenden Zellen. 2. Aufl. Stuttgart, 1906.

ANIMAL OXIDATIONS. 5

numerous toxicological observations, the unstability of the active protein mole-
cule is due to the simultaneous presence of aldehyde and unstable ainino groups.
These occur separated from each other in the active proteins, and when they
combine the protoplasm dies, the molecule being changed into a stable condition,
i.e., into dead protein. It is also a fact that all substances which react with
aldehyde and unstable amino groups are poisonous to the living cells.

LOEW has also shown, in conjunction with BOKORNY, that in many plants
a very unstable reserve-protein substance occurs, which to a certain extent occupies
an intermediate position between protein and organized living substance.

The explanation as to the oxidation process differs entirely accord-
ing to the conception of the structure of the unstable protoplasmic
molecule. If the living protoplasmic protein is not, like protein in the
ordinary sense,, indifferent to neutral oxygen, we can admit of a cleav-
age of the oxygen molecule by this change. The protein would be itself
oxidized, while on the other hand a secondary oxidation of other dif-
ficultly oxidizable substances could be brought about by the oxygen
atoms set free.

Another very widely diffused view exists in regard to the origin
of the activity of the oxygen, namely, that by the decomposition proc-
esses in the tissues, reducing substances are formed which split the
neutral oxygen molecule, uniting with one oxygen atom and setting
the other free.

The formation of reducing substances during fermentation and
putrefaction is generally known. The butyric fermentation of dextrose
in which hydrogen is set free — C6H12O6 = C4H8O2 + 2CO2 + 2H2 — is an
example of this kind. Another example is the appearance of nitrates
in consequence of an oxidation of nitrogen in cases of putrefaction,
which process is ordinarily explained by the statement that reducing,
easily oxidizable bodies are formed which split oxygen molecules, liberat-
ing oxygen atoms which afterward oxidize the nitrogen. It is assumed
also that the cells of the animal tissues and organs have the power, like
these lower organisms which produce fermentation and putrefaction,
of causing splitting processes in which easily oxidizable substances,
perhaps also nascent hydrogen (HoppE-SEYLER1), are produced.

In accordance with what has been stated above on the oxidations in
the animal body, primarily a cleavage of the organic constituents of
the body takes place with the formation of readily oxidizable substances.
The oxidation of these latter produces an activation of the oxygen
and hence may also cause a secondary oxidation of dysoxidizable sub-
stances. The products formed by these splittings and oxidations may
perhaps in part be burned within the body without undergoing further
cleavage, but more probably they must first undergo a further cleavage

1 Pfliiger's Archiv, 12.

6 INTRODUCTION.

and then succumb to consecutive oxidations, until after repeated cleav-
ages and oxidations the final products of metabolism are formed.

An activation of the oxygen may be produced according to O. NASSEX by a
hydroxylization of the constituents of the protoplasm with the splitting off of
molecules of water. If benzaldehyde is shaken with water and air, an oxidation
of the benzaldehyde into benzoic acid takes place, while oxidizable substances
present at the same time may also be oxidized. The simultaneous presence of
potassium iodide and starch or tincture of guaiacum causes a blue coloration
because the hydroxyl (OH) takes the place of the hydrogen in the aldehyde group,
and these two hydrogen atoms, one derived from the aldehyde and the other
from the water, have a splitting action on the molecular oxygen. NASSE and
ROSING 2 have also found that certain varieties of protein have the property of
being hydroxylized in the presence of water. According to NASSE a whole series
of oxidations in the animal body may be accounted for by the oxygen atoms set
free in hydroxylization similar to that of benzaldehyde. In opposition to this
view we must remark that the oxidation of benzaldehyde to benzoic acid may also
take place in other ways, thus by the intermediary formation of a peroxide (see
BAEYER and VILLIGER; ENGLER and WEISSBERG 3).

By quantitative methods, VAN'T HOFF and his pupils 4 have shown
that molecular oxygen can be divided in two parts by certain auto-
oxidation processes. One of these parts unites with the autooxidizer and
the other with a body simultaneously present but not directly oxidizable,
which, according to the suggestion of ENGLER,5 is called the acceptor.
VAN'T HOFF claims that the oxygen molecule dissociates at ordinary
temperatures into minimum quantities of positively and negatively
charged oxygen atoms,' the ions of similar charge uniting with the
autooxidizable substance, while the remaining ions oxidize the acceptor.
Such a division of the oxygen into halves has also been shown by
other investigators, such as MANCHOT, ENGLER, and his collaborators.6
These investigators nevertheless consider that autooxidation takes
place in another way, namely, by the formation first of peroxides by
the taking up of oxgyen molecules.

TRAUBE 7 has also expressed a similar view. According to him,
in autooxidation we have to deal, in the first place, not with a cleavage
of the oxygen, but with a splitting of water in which the hydroxyl groups
of the water combine with the oxidizable substance, while the hydrogen

1 O. Nasse, Rostocker Zeitung, No. 534, 1891, and No. 363, 1895.

2 E. Rosing, Untersuchungen iiber die Oxydation von Eiweiss in Gegenwart von
Schwefel. Inaug. Dissert. Rostock, 1891.

3 Baeyer and Villiger, Ber. d. d. chem. Gesellsch., 33; Engler and Weissberg, ibid., 33.

4 van't Hoff, Zeitschr. f. physikal. Chem., 16; Jorissen, Ber. d. d. chem. Gesellsch.,
30, and Zeitschr. f. physikal. Chem., 22; Ewan, ibid., 16.

5 Ber. d. d. chem. Gesellsch., 33.

6 Manchot, Ueber freiwillige Oxydation. Leipzig, 1900; Engler and Weissberg,
Ber. d. d. chem. Gesellsch., 33; Engler and Frankenstein, ibid., 34.

7 Ber. d. d. chem. Gesellsch., 15, 18, 19, 22, and 26.

ANIMAL OXIDATIONS.

atoms set free on the 'decomposition of the water unite with the neu-
tral oxygen, forming hydrogen peroxide, which may naturally also
have an oxidizing action.

According to the generally accepted view, which is based upon the
work of BACH, MANCHOT, ENGLER and collaborators,1 the oxygen acts
also in the autooxidation in the first place as an entirely inactive mole-
cule. According to ENGLER and his collaborators, at least in the
simplest cases (" direct autooxidation ;; according to ENGLER), the
oxygen molecules unite with the activating body (A) , forming a peroxide-
like substance which can give up one of the two oxygen atoms to an
acceptor (B) :

= AO2 and

The appearance of a peroxide has, at least in certain cases, been shown, as,
for instance, with dimethylfulvene C8H10 where the peroxide C8H10O4 occurs
(Engler). The autooxidation may also in other cases proceed otherwise, as
shown by Manchot2 in the oxidation of hydro-compounds like oxanthranol,
which proceeds according to the equation AH2 + O2 = A + H2O2, namely, with
the formation of a hydroperoxide. This is different from TRAUBE'S views.

If thio is so, still we do not know to what extent such peroxides are
formed in oxidation in the living cell. The possibility of a produc-
tion of peroxides, and also of hydrogen peroxide, in animal oxidation
is still generally admitted, and CHODAT and BACH 3 have indeed been
able to show a peroxide formation in plants. Still, if hydrogen peroxide
were formed in such oxidations it would have no further physiological
importance, according to LOEW, because the animal and plant cells
contain special enzymes, called by him catalases, which quickly decom-
pose the hydrogen peroxide with the production of molecular oxgyen.
According to LOEW 4 the physiological importance of the catalases is
to protect the cell from hydrogen peroxide, which acts as a protoplasmic
poison, a view which is accepted by many investigators but still dis-
puted by others.

1 Engler and Wild, Ber. d. d. chem. Gesellsch., 30; Bach, Le Moniteur scientifique,
1897, and Compt. rend., 124; Manchot, 1. c.

2 Verhandl. d. Phys. med. Gesellsch. zu Wiirzburg (N. F.), Bd. 39.

3 Ber. d. d. chem. GeselL, 35 u. 36.

4 Loew, U. S. Dept. of Agriculture, Rep. 68, 1901, and Ber. d. d. chem. Gesellsch.,
35; in regard to the opposed views see Chodat and Bach., 1. c., Bach, Monit. scientif.
(4) 20; Kastle and Loevenhart, Amer. Chem. Jour., 29; Herlitzka, Chem. Centralbl.,
1WX; Euler. Hofmeister's Beitrage, 7, and especially O. Loew, Centralbl. f. Bacteriol.,
21; Abt. 2, which contains the literature.

8 INTRODUCTION.

LOEW/ who has also opposed the view as to the oxygen becoming
active with the setting free of oxygen atoms, has sought for the reason
of the oxidations in the unstable properties of the protoplasmic pro-
teins. The active movement of the atoms within the active protein
molecule is transmitted to the oxygen and to the oxidizable substance,
and when this dissolution of the molecule has proceeded to a certain
point the oxidation occurs by virtue of the chemical affinity. The reason
for this unstable condition of living protein molecules has already been
given above.

ScHMiEDEBERG,2 who also denies the supposition that the oxygen
becomes active, io of the view that the tissues by the mediation of the
oxidations do not increase the oxidizing activity of the oxygen, but more
probably act on the oxidizing substances, making them more susceptible
to oxidation.

All the views presented thus far assume a continuous oxidation of
the primary active substance. The view has also been suggested that
animal oxidation may be brought about by oxygen -carriers, i.e., by bodies
which, according to the older views, without being oxidized themselves,
act in a manner analogous to that of the nitric oxide in the manufac-
ture of sulphuric acid by alternately taking up and giving off oxygen in
the oxidation of dysoxidizable bodies. TRAUBE many years ago ex-
plained the oxidations of the animal body in this way, and he called
these questionable oxygen -carriers oxidation ferments.3

It is a fact that in the animal and plant kingdom bodies have been
extensively found which can bring about oxidations and which in many
respects behave like ferments or enzymes. It is therefore necessary,
before we proceed further, that we discuss the peculiar and highly
important bodies that have been called ferments or enzymes. The
nature, properties and mode of action of these bodies will be discussed
somewhat in detail in a following chapter (II), but in order to under-
stand what follows, we will give a short summary of the subject.

Alcoholic fermentation by yeast and other processes of fermenta-
tion and putrefaction are dependent upon the presence of living organ-
isms, ferment fungi, and splitting fungi of different kinds. The ordinary
view, according to the researches of PASTEUR, is that these processes are
to be considered as phases of the life of these organisms. The name
organized ferments or ferments has been given to such micro-organisms,
of which ordinary yeast is an example. However, the same name has also
been given to certain bodies or mixtures of bodies of unknown organic origin

1 Die chem. Energie d. lebenden Zellen. 2. Aufl. Stuttgart, 1906.

2 Arch. f. exp. Path. u. Pharm., 14.

3 M. Traube, Theorie der Fermentwirkungen. Berlin, 1858.

FERMENTS AND ENZYMES. 9

which are products of the chemical work within the cell, and which after
they are removed from the cell still have their characteristic action.
Such bodies — for example, malt diastase, rennin, and the digestive fer-
ments— are capable in the very smallest quantity of causing a decom-
position or cleavage in very considerable quantities of other substances,
without entering into permanent chemical combination with the decom-
posed body or with any of the cleavage or decomposition products. These
formless or unorganized ferments are generally called enzymes, according
to KUHXE.

A ferment in a more restricted sense is therefore a living being,
while an enzyme is a product of chemical processes in the cell, a product
which has an individuality even without the cell, and which may be
active when separated from the cell. The splitting of invert-sugar into
carbon dioxide and alcohol by fermentation is a fermentative process
closely connected with the life of the yeast. The inversion of cane-
sugar is, on the contrary, an enzymotic process caused by one of the bodies
or a mixture of bodies formed by the living ferment, which can be separated
from this ferment, and still remain active even after the death of the latter.
Consequently ferments and enzymes are capable of manifesting a differ-
ent 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, 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 1
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
Munchen, 13, 1807, part 1, which also contains the discussion on this topic. See also
E. and H. Buchner and M. Hahn, Die Zymasegarung, Miinchen, 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.

10 INTRODUCTION.

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, HERZOG l) . 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.

All enzymes and ferments, both names having now the same signifi-
cance, are considered as organic substances formed in the cells but whose
chemical nature has not been determined for the present.

We have no characteristic reaction for all enzymes or ferments
in general, but each enzyme is characterized by its specific action and
by the conditions under which it operates. Of special importance is,
first, the fact that the enzymes do not form permanent chemical com-
binations in definite proportions by weight with the bodies upon which
they act, or their decomposition products; and, secondly, that an insig-
nificantly small amount of the enzyme can decompose a relatively enormous
amount of substance. For instance, 1 part of invertin can invert 100,000
parts of cane-sugar (O'SuLLiVAN and THOMPSON 2), and 1 part of chy-
mosin can in a short time decompose more than 400,000 parts of casein
(HAMMARSTEN 3). This does not exclude the possibility of a primary,
but temporary, combination of the enzymes with the substances acted
upon — a process which is highly probable from the numerous observa-
tions which will be discussed in Chapter II.

The specific action of the enzymes is of special importance, as 01 le
and the same enzyme acts only upon one substance or a definite group
of substances. Their action seems to be entirely dependent upon the
stereometric construction of the substance acted upon, which will be
discussed in Chapter II.

The relationship of the enzymes to the inorganic catalysts is also of

1 E. Buchner and J. Meisenheimer, Ber. d. d. chem. Gesellsch., 36; and Annal.
d. Chem. u. Pharm., 349; with Gaunt, ibid., 349; Herzog, Zeitschr. f. physiol. Chem., 37.
2' O' Sullivan and Thompson, Journ. of Chem. Soc., 57.
3 See Maly's Jahresbericht, 7.

ENZYMES. OXIDASES. 11

the greatest importance. The catalysts, like the enzymes, are not found
in the final products of the reaction, they are not used up in the process,
and the quantity of the active substance proportionate to the quantity
of substance transformed is infinitesimally small in enzyme action as well
as in catalysis. Without having positive proof, nevertheless we now
consider that enzyme action is not to be considered as the starting of a
reaction which would not of itself take place, but rather as an accelera-
tion of a slowly proceeding, often not noticeable, chemical change.
According to this conception enzyme action comes in line with catalysL,
as understood to-day (see Chapter II).

An enzyme is an organic substance formed in an animal or plant
cell, which is destroyed by heating its aqueous solution and which acts
like the catalytes, but only upon certain bodies. Some restriction must
be put to this, as the cells do not always produce a complete enzyme,
but more often only the mother-substance thereof. These mother-sub-
stances of the enzymes are called proenzymes or zymogens. The zymogens
are under certain conditions converted into enzymes, and in certain cases
this is brought about by the special action of bodies called kinases,
which have been little studied (see Chapter II, VI and IX).

According to their action most of the enzymes which have been
studied may be divided into two chief groups, namely, those having a
hydrolytic action and those having an oxidizing action.

After this short discussion of the enzymes we can now return to the
oxidations and the so-called oxidation ferments.

It has also been positively proven by the researches of JAQUET,
SALKOWSXI, SPITZER, ROHMANX, ABELOUS and BIARNES, BERTRAND,

BOURQUELOT, DE REY-PAILHADE, MEDWEDEW, PoHL, JACOBY, CHODAT

and BACH 1 and others that in the blood and different tissues of the ani-
mal body, as also in plant-cells, substances occur which have the prop-
erty of causing certain oxidations. For this reason these bodies have
been called oxidases, and they have been divided into two different
groups. The ferments of the first group, called primary or direct oxidases,
or simply oxidases, transfer the oxygen of the air directly to other
bodies. Those of the second group, the indirect oxidases or peroxidases,
are active only in the presence of a peroxide, as they set oxygen free

1 Jaquet, Arch. f. exp. Path. u. Pharm., 29; Salkowski, Centralbl. f. d. med. Wis-
sensch., 1892 and 1894, and Virchow's Arch., 147; Spitzer, Pfliiger's Archiv, 60 and
67; Spitzer and Rohmann, Ber. d. deutsch. chem. Gesellsch., 28; Abelous et Biarnes,
Arch, de physiol. (5), 7, 8, and 9, and Compt. rend. soc. biol., 46; Bertrand, Arch, de
physiol. (5), 8, 9, and Compt. rend., 122, 123, 124; Bourquelot, Compt. rend. soc.
biol., 48, and Compt. rend., 123; Medwedew, Pfliiger's Arch., 81; Jacoby, Ergebnisse
der Physiologic, Jahrg. I, Abt. 1, which contains the literature of the subject; Chodat
and Bach, 1. c.

12 INTRODUCTION.

from these latter by decomposition. Correspondingly the oxidases
turn tincture of guaiac blue directly, while the peroxidases only have
this action in the presence of a peroxide. The catalases do not give
any reaction with guaiac either directly or indirectly in the presence
of peroxides.

According to the investigations of BACH and CHODAT l the condi-
tions are otherwise. According to the observations they have made
upon plants, there exist no oxidases, and what has been described under
this name is only a mixture of oxygenases and peroxidases. The
oxygenases are of a protein nature, contain manganese or iron, and
are converted into peroxides after taking up oxygen. These per-
oxides themselves have only a slight oxidizing power, but are made
active by the peroxidases. The peroxidases, which do not have the
slightest oxidizing power in the absence of peroxides, are not proteins.
In oxidation, according to the hypothesis of BACH and CHODAT, the molec-
ular oxygen is first converted by the oxygenase into peroxide. This
peroxide is activated by the peroxidase and then has strong oxidizing
power. The oxidizing power of the so-called direct oxidases is brought
about by a combined action of the oxygenases and peroxidases.

The chemical nature of the oxidation enzymes is still unknown,
and the statements on this subject are very contradictory. Certain
oxidases are proteins, namely, nucleoproteins (SPITZER), others globu-
lins (ABELOUS and BIARNES), and still others, like the liver aldehydase
(JACOB Y) and laccase (BERTRAND), are of a non-protein nature. The
materials upon which the oxidation enzymes act may also be very dif-
ferent from each other. Thus the oxidases studied by ROHMANN and
SPITZER may by synthetical oxidation produce indophenol from a-naphthol
and p-phenylenediamine in the presence of alkali. The salicylase or
aldehydase detected in the liver and many other organs oxidizes many
aldehydes to their corresponding acids, but does not give the indophenol
reaction. The laccase isolated by BERTRAND from the juice of the lac-
tree has an oxidizing action upon polyhydric ;7-phenols, such as hydro-
quinone, but not upon tyrosine. The bodies called tyrosinases, first
found by BERTRAND 2 in certain fungi and later also found by BIEDER-
MANN, v. FURTH, and SCHNEIDER 3 in the animal kingdom, have, on the
contrary, an action upon tyrosine, converting it into colored com-
pounds. Another oxidase occurring in the liver and spleen, and called
xanthine-oxidase by BURIAN, has the property, as shown by SPITZER,

1 Biochem. Centralbl., 1, pp. 417 and 457.

2 In regard to the work of the various authors cited, see footnote 1, p. 11.
3Biedermann, Pfliiger's Archiv, 72; v. Fiirth and Schneider, Hofmeister's Bei-

trage, 1.

OXIDATION ENZYMES. 13

WIENER, SCHITTENHELM, and Bum AN,1 of transforming xan thine and
hypoxanthine into uric acid by oxidation.

Like other enzymes in general the so-called oxidation enzymes show,
from the above a more or less pronounced specificity, as a certain oxida-
tion enzyme acts only upon certain substances and does not oxidize
others. This behavior, which is difficult of reconciliation, that these
enzymes act as oxygen -carriers, indicate that in the oxidation the active
substances do not act upon the oxygen, but rather upon the substance
to be oxidized. We cannot at present give any statement as to the
extent of action of the oxidation enzymes in the oxidations of the ani-
mal body, and it is still a question whether we were actually dealing
with enzymes in all cases where oxidation enzymes have been claimed
to have been found.

In investigations with hydroperoxides and vegetable peroxidases BACH
and CnoDAT2 found that peroxides and peroxidases always took part
in the reaction in constant proportions, and that the peroxidases were
quickly used up, which certainly does not indicate that these bodies
have an enzymotic nature. Aso 3 has also shown that in certain cases
where an apparent oxidase action was present, we were very probably
dealing only with nitrites which were present; and finally, attention
must be called to the fact that manganese or iron, sometimes in consider-
able amounts, has been found in many oxidases. As manganous and
ferrous salts are active as catalytes in certain other oxidations, so also
in certain cases important rdles as oxygen -carriers have been ascribed
to these metals, for instance in laccase, which contains manganese (BER-
TRAND), and the oxidases containing iron (SPITZER'S nucleoprotein) .
MANCHOT 4 by his work on the autooxidation of ferrous sulphate has
called attention to the apparently great importance of iron for physio-
logical oxidations, and recently several investigators as TRILLAT, DONY-
HENAULT and J. WOLFF, have shown that several oxidase actions can be
brought about by colloidal inorganic catalytes or by mixtures of these
with organic colloids. Finally EULER and BOLIN 5 have shown that the
lucerne-laccase is not an enzyme at all, but that its action is due to the
presence of salts of organic acids. The question as to the nature of the
so-called oxidation enzymes surely requires a thorough investigation.

The assumption of special-oxidation enzymes does not at least give

1 Spitzer, Pfluger's Archiv, 56; Wiener, Arch. f. exp. Path. u. Pharm., 42; Schit-
tenhelm, Zeitschr. f . physiol. Chem., 42 and 43 ; Burian, ibid., 43.

2 Ber. d. d. chem. Gesellsch., 37.

3 Beihefte zum botan. Centralbl., 18.

4 Zeitschr. f. anorg. Chem., 27.

5 Trillat, Compt. rend., 137 and 138; Dony-Henault, Bull. Acad. Roy. Belg. 1908;
J. Wolff, Compt. rend., 146; H. Euler and J. Bolin, Zeitschr. f. physiol. Chem., 57.

14 INTRODUCTION.

us a sufficient explanation of the oxidative processes in the animal body,
and the various divergent views as to the nature of these show us
strikingly how little is positively known about these processes. There
is no doubt that the animal body possesses in the so-called oxidation
enzymes important means of bringing about oxidative decomposition
cf various substances, and the occurrence of numerous intermediary
metabolic products in the animal body teaches us that the oxidation of
the constituents of the body is not instantaneous and sudden, but takes
place step by step, and hand in hand with cleavages. For a long time
the oxidations in the animal body have been considered as a combus-
tion, and such a conception is easily reconcilable with the above-men-
tioned views. In combustion in the ordinary sense, as, for example,
the burning of wood or oil, we must not forget that the substances
themselves do not combine with oxgyen. It is only after the action of
heat has decomposed these bodies to a certain degree that the oxidation
of the products of such decomposition takes place and is accompanied
by the phenomenon of light. The above-mentioned specificity of the
oxidation enzymes and also the recent observation that indeed rather
simple cleavage products are burnt with difficulty in the organism and
also that of two optical antipodes of an amino-acid, for example of leucine,
the one (Z.-leucine) is readily burnt while the other (d-leucine) is burnt
with difficulty and only incompletely, seem to make it very probable that'
in many cases a very intense cleavage is necessary before oxidation occurs.

Most investigators are agreed that these decompositions are similar
to certain oxidations studied by DRECHSEL 1 outside the animal body,
where oxidations and reductions alternate in quick succession. The views
are divided in regard to the manner and origin of this cooperative action.2

As the oxidations are explained by the action of special enzymes,
so also special reducing enzymes, so-called reduclases or hydrogenases,
have also been accepted. Certain investigators claim that the so-called
philothions, which develop hydrogen sulphide in the presence of sulphur
and water, belong to this group, while others, on the contrary, do not
accept this view, but consider the enzymotic nature of the philothions
as doubtful.3 The investigations of NASSE and ROSING 4 on the oxi-

1 Journ. f. prakt. Chem. (N. F.), 22, 29, 38, and Festschrift fiir C. Ludwig, 1887.

2 See M. Nencki, Arch, des sciences biol. de St. Pe"tersbourg, 1, 483; Abelous and
Aloy, Compt. rend., 136, 137; Kastle and Elvove, Amer. Chem. Journ., 31; Underhill
and Closson, Amer. Journ. of Physiol., 13.

3 De Rey-Pailhade, Recherches expe"r. sur le Philothion, etc., Paris (G. Masson),
1891, and Nouvelles recherches sur le Philothion, Paris (G. Masson), 1892; Bull. soc.
chim. (4) I; Pozzi-Escot, Bull. soc. chim. (3), 27, and Chem. Centralbl., 1904, 1, p.
1645; Chodat and Bach, Ber. d. d. chem. Gesellsch., 36; Abelous and Ribaut, Compt.
rend., 137, and Bull. soc. chim., Paris (3), 31.

4 See footnote 2, p. G.

REDUCTIONS. 15

elation of protein in the presence of sulphur, speak against the enzymotic
nature of this formation of hydrogen sulphide and the recent investi-
gations of HEFFTER 1 have shown that certain reductions occurring in
the tissues cannot be produced by enzymes. He has shown that those
reductions which are not influenced by HCN, such as the reduction of
pigments (methylene blue) can be brought about by the unstable H of
sulphhydric compounds. For example, cysteine (see Chapter III), which
quickly reacts with sulphur with the formation of H2S, acts in this manner,
and he has also shown the presence of similarly acting substances in
various organs and organ extracts. Here we have a group of reductions
which are not enzymotic. The reduction of nitrates seems at least in
part, according to the investigations of VoGELSOHN,2 not to be brought
about by enzymotic substances.

There is no doubt that reductions occur to a great extent in the
animal body and often hand in hand with oxidations; nevertheless the
question as to how far special reduction enzymes take part in these reduc-
tions is still an open one. According to ABELOUS and ALOY 3 we have
indeed enzymes that have an oxidizing as well as a reducing action,
for they obtain the oxygen necessary for the oxidation of one body by
removing it from another substance through reduction. Still this question
requires further study.

The essential source of heat and mechanical work developed in the
organism is to be found in the oxidations. Chemical energy is trans-
formed into the above-mentioned forms of energy in cleavage processes,
where complicated chemical compounds are reduced to simpler ones,
and therefore the atoms change from an unstable to a stabler equi-
librium, and stronger chemical affinities are satisfied. The animal body
may also have a source of energy in the cleavage processes which are not
dependent on the presence of free oxygen. The processes taking place
in the active muscle are examples of this kind. A removed muscle,
which gives off no oxygen when in a vacuum, may, as HERMANN 4 has
shown, work, at least for a time, in an atmosphere devoid of oxygen,
and give off carbon dioxide at the same time.

The cleavage processes are not only of especially great importance
for the digestion of the foodstuffs and for their availability for the
animal body, but also are important for the metabolic processes. If

1 Mediz. naturw. Arch., 1, p. 81-104, Marburg; cited in Chem. Centralbl., 1907, p.
822.

2 Ueber die Einwirkung von Organextrakten auf Nitrate und Nitrite, Inaug.-
Dissert.. Bern, 1907.

3 Compt. rend., 136, 137 and 138. See also Abelous and Gerard, ibid., 129, on fhe
reductaaes; Pozzi-Escot, Bull. soc. chim. (3), 27.

4 Untersuch. iiber den Stoffwechsel der Menschen, Berlin, 1867.

16 INTRODUCTION.

a cleavage process is connected with a decomposition of water and a
taking up of its constituents it is called a hydrolytic cleavage. As an
example of such a cleavage we can mention the decomposition of starch
into sugar and the splitting of neutral fats into the corresponding fatty
acids and glycerin:

C3H5(OH) 3 + 3 (Ci8H36O2) .

Tristearin Glycerin Stearic acid

As a rule the hydrolytic cleavage processes as they occur in the ani-
mal body may be performed outside of it by means of higher tem-
peratures with or without the simultaneous action of acids or alkalies.
Considering the two above-mentioned examples, we know that starch
is converted into sugar when it is boiled with dilute acids, and also
that the fats are split into fatty acids and glycerin on heating them
with caustic alkalies or by the action of superheated steam. The heat
or the chemical reagents which are used for the performance of these
reactions would cause immediate death if applied to the living body.
Consequently the animal organism must have other means at its dis-
posal which act similarly, but in such a manner that they may work
without endangering the life or normal constitution of the tissues. The
animal body has in the enzymes such means and especially the second
chief group which have a hydrolytic action.

Among the hydrolytic enzymes we must mention, in, the first place
the proteolytic, or those which dissolve protein, whose best studied repre-
sentatives, pepsin and trypsin, occur in the animal kingdom; the lipolytic
or fat-splitting; and the amylolytic enzymes, or diastases which act
upon the complex carbohydrates. In this group we include the
amylases, which act upon starch, and the invertases, which split the
disaccharides into simpler forms of sugar. In close relation to those
enzymes we may mention the glucoside-splitting enzymes, which occur
especially in the higher plants. Among the hydrolytic enzymes of the
animal kingdom we must also include arginase, which splits arginine into
urea and ornithine ; the two deamidizing enzymes adenase and guanase,
which convert the two bodies adenine and guanine, with the splitting
off of ammonia, into hypoxan thine and xan thine respectively; and the
hippuric-acid-splitting histozyme and the urea-splitting urease. The
protein-coagulating enzymes, chymosin or casein-coagulating, and thrombin
or blood-coagulating enzyme, belong to a special though not clearly
defined group. These enzymes are also included by some investigators
among the proteolytic enzymes.

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

AUTOLYSIS. 17

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 act within the cells, hence are intracellular and therefore are called
intracellular enzymes or endoenzymes. Numerous enzymes besides the
yeast zymase belong to this group, and seemingly also oxidases and
enzymes having hydrolytic action. The best studied of this group
are the proteolytic enzymes, which were first observed by SALKOWSKI
and his pupils, and which bring about the self-digestion or autodiges-
tion of organs in the absence of micro-organisms. This autodigestion
has been the subject of numerous investigations, principally by the HOF-
MEISTER school and especially by JACOBY.1 The latter has given the
name autolysis to the process, and he has shown that the enzymes taking
part in this action do not come from the digestive tract and are not
pepsin or trypsin taken up by the cells. In autolysis we are not only
dealing with a proteolysis, but several other processes occur, such as the
splitting of fats and carbohydrates, oxidations and reductions, and
perhaps also syntheses.

We therefore generally designate as autolysis all the enzyme actions
which take place in removed organs or fluids without the aid of micro-
organisms, but it must not be forgotten that autolytic processes may
also occur intra vitam under certain conditions. The combined action
of various enzymes in autolysis also explains to us why, as especially
shown by LEVENE and by JONES, 2 the products obtained by the hydro-
lytic cleavage of an organ by means of an acid are somewhat different
from those products produced on autolysis.

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 processes, and conclusions must
be drawn very carefully from these results.

The post-mortem autolyses, as far as studied, are chiefly proteolyses,
but we must not forget that the enzymes taking part are in many cases
most active in acid reaction, while they have only a weak action or

1 A complete summary of the literature of intracellular enzymes and autolysis may
be found in Jacoby, Ueber die Bedeutung der intrazellulareri Fermente, etc., Ergeb-
nisse der Physiologic, Jahrg. 1, Abt. 1, 1902. See also Preti, Zeitschr. f. physiol. Chem.,
52; Arinkin, ibid., 53; Hedin, Hammarsten's Festschr., 1906.

2 Levene, Amer. Journ. of Physiol., 11 and 12, and Zeitschr. f. physiol. Chem., 41;
W. Jones, ibid., 42.

18 INTRODUCTION.

are inactive in neutral or alkaline reaction. The observations of LANE-
CLAYPTON and SCHRYVER/ that the autolysis of the liver and kidney
begins only after a latent period of from two to four hours subsequent
to the removal of the organ, are also of interest. The investigations
of WIENER 2 show that the post-mortem formation of acid is the important
factor in this, and according to HEDIN 3 it is due to the removal of the
retarding action. HEDIN has shown by experiments with various organs
that a preliminary treatment with acetic acid markedly helps the auto-
lysis in alkaline reaction and he has also shown, especially for the enzyme
3f the spleen (d-lienase) that an anti-body of the enzyme is hereby made
inactive.

The action of an enzyme can be retarded or arrested by another body
which has been called an antienzyme, but the action of these has not been
closely studied. They are perhaps of the greatest importance in life
in retarding or regulating the action of the mtra-cellular enzymes. Exper-
ience 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 accelerates it.
Autolysis may also be influenced by toxins (see Chapter II) (HESS
and SAXL 4) and indeed so that first a retardation of the cleavage of the
proteins takes place and then a strong acceleration.

It is difficult to judge of the importance of the autolytically active
proteolytic enzymes for the physiological life of the cells, but there does
not seem to be any doubt as to the importance of these enzymes in path-
ological 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, are examples of an
intra vitam autolysis which is considered by some as an abnormal rise
in the physiological autolysis. Another example is the solution of pneu-
monic infiltrations by the enzymes of the migrated and inclosed leu-
cocytes as studied by FR. MfJLLER,5 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 autol-
ysis, although not very marked, occurs in those organs or parts of organs
which have not been normally nourished because of a disturbance1 ir

1 Journ. of Physiol., 31.
2Centralbl. f. Physiol., 19, p. 319.

3 Hammarsten's Festschr., 1906.

4 Zeitschr. f. exp. Path. u. Therap., 5, and Wien. klin. Wochenschr., 21.

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

METABOLISM IN THE CELLS. 19

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 formation of bacter-
icidal bodies, as observed by CoNRADi,1 and of antitoxins (BLUM2)
by means of autolysis, we can consider this autolysis as a remedy and
perhaps also as a protective agent for the animal body.

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 idle. 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 (HOFMEISTER
and others 3) .

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 contraiy 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 researches into the chemical composition of the cells must there-
fore be of the greatest importance, but the difficulties which such investi-
gations entail are very striking. The chemical investigations of animal
cells must in most cases be connected with the study of those tissues
whose chief part they represent. Only in a few cases, such for example
as the investigation of pus or tissues very rich in cells, can the cells be
directly or by relatively simple manipulation, be isolated in a rather
pure form from the other parts of the tissues. Even in these cases the
chemical investigation does not give any positive results as to the cfln-
stituents of the living uninjured cells. If the physiological conditions
of life of the cells are removed, or injurious external influences such as the
action of high temperatures or of chemical agents are applied, then the
protoplasm dies. The protoplasm, which in the generative cell during
life forms a semi-solid mass which is contractile under certain conditions
and which readily changes, consists, with the exception of the water,
chiefly of protein substances, namely of colloids. On the death of the
cell these proteins coagulate, at least in part, and other chemical trans-
formations also occur in the cell. The alkaline reaction of the cells to

1 Hofmeister's Beitrage, 1.

2 Ibid., 5, p. 142.

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

20 INTRODUCTION.

litmus may change to an acid reaction due to the formation of paralactic
acid. Also on the death of the cell other new bodies may be produced,
or cell constituents, like glycogen, may be consumed and also constituents
may pass from the cell into the surrounding fluid and hence be lost for
the investigation.

There are still other difficulties that must not be underestimated.
The constituents occurring in the cells are not all of the same physio-'
logical importance. Some of these are essentially necessary for the
life of the cells, while others are only considered as stored-up reserve
material or as metabolic products. In this connection we have only
been able, thus far, to learn of certain substances which seem to occur
in every developing cell. Such bodies, called PRIMARY by KOSSEL/
are, besides water and certain mineral constituents, proteins, nucleo-
proteins or nucleoalbumins, phosphatides (lecithin), glycogen (?),
and cholesterin. Those bodies which do not occur in every developing
cell are called SECONDARY. Among these we have fat, glycogen (?),
pigments, etc. It must not be forgotten that it is still possible that
other primary cell constituents may exist, as yet unknown to us, and
we also do not know whether all the primary constituents of the cell
are necessary or essential for its life and functions.

Another important question is the division of the various cell con-
stituents between the two morphological components of the cell, namely,
the protoplasm and the nucleus. This is very difficult to decide, for
many of the constituents and even the division of the chief chemical
constituent, the proteins, between the protoplasm and nucleus, have been
but little studied. It is very probable that the nucleus (in abundance)
as well as the protoplasm contains the conjugated proteins, which have
been called nucleoproteins, and which will be discussed in detail in a
following chapter (III) . The nucleoproteins of the protoplasm are richer
in the protein component and poorer in the phosphorized component
as compared with the nucleoproteins of the nucleus, which also have a
more marked acid character.

The question as to the occurrence of a special external boundary
layer in the cells is of importance for the understanding of the metab-
olism of the cells, as well as for our knowledge as to the manner in which
the intake and output of bodies transpires in the cells. In this connec-
tion we must call attention to the fact that in certain cells an external
thick layer or a true membrane exists which seems to consist of protein
substance. Even cells in which no special external boundary layer
can be observed are still considered as having such a layer because of the
permeability conditions.

1 Verhandl. d. physiol. Gesellsch. zu Berlin, 1890-91, Nos. 5 and 6.

BOUNDARY LAYER OF CELLS. 21

NERNST 1 has shown by a special experiment that the permeability
of a membrane for a certain substance is essentially dependent upon the
solvent power of the membrane for the said substance. This point,
which is of the greatest importance in the study of osmotic phenomena
in living cells, has been specially investigated by OvERTON.2 The
behavior of the living cells toward dyestuffs, also the ready introduc-
tion into animal and plant protoplasm of such bodies as are insoluble
or only slightly soluble in water but readily soluble in fats or fat-like
bodies, has led OVERTON to conclude that the protoplasm-boundary
layer behaves like a substance layer whose solvent power is closely
related to the fatty oils. According to this investigator, the protoplasm-
boundary layer is probably impregnated with lipoids, i.e., bodies which
in regard to solubility and solvent power are more or less similar to the
fats. The lipoids are not a claSTof bodies that can be defined chemically.
The nature of certain of them is still not well known ; but the best-known
bodies, such as lecithin (the phosphatides especially) and cholesterin,
must be considered as of the greatest importance.

The occurrence of an accumulation of lipoids as a special external
boundary layer in the cells is not a sufficiently proven fact, and at least
does not apply to all animal cells, and is not necessarily important in the
explanation of the action of lipoids. Objections have also been made
by many investigators to OVERTON'S theory, which has received rather
general acceptation.3 It does not apply to all cases; for example,
according to COHNHEIM, it does not explain the absorption in the intestinal
canal, and according to MOORE and ROAF it does not explain certain prop-
erties of the cells, namely, the different composition of the electrolytes
within and without the cells and the selective taking up of certain
soluble substances, such as foodstuffs, therepeutic agents, toxines, and
antitoxines, on the part of the cells. The researches of these last investi-
gators are based essentially upon the behavior of mineral bodies, and they
show that the above theory presents certain difficulties in the understand-
ing of the very important exchange of mineral bodies between the cells
and the external fluid.

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

1 Zeitschr. f . physikal. Chem., 6.

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

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

22 INTRODUCTION.

about two-thirds water. If we also recall that water is of the very
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-
ucts, are connected with the presence of water, and that besides this the
water by its evaporation is an important regulator of temperature, if
is evident that water must be a necessity of life.

The mineral substances found habitually in the cells of higher plants
and of animate are potassium, sodium, calcium, magnesium, iron, phos-
phoric acid, sulphuric acid, 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. 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. Corresponding to this, the cells contain chiefly potas-
sium as phosphate, while they are less rich in sodium and chlorine com-
pounds. Still we have some exceptions to this rule, and it must be
remarked that BEEBE 2 has found considerably more sodium than
potassium in malignant tumors.

The division of the mineral bodies in the various parts of a cell cr a
tissue is very difficult to determine, but in certain cases by means of
micro-chemical reactions this can be determined in a way. For example,
MACALLUM 3 has been able to show such a division in the case of potas-
sium. According to him the potassium is absent in the cell nuclei and
in the head of the spermatozoa as well as in the nerve cells and the axis-
cylinders, while it occurs on the contrary in the medullary sheath and
especially in the region of the nodes of RANVIER.

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. Ohem., 42; Kunkel, ibid., 44. In regard to the barium see Schulze and
Thierfelder, Sitzimgsber. d. Gesellsch. naturforsch. Freunde, 1905, No. 1, and in
regard to lithium see Hermann, Pfluger's Arch., 109; and in regard to manganese
see Bradley, Journ. of Biol. Chem., 3.

2 Amer. Journ. of Physiol., 11 and 12.

3 Journ. of Physiol., 32.

MINERAL COXSTITTEXTS OF THE CELLS. 23

In the ordinary method of determining the mineral bodies in cells,
fluids and tissues, namely by incineration, no positive knowledge can
be acquired as to the division of the mineral bodies or their form of
combination. On incineration we obtain not only a mixture of the min-
eral bodies of the nucleus and protoplasm, but, as is true for all animal
fluids and tissues, the original relationship is markedly changed. The
combinations between the colloidal and mineral substances are destroyed,
carbon dioxide discharged, and sulphuric acid and phosphoric acid
may be produced from the organic bodies. The ordinary chemical
analysis is not sufficient for the study of the mineral constituents of the
fluids or tissues, their forms of combination and action; hence we must
resort to physical-chemical methods.

As a result of the investigations carried on by these methods, the
conclusion has been reached, irrespective of the importance of the min-
eral bodies for the osmotic tension in the cells and tissues, that the
part taken by the mineral bodies in cell life is essentially dependent
upon the action of the ions. The investigations of MAILLARD on the
toxic action of copper salts, and of PAUL and KROXIG 1 on that of mer-
cury salts, acids, and alkalies, offer examples. From these investiga-
tions it follows that the toxicity is dependent upon the dissociation,
and that it is not dependent upon the total amount of, for example,
copper or mercury salts present in the solution, but rather upon the
number of copper or mercury ions.

Important and conclusive investigations, which are considered as
beautiful and instructive examples of the importance of ions for cell
life, have been carried out by LOEB and his collaborators. Some of these
examples will be discussed in the next chapter (II).

The chief mass of the cells consists of colloids, and as the normal func-
tion of the cells is connected with a certain physical condition of the
protoplasm, it is natural to consider the action of ions in relationship
to the changes in the condition of the colloids. These colloids can be
precipitated by electrolytes, and it seems as if we are here dealing with
ion action. A physiologically balanced salt mixture suitable for the
normal functions may also be produced by the antagonism of the ion
action in a complex solution containing several salts (LOEB). Changes
in one or the other direction must correspondingly also bring about
changes in the state of the colloid by the action of the ions. The action
of ions in these cases, as well as the nature of colloids and the reasons
for the change in their conditions, is a very difficult question, and this
will be discussed in the following chapter (II).

1 MailUml, Journ. de Physiol. et Path., 1; Paul and Kronig, Zeitschr. f. physikal.
Chern., 12, and Zeitschr. f. Hygiene, 25.

24 INTRODUCTION.

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 complex 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.

The products produced by micro-organisms may be of very dif-
ferent kinds. Among the substances produced in the decomposition
of animal fluids and tissues by putrefactive organisms we find those
having a basic nature. To this class belong the cadaver alkaloids called
ptomaines, first found by SELMI in human cadavers and then specially
studied by BRIEGER and GAUTiER.1 Certain of these are poisonous,
designated as toxines, while others are non-poisonous. They all belong
to the aliphatic compounds and generally do not contain oxygen.
As an example of these basic substances 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 cystin-
uria.2 The putrefaction bases marcitine, CgHigNs, putrine, CnHoe^Oa,
and viridinine, CgHi 2^0.3 isolated by ACKERMANN, also belong to this
group. Of special interest is the bacterial poison isolated by FAUST3,
called sepsine, CsHu^Ojj, which is the substance producing the char-
acteristic 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.

Those substances of basic nature which are incessantly and rec;ularlv

1 Selmi, Sulle ptomaine od alcaloidi cadaveric! 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
appliquee a la physiologic, 2, 1873, and Compt. rend., 94.

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

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

LEUCOMAINES. 25

produced as products of the decomposition of the protein substances
in the living organism, and which therefore are to be considered as
products of the physiological metabolism, have been called leucomaines
by G'AUTIER l in contradistinction to the ptomaines and toxines pro-
duced by micro-organisms. These bodies, to which belong several well-
known animal extractives, are considered by many as being of certain
importance in Causing disease. It has been contended that when these
bodies accumulate on account of an incomplete excretion or oxidation
in the system, an autointoxication may be produced (BOUCHARD and
others) .2

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 antitoxines 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 in the next chapter (II).

1 Bull. soc. chim., 43, and A. Gautier, Sur le^ alcaloi'des derives de la destruction
bacterienne ou physiologique des tissues animaux, Paris, 1886.

2 Bouchard, Legons sur les auto-intoxications dans les maladies, Paris, 1887. See
also the various text-books of clinical medicine.

CHAPTER II.

PHYSICAL CHEMISTRY IN BIOLOGY.

I. OSMOTIC PRESSURE.

WHEN 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 dissolved
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 For example, such a membrane can be produced by care-
fully dropping a concentrated solution of copper sulphate into a dilute
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

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

26

OSMOTIC PRESSURE. 27

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 prevents 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, or the osmotic pressure of the solution. For dilute cane-
sugar solutions the osmotic pressure is approximately proportional to
the concentration, which is shown by the following figures.

Cone, (c) Osmotic Pressure (p)

1 per cent 53 . 5 cm. Hg. 53 . 5

2 " 101.6 " 50.8
4 " 208.2 " . 52.1
6 " 307.5 " 51.3

The osmotic pressure rises slowly with the temperature.

Experiments with other semipermeable membranes have also been
carried out by DE VRIES, and these will be discussed on page 30. 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.

VAX'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 pressure 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
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. GAY-LUSSAC'S law that the pressure of
a ,uus changes in proportion to the absolute temperature cannot be
absolutely proven for the osmotic pressure because of the inaccuracy
of the methods.

1 Osmotische Untersuchungen, Leipzig, 1877.

2 Zeitschr. f. physik. Chem., 1, 481, 1887.

28 PHYSICAL CHEMISTRY IN BIOLOGY.

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.

As the gas pressure, according to the kinetic theory of gases, is caused
by the impact of the gas molecule upon the walls of the vessel, so also
the osmotic pressure is considered as a pressure exerted by the dissolved
substance toward the outside upon the confining walls and the free
surface. Ordinarily this pressure never becomes evident on the out-
side, because it is always much surpassed by the reverse action of the
surface tension of the fluid. When the surface tension is removed, as is
the case when the solution is separated from the pure solvent by a semi-
permeable membrane, then the osmotic pressure becomes evident, as either
the membrane is moved by the pressure, or in case the membrane
is not movable and is not ruptured, pure solvent enters into the solution.
The pressure of the surface tension, which is transferred through the
membrane, is less than the sum of the surface tension of the solution
and the reverse-acting osmotic pressure; and the solvent is, therefore,
pressed against the membrane, with a force equal to the osmotic pressure
of the solution.

For physiological purposes it seems best to make use of the above
explanation, according to 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 directly 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 bodies which have been found
impermeable to the membranes. 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)
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.

OSMOTIC PRESSURE. 29

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 a 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+[tt — l]a). The expression (l + [n — 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,
if we find that A for a gram molecular solution of NaCl is 3.40° then we have
according to the above 1 : (l + [/i — 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-

1 Fuchs, Anleitung zu Molekulargewichtsbestimmungen. Leipzig, 1895; Ostwald-
Luther, Hand- und Hilfsbuch zur Ausfiihruiig physik.-chemischer Messung, 2. Aufl.,
1902.

30 PHYSICAL CHEMISTRY IN BIOLOGY.

duction of electricity, and the molecular conductivity ( = — -, -, — — r~r- — )

' v 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 POO the limit value which the molecular conductivity approaches in
infinite dilution and with ftv the molecular conductivity at some definite dilution

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

^00

The positively charged ions are called cations, and the negatively
charged 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 modi-
fied 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 bodies, changed their
appearance so - that the protoplasm retracted,1 DE VRIES studied
this phenomenon further.2 This phenomenon is called plasmolysis
by DE VRIES. The most important substances for bringing about
plasmolysis are the salts of the alkalies and alkaline earths, varieties of
sugars, monatomic 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
substance attracts water from the inner parts of the cell. The cell con-
tents 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 con-
centration, there must be a limit solution for every substance above
which all higher concentrations plasmolyze. The limit solution is called
isotonic with the cells; weaker solutions are called hypotonic, and stronger
hypertonic. DE VRIES, 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

1 Pflanzenphysiol. Untersuch., 1855.

2 Eine Analyse der Turgorkraft, Jahresber. f. Wissensch. Botanik, 14, 427, 1884.

OSMOTIC PRESSURE. 31

s found that the limit solution of analogously constructed salts had
the same molecular concentration. Thus the alkali salts of the type
NaCl (haloid salts, nitrate, acetate) plasmolyzed at one molecular con-
centration and the salts of the type Na2SO4 (sulphate, oxalate, diphos-
phate, 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.1

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-
BURGER 3 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 concentra-
tions of the same salt, all solutions whose concentration lies below a
certain limit cause the exudation of hemoglobin. On comparing the
molecular 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 osmotic action. From this it probably fol-
lows that the protective action of the salts upon the blood corpuscles
depends upon the same reasons 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 haemo-
globin. Those bodies, on the contrary, which do not cause plasmolysis,
act in aqueous solution in the same manner upon the blood corpuscles
as pure water. This has been especially shown by the investigations

Of GRYNS.4

Different investigators have attempted to perform plasmolytic

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

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

3 Arch. f. (Anat. u.) Physiol., 1887, p. 31; Zeitschr. f. Biol., 26, 414, 1889.

4 Pfliiger's Arch., 63, 86, 1896.

32 PHYSICAL CHEMISTRY IN BIOLOGY.

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,1 KOEPPE 2 and others.
It was found that the blood corpuscles swell in a weak salt solution and
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 freez-
ing-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: 3

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

The result -r-, 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 ancl pentatomic alcohols
do not pass into the blood corpuscles, or only to a slight degree. Fry-
thrite (tetratomic alcohol), passes slowly, and glycerin (triatomic) also
slowly, but faster than ery thrite. Ethylene glycol (diatomic alcohol)
passes rather rapidly, and the monatomic alcohols divide themselves
immediately equally in the serum and blood corpuscles. Ether, esters,
aldehyde, and acetone divide themselves so that the blood corpuscles con-

1 Skand. Arch. f. Physiol., 5, 207, 238, 377, 1895.

2 Arch. f. (Anat. u.) Physiol., 1895, 154.
3Pfliiger's.Arch., 68. 229, 1897; 70, 525, 1898.

OSMOTIC PRESSURE. 33

tain more than an equal volume of serum. These bodies are equally
absorbed by the blood corpuscles.

OVERTON had previously arrived at the same results, using plant
cells, but urea is probably more quickly taken up by the blood corpuscles
than by plant cells, and ammonium salts also seem to pass more easily
into the blood corpuscles than into the plant cells.

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

It must also be stated that according to HEDIN, only those bodies
which do not pass, or pass only sl6wly 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 CO2, 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 Na2C03 formed at the same time
gave the alkaline reaction to the solution. KOEPPE 3 as well as HAM-
BURGER and v. LIER 4 claim on the contrary that an exchange of HCO3-
ions and Cl-ions takes place between the blood corpuscles and the solu-
tion, 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. GURBER'S explanation is simpler and stands in accord with the
facts as found. The theory as to the permeability for anions does not
explain the fact that the anions in the blood corpuscles are so different
from those in the plasma.

Muscle Experiments. By investigations on the changes in the
weight (instead of the volume changes in the above-mentioned exper-
iments with plant cells and blood corpuscles) which frog muscles undergo
in solutions, various experimenters, NASSE,S LOEB,G and OVERTON/ have
tried to prove the ability of muscle to take up various substances. OVER-
TON 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

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

2 Sitzungsber. d. med. phys. Gesellsch. zu Wurzburg, 1805.

3 Pfliiger's Arch., 67, 189, 1897.

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

5 Pfliiger's Arch., 2, 114, 1869.

6 Ibid., 69, 1 ; 71, 457, 1898.

7 Ibid., 92, 115, 1902; 105, 176, 1904.

34 PHYSICAL CHEMISTRY IN BIOLOGY.

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 muscles l
in regard to permeability

Theories of Admissibility. On what docs the permeability or non-
permeability of cells for certain bodies depend? The discoverer of pre-~
cipitation membranes, M. TRAUBE, considered the membrane as a sort of
molecular sieve. The relation of the size of the particles passing and
the width of the pores of the membrane was important.2

OVERTON states that " the reason of the very variable permeability
of living protoplasm, or better the plasma membrane for various com-
pounds, lies in an impregnation of the membrane by bodies of similar
solvent ability like the high molecular monatomic alcohols, ether, olive
oil, etc. All compounds which are readily soluble in this impregnable
substance pass quickly into the cell, while those compounds which are
considerably less soluble therein than in water pass more slowly into the
cell, and indeed the slower the more the distribution coefficient changes in
favor of the water (page 48). Those compounds which are practically
insoluble in the impregnable substance do not pass into the cell." 3

Certain substances which are of the very greatest importance for
life processes and which probably are burnt to a great extent within the
cells, have, according to the above experiments, either no ability to
enter the cells, or only a limited ability. These bodies are the sugars
and the amino acids. Also the presence of salts within the cells is not
easily understood with the above theory. For this reason OVERTON
calls the above treated permeability (for which his theory attempts
an explanation) passive, and differentiates this from the active, in which
the protoplasm of the cells are active in one unknown way or another.4

MOORE and ROAF believe that the salts exist in the blood corpuscles
in the form of " adsorpates." The cells therefore do not take up added
salts, because the absorbing proteins have reached their saturation limit
(p. 48)6

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, or whether the substance which does not pass in has the ability
of attracting water or not. Therefore that part of the osmotic pres-

1 Verhandl. d. phys. med. Gesellsch. zu Wiirzburg, (N. F.), 38, 277, 1904.

2 Arch, f . Anat. Physiol. u. Med., 1867, 87.

3 Vierteljahrsschr. d. naturf. Gesellsch. zu Zurich, 44, 88, 1899, and Nagel's Hand-
buch d. Physiol. des Menschen. II, 817.

4 Nagel's Handbuch d. Physiol. des Menschen. II, 816.

5 Biochem. Journ., 3, 55, 1908.

OSMOTIC PRESSURE. 35

sure of body fluids which is caused by bodies not passing in is called
the effective osmotic pressure. These bodies include HEIDENHAIN'S lym-
phagogues of the second order (Chapter VII). Their action, according
to HEIDENHAIN, depends upon their ability to abstract water from the
tissues. To this group also belongs the salts of the alkalies and alkaline
earths and the sugars. 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 without 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
0.560,1 which corresponds to a 0.90 per cent NaCl solution and to an
osmotic pressure of about 6^ atmospheres. In lower animals A may
be slightly lower, for example, in the frog J = 0.46°. In inver-
tebrate sea animals the body fluid is equal to the osmotic pressure of the
surrounding sea water (^ = 2.3°) and varies with the quantity of salt
in the water (BOTTAZZI 2) . In lower fishes (Selachii) the osmotic pressure
of the blood is equal to the surrounding medium, and in higher fishes
(Teleostomi) lower (J=1.0°) (BOTTAZZI).

In sea fishes as well as fresh-water fishes, for example, the eel, a lower
osmotic pressure (^=0.41°) is found when kept in fresh water than
when kept in sea water (J = 0.55°)3. 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.4

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
having a low molecular weight.5 Milk and bile have the same
osmotic pressure as the blood,6 while saliva has a lower pressure.7

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

2 Archives ital. de Biol., 28, 61, 1897. •
3Dekhuisen, Arch, neerland, 10, 121, 1905; Quinton, Compt. rend. soc. biol., 57,

470, 513, 1904.

4 Physik. Chem. d. Zelle u. Gewebe, 2. AufL, 1906, 33.

5 Leathes, Journ. of PhysioL, 19, 1, 1895.

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

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

36 PHYSICAL CHEMISTRY IN BIOLOGY.

The urine of man and mammalia generally has a much higher
osmotic pressure than the corresponding blood.1 For human urine
A varies between 1.3 and 2.3°. After abundant drinking as well as
under pathological conditions (diabetes insipidus) the osmotic pres-
sure 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.2

H. 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.3 GRAHAM included soluble silicic acid among
the colloids and also analogous forms of stannic acid, titanic acid,
molybdic acid and tungstic acid, the aluminium hydroxide and analo-
gous metallic oxides, when they exist in the soluble form, and also starch,
dextrins, the gums, caramel, tannin, albumin and gelatin.

Some colloids are characterized by the fact that under certain
conditions they solidify in 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 ac4d
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 As2S3 and Sb2S3 can be obtained by passing H2S into dilute solutions
of the respective metallic oxide,4 and colloidal CuS can be prepared by washing
the precipitated compound with water, by which treatment the CuS finally becomes
soluble in water.5

The metals can be obtained as hydrosols, and indeed in two ways :

1 . By treating a salt with various reducing agents (for example formaldehyde,
hydrosulphurous acid, hydrazine, hydroxylamine) the various metals are obtained
in colloidal solution.8 As the solutions thus obtained are often very unstable,

1 Koranyi, Zeitschr. f. klin. Med., 33, 1, 1897; 34, 1, 1898.

2 Physikalische Chemie und Medizin. Leipzig, 1907.

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

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

5 Spring, Ber. d. d. chem. Gesellsch., 16, 1142, 1883.
« Miiller, Allg. Chemie d. Kolloide. Leipzig, 1907, 6.

COLLOIDS. 37

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 44.

2. BREDIG l has discovered a method which makes possible the obtainment
of pure metallic sols by the cathode spraying of metallic wires under water.
SVEDBEKG 2 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. Sols of all metals and
metalloids can be prepared practically in this way.

Among those bodies which 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 colloid con-
dition is due to physical properties, and this follows from the researches
of GRAHAM.

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 PERRINS and later accepted by HoBER,4
A. MULLER;S and Wo. OSTWALD,G although different authors use different
names for the two -classes. The classifications of HARDY 7 and ZSIG-
MONDY 8 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, and glycogen, belong to the
hydrophile colloids.

Contrary to the hydrophile colloids the colloids of the colloidal metal
type are called suspension colloids (suspenoids) 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 solution; besides this, the suspension col-

1 Anorganische Fermente. Leipzig, 1901, 24.

2 Ber. d. d. chem. Gesellsch., 38, 3616, 1905; 39, 1705, 1906.

3 Journ. de Chimie phy., 3, 84, 1905.

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

5 Allg. Chemie d. Kolloide, 1907, 187.

6 Zeitschr. f. Chem. u. Ind. d. Roll., 1, 331, 1907.

7 Proc. Roy. Soc., 66, 95, 1899.

8 Zur Erkenntnis d. Roll., 1905, 16.

38 PHYSICAL CHEMISTRY IN BIOLOGY.

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
behave m many respects like suspension colloids. Suspensions of mastic/
colophony,2 and cholesterin 3 belong to this class.

The hydrophile colloids stand closer to the crystalloids than to 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. These intermediary members are the colloidal
acids and metallic hydroxides, which correspond with the suspension col-
loids by being readily precipitated by electrolytes. In this case they
separate as gels which differ from the gels of the hydrophile colloids
by not being again soluble in water. 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 considered as contaminations which are difficultlv removed.

1 Zeitschr. f. physik. Chem., 57, 47, 1906. 3 Bioch. Zeitschr., 7, 152, 1908.

2 Ibid., 38, 385, 1901. 4 Bioch. Journ., 2, 34, 1906.

COLLOIDS. 39

For this reason it has been repeatedly stated that these salts are responsible
for the small differences in the osmotic pressure. By carefully washing
crystalline proteins from serum and egg-white, REID was able to prepare
bodies which gave finally no osmotic pressure in the osmometer.1 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.
STARLING,2 MOORE and PARKER,3 MOORE and RoAF4 and LILLIE, 5 using
protein preparations which had not been exposed to any strong treat-
ment before use (serum proteins, ovalbumin) , as well as REID 6 (with
hemoglobin) , 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 REID found a pressure of 3-4 mm. Hg. for a 1 per cent hemoglobin
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
aggregation 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
aggregation condition of the colloid, by the addition of salts it is brought closer
to its precipitation point and is probably united in large aggregations which
causes a lowering in the osmotic pressure.

Filterability. Large particles suspended in a liquid can be removed
from the fluid by filtering. The finer the suspended particles are the
thicker must the filter be. Extensive experiments on the filtering of
colloids have been carried out by BECHOLD.S He used paper filters
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 BECHOLD 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

1 Journ. of Physiol., 31, 438, 1904. 5 Amer. Journ. of Physiol., 20, 127, 1907.

2 Ibid., 19, 322, 1896. 6 Journ. of Physiol., 33, 12, 1905.

3 Amer. Journ. of Physiol., 7, 261, 1902. 7 Bioch. Journ., 3, 422, 1908.

4 Bioch. Journ., 2, 34, 1906. 8 Zeitsehr. f. physik. Chem., 60, 257, 1907.

40 PHYSICAL CHEMISTRY IN BIOLOGY.

organic colloids (gelatin, hemoglobin, seralbumin, proteoses, dextrin).
Still it must be remarked that according to ZSIGMONDY l 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
BECHOLD was able to show that the larger the particles of the proteoses,
the easier are they precipitable by ammonium sulphate.

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
solutions. Still there are colloid solutions (certain gold solutions, ZSIG-
MONDY), which do not give TYNDALL'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 ultramicroscope of SIEDENTOPF and ZSIGMONDY,
it has been made possible to directly see the colloidal particles.3 In
this apparatus the colloidal- particles are strongly illuminated by direct
light, so that no ray of the light directly falls in the eye of the observer.
The particles are hereby made visible on account of the formation of
diffraction disks which are visible by 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 called submicrons, while those that gradually
disappear on dilution are called amicrons.

If the quantity of metal and the number of particles are determined in the
unit volume of a metallic sol, then from this the size of the particles can be approx-
imately calculated under the assumption that the density of particles is the same
as the metal. The amicrons must not be considered in such measurements.
In this manner the following lineal dimensions have been found for the sub-
microns of certain metals, bearing in mind that 5 /*/* is about the lowest limit
of the ultramicroscope for these particles.

Gold 6-130 fifi*

Silver 50- 77 "

Platinum 44 "

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

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

3 Zsigmondy, Zur Erkenntniss der Koll. Jena, 1905, 83.

4 According to Zsigmondy (I.e. page 124) only those gold solutions are stable
whose average particles have at least a size of 60 /*//. When they are greater than
75 nn then the particles begin to settle.

COLLOIDS. 41

The investigations of ZSIGMONDY and others upon the growth of colloidal
metallic particles are also interesting. Thus the reduction of gold chloride by
formaldehyde, whereby colloid 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. l 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.2 In such processes the amicrons can
enlarge so that they can be observed by the ultramicroscope (submicrons).
According to the manner of preparation the colloids may have particles of different
sizes. (See page 39).

Submicrons have also been detected in solutions of organic colloids. The
•work of GATIN-GRUZEWSKA and Bnvrz,3 who used a specially pure glycogen,
must be especially mentioned. They found that the aqueous solution of glycogen
contained amicrons as \vell 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,

Internal Friction. The watery solutions of organic colloids are often character-
ized by their great thickness of viscosity. In a strictly scientific manner this
is expressed by the statement that the internal friction of the questionable solu-
tion is great. It seems generally accepted that the internal friction of suspen-
sion colloids is equal to that of the pure solvent, or differs from it only slightly.
The view is probably correct for various reasons, but as far as purely experi-
mental determinations are concerned it is based upon only a few experiments
carried out by FRIEDLANDER 4 with colloidal silver and suspensions of colophony.
The concentration of the " solutions " in these experiments was low, so that
positive conclusions could not be drawn from them.

Molecular Movement. R. BROWN 5 first found that small particles
suspended in water showed a quivering motion, and this phenomenon
has been called, from its discoverer, Brownian molecular motion. This
phenomenon has been observed since then by many investigators in
fluids having suspended solid particles as well as in substances dissolved
in colloid. It is believed by many that this movement is stopped by the
addition of electrolytes.

ZSIGMONDY has found in regard to the molecular movement of colloidal gold
that this cannot be caused by a change in concentration due to evaporation, and
also not influenced by the duration or intensity of the light applied. Small particles
of gold move much more actively than large ones, still sometimes large par-
ticles are met which have an active motion. The particles seem to somewhat
influence each other, as the activity of the motion generally diminishes on diluting
the gold solution. Old gold solutions (several months to 1^ years) may also show
active movement.

The molecular movement has been recently studied by SvEDBERG,6 who used
a new method. He has shown that the molecular motion of silver particles is
also perfectly normal in an isoelectric point, i.e., for perfectly uncharged particles
(page 46). Electrical charge can therefore not be responsible for the molec-

1 Zsigmondy, Zeitschr. f. physik. Chem., 56, 65, 1906.

2 Zsigmondy and Lottermoser, ibid., 56, 77, 1906.
3Pfluger's Arch., 105, 115, 1904.

4 Zeitschr. f. physik. Chem., 38, 430, 1901.

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

6 Studien zu Lehre von den kolloiden Losungen. Upsala, 1907, 128.

42 PHYSICAL CHEMISTRY IN BIOLOGY.

ular movement. SVEDBERG also experimentally proved the previously proposed
simple law that the distance traveled in a certain time is in inverse proportion to
the viscosity of the means of dispersion. Brownian molecular movement is con-
sidered by some as a manifestation of a general molecular movement of matter.

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-
pJwresis. Such movements have also been found in colloidal solutions.
According to BILTZ/ in dialyzed aqueous solution, the colloid metallic
hydroxides wander to the cathode, and the other colloids (metals,
metallic sulphides, acids) wander to the anode. The colloid 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 solution shows no cataphoresis. The addi-
tion of acid or alkali gives to the protein a positive or negative charge
respectively, hence an alkaline solution wanders to the anode and an acid
solution to the cathode (HARDY,2 PAULI 3) .

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 III). 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, protein 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 37), as the reversible colloids coincide in a measure with the
hydrophile colloids and the irreversible with the suspension colloids.

1 Ber. d. d. chem. Gesellsch., 37, 1095, 1904. 3 Hofmeister's Beitrage, 7, 531, 1906.

2 Journ. of Physiol., 24, 288, 1899. 4 Zur Erkenntniss der Koll., page 21.

COLLOIDS. 43

Electrolyte Precipitation of Suspension Colloids. It must be
remarked that for every precipitating electrolyte a certain minimal con-
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
millimolls (==-J^Q gram-molecule) per liter.

HARDY l 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 anion?.
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
and others.3 This valence rule becomes clear by the following experi-
ment of FREUNDLiCH.4 The figures give the lowest precipitation con-
centration expressed in millimolls per liter. The hydrosol was As2S3
(negative) and the valence of the cations is applicable chiefly for the
precipitating action.

K2S04 MgCl2 .................. 0.717

-~Y~ .............. b5'b MgS04 .................. 0.810

KCl ................ 49.5 CaCl2 ................... 0.649

KNO, .......... 50.0 SrCl2 ................... 0.635

NaCl ............ 51.0 BaCl2 ................... 0.691

Lid ................ 58.4 Ba(NO3)3 ............... 0.687

H2S04 ZnCl2 ................... 0.685

-%— .............. 30.1 UO2(N03), .............. 0.642

TTpi on o A1C13 ................... 0.0932

A1(N03)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

KXO, .............. 11.90 H2SO4 .................. 0.219

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

9.64 K2Cr2O7 ................. 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.

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

2 Journ. prakt. Chem. (2), 25, 431, 1882.

3 Proc. Roy. Soc., 66, 110, 1899.

4 Zeitschr. f. Chem. u. Ind. d. Roll., 1, 323, 1907.

44 PHYSICAL CHEMISTRY IN BIOLOGY.

Certain above-mentioned suspensions (mastic), as well as other particles
suspended in water, act the same as suspension colloids. ScnuLZE1 has found
that cloudiness due to clay particles on the addition of clarifying bodies (alum,
lime) give a voluminous deposition. SCHLOESSING 2 found that clay suspensions
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 role 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 already been stated, the colloids are considered as carriers
of electricity, and it has been proven that the oppositely charged col-
loids can act precipitatingly upon each other. This rule was first pro-
posed by LINDER and PiCTON,3 and subsequently has been substantiated
by many investigators. BILTZ 4 has made especially systematic investiga-
tions on this subject and finds that equally charged colloids do not pre-
cipitate each other. For the mutual complete precipitation of opposed
electrically charged colloids, a certain quantity relation 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 especially 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 5 have found with silver hydrosol that the presence
of protein prevented the flocking out by electrolytes. ZSIGMONDY&
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 Ice. 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 (As2S3, Sb2S3, CdS) are also
protected in the same manner against the influence of electrolytes
(A. MULLER and ARTMANN).?

1 Ann. Phys. (2), 129, 366, 1866. 5 Journ. prakt. Chem. (2), 56, 241, 1897.

2 Compt. rend., 70, 1345, 1870. 6 Zeitschr. analyt. Chem., 40, 697, 1901.

3 Journ. chem. Soc., 71, 572, 1897. 7 Oester. Chem. Ztg., 7, 149, 1904.

4 Ber. d. d. chem. Gesellsch., 37, 1095, 1904.

COLLOIDS. 45

Inorganic colloids can also serve as protective colloids. BILTZ 1 has
shown that zirconium hydroxide protects gold better than 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 QUINCKE 3 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 cataphoreis) were not
evident.

Electrolyte Precipitation of Hydrophile Colloids. The salts of
the alkalies precipitate the suspension colloids even in low concentra-
tions. The alkali salts behave differently with the organic col-
loids. This may in part be due to the fact that hydrophile colloids
have much less of a certain electric charge than the suspension colloids.
Egg-white by dialysis gradually loses its power of being influenced by the
electric current. For this reason the hydrophile colloids are often pre-
cipitated from their solution by alkali salt. 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 dif-
ferent alkali salts to act precipitatingly certain laws have been formu-
lated, but they cannot be arranged in a general rule.

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

1 Ber. d. d. Chem. Gesellsch., 35, 4431, 1902. 3 Ann. Phys. (3), 35, 580, 1888.

2 Zeitschr. f. physik. Chem., 60, 301, 1907. 4 Bioch. Zeitschr., 2, 251, 1907.

46 PHYSICAL CHEMISTRY IN BIOLOGY.

another time the same cation with different anions, PAULI has arranged the cations
and anions in the following order in increasing precipitation ability :

CNS < K Br < N03 < Cl< OCO.CH3 < HPO4 < SO4
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 HOBER* has recently
shown that the series KBr<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 (see page 42) .5 According to PAULI 6 dialyzed protein,
which had no electric charge, could not be precipitated by the addition of salts
of Zn, Cu, Hg, Fe, Pb, while the same protein in non-dialyzed form gave heavy
precipitates with these. PAULI believes that the native protein of the organism
on account of the OH ions originating from the tissue fluids has a negative charge.
In regard to the precipitation by salts of the heavy metals, the hydrophile col-
loids do not seem to differ essentially from the suspension colloids.7

Theories of Precipitation Phenomenon.

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.8 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
example, a trivalent ion is 3 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 44).

The mechanism of the precipitation of the isoelectric solution accepted
in HARDY'S theory is explained by BREDIG 9 as follows: At the boundary
between suspended particles and solvent a certain surface tension exists

1 Holfmeister's Beitrage, 3, 225, 1902. 8 Ibid., 7, 541, 1906.

2 Pfliiger's Arch., 78, 315, 1899. 7 Ibid., 6, 233, 1905.

3 Hofmeister's Beitrage, 4, 300, 1903. 8 Zeitschr. f. physik. Chem., 33, 385, 1900.

4 Ibid., 11, 35, 1908. 9 Anorganische Fermente, 1901, 15.

5 Ibid., 7, 531, 1906.

COLLOIDS. 47

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 great that it becomes evident 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.1

The findings of LINDER and PiCTON2 that when colloidal As2Ss is
precipitated with BaCl2 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 charges 3
(see also page 44) .

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
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

1 Zeitschr. f. physik. Chem., 45, 327, 1904; 51, 129, 1905.

2 Journ. Chem. Soc., 67, 63, 1895.

3 Zeitschr. f. physik. Chem., 51, 141, 1905.

48 PHYSICAL CHEMISTRY IN BIOLOGY.

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 two
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 c\ and c2, then it follows that — = k where & is a constant.1

£2

The first example where this law was shown to be correct was the
division of succinic acid between water and ether (BERTHELOT and JUNG-
FLEISCH 2). This law was also shown to be true for the division of
fi 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. For example, if we
are dealing with the absorption 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 absorbed fraction becomes continuously less so that
the absolute quantity taken up reaches a maximum which corresponds
to the greatest absorption ability of the solid body. This is expressed

Cin

by the formula -— =&, where Ci and c2 indicate the concentration of the

C2

solid body and in the solution; n and k are constants and indeed, n is
always >1. (If n=l then the formula would be — =k and we would

^2

be dealing with a so-called solid solution). The process here treated
is called adsorption.3

APPLEYARD and WALKER 4 have studied the adsorption of organic
acids from aqueous and alcoholic solutions by means of silk; the divi-

1 Nernst, 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.

4 Journ. Chem. Soc., 69, 1334, 1896.

COLLOIDS. 49

sion was found to correspond to the above formula for adsorption.
Recently FREUNDLICH l has carefully tested the adsorption of crys-
talloids 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 practically the same as with carbon. The influence of tem-
perature was slight.

According to KusTER,2 the combination between starch and iodine
is to be considered as an adsorption compound, and BILTZ 3 finds for the
division of As2C>3 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., the same attempts to diminish
the contact surface. The surface energy used thereby tends to be a
minimum potential energy. As the product from size of surface and
surface tension are the same, and as the first cannot change, the sur-
face energy can only be diminished by a reduction of the tension. If,
therefore, the tension is diminished by increasing the concentration
of a substance 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,4 FREUNDLICH5). In regard to the surface tension
of solid-fluid we only know that it is positive, but can otherwise show
great differences (OsTWALD,6 HULETT?). According to this theory
the facts are that certain solid substances possess the ability of adsorb-
ing . dissolved bodies, and for this reason the adsorbed substance
lowers the surface tension of the solid-fluid, and indeed, the more
the greater concentration in which it occurs. That especially carbon
and colloid substances are adsorption bodies lies in the fact that they
have an especially large surface due to their finely divided state or
porosity, which therefore, cet. par., must give then a great surface energy.

1 Ueber die Adsorption in Losungen, Leipzig, 1906.

2 Ann. d. Chem. u. Pharm., 283, 360, 1894.

3 Ber. d. d. chem. Gesellsch., 37, 3138, 1904.

4 Lehrb. d. allg. Chem., 2. Aufl., 2. Bd., 3. Teil, 237, 1906.

5 Ueber Adsorption in Losungen, 50-51.

« Zeitschr. f. physik. Chem., 34, 495, 1900.
7 Ibid., 37, 385, 1901.

50 PHYSICAL CHEMISTRY IN BIOLOGY.

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 1 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 this manner KC1 is taken up by colloidal
silicic acid.2 In certain cases undoubtedly chemical combinations
with quite positive conditions are formed.3

The precipitation of colloids by electrolytes has also been discussed
by FREUNDLiCH4 from the standpoint of the adsorption hypothesis.
Thus, for the precipitation ability of an electrolyte, the electric charge
of the precipitating ion comes first in consideration and secondly,
the ability of the precipitating colloid to adsorb the same. According
to MOORE and ROAF 5 the salts of the red corpuscles are retained as
adsorption 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-men-
tioned adsorption phenomena, as the combinations formed are in special
cases irreversible or gradually irreversible. It is well known that car-
bon takes up colloidal colored substances, and we have numerous examples
of the combination of dissolved colloids with solid colloids in technology.
BILTZ 6 has been able to show that many dyeing processes are to be
considered as adsorption phenomena, and later FREUNDLICH and LOSEV 7
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 47).

Tanning is also brought about by adsorption processes as the prepared skins
adsorb the tanning substance.8

1 Zeitschr. anorg. Chem., 23, 111, 321, 1900.

2 Schmidt, Zeitschr. f. physik. Chem., 15, 56, 1894.

3 v. Bemmelen, Journ. prakt. Chem. (2), 23, 324 and 379, 1880.

4 Zeitschr. f. Chem. u. Ind. d. Roll., 1, 321, 1907.

5 Bioch. Journ., 3, 55, 1908.

6 Ber. d. d. chem. Gesellsch., 37, 1766, 1904; 38, 2963, 2973, 4143, 1905.

7 Zeitschr. f . physik. Chem., 59, 284, 1907.

8 See Zeitschr. f. Chem. u. Ind. d. Koll., 2, 257, 1908.

COLLOIDS. 51

The precipitation of protein by adding finely divided solids (carbon,
kaolin J) or by suspended solids (mastic 2) precipitated in the liquid,, is
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) .3

BECHOLD,4 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 As2Ss,
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.

Especially with regard to the precipitation of hydrophile colloids we must
mention another theory suggested by SPIRO.S According to this the precipita-
tion depends upon a division of the colloid between two phases, . one of which
contains considerable water and salt and little colloid and the other much colloid,
but little water and salt.

Gels. We have already often mentioned gels or jellies (page 36).
Only certain colloids can occur in the form of gels. Certain gels are
spontaneously 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, concen-
trated 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
circumstances that the two phases cannot be analyzed separately.

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 role here. According to VAN BEMMELEN 7 the water is not chemi-
cally combined in definite proportions, but the quantity continually
changes with the temperature and the vapor pressure. On the other

1 Bioch. Zeitschr., 5, 365, 1907.

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

3 Zeitschr. f. physik. Chem., 47, 343, 1904.

4 Ibid., 60, 299, 1907.

5 Hofmeister's Beitrage, 4, 300, 1903.

c Zeitschr. f . physik. Chem., 33, 326, 1900.
'Zeitschr. anorg. Chem., 13, 233, 1896; 20, 185, 1899.

52 PHYSICAL CHEMISTRY IN BIOLOGY.

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 been recently 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 (SpiRO,3 Wo.
OSTWALD 4) . From the previously mentioned investigations of LILLIE,
on the osmotic tension of gelatin solutions, it was found that the addi-
tion of acids and alkalies increased it (page 39).

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.

CATALYSIS. 53

III. CATALYSIS.

When two bodies which can act chemically upon each other are
brought together the reaction generally takes place so fast that it
cannot 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
GULDBERG 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 molecular 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 ki, 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, i.e., two reactions occur simultaneously:
one between the alcohol and acetic acid, producing acetic ether and
water, and second, between acetic ether and water, reforming alcohol
and acetic acid. This is expressed as follows:

C2H5.OH + HO.CO.CH3 <=* C2H5.O.CO.CH3 + H2O.

\ "*

The velocity of reaction when it passes from left to right is called
Vi. If the velocity in the reverse reaction is called v2 and the molec-
ular concentration of the acetic ether and water is called CE and C^-,
then we obtain V2 = k2.CE.Cw. At the beginning when CE as well as
CW = Q} the velocity of the" ester formation is expressed by the formula
vi = ki.CA.C8; afterward it is expressed by the difference Vi — v2 or
k\.CA.Cs—k2.CE.Cw. When ki.CA.Cs = k2.CE.Cw is attained, then the
velocity of both reactions is the same; no measurable decomposi-
tion occurs and the system is in equilibrium. The equilibrium condi-
tion 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

ki.CA.Cs = k2.CE.Cw or -~ — -^ — = — = K.
CE-^W ki

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

54 PHYSICAL CHEMISTRY IN BIOLOGY.

equilibrium is present or from the velocity coefficient k\ and k% 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 che number of the kinds of molecules which diminish their
concentration thereby.1

BERZELius2 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. These phenomena were called
catalytic by BERZELIUS.

According to OSTWALD 3 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 undergo any appreciable change by the reaction.

Catalytic reactions have been studied, especially by WiLHELMY,4
VAN'T HoFF,5 OSTWALD,G ARRHENIUS 7 and BREDIG.S Of all other sub-
stances the acids and alkalies seem to act most catalytic. A well-known
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 -j-. According to the law

at

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

ar*-(c-*> a)

For practical use this equation is integrated into the following :

og. (2)

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

2 Berzelius, 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.

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

7 Zeitschr. f. physik. Chem., 4, 228, 1889.

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

CATALYSIS.

55

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

undecomposed cane-sugar = 1, then the equation becomes -rr = k, from

which it follows that k indicates the reaction velocity if the concen-
tration of the substrate could be kept the entire time at=l.

In these experiments k retains the same value. If in different
experiments 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
(ARRHENIUS 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 73) .

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.C2H5 + H2O = HO.CH2.CO.O.C2H5 + N2.

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.

CH

Cone, of the
//-ions by
Electric
Conductivity.

K

Velocity
Coefficient.

K
VH

Nitric acid

0.001820
0.000909
0.000909
0.000364
0.009900
0.003640
0.009090
0.018200

0.001820
0.000909
0.000909
0.000364
0.001680
0.001460
0.000724
0.000563

0.0703
0.0346
0.0356
0.0140
0.0632
0.0571
0.0285
0.0218

38.7
38.0
39.2
38.3
37.7
39.1
38.5
38.7

Picric acid

7w-Nitrobenzoic acid . .

Fumaric acid

Succinic acid ...

Acetic acid

As — — for the different acids and different quantities of acid is the same, then
CH

the velocity coefficient is here also proportional to the concentration of the K 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
ca^e of this kind was the transformation of hyoscyamine into the stable
atropine.4

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

2 Zeitschr. f. physik. Chem., 4, 226, 1889.

3 Ibid., 60, 202, 1907.

4 Ber. d. d. chem. Gesellsch., 21, 2777, 1888.

56 PHYSICAL CHEMISTRY IN BIOLOGY.

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

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.

r^tali/af Cone, of the Velocity

Catalyst- Catalyst. Constant.

Piperidine ............... 0.1090 0.038

Triethylamine ........... 0 . 4900 0 . 036

Ammonia ............... 0.5500 0.038

Tetraethylammonium / 0 . 0760 0 . 037

hydroxide ........... 1 0 . 0076 0 . 037

Sodium hydroxide ...... ^ g.086

By this a rule which VAN'T HOFF and OSTWALD 2 proved by thermo-
dynamic means, is substantiated, namely, that the equilibrium at constant
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 H2O2 in proportion to their concentration,3 and (2) cyan-
ions, which transform benzaldehyde into benzoin according to the following equa-
tion :

2C6H5.COH = CeH5.CO.CH (OH) .C6H6.<

The above-treated catalytic processes all occur in homogeneous sys-
tems, 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 reac-
tions 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 SO3 (from
SO2 and O), and the decomposition of H2O2 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
with the real chemical reaction it can be neglected. In these cases
the reaction velocity behaves similarly to a homogeneous system.5

1 Zeitschr. f. physik. Chem., 33, 129, 1900.

2 Van't Hoff, Vorlesungen, 1, 211.

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

4 Stern, ibid., 50, 513, 1905.

5 Goldschmidt, Zeitschr. f. physik. Cbem., 31, 235, 1899.

CATALYSIS. 57

2. The chemical reaction occurs at a rate which in comparison with
the time necessary for the accumulation can be neglected. In thivS
case the time necessary can be generally compared with a diffusion
process.1

The catalytic processes in heterogeneous systems have excited
interest since BREDIG 2 showed that the colloidal metals prepared by
him showed catalytic properties. The best-studied process is the
decomposition of H2O2 by colloidal platinum, gold, and other metals
or oxides (MnO2, PbO2). Attention must be called to the small quan-
tity of catalyst sufficient to decompose H2O2. The action of 1 gram
atom platinum in 70 million liters of reaction mixture has been detected.
The decomposition of H2O2 by platinum catalyst in nearly neutral or
faintly acid solution has been shown to be a monomolecular reaction.

In this connection we will give one experiment of BREDIG and v. BERNECK: 3

Time. Cone, of H2O2. 0.4343A;.

0 47.4

10 37.9 0.0097

20 30.0 0.0099

30 23.6 0.0101

40 18.2 0.0104

60 11.0 0.0106

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 H2O2 by platinum sols or by the enzyme hasmase,
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, ethyl butyrate,4 salicin and amygdalin5 are decom-
posed by platinum black as well as by enzymes.

1 Nernst and Brunner, ibid., 47, 52 and 56, 1904.

2 Anorganische Fermente, Leipzig, 42, 1901.

3 Zeitschr. f. physik. Chem., 31, 288, 1899.

4 Amer. Journ. of Physiol., 10, 191, 1904.

5 Ibid., 15, 148, 1906.

58 PHYSICAL CHEMISTRY IN BIOLOGY.

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 condi-
tions. Thus MENSCHUTKIN 1 found for the reaction

= (C2H5)4.N.L,
the following velocity in different solvents:

Hexane ........................ 0 . 00018

Heptane ....................... 0.000235

Xylene ......................... 0.00287

Benzene ............. .......... 0.00584

Ethyl alcohol ................... 0.03660

Benzyl alcohol .................. 0. 13300

Recently BKEDIG and FAJANS2 have been able to show that an opti-
cally 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 Z-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 tha catalyst
acts not only upon the velocity of the reaction. It is apparent that this does not
conform with OSTWALD'S definition of a catalyst (page 54).

IV. ENZYMES.

As the enzymes are active in very small amounts without under-
going any essential change, they have for a long time been included
among the catalytic substances.

In regard to the terminology it must be remarked that those bodies upon
which an enzyme acts is designated as the substrate, and often an enzyme is
named after the substrate (amylase, protease, lipase) ; in other cases the kind
of action determines the name (oxidase, reductase), and finally the name is
founded upon the product produced by the action (alcoholase) .

We have no general method for preparing the enzymes. 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.

1 Zeitschr. f. physik. Chem., 6, 41, 1890.

2 Ber. d. d. chem. Gesellsch., 41, 752, 1908.

ENZYMES. 59

In all these cases the enzymes are obtained strongly contaminated
with other bodies. No enzyme has thus far been obtained in a per-
fectly 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 col-
loids, from which they cannot be separated. The enzymes are character-
ized by the fact that they are readily taken up by finely divided sub-
stances (inorganic precipitates, carbon, kaolin, infusorial earth and other
colloids such as alumina, iron hydroxide, proteins1). 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,2 MICH.AELIS and EHREN-

REICH3).

All enzymes lose their specific action on sufficiently heating their
aqueous solutions, and even at ordinary temperature the enzymes are
gradually decomposed. 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 54) .4 The readiness
with which an enzyme is decomposed is nevertheless to a great extent
dependent upon the presence of other bodies (page 61).

Certain enzymes are also sensitive to light. According to SCHMIDT-
NiELSEN5 chymosin is injured by light and indeed, by the ultra-violet
rays. The experiments of JODLBAUER and TAPPEINER 6 with invertin
have led to the same results; the visible rays also can in certain cases
(peroxidase, hsemase) in the presence of oxygen or certain fluorescent
substances exert an injurious action.7

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. This seems
according to BUCHNER and collaborators 8 to be the case in alcoholic
fermentation, in which the dextrose is probably first split into lactic

1 Dauwe, Hofmeister's Beitrage, 6, 426, 1905.

2 Bioch. Journ., 2, 112, 1907.

3 Bioch. Zeitschr., 10, 283, 1908.

4 Arrhenius, Immunochemie, Leipzig, 1907, 5?§

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

6 Arch. f. klin. Med., 87, 373, 1906.

7 Bioch. Zeitschr., 8, 61 and 84, 1908.

8 Ber. d. d. chem. Gesellsch., 37, 419, 1904; 38, 620, 1905.

60 PHYSICAL CHEMISTRY IN BIOLOGY.

acid and this then into alcohol and carbon dioxide. In the same
manner the decomposition of protein into amino acids, with proteoses,
peptones, arid 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 (zymogens,
proenzymes). The best-studied example of a zymogen is trypsinogen,
which is contained in the pancreatic juice and converted into an active
enzyme by the enterokinase of the intestine. As this kinase is destroyed
by heat it also seems to be an enzyme-like body. (See Chapter IX.)
Trypsinogen can also be activated by lime salts. In certain other cases
the presence of bodies which resist temperature and are dialyzable
and therefore not enzymes, are necessary besides the real organic enzyme.
Thus the presence of an acid is necessary for the action of pepsin. R.
MAGNUS 1 has been able to separate by dialysis from a solution of liver-
lipase a body which is necessary for the action upon amyl salicylate.
Enzymes made inactive by dialysis can be activated again by the addi-
tion of boiled enzyme or the concentrated dialysate. HARDEN and
YOUNG 2 on filtering yeast-press juice through earthenware filters impreg-
nated 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 tempera-
ture, is added. According to BUCHNER and KLATTE 3 the constituent
of the zymase which is resistant to temperature is destroyed by lipase.
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
EuLER.4 Their action is probably different in different cases, and
differs also from the activating action of the kinases.

Laws of Action of the Enzymes. 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

1 Zeitschr. f . physiol. Chem., 42, 149, 1904.

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

3 Bioch. Zeitschr., 8, 520, 1908.

* Zeitschr. f. physiol. Chem., 57, 92, 1906.

ENZYMES. 61

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 67) of the substrate speaks essentially
for this view. The substrate also protects certain enzymes against destruc-
tive influences (heat, alkalies)1; casein is able to protect trypsin against
the retarding influence of seralbumin (HEDIN 2) . Trypsin adsorbed by
carbon is in part abstracted by proteins (HEDIN 3) . According to this
only that part of the added enzyme which is combined with the substrate
is active. We must now consider the following:

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. This applies to those cases where the chemical transforma-
tion 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 com-
parison 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,4 diastase,5 trypsin with casein.6

The second question, as to the division of the enzyme between different bodies,
we will discuss after we have spoken of the velocity of the real chemical reaction
as well as the retardation of enzyme action.

In regard to question 3, it must be recalled that the following laws
have been found for catalytic reactions:

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

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 Journ. of Physiol., 32, 390, 1905, also Zeitschr. f. physiol. Chem., 50, 497, 1907.

3 Bioch. Journ., 2, 81, 1906.

4 O'Sullivan and Thompson, Journ. Chem. Soc., 57, 926, 1890; Ducleau, TraitS
de microbiologie 11, 137; Brown, Trans. Chem. Soc., 81, 373, 1902; Armstrong, Proc.
Hoy. Soc., 73, 500, 1904.

5 Brown and Gliddinning, Proc. Chem. Soc., 18, 43, 1902.

6 Hedin, Journ. of Physiol, 32, 471, 1905.

62 PHYSICAL CHEMISTRY IN BIOLOGY.

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 invertin,1 lactase 2 and trypsin.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 65) 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
reactions 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.
With the aid of the formula for a monomolecular reaction

1 . C

t ° C-x

(page 54), constant values were obtained for the velocity coefficient k in
the following cases:

1. Decomposition of H2O2 by catalasc from blood (hsemase 5).

2. Decomposition of H2O2 by catalase from Boletus scaber.6

3. Decomposition of ethyl butyrate by an enzyme from pig fat.6

4. Decomposition of amyl butyrate by an enzyme from the pancreas.7

5. Decomposition of triolein and triacetin by an enzyme from the
castor oil seed.8

6. Decomposition of glycyl-glycine by erepsin.9

The reaction is in these cases monomolecular. Although probably
most enzymotic cleavage processes proceed with an excess of water
monomolecularly, still the proof of this is only exceptionally found.
Indeed such an apparently simple reaction as the inversion of cane-

1 Brown, Proc. chem. Soc., 18, 14, I'.HU.

2 Armstrong, Froc. Roy. Soc., 73, 500, 1904.

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

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

5 Senter, Zeitschr. f. physik. Chem., 44, 257, 1903.
" Miller, Hofmeister's Beitrage, 7, 1, IWti.

7 Dietz, Zeitschr. f. physiol. Chem., 52, 311, 1907.

8 Taylor, Journ. of biol. Chem., 2, 93, 1906; Nicloux, Compt. rend. soc. biol., ,">(».
840, 1904.

9 Euler, Zeitschr. f. physiol. Chem., 51, 213, 1907.

ENZYMES. G3

sugar cannot be expressed by the above formula.1 Recent investigations
of HUDSON 2 show, however, that the inversion of cane-sugar by enzyme
is a monomolecular reaction. The varying results of earlier investiga-
tors depend upon a disturbing action of the multirotation of the dextrose.
These results have been confirmed by EULER.S

The second law for catalytic reactions which we have formulated
that with constant quantities of substrate the reaction velocity is propor-
tional to the quantity of enzyme, has been*shown in certain cases where
the substrate was in excess (practically constant quantity) namely with
kephir lactase,4 trypsin with casein as substrate.5 In the just-mentioned
monomolecular enzyme reactions the velocity coefficient in a few cases was
found proportional to the quantity of enzyme (haemase,6 erepsin with
glycyl-glycine as substrate,7 pancreatic lipase 8) and in others not (catalase
from Boletus scaber,9 lipase from pig fat)9. It has been shown for several
enzymotic reactions that with the same substrate the same decomposi-
tion 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 : invertin (O'SuLLiVAN and THOMPSON under certain conditions10),
pepsin (SJOQVIST n), rennin (especially FULD 12), peptone-splitting enzyme
(VERNON13), fibrin ferment of snake poison (MARTIN14), trypsin (HEDiN15),
pepsin, rennin, trypsin, pyocyaneus protease (MADSENIG). On the
action of trypsin upon casein this law has been shown correct for different
stages in the reaction.17 This indicates that the progress of the entire
reaction remains the same with different quantities of enzyme, only
that the time for the same decomposition is inversely as the quantity

1 Henri, Zeitschr. f. physik. Chem., 39, 194, 1901; Brown, Trans. Chem. Soc.,
81, 373, 1902.

2 Journ. Amer. Chem. Soc., 1908.

3 Pflanzen Chemie, Braunschweig, 1908, 80.

4 Armstrong, Proc. Roy. Soc., 73, 500, 1904.

5 Hedin, Journ. of Physiol., 32, 471, 1905.

6 Senter, Zeitschr. f . physik. Chem., 44, 257, 1903.

7 Euler, Zeitschr. f. physiol. Chem., 51, 213, 1907.

8 Kastle and Loevenhart, Amer. Chem. Journ., 24, 491, 1900.
8 Euler, Hofmeister's Beitrage, 7, 1, 1906.

10 Trans. Chem. soc., 57, 926, 1890.

11 Skand. Arch. f. Physiol., 5, 358, 1895.

12 Hofmeister's Beitrage, 2, 169, 1902.

13 Journ. of Physiol., 30, 334, 1903.

14 Ibid., 32, 207, 1905.

15 Ibid., 32, 468, 1905; 34, 370, 1906.

18 Arrhenius, Immunochemie, Leipzig, 1907, 46.
17 Journ. of "Physiol., 32, 468, 1905; 34, 370, 1906.

64 PHYSICAL CHEMISTRY IN BIOLOGY.

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 assumptioa
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 division of the enzyme remains independent
of the quantity. In regard to the division of the enzymes REICHEL
and SPIRO 2 claim that rennin also during and after coagulation is divided
according to a constant factor between the whey and curd.

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 k is a constant, p the quantity of enzyme and t the
time of the action. This was first jshowri by SCHUTZ 3 for pepsin and indeed,
in this form, decomposition = k^p as the time (t) was constant. The form
decomposition = k\^pt was given by SCHUTZ, and HuPFERT.4 According to
PAWLOW this rule also applies to trypsin digestion.5 SCHUTZ'S 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 proven for the action of gastric and pan-
creatic lipase.6 According to ARRHENIUS 7 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 quantity of reac-
tion products.

Reversibility. 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 also synthetical processes can be produced
by enzymes.

The first example of such a reaction was given by CROFT-HILL.S
He treated a 40 per cent dextrose solution with maltase at 30° C. for a

1 Zeitschr. f. physiol. Chem., 57, 468, 1908.

2 Hofmeister's Beitrage, 6, 68, 1905.

3 Zeitschr. f . physiol. Chem., 9, 577, 1885.

4 Pfliiger's Arch., 80, 470, 1900.

5 Arbeit der Verdauungsdriisen, Wiesbaden, 1898, 33.

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

7 Immunochemie, 1907, 43.

8 Journ. of chem. Soc., 73, 634, 1898.

ENZYMES. 65

very long time and concluded from the change in rotation and reduction
power that some maltose was formed from the dextrose. EMMERLiNG1
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 2 emulsin splits isomaltose, but
not maltose, and therefore it can synthesize maltose from dextrose. A
similar reaction had previously been shown by E. FISCHER and ARM-
STRONG,3 that kefir-lactase produced isolactose and not lactose from
galactose and dextrose. According to CREMER4 yeast-press juice has
the power of forming glycogen from dextrose or levulose.

Of the enzymotic syntheses of protein-like substances nothing pos-
itive is known. Although it has been repeatedly claimed that the forma-
tion of plastein is a synthetical process, still the proofs for this are lack-
ing. It is just possible that we are here dealing with the simultaneous
precipitation of two or more digestion products.

A true reformation of a subtrate once split seems for the present
not to have been proven for the carbohydrates and proteins. Such a
synthesis of amygdalin is nevertheless known. Amygdalin is split by
maltase into mandelic acid nitrileglucoside and dextrose. EMMERLING 5
has been able to reconstruct amygdalin from these products with the aid
of maltase, and recently ROSENTHALER 6 reports that he has formed a
so-called asymmetrical synthesis whereby from benzaldehyde and hydro-
cyanic acid under the influence of emulsin the d-form of the mandelic
acid nitrile (C6H5CH(OH).CN) is formed. An undoubted synthesis
of fat and other ester-like combinations of fatty acids is also known.
KASTLE and LOEVENHART 7 have shown the formation of ethyl butyrate
fr,om ethyl alcohol and butyric acid under the influence of a pancreas
enzyme. In an analogous manner HARRIOT 8 obtained monobutyrin
from butyric acid and glycerin with blood serum. POTTEVIN 9 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.I()
The enzyme used by DIETZ was insoluble in water, and its action was

1 Ber. d. d. chem. Gesellsch., 34, 600 and 2207, 1901.

2 Proc. Roy. Soc. (ser. B), 76, 592, 1905.

3 Ber. d. d. chem. Gesellsch., 35, 3151, 1902.

4 Ibid., 32, 2062, 1899.

5 Ber. d. d. chem. Gesellsch., 34, 3810, 1901.
« Bioch. Zeitschr., 14, 238, 1908.

7 Amer. chem. Journ., 24, 491, 1900.

8 Compt. rend., 132, 212, 1901.

9 Ibid., 136, 1152, 1903; 138, 378, 1903; Ann. Inst. Past., 20, 901, 1906.

10 Zeitschr. f. physiol., Chem., 52, 279, 1907.

66 PHYSICAL CHEMISTRY IN BIOLOGY.

tested with i-amyl alcohol and n-butyric acid or the corresponding
ester. It was shown that the reaction took place in the insoluble phase
(enzyme). From the formula alcohol + acid fester -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 forma-
tion for a homogeneous system is — = ki.CA.Cs — k2-CE.Cw (see page 53),

dx

which equation can be simplified to —=ki.Cs—k2.CE as the alcohol

dt

and water were in excess and their concentration considered as con-
stant and included in the constants /bi and k-2- At equilibrium we

have kiCs=k2CE or j^-=~ = K (page 53). It follows that the same

k-2 Cs

equilibrium is attained irrespective of whether we start with alcohol -f-
acid, or ester +'H20. The equilibrium is also independent of the ante-
cedents 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 \/C 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 VcJ,
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 56), 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 another 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 dif-
ferent 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 proceeds further. These false equilibria
are generally caused by the enzyme being destroyed or put out of
action in other ways. In DIETZ'S experiment the equilibrium was true
in every regard, as this condition could be attained from both sides and
was independent of the quantity of enzyme.

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

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

ENZYMES. 67

(proteins, 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, invertin). 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 burnt with unequal facility in the organism was shown by E.
FISCHER, and that of the numerous aldohexoses only three, d-dextrose,
d-mannose and d-galactose, and of the ketohexoses only one, d-levulose
are fermentable; 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 (/?) 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 FISCHER also
found that amygdalin, which is split into benz aldehyde, hydrocyanic
acid and dextrose by emulsin, is split into mandelic acid nitrile-glucoside
and dextrose by yeast, and from these products the first can be further
decomposed by emulsin into benzaldehyde, hydrocyanic acid and dex-
trose.2 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,S 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 levo.ro-
tatory ester. Finally, we must mention the recent investigations of
FISCHER and ABDERHALDEN 4 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.

Retardation of Enzyme Action. In the first place the products
of enzymotic activity have a retarding action upon the enzymes. In

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

2 Ber. d. d. chem. Gesellsch., 28, 1508, 1896.

3 Journ. of Physiol., 30, 253, 1903; 32, 199, 1905.

4 Zeitschr. f. physiol. Chem., 40, 52, 1905; 51, 264, 1907.

68 PHYSICAL CHEMISTRY IN BIOLOGY.

certain cases this retardation can be explained by a reverse reaction (syn-
thesis) (page 59), in others no reverse reaction can be detected. That
the inversion of cane-sugar is retarded by invert sugar has been claimed
by many (HENRI/ A. J. BROWN,2 BARENDRECHT,3 ARMSTRONG4), and
Indeed BARENDRECHT claims that dextrose as well as levulose 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. ARM-
STRONG 5 found that invertin, maltase and lactase are retarded by just
those varieties of sugar which are produced by their activity.

The retarding action of amino-acids upon the decomposition of glycyl-
Z-tyrosine by yeast-press juice has recently been studied by ABDER-
HALDEN and GiGON.6 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 67).

There also exist proteins which act retardingly upon the digestion
of other proteins. The tryptic digestion of easily split protein bodies
is retarded by the difficultly digested white of egg (DELEZENNE and
PozERSKi,7 VERNON,S GOMPEL and HENRI,9 HEDiN.10 The white of
egg takes up a part of the enzyme and makes this partly inactive. At
this time we must also mention the retarding action which the proteo-
lytic primary cleavage products (proteoses, peptones) exert upon diges-
tion. These products are further split; a part of the enzyme is combined
with the products and in this way prevented from dissolving new pro-
tein (HEDIN10).

It has been known for a long time that blood-serum is able to retard
various enzymotic processes. Certain enzymes have their action retarded
even by normal serum. According to HAMMARSTEN and RODEN n normal
horse-serum retards the coagulation of milk, and the antitryptic action
of serum has been shown by several investigators (HAHN,12 CAMUS and

1 Zeitschr. f. physik. Chem., 39, 194, 1901.

2 Journ. Chem. Soc., 81, 382, 1902.

3 Zeitschr. f. physik. Chem., 49, 456, 1904.

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

5 Ibid., 79, 360, 1907.

6 Zeitschr. f . physiol. Chem., 53, 251, 1907.

7 Compt. rend. soc. biol., 55, 935, 1903.

8 Joum. of Physiol., 31, 495, 1904.

g Compt. rend. soc. biol., 58, 457, 1906.

10 Zeitschr. f. physiol. Chem., 52, 422, 1907.

11 Upsala lakare forenigs forh., 22, 546, 1887.

12 Ber. klin. Wochenschr., 34, 499, 1897.

ENZYMES. 69

,1 LANDSTEINER 2) . The serum can attain retarding action toward
other enzymes by repeatedly injecting the enzyme into an animal in the
same manner as the anti-sera are obtained for bacterial toxines. . In
this way HiLDEBRANDT3 first obtained an anti-enzyme, namely for
emulsin, and MORGENROTH 4 has produced in the same manner an anti-
rennin in goats' serum. According to EHRLICH the natural and the arti-
ficial anti-bodies are identical, which is not the case in the experiments
of MADSEN and WALBUM,S at least for the natural and artificially pro-
duced anti-rennin.

On studying the retardation of tryptic digestion, HEDIN 6 found that
in different cases a different process occurs. The retarding action of
normal beef serum is connected with the seralbumin (LANDSTEINER/
CATHCARTS). In this case the trypsin, according to HEDIN, is in some
way or other partly attached in an irreversible manner to the retarding
substance (see page 73), because much more trypsin is neutralized
when the mixture is made in the order seralbumin-trypsin-substrate,
than when the substrate is added to the trypsin before the seralbumin.
As above stated, egg albumin and the tryptic digestion products also act
retardingly upon tryptic digestion. In this case it is immaterial in
what order the bodies are mixed, and the trypsin is therefore taken up
in a perfectly reversible manner (enzyme deviation). On treating ser-
albumin with 0.1-0.2-per cent acetic acid at 37° C. it loses the power
of attaching trypsin; it has a less retarding action than before, and
now indeed in the same manner as egg albumin.

HEDIN 9 has also found that bone-charcoal has the remarkable ability
of adsorbing trypsin, and chiefly in an irreversible manner. From this
it follows that charcoal can retard the action of tiypsin in a very pro-
nounced manner; and many analogies between the retarding action
of native seralbumin and that of carbon can be detected.

Antigens and Anti-bodies. In connection with the retardation of
enzyme action we can also call attention to other similar processes. Under
the name antigen we include those substances which, when injected
into animals, cause the formation of bodies in the organism with which
they can in some way or another react. The bodies thus formed are

1 Compt. rend. soc. biol., 49, 845, 1897.

2 Centralbl. f. Bact., 27, 357, 1900.

3 Virchow's Arch., 131, 33, 1893.

4 Centralbl. f. Bakt. u. Parasitenk., 26, 349, 1899.

5 See Arrhenius Immunochemie, 180.

8 Zeitschr. f. physiol. Chem., 52, 412, 1907.

7 Centralbl. f. Bakt., 27, 357, 1900.

8 Journ. of Physiol., 30, 156, 1903.

9 Bioch. Journ., 1, 484, 1906; 2, 81, 1907.

70 PHYSICAL CHEMISTRY IN BIOLOGY.

called anti-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 associated with
colloids.

To the antigens 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.

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 them-
selves the immune bodies are inactive, but form with complements, sub-
stances occurring in normal serum, so-called cytotoxins, which destroy
the kind of cells active in their formation. These cytotoxins are called
bacteriolysins, hcemolysins, etc., according to the kind of cells used.
The immune bodies are specific and also stable against heat; the com-
plements can act together with different immune bodies and are very
unstable, as they are generally 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 formation. These anti-bodies are called agglu-
tinins.

The longest known (due to the epoch-making investigations of v.
BEHRING l) 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-
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 for measuring the quantity of toxin by injection into an ani-

1 Deutsch. med. Wochenschr., 1892; Zeitschr. f. Hygiene, 12, 1892.

KXZYMES. 71

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
(syn toxoids) . 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 can the addition of a certain quantity of
anti-body produce entirely different results.

In regard to the immune bodies, EHRLICH believes that they combine
with those kind of 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

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

72 PHYSICAL CHEMISTRY IN BIOLOGY.

the anti-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).

ARRHENIUS arid MADSEN oppose EHRLICH'S theory that the combina-
tion between toxin and anti-body is of a chemical nature, but claim that
their formation 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 53).

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 form-
ula, must always remain free.1 According to this theory the toxin is a
unit poison, as ARRHENIUS 2 recently admits with EHRLICH, that the poison
is gradually transformed into a non-toxic or only slightly toxic sub-
stance which has the same ability to combine with antitoxin as the
toxin itself.

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

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). On the

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.

IONS AND SALT ACTION. 73

contrary, HEDIN could not obtain free trypsin from trypsin neutralized
with native seralbumin, by treatment with very dilute acetic acid,
although the acid was shown by special experiments to be able to destroy
the binding properties of the seralbumin.1

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 and indeed, in an irreversible manner

(NERNST,2 BlLTZ,3 LANDSTEINER 4) .

V. IONS AND SALT ACTION.

We have already 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 5 has attempted to show a correspondence between
ion-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.

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 6 pancreatic juice dialyzed for a long time has no action
upon starch, but becomes active again on adding 'NaCl or other salts.
According to WOHLGEMUTH 7 the diastatic power of saliva is increased
ten-fold by the addition of NaCl. The anions are the active part in these
cases. The strong retarding action which NaFl exerts upon the enzy-

1 Bioch. Journ., 1, 479, 1906; Zeitschr. f. physiol. Chem., 50, 497, 1907.

2 Zeitschr. f. Elektrochem., 10, 379, 1904.

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

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

5 Zeitschr. f . physik. Chem. 36, 641 1901.

6 Compt. rend. soc. biol., 60, 479, 1906; 62, 432, 1907; Bioch. Zeitschr., 4, 1, 1907.

7 Bioch. Zeitschr., 9, 1, 1908.

74 PHYSICAL CHEMISTRY IN BIOLOGY.

motic cleavage of esters is also to be mentioned (LOEVENHART and
PIERCE, AMBERG AND LOEVENHART l).

Other actions of salts are also ascribed to ion-action. To these
belong the experiments of DRESSER^ 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 H ions the poisonous action of the first salt is ascribed to
the ions. PAUL and KRONIG 3 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 4 for copper salts.

In this connection it is interesting to mention the interesting inves-
tigations of J. LOEB 5 on the artificial fertilization of eggs of lower sea
animals. LOEB found that it is possible to obtain larvse from the unfer-
tilized eggs of the sea-urchin, Arbacia, which never develop in normal
sea-water. For this purpose it was only necessary to keep the eggs
for two hours in sea-water whose osmotic pressure had been raised 40-50 per
cent and then placing the eggs again in normal sea-water. It is immate-
rial how the osmotic pressure is raised, whether by the addition of NaCl,
KC1, MgCl2, urea, or sugar. It is evident that the bodies used are those
which cause an effective osmotic pressure. LOEB ascribes their action
to their ability to attract water. The development of the egg does not
take place exactly like an egg fertilized with spermatozoa. LOEB obtained
much better results by using the eggs of another sea-urchin, Strongy-
locentrotus, which he allows to lie first for J-l minute in sea-water slightly
acidulated -with a low fatty acid, foi instance formic acid, and then replaced
in normal sea-water. By this means chemical processes are introduced,
which when the eggs remain in sea-water causes death in less than 24
hours, but if the eggs, 5-10 minutes after the membrane formation which
takes place in sea-water, are kept for 20-45 minutes in hypertonic sea-
water, and then ag^in placed in normal sea-water, the development of
active larvse is the result. The presence of oxygen is necessary for the
result of the experiment. LOEB has also obtained larvae from other

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

2 Arch. exp. Pathol. u. Pharm., 32, 456, 1893.

3 Zeitschr. f. physik. Chem., 31, 411, 1896.

4 Compt. rend. soc. biol., 50, 1210, 1898.

5 Loeb, Vorlesungen iiber die Dynamik der Lebenserscheinungen, 1906, 243; Pfliiger's
Arch., 118, 572, 1907; Ueber den chemiscLen Charakter des Befruchtungsvorganges,
Leipzig, 1908.

IONS AND SALT ACTION. 75

animal forms by the aid of methods adhering more or less closely to the
above.

Certain ions or salts have an antagonistic action upon the animal
organs. NaCl has in general a beneficial influence upon animal tissues.
Nevertheless, pure NaCl solutions can have a poisonous action, for example
upon frog muscles. In such cases the toxic action is arrested by the
addition of small quantities of other salts, namely K and Ca salts. Upon
this depends the use, in experiments with animal organs, of the so-called
KIXGER-LOCKE 1 solution, which contains in a liter about the following
substances: 6.5-9.5 grams NaCl + 0.2 grams KC1 + 0.2-0.3 gram CaCl2,
and also 0.1 gram NaHCO3. The experiments of LOEB with the fertilized
eggs of the Fundulus heteroclitus are very interesting. These eggs
develop just as well in a remarkable manner in water free from salt as
in sea-water, and are completely insensible to osmotic influence. But
if the fertilized eggs are placed in a NaCl solution of the same osmotic
pressure as the sea-water they die; the toxicity of the NaCl solution
can be arrested by small quantities of nearly any salt with polyvalent
cations. Not only the salts of the alkaline earths, but also those of the
heavy metals can neutralize the toxicity of the NaCl in proper concen-
tration.2 Wo. OSTWALD has carried on similar experiments with
Gammarus pulex.3

1 Journ. of Physiol., 18, 318, 1895.

2 Pfliiger'S Arch., 88, 68, 1901.

3 Ibid., 106, 568, 1905.

CHAPTER III.
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 Trpoxrcvo, 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 burnt horn or wool. At the same time they
produce inflammable gases, water, carbon dioxide, ammonia, and nitro-
genized bases, besides many other substances, and leave a large quantity
of carbon. On hydrolytic cleavage they all yield, besides nitrogenous
basic substances, especially large amounts of a-monamino-acids of
different kinds.

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 diaminovalerianic acid as arginine, and
which has also been called the urea-forming group; (3) basic nitrogen
or diamino-acid nitrogen, which is precipitated by phosphotungstic
acid as basic products (to which also the guanidine residue of arginine

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 up to the year 1903 may be found in O. Cohnheim,
Chemie der Eiweisskorper, Braunschweig, 1904. See aslo Mann, Chemistry of the
Proteids, London, 1906, and Oppenheimer's Handbuch der Biochem. der Menschen
und der Tiere, 1908.

76

NITROGEN OF THE PROTEINS. 77

belongs); (4) monamino-acid nitrogen; and (5) the nitrogen 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 the plant gluten-proteids, 13-20
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 protein substances, 5-14 per cent
in zein and the gluten proteid, and up to 37 per cent in the plant globulins.
The chief quantity of the nitrogen, 55-76 per cent, occurs, with the excep-
tion of the protamines, as the monamino-acid groups. The results
for the melanoidin nitrogen vary so considerably that they will not be
mentioned. OSBORNE, LEAVENWORTH and BRAUTLECHT4 have recently
published the results of their investigations on the different forms of
binding of the nitrogen in the plant-proteins.

From the above results it follows that the nitrogen of most protein
bodies exists in such combination that the chief quantity appears in the
cleavage products as amino-compounds on hydrolytic cleavage by acids.
By the action of nitrous acid upon proteins only a very small part, 1-2
per cent, of the nitrogen is evolved,5 which seems to indicate that NH2
groups exist only to a slight extent hi protein substances. This assump-

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. ehem. Gesellsch., 34, 3214; Hofmeister, Ergebnisse der Physiol.,
Jahrgr. 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 Amer. Journ. of Physiol., 23.

5 See C. Paal, Ber. d. d. chem. Gesellsch., 29; H. Schiff, ibid., 1354; O. Loew,
Chemiker Zeitung, 1896; and O. Nasse, Pfliiger's Arch., 6.

78 THE PROTEIN SUBSTANCES

tion does not have sufficient foundation, for, according to LEVITES,*
the quantity of amide nitrogen is not diminished by the action of nitrous
acid upon the protein substances. SKRAUP and co-workers have in part
obtained similar results, and the deamidized proteins, the desamidoprotein
at least in certain cases, do not strikingly differ in elementary composi-
tion from the original mother-substance. The so-called deamidation
of proteins seems to be a rather complicated process whose nature is
not well understood,2 and therefore no positive conclusions can be drawn.

If by the action of HN02 only acid amide groups are attacked, then we should
expect that the corresponding deamidized protein would yield on hydrolysis
the same amino acids as the mother substance. If, on the contrary, the nitrous
acid reacts also with NH2 groups of the amino acids with an exchange of OH
for NH2, then it is apparent that on the hydrolysis of the diamino protein we
would find other cleavage products, namely, oxyacids instead of amino acids,
and either oxyacids or oxyamino acids instead of diamino acids. By the absence
of certain cleavage products and the occurrence of others, we can with probability
determine which of the cleavage products were so combined that one or the
other NH2 groups was free. From this standpoint SKRAUP and his co-workers
have studied the hydrolytic cleavage products of the diamino proteins, and have
found that these products, with the exception of lysin and in certain cases, A part
of the arginine, were the same qualitatively and quantitatively as from the mother
substance. Nearly all amino acids occur, it seems, in the investigated proteins
in a form of binding when the amino groups are not free.

The chief part of the nitrogen in the proteins exists as an imide-like
binding of the amino-acids, but other forms of bondage may occur at the
same time. This will be 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
bacterial proteids,3 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 bulphur of the protein substances is regularly, at least
in part, split off as cystine (K. MORNER) or, with bodies poorer in sul-
phur, as cystein (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 sulphuretted hydrogen (SIEBER and

1 Levites, Zeitschr. f . physiol. Chem., 43.

2 On the deamidation of proteins and their cleavage products see footnote 5, page
77; Treves and Salomone, Biochem. 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.

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

SULPHUR OF THE PROTEINS. 79

SCHOUBENKO, RuBNER), and a body having the odor of ethyl sulphide
(DRECHSEL) have been obtained.1

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, D ANILE WSKY, KRUGER,
FR. SCHULZ, OSBORNE, K. MoRNER 2) . What remains can be detected
only after fusing with potassium nitrate and sodium carbonate and test-
ing 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 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 protein
substances. If the quantity of lead-blackening sulphur in a protein
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.

According to RAIKOW 3 keratin-like proteins split off sulphur dioxide on
treatment with phosphoric acid at ordinary temperatures; hence it follows that
a part of the sulphur in the proteins, especially in the keratins, exists in direct
combination with oxygen and probably combined as in the sulphites.

The constitution of the protein bodies is still unknown, although
the great advances made in the last few years have brought us essentially
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 proteins have been
used that can be prepared in the crystalline form, first large atomic
complexes — proteoses and peptones — are obtained which still have

1 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. Pe"tersbourg, 1; Rubner, Arch. f. Hygiene, 19;
Drechsel, Centralbl. f. Physiol., 10, 529; Frankel, Sitzungsber. d. Wien. Akad., 112,
II b, 1903.

2 Fleitmann, Annal. der Chem. und Pharm., 66; Danilewsky, Zeitschr. f. physiol.
Chem., 7; Kriiger's, Pfliiger's Archiv, 34; F. Schulz, Zeitschr. f. physiol. Chem., 25;
Osborne, Connecticut Agric. Expt. Station Report 1900; Morner, 1. c.

3 See Biochem. Centralbl., 4, p. 353.

80 THE PROTEIN SUBSTANCES.

protein characteristics, and these then suffer further decomposition
until finally we obtain simpler, generally crystalline, or at least well-
characterized, end products.

On heating protein with barium hydroxide and water in sealed tubes to 150-
250° C., SCHUTZENBERGER l obtained a mixture of products among which were
ammonia, carbon dioxide, oxalic acid, acetic acid, and, as chief product, a mix-
ture of amino-acids. The conclusion he drew from this experiment, that the
protein is a complex ureide or oxamide, cannot be considered for several reasons.2

On fusing proteins with caustic alkali we obtain ammonia, methyl mercaptan,
and other volatile products; also leucine, from which then volatile fatty acids,'
such as acetic acid, valeric acid, and also butyric acid are obtained, and also
tyrosine, from which latter phenol, indol, and skatol are produced.

As to the products obtained by hydrolytic cleavage with mineral
acids, we have a number of investigations by various experimenters,
especially HLASIWETZ and HABERMANN, RITTHAUSEN and KREUSLER,
E. SCHULZE and his collaborators, DRECHSEL, SIEGFRIED, R. COHN,
KOSSEL and his pupils, K. MORNER, ABDERHALDEN and co-workers,
OSBORNE and CLAPP, SKRAUP, and recently E. FISCHER and his collab-
orators.3 The chief products thus obtained are monamino acids, such
as glycocoll, alanine, aminovaleric acid, leucine, phenylaminopropionic
acid, aspartic and glutamic acids, cysteine and its sulphide cystine; the
so-called hexone bases, lysine, arginine, and histidine, of which the first
two are diamino acids; tyrosine, oxymonamino acids, such as serine,
oxyaminosuccinic acid, and oxyaminosuberic acid; oxydiamino acids,
such as oxydiaminosuberic acid, oxydiaminosebacic acid, diaminotri-
oxydodecanoic acid, caseanic and caseinic acids; a-pyrrolidine and oxy-
pyrrolidine carboxylic acids; tryptophane (indolaminopropionic acid);
sulphuretted hydrogen, ethyl sulphide, leucinimide, ammonia, and
melanoidins,4 which latter seem to be secondary condensation products.

The proteins can be split into a large number of bodies by the proteo-
lytic enzymes, and these will be presented later. In the first place
proteoses and peptones are produced, also an abundance of monamino-
acids of different kinds, hexone bases, tryptophane, and finally oxy-
phenylethylamine, diamines, and a little ammonia and other substances.

A great many substances are produced in the putrefaction of pro-
teins. First the same bodies as are formed in the decomposition by

1 Annal. de chim. et phys. (5), 16, and Bull. Soc. chim., 23 and 24.

2 See Habermann and Ehrenfeld, Zeitschr. f. physiol. Chem., 30.

3 In regard to the literature see O. Cohnheim, Chemie der Eiweisskorper, Braun-
schweig, 1904, and F. Hofmeister, Ergebnisse der Physiologic, Jahrg. I, Abt. 1, 759,
1902; E. Fischer, Untersuchungen iiber Animosauren, Polypeptide und Proteine (1899-
1906), Berlin, 1906; also Mann, Chemistry of the Proteids, London, 1906. See also
special references.

4 See Samuely, Hofmeister's Beitrage, 2.

PUTREFACTIVE PRODUCTS OF THE PROTEINS. 81

means of proteolytic enzymes are produced, and then a further decom-
position occurs with the formation of a large number of bodies belonging
in part to the aliphatic and in part to the aromatic and heterocyclic series.
Of the first series we have ammonium salts of volatile fatty acids, such
as caproic, valeric, and butyric acids, also succinic acid, carbon dioxide,
methane, hydrogen, sulphuretted hydrogen, methyl mercaptan, and
others. The ptomaines also belong to these products, and are probably
in part formed by very different chemical processes, or even syntheses.

E. SALKOWSKI divides the putrefactive products of the aromatic
and heterocyclic series into three groups: (a) the phenol group, to which
tyrosine, the aromatic oxyacids, phenol, and cresol belong; (6) the phenyl
group, including phenylacetic acid and phenylpropionic acid; and
lastly (c) the indol group, which includes indol, skatol, indol propionic
acid and indol acetic acid. These various products are formed during
putrefaction with access of air. NENCKI and BOVET 1 obtained only
2>-oxyphenylpropionic acid, phenylpropionic acid, and skatolacetic acid
on the putrefaction of proteins by anaerobic schizomycetes in the
absence of oxygen. These three acids are produced by the action of
nascent hydrogen on the corresponding amino-acids, namely, tyrosine,
phenylaminopropionic acid, and skatolaminoacetic acid (indolamino-
propionic acid), and according to NENCKI these three last-mentioned
amino-acids exist preformed in the protein mplecule.

By the moderate action of chlorine, bromine, or iodine upon proteins,
these halogens enter into more or less firm combination with the mole-
cule (LOEW, BLUM, BLUM and VAUBEL, LIEBRECHT, HOPKINS and BROOK,
HOFMEISTER, KURAJEFF, and others), and according to the method
of procedure we can prepare derivatives having different but constant
amounts of halogens (HOPKINS and PINKUS). The proteins are so changed
that they do not split off sulphur on treatment with alkali, nor do they
respond to MILLON'S reaction, nor do they yield tyrosine as a cleavage
product. According to SCHMIDT, oxidations and cleavages may take
place, as secondary processes, but more probably a substitution of hydro-
gen by halogen occurs in the aromatic nucleus of tyrosine or phenylamino-
propionic acid and perhaps also in the indol nucleus of tryptophane
and the imidazol nucleus of histidine.2

1 Salkowski, Zeitschr. f. physiol. Chem., 12, 215, and 27, 302; Nencki and Bovet,
Monatshefte f. Chem., 10.

2 Loew, Journ. f. prakt. Chem. (N. F.), 31; Blum, Mimch. med. Wochenschr.,
1896; Blum and Vaubel, Journ. f. prakt. Chem. (N. F.), 57; Liebrecht, Ber. d. deutsch.
chem. Gesellsch., 30; Hopkins and Brook, Journ. of Physiol., 22; Hopkins and Pin-
kus, 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.

82 THE PROTEIN SUBSTANCES.

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, O 25.54
per cent, which is not a cleavage product, but an oxidation product in which
the group SH is changed into SO2.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 oxyprotein 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 the recent investigations of
v. FuRTH1 there exist at least three different peroxyproteic acids (from casein)
which differ from each other by a different division of the nitrogen in the mole-
cule. On treatment with baryta- water we find that they split off basic complexes
and oxalic-acid groups, and new bodies, the desaminoproteic acids, which give
the biuret reaction, are produced. These 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-amide-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 (KUTSCHER, ZlCKGRAF, SUBMANN, KuTSCHER and SCHENCK).2

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. By the action of ozone upon casein, HARRIES and
LANGHELD 3 found neither phenyl alanine nor tyrosine among the cleavage
products, which fact they explain by the destructive action of the ozone upon the
aromatic nucleus. Besides this, reducing bodies wrhich are not carbohydrates, but
which react with phenylhydrazine, are produced. JOLLES 4 claims to have obtained
large quantities of urea in the oxidation of various proteins by potassium per-
manganate in acid solution, but this has been disputed by other investigators
and the above statements in regard to the oxidation products of proteins are of
little interest.

Nitric acid gives various nitro-products such as trinitroalbumin, oxynitro-
albumin, xanthoprotein and others. A melanoidin substance, xanthomelanin,
has been obtained by v. FURTH. 5 HABERMANN and EHRENFELD 6 also obtained
oxyglutaric acid among other products. By the action of bromine under
strong pressure a number of products have been obtained: bromanil and
tribromacetic acid, bromoform, leucinimide, leucine, oxalic acid, tribromamino-

1 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; v. Fiirth, Hofmeister's Beitrage, 6.

2 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.

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

4 Zeitschr. f . physiol. Chem., 32 and 38.

5 See Maly's Jahresber., 30, 24.

6 Zeitschr. f. physiol. Chem., 35.

CARBOHYDRATE MOIETY OF THE PROTEINS. 83

benzoic acid, and other bodies. With aqua regia, fumaric acid, oxalic acid, chlor-
azol, and other bodies are obtained. The investigations of HABERMANN and
EHRENFELD and PANZER 1 upon the action of chlorine upon proteins and
closely related products are important.

By the dry distillation of proteins we obtain a large number of decomposition
products having a disagreeable burnt 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
MuLLER2 and his students. They have shown that it is always an
ammo-sugar, and generally glucosamine. 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,
WEYDEMANN, SEEMANN, FRANKEL, HOFMEISTER, and LANGSTEINS
have demonstrated that in these cases the carbohydrate is also glucos-
amine. A carbohydrate complex, although sometimes only to a very
slight amount, has also been detected in other proteins, ovoglobulin,
serglobulin, seralbumin, peaglobulin, albumin of the gramineae, yolk-
proteid, and fibrin. In other proteins, on the contrary, such as edestin
(of the hemp-seed) and casein, myosin, pure fibrinogen, end 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 complex or whether they are united with the protein only
as impurities. Several observations 4 show that in working with crys-
talline proteins a contamination with other protein substances is unfor-
tunately not excluded, and this must not be lost sight of, especially as
the quantity of carbohydrates obtained is often very small. In the
present state of our knowledge we are not warranted in considering the
carbohydrate groups as belonging to the carbon nucleus produced on the
destruction of the real protein complex.

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

2 Midler, Sitzungsber. d. Ges. d. Natuw. zu Marburg, 1896 and 1898, and Zeitschr.
f. Biologie, 42.

3 In regard to the literature on this subject see the work of Fr. Miiller, Zeitschr.
f. Biologie, 42, and Langstein, Ergebnisse der Physiologic, 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.

4 See Wichmann, Zeitschr. f. physiol. Chem., 27, and N. Schulz, Die Grosse des
Eiweissmolekiils, Jena, 1903, 51.

84 THE PROTEIN SUBSTANCES.

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,
valhie, leucine, and isoleucine. 3. Bibasic monamino-acids: Aspartic acid
and glutamic acid. 4. Oxymonamino-acids: serine oxyaminosuccinic 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.

II. The Nuclei belonging to the Carbocyclic Series.
Phenylaminopropionic acid 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, tyrosine, 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
that in the hydrolysis certain carbon nuclei may be secondarily formed
from others. We cannot exclude the possibility, as suggested by Losw,1
that in the hydrolysis a marked atomic displacement perhaps occurs
before cleavage, and for this reason two carbon nuclei, such as leucine
and lysine, or tyrosine and phenylalanine, may be produced from the
same atomic groupings, each according to the nature of the neighboring
groups. Such a possibility cannot be entirely excluded, but it has been
made rather improbable by recent investigations.

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

1 Loew, Die chem. Energie d. lebenden Zellen, Miinchen, 1898, and Hofmeister's
Beitrage, 1.

POLYPEPTIDES. 85

amino-acid unites with the carboxyl group of the other. In accord with
this behavior we can, as HOFMEISTER 1 has explained, but which was first
proven by the epoch-making investigations of EMIL FISCHER, consider
the Droteins as chiefly formed by the condensation of amino-acids, wrhere
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.CoH4(OH^ CH2.COOH C3H6.CH2.NH2

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

Closely connected with this conception is the question whether it is
possible to prepare protein-like substances synthetically. In this con-
nection we must mention that GRIMAUX and later also SCHUTZENBERGER
and PICKERING have been able to prepare substances, 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 synthetically the so-
called biuret base (triglycyl-glycine ethyl ester) and subsequently many
other bodies which were related to the proteins. The most important
work on the chaining of amino-acids has. been performed by E. FISCHER 2
and his pupils. They have prepared a large number of complex bodies
called polypeptid'es, 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-
alanine, leucyl tyrosine, propylalanine, diaminopropionic-acid dipepti.de,
lysyl-lysine, histidyl-histidine ; leucyl-histidine ; tripeptides: diglycyl-
glycine, leucyl-alanyl-glycine, dileucylcystine ; tetrapeptides: triglycyl-
glycine, dileucyl-glycyl-glycine ; pentapeptides : tetraglycyl-glycine, and
leucyl-tr;glycyl-glycine; hexa- and heptapeptides: leucyl-tetraglycyl-
glycine and leucyl-penta glycyl-glycine respectively. The most complex
polypeptide thus far prepared is an octodecapeptide with 15 glycocoll
and 3 leucine residues namely: /-leucyltriglycyW-leucyltriglycyl-Z-leucyl-
octoglycylglycine = NH2CH(C4H9)CO. [NHCH2CO]3. NHCH (C4H9) CO.
[NHCH2COl3.NHCH(C4H9)CO.fNHCH2CO]8.NHCH2COOH. with the sup-

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

3 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,
1S97; 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 Protei'ne (1899-1906) and Ber.
d. d. chem. Gesellsch., 39, 40, 41 and Annal. d. Chem. u. Pharm., 354, 357, 363.

86 THE PROTEIN SUBSTANCES.

position 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 Z-) dd, II, dl and Id.
Of these forms two can form a racemic dipeptide, thus d-alanyl-cZ-leucine +
Z-alanyl-Z-leucine and d-alanyl-Z-leucine + Z-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 CONH 2
and on total hydrolysis yields NH3, like most proteins. A polypeptide
of this kind is the tripeptide glycyl-Z-asparaginyl-Z-leucine prepared by
E. FISCHER and KOENIGS.

NH2CH2CO.NHCHCO.NHCH(C4H9)COOH

CH2CONH2

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 diketopiper-
azine, glycine anhydride, according to the following equation:

/CH2.COV

2(NH2CH2CO.O.C2H5) = 2C2H6OH + NH< >NH.

XCO.CH2/

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 principle other dipeptides can also be prepared.

SYNTHESIS OF POLYPEPTIDES. 87

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 accord-
ing to the following equation:

CH3CHBrCOCl + NH2CH2COOH = HC1 + CH3CHBrCO.NHCH2COOH
(brompropionyl glycine). On subsequent treatment with ammonia the
halogen (Br) is replaced by NH2 and the dipeptide alanylglycine

CH3CHNH2CO.NHCH2COOH

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.

The prolongation of the chain on the other side, namely, at the car-
boxyl, FISCHER has accomplished by chlorination of the amino-acids
by special treatment with phosphorus pentachloride. The carboxyl is
thus transformed into COC1, while the acid at the same time fixes a
molecule of HC1, for example CH3CHNH2HCL

COC1

Just as in the case of the carboxyl group of an amino-acid, so also can
a polypeptide 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-bromisocapronyldiglycyl glycine, first prepared a-brom-
isocapronyldiglycylglycyl chloride, and then with diglycylglycine he
obtained the heptapeptide leucyl-pentaglycylglycine,

C4H9CH(NH2)CO.(NHCH2CO)5.NHCH2COOH.

For the various combinations of the optically active amino-acids to
polypepteids 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 opti-
cal antipode the d-form. By the action of ammonia the d-amino-acid
is now obtained which in the above-mentioned manner can be retrans-
formed into the Z-form. Thus from d-leucine we first obtain Z-bromisoca-
proic acid and then by the action of ammonia Z-leucine and in the prepara-
tion of the polype*ptides the same occurs. Thus, for example, if by
reversion d-leucine is changed first into Z-bromisocapronyl chloride, if
this last is combined 'with Z-leucine, then*we obtain the dipeptide Z-leucyl-

88 THE PROTEIN SUBSTANCES.

Z-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.

A comparison of the artificially prepared polypeptides with the pro-
teins, and especially with the cleavage products of these last, the so-called
proteoses 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
octodecapeptide 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.

The behavior of the polypeptides with proteolytic enzymes is of
especially great interest. As far as known no artificial polypeptide
is split by gastric juice. On the contrary there are many, as shown by
FISCHER and especially by ABDERHALDEN and his collaborators, that
are split by pancreatic juice or intestinal juice or by yeast-press juice,
and by many enzymes occurring in animal tissues and fluids. The
constitution of the polypeptides is of the greatest importance in this
connection. Thus, for example, FISCHER and ABDERHALDEN,1 have found
with experiments with dog pancreatic juice that d-alanyl-d-alanine
and Z-leucyl-Z-leucine are split, but not d-alanyl-Z-alanine or Z-leucyl-
d-leucine. It sterns that only those polypeptides which are constructed
from amino-acids occurring in nature are hydrolyzable by enzymes.
If a racemic dipeptide is in part hydrolyzable then it is composed in part
of amino-acids occurring in nature. Only that part is split off and the
cleavage takes place asymmetrically. If for example, we take the two
racemic dipeptides from alanine and leucine mentioned on page 86,
we find that of the two racemic bodies only one (d-alanyl-Z-leucine + 1-
alanyl-d-leucine) , which contains the combination d-alanyl-Z-leucine, i.e.,
the two optical forms found in nature, is in part hydrolyzed while the
other (d-alanyl-d-leucine -f Z-alanyl-Z-leucine) is not attacked. The hydrol-
ysis of the first racemic body is in this case referable, it seems, to the
combination d-alanyl-/-leucine.

We will refer again, in Chapter IX, to this important and interesting
enzymotic cleavage of the polypeptides.' It is sufficient here to call atten-

1 Zeitschr. f. physiol. Chem., 51. The numerous works of Abderhalden and collab-
orators cannot be here especially cited, but they may be found in Zeitschr. f. physioL
Chem. Bdd., 48, 49, 51, 52, 53, 54, 55, -56, 57.

NATURAL POLYPEPTIDES. 89

tion to the fact that a hydrolysis of polypeptides as well as proteins by
the same enzymes with the splitting off of amino-acids is not sufficient
evidence that the proteins contain the same form of amino-acid chains
as the polypeptides.

We have also a very important support for this view in the fact that
polypeptides occur in the cleavage products of the proteins. This is,
to a certain extent, the reverse of the above-mentioned syntheses. After
FISCHER and BERGELL were able to isolate from the cleavage products
of silk fibroin a dipeptide in the form of an anhydride which seemed
to be a glycylalanine, FISCHER and ABDERHALDEN 1 further studied
this question, and by the partial hydrolysis of fibroin obtained not
only the above-mentioned polypeptide, • which they showed was glycyl-
d-alanine anhydride, but also glycyl-Z-tyrosine anhydride, and they
established the identity by the correspondence with the synthetically
prepared bodies. From elastin they obtained glycyl-Z-leucine anhydride,
Z-leucyl-d-alanine anhydride, glycyl-d-valine anhydride and a dipep-
tide anhydride which yielded d-alanine and Z-proline on hydrolysis.
They have also shown the presence of other polypeptides among the
hydrolytic products; we especially mention one tetrapeptide obtained
from fibroin which consisted of 2 molecules glycocoll, 1 molecule d~
alanine and 1 molecule Z-tyrosine. This tetrapeptide gave the biuret
reaction and also Millon's reaction. It was precipitated by tannic acid,
by saturated ammonium sulphate solution, and by NaCl in the presence
of free acid, and in many regards behaved like a proteose.

Polypeptides have also been found by other investigators in the hydro-
lytic cleavage products of proteins. Thus LEVENE with WALLACE and
BEATTY isolated a glycyl-Z-proline anhydride from gelatin after tryptic
digestion, and LEVENE and BEATTY have shown the presence of a lysyl-
glycyl peptide among the tryptic cleavage products of ovalbumin. OS-
BORNE and CLAPP2 obtained from a plant protein, gliadin, a crystalline
dipeptide which yielded phenylalanine and proline on cleavage.

We have therefore conclusive basis 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 bindings 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

1 Fischer and Bergell, see Bicohem. Centralbl., I, p. 84. Fischer and Abderhalden,
Sitz. Ber. d. k. Acad. d. Wissensch., 30 (1907) and Ber. d. d. chem. Gesellsch. 39 and 40.

2 Levene and Wallace, Zeitschr. f. physiol. Chem., 47; Levene and Beatty, Ber.
d. d. chem. Gesellsch., 39, and Biochem. Zeitschr., 4; Osborne and Clapp, Amer. Journ.
of Physiol., 18.

90 THE PROTEIN SUBSTANCES.

(diamino-valeric acid) by the imide binding. This imide binding
is 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 poly acidic 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 property of the proteins
is strongly influenced by the presence of other bodies, and under such
circumstances a proper classification, as demanded by science, is
impossible. On the other hand, a classification is important, and as the
ones used up to the present time were based upon the solubilities 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,
Albumins

^ Lactalbumin, and others.
f Fibrinogen,

Globulins i 0 7 , 7 .

[ Ser globulins, and others.

Phosphoproteins, (Nucleoalbu- J Ovovitellin,

mins) { Casein, and others.

Coagulated proteins.

Histones.

Protamines.

Zeitschr. f. physiol. Chem., 41.

PROTEIN CLASSIFICATION. 91

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.)

II. Compound Proteins
Nucleoproteins

f Mucin substances,
1 Ichthulin, and others.

f Hcemoqlobin,
Chromoprotems 1 „

[ 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 Amercian 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.

II. Conjugated Proteins.

A. Nucleoproteins.

B. Glycoproteins.

C. Phosphoproteins.
D Haemoglobins.
E. Lecithoproteins.

92 THE PROTEIN SUBSTANCES

III. 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.

III. 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.

SIMPLE PROTEINS. 93

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? a few contain also phosphorus. Iron is generally found
in traces in their ash, and it seems to be a regular constituent of a certain
group of the albuminous bodies, namely, the nucleoalbumins. The
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 50.6 —54.5 per cent.

H 6.5 -- 7.3

N 15.0 —17.6

S 0.3 -- 2.2

P 0.42— 0.85 "

O 21.50—23.50

The animal proteids are odorless, tasteless, and ordinarily amorphous.
The crystalloid spherules (D otter plattcheri) 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 proteids 2 have been prepared
from the seeds of various plants, and crystallized animal proteids (see
seralbumin and ovalbumin, Chapters VI and XIII) 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 footnote 3, p. 78.

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

94 THE PROTEIN SUBSTANCES.

tionable whether there exists any proteid body which is soluble in water
without the aid of mineral substances. Nevertheless it has not been
thus far successfully proven 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 are different, and the
maximum acid-combining power may perhaps also be used in the dif-
ferentiation of the various proteids (COHNHEIM, ERB, and others).

The acid-combining power of the proteins has been studied by means of
physical methods by SJOQVIST, BUGARSKY, and LIEBERMANN and with the aid
of chemical methods by SPIRO and PEMSEL, ERB, COHNHEIM and KRIEGER, and
v. RHORER. The methods pursued by COHNHEIM and KRIEGER consisted in pre-
cipitating the proteid from acid solution (HC1) with an alkaloid reagent (calcium
phosphotungstate) . The reaction takes place as follows : proteid hydrochloride
+ calcium phosphotungstate = proteid phosphotungstate + calcium chloride. The
acid remaining in the nitrate was determined, and when this quantity was sub-
tracted from the known original amount in the proteid solution, the difference
represented the acid combined with the proteid. If sodium picrate or potas-
sium-mercuric iodide is used instead of the phosphotungstate we have, accord-
ing to v. RnoRER,2 a method which is the best of all heretofore suggested.

The proteids can be salted out from their neutral solutions by neutral
raits (NaCl, Na2S04, MgS04, [NH4]2S04, 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 essentially
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 MgSO4 or (NH4)2SO4, is often made use of in the isolation of the
proteid, and special methods of separation are based upon fractional
precipitation. HASLAMS has recently shown that these methods may
lead to great errors, and give good results only under special conditions.

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-

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 Pfliiger's Arch., 90. In regard to the literature on this subject see Cohnheim,
Chemie der Eiweisskorper, 2. Aufl., pp. 107-109.

^See Cohnheiin, Chemie der Eiweisskorper, 2. Aufl., 1904, pp. 144-148; Pinkus,
Journ. of Physiol., 27; Pauli, Hofmeister's Beitrage, 3, p. 225; Halsam, Journ. of
Physiol., 32.

NATIVE AND MODIFIED PROTEINS. 95

tions in constant proportions, but are rather to be considered as loose
adsorption compounds of the proteid with the salt.1 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.

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 II. 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 solutions
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 slightly 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

1 See Galeotti, Zeitschr. f. physiol. Chem., 40, 42, 44, and 48 and Bonamartini and

Lombard!, ibid., 58.

96 THE PROTEIN SUBSTANCES.

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 various temperatures at
which coagulation of different proteids occurs in neutral solutions con-
taining 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 divided.1

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,2 and ether, by violent
shaking (RAMSDENS), 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 II in speaking of the gold equivalent. 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,S is based upon
this behavior.

Proteid solutions free from electrolytes have, according to PAULI,&
no electric charge, hence they do not migrate in an electric field. On the
addition of a trace of acid the proteid becomes electro-positive and moves
toward the cathode, while on the addition of alkali it becomes electro-
negative and wanders in the electric current to the anode. In the natural

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 1'Acad. 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 Miinchen, 1897; Pauli, Pfliiger's Arch., 78.

2 See Salkowski, Zeitschr. f. physiol. Chem., 31; Fr. Kriiger, Zeitschr. f. Biologic,
41; Loew and Aso, Bull. Coll. Agric., Tokio, 4.

3 Ramsden, Zeitschr. f. physik. Chem., 47 and Arch. f. (anat. u.) PhysioL,. 1894.

4 Biochem. Zeitschr., 5.

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

6 Hofmeister's Beitrage, 7.

PRECIPITATION REACTIONS. 97

fluids like the blood-serum it is electro-negatively charged, which fact
can be explained by the presence of an excess of OH ions.

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 very 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., depend-
ing on the amount of proteid present, and boiled before the addition of
each drop. If dilute nitric acid (25 per cent) be used, then to 10-15 cc. of
the previously boiled liquid 15-20 drops of the acid must be added. If
too little nitric acid be added, a soluble combination of the acid and pro-
teid is formed, which is precipitated by more acid. A proteid 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 idded 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 concen-
tration, are also geneial precipitants for the proteids.

1 See especially F. N. Schulz, Die Grosse des Eiweissmoleciile, Jena, 1903.

98 THE PROTEIN SUBSTANCES.

4. Precipitation by Metallic Salts such as copper sulphate, ferric
chloride, neutral and basic lead acetate (in small amounts), mercuric
chloride and others. On this is based the use of proteids as antidotes
in poisoning with metallic salts.

5. Precipitation by Mineral Acids 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. Pr-ecipitation by the so-called Alkaloid Reagents. To these belong
the precipitation by metaphosphoric 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
phosphomolybdic acid in the presence of free mineral acids; precipita-
tion by potassium-mercuric iodide or potassium-bismuth iodide in solu-
tions 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-
i'fied 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 acid 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, which depends on the presence
of the aromatic group in the proteid, is also given by tyrosine and 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

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 Pfliiger's
Arch., 83; see also Vaubel and Blum, Journ. f. prakt. Chem. (N. F.), 57.

COLOR REACTIONS. 99

a few drops of acetic acid are added. 2. Xanthoproteic Reaction. With
strong nitric acid the albuminous bodies give, on heating to boiling,
yellow flakes or a yellow solution. After making alkaline with ammonia
or alkalies the color becomes orange-yellow. 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 contact of the two liquids. Gela-
tin does not give this reaction.

A similar color is obtained, according to AGREE, with formaldehyde. About
0.1 gm. of the substance is treated with 0.1 cc. formaldehyde solution (1:5000)
and then after 2-3 minutes carefully treated with 0.5 cc. sulphuric acid. At
the boundary of the two layers a violet coloration occurs. According to ROSEN-
HEIM 2 this reaction occurs in the presence of oxidizing substance only, and further
investigations into this reaction are necessary.

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, producing
a violet color, and when they are previously boiled with alcohol and
then washed with ether (LIEBERMANN 3) they give a beautiful blue
solution. This blue color is due, according to CoLE,4 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, produced from the furfurpl formed by the action of the
concentrated hydrochloric acid upon the proteid. 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-dimethylaminobenzal-

1 Proceed. Roy. Soc., 68.

2Acree, Amer. chem. Journ., 37; Rosenheim, Chem. Centralbl., 1907, p. 1809.
See also Dakin, Journ. of Biol. Chem., 2.

3 Centralbl. f. d. med. Wissensch., 1887.

4 Journ. of Physiol., 30.

100 THE PROTEIN SUBSTANCES.

dehyde and concentrated sulphuric acid the proteids give a beautiful
reddish-violet or deep-violet coloration (O. NEUBATJER and E. RoHDE1).

Many of these color reactions are obtained as shown by SAT/KOWSI,Z by the
aromatic or heterocyclic cleavage products of the proteids. MILLON'S reaction
is given only by the substances of the phenol group ; the XANTHOPROTEIC reac-
tion by the phenol group and skatol or the indol group. LIEBERMANN'S reaction
depends, according to COLE, upon the indol group, and the reactions with sul-
phuric acid and sugar (COLE) and with diamethylaminobenzaldehyde (ROHDE)
are also caused by this group. ADAMKIEWICZ'S reaction is given only by the
bodies of the indol group. The biuret reaction is not only given by protein
substances, but also by many other bodies. According to H. SCHIFF 3 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 men-
tion several diamines or aminoamides, such as oximide, biuret, glycinamide,
«- and /?-aminobutyramide, aspartic-acid amide, etc., although we are not clear
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.4

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-
mine the quantity of acetic acid necessary to completely precipitate
the proteids in small measured portions of the neutralized liquid which

1 Zeitschr. f. physiol. Chem., 44.

2 Md., 12.

3 Ber. d. d. chem. Gesellsch., 29 and 30.

4 In regard to the precipitation and coloration reactions of proteids with aniline
dyes see Heidenhain, Pfliiger's Arch., 90, 96.

PRECIPITATION OF PROTEINS. 1 jl

have previously been heated on the water-bath, so that the filtiate epes
not respond to HELLER'S test. Xow 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 filtrate 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.

The precipitation by means of alcohol may also be used in the quan-
titative estimation of proteids. The liquid is first carefully neutralized,
treated with some NaCl if necessary, and then alcohol added until the
solution contains 70-80 vol. per cent anhydrous alcohol. The precipitate
is collected on a filter after 24 hours, extracted with alcohol and ether,
dried, weighed, incinerated, and again weighed. This method is only
applicable to liquids which do not contain any other substances, like
glycogen, which are insoluble in alcohol.

In both of these methods small quantities of proteid may remain in
the filtrates. These traces may be determined as follows: Concentrate
the filtrate sufficiently, remove any separated fat by shaking with ether,
and then precipitate with tannic acid. Approximately 63 per cent of
the tannic-acid precipitate, washed with cold water and then dried, may
be considered as proteid.

In many cases good results may be obtained by precipitating all the
proteid with tannic acid and determining the nitrogen in the washed pre-
cipitate by means of KJELDAHL'S method. On multiplying the quantity
of nitrogen found by 6.25 we obtain the quantity of proteid.

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 filtrates may be separated by boiling with freshty 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 the alter-
nate addition of potassium-mercuric iodide and hydrochloric acid (see
Chapter VIII, on Glycogen Estimation), or by trichloracetic acid as
suggested by OBERMAYER and FRAXKEL.2 Recently MICHAELIS and
RONA have suggested a method for the removal of proteids by using
kaolin, colloidal lerric hydrate or a mastic emulsion. The principle of
these methods has already been given on page 96 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.

1 Zeitschr. f. physiol. Chem., 2 and 4.

2 Obermayer, Wien. med. Jahrb., 1888; Frankel, Pfliiger's Arch., 52 and 55.

3 Zeitschr. f. physiol. Chem., 30.

102 THE PROTEIN SUBSTANCES.

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 and are not precipitated
by the addition of a little acid or alkali. They are 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 MgSO4 is added to satur-
ation to a neutral solution in water at the normal temperature 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-
cipitated by carbon dioxide, but the precipitate may be redissolved by
an excess of the precipitant. The neutral solutions of the globulins
containing salts are partly or completely precipitated on saturation 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.

According to J. STARKE l the globulins are not soluble in dilute salt solutions,
but form alkali proteid compounds whose solubility in salts is brought about by

1 Zeitschr. f. Biologic, 40 and 42. In regard to the various views on this subject
see Wolff and Smits, ibid., 41; Osborne, 1. c. Hammarsten, Ergebnisse der Physiologic,
Jahrg. I, Abt. 1.

PHOSPHOPROTEINS. 103

an increase in the free OH ions produced by the salts. This view is not
tenable for several globulins, and seems in fact not to be well founded.

That a sharp line cannot be drawn between the albumins and glob-
ulins follows from the fact that the albumins can be converted into glob-
ulins. The possibility of a conversion of ovalbumin into globulin is
based upon the observations of STARKE. That a transformation of
seralbumin into serglobulin with the aid of the weak action of alkali in the
warmth, with the splitting off of sulphur, can take place, has been more
conclusively shown by MOLL 1 by experimenting with blood-serum as
well as with crystalline seralbumin. According to MOLL, first pseudoglob-
ulin is formed from the seralbumin, and then euglobulin (see Chapter
VI) . The artificial globulins thus obtained had the same sulphur content
and properties as the natural products.

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 glob-
ulins, and not with a true transformation of the albumin into globulin.
This follows from the fact that by the action of weak alkali upon albumin,
which is free from glycocoll, we do not obtain globulin which contains
glycocoll. This is an instructive example of the subordinate importance
the solubility and precipitation properties have in the differentiation of
various groups of proteids.

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. OsBORNE,2 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 albumin-
ate 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 hi the animal and plant kingdoms and which include the
nucleoalbumins and the little-studied lecithalbumins.

Nudeoalbumins. These proteids behave like rather strong acids,
are nearly insoluble in water, but dissolve easily with the aid of a little
alkali and, in the entire absence of lecithin, 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 phosphorus. They stand

1 Hofmeister's Beitrage, 4 and 7.

2 Zeitschr. f. physiol. Chem., 33.

104 THE PROTEIN SUBSTANCES.

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 neucleoalbumins any proteid-
free pseudonucleic acids corresponding to the nucleic acids, but only
acids rich in phosphorus, which always give the proteid reactions (LEVENE
.and ALSBERG, SALKOWSKI, REH1). 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
lias been called para- or pseudonuclein. The claim made that the pseu-
donuclein is a combination of proteid with metaphosphoric acid has
been shown to be incorrect by the investigations of GiERTz.2 The nucleo-
albumins always seem to contain some iron.

The separation of pseudonuclein in peptic digestion is no doubt characteristic
of the nucleolabumin 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 WROBLEWSKI did not
obtain any pseudonuclein at all in the digestion of the casein from human milk.
WIMAN 3 has also shown in the digestion of vegetable nucleoalbumin that the
obtainment of considerable pseudonuclein or none is dependent upon the way in
which the digestion is performed. The most essential characteristic of this group
of proteids is that they contain phosphorus, and that the purine bases are absent
in their cleavage products.

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 a subsequent chapter (XIV).

Lecithalbumins. In the preparation of certain protein substances,
products are often obtained containing lecithin, and this lecithin can
be removed only with difficulty or incompletely by a mixture of alcohol
and ether. OvoviteTUn (Chapter XIII) is such a protein body con-
taining considerable lecithin, and HOPPE-SEYLER considers it a combina-
tion of proteid and lecithin. Similar substances occur in fish-eggs.
These last lecithalbumins often have the solubilities of the globulins;

1 Levene and Alsberg, ibid., 31; Salkowski, ibid., 32; Levene, ibid., 32; A. Reh,
Hofmeister's Beitrage, 11.

2 Giertz, Zeitschr. f. physiol. Chem., 28.

3 Salkowski, Pfliiger's Arch., 63; Wroblewski, Beitrage zur Kenntnis des Frauen-
kasei'ns, Inaug.-Diss., Bern, 1894; Wiman, Upsala Lakaref. Forh. (N. F.), 2.

LECITHALBUMINS. 105

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 considerable
amounts of lecithin, is readily soluble in dilute NaCl solution, but at
ordinary temperatures it is changed by 0.1 per cent HC1 nearly instanta-
neously and without splitting off lecithin, so that it becomes insoluble
in dilute salt solutions (HAMMARSTEN). LIEBERMANN 1 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
lecithaibumins. Further investigation of these bodies is desirable.

MAYER and TERROINE 2 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 lecithaibumins, 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
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, ovovitellin, although not quite
pure. The results are based on 100 parts of the substance.

The proteins occurring in the plant kingdom correspond in part to
the above-described three groups of animal proteids. Among these the
globulins are especially represented, and as an example we will specially
mention the crystalline edestin, occurring in the hemp-seed. It is not
clear whether the phosphorized plant proteids contain their phosphorus
as impurities or whether they are the same as the animal phospho-
proteins. There is no doubt that certain vegetable proteids cannot
be classified in the above groups, namely, gliadin of the wheat and zein
of the corn kernel, which are proteins soluble in alcohol. They are also
characterized by not yielding any lysine.

1 Hoppe-Seyler, Med. chem. Untersuch., 1868; also Zeitschr. f. physiol. Chem., 13,
479; Hammarsten, Skand. Arch. f. Physiol., 17; Liebermann, Pfliiger's Archiv, 50
.and 54.

2 Compt. rend. soc. biol., 62.

106

THE PROTEIN SUBSTANCES.

Lact-
albumin.1

Ser-
albumin.4

Ov-
albumin.5

Ser-
globulins.2

Fibrin.11

Casein.2

Vitellin.iO

Glycocoll

0 0

0 0

0 0

3 5

3 0

0 0

i i

Alanine
Valine . .

2.5

0 9

2.7

2.1

1 „-,«

2.2

_(-

3.6
1 0

0.9
1 0

+
9 4

Leucine
Serine

20.0
0 6

..,}"•

18.7

15.0
0 8

10.5
0 93

11.0

Aspartic acid ....
Glutamic acid. . . .
Cystin
Phenylalanine . . .
Tyrosine

1.0
10.1

2^4
0.85

3.1

7.72
2.533
3.1
2.1

1.5

8.0
0.33
4.4
2 43

2.5
8.5
1.513
3.8
2 5

2.0
10.4
1.173
2.5
3 5

1.2
11.1

0.073
3.2
4 5

0.5
12.2

'2.8
1 fi

Proline

4.0

1.04

2.25

2 8

3 6

3 1

3 3

Oxyproline
Tryptophane ....
Histidine

0.25
1.59

2 598

Arginine
Lysine

...

3.07
4.07

4.848
5.808

1 Abderhalden and H. Pribram, Zeitschr. f . physiol. Chem., 21.

2 Abderhalden, Lehrb. d. physiol. Chem., 1909.

3 K. Moraer, Zeitschr. f. physiol. Chem., 34.

4 Abderhalden, ibid., 37.

5 Abderhalden and Pregl., ibid.., 46.

6 Levene and Beatty, Bicohem. Zeitschr., 4.

7 Kutscher, Endprodukte der Trypsin Verdauung, Habit. Schrift., Marburg, 1899.

8 E. Hart, Zeitschr. f. physiol. Chem., 33.

9 Hopkins and Cole, Journ. of Physiol., 27.

10 Abderhalden and Hunter, Zeitschr. f. physiol. Chem., 48.

11 Abderhalden and Voitinovici, ibid., 52, p. 371.

In the tabulation on page 107 we give the cleavage products of certain
vegetable proteids; and in certain cases when the analytical results by
two investigators differ somewhat, we will give the results side by side.
The edestin originated from the hemp-seed, the legumin from the pea,
the hordein from barley, the gliadin from wheat and the zein from corn.

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 (RAMS-
DEN l), and in certain cases, as in the conversion of fibrinogen into
fibrin (Chapter VI), 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.

1 Arch. f. (Anat. u.) Physiol., 1894.

HISTONES.

107

Edestin. l

Legumin. 3

Hordein.

Gliadin.

Zein.

Glycocoll
\lanine

3.8
3.6

0.38
2.08

a4 65
0.0
0.43 1.34

a6 67
O.Q2 0.9
2.0 2 . 66

a8 69
0.0
2.23

Valine

+

l.O2

0.13 1.40

0.21 0.33

0.29

Leucine

20.9

8.0

5.67 7.0

5.61 6.00

18.60

Serine

0 33

0 53

. .. 0.10

0 13 0 12

0 57

A^partic acid

4 5

5 3

1.32

05 1 24

1 41

Ghitamic acid.

6 3

13.8

36.35 41.32

37.33 31.5

18 28

Cystine . . . •

0.25

0.45

Phenylalanine
Tyrosine

2.4
2.1

3.75
1.55

5.03 5.48
1.67 4.0

2.35 2.6
1.20 2.37

4.87
3.55 10.10

Proline

1.7

3.22

13.73 5.88

7 . 06 2.4

6 . 53

Oxyproline
Tryptophane
Histidine

2.0

+
1.1

+ "
2.42

+
i.28 0.51

' + ' i'.Q

0.61 1.7

6!6
0 . 43

Arginine
Lysine
Ammonia

11.7
1.0

10.12
4.29
1.49

2.16 3.14
0.0 0.0
4.87 4.34

3.16 3.4
0.0 0.0
5.11 ....

1.16
0.0 . .
3.61 . .

1 Abderhalden, Zeitschr. f. physiol. Chem., 37 and 40.

2 Abderhalden and Babkin, ibid., 47.

3 Osborne and Clapp, Journ. of Biol. Chem., 3. '

4 Osborne and Clapp, Amer. Journ. of Physiol., 19.

5 Kleinschmitt, Zeitschr. f. physiol. Chem., 54.

6 Osborne and Clapp, Amer. Journ. of Physiol., 17.

7 Abderhalden and Samuely, Zeitschr. f. physiol. Chem., 44, and Abderhalden,
Lehrbuch d. physiol. Chem., 1909.

8 Osborne and Clapp, Amer. Journ. of Physiol., 19.
• Kutscher, Zeitschr. f. physiol. Chem., 38.

Coagulated .proteins also seem to occur in animal tissues. We find,
at least in many organs such as the liver and other glands, proteins
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) ,

108 THE PROTEIN SUBSTANCES.

from the burbot, (lotahistone, EHRSTROM), and from the sea-urchin
(arbacin, MATHEws)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: The neutral salt-free solu-
tion first, 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 reap-
pears on cooling.

The different histones behave differently in 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 OsBORNE's2 edestan, also give these two
reactions; therefore it is impossible by qualitative tests alone to identify
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 rich in arginine, namely, 14.14 per cent (OSBORNE and CLAPP3). 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
RON A4 obtained from thymus histone the following: leucine ll.£,

1 Kossel, Zeitschr. f. physiol. Chem., 8, and Sitzungbers. der Gesellsch. zur BeforcL
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.

3 Amer. Journ. of Physiol., 19.

4 Zeitschr. f. physiol. Chem., 41

PROTAMINES. 109

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 l
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
pic rate. 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.

The parahistone found by FLEROFF in the thymus gland yields so little basic
nitrogen that it probably does not belong to the histone group (KossEL and
KUTSCHER 2).

Protamines. In close relation to the proteins stands a group of
substances, the protamines, discovered by MIESHER, 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,3 and the investigations of KOSSEL and
WEISS 4 have shown that the material from which the protamines are
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 fractious thereof. According to the generally accepted
view we will treat them as true proteids.

Protamine was discovered by MIESCHER 5 in salmon spermatozoa.

1 Zeitschr. f. physiol. Chem., 49.

2 Fleroff, Zeitschr. f. physiol. Chem., 28; Kossel and Kutscher, I.e.

3 Nelson, Arch. f. exp. Path. u. Pharm., 59, has recently shown that the body called
by him thymamin and prepared from the thymus glands, is a protamine, still he has
not given sufficient evidence of the protamine nature of the substance.

4 Zeitschr. f . physiol. Chem., 52.

5 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, and 44, and Sitzungsber. der
Gesellsch. zur Beford. der ges. Naturwiss. zu Marburg, 1897; Berl. klin. Wochenschr.,
1904; Kossel and Mathews, Zeitschr. f. physiol. Chem., 23 and 25; Kossel and Kutscher,
ibid., SI; Goto, ibid., 37; Kurajeff, ibid., 32; Morkowin, ibid., 28; Kossel and Dakin,
ibid., 40, 41, and 44; Maleniick, ibid.t 57; Nelson, Arch. f. exp. Path. u. Pharm., 59.

110 THE PROTEIN SUBSTANCES.

Later KOSSEL 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, scombine, sturine, cyprinine, cyclopterine, etc.

They differ essentially 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"'
moleculer weight.

The percentage composition of these bodies has not been satisfactorily
determined. As probable formulae we have for salmine C32H54Ni8O4
(MIESCHER, SCHMIDEBERG, NELSON), or C3oH57H17O6 (KOSSEL and GOTO),
for clupeine CaoH^N^Oo, and for sturine C36H69H19O7 (KOSSEL) or
C34H7iN17O9 (GOTO), or according to MALENUCK C27H55Hi3P7 for sturine
from Accipenser Guldenstadtii. On boiling with dilute mineral acids,
as also by tryptic digestion, the protamines first yield peptone-like sub-
stances called protones, from which simple products are derived on fur-
ther 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 salmine, clupeine
and scombrine the arginine nitrogen, according to KOSSEL and PRiNGLE,1
amounts to about 89 per cent of the total nitrogen. Sturine yields besides
this the two hexone bases lysine, 12 per cent, and histidine, 12.9 per cent.
Histidine has not been found in any other protamine. The carp pro-
tamine, cyprinine, occurs in two different modifications, namely, a-
and ^-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-carboxylic 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 proline. KOSSEL
believes that diarginide or polyarginide groups also occur in the pro-
tamines, and in clupeine we can accept the presence of diarginylalanine,
diarginylserine, diarginylprolme, and diarginylvaline (KOSSEL and
PRINGLE) .

The following summary according to KOSSEL 2 gives a view of the
cleavage products of the protamines thus far investigated :

1 Zeitschr. f . physiol. Chem., 49. 2 Ibid., 44.

ALBUMINOIDS.

Ill

Scorn-
brine.

Salmine.

Clupeine.

Sturine.

Cyclop-
terine.

a-C.yp-
nnine.

/9-Cyp-
rinine.

Alanine
Serine
Aminovaleric acid
Leucine
Arginine

+

0
0
0

+

0

+
+

0

+

+ 0 + + +

+
0
0

+
+

?

?

9
?

+

?

9

+
?

+

?
?

+
?

+

L/ysine

o

o

0

+

o

+

+

PlisticlinG

o

o

0

+

0

o

o

Prolin6

+

+

+

0

?

?

?

Tvrosine

o

0

0

0

+

0

o

Tryptophane

0

0

0

0

+

0

0

Solutions of these bases in water are alkaline and have the property
of giving precipitates with ammoniac al solutions of proteins or primary
proteoses, but the researches of HUNTER x 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. They give thebiuret
test beautifully, but with the exception of cyclopterine and /?-cyprinine
do not give MILLON'S reaction. The protamine salts are precipitated
in neutral or even faintly alkaline solutions by phosphotungstic acid,
picric acid, chromic acid, and alkali ferrocyanides.

The protamines are prepared, according bo 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 neces-
sary, 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 platinum
chloride. MIESCHER also precipitates the base as a double-platinum 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

Zeitschr. f. physiol. Chem., 53.

112 THE PROTEIN SUBSTANCES.

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 91.

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 l the organic
matrix of the eggshells of various vertebrate animals belongs in most
cases to the keratin group.

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 : 2

c H N s o

Human hair ... 50.65 6.36 17.14 5.00 20.95 (v. LAAR)

Nail 51.00 6.94 17.51 2.80 21.75 (MULDER)

Neurokeratin . . 56.11-58.45 7.26-8.02 11.46-14%.32 1.63-2.24 (KUHNE)

Neurokeratin. . . 56.61 7.45 14.17 2.27 (ARGIRIS)

Horn (average). 50.86 6.94 3.20 (HORBACZEWSKI)

Tortoise shell .. 54.89 6.56 16.77 2.22 19.56 (MULDER)

Shell membrane 49.78 6.64 16.43 4.25 22.90 (LiNDV ALL)

Egg membrane. 53.92 7.33 15.08 1.44 (PREGL)

(Scyllium)

MoHR3 has determined the quantity of sulphur in various keratin
substances. Sulphur is in great part in loose combination, and it is
chiefly removed 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 sulphuretted hydrogen or mercaptan
(BAUER), and the solution contains proteose-like substances (KRUKEN-

1 Kiihne and Ewald, Verb. d. naturhistor.-med. Vereins zu Heidelberg (N. F.), 1;
also Kiihne and Chittenden, Zeitschr. f. Biologic, 26; Neumeister, ibid., 31.

2 v. Laar, Annal. d. Chem. u. Pharm., 45; Mulder, Versuch einer allgem. physiol.
Chem., Braunschweig, 1844-51; Kiihne, Zeitschr. f. Biologie, 26; Horbaczewski, see
Drechsel in Ladenburg's Handworterbuch. d Chem., 3; Lindvall, Maly's Jahres-
bericht, 1881; Argiris, Zeitschr. f. physiol. Chem., 54; Pregl., ibid., 56.

3 Zeitschr. f. physiol. Chem., 20.

KERATINS. 113

BERG) called atmidkeratin and atmidkeralose by BAUER. 1 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,2 have recently found glycocoll, alanine, a-amino valeric acid,
proline, serine, phenylalanine, 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 was the first to positively prove 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 3 obtained the following amounts of cystine from the respec-
tive keratin formations, 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 and 2.17 per cent from pig hoofs. 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. GALIMARD 4
was able to get only a qualitative test for cystine in the keratin of the
adder eggs. SUTER, MORNER, and FRIEDMANN 5 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 114.

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

1 Krukenberg, Untersuch. iiber d. chem. Bau d. Eiweisskorper, Sitzunsber. d.
Jenaischen Gesellsch. f. Med. u. Naturwissensch., 1886; Bauer, Zeitschr. f. physiol.
Chem., 35.

2 Zeitschr. f. physiol. Chem., 3(>, which contains also the older literature.

3 Morner, ibid., 34 and 42; Emmerling, Ref. in Chemiker Zeitung, 1894; Buchtala,
Zeitschr. f. physiol. Chem., 52.

4 Chem. Centralbl. II, 1905.

5 Suter, Zeitschr. f. physiol. Chem., 20; Morner, ibid., 42; Friedmann, Hofmeister's
Beitriige, 2.

114

THE PROTEIN SUBSTANCES.

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
as thus far investigated do not chemically form a sufficient characteristic
group.

Keratin
from
Horse-
hair.i

Keratin
from
Sheep
Wool.4

Keratin
from
Goose
Feathers6

Keratin
from
Sheep
Horn.4

Shell
Mem-
brane
of the
Hen's
egg.6

,Fgg

Mem-
brane
of Seal-
hum
stellar e.*

Egg
Mem-
brane
of Tes-
tudo
graeca.g

Glycocoll

4 7

0 58

2 6

0 45

3 9

2 6

-f

Alanine

1 5

4 40

1 8

1 6

3 5

3 2

-j-

Valine

0.9

2 80

0 5

4 5

1 1

Leucine

7 1

11 5

8 0

15 3

7 4

5 8

-f

Serine

0.6

0 1

0 4

1 1

Aspartic acid

0.3

2 3

1 1

2 5

1 1

2.3

1.2

Glutamic acid 10
Cystine

3.7

7 982

12.9

7 3

2.3

17.2

7 5

8.1
7 6?7

7.2
?

2.9

Phenylalanine

0.0

0 0

1.9

3.3

+

Tyrosine

3 2

2 9

3 6

3 6

0 0

10 6

Proline

3 4

4 4

3 5

3 7

4 0

4 4

11 8

Histidine

0 613

1 7

Arginine

4 453

2 7

3 2

Lysine

1.12s

_

_

0.2

_

3.7

_

1 Abderhalden and Wells, Zeitschr. f. physiol. Chem., 46.

2 Buchtala, ibid., 52.

3 Argiris, ibid., 54.

4 Abderhalden and Voitinovici, ibid., 52.

5 Abderhalden and Le Count, ibid., 46.

6 Abderhalden and Ebstein, ibid., 48.

7 Korner, ibid., 34 and 42.

8 Pregl, ibid., 56.

9 Abderhalden and Strauss, ibid., 48.

10 Abderhalden and Fuchs, Zeitschr. f. physiol. Chem., 57, have recently shown that
the same variety of keratin, on ageing of the horn structure, becomes somewhat poorer
in glutamic acid.

Bodies occur in the animal kingdom which form to a certain extent
intermediate substances between coagulated protein and keratin. C.
TH. MORNER l has detected such a body (albumoid) 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-

1 See Maly's Jahresber., 18.

ELASTIN. 115

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. HEDENIUS this substance is insoluble in gastric
or pancreatic juice, and acts quite like keratin. According to K. B.
HOFMANN and PREGL,1 who call this substance koilin, it does not yield
any cystine on hydrolysis, or at least not a determinable quantity, and
differs from the keratins in this and other regards.

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 nuchae).

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
to prepare an elastin containing sulphur from the aorta by another method,
and this sulphur can be removed by the action of alkalies, without chang-
ing the properties of the elastin; and recently ZOJA, HEDIN, BERGH,
and RICHARDS and GIES 2 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:

1.

2.
3.

C
54.
54.
53.

32
24
95

H
6.99
7.27
7.03

N
16.
16.
16.

75
70
67

S
6.38

0
21.
21.

94
79

(HORBACZEWSKI 3

(CHITTEDDEN and
(H. SCHWARZ)

HART)

1 Hedenius, Skand. Arch. f. Physiol., 3; Hofmann and Pregl, Zeitschr. f. physiol.,
Chem., 52.

2 Chittenden and Hart, Zeitschr. f. Biologic, 25; Schwarz, Zeitschr. f. physiol.
Chem., 18; Zoja, ibid., 23; Bergh, ibid., 25; Hedin, ibid.; Richards and Gies, Amer.
Journ of Physiol., 7.

3 Zeitschr. f. physiol. Chem., 6.

116 THE PROTEIN SUBSTANCES.

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 was 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
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 quantity of hydrolytic cleavage products are given in the table
on page 124. 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). This fact and the very low sulphur content
make it questionable whether the elastin is a unit body.

Indol and skatol have not been found on the putrefaction of elastin,1
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. According
to RICHARDS and GIES, elastoses, especially protoelastoses, and true
peptones are formed, the latter only to a slight extent.

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 origins 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.

1 See Walchli, Journ. f. prakt. Chem. (N. F.), 17.

COLLAGEN.

117

On account of its great resistance to chemical reagents, elastin may
be prepared (best from the ligamentum nuchse) in the following way:
First boil with water, then with 1 per cent caustic potash, then again
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. Col-
lagen 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

c H N s o

Collagen 50 . 75 6 . 47 17 . 86 24 . 92 (HOFMEISTER)

Gelatin (commerical) .... 49.38 6.80 17.97 0.7 25.13 (CHITTENDEN)

Gelatin from tendons 50.11 6.56 17.81 0.26 25.26 (VAN NAME)

Gelatin from ligaments ... 50.49 6.71 17.90 0.57 24.33 (RICHARDS and GIES)

Fish glue (isinglass) 48 . 69 6 . 76 17 . 68 (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 contam-
ination with a substance rich in sulphur or to a splitting off of loosely
combined sulphur during the purification. C. Mo'RNER4 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,

1 Hoppe-Seyler, Physiol. Chem., p. 97.

2 Zeitschr. f. physiol. Chem., 2.

3 Hofmeiser, 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.

118 THE PROTEIN SUBSTANCES.

and some after treatment with sodium hydroxide and then carbonate, showed
somewhat different physical properties among each other, but had about the same
elementary composition, with 0.30-0.526 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 somehwat richer in sulphur, 0.53-0.712 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. 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 124. Of special
mention is the fact the gelatin contains no tyrosine but does yield con-
siderable glycocoll. This latter substance has, because of its sweet
taste, been called gelatin sugar. SKRAUP1 has obtained on the hydro-
lytic cleavage of gelatin a crystalline acid having the formula Ci2H25N5Oio,
which he calls glutinic acid. Gelatin yields considerable basic nitro-
gen, according to HAUSMANN,2 35.83 per cent of the total nitrogen.
DRECHSEL and FISCHER found lysine; HEDIN, KOSSEL and KUTSCHER 3
found arginine also, which amounted to 9.3 per cent (KossEL and
KUTSCHER). On putrefaction gelatin gives neither tyrosine, indol, nor
skatol.- According to SELTRENNY4 it yields phenylpropionic acid and
phenylacetic acid. The aromatic group in gelatin is therefore, as
directly shown by FISCHER and also by SPIRO,S represented by phenyl-
alanine.

On the oxidation of gelatin with potassium permanganate, SEEMANN obtained
besides volatile fatty acids (formic, acetic, butyric acids), benzoic acid, oxalic
acid, succinic acid, oxaluramide and probably also oxaluric acid. ZICKGRAF 8
produced guanidine from the arginine.

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

1 Monatshefte f. Chem., 26.

2 Zeitschr. f. physiol. Chem., 27.

3 Drechsel, Arch. f. Anat. u. Physiol., 1891; Hedin, Zeitschr. f. physiol. Chem., 21;
Kossel and Kutscher, ibid., 31.

4 Monatshefte f. Chem., 10.

5 Fischer, Levene and Aders, Zeitschr. f. physiol. Chem., 35; Spiro, Hofmeister's
Beitrage, 1.

8 Seemann, Zeitschr. f. physiol. Chem., 44; Zickgraf, ibid., 41.

GELATIN. 119

or heated with^ water above 70° C.1 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 RoNA2 have shown, various bodies may
have a different influence upon the gelatinization-point of a gelatin
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; by acetic acid and
common salt in substance ; mercuric chloride in the presence of HC1 and
NaCl ; by metaphosphoric 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'S. 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 /?-glutin by NASSE. According to NASSE
and KRUGER the specific rotatory power is hereby reduced from —167.5°
to about — 1360.3 On prolonged boiling with water, especially in the
presence of dilute acids, also in the gastric or tryptic digestion, thfe 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 hemi-
collin, are formed. The former is insoluble in alcohol of 70-80 per cent
and is precipitated by platinum chloride. The latter, which is not pre-
cipitated by platinum chloride, is soluble in alcohol. CHITTENDEN and
SOLLEY 4 have obtained in the peptic and tryptic digestion a proto- and

1 Kiihne and Ewald, Verb. d. Naturhist. Med. Vereins in Heidelberg, 1877, 1.

2 Hofmeister's Beitrage, 2.

3 Nasse and Kriiger, Maly's Jahresber., 19, p. 29. In regard to the rotation of
/?-glutin, see Framm, Pfliiger's Arch., 68.

4 Hofraeister, 1. c.; Chittenden and Solley, 1. c.

120 THE PROTEIN SUBSTANCES.

a deutero-gelatose, besides a true peptone. The elementary composition
of these gelatoses does not essentially differ from that of the gelatin.

According to LEVENE the proto- as well as the deuterogelatoses yield
a larger amount of glycocoll than the gelatin itself. On prolonged tryptic
digestion a further demolition takes place, so that the peptone yields
only about the same amount of glycocoll as the gelatin. Some leucine
and, as it appears, also some glutamic acid and phenylalanine are split
off. Quite a considerable splitting off of NH3 also takes place (LEVENE
ard STOOKEY).1

PAAL 2 has prepared gelatin-peptone hydrochlorides from gelatin by the
action of dilute hydrochloric acid. These salts are partly soluble in ethyl and
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 SCHEERMESSER and KRUGER and wThich
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 gela-
tin 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. 3

1 Levene, Zeitschr. f. physiol. Chem., 37; Levene and Stookey, ibid., 41.

2 Ber. d. deutsch. chem. Gesellsch., 25.

3 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.

RETICULIN, SKELETIXS. 121

Reticulin has the following composition: C 52.88; H 6.97; N 15.63;
S 1.8S; 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, sulphuretted hydrogen, ammonia, lysiner
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'S reactions, but not to
MILL-ON' s reagent.

According to TEBB reticulin is only a somewhat changed, impure collagen
but this is disputed by SIEGFRIED. 1

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 contamination
or as a combination with reticulin is removed. The thoroughly boiled
residue consists of reticulin.

Ichthylepidin is an organic compound, so called by C. MORNEE,* 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 with MILLON'S reagent,
the xanthoproteic reaction, and the biuret test. At least a part of the sul-
phur 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 than elastin
(ABDERHALDEN and VOITINOVICI 3).

As skeletins, KauKENBERG4 has designated a number of nitrogenized
substances which form the skeletal tissue of various classes of invertebrates.
These substances are chitin, spongin, conchiolin, byssus, cornein, and
crude silk (fibroin and sericiri). 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 pro-
tein group, and chitin will be discussed in another chapter.

1 Tebb, Journ. of Physiol., 27; Siegfried, ibid., 28.

2 Zeitschr. f . physiol. Chem., 24 and 37. See also Green and Tower, ibid., 35.

3 Zeitschr. f. physiol. Chem., 52, p. 368.

4 Grundziige einer vergl. Physiol. d. thier. Geriistsubst. Heidelberg, 1885.

122

THE PROTEIN SUBSTANCES.

H
6.54
6.88
6.30
6.35
5.90
6.27
6.50
6.18
6.32

N
16.60
17.86
16.20
16.40
16.81
18.31
19.20
18.30
17.14

S
0.85
0.31
0.50

(WETZEL)
(KRUKENBERG)
(CROOKEWITT)
(POSSELT)
(KRUKENBERG)
(CRAMER)
(VIGNON)
(CRAMER)

(BONDI)

The elementary composition of certain of the bodies belonging to
this group is as follows : 1

c
Conchiolin (from the shells of pinna) . . 52 . 70

(from snail eggs) 50 . 92

Spongin 46 . 50

" 48.75

Cornein 48 . 96

Fibroin 48.23

" 48.30

Sericin 44 . 32

" 44.50

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, lysin
3-4 per cent, and arginine 5.6 per cent.2 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 3 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 4 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 arid
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 6 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 7 and differs from
the pennatulin of the Pennatulidese 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

1 Krukenberg, Ber. d. d. chem. Gesellsch., 17 and 18, and Zeitschr. f. Biologie, 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.

2 Abderhalden and Strauss, Zeitschr. f. physiol. Chem., 48; Kossel and Kutscher,
ibid., 31, 20o.

3 Zeitschr. f. physiol. Chem., 24; Hundeshagen, Maly's Jahresber., 25, 394; see
also L. Scott, Biochem. Zeitschr., 1.

4 Biochem. Centralbl., ?.

J Zeitschr. f. physiol. Chem. 29, and Centralbl. f. Physiol., 13, 113.

• Zeitschr. f. physiol. Chem., 55.

7 Zeitschr. f . physiol. Chem., 51 and 55.

FIBROIN AND SERICIN. 123

KRUKENBERG, is nothing but iodine crystals, as shown by MORNER. After
DRECHSEL 1 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
is identical with the 3-5 di-iodo-tyrosine, HOI2C6H2.CH2.CHNH2 COOH,
synthetically prepared by WHEELER and JAMIESON.2 On acid
cleavage of gorgonin HENZE 3 obtained the three hexone bases, abundant
tyrosine and very little leucine. On cleavage with barium hydoxide 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,4 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 5 yielded fibroin but not sericin.

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'S reactions, the last only faintly. Fibroin
has an especially great interest because of the hydrolyses performed by
FISCHER and his co-workers, and especially by the finding of the pre-
viously 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 nearly complete absence of mohamino-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
koilin :

1 Zeitschr. f. Biol., 33.

2 Wheeler and Jamieson, Amer. Chem. Journ., 33; Wheeler, ibid., 38.

3 Henze, Zeitschr. f . physiol. Chem., 38 and 51.

4 Ibid., 34.

5 Ibid., 53.

124

THE PROTEIN SUBSTANCES.

Elastin.i

Gelatin. »

Koilin.3

Sericin.1

Fibroin.1

Spider
Silk.«

Glycocoll
Alanine

25.75
6.6

16.5

0.8

1.2

5 8

0.15
5 0

36.0
21 0

35.13
23 4

Valine

1.0

1.0

LeUCine

21 1

2 1

13 2

1 5

1 7fi

Serine

0 4

6 6

1 6

Aspartic acid. . .

-f?

0 56

2 3

_a_

Glutamic acid

0 8

0 88

5 2

11 70

Cystine

0 747

Phenylalanine

3 9

0 4

2 3

1 5

Tyrosine

0 34

0.0

5 4

5 0

10 5

8 20

Proline
Oxyproline

1.7

5.2
3.0

5.5

+

3.68

Trvptopliane

Histidine

0 4

0 03 4

4 0

_|_

1

Arginine

0 3

9 32

3 604

1 0

1 5 946

Lysiii6

2 75

1 644

4-

j

1 Cited from Abderhalden's Lehrbuch. d. physiol Chem., 1909.

2 Kossel and Kutscher, Zeitschr. f. physiol. Chem., 31.

3 K. B. Hofmann and F. Pregl, ibid., 52.
.4E. von Knaffl-Lenz, ibid., 52.

5E. Fischer, ibid., 53.

6 Calculated as arginine.

7 This figure is somewhat uncertain.

C. Cleavcage 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).

i. 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, whose
specific rotation is increased at the same time. If caustic alkali in sub-
stance or in strong solution be allowed to act on a concentrated proteid
solution, such as blood-serum or egg-albumin, the alkali albuminate
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

ALBUMINATES. 125

»

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 indeed
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 prefer to apply
the term syntonin to the acid albumin ate, 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 nearly insoluble in water and dilute common-salt
solution (see page 103), 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 CaCO3, 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
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

126 THE PROTEIN SUBSTANCES.

obtain an alkali albuminate by the action of an alkali upon an acid
albuminate; but we cannot obtain an acid albuminate by the reverse
reaction (K. MORNER x) . This does not exclude the possibility that by
the action of strong acids products can be obtained which perhaps
correspond to those products obtained by the action of less stronger
alkali.

The preparation of the albuminates has been given above. The cor-
responding albuminate obtained by the action of alkalies or acids upon
aproteid solution maybe 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 precipi-
tate, 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
nearly related albuminates are formed. The " alkali albumose " obtained by
MAAS 2 belongs to this class. The lysalbinic acid and protalbinic acid obtained
by PAAL 3 from ovalbumin are likewise alkali albuminates. Desaminoalbuminic
acid is an alkali albuminate which SCHMIEDEBERG 4 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 BLUM 5 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
products 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
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.

1 Pfliiger's Arch., 17.

2 Zeitschr. f. physiol. Chem., 30.

3 Ber. d. d. chem. Gesellsch., 35.

4 Arch, f . exp. Path. u. Pharm., 39.

5 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.

PROTEOSES AND PEPTONES. 127

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 circumstances
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 concep-
tion 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 there
possibly 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 proteoses 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
solution is saturated with NaCl in substance, the proteose is partly
precipitated 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.

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

1 Oppenheimer's Hanrlb. der Biochem., Bd. 1, 1908.

128 THE PROTEIN SUBSTANCES.

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' l 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 modem 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 amphopeptone
takes place into antipeptone and hemipeptone. 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 is at last obtained — the so-called
antipeptone.

1 Pfliiger's Archiv, 34.

2 See Kiihne, Verhandl. d. naturhistor. Vereins zu Heidelberg (N. F.), 3; J. Wenz,
Zeitschr. f. Biologic, 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; K'ihne, Verhandl. d.
naturhist. Vereins zu Heidelberg (N. F.), 1, and Kiihne and Chittenden, Zeitschr. f.
Biologic, 19. See also Paal, Ber. d. deutsch. chem. Gesellsch., 27.

PROTEOSES AND PEPTONES. 129

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
pepsi n digestion of fibrin 1 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. This precipitate is a
combination of the proteose with acid (HERTH2). The deuteropro-
teose is essentially the same thing that BRUCKE has designated as peptone.

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, albumoses, globuloses, vitelloses, caseoses, myo-
sinoses, elastoses, etc. These various proteoses are further distinguished,
as proto-, hetero-, and deuterocaseoses, for example. CniTTENDEN3 has
suggested the common name proteoses for the products formed inter-
mediary 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 gen-
eral term. Certain proteoses have also been obtained in a crystalline
state (SCHROTTER).

NEUMEISTER 4 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,

1 See Kiihne and Chittenden, Zeitschr. f . Biologic, 20.

2 Monatshefte f. Chein., 5.

3 Kiihne and Chittenden, Zeitschr. f. Biologic, 22 and 25; 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, Jourri. of
Physiol., 12.

4 Zeitschr. f. Biologic, 26. See also Chittenden and Meara, Journ. of Physiol.,
15, and Salkowski, Zeitschr. f. Biologic, 34 and 37.

130 THE PROTEIN SUBSTANCES.

which are closely allied to the peptones, he calls secondary proteases. As
essential differences between the primary and secondary proteoses he
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 secondary
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 pre-
cipitated 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 . Biologie, 24 and 2G.

2 Zeitschr. f. physiol. Chem., 24.

3 Frankel, Zur Kenntnis der Zerfallsprodukte des Eiweisses bei peptischer und
tryptischer Verdauung, Wien, 1896; Schrotter, Moiiatshefte f. Chem., 14 and 16.

PROTEOSES AND PEPTONES. 131

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, LANGSTEIN,*
and others have shown that by very lengthy digestion simpler products
can be produced, some whose nature is still unknown, while others are
known, such as alanine, leucine, leucinimide, valine, aspartic and glutamic
acids, phenylalanine, tyrosine, proline and lysine, and on further cleavage
indeed also oxyphenylethylamine, tetra- and pentamethylenediamine.
It has not been possible to cause a disappearance of the biuret reaction,
and the occurrence of tryptophane is somewhat disputed. MALFATTI
obtained tryptophane in peptic digestion only when he used a certain
apparently impure preparation of pepsin, and on using pepsin purified
according to PEKELHARING it was absent. According to PEKELHARiNG,2
purified pepsin also yields tryptophane when the solution is rich in pep-
sin, and also when the acidity is not too strong, in the presence of small
amounts of pepsin.

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 HzSOJ was continued for a very long time, indeed for an
entire year, without controlling the influence of the acid alone upon the proteoses.
The question as to the hydrolysis with the splitting off of amino-acids by the
continuous action of acid alone is still the subject of dispute.

KUHNE'S view that in tryptic digestion a peptone, so-called antipep-
tone, always remains which cannot be further split is not strictly true.
By sufficiently long autodigestion of the pancreas, KUTSCHER 3 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. In this connection we must remark that the. pure antipeptone
(see below), isolated by SIEGFRIED, could be split by trypsin only with
great difficulty, and also that the complete disappearance of the biuret
reaction in tryptic digestion does not show that a complete decomposi-
tion into amino-acids has taken place. According to E. FISCHER and
AfiDERHALDEN,4 polypeptide-like bodies are produced, especially in

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 Malfatti, Zeitschr. f. physiol. Chem., 31; Pekelharing, Archives d. scienc. biolog.
de St. Pe"tersbourg, 11; Pawlow Festband.

3 Zeitschr. f. physiol. Chem., 25, 26, 28, and Die Endprodukte der Trypsinver^
darning, Habilitationsschrift Strassburg, 1899.

4 Zeitschr. f. physiol. Chem., 39.

132 THE PROTEIN SUBSTANCES.

tryptic digestion, and these bodies resist the prolonged action of the
enzyme, but yield several different ammo-acids on hydrolytic cleavage by
acids. The same is probably also true for peptic digestion (see below) ,
and the difference in the digestive products between pepsin and trypsin
digestion consists, essentially, only in that, in the first case the cleavage
is slower and does not proceed so far, hence the biuret reaction remains
and generally no formation of tryptophane takes place.

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 recently been made
by UMBER, ALEXANDER, PFAUNDLER, PICK, ZUNZ, SIEGFRIED and his
pupils.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 digestion, 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 pri-
marily formed acid albuminate, PICK and ZUNZ have shown that several
proteoses, 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 heteroproteoses whose precipitation limit lies at 24-42 per c>ent 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 2, page 135.

2 Schiitz and Huppert, Pfliiger's Arch., 80.

3 F. Goldschmidt, Ueber die Einwirkung von Sauren auf Eiweissstoffe, Inaug.-
Diss. Strassburg, 1898.

PROTEOSES AND PEPTONES. 133

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, which should
contain secondary proteoses only, primary proteoses such as the gluco-
proteose (PICK), w.hich 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 in their investigation according to
different methods. For example RONA and MICHAELIS 2 find that certain
proteoses are precipitated by mastic emulsion while others are not. Those
that are precipitable 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 ZUNZ 3 like strong protection 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 pre-
cipitated 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 pro-
teoses 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 a
more detailed discussion of the properties of the various proteoses thus
far isolated is without 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 arginiiie and poorer in histidine than the proto-
proteose, and PICK has also found marked differences between the hetero-
and proto-proteose from fibrin. The hetero -proteose yields very little
tyrosine and indol but abundant leucine and glycocoll, and about 39
per cent of the total nitrogen in a basic form. The protoproteose,

1 Ueber Bau und Gruppirung der Eiweisskorper, Ergebnisse der Physiol., Jahrg. I,
Abt. 1, 783.

2 Biochem. Zeitschr., 3.

3 Arch, internal, d. Piiysiol., 1 and 5, and Bull. Soc. Scienc. med. et natur. Brux-
-elles, 64.

4 Journ. of Physiol., 37.

134 THE PROTEIN SUBSTANCES.

according to PICK, on the contrary yields considerable t}rrosine 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 ABLER l did not find the same results
as PICK in regard to the amounts of monamino-acids in the two pro-
teoses. These divergent results may be explained by the fact that they
were not working with pure substances, but rather with mixtures. Accord-
ing to HASLAM 2 the so-called protoproteoses is a mixture of two pro-
teoses which he designates a and /? protoproteose which have different
precipitation properties with alcohol and with one-half saturation with
ammonium sulphate.

According to PICK the heteroproteose is also more resistant toward
trypsin digestion than the protoproteose, a behavior which coincides
with KUHNE'S view of a resistant atomic complex, an antigroup, in the
protein bodies. KUHNE and CniTTENDEN3 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 has recently attracted further attention, because
as first found by D ANILE WSKY and other investigators, OKUNEW, SAW-
JALOW, LAWROW, and SALASKIN and KURAJEFF, have further shown,
that solutions of rennin, gastric juice, pancreatic juice, and papain cause
a coagulum in not too dilute proteose solutions. These coagula, called
plasteines (coagulum by rennin) by SAWJALOW, and coaguloses (coagu-
lum by papain) by KuRAJEFF,4 are similar in many respects to anti-
albumid, having a higher content of carbon (57-60 per cent) and nitro-
gen (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 Physiol., 32 and 36.

3 Kiihne and Chittenden, Zeitschr. f . Biologic, 19, 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 and 56; Kurajeff, Hofmeister's Beitrage, 1 and 2; see also Sacharow, Biochem.
Centralbl., 1, 233; Levene and v. Slyke, Biochem. Zeitschr., 13.

PROTEOSES AND PEPTONES. 135

is rather generally admitted that they are formed by a synthesis. Accord-
ing to SAWJALOW a plastein is not formed from a proteose alone, but
always from a mixture of these. LAWROW claims that they may be
produced from proteoses as well as from polypeptide substances, and cor-
respondingly we must differentiate between the coaguloses or coaguloso-
gens 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 products. Perhaps
the plasteinogen investigated by BAYER/ which essentially differs 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 ammonium
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.2

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 peptic-glutin peptone isolated
by SIEFGRIED and SCHEERMESSER yielded arginine, lysine, glutamic
acid and glycocoll. SIEGFRIED has given proof in several ways as to
the purity and unity of the peptones isolated by him.

1 Hofmeister's Beitrage, 4; see also L. Rosenfeld, ibid., 9; J. Lukomnik, ibid., 9
and F. Micheli, Biochem. Centralbl., 6, p. 562.

2 The works of Siegfried and his pupils, Fr. Miiller, Borkel, Miihler, Kriiger, Scheer-
messer and Neumann may be found in Arch. f. (Anat. u.) Physiol., 1894 and Zeitschr. f .
physiol. Chem., 21, 41, 43, 45, 48 and 50.

136 THE PROTEIN SUBSTANCES.

In another manner, namely by fractional precipitation with metallic
salts, especially with mercuric-potassium iodide and the preparation
of phenylisocyanate compounds, HOFMEISTER and his pupils STOOKEY,
RAPER and ROGOZINSKI l 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 warming with hydrochloric
acid, obtained a base, C2iH39NgO8, which can also be directly obtained
from gelatin. This he calls a kyrin, because it is to be considered as 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 belongs to the
bases and one-third to the amino-acids.

Recently he with O. PILZ on further hydrolysis has prepared a
^-glutokyrin, which only yielded arginine, lysine and glutamic acid.
Similar basic nuclei, protokyrins, have recently been obtained by SIEG-
FRIED 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 C23H47N908. It yields
arginine, lysine, and glutamic acid on cleavage. The basic nitrogen
amounts to about 85 per cent of the total nitrogen, and caseinokyrin,
whose unit nature is defended by SIEGFRIED 3 against the opinions of
SKRATJP, ZWERGER and Wiir,4 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 desig-
nate as proteins only those atomic complexes which contain, besides
chained monamino-acids, also arginine, or, more simply, show the pre-
viously mentioned imide bindings. Hence caseinokyrin, which yields
only arginine, lysine and glutamic acid, and scombrin (see below), which
yields only arginine, proline, and alanine, are the simplest known proteins.

Scombrin belongs to the previously mentioned group of prot amines
which, according to KOSSEL,S are formed by a successive cleavage of the
typical protein. The occurrence of basic protokyrins in the hydrolytic

1 Hofmeister's Beitrage, 7, 9, and 11.

2 Kgl. Sachs. Ges. d. Wiss., Math.-Phys. Klasse, 1903, and Zeitschr. f. physiol.
Chein., 43, with Pilz., ibid., 58.

3 Zeitschr. f. physiol. Chem., 48 and 50.

4 Monatash. f. Chem., 26 and 27.

5 Zeitschr. f. physiol. Chem., 44.

PROTEOSES AND PEPTONES. 137

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 these determinations have been made upor
impure substances or mixtures, the results l obtained are only of little
value. The same is true for the elementary analysis of the proteoses
and peptones.2

Besides the behavior in the salting-out process, attempts have been made to
find other points of difference between the peptones and proteoses. SCHROTTER
and FRANKEL 3 consider the sulphur content as a pronounced point of difference.
The peptones, according to them, are free from sulphur, while the proteoses,
on the contrary, contain sulphur. FRANKEL has been able to find only one pro-
teose (in KUHNE 's sense) which did not contain sulphur.

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 peptones
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 suggested
by PICK, but this method, as well as- that with ammonium sulphate,
gives good results only when the precautions suggested by HASLAM*
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 HOFMEISTER
and the SIEGFRIED schools. In regard to the literature on the detection
of proteoses and peptones in animal fluids we refer to Chapters VI and XV.

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 discussed
on pages 85-89, and of the cleavage products of the proteins only the
amino-acids remain to be discussed.

^abanejew, 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
Kuhne and Chittenden and their pupils, cited in footnote 3, p. 129; also by Herth,
Zeitschr. f. physiol. Chem., 1, and Moiiatshefte f. Chem., 5; Maly, Pfliiger's Arch.
9, 20; Henninger, Compt. rend., 86; Schrotter, 1. c., Paal, 1. c.

3 Schrotter, Monatshefte f. Chem., 14 and 16; Frankel, Zur Kenntnis der Zerfalls-
produkte des Eiweiss bei peptischer und tryptischer Verdauung, Wien, 1896. '

4 Journ. of Physiol., 32 and 36.

138 THE PROTEIN SUBSTANCES.

3. The Amino Acids.1

r^TT ^XTTT >\

Glycocoll (amino-acetic acid), C2H5NO2 = A^^TT , also called glycine

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. It is also formed in the decomposi-
tion of uric acid, xanthine, guanine, and adenine.

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 dis-
solves with difficulty in warm alcohol. It combines with acids and alkalies.
With the latter compounds we must mention those with copper and
silver. Glycocoll dissolves cupric 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 cool-
ing. The compound with hydrochloric acid is readily soluble in water
but less soluble in alcohol.

SoRENSEN2 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, SpiRO 4). The /9-naphthalene-sulpho-glycine with a melting-
point of 159°, the 4 nitro-tolulene-2-sulpho-glycine, melting at 180°,
the phenylisocyanate compound, melting at 195°, and the a-naphthyl-
isocyanate compound melting at 190.5-191.5° are also of importance.

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
the benzoic acid is removed, the filtrate concentrated, the remaining
benzoic acid removed by extracting with ether, the sulphuric acid pre-

1 In regard to the division of the animo-acids among the three chief groups of
organic compounds we refer to page 85.

2 Meddelelser, fraa Carlsberg-laboratoriet, 6, 1905.

3 Ber. d. d. chem. Geseilsch., 34.

4Ch. Fischer, Zeitschr. f. physiol. Chem., 19; Spiro, ibid., 28; Gonnermann,
Pfliiger's Arch., 59.

ALANIXE. VAL1NE. 139

cipitated by BaCOs, and the filtrate evaporated to the point of crystal-
lization. (In regard to its preparation from protein substances see below.)

CH3

Alanine (a'-aminopropionic acid, C3H7NO2 = CH(XH2). 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 Z-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 /-alanine by splitting with yeast
by WALDEN'S reversion (see page 87).

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=+2.7° and for a
solution in hydrochloric acid (9-10 per cent solution) is (a)D= + 10.3°.

The /?-naphthalene-sulpho-d-alanine melts at 79-81°, the phenyliso-
cyanate at 168°, and the naphthylisocyanate-alanine melts at 198° C.

CH3 CHs

\/

CH
Valine (a-amino-valeric acid) CoH

,TT/ATTT x , u
L/-ti(i\li2), has 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 DORPINGHAUS 2
5.7 per cent from horn substances. The acid isolated by H. and E.
SALKOWSKIS from putrefying proteid or gelatin seems to have been
a-amino-7i-valeric acid.

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 («)D= +6.42°. The hydrochloric acid solution (20 per cent) shows,
according to FISCHER, a rotation of (a)D=+28.8°. The copper salt,
which forms leaves which are rather soluble in water, is very easily soluble
in methyl alcohol (SCHULZ and WINTERSTEIN 4) .

1 Ber. d. d. chem. Gesellsch., 40.

2 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41; Fischer and Dorpinghaus,
ibid., 36.

8 Ber. d. d. chem. Gesellsch., 16 and 31.
4 Zeitschr. f. physiol. Chem., 35.

140 THE PROTEIN SUBSTANCES.

The phenylisocyanate melts at 147°, and on boiling with 20-per cent
hydrochloric acid for a short time, it is changed into d'-phenyliso-
propyl hydantoin, which melts at 131-133° C.

Leucine (aminocaproic acid, or; more correctly, a-aminoisobutylacetic

CH
acid), C6H13NO2= 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 SAMEC1).

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 prod-
ucts 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 poisoning).
Leucine occurs often in invertebrates and also in the plant kingdom. On
hydrolytic cleavage various protein substances yield different amounts
of leucine, as shown in the tables given on pages 106, 107, 114 and 124.
From the figures there given we call attention to the following: ERLEN-
MEYER and Sen OFFER obtained 36-45 per cent leucine from the cervical
ligament, COHN obtained 32 per cent from casein, E. FISCHER and
ABDERHALDEN 20 per cent from haemoglobin, and FISCHER and
DORPINHAUS 18.3 per cent from horn substance.2

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 HiiFNER3 from

1 Heckel, Monatsh. f. Chem., 29; Samec, ibid., 29.

2 Erlenmeyer and Schoffer, cited from Maly, Chem. d. Verdauungssafte, in Her-
mann's Handb. d. Physiol., 5, Theil 2, p. 209; Cohn, Zeitschr. f. physiol. Chem., 22;
Fischer and his collaborators, ibid., 36.

3 Journ. f. prakt. Chein. (N. F.), 1.

LEUCINE. 141

isovaleraldehyde, ammonia, and hydrocyanic acid is optically inactive.
Inactive leucine may also be prepared, as shown by E. SCHULZE and
BossHARD,1 by the cleavage of proteins with baryta at 160-180° C.,
because of a racemation, as the ordinary leucine is racemized on heating
with baryta at this temperature. The cW-leucine may be split into
the two components by various means, especially by the preparation
of the formyl combination.2

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.

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 layers 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 d-Z-leucine is much less soluble. According to HABER-
MANN and EHRENFELD 3 100 parts of boiling glacial acetic acid dissolve
29.23 parts of leucine. The specific rotation of Z-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= — 10.42°r
according to EHRLICH and WENDEL."*

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
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

1 See Zeitschr. f. physiol. Chem., 9 and 10.

2 Fischer and Warburg, Ber. d. d. chem. Gesellsch., 88.

3 Zeitschr. f. physiol. Chem., 37.

4 Fischer and Warburg, Ber. d. d. chein. Gesellsch., 38; Ehrlich and Wend el,
Biochem. Zeitschr., 8.

142 THE PROTEIN SUBSTANCES.

leucine-ethyl-ester hydrochloride, melting at 134° C., are formed (RoH-
MANN) . The same is produced by the action of gaseous HC1 upon leucine
in alcohol; and the free ethyl ester can be obtained from this by the
method suggested by E. FISCHER. 1

The picrate of the leucine ester melts at 128°. The phenylisocyanate
of d-Z-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 by the appearance of balls or knobs under the
microscope, by its action when heated (sublimation test), and by its com-
pounds, especially the hydrochloride and picrate of the ethyl ester and
the phenylisocyanate compound of the racemic leucine obtained on heat-
ing with baryta water, the a-naphthylisocyanate compound and the
/?-naphthalene-sulpho-leucine. According to the method suggested by
LIPPICH 2 the leucine can be transformed into isobutylhydantoin, having
a melting point of 205°, by boiling with an excess of urea and baryta water.
In the detection of leucine, it must be first isolated, and the preparation
of the ethyl ester and distillation of the same are important in this regard.
In order to avoid the difficulties which occur in the preparation of pure
leucine, EHRLICH and WENDEL 3 have suggested a new method.

P* TT r^fi ]V"fT f^O
Leucinimide, C12H22N2O2= 4 9';v/A /• nTT, was first obtained by RITT-

HAUSEN in the hydrolytic cleavage products on boiling proteins with acids, arid
subsequently by R. CORN. SALASKIN 4 obtained it in the peptic and tryptic
digestion of haemoglobin. As an anhydride of leucine (2.5-cliacipiperazine) 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 5 from leucine-ethyl
ester melted at 271° C.

\/

PTT

Isoleucine (methyl-ethyl-a-amino-propionic acid) C6H13NO2= .

CHJNH2

COOH

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

1 Rohmann, Ber. d. d. chem. Gesellsch., 30; E. Fischer, ibid., 34.

2 Ber. d. d. chem. Gesellsch., 39.

3 Biochem. Zeitschr., 8.

4 Ritthausen, Die Eiweisskorper der Getreidearten, etc., Bonn, 1872; R. Cohn,
Zeitschr. f. physiol. Chem., 22 and 29; Salaskin, ibid., 32.

5 Ber. d. d. chem. Gesellsch., 34.

SERINE. 143

recently it has been found by others l among the products of hydrolysis
of the proteins. It seems to be associated regularly with ordinary
leucine, forming mixed crystals, which give an impression of a chemical
combination and which are difficult 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. In the fermentation of sugar by
yeast it yields d-amyl alcohol, and on the other hand, it can also be
obtained, in a manner analogous to the synthesis of leucine, from
c?-amyl alcohol (as a mixture of isoleucine and alloisoleucine, the latter
is levogyrate and has a different stereometric configuration from the
isoleucine). The synthesis of isoleucine has been accomplished in
other ways by EHRLICH, by BRASCH and FRIEDMANN and by BOUVEAULT
and LocQinx.2

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=+9.74° 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.

CH2(OH)

Serine (a-amino-/?-oxypropionic acid) C3H7NO3 = 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 SKITAS from sericine;
KOSSEL and DAKIN 4 obtained a still larger amount from salmine, namely
7.8 per cent. The racemic serine is the one generally obtained. From
fibroin FISCHER 5 obtained a mixture of active and inactive serine anhy-
dride from which he finally prepared Z-serine by hydrolysis.

Synthetically d-Z-serine has been prepared by FISCHER and LEUCHS

1 Felix Ehrlich, Ber. d. d. chem. Gesellsch., 37; Winterstein ami Pantanelli, Zeitschr.
f. physiol. Chem., 45.

2 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.

3 Zeitschr. f . physiol. Chem., 35.

4 Fischer and Skita, Zeitschr. f. physiol. Chem., 35; Kossel and Dakin, ibid., 41.

5 Ber. d. d. chem. Gesellsch., 40.

144 THE PROTEIN SUBSTANCES.

from ammonia, hydrocyanic acid and glycol aldehyde, and recently in
other ways by ERLENMEYER, JR. and STOOP and by LEUCHS and GsiGER.1
FISCHER and JACOBS 2 have prepared Z-serine from cW-serine by the prepara-
tion of the alkaloid salt of the p-nitro-benzoyl combination. On reduc-
tion 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 formula:

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 /-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 Z-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
/?-naphthalene-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. (<*)D= —67.46°.

Isoserine (/9-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.3

COOH

f^TJ /TVTTT \

Aspartic acid (aminosuccinic acid), C4H7NO4= . , has been

COOH

obtained on the cleavage of protein substances by proteolytic enzymes as
well as by boiling them with dilute mineral acids in quantities given in the
tables on pages 106, 107, 114 and 124. This acid also occurs in secre-
tions of sea-snails (HENZE 4) 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. cW-Aspartic acid has been prepared syn
thetically from fumaric acid and alcoholic ammonia.

1 Fischer and Leuchs, Ber. d. d. chem. Gesellsch, 35; Erlenrneyer and Stoop, ibid*,
35; Leuchs and Geiger, ibid., 39.

2 Ber. d. d. chem. Gesellsch., 39.

3 Ellinger, Ber. d. d. chem. Gesellsch., 37; Neuberg and Silbermann, ibid., 37;
Neuberg and Mayer, Biochem., Zeitschr 3; Neuberg and Ascher, ibid., 0.

4 Ber. d. d. chem. Gesellsch., 34.

GLUTAMIC ACID. 145

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 («)D = + 25.7°;
in alkaline solution the acid is levo-rotatory. It forms with copper
oxide a crystalline compound which is soluble in boiling-hot water and
nearly insoluble in cold water, and which may be used in the preparation
of the pure acid from a mixture with other bodies.

The benzoyl-/-aspartic acid melts at 184-185°. For identification
we make use of the analysis of the free acid and the copper salt, as well
a-s of the specific rotation.

COOH
CH(NH2),

Glutamic acid (a-aminoglutaric acid) , C5H9NO4 = CH2 , is obtained

CH2
COOH

from the protein substances under the same conditions as the other mon-
amino-acids (see tables on pages 106, 107, 114 and 124) and from the pep-
tones (SIEGFRIED). HLASIWETZ and HABERMAXN 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.
ABDERHALDEN and FUNK 1 did not find any such differences in the hydro-
lytic action of the two acids, and obtained only 10-11 per cent glutamic
acid from casein. SKRAUP and TURK 2 obtained on the hydrolysis of casein
with 33 per cent sulphuric acid at boiling temperature for eighteen hours
about the same quantity of glutamic acid as on boiling for six hours with
fuming hydrochloric acid, namely, 20.3 and 22.3 per cent glutamic acid
hydrochloride. The considerable quantity of glutamic acid obtainable
from certain vegetable « proteins, as shown on page 107, is very remarka-
ble. LEVEXE and MANDEL 3 have found a strikingly large quantity of
glutamic acid, namely 25 per cent, from a nucleoprotein from the spleen.
c?-Glutamic acid crystallizes in rhombic tetrahedra or octahedra or in
small leaves. It dissolves in 100 parts water at 16° C., and the solution
tastes acid with a peculiar after-taste. It is insoluble in alcohol and in
ether.

In water it has a rotation of (a)D= +12.04° according to ANDRLiK.4
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. The

1 Hlasiwetz and Habermanu, Annal. d. chem. u. Pharm., 159; Kutscher, Zeitschr.
f. physiol. Chem., 28; Abderhalden and Funk, ibid., 53.
2Monatsh. f. chem., 80.

3 Biochem. Zeitschr., 5.

4 See Biochein. Centralbl., 3, p. 469.

146 THE PROTEIN SUBSTANCES.

d-glutamic acid forms a beautifully crystalline combination with hydro-
chloric acid, which is nearly 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. 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, CUH7NO5 occurs among the hydrolytic cleavage
products of proteids has been shown to be probable by SKRAUP. This acid has
been prepared synthetically by NEUBERG and SILBERMANN from diaminosuccinic
acid and barium nitrite -in sulphuric acid solution. Oxyaminosuberic acid,
C8H15NO5, has been detected by WOHLGEMUTH l in the cleavage products of a
liver nucleoprotein.

Cystine, C6H12N2S2O4 the disulphide of cysteine (a-amino-/?-thiolactic

CH2 O O CH2

acid), CH(NH2) CH(NH2), was first obtained with certainty as a

COOH COOH

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, 113 and 114.

According to NEUBERG and MAYER 3 two kinds of cystine occur in nature,
namely, stone-cystine, designated ,fl-cystine, and protein-cystine. Stone-cystine

CH2NH2 CH2NH2

is the disulphide of (5-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 cys-
tines is not improbable, from certain observations of MORNER, but FRIEDMANN
and BAER 5 have shown that these observations do not lead to this assumption^

1 Skraup, 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. Biologic, 27.

3 Zeitschr. f. physiol. Chem., 44.

4 Rothera, 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.

CYSTINE 147

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 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 1 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 FmEDMANN,2 and
he has also established the relation, between cystine and taurine.
Cystine is the disulphide of cysteine, which is a-amino-5-thiolactic acid.
From cysteine by oxidation FRIEDMANN obtained cysteinic acid,

CH2SO2OH
C3H7NSO5 = CH(NH2), from which taurine CH2(S02OH) is produced by

COOH CH2(NH2)

splitting off CO2.

a-Cystine has also been prepared synthetically. Starting from formyl
hippurate, ERLENMEYER, JR. and STOOP first prepared the benzoylserine
ester, and then with phosphorus pentasulphide they obtained the
benzoylcystine ester. On splitting the latter with HC1 they obtained
cysteine, and then on oxidation inactive cystine. GABRIEL has also
prepared an isocysteine by the cleavage of sulphocyanatedihydrouracil
with hydrochloric acid, and then inactive cystine by the oxidation of this
isocysteine, and recently FISCHER and RASKES have prepared cystine
from a-amino-/?-chlorpropionic acid (obtained from /-serine) by the
action of barium hydrosulphide and a subsequent oxidation in the air.

Z-a-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 levorotatory.
MORNER found it to be (a)D= —224.3°. On heating with hydrochloric
acid it can, according to MORNER, be changed into a modification crys-
tallizing 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 melt-
ing-point but slowly decomposes at 258-261°. On boiling cystine with

1 Zeitschr. f. physiol. Chem., 38.

2 Hofmeister's Beitrage, 3.

3 Erlenmeyer and Stoop, Ber. d. d. chem. Gesellsch., 36; Gabriel, ibid., 38; Fischer
and Raske, ibid., 41.

148 THE PROTEIN SUBSTANCES.

caustic alkali it decomposes and yields alkali sulphide, which can be
detected by lead acetate or sodium nitroprusside. According to MOR-
NER l 75 per cent of the total sulphur is separated. On treatment of
cystine with tin and hydrochloric acid it develops only a little sulphu-
retted hydrogen, and is converted into cysteine.

On heating upon platinum-foil cystine does not melt, but 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 evaporation 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 precipitation
with mercuric acetate is especially suited. The benzoyl cystine (BAUMANN
and GoLDMANN2) melts at 180-181°; the phenylisocyanate com-
pound at 160°. On boiling with 25 per cent hydrochloric acid this com-
pound passes to the anhydride, which is a hydantoin melting at 119° C.

Stone-cystine, according to NEUBERG and MAYER, differs in many respects
from the ordinary cystine, among which the following will 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 sulphur
reaction after boiling with alkali. As to its preparation from protein
substances see K. M6RNER.3 In regard to the detection of cystine in the
urine see Chapter XV.

CH2.SH
Cysteine (a-amino-/?-thiolactic acid), C3H7NSO2 = CH(NH2), is formed from

COOH

cystine by reduction with tin and hydrochloric acid. It is also produced in the
cleavage of protein substances, but this is considered by MORNER and PATTEN 4
as a secondary formation. Cysteine can be easily converted into cystine by
oxidation.

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) C3H6SO2 = CH(SH), has been found once

COOH

as a cleavage product of ox-horn by BAUMANN and SUTER. MORNER,
FRIEDMANN and BAER obtained it from cystine. It has been shown by FRIED-

1 Zeitschr. f. physiol. Chem., 34. 3 Zeitschr. f . physiol. Chem., 34.

2 Ibid., 12. 4 See footnote 1, page 79.

TAUI1IXE. 149

MAXN that this acid is a regular cleavage product of keratin substances, and
that it can also be obtained from the proteins. FRANKEL l obtained the acid
from haemoglobin. The pyroracemic acid obtained by MORNER as a decomposi-
tion product from several protein substances originates, according to MORNER,
only in part from the cystine.

Taurine (aminoethylsulphonic acid), C2H7NSO3= • ' , has

not been obtained as a cleavage product of protein substances ; still its
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 sul-
phides, 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. LANG2). 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 filtrate 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 dis-
solved in 5-per cent hydrochloric acid, and precipitated with 10 vols.
95-per cent alcohol. The crystals are readily purified by r eery st alii za-
tion from water.

The 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.

1 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, 6, 1903.

2 See Maly's Jahresber., (>.

150 THE PROTEIN SUBSTANCES.

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.

CeH5

CH2.
Phenylalanine (phenyl-a-aminopropionic acid), C9HnNO2 = CH(NH2),

COOH

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
given on pages 106, 107, 114 and 124. It has been prepared syntheti-
cally in several ways by ERLENMEYER JR., SORENSEN and E. FiscHER.2
The /-phenylalanine crystallizes in small, shining leaves or fine needles
which are rather difficultly soluble in cold water but readily soluble hi
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-phenylalanine melts at about 182° C.

C6H4(OH)

Tyrosine (p-oxyphenyl-a-aminopropionic acid), CgHnNO3=

COOH

is produced from most protein substances under the same conditions as
leucine, which it habitually accompanies. The largest quantity of tyro-
sine obtained from animal proteins was about 10 per cent (see tables,
pages 106, 107, 114 and 124). In gelatin and a few keratins tyrosine
is absent. It is especially found with leucine, in large quantities^ in old
cheese (Tv/oos), from which it derives its name. Tyrosine has not been
found with certainty in perfectly fresh organs. It occurs in the in-
testine in 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

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.

3 Erlenmeyer and Lipp, Ber. d. d. chem. Gesellsch., 15; Erlenmeyer and Halsey,
ibid., 30.

TYROSINE. 151

it yields p-oxybenzoic acid, acetic acid, and ammonia. On putrefaction
it may yield p-hydrocoumaric acid, oxyphenylacetic acid, and p-cresol.

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 1 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 (a)D=— 12.56 to 13.2° for the hydrochloric acid solution,
while SCHULZE and WiNTERSTEiN2 obtained higher results using tyrosine
from plants, namely, (a)D= — 16.2°. These investigators believe that
when lower results are obtained a contamination with racemic tyrosine
is the cause.

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 soluble
with difficulty in water, being dissolved by 2454 parts 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 (HABERMANN and EHREN-
FELD 3) . The Z-tyrosine-ethyl-ester crystallizes in colorless prisms which
melt at 10S-109°C. The naphthylisocyanate-Uyrosine 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 Chapter I). 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 BaCO3, 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.

1 Ber. d. d. chem. Gesellsch., 17.

2 See Hoppe-Seyler-Thierf elder, Handb. d. physiol. u. pathol. chem. Analyse, 8.
Aufl., 1909. Also E. Fi&cher, Ber. d. d. chem. Gesellsch., 32; Schulze and Winter-
stein, Zeitschr. f. physiol. Chem., 45.

3 Zeitschr. f. physiol. Chem., 37.

152 ' THE PROTEIN SUBSTANCES.

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
then the mixture boiled for some time, the liquid becomes a beautiful
red and then yields a red precipitate. Mercuric nitrate may first be
added, then, after this has boiled, nitric acid containing some nitrous
acid.

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.

Proline (a-pyrolidine carboxylic acid), C5H9NO2 = H2C CH.COOH,

NH

was first obtained by E. FISCHER and then by FISCHER and collaborators *
from several proteins. The proline here obtained was generally the
levo-rotatory modification. The largest quantity of proline was secured
from the vegetable protein hordein, namely, 13.7 per cent, and also from
the egg membrane of the Testudo grseca, 11.8 per cent (see table pages
106, 107, 114 and 124). KOSSEL and DAKIN 3 got 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 ornithine and proline.

SoRENSEN,4 by means of a general method of preparing a-amino-acids
synthetically, has prepared a-amino-o-oxy valeric acid from phthalimide-
malonic ester and has obtained proline from this by evaporating with
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-

STADTER.5

Z-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 phenylisooyanate
compound (melting-point 144°), and the picrate (ALEXANDROFF 6) ..

1 Deniges, Compt. rend., 130; C. Th. Morner, Zeitschr. f. physiol. Chem., 37.

2 E. Fischer, Zeitschr. f. physiol. Chein., 33 and 35. See also footnote 2, page 85.

3 Zeitschr. f. physiol. Chem., 41.

4 Zeitschr. f. physiol. Chem., 44; with A. C. Anderson, ibid., 56.

5 Ber. d. d. chem., Gesellsch., 33.

6 Zeitschr. f . physiol. Chem., 46.

TRYPTOPHANE. 153

The inactive acid and its compounds show somewhat varying
properties.

Oxyproline (oxy-a-pyrolidine carboxylic acid), C5H9NO3. This acid,
whose constitution is not understood was first obtained by E. FISCHER 1
on the hydrolysis of casein and of gelatin. It dissolves readily in water;
has a specific rotation of (a)D= — 81.04°, and the solution has a sweet
taste. Oxyproline crystallizes in beautiful colorless plates and gives-
a readily soluble copper salt. LEUcns2 has prepared two isomeric
inactive oxyprolines which were crystalline.

Tryptophane (indolaminopropionic acid),

C.CH2.CH(NH2)COOH
Ci iH12X202 = C6

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 3
considered tryptophane, which name is generally given to this acid,
as the mother-substance of various animal pigments.

Tryptophane was first prepared in a pure form by HOPKINS and
CoLE,4 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-£-tryptophane by ELLINGER and FLAMAND, 5
the nature of this substance as indolaminopropionic acid was established.

By condensation of ^-indolaldehyde with hippuric acid ELLINGER
and FLAMAND prepared the azlactone (lactimide) :

N

I I

co-o

1 Ber. d. d. chem. Gesellsch., 35 and 36.

2 Ber. d. d. chem., Gesellsch., 38. See also Leuchs and Felser ibid., 41.

3 In regard to tryptophane, see Stadelmann, Zeitschr. f. Biologic, 26; Neumeister,
ibid., 26; ^encki, Ber. d. d. chem. Gesellsch., 28; Beitler, ibid., 31; Kurajeff, Zeitschr.
f. physiol. Chem., 26; Klug, Pfiiiger's Arch., 86.

4 Journ. of Physiol., 27.

5 Ellinger, Ber. d. d. Chem. Gesellsch., 37 and 38. With Flamand, ibid., 40, and
Zeitschr. f. physiol. Chem., 55.

154 THE PROTEIN SUBSTANCES.

On boiling with dilute caustic soda, with the taking up of water, the
sodium salt of indoxyl-a-benzoylaminoacrylic acid,

C8H6N. CH:C.NH.COC6H6
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.COC6H5

| +H2+H20=C8H6N.CH2.CH.NH2 + C6H5COOH.

CCOH COOH

The trytophane formed in digestion is /-tryptophane, which is levoro-
tatory in aqueous solution (HOPKINS and COLE). Racemic cW-trypto-
phane has also been obtained by digestion in certain cases by ALLERS
and NEC BERG; this is probably formed from the /-tryptophane (ABDER-
HALDEN and L. BAUMANN J), which very readily undergoes racemization.

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 (^-/-trypto-
phane has a faint sweetish taste, and /-tryptophane a faint bitter taste.
The statements as to the optical behavior of tryptophane differ some-
what (HOPKINS and COLE, NEUBERG and POPOWSKY, ABDERHALDEN
and KEMPE, ELLINGER and FLAMAND, H. FISCHER 2) which, according
to ABDERHALDEN, is probably due to the readiness with which it under-
goes racemization. According to ABDERHALDEN and L. BAUMANN,
at 20° C. the aqueous solution has a rotation of (a)D= —30.33°. HOP-
KINS and COLE give (a)D= — 33° for the watery solution. It is dextro

N N N

rotatory in — or — XaOH as well as in — HC1.

Tryptophane yields indol and skatol when sufficiently heated. It gives
the AoAMKiEwicz-HopKiNS3 reaction and a rose-red color on the addi-
tion of chlorine or bromine water (tryptophane reaction). The compound
produced in this last reaction contains, according to NEUBERG, 4 1 atom
of bromine (or chlorine) while another compound containing 3
halogen atoms is yellow in color. If a pine stick previously moistened

1 R. Allers, Biochem. Zeitschr., 6; C. Neuberg, ibid., 6; Abderhalden and Baumann,
Zeitschr. f. physiol. Chem., 55. (Literature on the specific rotation.)

2 See Abderhalden and Baumann, Zeitschr. f. physiol. Chem., 55 (literature).

3 In regard to this reaction see also Dakin, Jourri. of Biol. Chem. 2, and O. Rosen-
heim, Biochem. Journ., 1.

4 Biochem. Zeitschr., 2 and 6. See also Levene and Rouiller, ibid., 4.

INDOL AND SKATOL. 155

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
/?-naphthalenesulphotryptophane and the naphthylisocyanatetrypto-
phane are according to ELLINGER and FLAMAND/ 185°, 180° and 158°
C. respectively. Several compounds of tryptophane have been prepared
by ABDERHALDEN and KEMPE.2 Among these we will mention the trypto-
phane chloride hydrochloride, because it is used as the starting material
for the synthesis of tryptophane polypep tides.

In regard to the rather complicated method for preparing trypto-
phane we must refer to the original work of HOPKINS and COLE, of NEU-
BERG, and of ABDERHALDEN and KEMPE.S

As shown by HOPKINS and CoLE,4 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, C8H7N = C6H4<^\CH, and Skatol, or /?-METHYLINDOL,

NH
C.CH3
C9H9N-C6H4</VCH^ are formed -m 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 (skatol obtained from
indigo is odorless). 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 without .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

2 Zeitschr. f. physiol. Chem., 52, and Ber. d. d. chem. Gesellsch., 40.

3 Hopkins and Cole, Journ. of Physiol., 27 and 29; Neuberg and Popowsky, Biochem.
Zeitschr., 2; Abderhalden and Kempe, Zeitschr. f. physiol. Chem., 52.

4 Journ. of Physiol., 29.

156 THE PROTEIN SUBSTANCES.

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 (SAL-
KowsKi2). 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 (KoNDO 3) . Skatol dissolves in concentrated hydrochloric
acid with a violet coloration. On warming skatol with sulphuric acid
a beautiful purple-red coloration is obtained (CIAMICIAN and MAGNANINI 4) .

For the detection of indol and skatol in, and their preparation from,
feces 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 abso-
lute alcohol and treated with 8-10 vols. of water. Skatol is precipitated,
but not the indol. The further treatment necessary for their separation
and purification will be found in other works.5

Skatosine, Ci0H16N2O2, is a base first obtained by BAUMmthe pancreas auto-
digestion and later studied by SWAIN. It develops an indol- or skatol-like odor
on fusing with potassium hydroxide. LANGSTEIN 6 obtained a substance which is
perhaps identical with skatosine, in the very lengthy peptic digestion of blood
proteins.

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; see also Dakin, Journ. of biol. Chem., 2.

4 Ber. d. d. chem. Gesellsch., 21, 1928.

5 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.

8 Baum, Hofmeister's Beitrage, 3; Swain, ibid.; Langstein, see Hofmeister, Ueber
Bau und Gruppierung der Eiweisskorper, in Ergebnisse der Physiologic, I, Abt. 1,
1902.

HISTIDIXE. 157

Histidine, C6H9N3O2, according to the investigations of H. PAULY,
.OOP and WINDAUS, and F. Kxoop1, is an a-amino-/?-imidazolpropionic
CH-NHv

C X^C

acid, = CH2

CH(NH2)

COOH

Histidine was first discovered by KOSSEL in the cleavage products of
sturine. It was then found by HEDIN in the cleavage products of pro-
teins by acid hydrolysis, and by KUTSCHER among the products of
tryptic digestion, and finally also as a cleavage product of different pro-
tein substances. It does not occur in the protamines, with the excep-
tion of sturine. Of the protein bodies globin (from horse-hsemoglobin)
seems to be richest in histidine, as ABDERHALDEN found 10.96 per cent.
It also occurs in germinating plants (E. SCHULZE 2) .

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 precip-
itated 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 levorotatoiy, (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) (FRANKEL3). On adding
sufficient bromine water and warming, a reddish coloration ensues

1 Pauly, Zeitschr. f. physiol. Chem., 42; Knoop and Windaus, Hofineister's Bei-
trage, 7 and 8; Knoop, ibid., 10.

2 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.

3 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 Hofineister's Bei-
trage, 8.

158 THE PROTEIN SUBSTANCES.

which turns deep wine-red, later becoming cloudy, due to the forma-
tion of dark amorphous particles (F. KNoop1).

It gives a very beautiful diazo-reaction with diazobenzenesulphonic
acid in solutions made alkaline with sodium carbonate, which according
to PAULYis deep cherry-red in dilutions of 1:20,000 and still markedly
red in 1 : 100,000 (tyrosine gives a similar reaction) .

Histidine is sometimes classified in a group, with the two diamino-
acids, arginine and lysine which KOSSEL has called the hexone bases.

Arginine (guanidine-a-amino valeric acid),

C6H14N402 = (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. GULLWITSCH has found arginine in the ox-spleen. 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 cleav-
age product of protein substances as a class. The greatest quantity was
obtained from the protamines; but the histones and certain plant pro-
teins, 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 KUTSCHER 2) .

On boiling with baryta-water, as well as by the action of an enzyme,
arginase, discovered by KOSSEL and DAKIN,S arginine yields urea and
ornithine. Arginine has been prepared synthetically frorn ornithine
(a-d-diamino-valeric acid) and cyanamide by SCHULZE and WINTER-

STEIN.4

Arginine crystallizes in rosette-like tufts, plates, or thin prisms, is readily
soluble in water with alkaline reaction and nearly insoluble in alcohol.
With several acids and metallic salts it forms crystalline salts and double
salts respectively. Its acidified watery solution is precipitated by
phosphotungstic acid. The most important salts are the copper-nitrate
(C6H14N4O2)2.Cu(N03)2 + 3H2O and the silver salts C6H14N4O2.HNO3 +

1 Hofmeister's Beitrage, 11

2 Schulze and Steiger, Zeitschr. f. physiol. Chem., 11; Schulze and Castoro, ibid., 41;
Gulewitsch, ibid., 30; Hedin, ibid., 20 and 21; Kossel and Kutscher, -ibid., 22, 25, 26.

3 Zeitschr. f. physiol. Chem., 41, and Dakin, Journ. of biol. Chem., 3.

4 Ber. d. d. chem. Gesellsch., 32 and Zeitschr. f. physiol. Chem., 34.

ORNITHINE, LYSINE. 159

AgN03 (the more readily soluble) and CeHuN^.AgNOs + ^H^O (the
mo re difficultly soluble), and its compound with picrolonic acid (STEUDEL1).
Arginine is dextrorotatory. In 9-10 per cent solution in the presence
of 1 mol. HC1 and at 20° C., it has a specific rotation («)D= + 10.7°.
The arginine obtained by KUTSCHER in the tryptic digestion of fibrin
was racemic arginine. KUTSCHER has prepared this racemic arginine
from d-arginine, and RIESSER 2 has obtained /-arginine by splitting
racemic arginine with arginase. On oxidation with permanganate
it splits off guanidine, which can be precipitated with sodium picrate.
ORGLMEISTER 3 bases his method for the quantitative estimation of argi-
nine in mixtures obtained by hydrolysis upon this fact.

CH2.(NH2)

//TTT \

Ornithine (a-^-diamino valeric acid), C5H12N2O2 = ATT/ATTT \ •

CH (NH 2) , is not a primary

COOH

cleavage product of proteins, but is formed from arginine on boiling with baryta-
water. JAFFE,* 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 recently SORENSEN 5 have prepared syn-
thetically yields, as shown by ELLINGER, putresdine (tetramethylenediamine) ,
C4H8(NH2)2, on putrefaction. A. LOEWY and NEUBERG 6 have shown that
ornithine is split into putrescine and CO2 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 dextrorotatory
a- d-dibenzoyldiamino valeric acid.

Diaminoacetic acid, C 2H6N 2O 2 = CH (NH 2) 2COOH was obtained by DRECHSEL 7
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 nearly insoluble in alcohol, and can be used in the isolation
of the acid.

CH2(NH2)

/PTT \

Lysine (a-e-diaminocaproic acid), C6H14N2O2= • , was first

CH(NH2)

COOH

1 Zeitschr. f. physiol. Chem., 37 and 44.

2 Kutscher, Zeitschr. f. physiol. Chem., 28 and 32; Riesser, ibid., 49.

3 Hofmeister's Beitrage, 7.

4 Ber. d. d. chem. Gesellsch., 10 and 11.

5 Fischer, ibid., 34; Sorensen, Zeitschr. f. pyhsiol. Chem., 44.

6 Ellinger, Zeitschr. f. physiol. Chem., 29; Loewy and Neuberg, ibid., 43.

7 Ber. d. sachs. Ges. d. Wissensch., 44.

160 THE PROTEIN SUBSTANCES.

ubtained 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 zein and gluten-protein. 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 l from the protamine a-cypririine.

Lysine has been synthetically prepared by E. FISCHER and WsiGERT.2
This lysine was inactive, 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 (a)D=+14° to 17.25° has been found. On heating with barium
hydroxide it is converted into the inactive modification. According
to ELLINGER 3 lysine yields cadaverine (pentamethylenediamine),
C5Hio(NH2)2, on putrefaction, and this base is formed from the lysine
in the organism of those with cystinuria and at the same time CO2 is
split off (A. LOEWY and NEUBERG).

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+C2H5dH. It gives two silver salts with AgNO3;
one has the formula AgNO3 + C6Hi4N2O2 and the other AgNOa-f-
C6Hi4N202.HN03. With benzoyl chloride and alkali, lysine forms
an acid, lysuric acid, C6Hi2(C7H5O)2N2O2 (DRECHSEL), which is homol-
ogous with ornithuric acid, and whose difficultly soluble acid barium
salt may be used in the separation of lysine.4 The rather insoluble
picrate, which is precipitated from a not too dilute solution of the hydro-
chloride by sodium picrate, may also be used in the detection of lysine.

KUTSCHER and LOHMANN 5 have found a lysine having somewhat different
properties in the final products of pancreas autolysis.

Lysatine or Lysatinine. The formula of this substance is either C,,H13N3O2 or

1 Drechsel, 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 and Kutscher, ibid., 31; Kutscher, ibid., 29; Schulze, ibid., 28; Winterstem,
cited in Schulze and Winterstein, Ergebnisse der Physiologic, I, Abt. 1, 1902; Kossel
and Dakin, Zeitschr. f. physiol. Chem., 40.

2 Ber. d. d. chem. Gesellsch., 35.

3 Zeitschr. f. physiol. Chem., 29.

4 Drechsel, Ber. d. d. chem. Gesellsch., 28; see also C. Willdenow, Zeitschr. f.
physiol. Chem., 25.

5 Zeitschr. f. physiol. Chem., 41.

OXYDIAMINO-ACIDS. 161

. In the first ease this base would appear to be a homologue of
creatine, C4H9N3O2, and in the other, case a homologue of creatiriine, C4H7N3O,
and this is the reason why this body is called lysatine as well as lysatinine. It
is still a question whether lysatine is a chemical individual or, as HEDIN suggests,
only a mixture of lysine and arginine.1

In the preparation of the above bases we can first precipitate all the
bases by phosphotungstic acid, when the monamino-acids remain in solu-
tion. 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 we must refer to the works of DRECHSEL and
HEDIN already cited. KOSSEL and KUTSCHER and recently WINTERSTEIN 2
have suggested a method of separating histidine and arginine as silver
compounds from lysine, and KOSSEL and PATTEN have proposed a method
of separating histidine from arginine by means of mercuric sulphate.

We give below a tabulation of the amounts of the three hexone bases
found in certain protein substances (in weight per cent) :

Arginine.

Lysine.

Histidine.

Sturine 3

58.2

12.0

12.9

Cyprinine (a) 7

4.9

28.8

0.0

Other protamines 3

. 62.5—87.4

0.0

0.0

Histories 3

. . 14.36—15.52

7.7—8.3

1.21—2.34

Casein 4

.. 4.70—4.84

1.92—5.80

2.53—2.59

Syntonin (from meat) 4

5 . 06

3.26

2.66

Heterosyntonose 4

8.53

3.08—7.03

0.37—1.12

Protosvntonose 4

4.55

3.08

3.35

Edestin 5

11.0—14.07

1.3

1.17

Proteid from coniferse seeds 5.. . .

10.9—11.3

0.25—0.79

0.62—0.78

Gluten casein 3

4.4

2.15

1.16

Gluten proteins 3.

2.75—3.13

0.0

0.43—1.53

Gelatin 3 and 4

7.62—9.3

2.49—6.0

0.40

Elastin 6

0.3

+

0.027

Of the oxydiamino-acids found on the hydrolysis of proteins we will
mention the following:

Oxydiaminosebacic acid, (?)C10H20N2O5, has been isolated by WOHLGEMUTH
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, C8HlnN206> has been obtained by SKRAUP 9 on the
hydrolysis of casein with hydrochloric acid. The copper salt crystallizes in

Hedin, Zeitschr. f. physiol. chem., 21.

Kossel and Kutscher, ibid., 31; Winterstein, ibid., 45; Kossel and Patten, 1. c.

Kossel and Kutscher, Zeitschr. f. physiol. Cheni., 31.

Hart, ibid., 33.

Schulze and Winterstein, ibid., 33; see also Kossel, Ber. d. d. chem. Gesellsch.,
34, 3236.

8 Kossel and Kutscher, Zeitschr. f . physiol. Chem., 25, and Richards and Gies,
Amer. Journ. of Physiol., 7.

7 Kossel and Dakiri, Zeitschr. f. physiol. Chem., 40.

8 Ber. d. d. chern. Gesellsch., 37, and Zeitschr. f. physiol.- chem. 44.

9 Zeitschr. f. physiol. Chem., 42.

162 THE PROTEIN SUBSTANCES.

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, C9H16N2O7, and caseinic acid, C12H24N2O5. 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, C12H28N205, is an acid obtained by FISCHER and
ABDERITALDEN * 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 : (a) D = about —9°. It crystallizes in plates, which grow into rosettes
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
crystalline copper salt.

In regard to the methods which are used in the separation and prep-
aration of the amino-acids and other products produced in the hydrolysis
of proteins, we refer the reader to larger handbooks and to E. FISCHER'S
work, " Untersuchungen iiber Aminosauren, Polypeptide und Proteine,"
Berlin, 1906.2

In order to quantitatively follow the proteolysis, especially the later
stages, one can, according to SORENSEN,S determine to advantage the
increase in the carboxyl groups which takes place during the progress
of the hydrolysis. For this purpose he has worked out a method which
is as follows: The amino groups are converted into methylene groups
by means of formaldehyde and then the carboxyl groups determined

N

acidimetrically by ^ Ba(OH)2 (or in certain cases by caustic alkali) using
o

phenolphthalein or thymolphthalein as indicator. By this method
not only can enzymotic products be titrated, but also products of acid
hydrolysis after decolorization by means of silver nitrate.

SIEGFRIED 4 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. For example glycocoll in the presence of lime
yields with carbon dioxide, calcium carbamino-acetic acid, CH2.NH.COO

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 boiling

CO
the filtered solution, then the quotient -~2 gives the number of N atoms

1 Zeitschr. f. physiol. Chem., 42.

2 See Hoppe Seyler-Thierfelder, Handb. d. physiol. in pathol. Chem. Analyse, 8
Aufl., Berlin, 1909, and also Abderhalden in Handb. der Biochem. by C. Oppenheimer,
Bd. 1, p. 347.

3 Biochem. Zeitschr., 7; with Hansen, ibid., 7.

4 Zeitschr. f. physiol. Chem., 44 and 46; with Neumann, ibid., 54; with -Lieber-
mann, ibid., 54.

GLYCOPROTEINS. 163

for every molecule €62 taken up. This quotient is equal to 1 for glycocoll
and the aliphatic amino-acids because these go over quantitatively into
carbamino-acids. With the diamino-acid arginine, which contains 4 nitro-
gen 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.

This behavior, which has been further studied by SIEGFRIED with
NEUMANN and LIEBERMANN, is valuable in determining whether we are
dealing with a mixture of protein cleavage products or a combination of
these, as the quotient becomes larger by the splitting of a peptide bind-
ing. As the quotient also increases during the progress of digestion
SIEGFRIED and his collaborators have attempted to control the progress
of proteolytic cleavage by determining this quotient.

II. Compound Proteins 1

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, nucleoproteins and chromoproteins. Of these the
last-mentioned group (haemoglobin and hsemocyanine) will be discussed
in a subsequent chapter (Chapter VI on the blood) .

a. Glycoproteins (glucoproteins).

Glycoproteins2 are those compound proteins which on decomposition
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

1 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
sense.

2 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 haemia 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»

164 THE PROTEIN SUBSTANCES

cleavage an amino-sugar, which has been shown to be glucosamine in all
but a few exceptions.1 In the chondroproteins, on the contrary, the
protein is united to chondroitin-sulphuric acid.

i. 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
of FR. MuLLER2 and his pupils, occurs, as 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 solu-
tions, 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 precipita-
tion 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 conditions
from the crystalline ovalbumin seem to indicate that we are dealing
with a contamination with a glycoprotein. This question requires fur-
ther study before we can designate these proteins as glycoproteins.

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 3) ,

1 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.

2 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.

3 Giacosa, Zeitschr. f. physiol. Chem., 7; Hammarsten, Pfliiger's Archiv., 36, and
Skand, Arch. f. Physiol., 17.

MUCINS. 165

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 LEVEXE and of CUTTER, and GiEs,1 contains
chondroitin-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 suffi-
ciently 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 (FR. MULLER 2)

Mucin from submaxillary 48.84 6.80 12.32 0.84 (HAMMARSTEN 2)

Mucin from snail 50.32 6.84 13.65 1 .75 (HAMMARSTEN 2)

Synovial mucin 51.05 6.53 13.01 1.34 (v. HOLST 3)

MULLER obtained 35 per cent glucosamine from mucous-membrane
mucin and 23.5 per cent from the submaxillary mucin.

By the action of superheated steam on mucin a carbohydrate, animal gum
(LANDWEHR), is split off. This has not been substantiated by other investigators,
such as HAMMARSTEN, FOLIN and FR. MULLER. 4 Instead of a non-nitrogenous
gum a nitrogenous carbohydrate derivative was always obtained.

On boiling mucin with dilute mineral acids, acid albuminate and
bodies similar to proteoses are obtained, besides a reducing substance
which is not free glucosamine (STEUDEL5). By the action of strong
acids upon mucins or mucoids OTORI 6 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. Certain
mucins, as the submaxillary mucin, are easily changed by very dilute

1 Levene, Zeitschr. f . physiol. Chem., 31 ; Cutter and Gies, Amer. Jourri. of Physiol., 6.

2 Fr. Miiller, Zeitschr. Biologie, 42; Hammarsten, Zeitschr. f. physiol. Chem., 12,
and Pfliiger's Arch., 36.

3 Zeitschr. f. physiol. Chem., 43.

4 Landwehr, Zeitschr. f. physiol. Chem., 8, 9; also Pfliiger's Arch., 39 and 40;
Folin, Zeitschr. f. physiol. Chem., 23; Fr. Miiller, Sitzungsber. d. Gesellsch. zur Beford.
•d. gesammt. Nat-urwiss. zu Marburg, 1896.

5 Zeitschr. f . physiol. Chem., 34.

6 Ibid., 42 and 43.

166 THE PROTEIN SUBSTANCES.

alkalies, as lime-water, while others, such as tendon-mucin, are not
affected. If a strong caustic-alkali solution, such as 5-per cent KOH
solution, is allowed to act on submaxillary mucin, we obtain alkali albu-
minate, bodies similar to proteoses and peptones and one or more sub-
stances of an acid reaction and with strong reducing powers.

On peptic digestion proteoses and peptone-like bodies, still containing
the carbohydrate group, are produced. On tryptic digestion still simpler
cleavage products are formed, namely, leucine, tyrosine, and trypto-
phane (POSNER and GIES 1) . The glucosamine, so far as we know, is
not split off by proteolytic enzymes, but only after strong hydrolysis with
acids, and this speaks against the assumption that the glucosamine group
exists as a glucoside-like combination in the mucin molecule (NEUBERG
and MiLCHNER2).

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 reactions
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, this
can now be carefully acidified with acetic acid without giving a precipitate.
Such acidified solutions are copiously precipitated by tannic acid; with
potassium ferrocyanide they give no precipitate, but on sufficient con-
centration they become thick or viscous. A neutral solution of alkali
mucin is precipitated by alcohol in the presence of neutral 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 submaxil-
lary 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 immedi-
ately precipitated, but dissolves on stirring. If this acid liquid is imme-

1 Amer. Journ. of Physiol., 11. 2 Berl. klin. Wochenschr., 1904.

MUCO1DS. 167

diately 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 (Fn. MULLER).

The precipitation by acetic acid, as shown by HAMMARSTEN,* is not applicable
in the preparation of submaxillary mucin, because another protein substance is
precipitated with the mucin, but remains in solution on using the hydrochloric-
acid method above described. POSNER and GIES 2 have by special experiments
shown the power of mucins of precipitating proteins, and this makes the ordi-
nary method of precipitating with acetic acid questionable.

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 pseudomucin, 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 3 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 substances
are included in this group. Certain of these hyalogens seem undoubtedly to
be glycoproteins. Neossin * of the Chinese edible swallow 's-nest, membranin 5
of DESCEMET'S membrane and of the capsule of the crystalline lens, and spiro-
graphin 6 of the skeletal tissue of the worm Spirographis, seem to act as such.
Others, on the contrary, such as hyalin 1 of the walls of hydatid cysts, and onu-
ptiin,6 from the tubes of Onuphis tubicola, do not seem to be compound proteins.
The so-called mucin of the holothurice 9 and chondrosin 10 of the sponge, Chondrosia
reniformis, and others may also be classed with the hyalogens. As the various
bodies designated by KRUKENBERG as hyalogens are very dissimilar, it is not
of much advantage to arrange these in special groups.

1 Zeitschr. f . physiol. Chem., 12.

2 Amer. Journ. of Physiol., 11.

3 Verb. d. physik.-med. Gesellsch. zu Wiirzburg, 1883; also Zeitschr. f. Biologie, 22.

4 Krukenberg, Zeitschr. f. Biologie, 22.

5 C. Th. Morner, Zeitschr. f . physiol. Chem., 18.

6 Krukenberg, Wiirzburg, Verhandl., 1883; also Zeitschr. f. Biologie, 22.

7 A. Liicke, Virchow's Arch., 19; also Krukenberg, Vergleichende physiol. Stud.,
Series 1 and 2, 1881.

8 Schmiedeberg, Mitth. aus d. zool. Stat. zu Neapel, 3, 1882.

9 Hilger, Pfliiger's Archiv, 3.

10 Krukenberg, Zeitschr. f. Biologie, 22.

168 THE PROTEIN SUBSTANCES.

2. Chondroproteins.

Chondroproteins are those glycoproteins which as primary cleav-
age products yield protein and an ethereal sulphuric acid containing a
carbohydrate, chondroitin-sulphuric acid. 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-sulphuric 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 (X). 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 appearing
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 nearly related to the amyloid substance, but not iden-
tical with it, as shown by NEUBERG.1

Very recently O. HANSSEN2 has studied the mechanically isolated
amyloid 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. On the other
hand, he 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 question as to what relation
this amyloid investigated by HANSSEN bears to such a chondroprotein
and to the substances studied by others and called amyloid, requires
further study, and the question as to the nature of the so-called amyloid
is still unsettled. The amyloid protein prepared by MAYEDAS did not
contain any chondroitin-sulphuric acid. It yielded histidine and did

1 Krawkow, Arch. f. exp. Path. u. Pharm., 40, which contains the literature; Neu-
berg, Verhandl. d. d. Pathol. Gesellsch., 1904.

2 Biochem. Zeitschr., 13.

3 Zeitschr. f. physiol. Chem., 58.

AMYLOID. 169

not, contrary to NEUBERG, behave like a histone. The quantity of
hexone bases was not greater than in the proteins from normal organs.
This amyloid protein yielded, like the amyloid degenerated tissues,
some histone-peptone, and he claims that we have no foundation for the
assumption that the amyloid protein has histone-like characteristics
or is rich in bases. Under these circumstances it must be remarked
that the following statements apply only to the results of older investiga-
tions.

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
per cent. The aorta amyloid of man and of the horse contained respec-
tively C 49.6 and 50.5; H 7.2; N 14.4 and 13.8; S 2.3 and 2.5 per cent.
According to NEUBERG, aorta amyloid differs from spleen and liver
amyloid by a different division of the nitrogen, which is evident from the
following :

Monamiiio-N. Diamino-N. Amide-N.

Liver amyloid 43.2 51.2 4.9

Spleen amyloid 30.6 57.0 11.2

Aorta amyloid 54.9 36.0 8.8

From liver amyloid NEUBERG obtained glycocoll 0.8; leucine 22.2;
glutamic acid 3.8; tyrosine 4.0; proline 3.1; arginine 13.9, and lysine
11.6 per cent.

By the action of alkali, amyloid splits into protein and chondroitin-
sulphuric acid (see Chapter X), and according to KRAWKOW it is there-
fore a firm, perhaps ester-like combination of this acid with protein.
The protein, from the investigations of NEUBERG, is of a basic nature
and most comparable to the histones. According to NEUBERG, amyloid
is a transformation product of the proteins, just as are the protamines,
and the differences between liver, spleen, and aorta amyloid indicate
various phases of this transformation.

Amyloid is an amorphous white substance, insoluble in water, alcohol,
«ther, dilute hydrochloric and acetic acids. It is soluble in concentrated
hydrochloric acid or caustic alkali with decomposition. On boiling 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 absorp-
tion is possible. The amyloid from the " sago " spleen studied by HANS-
SEN showed the same behavior with gastric juice as KRAWKOW found,
while trypsin, as well as autolysis for months, was without action.

170 THE PROTEIN SUBSTANCES.

Amyloid gives the xanthoproteic reaction and the reactions of MIL-
LON and ADAMKIEWICZ. 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 amyloid
obtained by HANSSEN from the " sago " spleen.

Amyloid may be prepared as follows, according to MODRZEJEWSKI
and KRAWKOW.1 The finely divided organ is exhausted first with water
and then with dilute ammonia, which leaves the insoluble amyloid and
removes the free or the combined chondroitin-sulphuric acid, besides
other substances. The product, after being washed with water, is
digested with pepsin for several days at 38° C. The residue, after wash-
ing with hydrochloric acid and water, is dissolved in dilute ammonia,
filtered, again precipitated with dilute hydrochloric acid, dissolved, if
necessary, in ammonia, precipitated a second time with hydrochloric
acid, washed with water, the precipitate dissolved in baryta-water, which
leaves the nucleins undissolved, and the barium filtrate precipitated
with hydrochloric acid, and then washed with water, 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, 2 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 phenyl-
hydrazine.

Another phosphoglycoprotein is helicoproteid, obtained by HAMMARSTEN 3
from the glands of the snail Helix pomatia. It has the following composition:
C 46.99; H6.78; N 6.08; S0.62; 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 4 in the spawn of
the frog probably belongs to this group, but instead of glucosamine it gives
galactosamine on cleavage.

1 Modrzejewski, Arch. f. exp. Path. u. Pharm., 1; Krawkow, 1. c.

2 Zeitschr. f. physiol. Chem., 15.

3 Hammarsten, Pfliiger's Arch., 36.

4 Zeitschr. f. physiol. Chem., 29.

NUCLEOPROTEINS. 171

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 nucleoproteins have
also been found in blood serum and other fluids.

The nucleoproteins may be considered as combinations of a protein
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. In cer-
tain cases this is histone, and the combinations between nucleic acid and
protamines are also sometimes classified as nucleoproteins. The com-
bination 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 nucleoprotamines.
The nucleoproteins differ not only according to the protein component
they contain, but also as to the nucleic acids, which vary among them-
selves. There are essentially different nucleic acids, some among which
contain a pentose carbohydrate while others contain a hexose carbo-
hydrate. 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 w^eak 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 the
precipitate dissolves with more or less difficulty and in some cases not
at all, in an excess of the acid. It dissolves, on the contrary, in very dilute
hydrochloric acid. In this respect nucleoproteins are similar to the nucleo-
albumins and the mucin substances, but differ from these two groups in
that they yield purine bases on hydrolysis. According to PLIMMER

1 Zeitschr. f. physiol. Chem., 55.

172 THE PROTEIN SUBSTANCES.

and SCOTT 1 the nucleoproteins differ from the nucleo-albumins by the
fact that with sodium hydroxide in 1-per cent solution the nucleo albumins
split off phosphoric acid while the nucleoproteins do not. The nucleo-
proteins give the color reactions of the proteins, but those which have
been investigated are dextrorotatory and not levorotatory (GAMGEE and
JONES 2) .

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 and a protein,
rich in phosphorus and poor in protein with strong acid character remains
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 nuclein (MIESCHER, HOPPE-
SEYLER 3) or true nuclein, remains undissolved.- As the nuclein is probably
nothing but a partly modified nucleoprotein poorer in protein, having a-
composition varying with the intensity of the cleavage, it seems unnec-
essary 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 neucleoproteins that the pseudo-
nuclein does to the nucleo albumins, we will give here 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, MILROY) 4. The nucleins
are rich in phosphorus, containing in the neighborhood of 5 per cent.
According to LIEBERMANN,S metaphosphoric acid can be split off from
true nucleins (yeast nuclein). The nucleins are decomposed into pro-
tein and nucleic acid by caustic alkali, and as different ^nucleic acids
exist, so also there exist different nucleins. As previously stated, pro-
teins may be precipitated in acid solutions by nucleic acids, and in this
way, as shown by MILROY, combinations of nucleic acid and proteins
may be prepared which behave quite like true nucleins. All nucleins
yield purine bases (so-called nuclein bases) on boiling with dilute acids.
The nucleins contain iron to a considerable extent. They act like rather
strong acids.

1 Plimmer and Scott, cited in Biochem. Centralbl., 8, p. 109.

2 Hofmeister's Beitrage, 4.

3 Hoppe-Seyler, Med. chem. Unters., 452.

4 Umber, Zeitschr. f. klin. Med., 34; Milroy, Zeitschr. f. physiol. Chem., 22.

5 Pfliiger's Arch., 47.

PSEUDONUCLEINS. 173

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,
washed and purified by alternately dissolving in very faintly alkaline
water and reprecipitating with an acid, washing with water, and treating
with alcohol-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 phos-
phatides must first be removed by treatment with acid, alcohol, and ether,
respectively. We must specially call attention to the fact, as shown by
LiEBERMANN,1 that it is very difficult to remove lecithin (the phospha-
tides) by means of alcohol -ether. No exact methods are known for the
quantitative 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 diges-
tion of certain nucleoalbumins. Pseudonucleins contain phosphorus,
which, as shown by LiEBERMANN,2 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 solu-
tion. They are readily split by barium hydroxide solution with the split-
ting off of phosphoric acid, and according to GIERTZ 3 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 pre-
cipitated from their solution in dilute alkalies by adding acid. They
give the protein reactions very strongly, but do not yield purine bases.

1 Pfliiger's Arch., 47.

2 Ber. d. d. chem. Gesellsch., 21, and Centralbl. f. d. med. Wissensch., 18S9.

3 Zeitschr. f. physiol. Chem., 28.

174 THE PROTEIN SUBSTANCES.

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 repre-
cipitating with acid.

Plastin. After the extraction of the nucleins from cell nuclei of certain plants
by dilute soda solution, a residue is obtained which is characterized by its great
insolubility. The substance which forms this residue has been called plastin.
This substance, of which the spongioplasm of the body of the cell and the nucleus
granules are alleged to be composed, is considered as a nuclein modification of
great insolubility, although its nature is not known.

Cleavage Products of the Nucleoproteins.
i. 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. Certain of them also contain pyrimidine bases. Not only
do the nucleic acids differ among themselves in regard to the occurrence of
different purine bases, but the opinions of authorities concerning this dif-
ference are conflicting. This last is perhaps due to the fact, which is now
admitted, that the two purine bases xanthine and hypoxanthine can be
produced secondarily from guanine and adenine. The statements as to
the occurrence of more than two purine bases in a nucleic acid require
further confirmation. 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.

Guanylic acid and inosinic acid contain only one purine base, namely
guanine and hypoxanthine respectively. The simplest nucleic acids also
contain no pyrimidine bases, which otherwise are found thus far in all
carefully investigated nucleic acids. Not all the nucleic acids are the
same in respect to the pyrimidine bases they contain. Although
thymine, cytosine and uracil are regularly obtained, and it is known that
the uracil can be produced secondarily from the cytosine, still the plant
nucleic acid, the triticonucleic acid, yields only cytosine and uracil, but
no thymine according to OSBORNE and HEYL/ who are confirmed by
WHEELER and JoHNSON.2 LEVENE and MANDELS have also found
cytosine and uracil, but no thymine, in the nucleic acid from the eggs of
the haddock.

1 Amer. Journ. of Physiol., 21.

2 Amer. Chem. Journ., 29.

3 Zeitschr. f. physiol. Chem., 49.

NUCLEIC ACIDS. 175

Pentoses have been found as the carbohydrate group in the guanylic
acid, the inosinic acid, and the plant nucleic acids (yeast and triticonucleic
acids), and a hexose has also been found in the yeast nucleic acid. In
the other animal thymo-nucleic acids, a hexacarbohydrate occurs, as
shown by the investigations of STEUDEL.1 We can differentiate between
two different groups of nucleic acids according to the kind of carbohy-
drate complexes they contain.

Another difference also exists, as above shown, in the number and
kind of purine and pyrimidine bases in the nucleic acids. According to
the number of these bases we can differentiate between the simple nucleic
acids with only one base and complex nucleic acids with several bases.
LEVENE and MANDEL2 designate the first group nucleotides or mononu-
cleotides and the second group poly nucleotides. As we are in the habit of
designating as nucleolin or nucleotinic acid (see below), only those
cleavage products of nucleic acids containing no purine base, but merely
pyrimidine bases, it is not proper, according to HAMMARSTEN, to desig-
nate as nucleotides such bodies as guanylic acid and inosinic acid, which
contain no pyrimidine, but only purine base. Until the nature of the
nucleic acids is completely explained, it would be better to differentiate
between mono- and polynucleic acids, or perhaps still better between
simple and complex nucleic acids. Yet we must bear in mind that the
complex nucleic acids have not been isolated from compound proteins,
but generally from organs, namely, from mixtures of several nucleo-
proteins; hence we cannot know whether these acids are chemical indi-
viduals or whether they are mixtures of closely related simple nucleic
acids. On the other hand, it is also possible that the simple nucleic
acids may be derived from more complex ones by cleavage. Such an
assumption does not apply to the guanylic acid, as its mother protein
contains only one base, namely guanine.

We generally admit of 4 atoms of phosphorus in the empirical' formula
of the various nucleic acids. The relationship of phosphorus to nitro-
gen in the thymus- and the salmo-nucleic acid is according to SCHMIEDE-
BERG 4 to 14, and according to STEUDEL 4 to 15. OSBORNE and HARRIS
found the relationship 4 to 16 in the triticonucleic acid, and BANG 3
found the relationship 4 to 20 in the guanylic acid.

All nucleic acids are amorphous, white, and have an acid reaction.
They are readily soluble in ammoniac al or alkaline water. They also
dissolve in concentrated acetic acid and form insoluble salts with copper

1 Zeitschr. f. physiol. Chem., Bd., 50, 52, 53, 55, 56.

2 Ber. d. d. chem. Gesellsch., 41.

3 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 3", 4'3, 57; Steudel, Zeitschr. f.
physiol. Chem., 49, 53, p. 14; Orborne and Harris, ibid., 36; Bang, ibid., 26 and 31,
and Hofmeister's Beitrage, 5 and Biochem. Centralbl., 1, p. 295.

176 THE PROTEIN SUBSTANCES.

chloride and salts of the heavy metals, and as a rule insoluble basic salts
with the alkaline earths. The /3-guanylic acid is soluble with difficulty
in cold water but rather readily in boiling water, from which it separates
on cooling. Guanylic acid is readily precipitated from its alkali compound
by an excess of acetic acid. The other nucleic acids are, on the contrary,
not precipitated from such compounds by an excess of acetic acid, but
by a slight excess of hydrochloric acid, especially in the presence of alcohol.
In acid solutions these latter nucleic acids give precipitates with pro-
teins, which are considered as nucleins. The behavior of guanylic acid
in this regard has not been shown on account of the great difficulty in
dissolving it in dilute acids. All nucleic acids are insoluble in alcohol
and ether. They do not give either the biuret test or MILLON'S reac-
tion. The nucleic acids are optically active and indeed dextrorotatory
{GAMGEE and JONES 1).

The proteolytic enzymes, such as pepsin and trypsin, decompose the
nucleoproteins more or less; the nucleic acids are not split by these
enzymes 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 which have been called nudeases (!WANOFF,
FR. SACHS). Micro-organisms can also bring about a more or less deep
cleavage of the nucleic acids (SCHITTENHELM and ScHROTER2).

The animal nucleic acids, with the exception of guanylic acid and
inosinic acid, are very closely related to each other, although they are
not identical. They all yield thymine as a cleavage product ; and as the
most studied representative are the nucleic acids of the thymus gland
(thymus nucleic acids) it is advisable for the present to treat them as
one group, which has received the common name of thymo-nucleic acids.

Thymonucleic Acids. The most important investigations on the
nucleic acids have been carried out by KOSSEL and his collaborators,

by MlESCHER, SCHMIEDEBERG, STEUDEL, LEVENE, and LEVENE and

MANDEL.S A. NEUMANN has isolated two nucleic acids, a- and ^-thymus

1 Proc. Roy. Soc., 72.

2 Nakayama, Zeitschr. f. physiol. Chem., 41; Iwanoff, ibid., 39; Fr. Sachs, " 1st die
Nuklease mit dern Trypsin identisch?" Inaug.-Dissert, Heidelberg, 1905; Schitten-
helm and Schroter, f. physiol. Zeitschr. Chem., 41.

3 The work of Kossel and his pupils on the nucleic acids can be found in: Arch,
f. (Anat. u.) Physiol, 1892, 1893; Sitz. Ber. d. Berl. Akad. d. Wiss., 18, 1894; Centralbl.
f. d. med. Wiss. 1893; Ber. d. d. chem. Gesellsch., 20 and 27; Zeitschr. f. physiol.
Chem., 22 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; Schmeideberg, ibid., 37, 43, and 57; Altman, Arch. f. (Anat. u.)
Physiol., 1889; 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; Steudel, ibid., 42, 43, 46, 49, 50, 52, 53, 55, 56.

THYMONUCLEIC ACIDS. 177

nucleic acid, from the thymus gland. The a -acid is soluble with difficulty,
and in proper concentration gives a sodium salt which gelatinizes,
and a barium salt which is precipitated by barium acetate in substance
(KosTYTSCHEw). The barium salt of the /?-acid is not precipitated by
barium acetate. The a-acid is designated as anhydric by SCHMIEDEBERG x,
and the /?-acid as hydrate, and the first can be transformed into the second
by heating. This transformation, according to KOSTYTSCHEW, is a decom-
position whereby two-thirds of the purine bases are split off.

According to SCHMIEDEBERG the thymus nucleic acid is identical
"with the salmo-nucleic acid (from salmon sperm), and also according
to STEUDEL probably with the acid from the herring sperm. Other
nucleic acids, at least 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 JERUSA-
LEM, STEUDEL), mammary glands and kidneys (LEVENE and MANDEL 2)
and from other organs.

We are at the present time not agreed as to the formula for the most
carefully studied thymonucleic acids (from the thymus, herring and sal-
m< n sperm). 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 relation
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
the formula C4oH56N14Oi6.2P2O5. According to STEUDEL for every 4
atoms of phosphorus we have 15 atoms nitrogen and from this he has
calculated the formula C43H57Ni5Oi2.2P2O5 for the thymonucleic acids.
The results obtained on elementary analysis are not sufficient to decide
between these two formula?.

On the decomposition of the nucleic acids the purine bases are first
more or less completely split off and correspondingly also various inter-
mediary products are obtained. One of these is the heminucleic acid
obtained by ALSBERG and containing only one-half of the purine bases,
and another body is thymic acid, which is obtained on heating the free
acid with water, when guanine, adenine and cytosine are simultaneously
split off. Thymic acid forms a barium salt soluble in water, having the
formula Ci6H23N3P2Oi2.Ba (KOSSEL and NEUMANN 3).

1 Neumann, Arch. f. (Anat. u.) Physiol., 1898 and 1899 Suppl.; Kostytschew,
Zeitschr. f. physiol. Chem., 39; Schmiedeberg, 1. c.

2 Alsberg, Arch. f. exp. Path. u. Pharm., 51; Noll, Zeitschr. f. phyisol. Chem., 25;
Mathews, ibid., 23; v. Fiirth and Jerusalem, Hofmeister's Beitrage, 10 and 11; Steudel,
Zeitschr. f . physiol. Chem., 53. See also foot-note 3, p. 176.

3 Alsberg, 1. c.; Kossel and Neumann, Zeitschr. f. physiol. Chein., 22.

178 THE PROTEIN SUBSTANCES.

On more energetic cleavage, ALSBERG was able to isolate a phosphorus-
free product, nucleotin, CaoH^N^is, which contained no purine bases
and which, according to SCHMIEDEBERG, is the ground substance of the
nucleic acid. On the other hand, as shown by STEUDEL, products may
be obtained by the action of nitric acid which contain nearly all of the
phosphoric acid in organic combination with the carbohydrate complexes.
Such a cleavage product containing phosphoric acid and carbohydrate
is of special interest, as it also contained thymine, has been obtained by
LEVENE and MANDEL on the acid cleavage of thymus nucleic acid, and
called glycophosphothymic acid. The relation of this substance to the
thymic acid has not been investigated.

KUTSCHER and SEEMANN obtained guanidine and urea, but no uric acid, as
products on the oxidation of nucleic acid with potassium permanganate. KUT-
SCHER and SCHENCK 1 besides these also obtained adenine, oxalic acid, acetic acid,
an acid having an unknown formula, and another acid which they call martamic
acid, besides guanidine and urea. Martamic acid has the formula C5H8N6O5 or
C4H10N6O5 and gives a silver salt which is soluble in ammonia or nitric acid, and
which crystallizes in tufts of leaves. The crystalline acid, which is soluble in
ether, sublimes at 150°, and does not give the murexide test or Weidel's test.

Nothing decisive is known as to the constitution of the thymonucleic
acids. According to SCHMIEDEBERG they are combinations of phos-
phoric acid with the above-mentioned nucleotin, a nucleotin-phosphoric
acid, with which the purine bases are combined in some way or another.

STEUDEL on the contrary considers them as a tetrametaphosphoric
acid, each phosphorus atom having a carbohydrate group (a hexa car-
bohydrate) and also one of the four nitrogenous cleavage products—
guanine, adenine, cytosine and thymine — attached . This view is undoubt-
edly very promising, and if the statement of LEVENE and MANDEL on the
formation of a glycophosphothymic acid as a cleavage product of the
thymus nucleic acid is substantiated by further research, then this view
of STEUDEL will receive further support. LEVENE and MANDEL admit
the existence of other such mononucleotides, namely, of glycophos-
pho-adeninic, -cytosinic, and -guaninic acids, which are combined with
each other, forming a complex nucleic acid, a polynucleotide. This view
is very similar to that held by STEUDEL. The question whether the com-
plex, sometimes called true nucleic acids, are chemical individuals or
are only mixtures of related, simpler nucleic acids has become of interest
by these investigations of LEVENE and STEUDEL.

Guanylic Acid. This acid, which was first prepared by BANG from
the pancreas, has, according to him, the composition C44H66N2oP4O34-
It has also been found by JONES and ROWNTREE in the spleen and by

1 Kutscher and Schenck, Zeitschr. f. physiol. Chem., 44; Kutscher and Seemann,
Ber. d. d. chem. Gesellsch., 36, and Centralbl. f. Physiol., 17.

GUANYLIC AND INOSINIC ACIDS. 179

LEVENE and MANDEL l in the liver. As positive cleavage products it
yields guanine, pentose (7-xylose according to NEUBERG) and phosphoric
acid. According to LEVENE and JACOBS 2 guanylic acid consists of a
combination of phosphoric acid and the nucleoside, guanosin, CioH13N2O5,
which is a glucoside-like combination of guanine and pentose (d-ribose).
As to the occurrence of glycerin among the cleavage products noth-
ing positive is known, and in fact certain investigations disprove the
occurrence of this substance.3 The acid first described by BANG, the
/?-acid, is formed from another acid, the a-guanylic acid, with a splitting
off of 1 molecule pentose, and the a-guanylic acid contains 1 molecule
pentose and 1 molecule guanine for every molecule of phosphoric acid.
Correspondingly, as pentose is split off in its formation, the /?-acid is some-
what richer in phosphorus and nitrogen than the a-acid, namely, 7.64 per
cent P and 18.21 per cent N as compared with 6.65 per cent P and 15.38
per cent N in the a-acid. This latter acid differs from the /?-acid in
being readily soluble in cold water.

According to BANG 4 the thymus also contains a simple nucleic acid,
namely, an adenylic acid; still this is not based on complete investigations.

Inosinic Acid, CioH13N4PO8 was first isolated by LIEBIG from the
flesh of certain animals and then closely studied by HAiSER.5 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.6
On hydrolysis it yields phosphoric acid, hypoxanthine and pentose,
according to the equation:

CioH! 3N4P08 + 2H20 = H3P04 + C5H4N4O + C5H1005.

The pentose is according to NEUBERG and BRAHN Z-xylose, but LEVENE
and JACOBS 7 have conclusively shown that the pentose is not Z-xylose,
but that it is d-ribose.

Inosinic acid is amorphous, and its solution, containing hydrochloric
acid is levogyrate: (a)D=— 18.5°. It gives crystalline salts, among

1 Bang, 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.

2 Ber. d. d. chem. Gesellsch., 42, 2469.

3v. Fiirth and Jerusalem, Hofmeister's Beitrage, 10 and 11; Steudel, Zeitschr. f.
physiol. Chem., 53. See also Bang, Hofmeister's Beitrage, 11.

4 Hofmeister's Beitrage, 5.

5 Liebig, Annal. d. chem. u. Pharm., 62; Haiser, Monatsh. f. chem., 16.

6 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.

7 Levene and Jacobs, Ber. d. d. chem. Gesellsch., 42.

180 THE PROTEIN SUBSTANCES.

which the barium salt, which is difficultly soluble in water, must bo
mentioned.

According to NEUMANN, the two thymus nucleic acids, a and (5, can
be obtained from the gland, after previously boiling the latter with water
containing acetic acid and then cutting it up fine. The finely divided
gland is boiled with about 3-per cent NaOH for one-half hour for the a-acid
and two hours for the /?-acid, and sodium acetate is added at the same
time. After neutralization with acetic acid, filtration and concentra-
tion, the product is precipitated with alcohol. The nucleic acids can
be obtained from the precipitated sodium nucleates by precipitating with
alcohol containing hydrochloric acid. In the separation of the two
acids, KOSTYTSCHEW makes use of the different behavior of the barium
salts on saturating their solution with barium acetate (see above).
LEVENE'S method consists, on the contrary, in treating the organs first
with 5-per cent sodium hydroxide or with 8-per cent ammonia in the cold,
then nearly neutralizing with acetic acid, precipitating the proteins with
picric acid, and treating the strongly acidified liquid (acetic acid) with
alcohol. In the presence of sufficient acetate the nucleic acids are pre-
cipitated. More recently LEVENE has suggested that the nucleic acid
be dissolved in strong acetic acid and then precipitated with copper
chloride or hydrochloric acid. SCHMIEDEBERG (in collaboration with
HERLANT) who has suggested a careful method for the preparation of
the nucleic acids as copper compounds, has recently l given very exact
and detailed instruction as to the preparation of the nucleic acids. In
regard to the details for the various methods of preparation we must
refer to the original publications cited below.

Guanylic acid may be best prepared, according to BANG and RAAS-
CHOU,2 by the following method: After treating the pancreas vvith 1-
per cent sodium-hydroxide solution for twenty-four hours at the room
temperature, it is dissolved by warming, then made faintly acid with
acetic acid, filtered, made faintly alkaline with ammonia, strongly con-
centrated, and precipitated with alcohol while hot. The proteoses
remain involution, and the precipitated guanylic acid («-acid) is purified
by repeated solutions in water and precipitations by alcohol.

In regard to the preparation of inosinic acid we refer to the works
of HAISER, of NEUBERG and BRAHN and of LEVENE and JACOBS cited on
page 179.

Plant Nucleic Acids. Those best known are the yeast nucleic acid and the
triticonucleic acid, C41H6,N18P4031, isolated by OSBORNE and HARRIS from the
wheat embryo, and which according to these investigators is identical with "the
yeast nucleic acid. Yeast nucleic acid has the probable formula C38H50N15P4O29,
according to LEVENE. 3 Instead of /-xylose it contains d-ribose as the pentose,

1 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 43 and 57; Herlant, ibid., 44; Neu-
mann, Arch. f. (Anat. u.) Physiol., 1899, Suppl.; Levene, Zeitschr. f. physiol.Chem.,
32 and 45; Kostytschew, ibid., 39.

2 Hofmeister's Beitrage, 4.

3 Biochem. Zeitschr., 17.

PURINE BASES. 181

and LEVENE and JACOBS l have prepared two nucleosides from this acid, guanotsin
and adenosin, which have an analogous structure to inosin, namely, consist of
pentose combined with the corresponding purine base, as indicated by the
name. All three nucleosides, inosin, guanosin and adenosin are levorotatory.
The plant nucleic acids are closely related to the thymonucleic acids, but differ
from them by the fact that in the thymonucleic acids the pyrimidine groups
are represented by uracil, cytosine, and thymine, and in the triticonucleic acid
by cytosine and uracil. This last acid, which is dextrorotatory, yields on hydrol-
ysis guanine, adenine, cytosine and uracil. (WHEELER and JOHNSON, OSBORNE
and HEYL.Z) As this last can be formed from the cytosine it is possible that only
the first three bases exist preformed in the acid. An acid with adenine, guanine
and two molecules cytosine also corresponds to the formula where the relationship
of P to N is 4 to 16. LEVENE has been able to prepare from the tubercle bacilli
nucleic acids wThose nature has not been closely studied.

Plasminic acid is an acid which was prepared by ASCOLI and KOSSEL 3 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.

In regard to the preparation of yeast and triticonucleic acid we must refer to
the works of ALTMANN, KOSSEL, OSBORNE and HARRIS. 4

2. Purine Bases.

The cleavage products obtained from the nucleic acids, the nuclein
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, 5 and he has
prepared many of the bodies synthetically. They can all be derived from
the synthetically prepared purine, CsH^N^, which has the formula given
below and which may be considered as a combination of a pyrimidine
ring with an imidazole ring.

N=CH N=CH

|| |

C C— NHv HC

HC-NH

HC C— NHv HC CH >CH

JU_x> UK HC-N/

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:

2C 5C—

N7

1 1 >•

5N— C— N9

1 Ber. d. d. chem. Gesellsch., 42, 2477 and 2703. 2 See foot-note 1, page 174.

3 Ascoli, Zeitschr. f. physiol., Chem. 28. 4 See foot-note 3, p. 176.

5 See E. Fischer, Untersuchungen in der Puringruppe (1882-1906) Berlin, 1907.

182 THE PROTEIN SUBSTANCES.

HN— CO

For example, uric acid, OC C — NHv , is 2, 6, 8-trioxypurine, adenine,

>CO

\J*u \J 11 ilS.

HN— C— NH/

N=C.HN2 HN— CO

HC C — NH\ , is 6-aminopurine, and heteroxanthine, OC C — N.CH3, is
II II >H | ||

N— C— W HN— C— N^L

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, 1-methylxan-
thine, paraxanthine, guanine, epiyuanine, hypoxanthine, episarkine, adenine
(and carnine?). The bodies theobromine, theophylline, and caffeine, occur-
ring 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:

Uric acid, C5H4N4O3 2, 6, 8-trioxypurine

Xanthine, C5H4N4O2 2, 6-dioxypurine

1-methylxanthine, C6H6N40 1-methyl

Heteroxanthine, C6H6N4O2 7 "

Theophylline, C7H8N4O2 1, 3-dimethyl

Paraxanthine, C7H8N4O2 1, 7- "

Theobromine, C7H8N4O2 3, 7- "

Caffeine, C8H10N4O2 1,3, 7-trimethyl

Hypoxanthine, C5H4N40 6-oxypurine

Guanine, C5H5N5O 2-amino

Epiguanine, C6H7N50 7-methyl " "

Adenine, C5H5N5 6-aminopurine

Episarkine, C4H8N4O3(?)

Carnine, C7H8N,O3

After SALOMON 1 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

1 Sitzungsber. d. Bot. Verein der Provinz Brandenburg, 1880.

PURINE BASES. 183

cells appear in such places as were before relatively poorly endowed.
As an example of this, the blood> in leucsemia, is extremely rich in leu-
cocytes. In such blood KOSSEL 1 found 1.04 p. m. purine bases, against
only traces in the normal blood. That these bases are also intermediate
steps in the formation of uric acid in the animal organism is probable,
and will be shown later (see Chapter XV).

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.

C5H4N4O.NH + HNO2 = C5H4N4O2 + N2 + H2O ;

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 2 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 (XV), is of very great interest. In this
connection we must also call attention to the fact, as shown by SUNDVIK,S
that by reduction of uric acid in alkaline solution two bodies may be
obtained which, although not quite identical with xanthine and hypo-
xanthine, are at least bodies very similar thereto.

According to BURIAN 4 the purine bases give beautiful red products
with diazo-compounds as long as the imide hydrogen in the 7th position
(see structural formula above) is not substituted. As the nucleic acids
do not react with the diazo compounds, BURIAN concludes that prob-

1 Zeitschr. f. physiol. Chem., 7.

2 See Chapter XV (uric acid formation) .

3 Zeitschr. f. physiol. Chem., 23.

4 Ber. d. d. chem. Gesellsch., 37 and Zeitschr. f. physiol. Chem., 42 and 51.

184 THE PROTEIN SUBSTANCES.

ably the nucleic-acid residue is combined with the imide hydrogen at
position 7.1

HANS FISCHER 2 has found that the combinations of the purine bases
with diazobodies are azo pigments, and that the diazo group enters at the
8th position. The purine bases substituted at the 8th position do not
combine with diazo bodies any more than those substituted at the 7th
position, and FISCHER concludes from this, contrary to BURIAN, that the
nucleic acid molecule, which also does not react with diazo bodies, with
the formation of pigments, can have the purine bases combined in the
8th as well as the 7th position.

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 XV) in the pres-
ence of a reducing substance such as hydroxylamine (DRECHSEL and
BALKE). Copper sulphate and sodium bisulphite may also be used to
advantage in their precipitation (KRiJGER).3 This behavior of the
purine bases serves just as well as the behavior with the silver solution
for their precipitation and preparation.

HN— CO

C—

Xanthine, CsI^N^, = OC C— NHv (2, 6-dioxypurine),4 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 (Jar vis
guano) .

Xanthine is amorphous, or forms granular masses of crystals, or may
also, according to HoRBACZEWSKi,5 separate as masses of shining, thin,
large rhombic plates with 1 mol. water of crystallization. It is very

1 In regard to the disputed views see Steudel, Zeitschr. f. physiol. Chem., 48;
Burian, ibid., 42 and 51.

2 Zeitschr. f . physiol. Chem., 60.

3 Balke, Zur Kenntnis der Xanthinkorper, Inaug.-Diss. Leipzig, 1893; Kriiger,
Zeitschr. f. physiol. Chem., 18.

4 In regard to the synthesis of xanthine and other purines see E. Fischer, foot-note
5, page 181.

5 Zeitschr. f. physiol. Chem., 23.

GUANINE. 185

slightly soluble in water, in 14,151-14,600 parts at 16° C., and in 1300-
1500 parts at 100° C. (ALMEN1). 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 acidr
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
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 nitric acid, 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 2
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

Guanine, C5H5N5O, = H2N.C C — NIL (2-amino-6-oxypurine) .

u_

Guanine is found in 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 epithelium
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 leuca?mic 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

1 Journ. f. prakt. Chem., 9ft, 2 Ber. d. deutsch. chem. Gesellsch., 30, 2236.

186 THE PROTEIN SUBSTANCES.

certain conditions appear in crystals similar to creatinine-zinc chloride.
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 WULFF 1 100 cc. of cold ammonia
solution containing 1, 3, or 5 per cent NH3 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 KOSSEL 2 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 oxidation
product of adenine and possibly occurs as a chemical metabolic 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). The composition of these and other guanine com-
pounds has been studied by KOSSEL and WULFF.S It also gives a com-
pound with picrolonic acid (LEVENE4). Guanine does not give WEIDEL'S
reaction .

HN— CO

Hypoxanthine,SARKiNE,C5H4N4O, = HC C — NEL = (6-oxypurine) .

This body has been found in all cellular organs and as a cleavage product
of inosinic acid. It is especially abundant in the sperm of the salmon

1 Zeitschr. f. physiol. Chem., 1".

2 Ueber die chem. Zusammensetz. der Zelle, Verh. d. physiol. Gesellsch. zu Berlin,
1890-91, Nos. 5 and 6.

3 Zeitschr. f. physiol. Chem., 17; Capranica, ibid., 4.

4 Biochem. Zeitschr., 4.

HYPOXANTHINE AND ADENINE. 187

and carp. Hypoxan thine occurs also in the marrow and in 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 forms very small, colorless, crystalline needles. It
dissolves with difficulty in cold water, but the claims concerning
solubility therein are very contradictory.1 It dissolves more readily
in boiling water, in about 70-80 parts. It is nearly 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 dilute alkalies and ammonia. The
silver compound dissolves with difficulty in boiling nitric acid. On
cooling, a mixture of two hypoxanthine silver-nitrate compounds possess-
ing an inconstant composition separates out. On treating this mixture
with ammonia and an excess of silver-nitrate and heating, a silver hypo-
xanthine is formed, which when dried at 120° C. has a constant com-
position, 2(C5H2Ag2N4O)H2O, and is used in the quantitative estima-
tion 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 nearly quan-
titatively precipitated as the compound CsHsAgN^.CeH^N^^OH.
Hypoxanthine does not yield an insoluble compound with metaphosphoric
acid. When treated, like xanthine, with nitric acid, it yields a nearly
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 a hypoxanthine solution becomes first ruby-red
and then brownish red in color on the addition of an excess of alkali
(KOSSEL). According to E. FISCHER 2 a red coloration occurs even in
the acid solution.

N=C.NH2

Adenine, CjHsNs, = HC C — NHX (6-aminopurine) , was first found

H

!UU

by KOSSEL 3 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 lucaemic urine (STADTHAGEN 4). It may be obtained
in large quantities from tea-leaves.

Adenine crystallizes with 3 molecules of water of crystallization in
long needles which gradually become opaque in the air, but much more

1 See E. Fischer, Ber. d. deutsch. chem. Gesellsch., 30.

2 Kossel, Zeitschr. f. physiol. Chem., 12, 252; E. Fischer, 1. c.

3 See Zeitschr. f . physiol. Chem., 10 and 12.

4 Virchow's Arch., 109.

188 THE PROTEIN SUBSTANCES.

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 difficultly
soluble in warm nitric acid, and deposits on cooling as a crystalline
mixture of adenine silver-nitrates. With picric acid adenine forms a
compound, CsHsNs.CeH^NC^sOH, which is very insoluble and which
separates more readily than the hypoxanthine picrate, and which 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
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, according to KOSSEL and his pupils, as follows:
The finely divided organ or tissue is boiled for three or four hours with
sulphuric acid of about 5 p. m. The filtered liquid is freed from protein
by basic lead acetate, and the new filtrate is treated with sulphuretted
hydrogen to remove the lead, again filtered, concentrated, and, after
adding an excess of ammonia, precipitated with ammoniacal silver nitrate.
The silver compound (with the addition of some urea to prevent nitri-
fication) is dissolved in not too large a quantity of boiling nitric acid of
sp. gr. 1.1, and this solution filtered boiling hot. On cooling, the silver
xanthine remains in the solution, while the double compounds of gua-
nine, hypoxanthine, and adenine crystallize out. The silver xan thine
may be precipitated from the filtrate by the addition of ammonia and
the xanthine set free by means of sulphuretted hydrogen. The three
above-mentioned silver-nitrate compounds are decomposed by sulphur-
etted hydrogen and the guanine separated from the adenine and hypo-
xanthine by treatment while hot with ammonia, in which the guanine in
difficultly soluble.

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 1 — we can advantageously precipitate
the purine bases with copper sulphate and hydroxylamine, following
the method suggested by KRUGER and ScniTTENHELM2 for the quanti-

1 See foot-note 3, page 184. 2 Zeitschr. f. physiol. Chem., 45.

CYTOSIXE. 189

tative determination of the purines in feces. Details for the above
methods may be found in complete hand-books. The same procedures
are followed in the quantitative estimation of the purine bases in animal

organs.1

3. Pyrimidine Bases.

These bodies are closely related to the purine? ; and pyrimidine,
N=CH

4H4N2, = HC CH.

UH

may be considered as the mother substance thereof.

The pyrimidine bases contained in the nucleic acids are cytosine, uracil
and thy mine.

HN— CNH2

II II
Cytosine, C4H5N3CV=OC CH (6 amino-2 oxypyrim! dine), was first

prepared by KOSSEL and NEUMANN from thymus nucleic acid, and then by
KOSSEL and STEUDEL and others from various animal nucleic acids, and
finally also by WHEELER and JOHNSON from triticonucleic acid. WHEELER
and JOHNSON 2 have also prepared it synthetically. It is transformed
into uracil by the action of nitrous acid.

The free base is difficultly soluble in water (129 parts) and crystallizes
in thin leaves with a mother-of-pearl luster. It is insoluble in ether
and difficultly soluble in alcohol. The double compound with platinum
chloride, the crystalline picrate, the nitrate, and the two sulphates 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 XV), and also the reaction
described by WHEELER and JOHNSON under uracil. In regard to
preparation see KOSSEL and STEUDEL 3 and also KuxscHER.4

1 See Burian and Hall, Zeitschr. f. physiol. Chem., 38; Kossel and Wulff, ibid., 17;
Bruhns, ibid., 14; His and Hagen, ibid., 30.

2 Amer. chem. Journ., 29.

3 Zeitschr. f. physiol. Chem., 37 and 38.

4 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.

190 THE PROTEIN SUBSTANCES.

HN— CO

I I
Uracil, C4H4N2O2, = OC CH (2, 6-dioxypyrimidine), was first

HN— CH

obtained by As COLI and KOSSEL from yeast nucleic acid and later obtained
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.1

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 JOHNSON :2
The uracil solution is treated with bromine-water until it is permanently
cloudy and then treated with baryta-water, when a purple or violet-col-
ored precipitate appears almost immediately. The coloration 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 isodi-
aluric and then dialuric acid is produced, both of which give the colora-
tion. In regard to the preparation of uracil see KOSSEL and STEUDEL.S

HN— CO

Thymine, C6HflN202, = 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
recently GERNGROSS4 have prepared it synthetically.

Thymine crystallizes in small leaves grouped in stellar or dendriform

1 Ascoli, Zeischr. f. physiol. Chem., 31; Kossel and Steudel, ibid., 37; Levene,
ibid., 38, 39; Levene and Maiidel, ibid., 49; E. Fischer and Roeder, Ber. d. d. chem.
Gesellsch., 34.

2 Journ. of biol. Chem., 3.

3 Zeitschr. f. physiol. Chem., 37.

4 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; Gerngrose, ibid., 38.

THYMINE. 191

clusters or, rarely, in short needles (GULEWITSCH l) . It melts at about
321° and sublimes. It is difficultly soluble 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.

In regard to the methods of preparation see KOSSEL and NEUMANN
and W. JoNEs.2

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 3 seem to contradict
such a view.

1 Zeitschr. f. physiol. Chem., 27.

2 Kossel and Neumann, 1. c., and W. Jones, Zeitschr. f . physiol. Chem., 29, 461.

3 Zeitschr. f. physiol. Chem., 51 and 53 (against Burian).

CHAPTER IV.
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 com-
binations with more complex molecules, forming compound proteins.
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 con-
sidered; because we not only have bodies, such as acetic acid and lactic
acid, which are not carbohydrates and still have their oxygen and hydro-
gen 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 car-
bohydrates 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 con-
taining 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 hydro-
lytic splitting.

192

MONOSACCHARIDES. 193

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 l 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.

i. 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, or more correctly oxygen atoms,
contained in the molecule .the monosaccharides 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
dextrose is an aldose, while ordinary fruit sugar (levulose) is a ketose.
The difference may be shown by the structural formulae of these two
varieties of sugar:

Dextrose = CH2(OH).CH(OH).CH(OH).CH(OH).CH(OH).CHO;
Levulose = 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 dextrose 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 Tollens' 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 Chernie der Zucker-
arten, Braunschweig, 1904.

194

THE CARBOHYDRATES.

Gluconic acid = 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 polyhydric
alcohols by nascent hydrogen. Thus ARABINOSE, which is a pentose,
CsKioOs, is transformed into the pentatomic alcohol, ARABITE, C5H12O5.
The three hexoses, DEXTROSE, LEVULOSE, and GALACTOSE, CeH^Oe,
are transformed into the corresponding three hexites, SORBITE, MANNITE,
and DULCITE, CeH^Oe. The ketoses, on the contrary, due to their
constitution, yield a mixture of two alcohols on the same treatment.
From <i-levulose for example we obtain a mixture of d-sorbite and l-
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
levulose, 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 a 1-, d- , and racemic
form, r or d-l form, which is a combination of the first two forms. 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 number of asymmetric
carbon atoms, then for the aldo-hexose, which contains 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 configuration formulae.

As these relations are readily conceived we will, for example, only
give the configuration formulae for the most important pentoses and
hexoses occurring in the animal body.

COH

COH

HOCH

HCOH

HCOH

HOCH

HCOH

HOCH

CH2OH

d-Arabinose

CH2OH

J-Arabinooe

COH

COH

HOCH

HCOH

HCOH

HOCH

HOCH

HCOH

CH?OH

d-Xylose

CH2OH

Z-Xylose

MONOSACCHARIDES.

195

COH

COH

COH

COH

HCOH

TiIOCH

HCOH

HOCH

HOCH

HCOH

HOCH

HCOH

HCOH

HOCH

HOCH

HCOH

HCOH

JJOCH

HCOH

HOCH

CH2OH

d-Gluco^e

CH2OH

VGlucose

CH2OH

d-Ga, la close

CH2OH

J-Galactose

CH2OH

CH2OH

'

CO

CO

HOCH

HCOH

HCOH

HOCH

HCOH

HOCH

CH2OH

d-Levulose

CH2OH

J-Levulose

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 J-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
levulose /-levulose, but d-levulose, showing its close relation to dextro-
rotatory c?-glucose. This designation is generally accepted, and the
above-mentioned signs only show the optical properties in certain cases.

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:

(«)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. In-
versely the per cent P of substance can be calculated, when the specific rotation

is known, 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)n = ± — j-r.

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

196 THE CARBOHYDRATES.

long time can be obtained immediately by boiling or on the addition of very
little ammonia.

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 dextrose 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 + 2H2O

= CH2(OH).[CH(OH)]4.CH(OH).COOH-fNH3.

By the action of nascent hydrogen on the lactone of this acid we obtain
glucoheptose, CyH^Oy 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)]4.CH:N.OH.
These compounds are of importance on account of the fact, as found by
WoHL,1 that they are the starting-point in the formation of one class
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 2 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-dextrose, d-levulose, and d-mannose, may take place (LOBRY DE BRUYX
and ALBERDA VAN EKENSTEINS), 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

1 Ber. d. d. chem. Gesellsch., 26, p. 730.

2 Ruff, Ber. d. d. chem. Gesellsch., 31 and 32; Neuberg, Biochem. Zeitschr., 7.

8 Ber. d. d. chem. Gesellsch., 28, 3078; Bull. soc. chim. de Paris (3), 15; Chem.
Centralbl.., 1896, 2, and 1897, 2.

DERIVATIVES OF SUGARS. 197

also occurs in the animal body. NEUBERG and MAYER l 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 (see 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.2 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 c?-arabinose, then d-glucosaminic acid and
finally from its lactone d-glucosamine, which occurs in the animal body.
In a similar manner they 3 obtained Z-glucosamine from /-arabinose.

KNOOP and WINDAUS 4 have obtained large amounts of methylimida-

CH3

zol, C — NH\ , from glucose by the action of ammonium-zinc hydroxide

CH— Nr

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 H.C.C— NH

| + + >CH= ||
COH NH3 0' 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

1 Zeitschr. f. physiol. Chem., 37.

2 Lobry de Bruyn, Ber. d. d. chem. Gesellsch., 28; E. Fischer, ibid., 35.

3 Ibid., 36 and 35, p. 3787.

4 Ibid., 38, and Hofmeister's Beitrage, 6.

198 THE CARBOHYDRATES.

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 oft
water and unite with other bodies, producing lactone-like combinations,
which have been called glucosides. These glucosides, which are generally
compounds with aromatic groups, occur widely distributed in the vegeta-
ble 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 dextrose. The
glucosides can be split into their components by chemical agents, boiling
with dilute mineral acids, as well as by the action of enzymes. The com-
plex 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 decomposition of this kind
is the splitting of amygdalin by the enzyme emulsin. It yields glucose,
benzaldehyde and hydrocyanic acid according to the equation:

C2oH27NOi i + 2H2O = 2C6Hi2O6 + C6H6COH + CNH.

With phenylhydrazine or substituted phenylhydraziries, the sugars
first yield hydrazones with the elimination of water, and then on the further
action of hydrazine on warming in an acetic-acid solution we obtain
osazones.

The reaction takes place with the aldoses as follows:

(a) CH2(OH).[CH(OH)]3.CH(OH).CHO + H2N.NH.C6H5= •

CH2(OH).[CH(OH)]3.CH(OH)CH:N.NH.C6H5

Phenylglucosehydrazone

(6) CH2(OH) [CH (OH)],.CH(OH) .CH : N.NH.C6H5 + H2N.NH.C6H5 =

CH2(OH).[CH(OH)]3.C.CH:N.NH.C6H5

N.NH.C6H5 +

Phenylglucosazone

and with the ketoses :

CH2(OH)[CH(OH)]3CO.CH2(OH)+H2N.NH.C6H5-

CH,(OH)[CH(OH)],C.CH8OH

N.NH.C6H5fH2O,
and CH2(OH)[CH(OH)]3.C.CH2(OH)

N.NH.C6H5 + H2N.NH.C6H5 =

CH2(OH) [CH(OH)],.C.CH : N.NH.C6H5 + H2O + H2.

N.NH.C6H5

The hydrogen is not evolved, but acts on a second molecule of phenylhy-
drazine and splits it into aniline and ammonia :

H2N.H.C6H5 + H2 = H2N.C6H5 + NH3.

OSAZONES 199

For the isolation of the sugars we often make use of the hydrazones
as well as the osazones obtained by the aid of substituted phenyl hydra-
zines instead of the phenyl hydrazine, because they yield difficultly soluble
compounds. 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 fuming
hydrochloric acid (for disaccharides still better with ben z aldehyde),1
the osazones yield so-called osones, which on reduction yield aldoses or
more often ketoses.

We can also pass from the osazones to the corresponding sugars in
other, indirect, ways. 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 yeast has been shown only for
the hexose group, and in fact all of 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 Worms 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 rf-levulose ferments while
the sorbose does not. Among the sugars containing nine atoms of carbon,
the nonoses, the mannonose does not ferment while the glucononose does.
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 and
WOHLGEMUTH 3 upon arabinose and of NEUBERG and MAYER 4 on man-

1 E. Fischer and Armstrong, Ber. d. d. chem. Gesellsch., 35.

2 Herzfeld, ibid., 28; Ruff and Ollendorff, ibid., 32.

3 Zeitschr. f. physiol. Chem., 35. 4 Ibid., 37.

200 THE CARBOHYDRATES.

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. These
observations as well as the varying ferment ability of the sugars are only
a few examples showing the great influence that chemical structure and
configuration have upon chemical compounds in making the material
changes in the animal and plant kingdom.

In the alcoholic fermentation the sugar is decomposed according
to the general equation C6Hi206 = 2C2H6O + 2CO2. The exact process
is not clear, and seems to be rather complicated. According to the inves-
tigations of BUCHNER and MEISSENHEIMER, STOKALSA and MAZE, l we are
•dealing here with the complex action of two enzymes, of which one,
the zymase (BUCHNER and MEISSENHEIMER), transforms the sugar
into lactic acid, while the other, the lactacidase (BUCHNER and MEISSEN-
HEIMER) splits the lactic acid into alcohol and carbon dioxide. Accord-
ing to certain investigators methyl glyoxal, CH^CO.COH, is formed
intermediary between the sugar and the lactic acid.2

As previously mentioned, the sugars also undergo other fermentations,
namely lactic acid and butyric acid fermentation.

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 ammoniac al 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, dex-
trose and levulose. They also occur in great abundance in nature as
more complex carbohydrates (di- and poly sac charides) ; 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

1 Buchner and Meissenheimer, Ber. d. d. chem. Gesellsch., 37 and 38; Stoklasa,
Ber. d. d. Botan. Gesellsch., 22, pages 358 and 460; Maze", Compt. rend., 138.

2 Buchner and Meissenheimer, 1. c. and Ber. d. d. chem. Gesellsch., 39. In regard
to the chemical processes in alcoholic fermentation see also Schade, Zeitschr. f.
physikal. Chem., 57, and Biochem. Zeitschr., 7; Wohl, ibid., 5; Slator, Ber. d. d. chem,
Gesellsch., 40.

PENTOSES. 201

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 by the hydrolytic 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 building up of certain plant con-
stituents. Methyl pentosans and methyl pentoses also occur in the
plants, and of these, the methyl pentose, rhamnose, 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,
and later by SALKOWSKI and others in human urine. Small quantities
of pentoses have been detected by KULZ and VOGEL l 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. 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 VIII). The pen-

1 Salkovvski 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.
Biologic, 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. Biologic, 29 and 42; Neuberg and Wohlgemuth,
Zeitschr. f. physiol. Chem., 35.

202 THE CARBOHYDRATES.

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.1
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, Sen ONE and TOLLENS seem to indicate that the pen-
toses are fermentable 2. 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

HC— CH

from this its adehyde, the furfurol HC C.CHO. The furfurol pass-

Y

ing over on distilling with hydrochloric acid can be detected by the aid
of aniline-acetate paper, which is colored beautifully red by furfurol.
In the quantitative estimation we can use the method suggested by TOL-
LENS, 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 3 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 reac-
tions, as suggested by TOLLENS, are especially applicable.

The orcin-hydrochloric acid test. Mix with the solution or the sub-
stance introduced into water an equal volume of concentrated hydro-
chloric 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-

1 See Ebstein, Virchow's Arch., 129; Tollens, Ber. d. deutsch. chem. Gesellsch., 20,
1208; Cremer, 1. c.; Lindemann and May, Deutsch. Arch. f. klin. Med., 56; Salkowski,
Zeitschr. f. physiol. Chem., 30.

2 Salkowski, Zeitschr. f. physiol. Chem., 30; Bendix, see Chem. Centralbl., 1.900, 1;
Schone and Tollens, ibid., 1901, 1.

3 Bendix and Ebstein, 1. c., which contains the literature; Jolles, Ber. d. d. chem.
Gesellsch., 39 and Zeitschr. f . anal. Chem., 46.

PENTOSES. 203

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 *) . In regard to the use of these tests in urine examina-
tion see Chapter XV.

Many modifications of these tests have been suggested. BRAT 2 performs
the orcin reaction by the addition of NaCl and heating to only 90-95°. RIAL 3
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 (1^-2 minutes),
when using this modification, a confusion with sugars of the six carbon series ma •
occur (BiAL, VAN LEERSUM).4 According to R. ADLER and 0. ADLER the phi »
roglucin and orcin tests can be made with glacial acetic acid and a few dro} -
hydrochloric acid instead of with the hydrochloric acid alone. These investigator^
also use a mixture of equal volumes of aniline and glacial acetic acid as a reagen*,
for pentoses. On the addition of a little pentose to the boiling mixture a beautiful
red color of furfurol-aniline acetate is obtained. A. NEUMANN 5 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, dextrose, and levulose give characteristic
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 6 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-ara,binose been
found (R. LUZZATTO). Z-Arabinose is said to pass into the urine after
partaking of certain fruits such as plums in large amounts (C. BARS-
ZCZEWSKI 7) .

1 Salkowski, Zeitschr. f. physiol. Chem., 27; Neuberg, ibid., 31.

2 Zeitschr. f. klin. Med., 47.

3 Deutsch. ined. Wochenschr., 1902 and 1903, and Zeitschr. f. klin. Med., 50.

4 Bial, Zeitschr. f. klin. Med., 50; van Leersum, Hofmeister's Beitrage, 5.

5 R. and O. Adler, Pfliiger's Arch., 106; A. Neumann, Berl. klin. Wochenschr.,
1904.

8 Fr. Sachs, Biochem. Zeitschr., 1 and 2; Jolles, ibid., 2, Centralbl. f. klin. Med.,
1907, and Zeitschr. f. anal. Chem., 46.

7 Neuberg, Ber. d. d. chem. Gesellsch., 33; Luzzatto, Hofmeister's Beitrage, 6,

204 THE CARBOHYDRATES.

The r-arabinose is crystalline, has a sweetish taste, and melts at
163-164° C. Its diphenylhydrazone, which, according to NEUBERG
and WoHLGEMUTH,1 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 phenylozasone 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 first pentose to be isolated from organs was Z-xylose
by NEUBERG from the pancreas neucleoproteins. NEUBERG and BRAHX
claim to have isolated Z-xylose from inosinic acid, but the investigations
of LEVENE and JACOBS 2 show that the pentose in this case is not Z-xylose,
but rather d-ribose. Z-Xylose occurs very extensively in the plant king-
dom, and is obtained from wood-gums by boiling with dilute acids.
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 3 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.

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 hexosesr
such as dextrose and levulose, 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.

and Arch. f. exp. Path. u. Pharm., 1908, Schmiedeberg-Festschr.; Barszczewski,
Maly's Jahrb., 27, p 733.

1 Zeitschr. f . physiol. Chem., 35.

2 Neuberg, Ber. d. d. chem. Gesellsch., 35; Neuberg and Brahn, Biochem. Zeitschr.,
5, and Ber. d. d. chem. Gesellsch., 41, p. 3376; Levene and Jacobs, Ber. d. d. chem.
Gesellsch., 41 and 42.

3Tollens and Muther, Ber. d. d. chem. Gesellsch., 37; Bertrand, Bull. soc. chim.
(3), 5.

HEXOSES. 205

All hexoses, as also their anhydrides, yield levulinic acid, C6H8Os,
besides formic acid and humus substances on boiling with dilute min-
eral acids. 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, DEXTROSE, and GALACTOSE, and to the other
IEVULOSE, and possibly 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 is given below.

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 1 later obtained a mixture
of several 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 a-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 fl-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 glyceric 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-levulose. With yeast one half, the levogyrate
c?-levulose ferments, while the dextrogyrate /-levulose remains. The r- and
/-levulose 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
/-man nose 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.

d-Levulose is obtained from d-mannose by the method given on page 196,
using the osazone as an intermediate step. The d- and /-mannonic acids are partly
converted into d- and /-gluconic acids on heating with quinoline, and d- or l-
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 c/-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.

1 Butlerpw, 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. 193.

206 THE CARBOHYDRATES.

BOKORNY l has shown, by special experiments on alga? 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.

Among the hexoses known at the present time only dextrose, levulose
and galactose are really of physiological-chemical interest; therefore of
the other hexoses only mannose will be incidentally mentioned.

Dextrose (d-glucose) — GLUCOSE, GRAPE-SUGAR, and DIABETIC SUGAR
— occurs abundantly in the grape, and also, often accompanied with
levulose (^-fructose), in honey, sweet fruits, seeds, roots, etc. It occurs
in the human and animal intestinal tract during digestion, also in small
quantities 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 dextrose
can be formed in the body from proteins or from fats is disputed and will
be discussed in a following chapter (VIII).

Properties of Dextrose. Dextrose 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, CeHioOg, at 170° C.
with the elimination of water. On strongly heating it is converted into
caramel and then decomposes.

Dextrose 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
+52.5° at 20° C.2 Dextrose dissolves sparingly in cold, but more freely
in boiling alcohol. 100 parts alcohol of sp. gr. O.S37 dissolves 1.95 parts
anhydrous dextrose at 17.5° C. and 27.7 parts at the boiling temperature
(ANTHON 3) . Dextrose is insoluble in ether.

If an alcoholic caustic-potash solution is added to an alcoholic solu-
tion of dextrose, an amphorous precipitate of insoluble sugar-potasn
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. Dextrose also forms compounds with lime and baryta.

1 Biolog. CentralbL, 12, pp. 321 and 481.

2 For further information see Tollens, Handbuch der Kohlehydrate, 2. AufL, 44.

3 Cited from Tollens' Handbuch.

DEXTROSE. 207

MOORE'S Test. If a dextrose 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 acidification.1

Dextrose forms several crystallizable combinations with NaCl, of
which the easiest to obtain is (C6Hi2O6)2.NaCl-f-H2O, which forms large
colorless six-sided double pyramids or rhomboids with 13.52 per cent
NaCl.

Dextrose in neutral or very faintly acid (organic acid) solution under-
goes alcoholic fermentation with beer-yeast : C6H12O6 = 2C2H5.OH -f 2CO2.
In the presence of acid milk or cheese the dextrose undergoes lac tic -
acid fermentation, especially in the presence of a base such as ZnO or
CaCOs. The lactic acid may then further undergo butyric-acid fermenta-
tion: 2C3H6O3 = C4H8O2-f-2CO2 + 4H.

Dextrose 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.2

TROMMER'S test is based on the property that dextrose possesses of
reducing cupric hydroxide in alkaline solution into cuprous oxide. Treat
the dextrose solution with about -J—J vol. caustic soda and then carefully
add a dilute copper-sulphate solution. The (fupric 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.

1 In regard to the products formed in this reaction, see Framm, Pfliiger's Arch., 64;
Neff, Annal. d. Chem. u. Pharm., 357; Buchner and Meissenheimer, Ber. d. d. chem.
Gesellsch., 39; Meissenheimer, ibid., 41.

2 In regard to the products produced see Neff, Annal. d. Chem. u. Pharm., 357.

208 THE CARBOHYDRATES.

According to BENEDICT * 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 dextrose 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
salt is dissolved as possible. If a dextrose solution is treated with about
-^o 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 dextrose 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 dextrose solution
gives a precipitate consisting of fine yellow crystalline needles which are
nearly 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 198). This com-
pounds melts when pure at 204-205° 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.3 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. Accord-
ing to NEUBERG4 this behavior can be used in the purification of the
osazone. The diphenylhydrazone and the methyl phenylhydrazone
are also of interest.

Dextrose is not precipitated by a lead-acetate solution, but is almost
completely precipitated by a solution of ammoniac al basic lead acetate.
On warming, the precipitate becomes flesh-color or rose-red (RUBNER'S
reaction 5) .

If a watery solution of dextrose is treated with benzoylchloride and
an excess of caustic soda, and shaken until the odor of benzoylchloride

1 Journ. of biol. Chem., 3.

2 Zeitschr. f . physiol. Chem., 8.

3 See E. Fischer, Ber. d. d. chem. Gesellsch., 41.

4 Ber. d. d. chem. Gesellsch., 32, 3384.

5 Zeitschr. f . Biologie, 20.

DEXTROSE. 209

has disappeared, a precipitate of benzoic-acid ester of dextrose will be
produced which is insoluble in water or alkali (BAUMANN J).

If J-l cc. of a dilute watery solution of dextrose is treated with a
few drops of a 10 per cent alcoholic solution (free from acetone) of a-
naphthol, the liquid is colored a beautiful violet on the addition of 1-2
cc. of concentrated sulphuric acid (MOLISCH). According to REINBOLD 2
this reaction depends first upon the formation of a volatile substance
which gives a bluish-violet color with a-naphthol and sulphuric acid
in the warmth. On further heating furfurol is also produced, which
gives a raspberry -red to ruby-red coloration.

DIAZOBENZENESULPHONIC ACID gives with a dextrose 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 dextrose and sodium carbonate, and this is converted into
indigo-white by an excess of sugar. An alkaline solution of dextrose is colored
deep red on being warmed with a dilute solution of PICRIC ACID. The behavior
of dextrose toward certain pentose reactions has already been given on page
203.

A more complete description as to the performance of these several
tests will be given in detail in a subsequent chapter (on the urine) .

Dextrose is prepared pure by inverting cane-sugar by the follow-
ing simple method of SOXHLET and TOLLENS, being a modification of
ScHWARz's3 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
dextrose 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 dextrose in animal fluids or extracts of tissues we may
make use of the above-mentioned reduction tests, the optical determina-
tion, fermentation, and phenylhydrazme tests. For the quantitative esti-
mation 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 dextrose. In regard to the difficulties
of operating with blood and serous fluids we refer the student to larger
works.

1 Ber. d. deutsch. chem. Gesellsch., 19; also Kueny, Zeitschr. f. physiol. Chem., 14,
and Skraup, Wien. Sitzungsber., 98, (1888).

2Molisch, Monatshefte f. Chem., 7, and Centralbl. f. d. med. Wissensch., 1887,
pp. 34 and 49; Reinbold, Pfliiger's Arch., 103.

3 Tollens, Handbuch der Kohlehydrate, 2. Aufl. I, 39.

210 THE CARBOHYDRATES.

Mannoses. d-Mannose, also called seminose, is obtained with d-levulose
on the careful oxidation of d-mannite. It is also obtained on the hydrolysis
of natural carbohydrates, such as salep slime and reserve cellulose (especially
from the shavings from 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.

Galactose (not to be mistaken for lactose or milk-sugar) is obtained
on the hydrolytic cleavage of milk-sugar and by hydrolysis of many other
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 dextrose. It is dextrogyrate, 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 andTmERFELDER), but not by
Saccharomyces apiculatus,2 which is of importance in 'physiological-
chemical investigations. Galactose reduces FEHLING'S solution to a
less extent than dextrose, 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
ot 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.

Levulose, also called ^-FRUCTOSE and FRUIT-SUGAR, occurs, as above
stated, mixed with dextrose extensively distributed in the vegetable
kingdom and also in 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 levulose in the urine. NEUBERG and STRAUSS 3
have detected levulose with positiveness in human blood-serum, and
exudates in certain cases.

Levulose crystallizes with comparative difficulty in coarse crusts
or warts or in fine needles. C. MORNER 4 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. Levulose

1 See C. Oppenheimer, Handb. d. Biocehm. 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.
4Svensk. Farmac. Tidskr, No. 6, 1907. See abo Maly's Jahresb., 37, p. 95.

LEVULOSE 211

is readily soluble in water, but nearly 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. Levulose ferments with
yeast, and gives the same reduction tests as dextrose, and also the same
osazone. It gives a compound with lime which is less soluble than the
corresponding dextrose compound. Levulose is not precipitated by
sugar of lead or basic lead acetate.

Levulose does not reduce copper to the same extent as dextrose.
Under similar conditions the reduction relationship is 100:92.08.

In detecting levulose and those varieties of sugar which yield levulose
on cleavage we make use of the following reaction, suggested by SELI-
WANOFF : 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 a substance precipitates
which is red in color and soluble in alcohol. According to OFNER l the
mixture must not contain more than 12 per cent HC1, and the boiling
must not be continued longer than twenty seconds, otherwise glucose,
mannose, and indeed maltose, may give a similar reaction. R. and O.
ADLER2 perform the test with glacial acetic acid and a drop of hydro-
chloric acid and some resorcinol, in which case a reaction with aldoses is
not obtained. SELIWANOFF'S reaction, which according to ROSIN may
"be made more delicate by a combination with the spectroscopic examina-
tion, is, as NEUBERG 3 has shown, a general reaction for ketoses.

The naphtho-resorcinol reaction as suggested by B. TOLLENS and
RoRiVE4 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 this
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, 5 methylphenylhydrazine is an excellent
substance to use for the separation and detection of levulose, as it gives
a characteristic levulose methylphenylosazone. This osazone when recrys-
tallized from alcohol melts at 153°. It shows a dextrorotation of 1° 40'

1 Monatshefte f. Chem., 25.

2 See foot-note 5, p. 203.

3 Zeitschr. f. physiol. Chem., 31; Rosin, ibid., 38.

4 Ber. d. d. chem. Gesellsch., 41, p. 1783 and Tollens, ibid., 41, p. 1788. See also
Mandel and Neuberg, Biochem. Zeitschr. 13.

5 Ber. d. d. chem. Gesellsch., 35; also Neuberg and Strauss, ibid., 36.

212 THE CARBOHYDRATES.

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 methylphenylhydrazine
in the detection of levulose. He has obtained the osazone from dex-
trose and methylphenlhydrazine, although the osazone is formed much
more quickly with levulose than with dextrose. Only when the separa-
tion of the osazone crystals with methylphenylhydrazine after the addi-
tion of acetic acid takes place within five hours at ordinary temperatures
is the presence of levulose positively proven (OFNER l).

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.

Levulose, as above stated, is best obtained by the hydrolytic cleavage
of inulin, by warming with faintly acidulated water.

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.

i

Appendix to the Monosaccharides.
a. Amino Sugars.

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 NEUBERG2 obtained the amino-acet aldehyde, 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 :

NH2

CH2 + CHO

CHO CH2 + O =
|

N

HC CH

II II +
HC CH

3H2O

NH2

\/

N

•

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-

1 Ber. d. cU chem. Gesellsch.. 37. and Zeitschr. f . physiol. Chem.. 45.
id., 41.

AMINO-SUGARS. 213

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 l 2,5-ditetraoxybutyl
pyrazine ( = fructosazine) is hereby produced according to the following
equation :

NH2 N

4H9C4.C C

O4H9C4.CH + CHO O4H9C4.C CH

| | +0= | || +3H20

CHO CH.C4H9O4 HC C.C4H9O4

/ V

NH2 . N

Fructosazine

The 2,5-ditetraoxybutyl pyrazine, which STOLTE obtained by LOBRY
DE BRUYN' s2 method from levulose 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-levulose and glycocoll (SriRO), 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.

According to OFFER 4, pentosamine occurs in the liver of the horse. Accord-
ing to OFFER, the pentose derivative, which he calls dipentosamine (C5H7O3.NH2)2 +
H2O and a second, perhaps a diacetyl-pentosamine 2(CH3CO)010H18N2O7 (?), 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 animal organs is glucosamine.

CH2OH

*,-,., . N r, TT AT/-. (CH.OH)s, whose synthet-
d-Glucosamme 5 (chitosarmne), C6H13N05, = .

CH.NH2
COH
ical preparation has already been given on page 197, was first prepared

1 Hofmeister's Beitrage, 11.

2 Cited by Stolte, Hofmeister's Beitrage, 11.

3Spiro, Hofmeister's Beitrage, 10, p. 283; Stolte, Biochem. Zeitschr., 12.

4 Hofmeister's Beitrage, 8.

5 According to E. Fischer's suggestion we shall use the term glucosamine instead
of the term chitosamine, which has lately been generally used.

214 THE CARBOHYDRATES.

by LEDDERHOSE l 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 83 and 164). Glucosamine is,
as E. FISCHER and LEUCHS 2 have shown, a derivative of glucose or d-
mannose (probably glucose), and is a a -ami no -sugar.

The free base, which can crystallize in needles, is readily soluble in
water with an alkaline reaction, and quickly decomposes. The charac-
teristic hydrochloride forms colorless crystals which are stable in the air
and readily soluble in water, difficultly soluble in alcohol, and insoluble in
ether. The solution is dextrorotatory, («) D = + 70. 15° to 74.64°, at various
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 phenylisocyajiate a compound which can be converted into
its anhydride by acetic acid, and is used in the separation and detection
of glucosamine (SxEUDEL4). On oxidation with nitric acid it yields
norisosaccharic acid, whose lead salt can be separated, and whose salts
with cinchonine or quinine are difficultly soluble in water and can also
be used very successfully in the detection of ghicosamine (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 previously been
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.7 In regard to it? prepara-
tion from protein substances we must refer to the works cited on page
83, footnote 3.

Albamine (diglucosamine), (CeHu04N)2 + H2O, is the name given by S. FRANKEL 8
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,

1 Zeitschr. f. physiol. Chem., 2 and 4.

2 Ber. d. d. chem. Gesellsch., 36.

3 See Hoppe-Seyler-Thierf elder's Handbuch, 8. Aufl.; Sundwik, Zeitschr. f. physiol.
Chem., 34.

4 Zeitschr. f . physiol. Chem., 34.

5 Ber. d. d. chem. Gesellsch., 34.

6 Mediz. Woche, 1901, No. 15; see Langstein, Ergebnisse der Physiol., I, Abt. 1, 88.

7 See Hoppe-Seyler-Thierf elder's Handbuch, 8. Aufl.
* Monatsh. f. Chem., 19.

GLUCURONIC ACID. 215

and reduces after boiling with acids. As hydrolytic cleavage product it yields
d-glucosamine.

Galactosamine is claimed to have been found by SCHTJLZ and DITTHORN in
a glycoprotein of the spawn of the frog. This claim is not generally accepted,
v. EKENSTEIN and BLANKSMA x obtained galactose on the hydrolysis of the slimy
envelope of frog eggs.

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 (XV). We here will only describe the
c?-glucuronic acid in connection with the carbohydrates.

CHO

d-Glucuronic acid (glycuronic acid) , C6H10O7 = (CH.OH) 4, is a derivative

COOH

of dextrose, and has been synthetically prepared by E. FISCHER and PILOTY 2
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 NEUBERG 3 have obtained Z-xylose from glu-
curonic acid by splitting off CO2 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,
phenol and probably also indoxyl- and skatoxylglucuronic acid (MAYER
and NEUBERG). It occurs to a much greater extent in urine as con-
jugated 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 campho glucuronic acid, and
then by v. MERING 4 from urochloralic acid by cleavage with dilute acids.
According to P. MAYER/ on the oxidation of dextroses partial formation
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 dextrose. Conjugated glucu-
ronic acids may also occur in the blood (P. MAYER, LEPINE and BOULUD 6),
in the feces, and in the bile.7 NEUBERG and NEIMANN 8 have prepared

1 Schulz and Ditthorn, Zeitschr. f. physiol. Chem., 29; v. Ekenstein and Blanksma,
Chem. Centralbl., 1907, 2, p. 1001.

2 Ber. d. d. chem. Gesellsch., 24.

3 Zeitschr. f. physiol. Chem., 36.

4 Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Schmiedeberg u. Meyer, ibid.,
3; v. Mering, ibid., 6.

5 Zeitschr. f. klin. Med., 4". See Chapter XV.

8 Zeitschr. f. physiol. Chem., 32; Lepine and Boulud, Compt. rend., 133, 134, 138.

7 See Bial, Hofmeister's Beitrage, 2, and v. Leersum, ibid., 3.

8 Zeitschr. f. physiol. Chem., 44.

216 THE CARBOHYDRATES.

certain conjugated glucuroric acids (see Chapter XV) 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 lac tone, glucurone, C6H8O6, 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 (NEUBERG1). Glucuronic
acid is dextrorotatory, while the conjugated acids are levorotatory ;
they behave like dextrose with the reduction tests, and do not ferment
with yeast. They give the pentose reactions with phloroglucin or orcin
and hydrochloric acid, and also a good reaction with naphthoresorcinol
and hydrochloric acid, (see page 211). 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 2 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. With the phenylhydrazine test it gives crystalline com-
pounds which are not sufficiently characteristic (THIERFELDER, P.
MAYERS). By the action of 3 mol. phenylhydrazine and the necessary
amount of acetic acid upon 1 mol. glucuronic acid at 40° for a few daysr
NEUBERG and NEIMANN obtained the glucuronic-acid osazone, which
was very similar to glucosazone and melted at 200-205°. With p-brom-
phenylhydrazine hydrochioride 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.4
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

1 Ber. d. d. chem. Gesellsch., 33.

2 Biochem. Zeitschr., 13.

3 Thierfelder, Zeitschr. f . physiol. Chem., 11, 13, 15; P. Mayer, ibid., 29.

4 See Neuberg, Ber. d. d. chem. Gesellsch., 32; and Mayer and Neuberg, Zeitschr,
f. physiol. Chem., 29.

DISACCHARIDES. 217

and LEFEVRE J have based their quantitative method for the estimation
of glucuronic acid.

Glucuronic acid is best prepared from euxanthic acid, which decomposes
on heating it with water to 120° C. for several hours. The filtrate from
the euxanthon is concentrated at 40° C., when the anhydride gradually
crystallizes out. On boiling the mother-liquor for some time and evaporat-
ing 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 NEI-

MANN.2

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
hydrolytic cleavage of complex carbohydrates. Isomaltose is also obtained
from dextrose by reversion (see page 219).

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 Ci2H22On. 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 + H2O = dextrose + levulose ;
Maltose + H2O = dextrose + dextrose ;
Lactose + H2O = dextrose -f galactose.

The configuration of the dissaccharides has not been determined with
positiveness.

The levulose turns the polarized ray more to the left than the dex-
trose 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

1 Ber. d. d. chem. Gesellsch., 40.

2 Tollens, Zeitschr. f. physiol. Chem., 44, which cites also the older work; Neuberg
and Neimann, ibid., 44; Neuberg, ibid., 45.

218 THE CARBOHYDRATES.

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-
dinarily 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 SciiEiBLER,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
changed by the presence of other inactive substances. The specific
xotation 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 hydrolysis. 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, and pancreatic juice. It is obtained from glyco-
gen under the same conditions (see Chapter VIII). 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,

1 See Tollen's Handbuch der Kohlehydrate, 2. Aufl. 1, 124.

ISOMALTOSE. 219

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 dextrose,1 and is generally given as
(a)D=-|-137 to 138°. Maltose ferments readily and completely with
yeast, and acts like dextrose in regard to the reduction tests. It yields
phenylmaltosazone on warming with phenylhydrazine for \\ hours.
This phenylmaltosazone melts at 205° C., and is more soluble in hot water
than the glucosazone. Maltose differs from dextrose chiefly in the fol-
lowing: It does not dissolve as readily in alcohol, has a stronger dextrorota-
tory power, and has a feebler reducing action on FEHLING'S solution; 10
cc. FEHLING'S solution are, according to SoxHLET,2 reduced by 77.8 milli-
grams anhydrous maltose in approximately 1-per cent solution.

Isomaltose. This variety of sugar, as has been shown by FISCHER,S
is produced, as are dextrin -like products, by reversion, and by the action
of fuming hydrochloric acid on dextrose. A re-formation of isomaltose
and other sugars from dextrose can also be brought about by means of
yeast maltase (HILL and EMMERLiNG4). It is also formed, besides
ordinary maltose, in the action of diastase on starch paste, and occurs
in beer and in commercial starch-sugar. The formation of isomaltose
in the hydrolysis of starch by malt diastase has been denied by many
investigators because they considered isomaltose as contaminated mal-
tose.5 It is produced, with maltose, by the action of saliva or pancreatic
juice (KULZ and VOGEL) or blood-serum (ROHMANN 6) on starch.

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) .

1 See Hoppe-Seyler-Thierf elder's Handbuch, 8. Aufl.

2 Cited from Tollens' Handbuch der Kohlehydrate, 2. Aufl. 1, 154.

3 Ber. d. deutsch. chem. Gesellsch., 23 and 28.

4 Emmerling, ibid., 34; Hill, ibid., 34, and 1. c., foot-notes 1 and 2, p. 65.

5 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,
Journ. of Chem. Soc., 1895; Pottevin, Chem. CentralbL, 1899, II, 1023.

6 Kiilz and Vogel, Zeitschr. f. Biologic, 31; Rohmann, CentralbL f. d. med. Wis-
sensch., 1893, 849.

220 THE CARBOHYDRATES.

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, AMYLUM (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
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 1 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, amylopectin, 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
with 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 rttrogradation by MAQUENNE and Roux, be
transformed into the solid modification, into " 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

*v. Nageli, Botan. MitteiL, 1863; Maquenne and Roux, Compt. rend., 138, 140,
142, 146, and Bull. Soc. chim. de Paris (3), 33 and 35.

STARCHES. 221

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), ID soluble 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
heating 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,1 it is con-
verted into soluble starch (AMYLODEXTRIN, AMIDULIN). Soluble starch
is also 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.2

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.3 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 dextrose. In
the conversion by means of diastatic enzymes we have, as a rule, besides
dextrin, maltose, and isomaltose, only very little dextrose. 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,4 by converting it into dextrose by hydrochloric acid and then
determining the dextrose by the ordinary methods.

Inulin, (C6Hi0O5)x + H2O, occurs in the underground parts of many
Composite, especially in the roots of the Inula helenium, the tubers

1 See Tollens' Handb., 191. In regard to other methods, see Wroblewsky, Ber. d.
deutsch. chem. Gesellsch., 30; Syniewski, ibid.

2 In regard to the compounds of soluble starch and dextrins with barium hydroxide,
see Billow, Pfluger's Arch., 62.

3 See Mylius, Ber. d. deutsch. chem. Gesellsch., 20, and Zeitsch. f . physiol. Chem., 11.
4Tollen's Handb., 2. Aufl., 1, 187.

222 THE CARBOHYDRATES.

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 aMevulose on boiling with dilute sulphuric acid. Diastatic
enzymes have no, or only a very slight, action on inulin.1

According to DEAN 2 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 rota-
tion of («)D= -38-40°.

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 3) .

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 dex-
trose, 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, a 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

Pollen's Handbuch, 208. 2 Amer. Chem. Journ., 32. 8 Upsala Lakaref. Forh., 28.

DEXTRINS AND GUMS. 223

formed, until finally we have sugar and a dextrin, maltodcxtrin, 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 dextrose 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 saccharine ation
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
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 en zymotic action.

The various dextrins are very harcl to isolate as chemical individuals
and to separate from each other. YOUNG 2 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.

The vegetable gums are soluble in water, forming solutions which are viscid
but may be filtered. We designate, on the contrary, as vegetable mucilages

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.

2 Journ. of Physiol., 22, which contains the older researches of Nasse, Kriiger,
Neumeister, Pohl, and Halliburton. Moreau, 1. c.

224 THE CARBOHYDRATES.

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.

The Cellulose Group (C6Hi0O5)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 sub-
stance 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 cellu-
lose 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 converted into dextrose. 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.

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 L25-per cent sulphuric
acid, arid of yielding arabinose, xylose, galactose, and mannose instead of dextrose.
Those hemi celluloses which serve partly as reserve food and partly as support-
substance, are very widely distributed in the plant kingdom.

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.

1 E. Schulze, Zeitschr. f . physiol. Chem., 16 and 19, with Castro, ibid., 36.

CHAPTER V.
ANIMAL FATS AND PHOSPHATIDES.

i. 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 C^Hs.Os.Rs. 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 cer-
tain fatty acids only little studied, the following non -volatile fatty acids,
as glycerides, namely, lauric acid, C^H^C^, myristic acid, C^H^C^,
and arachidic acid, C2oH4oO2. Of the unsaturated fatty acids, besides
oleic acid, we probably also have in small quantities glycerides of acids
of the linolic acid series CnR2n-^O2 and of the linolenic acid series,
CwH2n_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 abundantly. Besides these, oxyacids and high molecular alcohols

225

226 ANIMAL FATS AND PHOSPHATIDES.

have been found in many plant fats. The extent to which traces of
these oxyacids occur in the animal kingdom has not been thoroughly
investigated, but the occurrence of monoxystearic acid seems to have
been proven.^ The occurrence of high molecular alcohols, 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 TRISTEARIN, TRIPALMITIN, and TRIOLEIN, having
an average elementary composition of C 76.5, H 12.0, and O 11.5 per
<?ent. 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,
odorless 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
only after vigorous and prolonged shaking, but the emulsion is not per-

1 Erben, Zeitschr. f. physiol. Chem., 30; Bernert, Arch. f. exp. Path. u. Pharm., 49.

2 Guth, 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 Knopf elmacher, " Untersuch. iiber das Fett im Sauglingsalter," etc., Jahrbuch
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.

STEARIN. 227

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 = C3H3.CHO, when heated alone, or
more easily when heated with potassium bisulphate or with other
dehydrating substances.

The neutral fats may be split by the addition of the constituents of
water according to the following equation:

C3H5(OR) 3 + 3H2O - C3H5(OH) 3 + 3HOR.

This splitting may be produced by the pancreatic enzyme and
other enzymes occurring in the animal and vegetable kingdoms,
for example, the castor lipase. As shown by POTTEVIN and DIETZ l
the reverse action, namely, the synthesis of fatty acid esters, can be
brought about by enzymes, such as pancreatic lipase. 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 boil-
ing them with not too concentrated caustic alkali, or, still better (in
biochemical researches), with an alcoholic potash solution or with sodium
alcoholate. By this procedure, which is called saponification, 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 sapunification 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.C18H350

Stearin, or tristearin, C57HnoO6,==CH.O.C18H35O, occurs especially in
CH2.O.C18H350

1 Pottevin, Compt. rend., 138, and Bull. soc. chim. (3), 35; Dietz, Zeitschr. f.
physiol. Chem., 52.

228 ANIMAL FATS AND PHOSPHATIDES.

the solid varieties of tallow but also in the vegetable fats. Stearic acid,
CigHseC^, is found in the free state in decomposed pus, in the expectora-
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, less frequently as rhombic plates. The opinions
regarding the melting-point are somewhat varied. Pure stearin,
according 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, (CH2)i6, 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 69.2° C. Its barium salt contains 19.49
per cent barium, and its silver salt contains 27.59 per cent silver.

CH2.O.C16H310

Palmitin, or tripalmitin, C5iH98O6, = CH.O.C16H31O. Of the two solid

varieties of fats, palmitin is the one which occurs in predominant quan-
tities in human fat (LANGER 2) . 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,
Ci6H32O2, 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. Palmitic, like stearin,
has a variable melting- and solidify ing-point, depending upon the way it
has been previously treated. The melting-point is often given as 62° C.,
but some investigators3 claim that it melt? at 50.5° C.; solidifies on
further heating, and melts again at 66.5° C.

1 Annal. d. Chem. u. Pharm., 92.

2 Monatshefte f. Chem., 2; see also Jaeckle, Zeitschr. f. physiol. Chem., 36.

3 R. Benedikt, Analyse der Fette, 3. Aufl, 1897, p. 44.

OLEIN. 229

CH3
Palmitic acid, (CH2)14, crystallizes from an alcoholic solution in tufts

COOH

of fine needles. It melts at 62° C.; still the admixture with stearic acid,
as HEINTZ has shown, essentially changes the melting- and solidifying-
points according to the 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, chloro-
form, and benzene. Its barium salt contains 21.17 per cent barium, and
silver salt contains 29.72 per cent silver.

CH2.O.C18H33O

Olein, or triolein, €57^0^6, = CH.O..C18H33O, is present in all animal

fats, and in greater quantities in vegetable fats. It is a solvent for
stearin and palmitin. The oleic acid (elaic acid), Ci8H34O2, 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

PTT

Oleic acid, •• , is an unsaturated acid of the series CreHn_2 O2, and
CH

(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-
formed 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 monoxy stearic acid, found in animal fats in certair
instances, can be explained by this oxidation. Oleic acid on heating
yields, besides volatile fatty acids, sebacic acid, Ci0Hi8O4, which melts
at 127°; 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
odorless oily liquid which solidifies in crystals at about 4° C., which

230 ANIMAL FATS AND PHO3PHATIDES.

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 concentrated
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).1 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, DOECLIC ACID, which is solid at 4° C., liquid at
16° C., and soluble in alcohol, is found in the blubber of the Baloena rostrata.
According to BULL this acid is probably only a mixture of oleic acid and another
acid — gadoleic acid, r*20H38O2, having a melti rig-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 C16H3002. KURBATOFF 2 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 ZINK 3 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
previously made bluish-violet with tincture of alkanet, becomes red in
color. 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 various fatty acids from
each other, we must refer to complete!1 hand-books.

In addition to the methods already suggested there are other chemical meth-
ods 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

1 Zeitschr. f. physiol. Chem., 56.

2 Bull, Ber. d. d. rhem. Gesellsch., 39; Kurbatoff, Maly's Jahresb., 22.

3 Zeitschr. f. anal. Chem., 36.

ANIMAL FATS. 231

titrating 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 saponifi-
cation of 1 gram fat with N/2 alcoholic caustic potash. 3. KEICHERT-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 min-
eral 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 oleic 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 suggested
by KUMAGAWA 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 iso cholesterin 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
quantities of compound esters of lauric, myristic, and stearic acids with radicals
of the alcohols, LETHAL, C12H25.OH, METHAL; CuH^.OH, and STETHAL, Ci8HS7.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

Biochem. Zeitschr., 8.

232 ANIMAL FATS AND PHOSPHATIDES.

in water, but with an alcoholic solution it saponifies readily, and the above-men-
tioned alcohols are set free.

CH3

Ethal or cetyl alcohol, C16H34O. = (CH2),4, 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/ — 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 C16H30O2, occurs also, as found by LJUBARSKY, 2 in con-
siderable 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, Gael^D,,3 which occurs as cetyl
ether in Chinese wax and as free acid in ordinary wax. It dissolves in boiling
alcohol arid 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 rnelissyl (myricyl) alcohol, C30H61.OH. This alcohol is a silky, shining,
crystalline body melting, at 85° C. DUNHAM 4 has found carnaubic acid, C24H48O2,
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 THUDICHUMS 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
atoms of nitrogen in the molecule, and hence we differentiate between
monamido-(P:N = l:l), diamido- (P:N=1:2), triamido- (P: N = l : 3)
monophosphatides, etc. The lecithin group belongs to the monamido-
monophosphatides. The diamidomonophosphatides have been found,
besides in the brain, by THUDICHUM, in the bile (HAMMARSTEN), in the

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.

5 J. L. W. Thudichum, Die chemische Konstitution des Gehirns des Menschen, etc.,
Tubingen, 1901.

PHOSPHATIDES. 233

yolk of the egg (STERN and THIERFELDER), and in muscles (ERLANDSEN),
and seem to be widely distributed. A triamidomonophosphatide, called
neottin by S. FRANKEL and BoLAFFio,1 has been isolated from the yolk
of the egg, and according to THUDICHUM a tetramidomonophosphatide
occurs in ox bile. Among the monoamidodiphosphatides (P:N = 2:1)
we must mention cuorin, which was discovered and studied by ERLANDSEN,
and a phosphatide of the same type occurring in egg-yolk recently described
by MACLEAN.2 According to THUDICHUM non-nitrogenous phosphatides
are also possible (in the brain) . If this be true these bodies must not,
for the present at least, be classified as phosphatides.

The phosphatides seem to be closely related to each other; they
influence the solubility and precipitation properties of each other, 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 descriptions of their properties and composition must be accepted
with a certain reservation.

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. 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, always occurs in the phosphatides. The
phosphatides on this account always take up iodine. They are, as above
stated, autooxidizable, taking up oxygen from the air and being readily
changed (ERLANDSEN) . They also give a beautiful reaction with PETTEN-
KOFER'S bile-acid test. The nitrogenous base is generally choline; in
certain cases their nature is not known, and in others the statements are
somewhat contradictory.

The phosphatides are included in the group of lipoids, which are
difficult to characterize from a chemical standpoint, because they are
generally not soluble in water and because each phosphatide is dissolved by
at least one of the ordinary solvents of the fats. Among themselves they
may show quite a striking difference in behavior toward such solvents.
For example, one may be insoluble in cold alcohol or ether and another
soluble therein, and such differences are of importance in their prepara-
tion. They are generally all precipitated from their solution by acetone

1 Hammarsten, Zeitschr. f. physiol. Chem., 36; Stern and Thierfelder, ibid., 53;
Erlandsen, ibid., 51; Frilnkel (and Bolaffio), Biochem. Zeitschr., 9.

2 Thudichum, Virchow's Arch., 156; Erlandsen. 1. c.; MacLean, Zeitschr. f. physiol.
Chem., 57.

234 ANIMAL FATS AND PHOSPHATIDES.

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 cadmium 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 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 diamido
phosphatides which were 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.

In consideration of the uncertainty which exists as to the properties
and chemical individuality of the various phosphatides, we will here only
discuss in detail the most carefully studied phosphatides, namely, lecithin
and cuorin. The others will be discussed in their proper place.

Lecithins. These bodies are ester compounds l of glycerophosphoric
acid substituted by two fatty-acid radicals with a base called choline.
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 TnuDicHUM2 every true lecithin always
contains at least one oleic-acid radical. According to the investigations
of HENRIQUES and HANSEN, COUSIN and ERLANDSEN,S 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-
amidophosphatides, according to the following type:

CH2 — O — fatty-acid radical.
CH — 0 — fatty-acid radical.

CH2— O\

HO-^EO
yC2H4— O/
Nf(CH3)3
\OH

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-

1 Strecker, Annal. d. Chem. u. Pharm., 148; Hundeshagen, Journ. f. prakt. Chem.
(N. F.), 28; Gilson, Zeitschr. f . physiol. Chem., 12.

2 Thudichum, Die chemische Konstitution des Gehirns des Menschen, etc., Tubingen,
1901.

3Henriques and Hansen, Skand. Arch. f. Physiol., 14 (1903); Cousin., Compt.
rend., 137; Erlandsen, Zetischr. f. physiol. Chem., 51.

LECITHINS. 235

stearyl-lecithin studied by HOPPE-SEYLER and DiACONOW,1 which probably
has a different structure, has the formula C44H9oNPO9. ERLANDSEN
gives the formula C43H8oXPO9 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 only 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 2 claim that the
phosphoric acid is not bound on the middle unsymmetric CH group, but
rather at the end CH2 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 phosphorous in organic combinations, it must
be borne in mind that the above assertions relate chiefly to the occur-
rence of 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/* 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 ani-
mals. NERKING found 41.7 per cent lecithin in the bone marrow and
21.33 per cent in the suprarenal capsule of the urchin when calculated
on the living organs, while the corresponding results in the rabbit were
2.71 and 2.39 per cent, respectively. These determinations have also
shown that the amount of lecithin is especially higher in the new born, and
that the phosphatides to all appearances are of the greatest importance

1 Hoppe-Seyler, Med. chem. Unters., Heft 2 and 3.

2 Ber. d. d. chem. Gesellsch.. 37.

3 Physiol. Chem. Berlin, 1877-81, p. 57.

4 Siwertzow, see Biochem. Zeitschr., 2, p. 310; Glikin, Biochem. Zeitschr., 4 and
7; Nerking, ibid., 10.

236 ANIMAL FATS AND PHOSPHATIDES.

in development. The new born come into the world with a store of phos-
phatides which dimirishes during growth.

That the lecithins are of great importance in the development and
growth of living organisms, in fact for the bioplastic processes in general,
follows also from several investigations.1 We have in lecithin or the
phosphatides as a group, no doubt, very important material for the build-
ing up of the complicated phosphorized nuclein substances of the cell
and cell nucleus. The wide distribution of the lecithins, as also the fact
that they are primary cell constituents, gives great importance to these
substances.

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. MAYER3
claims to have prepared racemic lecithin from ordinary lecithin, and l-
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 XII). On warming this swollen mass or the concentrated
alcoholic solution, decomposition takes place with the production of a
brown color. On allowing the solution or the swollen mass to stand,
decomposition takes place and the reaction becomes acid. According
to the investigations of LONG 4 the lecithins seem to be much more

1 See Stoklasa, Ber. d. deutsch. chem. Gesellsch., 29; Wiener Sitzungsber., 104;
Zeitschr. f. physiol. Chem., 25; W. Danilewsky, Compt. rend., 121 and 123, and W.
Koch, Zeitschr. f. physiol. Chem., 37; P. Kyes, ibid., 41, and Berl. klin. Wochenschr.,
1904.

2 Chem. Centralbl., 1901, 2, p. 30 and 193.

3 Biochem. Zeitschr., 1.

4 Journ. of Amer. chem. Soc., 30, 1908.

LECITHINS. 237

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. GEPHART 1 by salts with monovalent cations, although
slowly. In putrefaction lecithins yield glycero phosphoric 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 bums, 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, which contains
3 molecules of lecithin and 4 molecules of cadmium chloride (ULPIANI 3)
is difficultly soluble in alcohol, but dissolves in a mixture of carbon
disulphide and ether or alcohol. The molybdenum compound must also
be mentioned (EHRENFELD 4) . A solution of lecithin in alcohol is not
precipitated by lead acetate and ammonia.

Lecithins are easily carried down during the precipitation of other
compounds such as the protein bodies, and may therefore very greatly
change the solubilities 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 chap-
ter, and attention is there called to the necessity for more thorough inves-
tigation of this subject. Further investigations of the so-called lecithin-
sugar (BING) is also desirable, as we know nothing positive as to its nature.
According to the investigations of WINTERSTEIN, HIESTAND and E.
ScHULZE,5 lecithins (phosphatides) containing carbohydrates occur in
the plant kingdom, and which contain about 20 per cent carbohydrate. We
are still not decided whether we are here dealing with combinations or
admixtures. The same is true for the iron content of the lecithins or
phosphatides as observed by GLIKIN.S

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 Chem. Centralbl., 1901, 2, p. 30 and 193.

4 Zeitschr. f. physiol. Chem., 56.

5 Winterstein and Hiestand, Zeitschr. f. physiol. Chem., 47 and 54; Schulze, ibid.,
52 and 55.

6 Ber. d. d. chem. Gesellsch., 41.

238 ANIMAL FATS AND PHOSFHATIDES.

Various methods have been suggested by STRECKER, HOPPE-SEYLER
and DIACONOW, THUDICHUM, GILSON, ZUELZER and BERGELL l for the
preparation of the lecithins. As none of these yields a positively pure
product we will only here 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 prepara-
tion 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 detec-
tion of choline is not evidence, as this base probably also occurs 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 precip-
tates only the cephalin.

Cephalin is also a monoamidophosphatide whose formula, based upon
the investigations of THUDICHUM and Kocn,4 is probably C42H82NPO13.
For the present cephalin must be classified with the lecithin group. The
views of these two investigators as to the constitution of this body,
which is difficult to purify, differ very considerably. According to THUDI-
CHUM, on cleavage it yields neurine, glycerophosphoric acid, stearic acid,
and a specific fatty acid, cephalic acid. According to KOCH it contains, on
the contrary, only one methyl group attached to nitrogen, and is therefore
probably dioxystearylmonomethyl lecithin, while according to COUSIN 5
it yields, like lecithin, stearic acid, an unsaturated fatty acid, glycero-
phosphoric acid and choline as decomposition products. Cephalin is
amorphous and swells up in water like lecithin. It is soluble in cold
ether, glacial acetic acid, and chloroform, but is insoluble in acetone and
in alcohol, either cold or warm. The alcoholic solution, as previously

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.

2 Erlandsen, 1. c.; Roaf and Edie, Thompson Yates Laboratory Reports, Vol. 6,
part I, 1905.

3 Koch and Woods, Journ. of biol. Chem., 1.

4 Thudichum, 1. c.; Koch, Zeitschr. f. physiol. Chem., 36.

5 Compt. rend. soc. biol., 62.

CHOLINE. 239

stated, is precipitated by an alcoholic lead acetate solution. Cephalin
is obtained from the brain, after dehydration with acetone, by extracting
with ether and precipitating the concentrated ethereal extract with alcohol.
The cephalin is perhaps identical with the myeline substance isolated by
ZuELZER1 from the brain. The different statements as to the cleavage
products of cephalin speak against its purity and chemical individuality.

Of the cleavage products of the lecithins choline is of especially
great interest.

Choline (trimethyloxy ethyl ammonium hydroxide),

/CH2.CH2.(OH)
C5H15N02, = HO.N<

\CH3)3,

stands in close relationship to the poisonous base neurine (trimethylvinyl

/(CH3)8,

ammonuim hydroxide), HO.N/ , which according to BRIEGER

N?H:CH2
can be formed from choline by the action of bacteria, and also to mus-

/(CH3)3
carine, HO.N< , which is the aldehyde of choline and occurs in the

XJHaCHO

>0v

fly agaric, and also to betaine, H3C.N<^ /CO, which may be considered

XJHa^

as the anhydride of the acid corresponding to choline. Muscarine
and betaine can be obtained from choline on oxidation. Choline yields
trimethylamine as 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,2 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 adrenaline (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 3), gives choline great physiological importance. It must

1 Zeitschr. f. physiol. Chem., 27.

2Mott and Halliburton, Philos. Trans., Ser. B, 191 (1899) and 194 (1901); Marino
Zuco, see Maly's Jahresber., 24, pp. 181 and 698.

3 Lohmann, Pfluger's Arch., 118 and 122; v. Fiirth and Schwarz, ibid., 124, which
also contains the literature.

240 ANIMAL FATS AND PHOSPHATIDES.

be remarked that MODRAKOWSKI l claims that the above action of choline
does not exist in pure choline, but is due to a .contamination with very
small amounts of another substance having powerful action. Choline
decomposes very readily and hence becomes easily contaminated. The
choline isolated from tissues is readily contaminated, hence the claims
made as to the physiological importance of choline in secretion require
further confirmation.

Choline is a syrupy fluid, readily misciblewith absolute alcohol. Hydro-
chloric acid gives with it a compound which is very soluble in water and
alcohol, but insoluble in ether, chloroform, and benzene. This compound
forms a double combination with platinum chloride, is soluble in water,
insoluble in absolute alcohol and ether, and crystallizing ordinarily in six-
sided orange-colored plates. This compound is used in the detection
and identification of this base. Choline also forms a crystalline double
compound with mercuric chloride and with gold chloride. Choline
is precipitated by potassium iodide and iodine (GULEWITSCH), and potas-
sium triiodide can be used for the quantitative estimation of this base
(STANEK2). On heating the free base it decomposes into trimethyl-
amine, 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
the above or the solid lecithin is boiled one hour with baryta -water,
filtered, and the excess of baryta precipitated by C02 ; 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.

CH2.OH

Glycerophosphoric acid, C3H9PO6 = CH.OH , is a bibasic acid which prob-

CH2— O\

OH-)PO

OH/

ably occurs in the animal fluids and tissues only as a cleavage product of lecithins.
According to WILLSTATTER and LUDECKE 3 the glycerophosphoric acid split off
from lecithins is optically active. Its barium and potassium salts are levorota-
tory, and behave in certain regards differently from the corresponding salts of
synthetically prepared glycerophosphoric acid.

The diaminomonophosphatides have unfortunately been little studied,
and the triamidomonophosphatide neottin will be discussed in a sub-

1 Pfl user's Arch., 124.

2 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.

3 Ber. d. d. chem. Gesellsch., 37.

CUORIN. 241

sequent chapter (XIII). The only carefully studied monamidodiphos-
pbatide is cuorin, discovered by ERLANDSEN.

Cuorin, CyiH^s^^C^i, is a monamidodiphosphatide prepared by
ERLANDSEN 1 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 but partly or entirely belonging to the series CnH2n— 40^
and CnH2n— 6^2', also glycerin, phosphoric acid and a base which is
rot well known, but 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 Zeitschr. f. physiol. Chem.; 51, where the method of preparation is described.

CHAPTER VI.
THE BLOOD

THE blood is to oe 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. We also find in the blood granules of
different kinds, which are to be considered as transformation products
of the form-elements.1

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, coagulates1
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, ARTHus2). 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.

1 See Latschenberger, Wien. Sitzungsber , 105.

2 Delezenne, Compt. rend. Soc. de biol., 49; Spangaro, Arch. ital. de Biol., 32;
Arthus, Journ. de Physiol. et Pathol., 4.

242

PREVENTION OF COAGULATION. 243

The coagulation of the blood may be prevented in other ways. After
the injection of pep tore, or, more correctly, proteose, solutions into
the blood (in the living dog), it does not coagulate on leaving the
veins (FANO, SCHMIDT-MULHEIM J). The plasma obtained from such
blood by means of centrifugal force is called peptone-plasma. According
to ARTHUS and HuBER2 the caseoses and gelatoses act like fibrin
proteose in dogs. Eel serum and certain lymph-forming extracts of
organs (see Chapter VII) 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 PAGES 4).
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

1 Fano, Arch. f. (Anat. u.) Physiol., 1881; Schmidt-Mulheim, ibid., 1880.

2 Arch, de Physiol. (5), 8.

3Haycraft, 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.

5 Journ. of Physiol., 19.

6 Journ. de Physiol. et Pharm., 3 and 4.

244 THE BLOOD

blood-clot (placenta sanguinis). If the blood is beaten during coagula-
tion, the fibrin separates in elastic threads or fibrous masses, and the
dcfibrinated blood which separates is sometimes called cruor,1 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 separates as insoluble fibrin.
The albuminous bodies of the plasma must therefore be first described.
They are, as far as we know at present, fibrinogen, nudeoprolein, ser-
globulins, and ser albumins.

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 arid probably other lymphoid organs such
as the spleen and lymph glands, and according to Do YON and NOLF,
the liver. The statement that the intestinal wall is a seat of formation
of fibrinogen, a view that had already 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. GAU-
TIER 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 formation
of fibrinogen in this tissue. The relation between the quantity of fibrin
and leucocytosis as shown by many investigators such as LANGSTEIN
and MAYER, MORAWITZ and REHN, also indicate such a 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

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. 245

strongly diminishes after the extirpation of the liver (NOLF), and that
fibrin ogen may indeed be entirely absent in the blood in phosphorus
poisoning (CoRix and ANSIAUX, JACOB Y, Do YON, MOREL, and KAREFF !),
and that the blood of the hepatic vein, according to DOYON, MOREL
and KAREFF,2 is richer in fibrinogen than the blood from other vessels.
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 XaCl in substance (thus differing from serglobulin) . A salt-free solution
of fibrinogen in as little alkali as possible gives with CaCl2 a precipitate
which contains calcium and soon becomes insoluble. In the presence
of NaCl or by the addition of an excess of CaCl2 the precipitate does not
appear.3 A neutral solution of fibrinogen is precipitated by a concentrated
solution of sodium fluoride when added in sufficient quantity. Fibrino-
gens from different kinds of blood behave somewhat differently in this
regard. According to HuiSKAMp4 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 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 protein 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 MITTELBACH.S

1 P. Muller, 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.

2 Journ. de Physiol., 8 (1906).

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. physio! .
Chem., 28.

5 Zeitschr. f . physiol. Chem., 19.

246 THE BLOOD.

Fibrinogen may be easily separated from the salt-plasma or oxalate-
plasma by precipitation with an equal volume of a saturated NaCl solution.
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. A neutralization of the plasma is not
necessary, and is not to be recommended. For further purification
the precipitate is pressed, redissolved in an 8-per cent salt solution, the
filtrate precipitated 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 dialysis with very faintly
alkaline water. The fibrinogen can be nearly 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, accord-
ing to REYE,1 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. From transudates we ordi-
narily obtain a fibrinogen which is strongly contaminated with lecithin
and which can hardly be purified without decomposing it. 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
srnaU, 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 tem-

1 W. Reye, Ueber Nachweis und Bestimmung des Fibrinogens, Inaug.-Diss. Strass-
burg, 1898.

FIBRIN. 247

perature, 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 carried
down by the fibrin or enclosed within the leucocytes (RuLOT2). Accord-
fng to GREEN and DASTRE 3 two globulins are formed in the solution
of fibrin in neutral salt solution, and according to RULOT also proteoses
(and peptones) on the solution of fibrin containing leucocytes. Fibrin,
like fibrinogen, decomposes hydrogen peroxide, due to a contamination
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 de-
scribed, 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 transvidates. For the preparation of pure fibrin,
as well as for the quantitative estimation of it, the spontaneously coagu-
lating liquid is at once, or the non-spontaneously coagulating liquid only
after the addition of blood-serum or fibrin ferment, thoroughly beaten
with a whalebone, 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 blood from which it was formed,
it partly dissolves (fibrinolysis — DASTRE5). This fibrinolysis must be
prevented in the exact quantitative estimation 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
(JACOBY and others), after extirpation of the liver (Nour), and also when
the coagulability of the blood has been reduced by the injection of pro-
teoses (NOLF, RULOT 6) .

A pure fibrinogen solution may be kept at the ordinary temperature
until putrefaction begins without showing a trace of fibrin coagula-

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 Zeitschr. f . Biologic, 28.

5 Archives de Physiol. (5), 5 and 6.

6 Jacoby, Zeitschr. f. physiol. Chem., 30; Nolf, Arch, intern, de Physiol., 3, 1905;
Rulot, 1. c.

248 THE BLOOD.

tion. But if to this solution is added a water-washed fibrin-clot or a
little blood-serum, it immediately coagulates, and may yield perfectly
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 BucHANAN,1 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. Although many investiga-
tors, especially English, consider fibrin ferment as a globulin, still more
recent experiments of PEKELHARING and others show that it is a nucleo-
protein which, according to HuiSKAMp;3 occurs in the thymus gland
partly as nucleohistone and partly in another form. Fibrin ferment
is produced, according to PEKELHARING, 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 prothrombin. The conversion
of this mother-substance into thrombin is a very complicated process,
which will be discussed under the coagulation of the blood. Thrombin
is 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 FULI>
has found that at least within certain limits an increase of double the
quantity of enzyme causes an increase in speed of coagulation of one and
one-half. This closely corresponds to SCHULTZ'S law; but it is true only
for experiments with bird plasma and tissue extracts. MARTIN 4 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 coagulation
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. the enzyme is destroyed
in neutral solution. 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; neverthe-

1 London Med. Gazette, 1845, 617. Cit. by Gamgee, Journal of Physiol., 1879.

2 Pfluger's Arch., 6; see also Zur Blutlehre, 1892, and Weitere Beitrage zur Blut-
lehre, 1895.

3 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.

4 Martin, Journ. of Physiol., 32; Hofmeister's Beitrage, 2; Loeb, ibid., 9.

THROMBIX. FIBRIN FORMATION. 249

less 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 i1
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 and by PEKELHARiNG.2

The preparation of a thrombin solution as free as possible from lime
may be accomplished by removing the lime salts from the serum by means
of an oxalate and precipitating the serum with alcohol and allowing it to
stand under alcohol for several months. The dried powder is rubbed with
water, and freed from soluble salts by repeated lixiviation with water and
by the use of centrifugal force. Then each gram of powder is allowed to
stand some time with 100-150 cc. water, is filtered, and in this way a
solution is obtained which contains only about 0.3-0.4 p. m. solids and
about 0.0007 p. m. CaO (HAMMARSTEN).

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, as shown by ALEX. SCHMIDT,
PEKELHARING, and HAMMARSTEN,S thrombin can transform fibrinogen
into typical fibrin in the absence of lime salts precipitable by oxalate.
The fibrin is not richer in lime than the fibrinogen used in its prepar-
ation 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 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.

1 Pfluger's Arch., 6.

2 Hammarsten, ibid., 18; Pekelharing, 1. c.

3 See Hammarsten, Zeitschr. f. physiol. Chem., 22, which also cites the works of
Schmidt and Pekelharing, and ibid., 28.

250 THE BLOOD.

This substance is called fibrin-globulin by HAMMARSTEN. The recent
investigations 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 of sodium fluoride besides the fibrinogen,
or perhaps in loose combination with fibrinogen. The view that a cleavage
takes place in the coagulation of the fibrinogen has not been supported
by these investigations.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 a physical process or a reaction
between colloids (IscovEsco, NOLF and others2). 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 3 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 (PANUM4), 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 substance,
but consists of a mixture of two or more protein bodies which 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.5

1 See 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 Iscovesco, Compt. rend. soc. biol., 60 and 61; Nolf, Arch, internat. d. Physiol.,
6 (1908).

3 Hofmeister's Beitrage, 8; Pekelharing and Huiskamp, 1. c. footnote 3, page 248.
4Kiihne, Lehrbuch d. physiol. Chem., Leipzig, 1866-68; Alex. Schmidt, Arch. f.

(Anat. u.) Physiol., 1861-62; Panum, Virchow's Arch., 3 and 4.

5 Mellanby, Journ. of Physiol., 36, claims that no separation of the two chief

SERGLOBULINS. 251

The nucleoproteir. has already been 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^SC^ even at 28 per
cent saturation.

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 1 also differentiates between,
a water-soluble globulin and one insoluble in water. According to the
recent 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 more difficultly precipitable fraction
(by 36-44 vols. per cent saturated solution). The first fraction is called
euglobulin and the second pseudoglobulin. According to FORGES and
SPIRO 3 the serglobulins can be separated by (NH4)2SO4 into three frac-
tions 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 HASLAM 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 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, 6 have the solubilities of a globulin
due to contamination with constituents of the serum, and K. MORNER 7 has also

groups of proteins, the globulins and albumins can be accomplished by saturation with.
MgSO4 of half-saturation with (NH4)SSO4.

1 Zeitschr. f . physiol. Chem., 28.

2 Hofmeister's Beitrage, 1,

3 Hofmeister's Beitrage, 3.

4 Zeitschr. f. physiol. Chem., 36.

5 Journ. of Physiol., 32.

8 See Hammarsten, Ergebnisse d. Physiol., 1, Abt. 1.
T Zeitschr. f. physiol. Chem., 34.

252 THE BLOOD.

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 excep-
tion of the enzymes, 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 pre-
cipitated 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)-D= —47.8° for
the serglobulin from ox-blood (FREDERICQ x). The various globulin
fractions do not differ essentially from each other in their coagulation
temperatures, specific rotation, refraction coefficient (REiss2), 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. MORNERS
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. LANGSTEiN4 has obtained several car-
bohydrates from the blood-globulin, namely, dextrose, glucosamine,
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 EICHHOLZS seem to show that the globulins are

1 Bull. Acad. Roy. de Belg. (2), 50. In regard to paraglobulin, see Hammarsten,
Pfluger's Arch., 17 and 18, and Ergebnisse d. Physiol., 1, Abt. 1.

3 Hofmeister's Beitrage, 4.

3 Zeitschr. f. physiol. Chem., 34.

4M6rner, 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 3, p. 83.

5 Zanetti, Chem. Centralbl., 1898, I, p. 624; Bywaters, Journ. of Physiol., 35, and
Bicohem. Zeitschr., 15; Eichholz, Journ. of Physiol., 23.

SERALBUMIXS. 253

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^SQ^ has been used chiefly. The
serglobulin from blood-serum is always contaminated with lecithin and
thrombin. A serglobulin free from thrombin may be prepared from fer-
ment-free transudates, as sometimes from hydrocele fluids, and this shows
that serglobulin and thrombin are different bodies. For the detection
and the quantitative estimation of serglobulin we may use the precipi-
tation by magnesium sulphate added to saturation (HAMMARSTEN), or by
an equal volume of a saturated neutral ammonium-sulphate solution (HoF-
MEISTER and KAUDER and POHL x). 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. The
accuracy of these methods is questionable, as shown by the researches
of HASLAM.

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
(from horse-serum) was first performed by GURBER. It crystallizes
with difficulty from other blood-sera (GRUZEWSKA). Even from horse-
serum only a portion of the albumins is obtained as crystals, and it is
also possible 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
serabumin is also a mixture, but this is disproved by the observations
of SCHULZ, WICHMANN, and KRiEGER.2 We know nothing as to the

1 Hammarsten, 1. c.; Hofmeister, Kauder and Pohl, Arch. f. exp. Path. u. Pharm., 20.

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.

254 THE BLOOD.

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,1 seem to indicate a
n on -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
nearly the same elementary composition (MICHEL) as the amorphous
mixture of albumin prepared from horse-serum (HAMMARSTEN and K.
STARKER. 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, wnich probably
exists only as cystine. LANGSTEIN 3 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 4 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= —61-61.2°, and by MAXIMOWITSCH on
the contrary («)D= -47.47°.

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 boiling
or on the addition of alcohol. On the addition of a little common salt
it coagulates in both cases.5

'-Halliburton, Journ. of Phyisol., 5 and 7; Hougardy, Centralbl. f. Physiol., 15,
665; Oppenheimer, Verhandl. d. physiol. Gesellsch., Berlin, 1902.

2 Michel, Verhandl. d. phys.-med. Gesellsch. zu Wiirzburg, 29, No. 3; K. Starke,
Maly's Jahresber., 11; K. Morner, 1. c.; Inagaki, Biochem. Centralbl., 4, p. 515.

3 K. Morner, 1. c.; Langstein, Hofmeister's Beitrage, 1.

4 Zeitschr. f. physiol. Chem., 41.

5 In regard to the relationship of neutral salts to heat coagulation, see J. Starke,
Sitzungsber. d. Gesellsch. f. Morph. u. Physiol. in Munchen, 1897.

SERALBUMINS. 255

Seralbumiii 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 contains 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 precipitating
with alcohol, which must be quickly removed. STARKE 1 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 acidification with acetic acid or sulphuric
acid the crystallization may be considerably accelerated.2 In the detec-
tion and quantitative estimation of seralbumin, the filtrate from the
globulin precipitated with magnesium sulphate can be heated to boil-
ing, after acidifying with a little acetic acid if necessary. The quan-
tity of seralbumin is best calculated as the difference between the total
proteins and the globulin.

Summary of the elementary composition of the above-mentioned and de-
scribed proteins (from horse-blood) :

C H N SO

Fibrinogen 52 . 93 6 . 90 16 . 66 1 . 25 22 . 26 (HAMMARSTEN)

Fibrin 52 . 68 6 . 83 16.91 1.10 22 . 48 "

Fibrin-globulin 52 . 70 6 . 98 16 . 06 "

Serglobulin 52.71 7.01 15.85 1.11 23.32

Seralbumin 53.08 7.10 15.93 1.90 21.96 (MICHEL)

Proteose-like substances have been found in blood-serum by several
investigators, and NOLF 3 has shown that after the abundant introduction
of proteoses into the intestine, they pass into the blood. BORCHARDT 4
has also been able to show that not only after the introduction of elastin-
proteose per os, but also after feeding dogs with not overabundant
quantities of elastin, a proteose, hemielastin, passes into the blood and
indeed can be eliminated by the urine. The question whether the pro-
teoses are normal constituents of the blood under ordinary conditions
is still much disputed. The difficulty in deciding this question lies in

1 Johansson, Zeitschr. f. physiol. Chem., 9; K. Starke, Maly's Jahresber., 11.

2 See Hopkins and Pinkus, Journ. of Physiol., 23; Krieger, Ueber die Darstellung
krystallinscher tierischer Eiweissstoffe, Inaug.-Dissert. Strassburg, 1899.

3 Bull. Acad. Roy. Belg., 1903 and 1904.

4 Zeitschr. f. physiol. Chem., 51 and 57.

256 THE BLOOD.

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 pre-
cipitated with the other bodies. The question as to the physiological
occurrence of proteoses in the blood or plasma must be considered as
still undecided.1

In close t 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-
WATERS2 11.9 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. 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:

Fat occurs from 1-7 p. m. in fasting animals. After partaking of
food the amount is increased to a great extent. Fatty adds, or soaps,
glycerin (NiCLOUX, FR. TANGL, and ST. WEISER 3) lecithin and cholesterin
are also found. Cholesterin occurs, according to HiJRTHLE,4 at least
in part, as fatty-acid esters (serolin according to BOUDET). According,
to LETSCHE 5 free cholesterin probably also occurs in the serum.

Sugar seems to be a physiological constituent of the plasma and

1 See especially Abderhalden, Zeitschr. f. physiol. Chem., 51, and Biochem. Zeitschr..,.
8 and 10, and E. Freund, ibid., 1 and 9, which also contains the literature.

2 Biochem. Zeitschr., 15.

3 Nicloux, Compt. rend. soc. biol., 55; Tangl and St. Weiser, Pfliiger's Arch., 115

4 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.

5 Zeitschr. f. physiol. Chem., 53.

BLOOD SERUM. 257

serum. According to the investigations of many workers 1 the sugar
found is dextrose. STRAUSS 2 has also detected levulose 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 pentose
(LEPINE and BOULUDS), in blood serum is still undecided. ASHER and
ROSENFELD and MICHAELIS* and RONA 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 4 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 conclusive-
ness of MICHAELIS and RONA'S experiment with 24-hour dialysis. A
further testing of this question is therefore very desirable. Besides
sugar, the blood-serum contains, as first shown by J. OTTO, another
reducing non-fermentable substance whose quantity in rabbits' blood
is about one-quarter of the total quantity of reducing substance (N.
ANDERSSON). The statements of JACOBSEN, HENRIQUES, and BiNG,5
that this substance is jecorin or lecithin sugar, do not have sufficient
foundation, and the identity with jecorin becomes more striking as the
existence of a jecorin is on the whole doubtful. The nature of another
carbohydrate in the blood, which is neither dextrorotatory nor reducing,
and which has been called virtual sugar by its discoverers, LEPINE and
BOULUD, 6 is also undetermined. The virtual sugar is more abundant in
the blood of the right ventricle than in the arterial blood, and this in
turn is richer than venous blood. In the passage of the blood through
the lungs the virtual sugar is converted into ordinary sugar; this may
also occur in the capillaries of the greater circulatory system.

Conjugated glucuronic acids, which probably originate from the
form-elements, have been shown to occur in blood by the researches of

1 See v. Mering, Arch. f. (Anat. u.) Physiol., 1877 (this article contains numerous
references); Seegen, Pfluger's Arch., 40; Miura, Zeitschr. f. Biologie, 32.

2 Fortschritte d. Mediz., 1902.

3 Pavy and Siau, Journ. of Physiol., 26; Lupine and Boulud, Compt. rend., 133, 135,
and 136.

4 Rosenfeld, Centralbl. f. Physiol., 19, p. 449; Lepine and Boulud, Compt. rend.,
143; Asher, Biochem. Zeitschr., 3; Michaelis and Rona, ibid., 14.

5 Otto, Pfliiger's Arch., 35 (a good review of the older literature on sugar in the
blood); N. Andersson, Biochem. Zeitschr., 12; Jacobsen, Centralbl. f. Physiol., 6, 368;
Henriques, Zeitschr. f. physiol. Chem., 23; Bing, Skand. Arch. f. Physiol., 9; see also
P. Mayer, Biochem. Zeitschr., 1 and 4 on jecorin and blood sugar.

6 Compt. rend., 137, 144, 147.

258 THE BLOOD.

P. MAYER, LEPINE and BOULUD l. The last two investigators find two
definite glucuronic acids in the blood, both of which are levorotatory.
One reduces FEHLING'S solution even at a temperature below 100°,
while the other reduces it at above 100°. Such large amounts of the first
acid often occur in the blood of dogs that the optical activity of the glucu-
ronic acid counteracts that of the glucose: The second acid also occurs
in larger quantities as compared with the sugar.

BERNARD2 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 DOYON and MOREL 3 it occurs only in the
serum but not in the plasma. According to LEPINE,4 it has some con-
nection with the pancreas. The glycolysis is, according to ROHMANN
and SPITZER and SiEBER,5 an oxidation which is produced, according
to the two last-mentioned investigators, by an oxidation ferment. There
is still some doubt whether this is a physiological process or not.6

The blood-plasma and the serum, as well as the lymph, also contain
enzymes of various kinds. According to ROHMANN, BIAL, HAMBURGER/
and others, diastases, which convert starch and glycogen into maltose or
isomaltose, as well as a maltase, are found in the blood. HANRIOT has
detected in the serum a lipase which decomposes butyrin, and which,
according to him, decomposes neutral fats and other esters. The occur-

1 Mayer, Zeitschr. f. physiol. Chem., 32; Lepine and Boulud, Compt. rend., 133,
135, 136, 138, 141, and Journ. de Physiol., 7 (cited from Biochem. Centralbl., 4, p. 421).

2 Lemons sur le diabete, Paris, 1877.

3 Arthus, Arch, de Physiol. (5), 3; Doyon and Morel, Compt. rend. soc. biol., 55.

4 In regard to the numerous memoirs of Lupine and Lepine and Barral, see Lyon
medical., 62 and 63; Compt. rendus, 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 med. expe"r. (Paris, 1892) ; Revue de medecine,
1895; Arthus, Arch, de Physiol. (5), 3, 4; Nasse and Framm, Pfliiger's Arch., 63,
Paderi, Maly's Jahresber., 26; see also Cremer, Physiologie des Glykogens in Ergebnisse
d. Physiol., l,Abt. 1.

5 Rohmann and Spitzer, Ber. d. d. chem. Gesellsch., 28; Spitzer, Pfluger's Arch.,
60 and 67; Sieber, Zeitschr. f. physiol. Chem., 39 and 44.

6 See Arthus, 1. c.; Colenbrander, Maly's Jahresber., 22; Rywosch, Centralbl. f.
Physiol., 11, 495.

7 Rohmann; 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

ENZYMES OF THE SERUM. 259

rence of a butyrinase is generally admitted, while the property of this
lipase of splitting olein and other neutral fats is not generally acknowledged
(ARTHUS, DOYON and MOREL x). 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 substances soluble in water, a
phenomenon first observed by CONNSTEIN and MICHAELIS and further
studied by WEIGERT. The occurrence of such a body is positively
denied by G. MANSFELD.2

Besides the above-mentioned enzymes and thrombin, several other
enzymes have been found in the blood-serum, namely, oxidates, catalases,
proteolytic enzymes, among which we must mention the polypeptide-
splitting enzymes studied by ABDERHALDEN and collaborators,3 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, hcemolysines,
cytotoxines, etc. It is also not within the scope of this book to discuss
the precipitines which can be used as a biological reagent on account of
their action upon various proteins. It may be sufficient to state that
the works of BORDET, EHRLICH, WASSERMANN, SCHUTZE, UnLENHAUT,4
and others have shown that the repeated injection into an animal of a
foreign protein body or of blood of a different species of animal so changes
the blood of this animal that it acquires precipitating properties toward
the injected protein or blood. In this manner we obtain a biological
reagent for various proteins and for blood of different animals. This
last behavior has become of great forensic importance, due to the work
of UHLENHAUT. The various enzymes and antienzymes, toxines and
and antitoxines, precipitines, etc., are as a rule precipitated with the
globulin, but differ among each other in that some are 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 especially
carefully studied by E. LETSCHE 5 and he has found, besides the previously
known bodies, that the serum contains several acids, among which there
are two nitrogenous acids whose nature has not been studied. These,
including other nitrogenous substances found by him, represent a part

1 Hanriot, Compt. rend. soc. biol., 48 and 54; Compt. rend., 123 and 132; Arthus,
Journ. de Physiol. et de Pathol., 4; Doyon and Morel, Compt. rend. soc. biol., 54;
Achard and Clerg (Lipase in Disease), Compt. rend., 129, and Arch. d. med. exp6r., 14.

2 Connstein and Michaelis, Pfliiger's Arch., 65 and 69; Weigert, ibid., 82; Mansfeld,
Centralbl. f. Physiol., 21.

3 Zeitschr. f . physiol. Chem., 51, 53, 55.

4 The literature on this subject may be found in bacteriological journals and works.
See also L. Michaelis, Biohcem. Centralbl., 3, p. 693.

5 Zeitschr. f. physiol. Chem., 53.

260 THE BLOOD.

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 acid, ammonia, hippuric
acid, phosphocarnic acid (PANELLA1), traces of indol (HERViEUX2),
perhaps also uric acid found by ABELES 3 in human blood, while LETS CHE
could not find any in horse-blood. LETSCHE could not find any mon-
amino- or diamino -acids and purine bases which, like lysine (NEUBERG
and RiCHTER4), leucine, tyrosine and bile-acids are found in blood serum
under pathological conditions.

According to BROWINSKI 5 proteic acids (see Chapter XV) occur in
the serum. As above stated, the occurrence of proteoses is disputed.
We have several investigations on the occurrence of amino-acids (v.
BERGMANN, HOWELL, LETSCHE and others °) which make the occurrence
of these very probable, and recently BiNGEL7has been able to show the
presence of glycocoll in normal ox-blood. That the quantity of rest
nitrogen is larger during digestion than in starvation requires further
confirmation (V. BERGMANN and LANGSTEIN, HOHLWEG and H. MEYER 8).

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 RANC is the only pigment of the serum of this
animal. This pigment occurs according to BIFFI and GALLI in especially
large quantities in the blood of new-born.9 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 KRUKEN-
BERG 10 was able to isolate with amyl alcohol a so-called lipochrome 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 phos-
phoric acid is removed on the coagulation of the fibrin. By means of

1 Panella, cited in Virchow's Jahresb., 1902, 150.

2 Compt. rend. soc. biol., 56.
8 Wien. raed. Jahrb., 1887.
4Deutsch. med. Wochenschr., 1904.

6 Zeitschr. f. physiol. Chem., 54 and 58.

8 v. Bergmann, Hofmeister's Beitrage, 6; Howell, Amer. Journ. of Physiol., 17;
Letsche, 1. c.

7 Zeitschr. f. physiol. Chem., 57.

8 v. Bergmann and Langstein, Hofmeister's Beitrage, 6; Hohlweg and Meyer, ibid.,
11.

9 Hammarsten, see Maly's Jahresb., 8 (1878); Ranc, Compt. rend. soc. biol., 62;
Biffi and Galli, Journ. de Physiol. et Path., 9 (1907).

10 Sitz. Ber. d. Jen. Gesellsch. f. Med., 1885.

MINERAL BODIES OF THE SERUM. 261

dialysis, the presence of sodium chloride, which forms the chief mass or
60-70 per cent of the total . mineral bodies, lime-salts, sodium carbonate,
and traces of sulphuric and phosphoric acids and of potassium, may be
directly shown in the serum.1 Traces of silicic acid, fluorine, copper,
iron, manganese, and ammonia 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 com-
pounds, carbonate and phosphate, but as non-diffusible compounds,
protein combinations. According to HAMBURGER 2 37 per cent of
the alkali of the serum from horse-blood was diffusible and 63 per
cent non-diffusible.

Iodine, which seems to be habitually found, is also considered as
a mineral constituent of the plasma or serum (GLEY and BQURCET),
while arsenic, although not found in all blood, occurs in human blood
(GAUTIER, BOURCETS). 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 consist 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.4 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

Seralbumiii 24 . 6

Fat 1.2]

Extractive substances 4.0

Soluble salts 6.4 [

Insoluble salts 1 . 7 I

1 See Giirber, Verhandl. d. phys.-med. Gesellsch. zu Wiirzburg, 23.

2 In regard to method, see Arch. f. (Anat. u.) Physiol., 1898.

3 Gley and Bourcet, Compt. rend., 130; Bourcet, ibid., 131; Gautier, ibid., 131.

4 Cit. from v. Gorup-Besanez's Lehrbuch der physiol. Chem., 4. Aufl., 346.

262 THE BLOOD.

LEWINSKY 1 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
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,2 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 rela-
ship 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 recently
substantiated by GITHENS,S that the quantity of globulins relative to
that of albumin is increased. 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 microorganisms (LANGSTEIN and MAYER4). The total pro-
tein content is raised in nearly all cases. The amount of fibrinogen in
the plasma is especially increased by pneumococci, streptococci, and
pus-staphylococci (P. MuLLER5).

The quantity of mineral bodies in the serum, has been determined by
many investigators. The conclusion drawn from the analyses is that there

1 Pfluger's Arch., 100.

3 Abderhalden, Zeitschr. f. physiol. Chem., 25; Hammarsten, Pfluger's Arch., 17;
Halliburton, Journ. of Physiol., 7; Rubbrecht, Travaux du laboratoire de 1'institut
de physiologic de Liege, 5, 1896.

3 Burckhardt, Arch. f. exp. Path. u. Pharm., 16; Githens, Hofmeister's Beitrage, 5;
see also Morawitz, ibid., 7, and Inagaki, Zeitschr. f. Biol., 49.

4 Hofmeister's Beitrage, 5.

5 Ibid., 6.

OSMOTIC PRESSURE OF THE SERUM. 263

exists a rather close correspondence between human and animal blood-
serum, and it is therefore sufficient to give here the analysis of C. SCHMIDT 1
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

P2O5 (inorg.) 0.052-0.085

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 explanation
of the number of different ions present in the serum or in other fluids, a
question which is of the greatest physiological importance. AD answer
to these questions is obtainable only by physico-chemical investigations,
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 already given in Chapter II, closely to a 9 p. m. (J= —
0.551-0.561°) 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 II) 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 BUGARSKY
and TANGL, on an average of about 0.320 mol. per liter. They also

1 Cit. from Hoppe-Seyler, Physiol. Chem., 1881, p. 439.

2 Osmotisher Druck und lonenlehre, Wiesbaden, 1902-1904, where the literature
on the physical chemistry of the blood can be found.

264 THE BLOOD.

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
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 CO3=, depending upon the dilution. The C03= 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,
then the equilibrium is disturbed, a new quantity of Na2CO3 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 HOBER has worked out a physical-chemical method of
determining alkalinity, based upon NERNST'S theory of liquid chains.
This method was used later by FARKAS, FRANCKEL, and HOBER after
a few changes. The investigations of these last-mentioned experimenters
show that the concentration of the hydroxyl ions in blood-serum and
blood is nearly the same as in distilled water, and that these fluids are
nearly neutral in behavior, which is accounted for by the presence of car-
bonic acid. FRiEDENTHAL,1 by testing serum with phenolphthalein, arrived
at similar results.

II. THE FORM-ELEMENTS OF THE BLOOD.
The Bed 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 Cy clostoma) , the corpuscles
have in general a nucleus, are biconvex and more or less ellipitical. The
size varies in different animals. In man they have an average diameter
of 7 to 8 /j. (/z^O.OOl mm.) and a maximum thickness of 1.9 /.«. They
are heavier than the blood-plasma or serum, and therefore sink in these

1 Hober, Pfluger's Arch., 81 and 99; Farkas, see Biochem. Centralbl., 1, 626;
Franckel, Pfluger's Arch., 96; Friedenthal, Zeitschr. f. allg. Physiol., 1 and 4.

RED BLOOD-CORPUSCLES. 265

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 the 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 hac 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 corpuscles
in 1 c.mm., and in woman 4 to 4.5 million.

The blood-corpuscles consist essentially of two chief constituents,
the stroma, which forms the real protoplasm, and the intraglobular
contents, whose chief constituent is hemoglobin. We cannot state
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 haemo-
globin 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 according to HAMBURGER the stroma forms a protoplasmic
net in whose meshes there exists a red fluid or semi-fluid mass which
consists in great measure of hemoglobin. 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 wrater but not permeable to certain crystalloids. The
researches of KOPPE, ALBRECHT, PASCUCCI, Rywoscn,1 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 choles-
terin, 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 isotonic
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 up, due to
the taking up of water, and this swelling may. be so great, as on diluting

1 See 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.

266 THE BLOOD.

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 II).

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 hsemolytic action. Of special interest in this
regard are the haemoly sines, which act like toxines. These haerholysines
may be metabolic products of bacteria and may be formed by higher
plants and by animals, such as snakes, toads, bees, spiders, and others.
Finally, the hsemolysines or globulicidal bodies, occurring normally in
blood-sera or produced in the immunization of the blood, also belong here.

It seems that haemolysis is brought about in various cases in different ways.
In the haemolysis by means of water we are probably dealing with a destruction
or rupture of the boundary-layer, while such bodies as ether, chloroform, alkalies,
bile-acids, and saponin substances, which dissolve lipoids or form combinations
therewith, in this way cause the passage of the haemoglobin to the outside
(ROPPE, RANSOM and ROBERT, PESKIND, PASCUCCI). The action of other
haemolysines, such as snake-venom and tetanotoxine, seems to be an action con-
nected with the lecithin (KEYES, PASCUCCI 1).

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 (KOPPE), and
attempts have been made to isolate it for chemical investigation. In the
following pages we mean by the name stroma only that residue that
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

1 Roppe, 1. c.; Peskind, Amer. Journ. of Physiol., 12; Ransom and Robert, cited
by Pascucci, Hofmeister's Beitrage, 6; Reyes, Zeitschr. f. physiol. Chem., 41, and Berl.
klin. Wochenschr., 1904.

STROMA OF THE BLOOD-CORPUSCLES. 267

accelerated by centrifugal force, and the liquid which separates therefrom
is very carefully treated with a 1 per cent solution of KHSO4 until it is
about as dense as the original blood. The separated stromata are col-
lected on a filter and quickly washed. PASCUCCI/ 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 probably 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 eere-
broside), and two-thirds protein substances and mineral bodies. The
nucleated red blood-corpuscles of the bird contain, according to PLOSZ
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 be closely
related to the hyaline substance (hyaline substance of ROVIDA, see page 295)
occurring in the lymph-cells. In the mass extracted by alcohol from the
blood-corpuscles of the hen, ACKERMANN found 3.93 per cent phosphorus
and 17.2 per cent nitrogen, which on calculation gave 42.10 per cent nucleic
acid and 57.82 per cent histone. PIETTRE and VILAS 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 are rich in hemoglobin; the nucleated corpuscles are richer in protein
and poorer in haemoglobin than the non-nucleated. Several enzymes
probably also occur as constituents of the stromata and among these
occurs the proteolytic enzyme studied by ABDERHALDEN and collaborators.4
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

1 Hofmeister's Beit rage, 6.

2Wooldridge, Arch. f. (Anat. u.) Physiol., 1881, 387; Halliburton and Friend,
Journal of Physiol., 10; Halliburton, ibid., 18; Plosz, Hoppe-Seyler's Med. chem.
Untersuch., 510.

3 Ackermann, Zeitschr. f. physiol. Chem., 43; Piettre and Vila, Compt. Rend., 143.

4 Zeitschr. f. physiol. Chem., 51, 53 and 55.

268 THE BLOOD.

the blood, or on dissolving the blood-corpuscles of one kind of animal
in the serum of another (LANDOIS, stroma-fibrin) ; i.e., in the so-called
hcrmagglutination, a clumping of the red blood-corpuscles into clusters
takes place. This agglutination can be brought about by bodies
similar to the ha3molysines and also by serum constituents pro-
duced normally or by immunization. It has not been shown that
a fibrin formation from the stroma takes place. Fibrinogen has only
been detected in the red corpuscles of frog's blood (ALEX. SCHMIDT and

SEMMER1).

Closely related to the anatomical and chemical structure of the erythro-
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 taking
up substances of different kinds. This question as well as the permeability
of the blood-corpuscles for anions under the influence of carbon dioxide
has been discussed in a previous chapter (II, page 33) .

The mineral bodies of the red corpuscles will be treated in connection
with their quantitative constitution.

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 substance.
The crystalline coloring-matter, the hemoglobin or oxyhsemoglobin,.
which may be isolated from the blood, is considered, according to HOPPE-
SEYLER, as a cleavage product of this compound, and 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 resistance 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 investiga-
tors, such as H. IT. KOBERT and BoHR,2 the latter calling the pigment

1 Landois, Centralbl. f. d. med. Wissensch., 1874, 421; Schmidt, Pfluger's Arch.,
11, 550-559.

2 Hoppe-Seyler, Zeitschr. f. physiol. Chem., 13, 479; H. U. Robert, Das Wirbeltier-
blut in mikro-kristallogr. Hinsicht, Stuttgart, 1901; Bohr, Cenrtalbl. f. Physiol., 17,
p. 688.

BLOOD-PIGMENTS. 269

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 pig-
ment, the hemoglobin.

The color of the blood depends in part on hemoglobin and in
part on a molecular combination of this substance with oxygen,
the oxyhcemoglobin. We find in blood after asphyxiation almost
exclusively haemoglobin, in arterial blood disproportionately large
amounts of oxyhaemoglobin, and in venous blood a mixture of
both. Blood-coloring matters are also found in striated as well
as in certain smooth muscles, and lastly in solution in different
invertebrates. 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, hcemo-
chromogen (about 4 per cent), containing iron, which in the presence of
oxygen is easily oxidized into h&matin.

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
haematoporphyrin. By the investigations of NENCKI in conjunction with
MARCHLEWSKI and ZALESKI, J it was shown that haemopyrol could be
prepared from the derivatives of both the leaf-pigment and the blood-
pigments by reduction. The fact that chlorophyll and blood-pigments
are closely related and are constructed from the same mother-substance,
is of the greatest biological importance.

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 :

1 Schunck and Marchlewski, Annal. d. Chem. u. Pharm., 278, 284, 288, 290; NenckL
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.

270 THE BLOOD.

Hcemoglobin from the C H N S Fe O PoOs

Dog 53.85 7.32 16.17 0.390 0.430 21.84 (HOPPE-SEYLER)

" 54.577.2216.380.5680.33620.93 (JAQUET)

Horse 54.87 6.79 17.31 0.650 0.470 19.73 (KOSSEL)

" :.. 51.156.7617.940.3900.33523.43 .... (ZINOFFSKY)

Ox 54.66 7.25 17.70 0.447 0.400 19.543 .... (HUFNER)

Pig 54.17 7.38 16.23 0.660 0.430 21.360 .... (OTTO)

" 54.71 7.38 17.43 0.479 0.399 19.602 .... (HUFNER)

Guinea-pig 54.12 7.36 16.78 0.580 0.480 20.680 .... (HOPPE-SEYLER)

Squirrel 54.097.3916.090.4000.59021.440

Goose 54.267.1016.210.5400.43020.6900.770

Hen 52.477.1916.450.8570.33522.5000.197 (JAQUET)

That the repeatedly observed quantity of phosphorus in the haemo-
globin of birds (Inoko and others) is due to a contamination has been
proven 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 (JAQUET) the relation is 1 to 3. From the data of the elementary
analysis, as also from the amount of loosely combined oxygen, HUF-
NER 1 has calculated the molecular weight of dog-haemoglobin as 14,129,
and the formula CeseHK^Ni^FeSsOigi. According to the more recent
determinations of HUFNER and JAQUET,2 ox-haemoglobin contains an
average of 0.336 per cent iron, from which a molecular weight of 16,669
may be calculated. HUFNER and GANSSERS have attempted to learn
the size of the molecular 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 crystallization; 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 crystallization, 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 observa-
tions that the ordinary haemoglobin consists of a mixture of different
haemoglobins. In opposition to this statement, HUFNER 4 has shown

1 Hoppe-Seyler, Med. chem. Untersuch., 370; Jaquet, Zeitschr. f. physiol. Chem.,
14, 296; Kossel, ibid., 2, 150; Zinoffsky, ibid., 10; Hufner, 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.

2 Arch. f. (Anat. u.) Physiol., 1894.

3 Arch. f. (Anat. u.) Physiol., 1907.

4 Bohr, " Sur les combinaisons de 1'hemoglobine avec 1'oxygene," Extrait du
Bulletin de l'Acad6mie Royale Danoise des sciences, 1890; also Centralbl. f. Physiol.,
1890, 249. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 2; Hufner, Arch. f. (Anat. u.)
Physiol., 1894.

OXYH^MOGLOBIN. 271

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 G AN SSER, and the amount of loosely combined oxygen which is united
to 1 gram of haemoglobin (of the ox) has been determined by HUFNER 1
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
a~j P~t T- and ^-oxyhaemoglobin, 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--oxyhsRmoglobin is the ordinary one obtained by the customary method of
preparation. BOHR designates as a-oxyhaemoglobin the crystalline powder
obtained by drying 7--oxyhaemoglobin in the air. On dissolving a-oxyhsemo-
globin in water it is converted into /?-oxyhsemoglobin without decomposition, and
the quantity of iron is increased. On keeping a solution of y-oxyhsemoglobin
in a sealed tube it is transformed into ^-oxyhaemoglobin, although the exact
conditions under which this change takes place are not known. According to
HUFNER 2 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
molecule of haemoglobin is combined with 1 molecule of oxygen. Still
this reaction is reversible according to the type l(Hb) + l(O2)«=H(OHb),
and with diminished oxygen pressure a dissociation must take place,
with the giving up of oxygen and a re-formation of haemoglobin. The
equilibrium between oxyhaemoglobin, haemoglobin, and oxygen is deter-
mined according to the law of mass-action, and according to the investiga-
tions of HUFNER it is possible to calculate the relationship between
oxyhaemoglobin (OHb) and haemoglobin (Hb), at every desired partial
pressure of the oxygen, by a formula suggested by him. According to
BOHR 3 this formula does nojb have sufficient basis and does not correspond
to the facts. BOHR found, in opposition to HUFNER' s statements, that
with the same oxygen tension the absorption of oxygen by a haemoglobin
solution changes with the concentration, and that a dilute solution

1 Arch. f. (Anat. u.) Physiol., 1901, Suppl.

2 Arch. f. (Anat. u.) physiol., 1894.

3 Bohr, Centralbl. f. Physiol., 17, pp. 682 and 688.

272 THE BLOOD.

combines with more oxygen, calculated per 1 gram haemoglobin, than a
concentrated solution. BOHR suggested another formula expressing the
relationship between the oxygen absorption and the oxygen tension,
based upon the assumption that, besides the dissociation of the oxygen-
hemoglobin compound, a dissociation of the haemoglobin into a part
containing iron and a part containing no iron also takes place. This
formula, which in fact accords well with BOHR'S findings, is nevertheless
only true for a hemoglobin solution and not for blood, as, according to
BOHR, the blood-pigment in the blood-corpuscles (the hemochrom) is
changed on being converted into haemoglobin. HENRI 1 also finds that
HUFNER'S formula for the dissociation of oxyhemoglobin is not useful.

The native pigment, the ha?mochrom, combines, according to BOHR,
in maximum with the same quantity of oxygen as the corresponding
haemoglobin, when the latter is prepared without the use of violent
methods; still from this it does not follow that the oxygen combina-
tion in hemochrom is identical with that in haemoglobin. According
to BOHR this is not the case, at least with diminished pressure, for with
low oxygen tension more oxygen is taken up by the blood than by a
corresponding hemoglobin solution. The curve showing the oxygen
absorption is lower in this case for a haemoglobin solution than for blood.
The reason for this lies, according to BOHR, in the fact that the tension
curve is influenced by the form of union of the part of the haemoglobin
containing iron with the iron-free part, and that this union is changed
because of changes in the iron-free part, as by the splitting off of lecithin,
etc. The tension curve of the oxygen in the blood can, according to
BOHR, be determined only by direct experiments on the blood itself and
not by experiments upon hemoglobin solutions.

The elucidation of these conditions is of the very greatest importance,
as the dependence of the reaction between OHb, Hb, and O upon the
law of mass-action is naturally of the very greatest moment for the
taking up of oxygen in the lungs and the giving up of the same to the
tissues. The dissociation of the oxyhemoglobin makes it also possible
to completely expel the oxygen from a hemoglobin solution or from
blood by means of a vacuum or by passing an indifferent gas through the
blood.

Oxyhemoglobin, which is generally considered as a weak acid, is
dextrorotatory, according to GAMGEE.2 The Specific rotation for light
of medium wave-length of C is (a) C = about +10°, which corresponds
also for carbon-monoxide hemoglobin. The hemoglobin is also, like
carbon-monoxide hemoglobin (COHb) and methemoglobin (MHb),
diamagnetic, while the hematin, which is richer in iron, is strongly mag-

1 Henri, Compt. rend. soc. biolog., 56. 2 Hofmeister's Beitrage, 4.

OXYH^MOGLOBIN. 273

netic (GAMGEE l) . 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 (GAMGEE2). This transportation of the
colloidal haemoglobin may also be made to take place through an animal
membrane or through parchment paper. 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 oxy haemoglobin 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.3 The quantity of water of crystallization varies between 3-10
per cent for the different oxybaemoglobins. When completely dried
at a low temperature over sulphuric acid the crystals may be heated
to 110-115° C. without decomposition. At higher temperatures, some-
what above 160° C., they decompose, giving an odor of burnt horn,
and leave, after complete combustion, an ash consisting of oxide of
iron. The oxyhaemoglobin crystals from difficultly crystallizable kinds of
blood, for example from such as ox's, human, and pig's blood, are
easily soluble in water. The oxyhaemoglobins 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 oxyhaemogiobin 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 quickly decompose. The crystals are
insoluble in absolute alcohol without decolorization. According to
NENCKi,4 it is hereby converted into an isomeric or polymeric modifica-
tion, called by him parahoemoglobin. Oxyhaemoglobin 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 haematin when sufficiently heated. It is also readily

1 Proceedings of Roy. Society, 68.

2 Ibid., 70.

3 The observation of Uhlik (Pfliiger's Arch., 104) that the haemoglobin from horse-
blood can also crystallize in hexagonal six-sided plates seems to be due to the fact that
he had haemoglobin and not oxyhaemoglobin.

4 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.

274 THE BLOOD.

decomposed by acids, alkalies, and many metallic salts. It gives the
ordinary reactions for proteins with those protein reagents which first
decompose the oxy haemoglobin with the splitting off of protein. Oxy-
ha3moglobin, 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 FRAUNHOFER
lines D and E (spectrum plate 1). The one band, a, which is narrower
but darker and sharper, lies on the line D; the other, broader, less defined
and less dark band, /?, lies at E. The middle of the first band corresponds
to a wave-length ^ = 579 and the second ^=542. On dilution the band
ft 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 by the aid
of special appliances (L. LEWIN, MIETHE, and STENGER) the band
described 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 oxyhffimoglobin bands are still detectable in a dilution of
1 : 14,700 the violet band may be seen, according to LEWIN, MIETHE and
STENGER 1 in a dilution of 1 : 40,000.

The observation of PIETTRE and VILA that so-called laky blood and oxyhsemo-
globin solutions in thick layers also show a third band in the red (A =-634) depends
in all probability, as also claimed by VILLB and DERRIEN, upon a partial forma-
tion of metha3moglobin 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.
water and then shaken with ether. After decanting the ether and allow-
ing 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

1 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.

HAEMOGLOBIN. 275

containing alcohol (one-quarter vol. alcohol) and dried in vacuum at 0°

C. or a lower temperature.1

For the preparation of oxyhaemoglobin crystals in small quantities
from easily c ryst alii z able 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 HEMOGLOBIN or PURPLE CRUORIN
(STOKES 2), occurs only in very small quantities in arterial blood, in larger
quantities in venous blood, and is nearly the only blood-coloring matter
after asphyxiation.

Haemoglobin is much more soluble than the oxyhaemoglobin, and
it can therefore be obtained as crystals only with difficulty. These
crystals are as a rule isomorphous with the corresponding oxyha3moglobin
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 3 in hexagonal six-sided plates. Its solu-
tions in water are darker and more violet or purplish than solutions of
oxyhaemoglobin 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 solu-
tion shows a spectrum with one broad, not sharply defined band between
D and E, whose darkest part corresponds to the wave-length ^ = 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. A haemoglobin solution actively absorbs oxygen from the air and is
converted into an oxyhaemoglobin 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 ammoniac al ferrous-tartrate solution (STOKES' reduction
liquid). If an oxyhaemoglobin solution or arterial blood is kept in a
sealed tube, we observe a gradual consumption of oxygen and a reduction
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 4).

1 In regard to the preparation of oxyhaemoglobin, see also Hoppe-Seyler-Thier-
felder's Handbuch, 8. Aufl.; also the works cited in footnote 1, p. 270; also Schuur-
manns-Stekhoven, Zeitschr. f. physiol. Chem., 33, 296; see also Bohr, Skand. Arch,
f . Physiol., 3.

2 Philosophical Magazine, 28, No. 190, Nov., 1864.

3 Pfliiger's Arch., 104.

4 Zeitschr. f. physiol. Chem., 4; see also Uhlik, 1. c. -

276 THE BLOOD..

Pseudohaemoglobin. LUDWTG and SIEGFRIED l have observed that blood
which has been reduced by hyposulphites so completely that the oxyhaemoglobin
spectrum disappears and only the haemoglobin spectrum is seen, yields large
amounts of oxygen when exposed to a vacuum. Blood which has been reduced
by the passage of a stream of hydrogen through it until the oxyhaemoglobin
spectrum disappears acts in the same manner. Hence a loose combination of
hemoglobin and oxygen exists which gives the haemoglobin spectrum, and this
combination is called pscudohaemoglobin by LUDWIG and SIEGFRIED. Pseudo-
haemoglobin, whose presence has been detected in asphyxiation blood from dogs,
is considered by HAMMARSTEN as an intermediate step between hsemoglQbin and
oxyhsemoglobin on the reduction of the latter. The occurrence of pseudohsemo-
globin does not seem to have been positively proven.2

Methaemoglobin. This name has been given to a coloring-matter
which is easily obtained from oxyhsemoglobin as a transformation product
and which has been correspondingly found in transudates and cystic
fluids containing blood, in urine in hsematuria or hsemoglobinuria, and
also in urine and blood on poisoning with potassium chlorate, amyl
nitrite or alkali nitrite, and many other bodies.

Methsemoglobin does not contain any oxygen in molecular or dis-
sociable combination, but still the oxygen seems to be of importance in
the formation of methsemoglobin, 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
methsemoglobin is formed. The same occurs on the addition of a sir all
quantity of acid to the blood. By the spontaneous decomposition of
blood some methsemoglobin is formed, and by the action of ozone, potas-
sium permanganate, potassium ferricyanide, chlorates, nitrites, nitro-
benzene, pyrogallol, pyrocatechin, acetanilide, and certain other bodies
on the blood an abundant formation of methsemoglobin takes
place.

According to the investigations of HUFNER, KULZ, and OTTO 3
methsemoglobin contains just as much oxygen as oxyhsemoglobin, but
it is more strongly combined. By the action of .potassium ferricyanide
or potassium permanganate upon oxyhsemoglobin first 1 molecule oxygen
(i.e., the entire quantity of loosely combined oxygen) is split off, and in
the subsequent methsemoglobin formation either two oxygen atoms
(HALDANE) or two hydro xyl groups are combined (HUFNER, v. ZEYNEK 4) .
Methsemoglobin solutions are reduced to hsemoglobin by reducing agents.
JADERHOLM and SAARBACH claim that methsemoglobin is first converted

1 Arch. f. (Anat. u.) Physiol., 1890; see also Ivo Novi, Pfliiger's Archiv, 56.

2 See Hiifner, Arch. f. (Anat. u.) Physiol., 1894, 140.

3 See Otto Zeitschr. f. physiol. Chem., 7.

4Haldane, Journ. of Physiol., 22; v. Zeynek, Arch. f. (Anat. u.) Physiol., 1899;
Hiifner, ibid.

METH^MOGLOBIN. 277

into oxyhsemoglobin and then into haemoglobin by reducing substances,
while others (HOPPE-SEYLER and ARAKI 1) dispute this.

According to HUFNER and REINBOLD - \ 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
ammonia. The absorption-spectrum of a watery or acidified solution
of methsemoglobin is, according to JADERHOLM and BERTIN-SANS, very
similar to that of haBmatin in acid solution, but is easily distinguished from
the latter since, on the addition ol a little alkali ana a reducing substance,
the former passes over to the spectrum of reduced hemoglobin, while
a hsematin solution under the same conditions gives the spectrum of an
alkaline ha3mochromogen solution (see below). According to ARAKI
and DITTRICH, a neutral or faintly acid methaBmoglobin solution shows
only one characteristic band, a, between C and D, whose middle corresponds
to about ^ = 634. The two bands between D and E are only due to con-
tamination with oxyha3moglobin (MENZIES, LEWIN, MIETHE and STEN-
GER.3 MethaBmoglobin in alkaline solution shows two absorption-bands
which are like the two oxy hemoglobin bands, but they differ from these
in that the band /? 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. (Spectrum Plate 4) .

The claims as to the action of sodium fluoride upon haemoglobin and methaeino-
globin are somewhat contradictory.4

Crystallized methemoglobin may be easily obtained by treating a con-
centrated solution of oxyhsemoglobin with a sufficient quantity of con-
centrated 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.

Cyanmethaemoglobin (cyanhaernoglobin) is, according to HALDANE, identical
with photomethsemoglobin (BOCK), which is produced by the influence of sun-
light upon a methsemoglobin solution containing potassium ferricyanide. It
was first carefully described by R. ROBERT and obtained in a crystalline form

1 Jaderholm, Zeitschr. f. Biologie, 16; Saarbach, Pfluger's.Arch., 28; Araki, Zeit-
schr. f. physiol. Chem., 14.

2 Arch. f. (Anat. u.) Physiol., 1904. Suppl.

3 Jaderholm, 1. c.; Bertin-Sans, Comp. rend., 106; Dittrich, Arch. f. exp. Path. u.
Pharm., 29; Menzies, Journ. of. Physiol, 17; Lewin and collaborators, footnote 1, page
274. Important references on methaemoglobin are given by Otto, Pfluger's Arch., 31.

4 Piettre and Vila, Compt. rend., 140; Ville and Derrien, ibid., 140.

278 THE BLOOD.

by v. ZEYNEK.1 It is immediately formed in the cold by the action of a hydro-
cyanic-acid solution upon met haemoglobin, but is formed by its action upon
oxy haemoglobin only at the body temperature. The neutral or faintly alkaline
solutions show a spectrum which is very similar to the haemoglobin spectrum.
Acid haemoglobin is a coloring-matter produced by the action of very weak
acids upon oxyhaemoglobin, which according to HARNACK 2 is not, as used to be
admitted, identical with methaemoglobin.

Carbon-monoxide Haemoglobin 3 is the molecular combination between
1 molecule of haemoglobin and 1 molecule of CO, according to HtJFNER,4
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 oxyhaemoglobin 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,S whose results
are tabulated.

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 solution
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 6).

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 oxyhsemoglobin. These crystals are iso-
morphous with the oxyhaemoglobin crystals, but are less soluble and more
stable, and their bluish-red color is more marked. For the detection
of carbon-monoxide haemoglobin, its absorption-spectrum is of the greatest
importance. This spectrum shows two bands which are very similar
to those of oxyhaemoglobin, but they occur more toward the violet

1 Haldane, Journ. of Physiol., 25; Bock, Skand. Arch. f. Physiol., 6; Robert,
Pfluger's Arch., 82; v. Zeynek, Zeitschr. f . physiol. Chem., 33. See also Leers, Biochem.
Zeitschr., 12.

2 Zeitschr. f. physiol. Chem., 26.

3 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.

4 Arch. f. (Anat. u.) Physiol., 1894. On the dissociation constant of carbon-
monoxide hemoglobin, see ibid., 1895. In regard to the contradictory statements of
Saint-Martin and others and their disapproval, see Hufner, Arch. f. (Anat. u.) Physiol.,
1903.

5 Arch. f. exp. Path. u. Pharm., 48.
.6 Journ. of Physiol., 22.

CARBON-MONOXIDE HAEMOGLOBIN. 279

part of the spectrum. The middle of the first band corresponds to
/I = 570, and the second to A =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 hemoglobin, and oxy hemoglobin. If the blood contains oxy-
hemoglobin and carbon-monoxide hemoglobin at the same time, we
obtain on the addition of a reducing substance (ammoniacal ferro-t art rate
solution) a mixed spectrum originating from the hemoglobin and carbon-
monoxide hemoglobin. Carbon-monoxide hemoglobin also gives a band
in the violet A =416.

A great many reactions have been suggested for the detection of car-
bon-monoxide hemoglobin 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 modifications of this
test have been proposed. Another ,very good reagent is tannic acid,
which gives with dilute normal blood a brownish-green precipitate and
with carbon-monoxide blood a pale crimson-red precipitate.1

As according to BOHR there are several oxyhaemoglobins, so also, according
to BOHR and BocK,2 there are several carbon-monoxide haemoglobins, 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 haemoglobin,
but this is contradicted by BERTIN-SANS and MorrasssnCB.1 Sulphur methaemo-
globin is the name given by HOPPE-SEYLER to that coloring-matter which is
formed by the action of sulphuretted 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.
According to HARNACK the conditions are different when H2S is passed through
an oxygen-free solution of haemoglobin (or carbon-monoxide haemoglobin). The
sulphaemoglobin thus formed shows one band in the red between C and D.
According -to CLARKE and HURTLEY 4 the formation of sulphaemoglobin takes
place after the reduction to haemoglobin.

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.

4 Hoppe-Seyler, Med.-chem. Untersuch., 151. See Araki, Zeitschr. f. physiol. Chem.,
14; Harnack, 1. c.; Clarke and Hurtley, Journ. of Physiol., 38.

280 THE BLOOD.

Carbon-dioxide Haemoglobin, Carbohcemoglobin. Haemoglobin, accord-
ing to BOHR and ToRUP,1 also forms a molecular combination with car-
bon dioxide whose spectrum is similar to that of haemoglobin. Accord-
ing to BOHR there are three different carbohaemoglobins, namely, a-,
/?-, 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. BOHR has given an equilibrium formula
for the carbon-dioxide absorption of haemoglobin at different carbon-
dioxide tensions, and the results obtained on calculation, using this for-
mula, correspond very well with the results obtained directly. 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 disappear
on the addition of reducing bodies. Haemoglobin also forms a molecular
combination with acetylene.

Hsemorrhodin 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 2 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 ta
60° C. we obtain, according to v. KLAVEREN, a pigment which he calls kathoemo-
ylobin, but called by ARNOLD,* who first obtained it, neutral hoematin, 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
hspmatin.

Decomposition products of the blood-pigments. By its decomposi-
tion haemoglobin yields, as previously stated, a protein, which has been

1 Bohr, Extrait du Bull, de 1'Acad. Danoise, 1890; Centralbl. f. Physiol., 4 and
17; Torup, Maly's Jahresber., 17.

2 K. B. Lehmann, Sitzungsber. d. phys.-med. Gesellsch. Wurzburg, 1899; Lewin,.
Compt. rend., 133.

3 v. Klaveren, Zeitschr. f. physiol. Chem., 33; Arnold, ibid., 29.

H^MOCHROMOGEN. 281

called globin (PREYER, SCHULZ), and a ferruginous pigment as chief prod-
ucts. According to LAWROW 94.09 per cent protein, 4.47 per cent
haBmatin, and 1.44 per cent other bodies are produced in this decomposi-
tion. The globin, which was isolated and studied by ScHULZ,1 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 coag-
ulum is readily soluble in acids. Because of these reactions it is con-
sidered as a histone by SCHULZ.

On hydrolytic cleavage globin (from horse-blood) yields, according
to ABDERHALDEN,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, haBmochromogen is quickly
oxidized to haBmatin, and there is therefore obtained in this case h&matin
as a colored decomposition product. As haBmochromogen is easily
converted by oxygen into hsematin, so this latter may be reconverted
into haBmochromogen by reducing substances.

Haemochromogen was discovered by HOPPE-SEYLER.S It is, accord-
ing to HOPPE-SEYLER, the colored atomic group of hemoglobin 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 haBmochromogen, and it is also this atomic group which binds in
the oxy hemoglobin 1 molecule of oxygen and in the carbon-monoxide
haemoglobin 1 molecule of carbon monoxide with 1 atom of iron. * HaBmo-
chromogen is produced in an alkaline solution of haBmatin by the action
of reducing bodies. By the reduction ofhaBmatmin alcoholic ammoniacal
solution by means of hydrazine v. ZEYNEK 4 was able to obtain the solid
brownish-red ammonia combination.

Hsemochromogen also combines, as HOPPE-SEYLER first showed, with
carbon monoxide. This compound, which in aqueous solution gives
a spectrum similar to oxyhaBmoglobin, has been obtained by PREGL 5 in

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. 5 Ibid., 44.

282 THE BLOOD.

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.1

An alkaline hsemochromogen 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
A = 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
corresponds to A=526 to 530 according to LEWIN, MIETHE and STENGER.
In acid solution hsemochromogen shows four bands, which, according
to JADERHOLM,2 depend on a mixture of hasmochromogen and hsemato-
porphyrin (see below), this last formed by a partial decomposition
resulting from the action of the acid.

MiLROY,3 from an alcoholic solution of hsematin containing oxalic
acid, after driving out the air by means of hydrogen gas, gradually obtained
an acid solution of reduced hsematin (hsemochromogen) by means of
.zinc dust. This solution showed one absorption-band between D and E.

Hsemochromogen may be obtained as crystals by the action of caustic
soda on hsemoglobin at 100° C. in the absence of oxygen (HOPPE-SEYLER).
By the decomposition of hsemoglobin by acids (of course in the absence
of air) we obtain hsemochromogen contaminated with a little hsematopor-
phyrin. An alkaline hsemochromogen solution is easily obtained by the
action of a reducing substance (STORES' reduction liquid) on an alkaline
hsematin solution. An ammoniac al solution of hsematin on reduction
with hydrazine yields hsemochromogen very easily. An alcoholic, alkaline
hydrazine solution is also recommended by RIEGLER 4 as a reagent for
blood-pigments, converting them into hsemochromogen.

Haeniatin, also called OXYH.EMATIN, is sometimes found in old transu-
dates. It is formed by the action of the gastric or pancreatic juices on
oxyhtemoglobin, and is, therefore, also 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-
retted hydrogen. As shown above, the hsematin is formed by the decom-
position of oxyhsemoglobin, or at least of hsemoglobin, in the presence
of oxygen.

1 Hiifner, and Kuster, Arch. f. (Anat. u.) Physiol., 1904, Suppl. Pregl, 1. c.

2 Nord. Med. Arkiv., 16.

3 Journ. of Physiol., 32.

4 Zeitschr. f . anal. Chem., 43.

H^MATIN. 283

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 somewhat different composition, dependent upon various conditions.
According to HOPPE-SEYLER hsematin has the formula C34H34X4FeO5,
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. Ac-
cording to CLOETTA, and also ROSENFELD/ hsematin has the formula
C3oH34N3FeO3, with 1 atom of iron for every 3 atoms of nitrogen. The
question whether the hsematins obtained under different conditions
are identical or not is still undecided (v. ZEYNEK, EppiNGER2).

.Hsematin is very resistant toward boiling concentrated caustic
potash as well as toward boiling hydrochloric acid. It dissolves in
concentrated sulphuric acid, and is converted into hsematoporphyrin
with the splitting off of iron. On heating dry haimatin it yields abundant
pyrrol. On reduction with tin and hydrochloric acid a body similar
to urobilin is formed. As an oxidation product of hsematin in glacial
acetic acid with potassium bichromate or chromium trioxide, KUSTER
obtained the imide of the tribasic hsematinic acid, CgH9NO4, which is
also produced on the oxidation of hsematoporphyrin and bilirubin. On
reduction with phosphonium iodide it yields hsemopyrrol (see hsemato-
porphyrin) .

The imide of the tribasic hsematinic acid, which is a derivative of malei'c acid

and probably has the formula C5H7(COOHX /NH, is readily transformed into

NXK
the anhydride of the tribasic haematinic acid, C8H805, having the probable formula

CH3.C.C(X

;O. On heating the imide with alcoholic ammonia to
COOH.CH2.CH2.C.CXK

130° C. it splits off carbon dioxide, and the imide of the bibasic haBmaJinic acid
C7H9NO2 is obtained. From this imide on saponificatiori with baryta-water we
obtain the barium salt of an acid whose anhydride is methyl-ethyl maleic-acid

C2H5.C.
anhydride,

CH..C.C

On heating hrematinic acid ester with alcoholic ammonia in a tube to 130°
KUSTER obtained a colored product whose bluish-violet aqueous solution gave
a spectrum with two bands which in position were similar to the oxyhffimoglobin
spectrum. He has also prepared and studied various salts, esters and aniline
derivatives of the haematinic acids and condensation products of their esters.

1 Hoppe-Seyler, Msd.-chem. Untersuch., p. 525; Cloetta, Arch. f. exp. Path. u.
Pharm., 36; Rosenfeld, ibid., 40.

2 v. Zeynek, Zeitschr. f. physiol. Chem.. 49; P. Eppinger, Unters. uber den Blut-
farbstoffe Dissert. Miinchen, 1907.

284 THE BLOOD.

Based upon his investigations on the hsematinic acids and hsemopyrrol,
KUSTER 1 believes that the hsematin in part contains a group which is
readily changed into hsematinic acid but not into hsBmopyrrol, and a
part which can be changed into both hsematinic acid and pyrrol.

PiLOTY2 has published important investigations on the constitution
of hsematin and on the origin of KUSTER'S hsematinic acid. On warming
hsematoporphyrin with hydrochloric acid and tin chloride he obtained
three products, namely, hsemopyrrol, hsemopyrrol carboxylic acid, and
desoxyhsematoporphyrin. Ha?mopyrrol seems to be a unit body, and
is considered as 3-methyl-3-n-propyl-pyrrol. The hsematinic acid is
not formed from hsemopyrrol, but rather from the crystalline hsemopyrrol
carboxylic acid, CgH^NOg, which is formed in addition to hsemopyrrol in
the reduction of hsematoporphyrin, and which is transformed into hsematinic
acid, CgHgNO^ on oxidation. The desoxyhsematoporphyrin, C34H38X4O5,
which differs from the hsematoporphyrin by containing one atom of
oxygen less, yields on further reduction hsemopyrrol, hsemopyrrol car-
boxylic acid, and hsematopyrrolidinic acid. This last, which has not
been obtained pure, yields on further cleavage and oxidation, hsematinic
acid and a basic oil having a piperidine-like odor. These investigations
are in accord with KUSTER'S observations that a part of the hsematinic
acid is relatively easily split, while another part, on the contrary, is only
split with difficulty and gradually. They do not, on the contrary, agree
with the above statement of KUSTER that hsematin contains a group
which is readily changed into hsematinic acid but not into haemopyrrol,
and another group which yields hsematinic acid as well as pyrrol.

Hsematin is amorphous, dark brown or bluish black. It may be
heated to 180° C. without decomposition; on burning.it leaves a residue
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 hsematin 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

1 Beitrage zur Kenntnis des Hamatins, Tubingen, 1896; Ber. d. d. chem. Gesellsch.,

27, 30, 32, and 35; Annal. d. Chem. u. Pharm., 315, and Zeitschr. f. physiol. Chem.,

28, 40, 44, 54, and 55.

2 Annal. d. Chem. u. Pharm., 366.

ELEMIN. 285

D and F a second, much broader, less sharply denned band occurs, which by
>roper 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. Hsema-
tin may thus in acid solution show four absorption-bands; ordinarily
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 hsema-
tin (spectrum Plate 5), shows 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 ha?matin
bands in the spectrum is quite variable, the exact wave-lengths correspond-
ing thereto cannot be given exactly.

Haemin, H.EMIN CRYSTALS, or TEICHMANN'S CRYSTALS. Hsemin is
the hydrochloric-acid ester of hsematin, and is the starting-point in the
preparation of the latter.

Opinions as to the composition of haBrrn'n are just as variable
as those for hsematin, which is partly due to the fact, as shown by NENCKI
and ZALESKI, that the hsematin, which contains two hydroxyls in the
molecule, may form ethers with acids and alkyl radicals, which also yield
addition products with indifferent compounds. Thus the hsemin pre-
pared according to NENCKI and SIEBER'S method contains amyl alcohol.
SCHALFEJEFF'S hsemin, having the formula C34H33N4FeO4Cl, is supposed
to contain an acetyl group, and hence is called acethsemin. MORNER'S
haBmin, CssHss^FeC^Cl, is considered as a monoethyl ether of acethaBmin.
The investigations of ZALESKI, HETPER and MARCHLEWSKI, K. MOR-
NER, and especially those of KUSTER, have given explanations of these
conditions. The so-called acethaBmin does not contain any acetic-acid
radical, hence its name is incorrect. KUSTER, by a new method of purifica-
tion and recrystallization, has shown that the older various kinds of
haBmins were not chemical individuals, and that we have only one haBmin.
This view is now accepted by MORNER and most of the other investigators,
and the formula Ca^asC^NJfeCl is now given to haBmin. PIETTRE
and VILA l dispute this formula, and they claim to have prepared chlor-
ine-free hsemin from pure crystalline oxyhaBmoglobin.

HaBmin crystals form in large masses a bluish-black powder, but are
so small that they can be seen only by aid of the miscrocope. They

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 with
Nencki and Zaleski, 1. c.; Bialobrzeski, Arch, des scienc. biol. de St. Petersbourg, 5;
K. Morner, Nord. Med. Arkiv, Festband; 1897, Nos. 1 and 26, and Zeitschr. f. physiol.
Chem., 41; Zaleski, ibid., 37; Hetper and Marchlewski, ibid., 41 and 42; Ktister, ibid.,
40; Piettre and Vila, Compt. rend., 141, p. 734.

286 THE BLOOD.

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 EPPINGER and then also by v. SiEWERT,1 crystalline hasmin
can be reobtained from the haBmatin.

On shaking with cold aniline and treating first with acetic acid and then
with ether, KUSTER obtained a product, dehydrochloride haemin, which was
poor in the elements of hydrochloric acid, and which again took up HC1 and was
converted into haxnin. 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 haBmin 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 -|-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° 0., 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. For particulars as to the various methods of preparation and
purification we refer the reader to the above-cited works of NENCKI
and SIEBER, CLOETTA, MORNER, ROSEXFELD, NENCKI and ZALESKI
(SHALFEJEFF), and especially to KUSTER. 2

Hsematin is obtained on dissolving the haamin crystals in very dilute
caustic alkali and precipitating with an acid.

In preparing ha3min 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 precaution 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 ha3min crystals of varying forms will be seen.

1 Eppinger, 1. c.; v. Siewert, Arch. f. exp. Path. u. Pharm., 58.

2 Kiister, Zeitschr. f. physiol. Chem., 40.

H^EM ATO PORPH YRIN. 287

In regard to the preparation and properties of the iodine-, bromine-,
and acetone-haemin we refer to the work of STRZYZOWSKI, MERUNOWICZ
and ZALESKi.1

By the action of acids upon haemochromogen, haematin, or haemin, a
new iron-free pigment, which was first closely studied by HOPPE-SEYLER
and called hcem,atoporphyrin, is produced. According to the method of
preparation haematoporphyrins 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 haema-
toporphyrin is the one obtained according to NENCKI and SIEBER'S
method, by the action of glacial acetic acid saturated with hydrobromic
acid upon haemin crystals, best at the temperature of the body (NENCKI
and ZALESKi2).

Haematoporphyrin, C16H18N2O3, or C34H38N4O6 according to ZALESKI,S
is a pigment which according to MAcMuNN,4 occurs as a physiological
pigment in certain animals. It occurs, as shown by GARROD and
SAILLET, as a normal constituent, although only as traces, in human
urine. It occurs in greater quantities in human urine after the use of
sulfonal (see Chapter XV).

The formation of hsematoporphyrin from haematin can be expressed
by the following equation if we start with the above formula for haemin
and ZALESKI'S formula for haematoporphyriii :

C34H33N404FeCl + 2HBr + 2H2O = C34H38N4O6 + FeBr2 + HC1 .

On heating haematoporphyrin it generates an odor of pyrrol. On oxida-
tion with bichromate and glacial acetic acid it yields hsematinic acid (see
page 283). A pigment closely allied to the urinary pigment, urobilin,
has been obtained by the action of reducing substances on haematopor-
phyrin (HOPPE-SEYLER, NENCKI and SIEBER, LE NOBEL, MACMUNN).
On the administration of hacmatoporphyrin to rabbits, NENCKI and
RoTSCHY5 observed that a part was reduced to a substance similar to
urobilin.

Of especial interest are the investigations of NENCKI, MARCHLEWSKI,
and ZALESKI 6 upon the reduction products of haematoporphyrin and

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 Zeitschr. f. physiol. Chem., 37, 54.

4 Journ. of Physiol., 7.

5 Hoppe-Seyler, 1. c., 523; Le Nobel, Pfluger's Arch., 40; MacMunn, Proc. Roy.
Soc., 30, and Journ. of Physiol., 10; Nencki and Rotschy, Monatshefte f . Chem., 10.

6 See footnote 1, page 269.

288 THE BLOOD.

their relation to the chlorophyll derivatives. By the action of glacial
acetic acid containing HI and of iodophosphonium upon haemin or
haomochromogen NENCKI and ZALESKI obtained a markedly char-
acteristic pigment, mesoporphyrin, having the formula C16H18N202, or,
according to ZALESKI, x C34H38N4O4, and which stands in a certain measure
between haematoporphyrin, Ci6H18X2O37 and the chlorophyll derivative
phylloporphyrin, Ci6H18N2O, which is very similar to hsematoporphyrin.
By the action of the same reducing agent upon haemin or haemochro-
mogen, but under other conditions, we obtain haemopyrrol, C8H13N, a
colorless oil, which in the air gradually changes into urobilin. Haemopyr-
rol is produced by the action of the same reducing agents upon the
chlorophyll derivative phyllocyanin (NENCKI and MARCHLEWSKI),
which, as above remarked, shows a close relation between the blood-
pigment and chlorophyll.

We have numerous investigations by NENCKI and ZALESKI, KUSTER
and HAAS, MARCHLEWSKI and BURACZEWSKI and ST. MOSTOWSKI,
RETINGER, GOLDMANN and HETPER2 on hsemopyrrol. These investi-
gations have not led to any conclusive results, but show that haemo-
pyrrol is not a unit body. KUSTER obtained methyl-ethyl maleinamide
as an oxidation product and he considers haemopyrrol as a mixture of
two methyl-ethyl pyrrols. According to MARCHLEWSKI it is, on the
contrary, a mixture of several substances whose chief constituent is methyl-
propyl pyrrol.

Haematoporphyrin is, according to NENCKI and SIEBER, isomeric with
the bile-pigment bilirubin, and like this latter gives a play of colors —
green, blue, and yellow — when treated with fuming nitric acid.

The hydrochloric-acid compound 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,
and chloroform, but readily soluble in ethyl alcohol, alkalies, and dilute
mineral acids. The compound with sodium crystallizes as small tufts
of brown crystals. The acid alcoholic solutions have a beautiful purple
color, which becomes violet-blue on the addition of large quantities of
acid. The alkaline solution has a beautiful red color, especially when
not too much alkali is present.

An alcoholic solution of haematoporphyrin, 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

1 The works of Kiister and collaborators may. be found in Ber. d. d. chem. Gesellsch.,
37 and 40, and Zeitschr. f. physiol. Chem., 55, and of Marchlewski and co-workers,
in Zeitschr. f. physiol. Chem., 43, 45, 51, 56, and Biochem. Zeitschr., 10.

2 See footnote 1.

HSEMATOPORPHYRIN. 289

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 addition
of an alkaline zinc-chloride solution the spectrum changes more or less
rapidly/ 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 pig-
ment 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 corre-
spond 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 char-
acter 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 288.

Haematinogen 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 h&matin. 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
hydrochloride 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.
ZALESKI considers this pigment as a hydrogenized hsemin. A regenera-
tion of ha?matin from hsematoporphyrin has been performed by LAIDLAW.
If hsematoporphyrin is dissolved in dilute ammonia and warmed with
STOKES' solution and hydrazine hydrate, iron is taken up again and hsemo-
chromogen is produced, which is changed into hsematin by shaking with
air. According to HAM and BALEAX,4 it is possible to produce hsemo-

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.

4 Zaleski. Zeitschr. f. physiol. Chem., 43; Laidlaw, Journ. of Physiol., 31; Ham
and Balean, ibid., 32.

290 THE BLOOD.

globin from hsemochromogen 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 blooa extrav-
asations, and whose origin from the blood-coloring matters seems to
be established (LANGHANS, CORDUA, QUINCKE, and others l) . A solu-
tion of hsematoidin shows no absorption-bands, but only a strong absorp-
tion from the violet to the green (EWALD 2). 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,3 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, methsemoglobin
or ha3matin, then the preparation of hsemin crystals is an absolutely
positive test. In regard to the detection of blood in urine see Chapter
XV and for the detection of blood in intestinal contents, in pathological
fluids and in chemico-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 haemoglobin may be calculated. JOLLES 4 has suggested
a clinical method based on this procedure.

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 oxyha?mo-
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
sides containing a layer of liquid 1 cm. thick (HOPPE-SEYLER'S ha?matinom-
eter). The use of HOPPE-SEYLER'S colorimetric double pipette is more
advantageous. Other good forms of apparatus have been constructed
by GIACOSA and ZANGERMEISTER.S Instead of an oxyhsemoglobin

1 A comprehensive review of the literature pertaining to hsematoidin may be found
in Stadelmann, Der Icterus, etc., Stuttgart, 1891, pp. 3 and 45.

2 Zeitschr. f . Biologic, 22, 475.

3 Cit. from Gorup-Besanez, Lebrbuch d. physiol. Chem., 4. Aufl., 1878.

4 Jolles, Pfluger's Arch., 65; Monatshefte f. Chem., 17. See also Oerum, Zeitschr. f
anal. Chem., 43; and the works cited in Maly's Jahresber., 33.

5 F. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 16; G. Hoppe-Seyler, ibid., 21;
Winternitz, ibid.; Giacosa, Maly's Jahresber., 26; Zangermeister, Zeitschr. f. Biolo-
gic, 33.

QUANTITATIVE ESTIMATION OF THE BLOOD PIGMENTS. 291

solution 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.1

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 spectro photo metric 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=Ci:El, when C and
Ci represent the different concentrations and E and EI the corresponding

C C
coefficients of extinction. From the equation -=,= j^. it follows that for

£j E/l

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 (VlERORDT and
HUFNER 2) 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 oxyhaemoglobin, especially between the wave-lengths
,554 fifi and 565 fifi, and (b) the region of the second band, especially the inter-
val between the wave-lengths 531.5 ft ft and 542.5 fin. 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 is

0 = 100.7. 4.#and
C = 100. V.A'.E'.

The absorption ratio or the constants in the two above-mentioned regions
of the spectrum have been determined for oxyhsemoglobin, haemoglobin, carbon
monoxide haemoglobin, and methaemoglobin, as follows:

Oxyhsemoglobin A0 =0.002070 and A'0 =0.001312

Hemoglobin Ar =0.001354 and A'r =0.001778

Carbon-monoxide haemoglobin . ,AC =0.001383 and A'c =0.001263
Methsemoglobin A ,„ = 0.002077 and A'm = 0.001754

The quantity of each coloring matter may be determined in a mixture of
.two blood-coloring matters by this method ; this is of special importance in
the determination of the quantity of oxy haemoglobin and haemoglobin present
in blood at the same time.

In order to facilitate these determinations, HUFNER 3 has worked out tables
which give the relation between the two pigments existing in a solution contain-

1 See Haldane, Journ. of Physiol., 26.

2 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, Pfluger's Arch., 31 and 36.

3 Arch. f. (Anat. u.) Physiol., 1900.

292 THE BLOOD.

ing oxyhaemoglobin and another pigment (haemoglogbin, methacmoglobin, or car-
bonmonoxide haemoglobin), and thus allowing of the calculation of the absolute
quantity of each pigment.

Among the many apparatus constructed for clinical purposes for the
quantitative estimation of haemoglobin, FLEISCHI/S kcemometer, which has
undergone numerous modifications. HEXOCQUE'S hcematoscope, and
SAHLI'S h&mometer are to be specially mentioned. In regard to these
apparatus 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 Arachnid®, Crustacea, Gasteropodae
and Cephalopodae 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 haemocyanin 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; O 21.67 per cent, On hydrolytic
cleavage with hydrochloric acid HENZE found the following division of the nitro-
gen in haemocyanin: 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 Chaetopodae.
H center ythr in, so called by KRUKENBERG but first observed by SCHAWLBE, is a
red coloring matter from certain Gephyrea. Besides haemocyanin 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
by MAcMuNN,1 is a brown coloring matter occurring in the perivisceral fluid of
a variety of echinoderms.

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 A~A; °f the dried substance consists of haemoglobin (in
human and mammalian blood).

According to the analyses of HOPPE-SEYLER 2 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

Dogis " 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-cor-
puscles 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. ;

1 Fredericq, 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 Med.-chem. Untersuch., 390 and 393.

WHITE BLOOD-CORPUSCLES. 293

protein, 5.32 (clog) -78. 5 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 hemoglobin 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 corpuscles 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 con-
tain, according to BOTTAZZI and CAPPELLi,1 considerably more potassium
than sodium. Lime is claimed to be absent in the blood-corpuscles, and
magnesia occurs only in small amounts: 0.016 (sheep)-O.loO 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.2

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 polynuclear
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

1 Bunge, Zeitschr. f. Biologic, 12, and Abderhalden, Zeitschr. f. physiol. Chem., 23
and 25; Wanach, Maly's Jahresber., 18, 88; Bottazzi and Cappelli, Arch. Ital. de Biolo-
gie, 32.

2 Hamburger, Arch. f. (Anat. u.) Physiol., 1898; Koeppe, ibid., 1899 and 1900.

294 THE BLOOD.

gravity than the red corpuscles, move in the circulating 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 l 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 leu-
cocytes than the circulating blood. The correctness of this statement
has been denied by other investigators.

From a histological standpoint we generally, as above indicated, dis-
criminate 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 movements
which serve partly to make possible the wandering of the cells, and partly
to aid in the absorption of smaller grains or foreign bodies. On these
grounds the occurrence of myosin in them has been admitted even without
any special proof thereof. We know nothing with positiveness 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 chiefly
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
chief 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.

1 Pfliiger's Arch., 11 and Kriiger, Arch. f. exp. Path. u. Pharm., 51.

2 See Halliburton, On the chem. Physiol. of the animal cell. King's College, London,
Physiol. Labor. Collected papers, 1893.

BLOOD-PLATES. 295

On digesting the leucocytes with water, a. solution of a protein body
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
praglobulin (A LEX. SCHMIDT) or nudeohistone (KossEL and LILIENFELD 1)
and consists; chiefly at least, of nucleoprotein. The ordinary view that
this is nudeohistone does not seem to be correct, according to the recent
investigations 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, odd (in pus corpuscles, MANDEL and LEVENES); pu^ine 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 6 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. In regard to the other Constituents of the leucocytes
we refer to Chapter VII, on pus.

The blood-plates (BIZZOZERO), hsematoblasts (HAYEM), whose nature,
preformed occurrence, and physiological importance have been much
questioned, are pale, colorless, gummy disks, round or somewhat oval in
shape, and generally with a diameter one-half or one-third that of the
blood-corpuscles. By 'the action of different reagents the blood-plates
are separated into two substances, one of which is homogeneous and non-
refractive, while the other is highly refractive and granular. Blood-
plates readily stick together and attach themselves to foreign bodies.

1 See Wooldridge, Die Gerinnimg 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 tfukleoproteider, Kristiania, 1902.

3 Biochem. Zeitschr., 4.

4 In regard to the literature on Glycogen see Chapter VIII.

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 See Erben, Jochmann and E. Miiller, Jochmann and Lockemann, Hofmeister's
Beit rage, 11, which contains the literature. Opie, Journ. of exper. Medicine, 8; with
Barker, ibid., 9.

296 THE BLOOD.

According to the researches of KOSSEL and of LILIENFELD l 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, and especially in regard to the manner in which
these plates act in coagulation, are unfortunately very divergent.

III. 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 when about two years
old and in pregnant women. The determinations of LLOYD JONES,
HAMMERSCHLAG,2 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 most accurately done 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 deter-
mine 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.S

The reaction of the blood is alkaline toward litmus. The quantity
of alkali, calculated as Na^COs, in fresh, non-defibrinated blood from the
dog, horse, and man, is, according to LOEWY, 4.93, 4.43, and 5.95 p. m.
respectively. According to STRAUSS the average for normal human blood
may be calculated as about 4.3 p. m. Na2CO3. Quantities below 3.3
p. m. and above 5.3 p. m. are, according to him, to be considered as

1 In regard to the literature of the blood -plates, see Lilienfeld, Arch. f. (Anat. a.)
Physiol., 1892, and " Leukocyten und Blutgerinmmg," Verhandl. d. physiol. Gesellsch.
zu Berlin, 1892; and also Mosen, Arch. f. (Anat. u.) Physiol., 1893, and Maly's Jahres-
ber., 30 and 31.

2 Lloyd Jones, Journ. of Physiol., 8; Hammerschlag, Wien. klin. Wochenschrift,
1890, and Zeitschr. f. klin. Med., 20.

3 Zuntz, Pfluger's Arch., 66; Levy, Proceed. Roy. Soc., 71.

REACTION OF THE BLOOD. 297

pathological, v. JAKSCH found the quantity of alkali in man to vary
} etween 3.38 and 3.90 p. m., and BRANDENBURG found 3 p. m. NaOH
( = 3.98 p. m. Na2CO3). The alkaline reaction diminishes outside of the
boch', 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,
COHXSTEIN), and it is also decreased after the continuous ingestion of
acids (LASSAR, FREUDBERG J) . According to the investigations of
ALLERS and BONDI 2, on poisoning rabbits with hydrochloric acid, the
relation of the lime to the other bases changes by a relative increase in
the lime.

Numerous investigations have been made in regard to the alkalinity
of the blood in disease, but as there is at present no trustworthy method
for estimating the alkalinity of the blood, and as the results are dependent
upon the indicator used, these investigations, as also the claims in
regard to the physiological alkalinity, require further substantiation.3
Attention must also be called to what was stated (page 264) in regard
to the determination of the alkalinity of blood-serum — that determina-
tions are made only of the titratable alkali and not of the true alkalinity
-caused by hydroxyl ions. This alkalinity, as previously remarked, is
so very slight that the blood is considered as a nearly neutral fluid. As
explained by L. HENDERSON,4 the carbon dioxide, the alkali carbonate
and the mono- and di-phosphates are of especially great importance for
this condition as well as for the regulation of the reaction of the blood.

The alkali of the blood exists in part as alkaline salts, carbonate
and phosphate, and partly in combination with protein or hemoglobin.
The first are often spoken of as readily diffusible alkalies, while the others
are not or are only diffusible with difficulty (see page 261). The quan-
tity of the first in human blood is about one-fifth of the total alkali

1 Loewy, Pfliiger's Arch., 58, which also contains the references to the literature;
H. Strauss, Zeitschr. f. klin. Med., 30; v. Jaksch, ibid., 13; Peiper, Virchow's Arch.,
116; Cohnstein, ibid., 130, which also cites the works of Minkowski, Zuntz, and Gep-
pert; Freudberg, ibid., 125. See also Weiss, Zeitschr. f. physiol. Chem., 38; Branden-
burg, Zeitschr. f. klin. Med., 45.

2 Biochem. Zeitschr., 6.

3 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, 1898. See also Maly's Jahresber., 29, 30, and 31; Salaskin
and Pupkin, Zeitschr. f. physiol. Chem., 42, and O. Folin, ibid., 43; Laitinen, Ham-
marsten's Festschr., 1906; Westenrijk, Arch. f. exp. Path. u. Pharm. Suppl., 1908,
Sclimiedeberg-Festschrift.

4 Amer. Journ. of Physiol., 21 (1908).

298 THE BLOOD.

(BRANDENBURG). The readily as well as the difficultly diffusible alkali
is divided between the blood-corpuscles and plasma, and the blood-cor-
puscles 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,
LoEWYand ZUNTZ, HAMBURGER, LIMBECK, and GURBER/ 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 equilibrium 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. KORANYI and
BENCE,2 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 viscosit}^ 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 cofficient of the serum takes place, and
these same changes can be brought about by CO2 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 2 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 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

1 Zuntz, in Hermann's Handbuch der Physiol., 4, Abt. 2; Loewy and Zuntz,
Pfluger's Arch., 58; Hamburger, Arch. f. (Anat. u.) Physiol., 1894 and 1898, and
Zeitschr. f. Biologic, 28 and 35; v. Limbeck, Arch. f. exp. Path. u. Pharm., 35; Giirber,
Sitzungsber. d. phys. med. Gesellsch zu Wiirzburg, 1895.

2 Pfluger's Arch., 110.

3 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

COAGULATION OF THE BLOOD. 299

matters occur in the blood-corpuscles. For this reason blood is opaque
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 266), the blood becomes transparent and has then a "lake
color." 1 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, better,
on the different amounts of oxyhaemoglobin and hemoglobin 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 " huffy coat," is not char-
acteristic 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 ot 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., 1*9, p. 65.

300 THE BLOOD.

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 dis-
solved 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 circulation.
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 uninjured
blood-vessels exert upon it. These views are derived from the observa-
tions 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. BRUCKE2 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 oanula 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
to FREUND 3 it is this adhesion between the blood and a foreign substance

1Delezenne, Arch, de Physiol. (5), 8; Wright, Journ. of Physiol., 28; Arthus,
Journ. de Physiol. et Pathol., 4.

2 Hewson'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; Brucke, Virchow's Arch., 12,

3 Freund, Wien. med. Jahrb., 1886; Haycraft and Carlier, Journ. of Anat. and
Physiol., 22.

COAGULATION OF THE BLOOD. 301

— and the diseased walls of the vessel also act as such — that gives the
impulse toward coagulation, while the lack of adhesion prevents the
blood from coagulating. BORDET and GENGOU 1 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-
hig 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 coagu-
lation. That this adhesion of the form-elements is of great importance
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 2 and the Dorpat school an
abundant 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 Lowrr.3
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 4) . Great
importance has also been ascribed to the blood-plates in coagulation, as
certain investigators (BIZZOZERO, LILIENFELD, SCHWALBE, MORAWITZ,
BURKER) found that they cause or accelerate coagulation, while others
(PETRONB) on the contrary find a retarding action5.

WOOLDRIDGE 8 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-

1 Annal. de 1' Institute Pasteur, 17.

2 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.

3 Wien. Sitzungsber., 89 and 90, and Prager med. Wochenschr., 1889, referred
to in Centralbl. f. d. med. Wissens£i., 28, 265.

4 Morawitz, Hofmeister's Beitrage, 5; Arthus, Compt. rend. soc. biolog., 55; Dastre,
ibid., 55.

5 See foot-note 1, p. 296. Also Schwalbe, Unters. z. Blutgerinnung, etc., Braun-
schweig, 1900; Morawitz, Deutsch. Arch. f. klin. Med., 79, and Hofmeister's Beitrage,
4 and 5; Biirker, Pfluger's Arch., 102, and Centralbl. f. Physiol., 21; Petrone, Maly's
Jahresber., 31, p. 170.

6 Die Gerinnung des Blutes (published by M. v. Frey, Leipzig, 1891).

302 THE BLOOD.

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-
DKIDGE'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,
while 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 suppressed by the addition of zymoplastic sub-
stances, and the yield of fibrin on coagulation 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 fibinogen from the paraglobulin and
then converts the fibrinogen into fibrin. The assumption that fibrinogen
can be split from paraglobulin 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-

COAGULATION OF THE BLOOD. 303

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 coagu-
lation accelerators are bodies soluble in alcohol, and the compound pro-
teins exhausted with alcohol act only retardingly on coagulation, LILIEN-
FELD states that the substance which acts acceleratingly and retardingly
on coagulation are contained in a nucleoprotein, namely, nucleohistone.
Nucleohistone readily splits into leuconuclein and hist one, 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.

LILIENFELD'S theory that the leuconuclein splits off a protein substance,
thrombosin, from the fibrinogen, and that this thrombosin forms an insoluble com-
pound with the lime present, producing thrombosin lime (fibrin), which separates,
is incorrect according to HAMMARSTEN, SCHAFER, and CRAMER. 3 LILIENFELD'S
thrombosin is nothing but fibrinogen which is precipitated by a lime salt from a
salt-poor or salt-free solution.

According to PEKELHARING 4 thrombin is the lime compound of

1 Hammarsten, Nova Acta reg. Soc. Sclent. Upsal. (3), 10, 1879; Green, Journ. oj
Physiol., 8; Ringer and Sainsbury, ibid., 11 and 12; Arthus et Pages and Arthus,
see footnote 6, p. 243; 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.

3 Hammarsten, 1. c.; Schafer, Journ. of Physiol., 17; Cramer. Zeitschr. f. physiol.
Chem., 23.

4 See footnote 3, p. 248, and especially Virchow's Festschrift, 1, 1891.

304 THE BLOOD.

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. Of the objections
to this theory can be mentioned, among others, the fact that fibrin has
not been obtained absolutely free from lime, but still so poor in lime
(HAMMARSTEN l) that if the lime belongs to the fibrin, its molecule
must be more than ten times greater than the hemoglobin 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 con-
tradict 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 or 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
appreciable amounts of prothrombin. This body, according to him,
passes before coagulation from the form-elements into the plasma, and

1 Zeitschr. f. physiol. Chem., 28.

COAGULATION OF THE BLOOD. 305

is there converted into thrombin by the calcium salts. The observation
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, according to MORAWITZ,
is an antibody toward thrombin and quantitatively neutralizes it. On
the addition of prothrombin, new thrombin may be formed, which may
act if the hirudin is not present in too great an excess.

The behavior of sodium -fluoride plasma shows more conclusively
the absence of prothrombin in the circulating plasma. Such plasma,
according to ARTHUS, contains no prothrombin, a statement which has
been partly substantiated by MORAWITZ, who finds that fluoride-plasma
contains more or less prothrombin, dependent upon the greater or less
change the blood undergoes before it flows into the sodium-fluoride
solution. One can obtain, according to MORAWITZ, at least sometimes,
a fluoride-plasma which contains no prothrombin. The observations
of FULD and of SCHITTENHELM and BODONG contradict the statement
that fluoride-plasma contains prothrombin. As BORDET and GENGOU 1
have shown that prothrombin can be carried down by the precipitate
produced in fluoride-plasma, it seems as if the observations of ARTHUS
and MORAWITZ on this point are not conclusive, and MORAWITZ is now
also of the opinion that the prothrombin occurs preformed in the
plasma. NoLF2 also holds this view, ana for the present we generally
believe that prothrombin, or as it is also now designated thrombogen
{MORAWITZ, NOLF), is a preformed constituent of the plasma. The
absence of prothrombin, as observed by ARTHUS, in peritoneal transu-
dates in the horse, can hardly be considered as sufficient evidence as to
the non-occurrence of this body in blood-plasma.

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 accelerating
action upon coagulation, of different tissue extracts, an action which has
been known for a long time and was especially studied by DELEZENNE
on the plasma from bird's blood. Unfortunately we. are not in accord

1 Arthus, Journ. de Physiol. et Pathol., 3 and 4, and Compt. rend. soc. biol., 56.
The works of Morawitz may be found in Hofmejster'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, Annal. Institut
Pasteur, 18. For more recent literature see Loeb, Biochem. Centralbl., 6, p. 907.

2 Arch, internat. de Physiol., 6, 1908.

306 THE BLOOD.

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 (thrombogen 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 necessary, accord-
ing 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 transforms the
thrombogen into a-prothrombin, which latter then is converted into
thrombin (a) by the lime-salts.

The thrombokinase also 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 l 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 chief 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, PiiGLiESE2 and others.

According to the theory of MORAWITZ, FULD and SPIRO, 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 irritating action upon
the form-elements, and therefore no mentionable quantity of kinase is given off
under these circumstances. Su^h an elimination 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
thrombin then transforms the fibrinogen into fibrin.

1 Centralbl. f. Physiol., 17. See also Fuld and Spiro, Hofmeister's Beitrage, 5.
*Fuld and Spiro, 1. c.; Morawitz, 1. c.; Loeb, Hofmeister's Beitrage, 5; Nolf, Arch,
internat. de Physiol., 6; Pugliese, Biochem. Centralbl., 5, p. 930.

COAGULATION OF THE BLOOD. 307

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 (/5) is produced which is somewhat different from
tt-thrombin. The /?-thrombin is produced from a special /9-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 (/?-pro-
thrombin), which is then transformed by the alkali or acid into neozym ( = /?-
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
PEKELHARING 1 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, Huis-
KAMP, DELEZENNE and LOEB 2) they are thrombin, or at least bodies hav-
ing an analogous action.

L. LoEB,3 who has carried out complete investigations on the coagu-
lation of blood, especially of Crustacea}, has arrived at the following
view: The coagulation in the Crustacese can, according to him, be of two
kinds. It may in part be an agglutination of the amoebocytes and
in part a fibrin formation from a fibrinogen of the plasma. This latter
coagulation is essentially the same as occurs in vertebrates. The sub-
stance 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, 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

1 Bioch. Zeitschr., 11.

2 Huiskamp. Zeitschr. f. physiol. Chem., 3-4, 89; Delezenne, Arch, de physiol.,
1897; Loeb, Biochem. Centralbl., 6, pages 829 and 889.

3 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.

308 THE BLOOD.

they would act as kinases in the vertebrates. Under favorable condi-
tions 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, is, in LOEB'S opinion, not proven.

The coagulins of the blood are, as above stated, according to LOEB, different
from the tissue coagulins. The first show no specific action, i.e., between inver-
tebrates and vertebrates. The tissue coagulins, on the contrary, have by their
action upon the blood a certain specificity, at least in animals widely separated
from one another.

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 /ymoplastic substances (ALEX.
SCHMIDT) .

According to NOLF 1 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 proportions
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 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 la fibrine insuffisamment pourvue de fibrinogene. Dans la

1 Arch, internal, de Physiol., 6, Fasc., 1, 2 and 3.

COAGULATION OF THE BLOOD. 309

coagulation du fibrinogene par la thrombine les affinites restees libres
de celle-ci peuvent s'assouvir; le compose moins sature se transforme
en un compose plus sature.") The formation of fibrin from fibrinogen
is not, according to NOLF, an enzymotic process.

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. It is also a normal
constituent of the blood plasma circulating in the living body. Most
tissues, on the contrary, 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. The circulating blood plasma
contains all the bodies directly necessary in the coagulation, namely,,
fibrinogen, thrombogen, thrombozym and lime salts. Besides these it
also contains a substance that inhibits coagulation, antithrombin, which
is formed in the liver and which NOLF now considers as a special sub-
stance and not as he formerly believed, an excess of thrombogen. 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; but unfortunately it is not clear how this thrombo-
plastic 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
it has a proteolytic action only upon fibrin and not upon fibrinogen.
Apcording 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
fibrinoylsis, 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

310 THE BLOOD.

in regard to his remarkable investigations or the foundations on which
he bases his theory and the objections which can be raised against it.

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 arid 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 proven, 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-
tion of thrombin by the action upon the thrombokinase; the hirudin l
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 (MARTIN 3
and others) or certain of the protein-like colloid substances, synthetically
prepared according to GRIMAUX'S method (HALLIBURTON and PICKER-
ING 4). If too little of the above-mentioned bodies be injected, then we
observe only a marked retarding tendency in the coagulation of the

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.

INTRAVASCULAR COAGULATION. 311

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 formation
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 dif-
ferent ways. The best studied is the action of proteoses, but no conclusive
results have been obtained thus far. The assertion of PICK and SPTRO
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-
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 retarding substances by different investigators, such as
GROSJEAN, LEDOUX, CONTEJEAN, DASTRE, FLORESCO, ATHANASIU, CAR-
WALLO, GLEY, PACHON and GLEY, SPIRO and ELLINGER, FULD and SPIRO,
MORAWITZ and NOLF, but those of DELEZENNE 2 are of the greatest impor-
tance. We can say with certainty that the action is indirect and that
the liver is important for the process. The non-coagulability of " pep-
tone-blood " seems to be due to several reasons, but it has not been
thoroughly explained. On the one hand such blood contains an anti-
thrombin 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

1 Pick 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.

2 Grosjean, Travaux du lahoratoire de L. Fredericq, 4, Liege, 1892; Ledoux, ibid.,
5, 1896; Nolf, Bull. 1'Acad. roy. de Belgique, 1902 and 1905, and Biochem. Centralbl., 3;
Spiro and Ellinger, Zeitschr. f. physiol. Chem., 23; Fuld and Spiro, 1. c.; Morawitz,
1. c. The works of the above-mentioned French investigators 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.

312 THE. BLOOD.

water or the addition of a little acid. Opinions in regard to the occurrence
of an antithrombin in the peptone-plasma seem to be unanimous, and we
have collected rather considerable experience in regard to the formation
of this antithrombin. According to NOLF, the peptone (more correctly
the proteoses) causes an alteration 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 accelerat-
ing coagulation and another having a retarding action are set free. The
first is destroyed by the liver, and hence the action of the retarding sub-
stance (the antithrombin) is obtained. The only thing that is positively
proven is the part taken by the liver in this retardation of coagulation,
as shown by GLEY and PACHON; the non-appearance of the thrombin
formation is not explained by the above theories.

The reason of the slow coagulation of the blood in hemophilia is not-
well known. Recent investigations of MORAWITZ and LOSSEN 1 seem
to show that a lack of thrombokinase plays an important part in this
condition. This explains 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 the fact that it contains no fibrinogen, due to a fibrinolysis.

The gases of the blood will be treated of in Chapter XVII (on respira-
tion) .

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.
BLEiBTREu3 against which important objections have been raised by

1 Deutsch. Arch. f. klin. Med., 94.

2 Hofmeister's Beitrage, 8.

3 Pfltiger's Arch., 51, 55, and 60.

QUANTITATIVE COMPOSITION OF THE BLOOD. 313

several investigators, such as EYKMAN, BIERNACKI and HEDIX, * must
be especially mentioned. In regard to this as well as to the method
of St. BUGARSKY and TAXGL, which is based upon a difference in the
electrical conductivity of the blood and the plasma, and STEWART'S 2
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, con-
structed by BLIX and described and tested by HEDIX. A measured
quantity of blood is mixed with a known volume (best an equal volume)
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,
HEDIX 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 3 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 4 has shown that in centrifuging blood very rapidly, more
than 5000 times 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 6, then the amount of x in the plasma from 100 parts of blood is

100.6
is x = .

1 Biernacki, Zeitschr. f. physiol. Chem., 19; Eykman/Pfliiger's Arch., 60; Hedin,
ibid., and Skand. Arch. f. Physio].. 5.

2 Bugarsky and Tangl, Centralbl. f. Physiol., 11; Stewart, Jouni. of Physiol., 24.

3 Hedin, Skand. Arch. f. Physiol., 2, 134 and 361, and 5; Pfltiger's Arch., 60; Daland,
Fortschritte d. Med., 9.

4 Pfliiger's Arch., 107.

314 THE BLOOD.

Such a substance, which occurs only in the plasma, is fibrin according
to HOPPE-SEYLER, sodium according to BUNGE (in certain kinds of blood),
and sugar according to OTTO.1 The experimenters just named have
tried to determine the amount of the plasma and blood-corpuscles, respec-
tively, in different kinds of blood, starting from the above-mentioned
substances.

Another method suggested by HOPPE-SEYLER is to determine the
total amount of hemoglobin and proteins in a portion of blood, and on
the other hand the amount of haemoglobin 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
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 determinations. 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
of the blood, plasma and blood-corpuscles may be ascertained. In
the table opposite are given analyses of the blood of various animals by
AsDERHALDEN2 according to HOPPE-SEYLER'S and BUNGE'S methods.
The analyses of human blood by C. SCHMIDT 3 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 consider-
ably under different circumstances even in the same species of animal.
In animals, in most cases considerably more plasma is found, sometimes
two-thirds of the weight of the blood.4 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 5 found 349.6
arid 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 recent 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 bloocl-cor-

1 Hoppe-Seyler, Handb. d. physiol. u. path. chem. Analyse, 6. Aufl.; Bunge, Zeit-
schr. f. Biologie, 12; Otto, Pfluger's Arch., 35.

2 Zeitschr. f. physiol. Chem., 23 and 25.

3 Cited and in part recalculated from v. Gorup-Besanez, Lehrb. d. physiol. Chem.,
4. Aufl., 345.

4 See Sacharjin in Hoppe-Seyler's Physiol. Chem., 447; Otto, Pfliiger's Arch., 35;
Bunge, 1. c.; L. and M. Bleibtreu, Pfluger's Arch., 51.

5 Arronet, Maly's Jahresber., 17; Schneider, Centralbl. f. Physiol., 5, 362.

ANALYSES OF BLOOD.

315

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42.89

34.05
0.74
0.37
0.98
0.91
0.70

0.01

2.39
0.14

0.06
0.03
2.31
0.14
0.05

206.81
127.50
106.40
15.38

0.610
0.953

0.0194

0.839
0.233
0.562

0.009
0.628
0.236
0.133

608.03
5V. GO

46.41
0.679
0.599
1.244
2.357
0.494

0.0089

2.873
0.174

0.073
0.027
2.453
0.156
0.041

200.03
11S.82
102.80
12.80

1.147
1.329

0.0235
0.760
0.236
0.545

0.006
0.575
0.228
0.088

624.16
56.63

46.56
0.708
0.891
1.088
0.859
0.4908

0.0109

2.917
0.172

0.080
0.027
2.516
0.163
0.057

Solids
Haemoglobin
Protein

Sugar

Cholesterin
Lecithin

Fat
Fatty acids

Phosphoric acid \
as nuclein /
Soda

Potash . .

Iron oxide
Lime

Magnesia
Chlorine
Phosphoric acid . .
Inorganic PzOb . . .

Goat-blood.

Cat-blood.

Rabbit-blood.

Human Blood,
Man.

Human Blood,
Woman.

B

Jb

~3 £Ti>

o o •<**

.2 oco

a

.00

S^i
310

r

1

• 1°'

-C E?Tf

o oco
,2 w^

3

-°!
I"3

J

•ge£

o o t^
,2 oco

a

fi

03

Blood-
corpuscles,
513.02.

Serum,
486.98.

Blood-
corpuscles,
396.24.

Serum,
603.76.

Water. . .
Solids
Haemoglobin . .
Protein
Sugar
Cholesterin
Lecithin
Fat

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

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

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
I 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

272.56
123.68

120.13

3.55

0.65
1.41

0.36

551.99
51.77

46.70

5.07

1.92
20.20

0.14

Fatty acids

Phosphoric acid\
as nuclein /
Soda
Potash

Iron oxide
I ime

Magnesia
Chlorine
Phosphoric acid . .
Inorganic P2Os . .

puscles as well as in the plasma, of man with diabetes mellitus is
increased. They found the same in dogs with alimentary glycosuria.1
HoLLiNGER2 also found that in man with normal quantity of sugar
in the blood the sugar was distributed nearly equally between the
blood-corpuscles and the plasma, and in most of the cases of hypergly-
caemia investigated the blood-corpuscles contained much more of the

Biochcm. Zeitschr., 16 and 18.

2 Biochm. Zeitschr., 18.

316 THE BLOOD.

abnormally high sugar content than the serum. As the blood-corpuscles
of drawn blood are impermeable to sugar, a fact further substantiated
by special experiments by MICHAEL-IS and RONA, the reason for the
permeability of the corpuscles in life must be investigated. According
to ABDERHALDEN lime, fat and perhaps also fatty acids occur only in the
serum. The small traces of bile-acids found in normal blood are, accord-
ing to CROFTAN,1 contained in the leucocytes. The division of the
alkalies between the blood-corpuscles and the plasma is different, as the
blood-corpuscles from the pig, horse, and rabbit contain no soda, those
from human blood are richer in potassium, and the corpuscles from ox-,
sheep-, goat-, dog-, and cat-blood are considerably richer in sodium than
potassium. Chlorine exists in all blood to a greater extent in the serum
than in the blood-corpuscles. Iodine is found only in the serum, while
iron occurs chiefly in the form-elements, especially in the erythrocytes.
As the nucleoproteins contain iron, some iron always occurs in the leu-
cocytes, and traces of iron are also found in the serum. This amount
under normal conditions is very small, while in disease the relation between
hsemoglobin-iron and other blood-iron does not seem to change very
much. There are also found in the blood manganese and traces of
lithium, copper, lead, silver, and in menstrual blood arsenic has also been
noted. The blood as a whole contains in ordinary cases 770-820 p. m.
water, with 180-230 p. m. solids; of these 173-220 p. m. are organic and
6-10 p. m. inorganic. The organic consists, deducting 6-12 p. m. of
extractive bodies, of proteins and hemoglobin. The amount of this last-
mentioned body in human blood is about 130-150 p. m. In the dog,
cat, pig, and horse the quantity of haemoglobin is about the same, but
is lower in the blood from the ox, bull, sheep, goat, and rabbit (ABDER-
HALDEN).

The amount of sugar in the blood is, according to most statements,
on an average 1 p. m. It seems to be independent of the composition
of the food, but feeding with large amounts of sugar or dextrin causes a
considerable increase in the sugar of the blood, as observed by BLEILE.
This condition does not seem to be the same for all animals, as shown by
OPPLER and RONA; the amount of blood-sugar varied quite considerably
in rabbits, while on the contrary in dogs it was very constant even under
various conditions of life. When the quantity of sugar amounts to more
than 3 p. m., then, according to CL. BERNARD,2 sugar occurs in the urine
and a glycosuria' appears. An increase in the quantity of sugar takes
place, as first observed by BERNARD and substantiated by FR. SCHENCK,

1 Pfluger's Arch., 90.

2 Bleile, Arch. f. (Anat. u.) Physiol., 1879; Oppler and Rona, Bioch. Zeitschr., 13;
Bernard, Legons sur le diabete, Paris, 1877.

EXTRACTIVES OF THE BLOOD. 317

after removal of blood. According to HENRIQUES 1 this increase of the
reducing power, at least in dogs, is not due to sugar, but chiefly to jecorin,
which substance in normal blood is the cause of more of the reduction
than the sugar. It is difficult to judge of the value of many reports
as to the amount of sugar and the reducing power of the blood, because
the experimenters generally have not considered the presence of a certain
quantity of jecorin or conjugated glucuronic acids, or they were unable
to detect them. The investigations of ANDERSSON2 seem to indicate
that at least in rabbits, on drawing blood, the fermentable sugar, as well
as the non-fermentable " sugar rest," increases to about the same extent.3

The quantity of urea, which, according to SCHONDORFF, is equally
divided between the blood-corpuscles and the plasma, is greater on taking
food than in starvation (GREHANT and QUINQUAUD, SCHONDORFF) and
varies between 0.2 and 1.5 p. m. In dogs SCHONDORFF found in starva-
tion 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. In man v. JAKSCH 4 found 0.5-0.6 p. m. urea
in normal blood. The quantity of urea is somewhat increased in fever,
and in general in augmented protein metabolism the increased urea for-
mation is dependent thereon. 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 infant-urn 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 5
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,6 who worked with the improved NENCKI
and ZALESKI method, the quantity in arterial dog-blood was 0.41 milli-

1 Schenck, Pfliiger's Arch., 57; Henriques, Zeitschr. f. physiol. Chem., 23. See also
Kolisch and Stejskal, Wien. klin. Wochenschr., 1898.

2 Bioch. Zeitschr., 12.

3 In regard to the recent methods for determining sugar in the blood see Bang,
Bioch. Zeitschr., 7; Michaelis and Rona, ibid., 7; Oppler and Rona, ibid., 13.

4 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; v. Jaksch, Leyden-Festschr., I, 1901.

5 v. Schroder, Zeitschr. f. physiol. Chem., 14; Baglioni, Centralbl. f. Physiol., 19.
8 Zeitschr. f. physiol. Chem., 35, which also gives the older literature.

318 THE BLOOD.

gram in 100 grams of blood. The blood of the portal vein contains
considerably more than the blood of the arteries, being 3-4.5 times richer;
this, however, is disputed by BIEDL and WiNTERBERG.1 The blood from,
healthy persons contains on an average 0.90 milligram per 100 cc.,
according to WiNTERBERG.2 The quantity of uric acid may be 0.1 p. m.
in bird's blood (v. SCHRODER 3) . Uric acid has not been detected
with positiveness in human blood under normal conditions, while it has
been found in the blood in gout, croupous pneumonia, and certain other
diseased conditions. 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. SAITO and KATSUYAMA* 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.

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 contain-
ing different amounts of gas and different amounts of oxyhaBmoglobin
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 'moro 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 hemoglobin than the arterial blood; but this is denied by
modern investigators. According to KRUGER 5 and his pupils the quantity
of dry residue and hemoglobin in blood from the carotid artery and from
the jugular vein (in cats) is the same. ROHMANN and MUHSAM 6 could
not detect any difference in the quantity of fat in arterial and venous 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 quan-
tity of blood circulating through the liver in a given time, we can hardly
expect to detect by chemical analysis a positive difference in the. com-
position between the blood of the portal and hepatic veins. The state-

1 Pfluger's Arch., 88.

2 Wien. klin. Wochenschr., 1897, and Zeitschr. f. klin. Med., 35.

3 Ludwig's Festschrift, 1887.

4 Irisawa, Zeitschr. f. physiol. Chem., 17, which also gives the older literature; Saito
and Katsuyama, ibid., 32.

5 Zeitschr. f. Biologic, 26. This also gives the literature on the composition of the
blood in different vascular regions.

8 Pfluger's Archiv, 46.

BLOOD OF THE SPLENIC VEIN. 319

ments in regard to such a difference are in fact contradictory. For
example, DROSDOFF found more hemoglobin in the hepatic than in
the portal vein, while OTTO found less. KRUGER finds that the quantities
of hemoglobin, as well as of the solids, in the blood from the vessels
passing to and from the liver are different, but a constant relation
cannot be determined. The hepatic vein, according to DOYON and
collaborators,1 is richer in fibrinogen than the blood from the portal
vein. The disputed question as to the varying quantities of sugar in
the portal and hepatic veins will be discussed in a following chapter (see
Chapter VIII, 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 dextrose, but may contain also dextrin and other carbohydrates
(v. MERING, OTTO 2) . The amount of urea in the blood from the hepatic
vein is greater than in other blood (GREHANT and QuiNQUAUD3). In
legard to the quantity of ammonia, see page 318.

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, 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 hemoglobin
than arterial blood. KRUGER 4 and his pupils found that the blood
from the vena lienalis is generally richer in hemoglobin 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 retention of the blood-clot by

1 See footnote 2, page 245.

2 Drosdoff, Zeitschr. f. physiol. Chem., 1; Otto, Maly's Jahresber, 17; v. Mering,
Arch. f. (Anat. u.) Physiol, 1877, 214.

3 I.e.

4 v. Middendorff, Centralbl. f. Physiol., 2, 753; Kriiger, 1. c.

320 THE BLOOD.

the womb and the vagina, so that only fluid cruor is at times eliminated,
and in part on a contamination with vaginal mucus, which disturbs the
coagulation. Menstrual blood, according to GAUTIER and BOURCET,
contains arsenic and is also richer in iodine than other blood (see Blood-
serum, page 261).

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 arid haemoglobin is somewhat smaller in woman's
blood. The amount of hemoglobin 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 normal
again. 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 l found in most cases an
increase in the amount of haemoglobin in pregnancy in the last months.

The Blood at Different Periods of Life. Foatal and infant blood is
richer in erythrocytes and haemoglobin than the blood of the mother.
The highest percentage of haemoglobin in the blood has been observed
by several investigators, such as COHNSTEIN and ZUNTZ, OTTO, WINTER-
NITZ, 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-cqr-
puscles 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, Orro2). According to earlier reports, the blood at

1 Nasse, Maly's Jahresber., 7; Becquerel and Rodier, Traite de chim. pathol.,
Paris, 1854; Cohnstein, Pfliiger's Arch., 34, 233; Mollenberg, Maly's Jahresber., 31,
185. See also Payer, Arch. f. Gynak., 71.

2 Cohnstein and Zuntz, Pfliiger's Arch., 34; Winternitz, Zeitschr. f. physiol. Chem.,.

INFLUENCE OF FOOD ON THE BLOOD. 321

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, BUNTZEN 1), 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.
In rabbits and to a less extent in dogs, PO?EL 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 2) .

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, LEICHTENSTBRN) . 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. The composition
of the food acts essentially on the amount of haemoglobin in the blood.
The blood of herbivora is generally poorer in hemoglobin than that of
carnivora, and SUBBOTIN has observed in dogs after a partial 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 3
has also shown in experiments on rabbits and dogs that with an insuf-
ficient diet of bread and potatoes, where the body gave up protein and
contained relatively considerable carbohydrate, the amount of haemoglobin
decreased and the blood became richer in water. According to LEICH-
TENSTERN. a gradual increase in the amount of haemoglobin is found to
take place in the blood of human beings on enriching the food, and accord-
ing to the same investigator the blood of lean persons is generally some-

22; Leichtenstern, Untersuch. uber den Hamoglobingehalt des Blutes, etc., Leipzig,
1878; Otto, Maly's Jahresber., 15 and 17; Abderhalden, Zeitschr. f. physiol. Chem.,
34; Schwinge, Pfluger's Arch., 73 (literature). See also Fehrsen, Journ. of Physiol.,
30.

1 Panum, Virchow's Arch., 29; Subbotin, Zeitschr. f. Biologic, 7; Otto, 1. c.; Worm
Muller, Transfusion und Plethora, Christiania, 1875; Buntzen, see Maly's Jahresber.,
9; Hermann and Groll, Pfluger's Arch., 43; Luciani and Bufalini, Maly's Jahresber.,
12.

2 Popel, Arch, des scienc. biol. de St. Pe"tersbourg, 4, 354; Schulz, Pfluger's Arch.,
65; Daddi, Maly's Jahresber., 30.

3 Subbotin, 1. c.; Tsuboi, Zeitschr. f. Biologie, 44.

322 THE BLOOD.

what 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.1 There does not seem to be
any doubt that the iron contained in the food in the form of organic com-
pounds 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 sulphuretted hydrogen of the intestinal canal and
thereby prevent the iron associated in the food as assimilable protein
compounds from being eliminated as iron sulphide (BUNGE), a view which
is now generally discarded?

An increase in the number of red corpuscles, a true " plethora poly-
cythcemia," takes place after transfusion of blood of the same species
of animal. According to the observations of PANUM and WORM MiJLLER,2
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 transudation from the blood, as in
cholera arid 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 consider-
able increase in the number of red corpuscles, in his own case 5-8 millions.
A similar increase of the red blood-corpuscles, as also an increase in the
quantity of haemoglobin under the influence of diminished pressure,
has been observed by many other investigators 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 diminished 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

1 See Bunge, Zeitsehr. 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, Pfluger's Arch., 61;
Macallum. Journal of Physiol., 16; Abderhalden, Zeitsehr. f. Biologie, 39.

2 Pan urn, Virchow's Arch., 29; Worm Miiller, 1. c.

NUMBER OF RED CORPUSCLES. 323

in the capillaries, from which region the blood lias 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 contrac-
tion of the vascular system with the pressing out of plasma (BuNGE,
ABDERHALDEN l). In connection with these experiments, it must be
remarked that several trustworthy observations show that under the
influence of diminished blood-pressure an actual increase in the red
blood-corpuscles takes place, and ZUNTZ 2 and his co-workers have also
shown that the activity in the red bone-marrow is increased.

A decrease in the number of red corpuscles occurs in anemia 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 a hydraemia. The amount of protein then
gradually increases again ; but the reformation of the red blood-corpuscles
is slower, and after the hydraemia follows also an oligocythsemia. After
a little time the number of blood-corpuscles rises to normal. INAGAKI 3
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 detail as to the results, but simply to state that they
substantiate the previously known observation that during regenera-
tion irregularities may occur in the relation between the quantity of
hemoglobin and the number of erythrocytes. This he explains by the
fact, as claimed by BOHR (see page 271), that there exist different
haemoglobins containing different quantities of iron. 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 character-
istic 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.4

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.

3 Zeitschr. f. Biol., 49.

4 Complete analyses of chlorotic blood may be found in Erben, Zeitschr. f. klin.
Med., 47.

324 THE BLOOD.

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 (|— iV)
occurs in pernicious anaemia (HAYEM, LAACHE, and others). 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 corpus-
cles often, but not 'always, show in pernicious anaemia remarkable
and extraordinary irregularities of form and size, which QUINCKE 1 has
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 increased to a considerable extent in leucaemia. Leuca?mic
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 XIII) 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,2 a digestive product which
is 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 recently been made by ERBEN.S

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 concerning
the composition of the blood of diseased human beings, it is impossible

1 Laache, Die Anamie (Christiania, 1883), which also contains the literature;
Quincke, Deutsch. Arch. f. klin. Med., 20 and 25. A complete chemical analysis of
the blood has been made by Erben, Zeitschr. f. klin. Med., 40.

2 Erben, Zeitschr. f . Heilkimde, 24, and Hofmeister's Beitrage, 5. See also Schumm,
ibid., 4 and 5. See also footnote 6, page 295.

3 Zeitschr. f. klin. Med., 66 (1908).

QUANTITY OF BLOOD. 325

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 fa-^ °f tne weight of the
body, and in new-born infants about -fa. HALDANE and LORRAIN SMITH/
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 OERTJM 2 has determined the quantity
of blood in men as about -fa and in wo^nen -^ 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 3), 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-
sinking of the blood-pressure in the arteries, because the smaller arteries
accommodate themselves to the small quantities of blood by con-
tracting (WORM MtJLLER4). 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, AVoRM
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 5), a polycythsemia is gradually produced.

1 Journ. of Physiol., 25.

2 Deutsch. Arch. f. klin. Med., 93 (1908).
3Virchow's Arch., 29.

4 Transfusion und Plethora, Christiania, 1875.

5 Panum, Nord. med. Ark., 7; Virchow's Arch., 63; Landois, Centralbl. f. d. med.

326 THE BLOOD.

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.

Wissensch., 1875, and Die Transfusion des Blutes, Leipzig, 1875; Worm Mailer,
Transfusion und Plethora; Ponfick, Virchow's Arch., 62; Tsehirjew, Arbeiten aus
der physiol. Anstalt zu Leipzig, 1874, 292; Forster, Zeitschr. f. Biologic, 11; Panum,
Virchow's Arch., 29.

1 Die Blutvertheilung und der Thatigkeitswechsel des Organe, Leipzig, 1871.

CHAPTER VII.

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 HEIDENHAIN, differentiate between blood -lymph and tissue-lymph
according to origin. It is impossible at the present time to completely
separate that which comes from the one or the other source.

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 110 doubt of a quantitative nature.
This difference consists chiefly in that the lymph is poorer in proteins.
No essential chemical difference has been found between the lymph and
the chyle of starving animals. After fatty food the chyle differs from
the lymph in its wealth of minutely divided fat-globules, which gives it
a milky appearance; hence the old name "lacteal fluid."

Chyle and lymph, like the plasma, contain seralbumin, serglobulins,
fibrinogen, and fibrin ferment. The two last-mentioned bodies occur only
in very small amounts; therefore the chyle and lymph coagulate slowly
(but spontaneously) and yield but little fibrin. Like other liquids poor
in fibrin ferment, chyle and lymph do not at once coagulate completely,
but repeated coagulations take place.

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

1 Zeitschr. f. Biologie, 36.

327

328 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

the blood-serum, but in larger quantities than in the blood; this depends
on the fact that the blood-corpuscles contain no sugar. The glycoyen
detected by DASTRE 1 in the lymph occurs only in the leucocytes. Accord-
ing to ROHMANN and BIAL, lymph contains a diastatic enzyme similar
to that in blood-plasma, and LEPINE 2 found that the chyle of a dog
during digestion has great glycolytic activity. 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.

As form-elements, -leucocytes and 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, MuNK4 found
that the chyle contained chiefly neutral fat with only small amounts of
fatty acids or soaps.

The gases of the chyle have not been studied, and it seems that the
gases of an entirely normal human lymph have not thus far been investi-
gated. The gases from dog-lymph contain, according to HAMMARSTEN,
only traces of oxygen, and consist of 37.4—53.1 per cent CO2 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 combination.
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.6 The analyses thus far made refer only to that mixture of chyle
and lymph which is obtained from the thoracic duct. The specific
gravity varies between 1.007 and 1.043. As an example of the compo-
sition 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

1 Compt. 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; Lupine, 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.

f Hammarsten, Die Gase der Hundelymphe, Arbeiten aus d. physiol. Anstalt zu
Leipzig, 1871 ; Strassburg, Pfliiger's Archiv, 6.

6 See also Panzer, Zeitschr. f. physiol. Chem., 30.

CHYLE AND LYMPH. 329

HOPPE-SEYLER/ of the chyle in a case of rupture of the thoracic ciuct.
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 7.23fat

2.35 soaps

Remaining organic bodies 10.8

0.83 lecithin

1 . 32 cholesterin

3 . 63 alcohol extractives

0 . 58 water extractives

g lt 44 / 6. 80 soluble salts

\ 0.35 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. RosENSTEiN2 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 weighing
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 ^ of the neutral fats. ScnuMM3 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
chief 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 from the upper part of the thigh of a woman aged
thirty-nine; and 3, made by v. SCHERER, is an analysis of lymph from the
sac-like dilated lymphatic vessels of the spermatic cord. No. 4 was made

1 Owen-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.

330 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

by C. SCHMIDT/ the data being obtained from lymph from the neck
of a colt. The results are expressed in parts per 1000.

1234

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 34.71

Fat, cholesterin, lecithin 3.8 9.2 I 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 fol-
lowing 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;
Na2CO3 2.17; K2HPO4 0.28; . Ca3(PO4)2 0.28; MgsCPOJa 0.09; and
Fe (P(>4) 0.025. CARLSON, GREEN and LUCKHARDT 2 have recently made
comparative 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.

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 3
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

1 Gubler and Quevenne, cited from Hoppe-Seyler's Physiol. Chem., 591 ; v. Scherer,
ibid., 591; C.' Schmidt, ibid., 592.

2 Amer. Journ. of Physiol., 22 (1908).

3 Hensen, Pfliiger's Arch., 10; Lang, see Maly's Jahresber., 4.

FORMATION OF LYMPH. 331

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 in the thoracic duct of animals. According 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. KOEL-PATON l 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 2 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 BARBERA 3 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 on preventing the flow of blood by means of ligatures,
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 4), and the quan-
tity of solids in the lymph is also increased.

The bodies inciting lymph-flow, the so-called lymphagogues, are of

1 Journ. of Physiol., 11.

2 Cited from Hoppe-Seyler, Physiol. Chem., 593.

3 The works of Asher and collaborators, Barbera, Gies, and Busch, upon lymph
formation may be found in Zeitschr. f . Biologic, 36, 37, 40.

4 Lesser, Arbeiten aus der physiol. Anstalt zu Leipzig, Jahrgang 6; Paschutin,
ibid., 7.

332 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

especially great interest, and they may, according to HEiDENHAiN,1 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'bef ore.
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 lymphy
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 endotheJium, assum-
ing that they take part in the formation of lymph in a secretory manner.

Another view, which besides the physical processes is also of especial
physiological moment in the explanation of lymph formation, was sug-
gested by ASHER and his collaborators (BARBERA, GIES, and BUSCH),
According to them the lymph is a product of the work of the organs;:
its amount is dependent upon an increased or diminished activity of the
organs, 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

1 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.

FORMATION OF LYMP,H. 333

of lymphagogues on blood coagulation and liver activity (DELEZENNE
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 l) .

The extent of organ work certainly essentially influences the quan-
tity and properties of the lymph. Still from this we cannot draw any
positive 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 called in the first
place to the fact that the important works of HEIDENHAIN, HAMBURGER,
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 the part played by filtration as compared with that of the osmotic
force is only very trivial has been conclusively shown by the adherents
of the physico-chemical theory of lymph formation.

Several investigators (KoRANYl, STARLING, ROTH, ASHER, and others)
have clearly shown that the work in the glands and tissue-cells must cause
a difference in the osmotic pressure upon the two sides of the capillary
wall. That this is so follows from several circumstances, and especially
from the fact that, in disassimilation 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

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.

334 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

are alone sufficient to explain the lymph formation (COHNSTEIN, ELLINGER)
remains an open and disputed question.1

II. 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 subcutaneous
tissues, or under the epidermis; and in this way pathological transudates
are formed. Such true transudates, which are similar to tymph, 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 of 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
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 formation
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

1 On this question see Ellinger, " Bie Bildimg der Lymphe," Ergebnisse der Phy-
siologic, I, Abt. 1, 355, and Asher, Biochem. Centralbl., 4, pp. 1 and 45.

TRANSUDATES. 335

interesting observation made by PAUKULL,1 that in those cases in which
an inflammatory irritation nab 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 tne 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 hydra^mia the amount of protein in
the transudate is very small. With the increase in the age of a tsansudate,
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, witnout any previous hemorrhage, is
even greater than in the blood-serum.

The proteins of transudates are chiefly seralbumm, serglobulin, and
a little fibrinogen. Proteoses and peptones do not occur, excepting
perhaps in the cerebrospinal fluid, and in these cases where an autolysis
has taken place in the liquid.3 The non-inflammatory transudates do not
as a rule undergo spontaneous coagulation only very slowly, or not at all.
On the addition of blood or blood-serum they coagulate. Inflammatory
exudates coagulate spontaneously, and PAIJKULL has shown that these
often contain nucleoprotein (or nucleoalbumm). In inflammatory
exudates a protein substance has been habitually observed which is pre-
cipitated 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 MORITZ, STAEHELIN, UMBER, and RIVALTA, is claimed
by the first three observers to be free from phosphorus, while RIVALTA

1 See Maly's Jahresber., 22.

2 Cited from Hoppe-Seyler, Physiol. Chem., 607.

3 Umber, Munch, med. Wochenschr., 1902, and Berlin, klin. Wochenschr., 1903.
In regard to the autolysis in transudates, see also Galdi, Biochem. Centralbl., 3; Ep-
pinger, Zeitschr. f. Heilkunde, 35 and Zak, Wien. klin. Wochenschr., 1905.

336 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

considers it to be a phosphorized pseudoglobulin. UMBER calls it sero-
samucin, although it yields only very little reducing carbohydrate.
According to JOACHIM 1 it is only a part of the globulin, a view which can-
not be correct for all cases, v. HoLST2 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 circum-
stances, although the globulins form besides seralbumin ordinarily the
chief 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 PAUKULL3 to be regular
constituents of transudates and are closely related to the above-men-
tioned serosamucin.

There are numerous investigations on the relation between glob"
ulin and seralbumin, and JOACHIM has recently determined the rela-
tion between euglobulin and the total globulin. No conclusive results
can be drawn from these determinations. The relation between globulin
and seralbumin varies very much in different cases, but, as HOFFMANN
and PIGEAND 4 have shown, the variation is in each case the same as in
the blood-serum of the individual.

The specific gravity runs nearly parallel with the quantity of protein.
The varying specific gravity has been suggested as a means of differentia-
tion between transudates and exudates by Rsuss,5 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

1 Paijkull,4!. c.; Moritz, Munch, med. Wochenschr., 1903; Staehelin, ibid., 1902;
Umber, Zeitschr. f. klin. Med., 48; Rivalta, Biochem. Centralbl., 2 and 5; Joachim,
Pfluger's Arch., 93,

2 Zeitschr. f . physiol. Chem., 43.

3 Hamrnarsten, ibid., 15; Paijkull, 1. c.

4 Joachim, 1. c.; Hoffmann, Arch. f. exp. Path. u. Pharm., 16; Pigeand, see Maly's
Jahresber., 16.

5 Reuss, Deutsch. Arch. f. klin. Med., 28. See also Otto, Zeitschr. f. Heilkunde, 17.

TRANSUDATES. 3^7

due to other bodies, as in blood-serum. A reducing, non-fermentable
substance has been found by PICKARDT in transudates. The sugar is
generally dextrose, but levulose seems to have been found l in several
cases. Sarcolactic acid has been found by C. KULZ 2 in the pericardial
fluid from oxen. Succinic acid has been found in a few cases in hydrocaie
fluids, while in other cases it is entirely absent. Leucine and tyrosine
have been found in transudates from diseased livers and pus-like trans-
udates wrhich have undergone decomposition, and after autolysis. Among
other extractives found in transudates must be mentioned allantoin
(MoscATELLi3), uric acid, purine bases, creatine, inosite, and pyrocate-
chin (?).

The division of the nitrogenous substances in human transudates
and exudates has so far been little studied. OTORi4 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 amino-acid nitrogen and purine
nitrogen. The amino-acid nitrogen and the urea nitrogen in pus is
greater as the specific gravity rises, but not in serous exudates and transu-
dates.

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 sometimes
rather divergent results have been found. The concentration of the
electrolytes shows according to BoDON,5 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 (Bo DON) .

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

1 Pickardt, Berl. klin. Wochenschr., 1897. See also Rotmann, Munch, med. Woch-
enschr., 1898; Neuberg and Strauss, Zeitschr. f. physiol. Chem., 36.

2 Zeitschr. f . Biologic, 32.

3 Zeitschr. f. physiol. Chem., 13.
* Zeitschr. f. Heilkunde, 25.

5 Pfluger's Arch., 104, where the literature on this subject may be found.

338 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

following, relative to the quantitative composition, chief 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 Hopps-SEYLER,1
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

f Fibrin 0. 31

Proteins 28 . 60 \ Globulin 5 . 95

[Albumin... . 22.34

Soluble salts 8.60 NaCl 7.28

Insoluble salts 0.15

Extractive bodies 2 . 00

FRIEND 2 found nearly the same composition for a pericardial fluid
from a horse, with the exception that this liquid was relatively richer
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, HASEBROEK3 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 nucleoal-
bumin or nucleoprotein (the pyrin of early writers) may be obtained on

1 v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 401; Wachsmuth, Vir-
chow's Arch., 7; Hoppe-Seyler, Physiol. Chem., 605.

2 Halliburton, Text-book of Chem. Physiol., etc., London, 1891.
8 Zeitschr. f. physiol. Chem., 12.

PERITONEAL FLUID. 339

the addition of acetic acid. This precipitate is soluble with difficulty
in an excess of acetic acid.

Numerous analyses, by many investigators,1 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 abun-
dant 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. (HAMMAR-
STEN). Mucoid substances have also been detected in pleural fluids by
PAIJKULL. Cases of chylous pleurisy are also known; in such a case
MEHU 2 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
conditions (ascitic fluid). The color, transparency, and consistency of
these may vary greatly.

In cachectic conditions or a hydraemic condition of the blood the fluid
has little color, is milky, opalescent, watery, does not coagulate spon-
taneously, has a very low specific gravity, 1.005-1.010-1.015, and is nearly
free from form elements. The ascitic fluid in portal stagnation, or in
general venous congestion, has a low specific gravity and ordinarily
less than 20 p. m. protein, although in certain cases the quantity of pro-
tein 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

1 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.

2 Arch. gen. de m6d., 1886, 2, cited from Maly's Jahresber., 16.

340 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

found in the ascitic fluid (GUINOCHET, HAY1), and even 17-43 p. m. has
been found by MINKOWSKL

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 the fluid from an ovarian cyst the
former may sometimes contain pseudomucin (see Chapter XIII). 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 sy philitica in men. According to the
investigations of PAIJKULL, these substances seem to occur often and per-
haps habitually in the ascitic fluids.

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 3 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 cir-
rhosis 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 4 also a lipase.

Hydrocele and Spermatocele Fluids. These fluids differ e'ssentially
from each other in various ways. The hydrocele fluids are generally
colored light or dark yellow, sometimes brownish with a shade of green.

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.

3 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.
4 Arch. f. (Anat. u.) Physiol., 1900, 433.

CEREBROSPINAL FLUID. 341

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 HAMMAKSTEN.1

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 ]

Soluble salts 8.60 f 10.76

Insoluble salts 0 . 66 J

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 DsviLLARD,2 sometimes contain paralbumin or metalbumm (?).
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 3 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. According to HALLI-
BURTON the protein of the cerebrospinal fluid is a mixture of globulin
and proteose; occasionally some peptone occurs, and more rarely, in special
cases, seralbumin appears. The conclusions of HALLIBURTON on the
occurrenc'e of proteose do not coincide with the observations of other
investigators (PANZER, SALKO WSKI 4) . In general paralysis HALLI-

1 Upsala Lakaref. Forh., 14, and Maly's Jahresber., 8, 347.

2 Bull. Soc. chim., 42, 617.

3 Bioch. Zeitschr., 2.

4 Halliburton' s Text-book; Panzer, Wien. klin. Wochenschr., 1899; Salkowski,
Jaffe Festschrift, 265.

342 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

BURTON 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 1). Dextrose, 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
by NAWRATZKi,2 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 BAUMGARTEN3 in many pathological cases. The variable
relation between potassium and sodium is probably due. according to
SALKowsKi,4 to the absence or presence of fever during the formation
of the exudate; the amount of potassium is high in the acute cases and
low in the chronic ones. According to LANDAU and HALPERN 5 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 conclusion that the cerebrospinal fluid is
formed by a true secretory process.

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 0.8-1.02 p. m. The protein
consists of seralbumin and globulin and very little fibrinogen and mucin.
According to GRUENHAGEN it contains paralactic acid, another dextrogyrate
substance, and a reducing body which is unlike dextrose or dextrin.
PAUTZ 7 found urea and sugar in the aqueous humor of oxen.

Blister-fluid. The content of blisters caused by burns, and of vesicatory
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

1 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.

2 Zeitschr. f. physiol. Chem., 23. See also Rossi, ibid., 39 (literature).

3 Zeitschr. f. exp. Path. u. Therap., 4 (literature).

4 See Salkowski, 1. c. New quantitative analyses of cerebrospinal and hydro-
cephalus fluids may be found in the cited works of Nawratzki, Panzer, and SalkowskL

5 Bioch. Zeitschr., 9.

6 See Mary's Jahresber., 22, 346, and Centralbl. f. Physiol., 15, 216.

7 Gruenhagen, Pfluger's Arch., 43; Pautz, Zeitschr. f. Biologic, 31.

SYNOVIAL FLUID. 343

contents of vesicatory blisters. In a burn-blister K. MORNER 1 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 reac-
tion. A wound secretion collected by LiEBLEiN2 under aseptic condi-
tions 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
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. in., — according to HOFFMANN 1-8 p. m., — and in serious affec-
tions of the kidneys, generally with amyloid degeneration, less than 1
p.m. has been shown (HOFFMANN 3) . The cedematous fluid also habitually
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 7p.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 4) . In pathological
synovia HAMMARSTEN found a mucin-like substance which is not mucin.
It behaves like a nucleo albumin or a nucleoprotein, and gives no reducing
substance on boiling with acids. SALKOWSKI 5 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,

1 Skand. Arch. f. Physiol., 5.

a Habilitationsschrift Prag, 1902, printed by H. Laupp, Tubingen.

'Deutsch. Arch. f. klin. Med., 44.

4 Zeitschr. f . physiol. Chem., 43.

a Hammarsten, Maly's Jahresber., 12; Salkowski, Virchow's Arch., 131.

344 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

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.1 The figures repre-
sent parts per 1000.

I. Synovia from II. Synovia from
a Stall-fed Ox. a Field-fed Ox.

Water 969.9 94-8.5

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

The synovia of new-born babes corresponds to that of resting animals.
The fluid of the bursse mucosse, 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. It may become strongly alkaline when putrefaction
occurs with the formation of ammonia.

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 maceration.
The pus-serum contains, moreover, at least in many cases, no fibrin

1 Wagner's Handworterbuch, 3, Abt. 41, 63.

PUS. 345

ferment. According to the analyses of HOPPE-SEYLER : the pus-serum
contains in 1000 parts:

i. n.

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

The ash of pus-serum has the following composition, calculated to
1000 parts of the serum.

i. ii.

NaCl 5.22 5.39

Na2SO4 0.40 0.31

NaJIP04 0 . 98 0 . 46

Na2GO3. . 0.49 1 . 13

Ca3(PO4)2 0.49 0.31

Mg3(P04)2 0.19 0.12

PO4 (hi 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 2 seems to stand in close relation, we also have a globulin
which coagulates at 48-49° C., as well as serglobulin (?), seralbuminy
a substance similar to coagulated protein (MIESCHER), and lastly peptone

Med.-chem. Untersuch., 490. 2 Bioch. Zeitschr., 16.

346 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

or protease (HOFMEISTER l) . It is very remarkable that no nucleo-
histone or historic 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, glucothionicacid,2 purine bodies, fat, and soaps.
HOPPE-SEYLER has found cerebrin, a decomposition product of a pro-
tagon-like substance, in pus (see Chapter XII). KOSSEL and FREYTAG 2
have isolated from pus two substances, pyosin and pyogenin, which
belong to the cerebrin group (see Chapter XII). HOPPE-SEYLER 4
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 nucleoproteins.

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 5).

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. ii.

Proteins 137 .62 ]

Nuclein 342.57 (-685.85 673.69

Insoluble bodies 205 . 66 J

Lecithin . . \ -, , 0 oo 75 . 64

Fat ./ 75.00

Cholesterin. . 74 .00 72 .83

Cerebrin 51 .99

Extractive bodies 44 . 33

102.84

1 Miescher in Hoppe-Seyler's Med.-chem. Untersuch., 441; Hofmeister, Zeitschr. f.
physiol. Chem., 4.

2 Mandel and Levene, Bioch. Zeitschr., 4.

3 Zeitschr. f. physiol. Chem., 17, 452.

4 Hoppe-Seyler, Physiol. Chem., 790.

5Fr. Miiller, Verhandl. Nat. Gesellsch. zu Basel, 1901; O. Simon, Deutsch. Arch,
f. klin. Med., 70.

LYMPHATIC GLANDS. 347

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

P04 9.16

Na 0.68

K Traces (?)

MIESCHER obtained other results for the alkali compounds, namely,
potassium phosphate 12, sodium phosphate 6.1, earthy phosphate and iron
phosphate 4.2, sodium chloride 1.4, and phosphoric acid combined with organic
substances 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 volatile
fatty acids, such as formic acid, butyric acid and valeric acid. There
are also found chrondrin (?) and glutin (?), urea, dextrose (in diabetes),
bile-pigments, and bile-acids (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 acid and chlorrhodinic acid, 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 (Bacillus
pyocyaneus). From such pus FORDOS and LUCRE 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, etc.

The Lymphatic Glands. The cells of the lymphatic glands are
found to contain the protein substances generally occurring in cells
(Chapters I and VI). According to BANG2 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),
thymine, 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, sarco-
lactic acid, purine bases, and leucine. In the inguinal glands of an old

1 Fordos, Compt. rend., 51 and 56; Lucke, 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.

348 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

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 mesenteral lymphatic
glands of oxen BANG1 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 Tvith acetic acid and then further purifying, a
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 leuconuclein. 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, and HuiSKAMP2 upon nudeo-
histone all show that this nucleoprotein is not a unit substance, but a
mixture of at least two bodies. The views of the investigators mentioned
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.

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 as one cleavage product and free nucleic acid

»L.c.

3 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.

THYMUS. 349

as the other, according to the unanimous opinion of the above-mentioned
investigators. According to BANG, whose statements on this point
have been substantiated by MALENGREAU, it splits on saturating with NaCl
into nucleic acid and histone without yielding any other protein. On
this account BANG does not consider this body as nucleohistone in the
ordinary sense, i.e., not as a nucleoprotein, but as a histone nucleate.
The nucleohistone behaves like an acid, whose salts, especially the cal-
cium 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 therefore ionized in the solution. The nucleic acid-cal-
cium histone-compound 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 question as to what
compound contains the A-histone remains to be investigated.

The nucleohistone prepared by HUISK AMP'S method of precipitating with
CaCl2 is, according to him, a mixture of two nucleohistones, of which one, the
«-nucleohistone, contains 4.5 per cent phosphorus, and the other, /?-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 question
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, leucine, succinic
add, lactic acid, 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

1 Zeitschr. f . physiol. Chem., 39. 3 Compt. rend., 129.

2 See foot-note 1, p. 295. 4 Zeitschr. f . physiol. Chem., 13.

350 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

dry substance, and guanine. The bodies ihymine and (uracil ?) obtained,
besides lysine and ammonia, by KUTSCHER, as products of autodigestion
of the gland, probably have a similar origin. LILIENFELD l found
inosite and protagon in the cells of the thymus. Among the enzymes,
besides arginase, guanase, and adenase, we must especially mention the
enzyme studied by JoNEs,2 which acts like a nudease, splitting off phos-
phoric acid and purine bases, from the nucleoproteins. 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 of 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 KH2PO4 among the bodies soluble in
alcohol.

Attention must be called to the analyses of BANG,S 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. OIDTMANN*
found 807.06 p. m. water, 192.74 p. m. organic and 0.2 p. m. inorganic
substances in the gland of a child two weeks old.

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 MANDELS is to be considered as a true
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 6 was able
to isolate a histone-peptone as sulphate from the spleen. The ferruginous

1 Kutscher, Zeitschr. f. physiol. Chem., 34; Lilienfeld, ibid., 18.

2 Zeitschr. f. physiol. Chem., 41.
3 1. c., Arch. f. Math., etc.

4 Cited from v. Gorup-Besanez, Lehrb. d. physiol. Chem., 4. Aufl., p. 732.

5 Bioch. Zeitschr., 5.

8 Zeitschr. f. physiol. Chem., 49.

SPLEEN. 351

albuminate has been considered as a spleen constituent for a long time,
and especially also a protein substance which does not coagulate on boiling
and which is precipitated by acetic acid and yields an ash containing
much phosphoric acid and iron oxide.1

The pulp of the spleen, when fresh, has an alkaline reaction, but
quickly turns acid, due partly to the formation of free paralactic acid
and partly perhaps to glycerophosphoric acid. Besides these two acids
there are found in the spleen also volatile fatty acids, as formic, acetic,
and butyric acids, as well as succinic acid, neutral fats, cholesterin, traces
of leucine, inosite (in ox-spleen), scyllite, a body related to inosite (in the
spleen of plagiostoma) ; glycogen (in dog-spleen), uric acid, purine bases,
and jecorin. LEVENE found in the spleen a glucothionic acid, 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 work of MANDEL
and NEUBERG and LEVENE and JACOBS.2

Many enzymes are found in the spleen, and certain of these are of
special interest. To these belong the uric-acid-forming enzyme, the
xanthine oxidase (Bum AN), which occurs in the spleen of oxen and horses,
but not in man, dogs, and pigs (SCHITTENHELM), and which transforms
the oxypurines, hypoxanthine, and xanthine into uric acid; also the
hydrolytically active deamidizing enzymes 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 (SCHITTEN-
HELM), in the pig-spleen.3 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, /?-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 pro-
teins of the spleen, but they also dissolve fibrin and coagulated blood-
serum. In. the autolysis of the spleen LEATHES found among the
cleavage products, proteoses, lysine, arginine, histidine, leucine, amino-
valeric acid, aspartic acid, and tryptophane. ScnuMM4 found, in the

1 See v. Gorup-Besanez, Lehrbuch, 4. Aufl., p. 717.

2 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.

3 See chapter XV for the literature.

4 Hedin and Rowland, Zeitschr. f . physiol. Chem., 32, and Hedin, Journ. of Physiol.,

352 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

autolysis of a leucsemic spleen, besides leucine and tyrosine, relatively
large quantities of ammonia, also r-alanine, histidine, and lysine (but
no arginine), guanine, xanthine, hypoxanthine, thymine, and p-lactic
acid. The autolysis of the leucaBmic spleen was much more extensive
than the normal.

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 l 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. P2O57 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 in com-
parison with sodium and phosphoric acid the amount of potassium and
chlorine is small. The amount of iron in new-born and young animals
is small (LAPICQUE, KRUGER, and PERNOTJ), 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 2 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.

The quantitative analyses of the human spleen by OIDTMANN 3 give the
following results: In men he found 750-694 p. m. water and 250-306
p. m. solids. In that of a woman he found 774.8 p. m. water and 225.2
p. m. solids. The quantity of inorganic bodies was in men 4.9-7.4 p. m.,
and in women 9.5 p. m.

In regard to the pathological processes going on in the spleen we must
specially recall the abundant re-formation of leucocytes in leucaBmia and
the appearance of amyloid substance (see page 168).

30; and Hammarsten's Festschrift, 1906; Leathes, Journ. of Physiol., 28; Schumm,
Hofmeister's Beitrage, 3 and 7.

1 Maly's Jahresber., 19, p. 315.

3 Lapicque, ibid., 20; Lapicque and Guillemonat, Compt, rend, de soc. biol., 48r
and Arch, de Physiol. (5) 8: Kriiger and Pernou, Zeitschr. f. Biologic, 27; Nasser
cited from Hoppe-Seyler, Physiol. Chem., 720.

3 Cited from v. Gorup-Besanez, Lehrbuch, 4. Aufl., p. 719.

SPLEEN. 353

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-cor-
puscles, 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 GROSSENBACHER 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.
GROSSENBACHER and ZIMMERMANN 1 found that a splenectomized "dog
eliminates more iron than a normal dog even 10-11 months after the
operation. This shows that no compensation occurs even after this long
time. The spleen seems to be of little importance in taking up artificially
introduced iron. The destruction of blood-corpuscles caused by pyro-
dine (acetylphenylhydrazine) increases the elimination of iron by nor-
mal dogs ana also in splenectomized dogs, but in these latter to a much
higher degree. The destruction of body substance produced exper-
imentally by insufficient food or iron-free food causes a strongly increased
elimination of iron and comparatively much stronger in splenectomized
animals as compared with normal animals. This speaks in favor of
ASHER'S view that the spleen is an organ which works up the iron set
free in the destruction of the body material containing iron.

The spleen has also been claimed to play a certain part in digestion.
This organ is said by SCHIFF, HERZEN, and others to be of importance in
the production of trypsin in the pancreas. The investigations of HER-
ZEN seem to confirm this relation, but the recent work of PRYM2 has
made the assumption doubtful.

An increase in the quantity of uric acid eliminated in splenic leucaemia
has been observed by many investigators (see Chapter XV), 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 formation of uric acid. This relation has been studied by
HORBACZEWSKT. He has shown that when the spleen-pulp and blood of
calves are allowed to act on each other, under certain conditions and tem-
perature, in the presence of air, large quantities of uric acid are formed.
Under other conditions he obtained from the spleen-pulp only purine
bases with very little or no uric acid. HORBACZEWSKI 3 has also shown
that the uric acid originates from the nucleins of the spleen, which yield

1 Asher and Grossenbacher, Centralbl. f. Physiol., 22, 375, and Grossenbacher and
Zimmermann, Bioch. Zeitschr., 17.

2 Schiff, cited by Herzen, Pfliiger's Arch., 30, 295, 308, and 84, and Maly's Jahr-
esber., 18; Prym, Pfliiger's Arch., 104 and 107; see also Chapter IX.

3 Monatshefte f. Chem., 10, and Wien. Sitzungsber. Math. Nat. Klasse, 100, Abt. 3.

354 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

uric acid or purine bases according to the experimental conditions.
This behavior is explained by the above-mentioned investigations 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 XV).

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.
One of these, iodolkyreoglobulin, behaves like a globulin, while the other
is a nucleoprotein (see also GouRLAY1). The iodine present in the gland
occurs chiefly in the first body, while the arsenic, which has been shown
to be a normal constituent by GAUTIER and BERTRAND,2 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, xanihine, hypoxanihine, choline,3 iodothyrine, lactic and
succinic acids occur in the thyreoidea. OIDTMANN 4 found in the thyroid
gland of an old woman 822.4 p. m. water, 176.6 p. m. organic and 0.9
p. m. inorganic substances. He found 772.1 p. m. water, 223.5 p. m.
organic and 4.4 p. m. inorganic substances in an infant two weeks old.

In " STRUM A CYSTIC A " HoPFE-SEYLER found hardly any protein in the smaller
glandular vessels, but an excess of mutin, while in the larger he found a great
deal of protein, 70-80 p. m.5 Cholesterin 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 haematin?). 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 XIII) .

1 Gourlay, Journ. of Physiol., 16; Oswald, Zeitschr. f. physiol. Chem., 32, and
Biochem. Centralbl., 1, 429.

2 Gautier, Compt. rend., 129. See also ibid., 130, 131, 134, 135; Bertrand, ibid.,
134, 135.

3 v. Fiirth and Schwarz, Pfluger's Arch., 124.
4 1. c., 732.

5 Physiol. Chem., p. 721.

THYROID GLAND. 355

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, 6f 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 GENERALI 1 upon various animals have shown
that for the success of the operation it is of the greatest importance
whether the parathyroids, discovered by SANDSTROM,2 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 myxoedema.

All these conditions indicate that the thyroids belong to those glands
with internal secretion, so called endocrinogenic 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. A
further proof of practical importance is that the injurious results from
removal of the thyroids can be counteracted by the introduction of arti-
ficial 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 (athyreoiclismus 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 3) . The reverse is observed when large quantities
of the thyroid gland substance is fed, namely, a strong increase in the
protein metabolism, besides certain other symptoms. BASEDOW'S disease

1 Gley, Compt. rend. soc. biol., 1891, and Arch, de Physiol (5), 4; Vassale and
General!, Arch. Ital. d. Biol., 25 and 26.

2 Upsala Lakaref. Forh., 15 (1880).

3 Eppinger, Falta and Rudinger, Zeitschr. f. klin. Med., 66.

356 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

is also considered as a form of hyperthyreoidisrnus which, by an increased
activity of the glands, brings about an overproduction of the specific
secretion.

It is impossible for the present to state anything about the kind of
bodies here, having a specific action or anything about the importance of
the bases found by certain investigators, such as S. FRANKEL, DRECHSEL,
and KocHER1 ; these bodies have not been characterized sufficiently. It
seems proven that the specifically active substance is, as first shown
by NOTKIN 2 and OSWALD;S a protein substance: NOTKIN'S thyreoproteid,
OSWALD'S thyreoglobulin. This does not conflict with the views of
BAUMANN and Roos that the active substance is iodothyrin, as this can
be produced as a cleavage product from the iodothyreoglobulin. In
fact OSWALD 4 has found in the tryptic digestion of iodothyreoglobulin
that a substance similar to iodothyrin is produced; for several reasons it
seems that 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 5 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 transfor-
mation 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 sulphate.
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

1 Frankel, Wien. med. Blatter, 1895 and 1896; Drechsel and Kocher, Centralbl.
f. Physiol., 9, 705.

2 Wien. med. Wochenschr., 1895, and Virchow's Arch., 144, Suppl., 224.

3 Zeitschr. f . physiol. Chem., 32, and Bioch. Centralbl., 1, 249.

4 Arch, f . exp. Path. u. Pharm., 60.

5 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, Pfluger'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 destruc-
tion and metabolism, see F. Voit, Zeitschr. f. Biologic, 35; Schondorff, Pfluger's Arch.,
67, and Anderson and Bergman, Skand. Arch. f. Physiol., 8; Magnus-Levy, Zeitschr.
f. klin. Med., 32.

ADRENAL BODIES. 357

•cent in man. In the iodothyreoglobulin of the ox, NUREMBERG* 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 iodothyreo-
globulin. By introducing iodine salts the iodine content of the iodo-
thyreoglobulin 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.

JOLINT 2 has examined a large number of thyroid glands from healthy and
diseased persons (in SWEDEN), for their iodine content. In 28 children, ages
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. m. iodine. In glands from persons after using
iodine preparations (34 cases) the iodine content was 2.56 p. m.

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.3

The Adrenal Bodies. Besides proteins, substances of the con-
nective tissue, and salts, there occur in the suprarenal capsule inosite,
purine bases, especially ocanthine (OKER-BLOM), a protagon-like substance
(ORGLER), relatively considerable lecithin and choline, and glycerophos-
phoric add, which are probably decomposition products of the lecithin.
The earlier accounts of the occurrence of benzoic acid, hippuric acid,
and bile-acids are, on the contrary, doubtful, and are not substantiated
by recent investigations (STADELMANN) . Earlier investigators, like VUL-
PIAX and ARNOLD,4 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 LOH-
MANN 5 has shown that it is formed in the cortical substance of the adrenals.
That the watery extract of the adrenals has a blood -pressure raising
action was shown by OLIVER and SCHAFER, CYBULSKI and SZYMONO-
wicz.6 The substance which is here active was formerly called sphyg-

1 Bioch. Zeitschr., 16.

2 Hammarsten's Festschr., 1906.

3 A summary of the thyroid literature may be found in Maly's Jahresber., 24 and
25. See also the works of Blum and Oswald, cited by Oswald in Biochem. Cen-
tralbl., 1, 249.

4 Oker-Blom, Zeitschr. f. physiol. Chem., 28; Stadelmann, ibid., 18, which also
contains the literature on this subject; Orgler, Salkowski's Festschrift, 1904.

5 Centralbl. f. Physiol., 21, and Pfluger's Arch., 118.

6 Oliver and Schiifer, Proceed, of Physiol. Soc., London, 1895. Further literature
•on the function of the adrenals may be found in Szymonowicz, Pfluger's Arch., 64.

358 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.

mogenin 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. This body has been chemically investigated by several
experimenters such as v. FURTH, ABEL, TAKAMINE, ALDRICH, JOWETT,
PAULY, ABDERHALDEN and BERGELL, FRIEDMANN and STOLZ.1 v. FURTH
calls it suprarenin, ABEL epinephrin, and TAKAMINE adrenalin. This
last name seems to be the most generally accepted one.
Adrenalin (suprarenin epinephrin), CgHiaNOs,

CH

(HO)C C.CH(OH)CH2.NHCH3
(HO)C CH

v

CH

The constitution of adrenalin has been essentially proven by FRIEDMANN,2
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 :

which yields adrenalin on reduction.

The synthetically prepared adrenalin is optically inactive d-Z-adrenalin,
while that from the adrenals is optically active Z-adrenalin. FLACHER has
recently 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. MiJLLER.4
These last investigators also found that the ^-adrenalin had at least 15
times as strong an action upon the blood-pressure as the d-adrenalin, and
later ABDERHALDEN with THIES and SLAVU found that the Z-adrenalin
had also in other respects a much stronger action than d-adrenalin.

1 The literature on this subject may be found in v. Fiirth, Zeitschr. f . physiol. Chem.,
23, 26, 29 and Wien. Sitzungsber. Math. Nat. Kl., 112, 1903. See also Abel, Zeitschr.
f. physiol. Chem., 28; Amer. Journ. of Physiol., 1899, and The Johns Hopkins Hospi-
tal Bull., No. 76 (1897), 90 and 91 (1898), 120 and 128 (1901), 131 and 132 (1902);
Ber. d. d. chem. Gesellsch., 36; Abel and Taveau, Journ. of Biol. Chem., 1, and Fried-
mann, Hofmeister's Beitrage, 6 and 8.

" Hofmeister's Beitrage, 8.

' Ber. d. d. chem. Gesellsch., 37.

4 Flacher, Zeitschr. f. physiol. Chem., 58; Abderhalden and Franz Muller, ibid.,
58; with Thies, ibid., 59; with Slavu, ibid., 59.

ADRENALIN. 359

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 *).
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.

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 formation
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 GUG-
GENHEIM 2 with tyrosinase. This would indicate a close relation
between adrenalin and tyrosin, which also gives melanin with the sepia
enzyme, and indeed tyrosin has been considered as the probable mother
substance of adrenalin (HALLE3). From the above-mentioned exper-
iments as well as from the investigations of ABELOUS, SOULIE and TOUJAN 4
on the formation of adrenalin in the adrenals under the coaction of autory -
tic products of other organs or organ extracts, no positive conclusions
can be drawn.

Besides the action of producing a rise in the blood-pressure, adrenalin
is also of special interest because, as first shown by BLUM,S 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.

1 Zeitschr. f. physiol. Chem., 57.

2 Neuberg, Bioch. Zeitschr., 8; Abderhalden and Guggenheim, Zeitschr. f. physioi.
Chem., 57.

3 Halle, Hofmeister's Beitrage, 8.

4 Compt. rend. soc. biol., 58, 59, 60.

5 Deutsch. Arch. f. klin. Med., 91, and Pfliiger's Arch., 90.

CHAPTER VIII.
THE LIVER.

THE liver, which is the largest gland of the body, stands in close
relation to the glands mentioned in Chapter VII. The importance
of this organ for the assimilation of the food-stuft's 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. We are not clear as to what extent an
assimilation of the absorbed digestion products of the proteins brought
to the liver by the portal blood takes place in this organ; for the car-
bohydrates it has been proven that glycogen is formed from the dextrose.
By this glycogen formation the liver becomes the real reserve-organ for
the carbohydrates: it is also a storage-organ for fat, and perhaps also
for proteins.1

The formation of glycogen from dextrose is a synthesis, and the
occurrence of other syntheses in the liver has been repeatedly shown
by special observations. For example, in the liver certain ammonia
combinations are converted into urea or uric acid (in birds) (see Chapter
XV) , while certain products of putrefaction in the intestine, such as phenols,
may be converted by synthesis into ethereal sulphuric acids by the
liver (PFLUGER and KOCHS, EMBDEN and GLAESSNER), probably also
converted into conjugated glucuronic acids (EMBDEN2). The liver has
also the property of removing and retaining heterogeneous bodies from
the blood, and this is true not only of metallic salts, which are often
removed by this organ, but also, as SCHIFF, HEGER, and others, but espe-
cially ROGER, have shown, the alkaloids are retained, and are probably
also partially decomposed in the liver. Toxines are also withheld by the
liver, and hence this organ has a protective action against poisons.3

1 See Seitz, Pfliiger's Arch., Ill, and Asher and Boehm, Zeitschr. f. Biol., 51.

2 Pfluger and Kochs, Pfliiger's Arch., 20 and 23; Embden and Glaessner, Hof-
meister's Beitrage, 1; Embden, ibid., 2.

3 Roger, Action du foie sur les poisons (Paris, 1887), which also contains the older
literature; Bouchard, Legons sur les autointoxications dans les maladies (Paris, 1887) ;
and E. Kotliar in Arch, des sciences biologiques de St, Petersbourg, 2. See also de

360

PROTEINS OF THE LIVER 361

Even though the liver is of assimilatory importance and purifies the
blood coming from the digestive tract, it is at the same time a secretory
organ which eliminates a specific secretion, the bile, in the production
of which the red blood-corpuscles are destroyed, or at least one of their
constituents, the haemoglobin. It is generally admitted that the liver
acts contrariwise during fostal life, at that time forming the red blood-
corpuscles.

There is no doubt that the chemical operations going on in this
organ are manifold and must be of the greatest importance to the
organism. Our knowledge on this subject has been essentially advanced
by recent investigations, but nevertheless it must be admitted that
we know little of the character and extent of these changes. Among
the products of these chemical processes there are two which are
especially important and must be treated in this chapter, namely, the
glycogen and the bile. Before the study of these products is taken up,
a short discussion of the constituents and the chemical composition of
the liver is necessary.

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 l) . 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 PiAsz.
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 nudeoalbumin 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 pos-
sessed 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
(0.28-1.3 per cent) of this globulin as well as the insolubility of the pre-

Vamossy, Centralbl. f. Physiol., 18, and Rothberger, Wien. klin. Wochenschr., 1905,
Rothberger and Winterberg, Biochem. Centralbl., 4.

1 Morishima, Arch. f. exp. Path. u. Pharm., 43 ; Magnus-Levy, Hofmeister's Beitrage,2.

2 Plosz, Pfliiger's Arch., 7; Halliburton, Journ. of Physiol., 13, Suppl. 1892.

362 THE LIVER.

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 nearly complete digestibility
with pepsin-hydrochloric acid does not controvert this assumption,
because, as is known, nucleoproteins may on digestion yield no residue
(see Chapter III). Nor can we be positive concerning the nature
of the liver-globulin found by DASTRE,* having a coagulation tem-
perature of 56°. The proteins extractable from the liver without modifi-
cation 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 PLOSZ). 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 SCHMIE DE-
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 Z-xylose, the four nuclein bases, and also arginine, lysine (and
histidine?), tyrosine, leucine, glycocoll, alanine, a-proline, glutamic acid,
aspartic acid, phenylalanine, oxyaminosuberic acid, and oxydiamino-
sebacic acid (see Chapter III) . 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 thymonucJeic 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 SCHAFFIDI and SALKOWSKI. 6

The yellow or brown pigment of the liver has been little studied. DASTRE
and FLORESCO 7 differentiate, in vertebrates and certain invertebrates, between a

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,
Pfluger's Arch., 67.

3 Arch. f. exp. Path. u. Pharm., 33; see also Vay, Zeitschr. f. physiol. Chem., 20.

4 Wohlgemuth, 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 Schaffidi, Zeitschr. f. physiol. Chem., 58; Salkowski, ibid., 58.

7 Arch, de Physiol. (5), 10.

FAT AND PHOSPHATIDES. 363

ferruginous pigment soluble in water, ferrine, and a pigment soluble in chloro-
form 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 l). 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 content.
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, BoTTAZZi)2 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 3 observed the
opposite condition on feeding dogs with mutton-fat.

The deductions as to the quantity of glycerides with stronger unsaturated
acids than oleic acid in liver fat are to be accepted with caution, as these acids
originate from contaminated phosphatides.

Phosphatides, which were formerly designated lecithin, and whose
quantity is generally calculated as such, also belong to the normal constit-
uents of the liver. The quantity (as lecithin) amounts to over 23.5 p. m.
according to NoEL-PATON.4 In starvation the lecithin, according to NOEL-

1 Xoel-Paton, Journ. of Physiol., 19; Leo, Zeitschr. f. physiol. Chem., 9; Lebedeff
Pfliiger's Arch., 31; Athanasiu, Pfliigers 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. Wochens^hr.,
1904; Schwalbe, Centralbl. f. Physiol.', 18, p. 319.

2 Arch. Ital. d. Biol., 48 (1908), cited in Bioch. Centralbl., 7, p. 833.

3 Cited by Lummert, Pfliiger's Arch., 71. In regard to the liver-fat of children, see
Thiemich, Zeitschr. f. physiol. Chem., 26.

4 1. c. See also Heffter, Arch. f. exp. Path. u. Pharm., 28.

364 THE LIVER.

PATON, forms the greater part of the ethereal extract, while with
food rich in fat, on the contrary, it forms the smaller part. The phos-
ghatides are undoubtedly of various kinds, but they have not been closely
studied. Among others, we have lecithin and the so-called jecorin.1
Cholesterin is also a constituent of the liver, although only in small
quantities.

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 dextrose as osazone in the carbohydrate complex of Jecorin.

The statement by BING that Jecorin is a combination of lecithin and dextrose
does not follow from the analyses of Jecorin thus far known. Jecorin contains sul-
phur, 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 recent
investigations of BASKOFF the liver Jecorin, prepared according to DRECHSEL'S
suggestion, and when it is so pure that it is completely soluble in ether, and quan-
titatively precipitated by alcohol, from this solution is a rather constant compound
at least in regard to the N, P and dextrose content. BASKOFF found as average
2.55 per cent N, 2.87 per cent P, and about 14 per cent dextrose. The relation
P : N was nearly 1 : 2 and therefore Jecorin is correspondingly a diamidomono-
phosphatide.

The variable composition and divergent properties of the Jecorin isolated and
analyzed by various investigators 2 depends, according to BASKOFF, upon imper-
fect 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.

Another phosphatide, which does not reduce directly or after boiling with acid,
has been called heparphosphatide by BASKOFF. In certain regards 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 3 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, leucine, and cystine. In pathological cases inosite and amino-acids
have been detected. The occurrence of bile-coloring matters in the liver-

1 See Baskoff, Zeitschr. f. physiol. Chem., 57.

2 Drechsel, Ber. d. sachs. Gesellsch. d. Wissensch., 1886, p. 44, and Zeitsch. L Biol-
ogic, 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;
Meinertz, Zeitschr. f. physiol. Chem., 46; Siegfried and Mark, ibid.', Paul Mayer,
Bioch. Zeitschr., 1, and Baskoff, Zeitschr. f. physiol. Chem., 57.

3 Zeitschr. f. physiol. Chem., 8.

ENZYMES OF THE LIVER. 365

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, the glycolytic enzymes, which will be spoken of later, the
enzymes taking part in the formation of uric acid and destruction of uric
acid (Chapter XV), the arginase, which forms urea, and the diastase
acting upon glycogen, the ester-splitting Upases and the proteolytic
enzymes.1

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
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. LoEWY2), it is desirable
that a greater number of observations be made on 'this subject. The
increased consumption of glycogen under the above-mentioned patholog-
ical conditions 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 3 to be considered only as a more pronounced
appearance of the fat previously occurring in the organ.

Besides the above-mentioned organic constituents in the liver we must
mention the glucothionic acid found by MANDEL and LEVENE, whose
chemical individuality is doubted, as well as the nitrogenous carbohy-
drate found in the liver by SEEGEN and NEIMANN which also requires
further investigation, and whose occurrence in the liver could not be
substantiated by TtJRKEL.4

1 Granstrom, Hofmeister's Beitrage, 11, claims to have found in the liver an enzyme
which he calls glyoxylase, which destroys glyoxylic acid.

2 Neuberg and Richter, Deutsch. med. Wochenschr., 1904; Wakeman, Zeitschr. f.
physiol. Chem., 44; Wells, Journ. of Exper. Med., 9; Glikin and Loewy, Bioch. Zeit-
schr., 10.

3 Hofmeister's Beitrage, 10.

4 Mandel and Levene, Zeitschr. f. physiol. Chem., 45; Seegen, Centralbl. f . Physiol.,

366 THE LIVER.

The mineral bodies of the liver consist of phosphoric acid, potassium,
sodium, 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. BUNGE 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. Calculated
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 GUILLEMONAT 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 more, and in
women less, than 0.20 p. m. (calculated on the fresh moist organ). Above
0.5 p. m. is considered as pathological. According to BIELFELD,! who
also finds a greater iron content in men, this difference appears only after
the first 20-25 years. At this age (20-25 years) the iron content is
smallest.

The quantity of iron in the liver can be increased by drugs containing
iron, as also by inorganic iron salts, and the largest deposition of iron
was observed by Novi2 after the hypodermic injection of iron. The
quantity of iron may also be increased by an abundant destruction of
red blood-corpuscles, which will result from the injection 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.s A destruction of blood-pigments, with a splitting
oft 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
FLORESCO4 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,

12 and 13, with Neimann, Wiener Sitzungsber. Math. Klasse, 112; Turkel, Hofmeister's
Beitrage, 9.

1 Bunge, Zeitschr. f. physiol. Chem., 17, 78; Guillemonat and Lapicque, Compt.
rend, de soc, biol., 48, and Arch, de Physiol. (5), 8; Bielfeld, Hofmeister's Beitrage,
2; see also Schmey, Zeitschr. f. physiol. Chem., 39; Scaffidi, ibid., 54.

2 Se§ Centralbl. d. Physiol., 16, 393.

3 See Lapicque, Compt. rend., 124, and Schurig, Arch. f. exp. Path. u. Pharm., 41.

4 Arch, de Physiol. (5), 10.

MINERAL BODIES OF THE LIVER. 367

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-fostus 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 l 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.

Z ALE£Kl) .

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
about the same as in the human liver. The amount of magnesium
oxide was remarkably high, namely, 0.168, 0.198, and 0.158 p. m., in
the livers of the horse, ox, and pig respectively. KRUGER 2 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 foetus are antagonistic; that is, an increase in the
quantity of calcium in the liver causes a diminution 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 (HENZE3). Foreign metals, such as lead, zinc, arsenic,
and others (also iron), are easily taken up and combined by the liver
(SLOWTZOFF, v. ZEYNEK, and others 4) .

v. BIBRA 5 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.

PROFITLICH 6 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).

1 St. Zaleski, I.e.; Kriiger and collaborators, Zeitschr. f. Biologie, 27; Lapicque,
Maly's Jahresber., 20.

2 Zeitschr. f. Biologic, 31; Toyonaga, Bull, of the College of Agriculture, Tokio, 6.

3 Zeitschr. f. physiol. Chem., 33.

1 Slowtzoff, Hofmeister's Beitrage, 1; v. Zeynek, see Centralbl. f. Physiol., 15.

5 See v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., p. 711.

6 Pfliiger's Arch., 119.

368 THE LIVER.

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 l 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. It is also difficult to decide as to
how far in these experiments we are dealing with an increase in the
number or the size of the liver-cells or with a deposition of reserve
protein in the same sense as of glycogen or excessive fat.

There is a unanimous belief that the liver is an especially important
storage-organ for glycogen.

Glycogen and its Formation.

Glycogen was first discovered by BERNARD. It is a carbohydrate
closely related to the starches or dextrinaf with the general formula
m(C6H10O5). Its molecular weight is unknown, but seems to be very
large (GATIN-GRUZEWSKA and v. KNAFFL-LENZ 2) . The largest quan-
tities are found in the liver -of adult anima^ 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,
(see also MENDEL and LEAVEN WORTH 3), 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

1 Pfluger's Arch., 111.

2 Gatin-Gruzewska, Pfluger's Arch., 103; v. Knaffl-Lenz, Zeitschr. f. physiol. Chem.,
46.

3 Amer. Journ. of Physiol., 20.

GLYCOGEN. 369

(Bizio), Tsenia and Ascaridse (WEiNLAND1), 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 PFLUGER,2 it never entirely
disappears in starvation, as a reformation 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 SCHOXDORFF 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 3 in the frog. The
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. (BoTTAZzi4 ) . 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
introduction 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 tlie1 liver-glycogen. KULZ, ZUNTZ and VOGELIUS,
FREXTZEL, 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. 5 the liver and muscles in rabbits can be made glycogen-

1 Zeitschr. f. Biologic, 41. The extensive literature on glycogen may be found in
E. Pfiiiger, 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
M'orks.

2 Pfluger's Arch., 119, which contains the literature.

3 Pfluger's Arch., 121.

4 Arch. Ital. d. Biol., 48; cited in Bioch. Centralbl., 7, 833.

5 Compt. rend., 142.

370 THE LIVER.

free after 36-40 hours by first starving one day and then injecting
adrenalin.

Glycogen forms an amorphous, white, tasteless, and inodorous 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). Its aqueous solution is dextrorotatory, and HUPPERT
found it to be (a)D= + 196.63°. GATIN-GRUZEWSKA has recently obtained
the same result by using a perfectly pure solution of glycogen.1 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 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
magnesium or ammonium sulphate. slt is not precipitated by sodium
chloride, or by half saturation with ammonium sulphate (NASSE, NEU-
MEISTER, HALLIBURTON, YOUNG 2). On boiling with dilute caustic
potash (1-2 per cent) the glycogen 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 dextrose, depending upon the nature
of the enzyme. It is transformed into dextrose by dilute mineral acids.
According to TEBB 3 various dextrins appear as intermediary steps in
the saccharification of glycogen, depending on whether the hydrolysis
is caused by mineral acids or enzymes. The question whether the gly-
cogen from various animals and different organs is the same in this regard
has not been sufficiently investigated. Nor has it been decided whether

1 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.

2 Young, Journ. of Physiol., 22, citing the other investigators.

3 Journ. of Physiol., 22.

GLYCOGEN. 371

all the glycogen in the liver occurs as such or whether it is in part
combined with protein (PFLUGER-NERKING). The investigations of
LOESCHCKE l 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 purification,
as suggested by BRUCKE, it is better, for quantitative determinations of
glycogen, to precipitate it directly from the alkaline solution by alcohol

(PFLUGER2).

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 cent KOH;
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 3
and BANG.4

Numerous investigators have endeavored to determine the origin
of glycogen in the animal body. It is positively established by the
unanimous observations of many investigators 5 that the varieties of
sugars and their anhydrides, dextrins and starches, have the property of

iger's Arch., 102.

also the method suggested by Gautier, Compt. rend., 129.
Pfliiger's Arch., 103, 104, 121.

4 Hammarsten's Festschr., 1906.

5 In reference to the literature on this subject, see E. Kiilz, Pfluger's Arch., 24,
and Luclwig, Festschrift, 1891 ; also the cited works of Pfliiger and Cremer, foot-note
1, p. 3C9.

372 THE LIVER.

increasing the quantity of glycogen in the body. The action of inulin
seems to be somewhat uncertain.1 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
NEUBERG and WOHLGEMUTH 2 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. VoiT3 and his pupils have shown that dextrose has a
more powerful action than cane-sugar, while milk-sugar is less active
(in rabbits and hens) than dextrose, levulose, 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, according 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, glycocoll, and asparagine may similarly r
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, pro-
duce an increase in the glycogen of the liver. This action of the anti-
pyretics (especially antipyrine) had been shown by LEPINE and PoRTERET.4

PFLUGER has conclusively shown that we have no positive proof
as to the action of these various bodies as glycogen-formers. That
glycerin may in a positive sense influence the amount of glycogen in the
liver is not to be doubted from the experiments of WEISS and LUCHSINGER
on glycogen formation, which will be mentioned in connection with the
experiments on the relation of glycerin to the formation of sugar.

The fats, according to BOUCHARD and DESGREZ, increase the glycogen
content of the muscles but not of the liver, while COUVREUR 5 believes that
the glycogen is increased at the expense of the fat in the silkworm larva

1 See Miura, Zeitschr. f . Biologic, 32, and Nakaseko, Amer. Journ. of Physiol., 4.

2 Salkowski, Zeitschr. f. physiol. Chem., 32; Neuberg and Wohlgemuth, ibid.
See also Pniiger, 1. c., and Cremer, 1. c.

3 Zeitschr. f . Biologic, 28.

4R6hmann, Pfluger's Arch., 39; Nebelthau, Zeitschr. f. Biologic, 28; Lupine and
Porteret, Compt. rend., 107.

5 Bouchard et Desgrez, Compt. rend., 130; Couvreur, Compt. rend, de soc. biol., 47.

FORMATION OF GLYCOGEN. 373

ao it changes into a chrysalis. 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. PFLUGER explains this by the fact that the
extent of fat metabolism is not dependent upon the quantity of fat
supplied, but upon the amount of fat required by work. If more
fat is supplied, then it is not destroyed, but is stored up. Even
if sugar is continuously formed from the fat, in metabolism this is
immediately burned and does not yield any material for the formation
of the reserve substance glycogen.

Opinions in regard to the influence of the proteins are somewhat
contradictory. From several investigations the conclusion has been
drawn that the proteins cause an increase in the glycogen of the liver.
Among these investigations must be included certain feeding experi-
ments writh boiled beef (NAUNYN) or blood-fibrin (v. MERING), and espe-
cially the very careful experiments made by E. KULZ on hens, with
pure proteins, such as casein, ser albumin, and ovalbumin. The value
of these experiments is disputed by PFLUGER, and as a direct proof against
the formation of glycogen from protein he refers to SCHONDORFF'S inves-
tigations when feeding carbohydrate-free protein (casein) to frogs without
finding the least increase in the total glycogen. Later BLUMENTHAL
and WOHLGEMTJTH arrived at similar results. They found no glycogen
accumulation in frogs after feeding with casein or gelatin, but did find
it after feeding with ovalbumin, which contains a carbohydrate group.
On the contrary, BENDIX was able to show an increase in the glycogen
in dogs by feeding casein and gelatin, as well as ovalbumin, and in fact
a greater increase by casein than by ovalbumin. STOOKEY l arrived at
similar results in hens, as he found a glycogen formation after- feeding
casein, while he obtained no positive results after feeding glucoproteids.
It seems as if the conditions in cold-blooded animals were different from
those in warm-blooded ones. According to PFLUGER, the experiments
of BENDIX are not conclusive, and he doubts the formation of glycogen
from protein.

Many investigators are still of the opinion that an increase in the
glycogen of the liver as well as of other organs can be' brought about by
feeding animals with carbohydrate-free proteins. The circumstance
that, as shown by PFLUGER,2 the glycogen by long-continued starvation
entirely disappear from the body but is being reformed, and that

1 Schondorff, Pfluger'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.

9 Pfliiger's Arch., 119.

374 THE LIVER.

frogs which had starved 13 months still contained remarkably large
amounts of glycogen due to this reformation (PFLUGER), makes the
formation of glycogen from protein very probable.

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
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 glycogen
has given rise to the opinion that the glycogen in the liver is produced
from sugar by a synthesis in which water separates with the formation
of an anhydride (LUCHSINGER and others). This theory (anhydride
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.1 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 2) .

In opposition to this theory C. and E. VOIT and their pupils have
shown that the carbohydrates are " true " glycogen-formers. Aftei
taking of large quantities of carbohydrates, the amount of glycogen'
up in the body is sometimes so great that it cannot be covered

1 See Pfliiger in his Arch., 121.

2 In regard to these two theories, see especially Wolffberg, Zeitschr. f . Biologic, 16.

FORMATION OF GLYCOGEN. 375

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 gly cog en-formers. For the present, only
dextrose, levulose, galactose (WEINLAND *), and perhaps also d-mannose
(CREMER) are designated as true glycogen-formers. Other mono-
saccharides may indeed, according to CREMER influence the formation
of glycogen, but they are not converted into glycogen, and hence are
called only pseudogly cog en-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
be inverted in the intestine. These two varieties -of sugar, therefore,
cannot, like dextrose and levulose, 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. VoiT2).

The ability of the liver to form glycogen from monosaccharides has
also recently been shown by K. GRUBE 3 in a very interesting and direct
manner, by perfusion experiments with solutions of various carbohydrates.
In these perfusion experiments on tortoise livers, dextrose produced an
abundant glycogen formation, while with levulose and galactose it was
less abundant. Pentoses, disaccharides, casein and amino-acids (glycocoll,
alanine and leucine) were inactive while on the contrary glycerin and
also formaldehyde acted as glycogen-formers.

After PAVY4 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 III), 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

1 E. Voit, Zeitschr. f. Biologie, 25, 543, and C. Voit, ibid., 28. See also Kausch
and'So.cin, Arch. f. exp. Path. u. Pharm., 31; Weinland, Zeitschr. f. Biologie, 40 and
38; Cremer, ibid., 42, and Ergebnisse der Physiol., 1.

"••2 Dastre, Arch, de Physiol. (5), 3, 1891; Dastre and Bourquelot, Compt. rend., 98;
Fritz Voit, Verhandl. d. Gesellsch. f. Morph. u. Physiol. in Munchen, 1896, and Deutsch.
Arch. f. klin. Med., 58.

3 Pfluger's Arch., 118, 121, 122 and 126.

4 The Physiology of the Carbohydrates, London, 1894,

376 THE LIVER.

can be drawn from this on the behavior of the carbohydrate groups,
which exist not as free groups but combined with 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/ have yielded no proofs for
the theory that glycogen is formed from glucosamine.

Whether or not, or to what extent, the glucoproteins take part in
the sugar or glycogen formation in the animal 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 meagre.

If the proteins are to be counted among those bodies which can
Increase the glycogen of the body, then we must ask the question: Do
the proteins act only indirectly as pseudoglyeogen-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 f at, 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? 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 positiveness, and the
investigations on this subject by different experimenters have given
varying results. The experiments of KiJLZ,2 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 dextrose seem to indicate a glycogen
formation from sugar in the muscles. The investigations of DE FILIPPI 3
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

1 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.

2 See Minkowski and Laves, Arch. f. exp. Path. u. Pharm., 23; Kiilz, Zeitschr. f..
Biologic, 27; Hatcher and Wolff, Journ. of Biol. Chem., 3.

3 Ze'tschr. f. Biol., 49 and 50.

FORMATION OF SUGAR. 377

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
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 ARTHUS and
HUBER, PAVY, PICK and BiAL2 proved, by a diastatic enzyme which,
according to ROHMANN and BORCHARDT,S is identical with a diastatic
enzyme of the blood.

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, HITTER, SCHIFF, EULENBERG, LUSSANA, MOSSE, N. ZUNTZ
and others,4 and in regard to the sugar content in the two kinds of

1 See Dastre, Compt. rend, de soc. biol., 47, 280, and Kaufmann, ibid., 316.

2 Arthus and Huber, Arch, de Physiol. (5), 4, 659; Pavy, Journal of Physiol., 22;
Pick, Hofmeister's Beitr., 3; Bial. Arch. f. (Anat. u.) Physiol., 1901.

3Rohmann, Verh. d. Ges. deutsch. Naturf. u. Aerzte, Breslau, 1903; Borchardt,
Pfliiger's Arch., 100.

4 In regard to the literature on sugar formation in the liver see Bernard, Legons sur

378 THE LIVER.

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.1

The circumstance that the blood-sugar rapidly sinks to £-J of its
original quantity, or even disappears when the liver is cut out of the cir-
culation, indicates a vital formation of sugar in the liver (SEEGEN. BOCK
and HOFFMANN, KAUFMANN, TANGL and HARLEY, PAVY). In geese
whose 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
quantity of sugar in the blood is not increased (ScHENCK2). An
important proof of the possibility of a vital formation of sugar from
the liver glycogen lies in the fact that we shall learn below of certain
poisons and operative changes which may cause an abundant elimina-
tion 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 3 and others explain it by a special activity of the protoplasm.

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 import-
ant 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 dextrose 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 dextrose. When the quantity of
sugar in the blood rises to 3 p. m. or above, then sugar passes into the
urine, but not always.4 The kidneys have the property to a certain

le diabete, Paris, 1877; Seegen, Die Zuckerbildung im Tierkorper, 2. Aufl., Berlin,
1900; M. Bial, Pfliiger's Arch., 55, 434.

1 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.

2 Seegen, Bock, and Hoffmann, see Seegen, I.e.; Kaufmann, Arch.de Physiol. (5),
8; Tangl and Harley, Pfluger's Arch., 61; Pavy, Journ. of Physiol., 29., Minkowski,
Arch. f. exp. Path. u. Pharm., 21; Schenck, Pfluger's Arch., 57.

3 McGuigan and Brooks, Amer. Journ. of Physiol., 18. In regard to the literature
see Pick, Hofmeister's Beitrage, 3, 182.

4 See Mohr, Zeitschr. f . exp. Path. u. Therap., 4.

PHLORHIZIN DIABETES. 379

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, 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 car-
bohydrates, 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
(MIXKOWSKI), but this is disputed by PAVY. This last investigator
found, although only to a slight degree, that the sugar in the blood was
increased, but he holds the same view that v. MERIXG does, that phlor-
hizin diabetes is a kidney diabetes. 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 arid more abundantly
than the urine from the other kidney (ZUNTZ), tends to favor this
view. The experiments especially performed by PAVY, BRODIE, and
SIAU l upon blood containing phlorhizin and surviving kidneys also
indicate the same, namely, that the phlorhizin acts upon the kidneys.
While v. MERING believes in an increased permeability of the kidneys
for sugar, produced by the phlorhizin, PAVY is, on the contrary, of the
opinion that the kidneys, under the influence of the phlorhizin, split
off sugar from a substance circulating in the blood, perhaps from a pro-
tein with loosely combined carbohydrate groups.

Another form of glycosuria which seems to be connected with a
changed permeability of the kidneys is the glycosuria first observed by
BOCK and HOFFMANN 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, it can be again arrested by an injection of

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 Ritter,
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, and the
monographs upon diabetes.

380 THE LIVER.

a salt solution containing CaCl2. According to the investigations of
UNDERBILL and CLOSSON 1 on rabbits, the injection of salt into the carotid
artery brings about a hyperglycsemia by a disturbance in respiration;
the injection of the salt solution into the ear vein causes on the contrary
a glycosuria with polyuria and a hypoglycaimia; and they account for
the salt-glycosuria produced in this manner by pointing to an increased
permeability of the kidneys.

With the exception of these two forms of glycosuria, the phlorhizin
diabetes and the salt-glycosuria; and also the glycosuria produced by
uranium salts, all other forms of glycosuria or diabetes, as far as known
at present, depend on a hyperglyccemia.

A hyperglycsemia 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 IX, on absorption) is overreached, then the excess of absorbed
sugar passes into the urine. This form of glycosuria is called alimentary
ylycosuria,2 and it 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
dextrose), 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
diabetes, where the sugar disappears from the urine when the carbohy-
drates are cut off as much as possible for the food, to this class of gly-
cosuria.

A hyperglycaBmia 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 (glycogen) occurs.
The so-called piqure of BERNARD, and probably also those glycosurias

1 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.

2 In regard to alimentary glycosuria see Moritz, Arch. f. klin. Med., 46, which also
contains the earlier literature; B. Rosenberg, Ueber das Vorkommen der alimentiiren
Glykosurie, etc. (Inaug.-Dissert. Berlin, 1897); van Oondt, Munch, med. Wochen-
schr., 1898; v. Noorden, Die Zuckerkrankl.eit, 3. AufL, 1901.

GLYCOSURIAS. 331

which occur after other lesions of the nervous system, belong to tha
above group of glycosurias. The glycosuria produced on poisoning
with carbon monoxide, ether, chloroform, curare, strychnine, morphine,
piperidine, etc., also belongs to this group. How these glycosuriab
are brought about is not known with certainty, and the conditions
become complicated because in most cases the appearance of the gly-
cosuria is connected with an insufficient supply of oxygen. UNDERBILL
has shown for the piperidine-glycosuria and PENZOLDT and FLEISCHER
and SAUER : for the curare-glycosuria that one can prevent the appear-
ance of the glycosuria (on poisoning with piperidine) and the hyper-
glycsemia as well by supplying oxygen. MACLEOD 2 has also shown that
the irritation of the central end of the cut vagus, or irritation of the spinal
marrow, produces glycosuria and hyperglycsemia only with simultaneous
dyspnoea; with sufficient oxygen supply the glycosuria remains absent.
This does not conflict with the ordinary assumption that in this group
of glycosurias an increased formation of sugar occurs from the gly-
cogen. The investigations of BANG, LJUNGDAHL and BOHM;S in which
the extent of enzymotic decomposition of glycogen in the liver was
determined, also indicate positively that in the piqure as well as in the
asphyxia of strychnine and morphine poisoning an increased decomposi-
tion of glycogen takes place.

The material from which- the sugar is formed is glycogen in most
cases. That the glycosuria produced after piqure is due to an increased
transformation of the glycogen 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.
In other cases, as in carbon-monoxide poisoning, the origin of the sugar
is less clear. In the last-mentioned case a sugar formation from pro-
teins has indeed been accepted, as this glyqosuria appears only in
those cases when the poisoned animal has a sufficient quantity of protein
at its disposal (STRAUB and ROSENSTEIN 4) . Protein starvation with a
simultaneously abundant supply of carbohydrates causes this glyco-
suria to disappear. In the glycosuria produced by irritation of the
vagus, which as above remarked, according to MACLEOD only appears
with insufficient supply of oxygen, the hyperglycamia (in rabbits)

1 Underbill, Journ. of biol. Chem., 1; Penzoldt and Fleischer, Virchow's Arch., 87;
Sauer, Pfluger's Arch., 49, 425, 426.

2 Macleod, Amer. Journ. of Physiol., 19, with Briggs, Cleveland Med. Journ., 1907.

3 Hofmeister's Beitrage, 9 and 10.

* See Dock, Pfluger's Arch., 5; Bock and Hoffmann, Exp. Studien tiber Diabetes
(Berlin, 1874); Cl. Bernard, Legons sur le diabete (Paris); T. Araki, Zeitschr. f. physiol.
Chem., 15, 351; Straub, Arch. f. exp. Path. u. Pharm., 38; Rosenstein, ibid., 40;
Pfluger, Pfliiger's Arch., 96.

382 THE LIVER.

depends, according to BANG l and collaborators, upon an increased
decomposition of the glycogen of the muscles and not of the liver.

A hyperglycsemia and glycosuria may also be caused by a decreased
ability of the animal to consume or to utilize the sugar or to trans-
form 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.2 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, d-saccharic acid, glucosamine,
mucic acid, and others, and he found that diabetics destroyed or burnt
these bodies to the same extent as healthy individuals. Besides this
it must be remarked that the two varieties of sugar, dextrose and levulose,
which are oxidized with the same readiness, act differently in diabetics.
According to KULZ and other investigators levulose is, contrary to
dextrose, utilized to a great extent in the organism, and may, according
to MiNKOWSKi,3 even cause a deposit of glycogen in the liver in animals
with pancreas diabetes (see below). 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 dextrose suffers diminution, and the explanation of this
has been sought in the fact that the dextrose is not previously split before
combustion.

The variation in the respiratory quotient, i.e., the relation -~, seems

to show an insufficiency of the dextrose combustion in the tissues in
diabetes. As will be thoroughly explained in a following chapter, this
quotient is greater the more carbohydrates are burnt in the body, and
it is correspondingly smaller when protein and fat are chiefly burnt.
The investigations of LEO, HANRIOT, WEINTRAUD and LAVES,4 and

1 Bang, Ljungdahl and Bohm, Hofmeister's Beitrage, 10.

2 Schultzen, 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.

3 Kiilz, Beitrage zur Path. u. Therap. des Diabetes mellitus (Marburg, 1874), 1;
Weintraud and Laves, Zeitschr. f. physiol. Chem., 19; Haycraft, ibid.', Minkowski,
Arch. f. exp. Path. u. Phann., 31.

4 See. v Noorden, Die Zuckerkrankheit, 3. Aufl., 1901.

PANCREAS DIABETES. 383

others have shown that in severe cases of diabetes, in the starving con-
dition, the low quotient is not raised after partaking of dextrose, as in
healthy individuals, but that it is raised after feeding levulose, which is
also of value to diabetics (WEINTRAUD and LAVES). The poverty of the
organs and tissues of diabetics in glycogen indicates that it is perhaps
not a diminished combustion of the dextrose which is essential, but
more likely an inability of the body to transform the dextrose into
giycogen or to utilize it at all.

The relation of the pancreas to diabetic glycosuria is of the greatest
importance for its proper understanding.

The investigations of MINKOWSKI, v. MERING, DOMINICIS, and later
of many other investigators/ 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.2 It is generally accepted that in pancreas diabetes
a diminished consumption exists, i.e., diminished utilization, which
does not exclude an increased sugar formation. There are also certain
investigators who explain this form of diabetes as not entirely due to a
diminished combustion of sugar, but to a pathological increase in the
sugar formation.

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
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.

1 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, Legons sur le diabete (Paris); Seegen, Die Zuckerbildung im Thierkorper
(Berlin, 1890), and Pfluger, Des Glykogen, 2. Aufl., 1905, and especially v. Noorden's
Hamb. d. Pathol. des Stoffwechsels, 2. Aufl., 1907, Bd. 2, Chapter I.

2 See Falta " Ueber den Eiweissumsatz beim Diabetes mellitus." Berl. klin. Woch-
enschr., 1908, and Zeitschr. f. klin. Med., <>6.

384 THE LIVER.

It seems as if not only does the liver stand in close relation to pan-
creas diabetes, but to other organs also. PFLUGER found that in
frogs, after total extirpation of the duodenum, a strong and continuous
glycosuria is the result, and HERLITZKA found the same after poison-
ing the central nerves of the duodenum of the frog by means of nicotine.
According to PFLUGER we can explain this glycosuria by the assumption
that the pancreas has an antidiabetic action which is influenced by the
nerve centers of the intestine. This relation of the duodenum to pan-
creas diabetes has not been generally admitted, and in warm-blooded
animals (at least in dogs) the occurrence of a duodenal diabetes has not
been shown. Still REALE and DE RENZI observed a glycosuria in
dogs after duodenal resection, but others have not been able to confirm
this, or at least only in part. PFLUGER, on carefully repeating REAL'S
and DE RENZI' s experiment, observed only a very slight or no glycosuria
at all, and previously EHRMANN, ROSENBERG and MINKOWSKI, in an
especially convincing manner, after the total extirpation of the duodenum
in dogs, obtained completely negative results. The positive results of
REAL and DE RENZI are explained by PFLUGER 1 by the healing up of
the intestinal tube and the disturbance produced in its neighborhood.
No positive conclusion can be drawn from the glycosuria observed by
GAULTIER and by ZAK after corrosion of the duodenal mucous membrane,
as a glycosuria can also be produced by the corrosion of other parts of
the intestine (EICHLER and SILBERGLEIT 2) . The occurrence of a duo-
denal glycosuria in dogs has thus far not been proven.

There seems, on the contrary, to exist a relation between pancreas
diabetes and the function of the adrenals. As first shown by BLUM,
adrenalin produces a strong glycosuria which apparently brings about
an increase in the destruction of glycogen with hyperglycsemia by an
abundant " mobilization of the carbohydrates." This glycosuric action
of adrenalin could be prevented by ZUELZER by the injection of pan-
creas extracts, and this statement is confirmed by FRUGONI by exper-
iments with pancreatic juice. Further proof of the relation of the
adrenals to the pancreas has been given by EPPINGER, FALTA and RUDIN-
GER.3 According to the last-mentioned investigators there is evidence
of a certain relation existing in pancreas diabetes between the pancreas,
adrenals and thyroids. According to them a mutual retarding action
exists between the pancreas and the thyroids as well as between the

1 Pfluger in his Archives, 118, 119, 122, 124; Minkowski, Arch. f. exp. Path! u.
Pharm., 58. The other works cited are found in the above literature.

2 Gaultier, Compt. rend. soc. biol., 64; Zak, Wien. klin. Wochenschr., 21; Eichler
and Silbergleit, Berlin, klin. Wochenschr., 1908.

3 Frugoni, Berl. klin. Wochenschr., 1908; Eppinger, Falta and Rudinger, Zeitschr^
f. klin. Med.. 66, which also contains the literature on adrenalin diabetes.

PANCREAS DIABETES. 385

pancreas and the adrenals, while between the thyroids and the adrenals
a mutual accelerating action exists. In depancreatized dogs the retard-
ing action of the pancreas upon the thyroids is removed, and in this
way we explain the strong increase in the protein, fat (Menu) and salt-
metabolism (FALTA and WHITNEY J) observed in pancreas diabetes. By
the removal of the retarding action of the pancreas upon the adrenals,
the mobilization 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-men-
tioned authors, but we cannot enter more into detail in regard to
the interesting question, which requires further study. Nevertheless
we must mention that according to PICK and PINELES 2 the extirpation
of the thyroid glands in young goats, but not in rabbits, prevents
the appearance of adrenalin-glycosuria. The negative results with
rabbits probably depend upon the fact that in rabbits the parathyroids
remain completely intact. R. HIRSCH has observed in dogs that com-
plete thyroidectomy, but not the removal of the chief thyroid glands
alone, itself brings about an alimentary glycosuria.

The conditions in pancreas diabetes are certainly very complicated,
and the reasons for this are still very dark. Most investigators are of
the view 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 others3
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. PFLUGER has made important objections to the value of the
results of these experiments, and on the other hand ZUELZER, DOHRN and

1 Mohr, Zeitschr. f. exp. Path. u. Therap., 4; Falta and Whitney, Hofmeister's
Beitrage, 11.

2 Pick and Pineles, Bioch. Zeitschr., 12; R. Hirsch, Zeitschr. f. exp. Path. u.
Therap., 5.

3 See Mmkowski, Arch. f. exp. Path. u. Pharm., 31; He"don, Diabete pancre"atique,
Travaux de Physiologic (Laboratoire de Montpellier, 1898), and the works on diabetes.

386 THE LIVER.

MARXER l have recently in an important work given the theory of internal
secretion a strong support, if their work is substantiated. These investi-
gators have not only given further proof of the antagonism between
the adrenals and the pancreas, but they have obtained a preparation
from the pancreas, in a manner not described in detail, which causes
in dogs as well as in man a diminution in the elimination of sugar (and
acetone bodies) in diabetes, and an improvement in the general condition.

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 LEPINE 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 (STOKLASA) considers
the glycolysis as analogous to alcoholic fermentation, where we have
processes brought on by special tissue zymases.

The prevailing opinions hold that (Chapter IV) alcoholic fermenta-
tion takes place in two steps. In the first step lactic acid is produced
from the sugar and in the second the lactic acid splits into carbon
dioxide and alcohol. According to STOKLASA 2 and his collaborators
a decomposition of sugar occurs in the animal tissues in a similar
manner by the analogous action of enzymes. Many objections have
been advanced from various quarters against these investigations, which
seem to indicate that in these cases the experimenters were dealing with
the action of micro-organisms.3 According to HAMMARSTEN the claims
of STOKLASA and his collaborators are not disproved, and we cannot
dispute the possibility that in the animal tissues as well as in the
plant 4 tissues in anaerobic respiration, an alcoholic fermentation may

1 Deutsch. med. Wochenschr., 1908.

2 Hofmeister's Beitrage, 3, Centralbl. f. Physiol., 16, 17, 18; Ber. d. d. chem.
Gesellsch., 38; also with Czerny, ibid., 36; with Jelinek, Simacgk and Vitek, Pfluger's
Arch., 101.

3 See the works of O. Cohnheim, Zeitschr. f. physiol. Chem., 39, 42, 43; Batelli,
Compt. rend., 137; Portier, Compt. rend. soc. biol., 57.

4 See Palladin, Zeitschr. f. physiol. Chem., 55 and 56.

GLYCOLYSIS. 387

occur. Recently VAHLEN 1 found a substance in the pancreas which
accelerates catalytically the alcoholic fermentation of sugar by yeast.

That lactic acid can be an intermediary step in the destruction of
sugar in the animal body follows from the several circumstances as to
the origin of lactic acid, which will be mentioned in a subsequent chap-
ter (XI, Muscle,) and also the observation of A. R. MANDEL and LUSK 2
on the relation of lactic acid to diabetes. These experimenters showed
after phosphorus poisoning in dogs, that the blood and urine con-
tained abundance of lactic acid, and on producing phlorhizin-diabetes
it disappeared from these fluids, and also that phosphorus poisoning
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 are not agreed 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 found that a cell-free fluid can be
obtained from a mixture of pancreas and muscle, which destroys dextrose,
while the pancreas alone does not have this action, and the muscle only
to a slight extent. The pancreas does not contain, according to COHN-
HEIM, a glycolytic enzyme, but a substance resistant to boiling tem-
peratures, 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. DE MEYER 3 holds an almost similar view, but
with this exception, that he does not consider the activating substance
coming from the muscles, but from the leucocytes. This proenzyme is
activated by the internal secretion of the pancreas.

The findings of COHNHEIM have not been fully confirmed by other
investigators.4 Certain of these investigators come to more or less sim-
ilar conclusions while, on the contrary, others cannot substantiate his
deductions at all, and for the present our knowledge of the mode of
action of the pancreas in the sugar metabolism in the animal body is very
meager and incomplete.

1 Zeitschr. f. physiol. Chem., 59.

2 Amer. Journ. of Physiol., 16.

3 Cohnheim, Zeitschr. f. physiol. Chem., 39, 42, 43, and 47; De Meyer, Arch, intern.,
de Physiol., 2, cited from Biochem. Centralbl., 3, and Centralbl. f. Physiol., 20.

4 Stocklasa and collaborators, Centralbl. f. Physiol., 17, and Ber. d. d. chem..
Gesellsch., 36 and 38; Feinschmidt, Hofmeister's Beitrage, 4; Hirsch, ibid.; Glaus and:
Embden, ibid., 6; Arnheim and Rosenbaum, Zeitschr. f. physiol. Chem., 40; Braun-
stein, Zeitschr. f. klin. Med., 51.

388 THE LIVER.

Where does the sugar eliminated in diabetes originate? Does it
depend entirely upon the carbohydrates of the food or the store of car-
bohydrate 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 pancreas
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 PFLiJGER,1 with the
results that the power of the animal body to produce sugar from non-
carbohydrate material is now definitely proven.

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.2
The actual relation between dextrose 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 formed,
as well as the origin of the sugar, but according to the views of HAMMARS-
TEN such conclusions are mostly very uncertain. The sugar eliminated
by the urine represents the difference between the total sugar produc-
tion 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 quantity of sugar produced:
it is not known how far this supposition can be applied in the various
forms of diabetes. Still several observations seem to show that in tne
different forms of diabetes variable amounts of the sugar are burned,

je, Deutsch. Arch. f. klin. Med., 79, and Pfliiger's Arch., 106; Pfliiger,
Pfliiger's Arch., 108.

2 See Falta, Zeitschr. f. klin. Med., 65.

RELATION OF PROTEINS TO CARBOHYDRATE METABOLISM. 389

and only in special cases can we draw approximately accurate conclu-
sions. When, for example, the quotient in a case was especially high,
we could conclude that sugar was formed from fat ; if no nitrogen reten-
tion existed, with a carbohydrate-free diet.

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 by 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, whose 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 123.6 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-
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
protein than from fat.

1 Deutsch. Arch. f. klin. Med., 79.

390 THE LIVER.

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 recent investigations of
NEUBERG and LANGSTEIN, where in feeding experiments with alanine
they found abundance of lactic acid in urine, and P. MAYER observed
gly eerie acid in the urine after the subcutaneous injection of diamino-
propionic acid. Finally, LANG 1 has shown that various organs in anti-
septic autolysis have the power of deamidizing amides and amino-acids.
As from amino-acids by deamidation it is possible to produce oxyfatty
acids according to the formula — CH.NH2 + H2O = — CH(OH)+NHX,
it was interesting to test the action of amino-acids upon carbohydrate
metabolism. Several investigations 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 probable 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 car-
bohydrate. LUSK 2 has shown the same with glutamic acid when fed
to dogs poisoned with phlorhizin. It is still an open question whether
the amino-acids are only indirectly active in this, or whether they form the
material from which the sugar is formed. In general we consider the forma-
tion of sugar with amino-acids as intermediary bodies as very probable.

The investigations of WEINLAND 3 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 which
the sugar was formed.

If we assume a formation of sugar from fat, we must differentiate
between the two components of neutral fats, that is, between the gly-
cerin %and the fatty acids. A formation of sugar from glycerin can
be considered as proven by the investigations of CREMER, and espe-
cially those of LtiTHjE,4 and in the following we will discuss only the
formation of sugar from the fatty acids.

1 Baumann, Zeitschr. f. physiol. Chem., 4; Blendermann, ibid., 6; Neuberg and
Langstein, Arch. f. (Anat. u.) Physiol., 1903, Suppl.; Mayer, Zeitschr. f. physiol. Chem.,
42; Lang, Hofmeister's Beitrage, 5..

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.

3 Zeitschr. f. Biol., 49 (N. F., 31).

4 Cremer, Sitzungsber. d. Ges. f. Morph. u. Physiol. Munchen, 1902; Liithje, Deutsch.
Arch. f. klin. Med., 80.

FORMATION OF SUGAR. 391

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 PFLUGER and several French
observers, among whom we must specially mention CHAUVEAU and

KAUFMANN.1

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 other-
wise free from error we can admit of a formation of sugar from fat. Sev-
eral such cases of diabetes in man have been published (RUMPF, ROSEN-
QVTST, MOKR, v. NooRDEN, ALLARD, FALTA and co-workers and others),
and also in animals (HARTOGH and ScnuMM2) . Although these researches
are not fully conclusive, still certain of them indicate a probable forma-
tion of sugar from fat. We also have several conditions which indicate
the same, namely, that in phlorhizin diabetes after the disappearance
of the liver-glycogen the fat which migrates to the liver serves as mate-
rial for the formation of sugar (PFLUGER) ; still this is not sufficient for
a positive proof.

On the other hand there are also 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 recently published results of experiments 3 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.4 As the quotient D:N

1 Kaufmaim, Arch, f . Physiol. (5), 8, where Chauveau's work is cited.

2Rumpf, 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.

3 Amer. Journ. of Physiol., 22.

4 Magnus-Levy, Zeitschr. f. klin. Med., 56; Pfliiger, Pfluger's Arch., 108; Mohr,
Zeitschr. f. exp. Path. u. Therap., 4.

392 THE LIVER.

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
•conclude from the respiratory quotient that sugar is formed from the
fats as from the proteins.

We have no complete proofs of a sugar formation from fat or from
protein alone, nevertheless we have proofs of the possibility of a formation
from both of these. There is really no objection to the assumption that
the body has the power of producing sugar from protein as well as from
fat. The observations on the formation of sugar or on the carbohydrate
metabolism in diabetes do not give any positive explanations as to the
question whether proteins are direct glycogen-formers or not.

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
DASTRE and PAWLOW l, 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. RANKE found (using a method which is not free
from criticism) a secretion of 14 grams of bile with 0.44 gram of solids
per kilo in twenty-four hours. NOEL-PAYTON, MAYO-ROBSON, HAM-
MARSTEN, 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

'Schwann, 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 Ranke, Die Blutvertheilung und der Thatigkeitswechsel der Organe (Leipzig,
1871); Noel-Paton, 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., 1897; Brand, Pfliiger's Arch., 90.

BILE AND ITS FORMATION. 393

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/ subject to such great variation, even under physiological
conditions, that the study of those circumstances which influence the
secretion is very difficult and uncertain. The contradictory statements
by different investigators may probably be explained by this fact.

In starvation the secretion diminishes. According to LUKJANOW
and ALBERTONi,2 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 contra-
dictory in regard to the time necessary, after partaking of food, before
the secretion reaches its maximum. After a careful examination and
compilation of all the existing reports, HEiDENHAiN3 has come to
the conclusion 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 BARBER A4 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 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 author-
ities are by no means agreed 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 BARBERA
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 5 — has not been proven.

As BARBERA has shown, a close relation exists between the bile
secretion and the quantity of urea formed, as an increase in the first

1 Stadelmann, Der Icterus, etc., Stuttgart, 1891.

2 Lukjanow, Zeitschr. f. physiol. Chem., 16; Albertoni, Recherches sur la secretion
biliaire, Turin, 1893.

3 Hermann's Handb., 5, and Stadelmann, Der Icterus, etc.

4 Centralbl. f. Physiol., 12 and 16.

5 Barbera, Bull, della scienz. med. di Bologna (7), 5, Maly's Jahresber., 24, an 1
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.

394 THE LIVER.

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
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 proven fact by the investigations of several experimenters.1
Sodium salicylate is also perhaps a cholagogue (STADELMANN, DOYON

and DUFOURT, WlNOGRADOW2).

Acids, and especially, under normal conditions, hydrochloric acid,
seem to be physiological excitants for bile secretion. According to FAL-
LOISE and FLEIG the acids act upon the duodenum and the upper part
of the jejunum, and the action is brought about by a secretin formation
similar to the action of acids upon the secretion of pancreatic juice (see
Chapter IX) . 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 mucus 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

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 Arch, f . (Anat. u.) Physiol., 1908. See also foot-note 5, page 393.

3 Falloise, Bull. Acad. Roy. de Belg., 1903; Fleig, ibid., 1903.

BILE SALTS. 395

or bluish green. Bile obtained trom an executed person immediately
after death is golden yellow or yellow with a shade of brown. Still cases
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
nmcin (PAIJKULL). The bile from the animals investigated by HAM-
MARSTEN showed a similar behavior. HAMMARSTEN 1 has, on the con-
trary, found a true mucin in human bile. To all appearances this mucin
originates from the biliary 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 nucleoalbumin.

The specific constituents of the bile are bile-acids combined with alkalies,
bile-pigments, and, besides small quantities of lecithin and phosphatides,
cholesterin, soaps, neutral fats, urea, ethereal sulphuric acid, traces of con-
jugated glucuronic acids, enzymes and mineral substances, chiefly chlorides,
besides phosphates of calcium, magnesium, and iron. Traces of copper
also occur.

Bile-salts. The bile-acids wrhich thus far have best been studied
may be divided into two groups, the glycocholic and taurocholic acid
groups. As found by HAMMARSTEN,S 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 acid
by HAMMARSTEN, yields a.s cleavage products sulphuric acid and a non-nitrogenous

1 Paijkull, Zeitschr. f. physiol. Chem., 12; Hammarsten, 1. c., Nova Act. (3), 16,
and Ergebnisse der Physiol., Bd. 4.

2 Maly's Jahserber., 32.

3 Hammarsten, Zeitschr. f. physiol. Chem., 24.

396 THE LIVER.

substance, scymnol (C27H4805), which gives the characteristic color reactions of
cholic acid.

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 (ZANETTI *) . In the bile of certain animals we
find almost solely glycocholic acid, in others only taurocholic acid, and
in other animals 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 alcohgl 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 arer
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.S 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 containing
the bile-acids is mixed with concentrated sulphuric acid, taking special
care in both cases that the temperature does not rise higher than 60-7CP
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 indicated
by the production of a beautiful red liquid, whose color does not disap-
pear at the ordinary temperature, but becomes more bluish violet in

1 See Chem. Centralbl., 1903, 1, 180.

2 Zeitschr. f . physiol. Chem., 41.
" Zeitschr. f. physiol. Chem., 45.

GLYCOCHOLIC ACID. 397

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.

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 doubtful
cases the spectroscopic examination of the red solution must not be
forgotten.

PETTENKOFER'S test for the bile-acids depends essentially on the
lact that furfurol is formed from the sugar by the sulphuric acid, and
this body can therefore be substituted for the sugar in this test (MYLIUS) .
According to MYLIUS and v. UDRANSZKY l 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 -^ to •£$ milligram cholic acid (v. UDRANSZKY).
Other modifications of PETTENKOFER'S test have been proposed.

The assumption that PETTENKOFER'S reaction is due to the production" of
furfurol from the sugar is not sufficiently proven, and according to certain investi-
gators, such as BARDACHZI and VILLE 2 the spectrum is somewhat different
when using furfurol.

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 C26H43NO6. Glycocholic acid is absent, or nearly sor
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 MtJLLER3 prepared first cholic-acid hydrazide, and then,
by the action of nitrous acid upon this, they obtained the cholic-acid
azide, C23H39O3CO.N3, and finally from this last in alkaline solution
with glycocoll they synthetically prepared the alkali salt of glycocholic
acid, at the same time splitting off nitrogen.

1 Mylius, Zeitschr. f. physiol. Chem., 11; v. Udranszky, ibid., 12.

Bardachzi, Zeitschr, f. physiol. Chem., 48; Ville, cited from Chem. Centralbl.,
1907, 2, p. 1712

Zeitschr f . physiol. Chem., 47.

398 THE LIVER.

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 1 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. EMicn2
found the melting-point 132-134° for the acid crystallized out of water.
The salts cf 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.

Glycocholeic Acid is a second glycocholic acid, first isolated by WAHL-
GREN 3 from ox-bile, and has the formula C26H43NO5 or C27H45NO5.
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 (HAM-

MARSTEN 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
by the salts of the heavy metals, but also by the salts of barium, cal-
cium and magnesium.

The principle of the preparation of the pure glycocholic acids consists
in treating a 2-3 per-cent solution of bile free from mucus, when rich in
glycocholic acid (so-called HUFNER'S bile 5), 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.

1 Zeitschr. f. physiol. Chem., 53.

2 Monatsh. f. Chem., 3.

8 Zeitschr. f. physiol. Chem., 36.

4 Ibid., 43.

5 Hiifner, Journ. f. prakt. Chem. (N. F.), 10, 19, and 25.

TAUROCHOLIC ACID. 399

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, C27H43NO5, 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 CaCl?, BaCl2, and MgCL, and may be salted out like a soap by
Na2SO4 when added in sufficient quantity. By precipitation with NaCl in such
quantity that the precipitate redissolves on warming, HAMMARSTEN l 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 2).

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 C26H45NS07. 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,3 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
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.

1 Not published.

2 Zeitschr. f physiol. Chem., 12 and 13.
- Zeitschr. f. physiol. Chem., 43.

400 THE LIVER.

Taurocholeic Acid is a second taurocholic acid, detected by HAMMAR-
STEN in dog-bile and isolated by GULLBRING 1 from ox-bile, and has the
formula C26H45N£O6 or C27H47NS06. 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 Na2COs, and then the faintly alkaline filtrate satur-
ated with NaCl. The taurocholate separates out and after further puri-
fication 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 C29H4PNSO6. 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
can be precipitated from the solution of the alkali salts by the addition
of mineral acids, like glycocholic acid (HAMMARSTEN 2).

As repeatedly mentioned above, the two bile-acids split on boiling
with acids or alkalies into non-nitrogenous cholic acids and 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-
ing to STRECKER and nearly all recent investigators, the constitution

1 Hammarsten, Zeitschr. f. physiol. Chem., 43; Gullbring, ibid., 45.

2 Not published.

CHOLIC ACID. 40'

rCHOH
C24H4005, = C20H31 | (CH2OH)2. According to MYLius,1 cholic acid is c.

ICOOH

monobasic alcohol-acid with one secondary and two primary alcohol
groups. CURTIUS 2 has shown by preparing the cholamine, C23H->9O3.NH2,
from the above-mentioned (p. 397) cholic-acid azide, with choHc-acid
urethane as an intermediary step, that the carboxyl group is not imme-
diately connected with the GHOH group, but is combined with the chief
nucleus without the neighboring secondly alcohol group. On oxida-
tion it first yields dehydrocholic arid. C24H3105 (HAMMARSTEN). On
further oxidation bilianic acid, O;.4fl34O8 (CLEVE), is obtained, or, more
correctly, according to LATSC«HNOFF, LASSAR-COHN and PREGL, a mix-
ture of bilianic and isobilianic acids discovered by LATSCHINOFF. On
oxidation, bilianic acia )rields cilianic acid (LASSAR-COHN), whose form-
ula, according to PREGL,S is C20H28C8. On reduction (by putrefaction)
MYLIUS obtai^d desoxy cholic acid, C24H4004 from cholic acid.

On stronger oxidation it yields cholesterinic acid, which has not been carefully
studied, and finally phthalic acid, as maintained by SENKOWSKI, but not sub-
star aated by BULNHEIM or PREGL.* On reduction with hydriodic acid and red
p1 /sphorus, PREGL obtained a product which he considers as a mono-carboxylic

fCH2
,cid, with the formula C20H31 j (CH3)2. SENKOWSKI ° obtained an acid with the

ICOOH

formula C24H4002, cholylic acid, on the reduction of the anhydride.

As above-mentioned, PREGL 6 obtained, by the action of concen-
trated sulphuric acid upon cholic acid> a fluorescent substance which
he calls dehydrocholon. This is produced by oxidation, and at the same
time, water is eliminated. It has probably the formula C24H38O. Dehy-
drocholon is nitrated by nitric acid, while the cholic acid is not. From
this behavior, as well as from the determination of the molecular refrac-
tion and dispersion of both bodies, PREGL finds it probable that cholic
acid belongs to the hydrated carbocyclic compounds. This view has
received further support by PANZER/ who obtained a homologue of
benzene having the formula CnH16 from a cholic acid derivate, the chole-

1 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.

2 Ibid., 39.

3 Hammarsten, Ber. d. deutsch. chem. Gesellsch., 14; Cleve, Bull. Soc. chim., 35;
Latschinoff, Ber. d. d. chem. Gesellsch., 15; Lassar-Cohn, Ber. d. d. chem. Gesellsch.,
32; Pregl, Wien. Sitzungsber., Ill, 1902.

4Se"nkowski, Monatsh. f. Chem., 17; Bulnheim, Zeitschr. f. physiol. Chem., 25
which also contains the literature on cholesterinic acid.

5 Mylius, 1. c.; Pregl, Pfliiger's Arch., 71; Senkowski, Monatshefte f. Chem., 19.

6 Zeitschr. f. physiol. Chem., 45.

7 Zeitschr. f. physiol. Chem., 48; Latschinoff, Ber. d. d. Chem. Gesellsch., 12 and 13

402 THE LIVER.

campheric acid of LATSCHINOFF, by distillation with soda-lime and whicli
can be considered as formed from a hydro-aromatic ring by splitting
off of water and formation of double bonds.

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 (MYLIUS). 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° C. According to BONDI and MULLER the melting-point of the per-
fectly pure acid is 198°. 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 (HAMMARSTEN).

The alkali salts are readily soluble in water, but when treated with a
concentrated caustic or carbonated alkali solution they may then be sepa-
rated 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 l
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 (C25H42O4, LATSCHINOFF) is another cholic acid which,
according to LASSAR-CoHN,2 has the formula C24H40O4. This acid,
which occurs in varying but always small quantities in ox-bile, yields
dehydrocholeic acid, C24H34O4, and then cholanic acid C24H34O7, and
isocholanic acid on oxidation.

Choleic acid crystallizes when free from water in hexagonal vitreous
prisms with pointed ends, melting at 185-190° C. The crystalline
acid containing water melts at 135-140° C. (LATSCHINOFF). The acid

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.

DESOXYCHOLIC ACID. 403

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, C^H^O^ is the name given by MYLIUS to a
cholic acid isolated by him from putrid ox-bile, and which is formed from
the cholic acid (on the putrefaction of the bile) by reduction. This last
is still veiy 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
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 what is more probable is that we have two different
acids which are probably isomeric with each other. Both acids can also
be detected in perfectly fresh ox-bile as shown by LANGHELD, and also
by HAMMARSTEN.1 According to PREGL and to HAMMARSTEN, desoxy-
cholic acid is a preformed acid of the fresh bile.

The acid crystallizes from glacial acetic acid in needles with 1 mole-
cule acetic acid, having a melting-point of 144-145°. The melting-point
of the acid crystallized from alcohol-ether is 135-1 55°;2 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

1 Mylius, Ber. d d. chem. Gesellsch., 19 and 20; Ekbom, Zeitschr. f. physiol. Chem.,
50; Pregl, Wien. Sitz.-Ber., Ill Math. Naturw. Kl., 9102; Latschinoff, Ber. d. d.
chem. Gesellsch., 20; Langheld, ibid., 41; Hammarsten, unpublished investigations.

2 See Pregl, I.e.

404 THE LIVER.

by hydrochloric acid, dissolved in ammoniacal water and precipitated
by BaCl2- The precipitate contains essentially choleic and desoxy-
cholic acids, while the filtrate 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 sug-
gested for the preparation of the pure cholic acids, we must refer to com-
pleter hand-books.

Fellic Acid, CsaH^CX 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-
stances various conjugate bile-acids. In some cases the bile-salts of
human bile are precipitated by BaCl2 and in others not. According to
the statements of LASSAR-CoHN1 three cholic acids may be prepared
from human bile, namely, ordinary CHOLIC ACID, CHOLEIC ACID, and

FELLIC ACID.

Lithofellic Acid, C2oH36O4, 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

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 hippotamus-
bile, which stands very close to the pig-bile (HAMMARSTEN 3) . In the
polar bear a third cholic acid exists besides cholic. and choleic acids,
It is called ursocholcic acid, Ci9H3oO4 or C18H8204 (HAMMARSTEN4).
The bile of other animals (walrus, seal) contains special cholic acids
(HAMMARSTEN 5).

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, C24H36O3, corresponding to ordinary cholic
acid, which occurs in faeces, is amorphous, insoluble in water and alkalies.
Choloidic acid, C24H38O4, 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.

1 Schotten, Zeitschr. f. physiol. Chem., 11; Las<??,r-Cohn, Ber. d. deutsch. chem.
Gesellsch., 27.

2 See Jiinger and Klages, Ber. d. deutsch. chem. Gesellsch. 28 (older literature).

3 Investigations not published.

4 Zeitschr. f. physiol. Chem., 36.

5 Investigations not published.

BILE PIGMENTS. 405-

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 fat 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, distill the alcohol, and evaporate to dryness. The residue is
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 dryness, 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 crystal-
lization ; 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 reac-
tion, 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 ki the normal bile, but occur
either in post-mortem bile or principally in the bile concrements. The
pigments which occur under physiological conditions are the reddish-
yellow bilirubin, the green biliverdin, and sometimes also urdbilin (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 choletelin?) . 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 some-
times greenish color to the bile; or when, as is most frequently the case
in ox-bile, the two pigments are present in the bile at the same timer
they produce the different shades between reddish-brown and green.

Bilirubin. This pigment has the formula Ci6Hi8N2O3, or accord-
ing to ORNDORFF and TEEPLE and KtisTER,1 more correctly C32H36N4O6r
and is designated by the names CHOLEPYRRHIN, BILIPH^IN, BILIFULVIN,
and KLEMATOIDIN. 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,

1 Orndorff and Teeple, Salkowski's Festschrift, Berlin, 1904; Ktister, Zeitschr. f.
Physiol. Chem., 59.

406 THE LIVER.

in old blood extravasations (as hsematoidin), and in the urine and the
yellow-colored tissue in icterus. It is converted into hydrobilirubin,
C32H40N407 (MALY), by hydrogen in a nascent state, and then shows
great similarity to the urinary pigment, urobilin, as well as to stercobilin
found in the contents of the intestine (MASIUS and VANLAin1). On
careful oxidation bilirubin yields biliverdin and other coloring-matters
(see below).

Bilirubin is derived from the blood-pigment. It has the same per-
centage composition as hsematoporphyrin, and like hsematin it yields
hsematimc-acid imide as an oxidation product (KUSTER). On reduction
with zinc powder or with nascent HI it yields hsemopyrrol, according to
ORNDOKFF and TEEPLE.2

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 gly-
cerin. 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
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 re-adily. 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 (1:500,000), in a layer 1.5 cm. thick. If a dilute solution of

1 Maly, Wien. Sitzungsber., 57, and Annal. d. Chem., 163; Masius and Vanlair,
Centralbl. f. d. med. Wissensch., 1871, 369.

2 I.e.

3 Ber. d. d. chem. Gesellsch., 30 and 35, and Zeitschr. f . physiol. Chem., 47.

BILIRUBIN. 407

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 compounds of bilirubin with alkalies are insoluble in
chloroform, and bilirubin may be separated from its solution in chloro-
form by shaking with dilute caustic alkali (differing from lutein). Solu-
tions of alkali bilirubin in water are precipitated by the soluble salts of
the alkaline earths and also by metallic salts.

As EHRLICH first showed, bilirubin forms combinations with diazo
compounds, which have been closely studied by PROSCHER, ORNDORFP
and TEEPLE.1 A test suggested by EHRLICH for bilirubin is based upon
this behavior with diazobenzenesulphonic acid.

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 hsematinic acid. Biliverdin is formed only
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,2 biliverdin is
produced by the use ot HUBL/S iodine solution, while according to others
(THUDICHUM, MALY 3) 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 biliru-
bin. 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 reaction
takes place too quickly and it does not become typical. Alcohol must

1 Ehrlich, Zeitschr. f. anal. Chem., 23; Proscher, Zeitschr. f. physiol. Chem., 39;
Orndorff and Teeple, 1. c.

2 Kiister, Ber. d. d. chem. Gesellsch., 35 and 59; Jolles, Journ. f. prakt. Chem. (N. P.),
59, and Pfliiger's Arch., 75.

3Thudichum, Journ. of Chem. Soc. (2), 13, and Journ. f. prakt. Chem. (N. P.),
53; Maly, Wien. Sitzungsber., 72.

408 THE LIVER.

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 XV (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
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
with 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 l obtained a product
which he calls choletelin, which is quite similar to urinary urobilin, to-
be discussed later.

1 Heinsius and Campbell, Pfliiger's Arch., 4; Stokvis, Centralbl. f . med. Wis-
sensch., 1872, 785; ibid., 1873, 211 and 449; Jaff<§, ibid., 1868; Maly, Wien. Sitzungs-
ber., 59.

BILIVERDIN. 409

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 and bile-acids. In order to remove the mineral con-
stituents it is better to use 10-per cent acetic acid instead of hydrochloric
acid (KtisTER1). 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 boiling
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.2

The quantitative estimation of bilirubin may be made by the spectro-
photometric method, according to the steps suggested for the blood-
coloring matters.

Biliverdin, C16H18N204 or C32H36N408. 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'

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 3) .

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.

1 Zeitschr. f. physiol. Chem., 47.

2 Zeitschr. f. physiol. Chem., 59.

3 Centralbl. f. Physiol., 3, 222, and Zeitschr. f. physiol. Chem., 14.

410 THE LIVER.

HUGOUNENQ and DOYON 1 prepared biliverdin from bilirubin by the
action of sodium peroxide and a little hydrochloric acid.

Choleprasin is a green pigment isolated by KUSTER 2 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, 3 is an amorphous brown pigment soluble
in alcohol and alkalies, nearly 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 for the bilifuscin
prepared by v. ZUMBUSCH/ which is more like a humin substance, and the formula
of which is C64H96N7Oi4. 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,5 on the
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 oxidation
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 hsematin 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.
Phylloerythrin has been detected by MARCHLEWSKI a 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

1 Hugounenq et Doyon, Arch, de Phyisol. (5), 8; Kiister, Zeitschr. f. physiol.
Chem., 59.

a Zeitschr. f. physiol. Chem., 47.

3 Cited from Hoppe-Seyler, Physiol. u. Path. chem. Analyse, 6. Aufl., p. 225.

4 Zeitschr. f. physiol. Chem., 31.

5 Arch, de Physiol. (5), 9.

6 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.

CONSTITUENTS OF THE BILE. 411

blood, according to HEDEXius,1 by precipitating die proteins with alcohol,
filtering and acidifying the filtrate with hydrochloric or sulphuric acid,
and boiling. The liquid becomes of a greenish color. Serum arid serous
fluids may be boiled directly with a little acid after the addition of alcohol.

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 acid (LASSAR-COHN 2) , soaps, ethereal
sulphuric acids, conjugated glucuronates, diastatic and proteolytic enzymes.
Choline and glycerophosphoric acid, when they are present, may be con-
sidered as decomposition products of lecithin. Urea occurs, though
only in traces, as a physiological constituent of human, 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.3 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 (YouNG4). 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 formation
and destruction of blood. According to BECCARI 5 the iron does not
disappear from the bile in inanition, and the percentage shows no con-
stant 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,G deny any such
elimination of iron by the bile.

1 Upsala. Lakaref . Forh., 29, and Maly's Jahresber., 24.

2 Zeitschr. f. physiol. Chem., 17; Hammarsten, ibid., 32, 36 and 43.

3 Hammarsten, ibid., 24.

4 Journ. of Anat. and Physiol., 5, 158.

6 Novi, see Maly's Jahresber., 20; Dastre, Arch, de Physiol. (5), 3; Beccari, Arch,
ital. de Biol., 28.

5 Kunkel, 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 1, p. 322.

412 THE LIVER.

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.1
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.

FRERICHS. 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\ A7 ~ Qn Q

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 repeatedly
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 2 observed still higher figures, more than 40 p. m., in a couple
of 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,S is nearly always 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.4

Human bile sometimes, but not always, contains sulphur in an ethereal

*See Hoppe-Seyler Physiol. Chem., 301; Socoloff, Pfluger's Arch., 12; Trifanow-
ski, ibid., 9; Frerichs in Hoppe-Seyler's Physiol. Chem., 299; v. Gorup-Besanez, ibid.

2 Jacobsen, Ber. d. deutsch. chem. Gesellsch., 6; Hammarsten, Nova Acta Reg.
Soc. Scient. Upsala, 16; Brand, Pfluger's Arch., 90.

3 Brand, 1. c.; Bonanni, Biochem. Centralbl., 1; Strauss, Berl. klin. Wochenschr.,
1903.

4 See Brand, 1. c.; Hammarsten, 1. c.

COMPOSITION OF HUMAN BILE. 413

sulphuric-acid-like combination (HAMMARSTEN, OERUM, BRAND). The
quantity of such sulphur may even amount to J-J of the total sulphur.
We do not know the nature of these ethereal sulphuric acids. According
to OERUM 1 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 110 taurocholic acid.

As an example of the composition of human liver-bile the following
results of three analyses made by HAMMARSTEN are given. The results
are calculated in parts per 1000.2

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 1.600' 1.500

Lecithin . . . 1 n OOA 0 . 574 0 . 650

Fat.. 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 3 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. taurocholate) ; 3.4 p. m. cholesterin;
6.7 p. m. fat, and 2.8 p. m. leuci'ne.4

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. More
exact results obtained by spectrophotometric methods are on record
for dog-bile. According to STADELMANN 5 dog-bile contains on an average

1 Skand. Archiv. f. Physiol., 16.

2 Recent quantitative analyses may be found in Brand, I.e.; v. Zeynek, Wien.
klin. Wochenschr., 1899; Bonanni, 1. c.

3 Verhandl. d. physiol. Gesellsch. zu Berlin, 1894-95.

4 Analyses of bile from children may be found in Heptner, Maly's Jahresber., 30.

5 Noel-Paton, Rep. Lab. Roy. Soc. Coll. Phys. Edinburgh, 3; Stadelmann, Der
Icterus.

414 THE LIVER.

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
prevailing acid in carnivorous mammals, birds, snakes, and fishes.
Among the herbivora, sheep and goats have a predominance of tauro-
cholic 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, kan-
garoo, 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 dif-
ferences in diet has not been detected. RITTER 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 exact
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
(HAMMARSTEN 4) .

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 5) .

The gases of the bile consist of a large quantity of carbon dioxide,

1 Investigations not published. See Ergebnisse der Physiol., 4.

2 Cited from Maly's Jahresber., 6, 195.

3 Hammarsten, Zeitschr. f. physiol. Chem., 32, and Ergebnisse der Physiol., 4.

4 Zeitschr. f. physiol. Chem., 36, and Ergebnisse der Physiol., 4.

5 Arch, de Physiol. (5), 8.

CHEMICAL FORMATION OF THE BILE. 415

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 properties 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 RITTER 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
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 haemoglobin injection (WERTHEIMER
and MEYER, FILEHNE, STERN 2). Albumin can pass into the bile after the intra-
venous injection ot a foreign protein (casein) (GURBER and HALLAUER), as well
as after poisoning writh 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 WAHLGREN,4 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

1 Ritter, 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.

3 Curber and Hallauer, Zeitschr. f. Biologie, 45; Pilzecker, Zeitschr. f. physiol.
Chem., 41; Brauer, ibid., 40.

4 See Maly's Jahresber., 32.

5 Winternitz, Zeitschr. f. physiol. Chem., 21; Sollmann, Amer. Medicine, 5 (1903).

416 THE LIVER.

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
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. Thes"e claims have not been con-
firmed 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 indubitably proven 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 290). LATSCHEN-
BERGER 3 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
ori 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 elimin?/non and formation of bile-pigments. In this regard

1 Kobner, see Heidenhain, Physiologie 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'B .Tp,hresber., 16, and Monatshefte f . Chem., 9.

CHEMICAL FORMATION OF THE BILE. 417

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 lately confirmed by the investigations of Vossius.1

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
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 NAUNYN2 also found that poisoning
with arseniuretted 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.

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 FRIEDMANN
(see Chapter III), and recently v. BERGMANN 3 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 formerly considered as originating from
the fats, nothing is positively known.

The blood-coloring matters are considered as the mother-substances
of the bile-pigments. If the identity of haBmatoidin and bilirubin was
settled beyond a doubt, then this view might be considered as proven.
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

1 Tarchanoff, Pfliiger's Arch., 9; Vossius, cited from Stadelmann, Der Icterus.

2 Stern, Arch. f. exp. Path. u. Pharm., 19; Minkowski and Naunyn, ibid., 21.

3 Hofmeister's Beitrage, 4. See also Wohlgemuth, Zeitschr. f. physiol. Chem., 40.

418 THE LIVER.

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). A further proof
of the formation of the bile-pigments from the blood-coloring matters
consists in the fact that haematin on reduction yields urobilin, which is
identical with hydrobilirubin (see Chapter XV). Further, haBmato-
porphyrin (see page 288) and bilirubin are isomers, according to NENCKI
and SIEBER, and closely allied. The formation of bilirubin from the blood -
coloring matters is shown, according to the 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.

If bilirubin, which contains no iron, is derived from hsematin, which
contains iron, then iron must be split off. This process may be repre-
sented by the following equation : C32H34N4O5Fe + H20 — Fe = C32H36N4O6.
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 hsematin contain about
9 parts iron. MINKOWSKI and BASERIN 3 also found that the abundant
formation of bile-pigments occurring in poisoning by arseniuretted hydro-
gen 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 researches of several investi-
gators 4 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

1 See Stadelmann, Der Icterus, etc., Stuttgart, 1891.

2 See Stadelmann, ibid.

3 Kunkel, Pfliiger's Arch., 14; Minkowski and Baserin, Arch. f. exp. Path. u.
Pharm., 23.

4 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. 362.

BILE CONCRETIONS. 419

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 haemoglobin 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, arseniuretted hydrogen, phosphorus,
or toluylenediamine, and in other cases. This also occurs in man in
severe infectious diseases. 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 by the important researches of MINKOWSKI and NAUNYN,
AFANASSIEW, SILBERMANN, and especially of STADELMANN/ and in some
of the above-mentioned cases, as after poisoning with phosphorus, toluy-
lenediamine, and arseniuretted hydrogen, it has been disproven by direct
experiment.

The icterus is also in these cases hepatogenic; it depends upon an
absorption of bile-pigments from the liver, and this absorption seems to
originate in various cases in somewhat different ways. Thus the bile
may be viscous and cause a congestion of bile by counteracting the low
secretion pressure. In other cases the fine biliary passages may be
compressed by an abnormal swelling of the liver-cells, or a catarrh of the
bile-passages may occur, causing a congestion of the bile (STADELMANN).

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 chief mass. One group of
gallstones 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

1 The literature belonging to this subject is found in Stadelmann, Der Icterus, etc.,
Stuttgart, 1891.

420 THE LIVER.

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 cases they consist chiefly of calcium-bilirubin with little or no
biliverdin. 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. Manganese 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. (RiTTER1). 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 C27H46O (OBERMULLER) or C27H44O
(MAUTHNER and SUIDA).

Because of the fact that from cholesterin, hydrocarbons which
have been called cholesteriline, cholesterone and cholesterilene, can be
prepared in different ways, 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.2 From these investigations
we conclude that cholesterin is a monoatomic, unsaturated, secondary
alcohol whose hydroxyl group exists in a hydrogenized ring, indeed
between two methyl groups, and which also contains an isopropyl group.
It is also generally admitted that cholesterin contains only one double

1 Journ. de 1'anat. et de la physiol. (Robin), 1872.

2 The literature on cholesterin can be found in Windaus, Arch. d. Pharm., 246,
Hft. 2, and especially in Glikin, Bioch. Centralbl., 7, 372-377.

CHOLESTERIN. 421

bond, which occurs in a vinyl group CH:CH2 at the end. Contrary to
this MOLINARI and FENAROLi,1 basing their opinions upon the behavior
of cholesterin with ozone, believe the cholesterin has two double bonds.
Under these circumstances no constitutional formula for cholesterin
can be given; still there is no doubt 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, C27H48O. On treating cholestenon, the ketone of
cholesterin, DIELS and ABDERHALDEN obtained a second dihydrocholesterin,
the fl-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 LiNN2 /?-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. For the
present this question must be answered in the negative.

On heating cholesterin, when contaminated with iron, to 300-320°, according
to DIELS and LINN,S 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.

1 Ber. d. d. chem. Gesellsch., 41.

2 Willstatter and Mayer, Ber. d. d. chem. Gesellsch., 41; Diels and Linn, ibid., 41.

3 Ibid., 41.

422 THE LIVER.

Cholesterin is insoluble in water, dilute acids, and alkalies. It is
neither dissolved nor changed by boiling caustic alkali. It is easily
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 (GERARD1). 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
C2H5.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 fact 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.

SALKOWSKi's2 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 first 10 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.

NEUBERG-RAUCHWERGER'S 4 Reaction. With rhamnose, or better
still with d-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, somo

1 Compt. rend. soc. biol., 58.

2 Pfluger'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.

VARIOUS STERINS. 423

camphor derivatives, abietinic acid, and a hydride of retene. For details
of its performance, see original publication.

LIFSCHUTZ'S Reaction.1 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 (QBERMULLER 3).

SCHIFF'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).

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 C27H48O, 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.

Hippokoprosterin is another cholesterin richer in hydrogen, which BONDZYNSKI
and HUMNICKI found in the feces of the horse. Its formula is C27H54O. 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 ScnuLZE,5 with the formula

1 Ber. d. d. chem. Gesellsch., 41.

2 Zeitschr. f. physiol. Chem., 50, 53, 58, and Ber. d. d. chem. Gesellsch., 41.

3 Zeitschr. f . physiol. Chem., 15.

4 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; Kusu-
moto, Bioch. Zeitschr., 14; Doree and Gardner, Proc. Roy. Soc. London, 80, Ser. B.

5 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.

424 THE LIVER.

C26H43OH, 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= +60°.

Spongosterin is the name given by HENZE l 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 2 to a cholesterin
isolated by them from the chrysalis of the silkworm, which has a melting-point
of 148° and a specific rotation of (a)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 IX), 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 3 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 body. This action of
the cholesterins in regard to inhibiting the hsemolytic action of saponin,
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.4

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

prakt. Chem. (N. F.), 25, 159. In regard to the formula for isocholesterin, see Darm-
staiterand Lifschiitz, Ber. d. deutsch. chem. Gesellsch., 31, and E. Schulze, ibid., 1200.

1 Zeitschr. f . physiol. Chem., 41 and 55.

2 Cited from Chem. Centralbl., 1908.

3 Zeitschr. f. physiol. Chem., 57.

4 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.

PREPARATION OF CHOLESTERIN. 425

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 HITTER. J The essential points in his method
consist in saponifying the fat with sodium alcoholate, removing the alcoho
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.

1 Zeitschr. f. physiol. Chem., 34.

CHAPTER IX.
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 sub-
lingual and sub-maxillary glands of many animals), and partly mixed
glands (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 OIDTMANN 1 the salivary glands of a dog contain 790 p. m. water, 200
p. m. organic and 10 p. m. inorganic solids. Among the solids we find
mucin, proteins, nucleoproteins, nuclein, enzymes and their zymogens,
besides extractive bodies, leucine, purine bases, and mineral substances.

1 Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 732. The
figures there given amount to 1010 parts instead of 1000 parts.

426

STJBMAXILLARY SALIVA. 427

The occurrence of a mucinogen has not been proven. 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 submaxillary 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 (ECKHARD 2) . 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,S between
J= -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.
Other investigators, such as ASHER and CUTTER, and JAPPELLI4 also
found that the osmotic pressure of the submaxillary saliva is con-
siderably lower than that of the blood. On changing the osmotic
pressure of the blood the osmotic pressure of the saliva, according to
JAPPELLI, changes in the same direction; the difference between the
pressure of both fluids remains constant. The gases of the chorda-
saliva have been investigated by PrLUGER.5 He found 0.5-0.8 per cent
oxygen, 0.9-1 per cent nitrogen, and 64.73-85.13 per cent carbon

1Upsala 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 Asher and Cutter, Zeitschr. f . Biol., 40; Jappelli, ibid., 48 and 51.
6 Pfluger's Arch., 1.

428 DIGESTION.

dioxide — all results calculated at 0° C. and 760 mm. pressure. The greater
part of the carbon dioxide was chemically combined.

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.1 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,2 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, 3 Q.9S 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-
tation of the glandular nerves, either directly (in animals) or reflexly,
by mechanical or chemical irritation of the mucous membrane of the
mouth. Among the chemical irritants the acids take first place. Mas-

1 See Maly's " Chemie der Verdauungssafte und der Verdauung," in Hermann's
Handb., 5, part II, 18. This article contains also the pertinent literature.

2 Studien d. physiol. Instituts zu Breslau, Heft 4.

3 Die Verdauungssafte und der Stoffwechsel (Mitau and Leipzig, 1852), p. 5.

PAROTID SALIVA. 429

\

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 l 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, as PAWLOW'S 2 school has shown,
is greatly dependent in dogs upon the psychical excitement, 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. Under the influence of hard and dry food the glands secrete
an abundance of saliva, while with food rich in water the secretion
is considerably less and accommodates itself to the quantity of water
in the food. Milk is an exception to this rule, as it causes a more abundant
secretion of saliva than meat. This is of importance in digestion of milk,
as in the stomach the mixture of milk and saliva does not coagulate to
a compact mass, but separates in a finely divided, readily digestible con-
dition. By the action of strong chemical bodies the saliva is secreted in
proportion to the strength of the irritant. According to POPIELSKI 3
this is true only for irritants of medium strength, as after stronger irrita-
tion, for example with capsicin solution, the quantity of saliva decreases
with the increase in the amount of irritant. The irritating substance
is diluted by the saliva and the mouth is washed out at the same time
(PAWLOW). The partaking ©f acid produces, according to PAWLOW, a
thin saliva, poor in mucin, in quantities sufficient to neutralize the acid.
This claim does not agree with the observations of POPIELSKI, who found
that isomolecular acid solutions produced the secretion of the same
amount of saliva, and that on using isopercentage acids the quantity of
saliva was in inverse proportion to the molecular weight of the acid.

1 Zeitschr. f . Biologie, 23s

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.

3 Pfliiger's Arch., 127.

430 DIGESTION.

POPIELSKI also disputes the assumption of PAWLOW that the secretion of
saliva (in dogs) accommodates itself to the kind of irritant and to the
kind of food ; although the theory that in man there is an accommodation
of the secretion to circumstances has found supporters, yet belief in it
is not general.1

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 consisting
of calcium carbonate and a small quantity of an organic substance, or
it gradually becomes cloudy. Its reaction is generally alkaline to litmus.
The degree of alkalinity varies considerably not only in different indivi-
duals but also in the same individual during different parts of the day,
so that it is difficult to state the average alkalinity. According to CHITTEN-
DEN and ELY it corresponds to the alkalinity of 0.8 p. m. Na£CO3 solu-
tion, or to 0.2 p. m. solution according to COHN. According to FOA the
the actual alkalinity (OH-ion concentration) is always considerably
less than that found by titration, and the reaction determined electro-
metrically 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. Accord-
ing to CoHN,2 J= —0.20° on an average, and the amount of NaCl is 1.6
p. m. The solids, irrespective of the form-constituents mentioned,
consist of protein, mucin, oxidases,3 two enzymes, ptyalin and maltase,
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 ordinary
smokers the quantity of sulphocyanides may rise to 0.2 p. m. (FLECK-
SEDER 4).

1 See Zebrowski, Pfliiger's Arch., 110; Neilson and Lewis, Journ. of biol. Chem., 4,
and with Scheele, ibid., 5.

2Chittenden 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.

3 Bogdanow-Beresowski, cited from Biochem. Centralbl., 2, 653.

4 Munk, Virchow's Arch., 69; Schneider, Amer. Journ. of Physiol., 5; Kriiger,
Zeitschr. f. 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.

PTYALIN. 431

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 l method.

Ptyalin, or salivary diastase, is the arnylolytic enzyme of the saliva.
This enzyme is found in human saliva,2 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 3 claims that it is present not only in the parotid gland of children,
but also in the mucin glands.

According to H. GOLDSCHMIDT 4 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 5 method, which consists
in carrying the enzyme down mechanically with a calc'um-phosphate
precipitate and washing the precipitate with water, which dissolves the
ptyalin, and from wrhich 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 223) . The nature of the sugar
thus produced is known with certainty. For a long time it was con-
sidered that dextrose was the sugar formed from starch and glycogen,
but SEEGEN and O. NASSE have shown that this is not true. MUSCULUS
and v. MERIXG have shown that the sugar formed by the action of saliva,

1 Gscheidlen, Maly's Jahresber., 4; Solera, see ibid., 7 and 8; Munk, Virchow's
Arch., 69; Ganassini, Biochem. Centralbl., 2, p. 361

2 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.

3 Zweifel, Untersuchurigen iiber den Verdauungsapparat der Neugeborenen (Berlin,
1874); Berger, see Maly's Jahresber., 30, 399.

4 Zeitschr. f . physiol. Chem., 10.

5 Virchow's Arch., 28.

432 DIGESTION.

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. VOGEL l have also demonstrated that in the saccharin" ca-
tion of starch and glycogen, isomaltose, maltose, and some dextrose are
formed, the varying quantities depending upon the amount of ferment
and the length of its action. The formation of dextrose is claimed by
TEBB, ROHMANN, and HAMBURGER2 to be only a product of the inver-
sion of the maltose by the maltase.

The action of ptyalin in Various reactions has been the subject of
numerous investigations.3 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, NYLEN,
and others, may entirely destroy the enzyme. This is important in regard
to the physiological significance of the saliva. According to ROGER
and SIMON 4 ptyalin is not destroyed by gastric juice, but its action is

1 Seegen, tentralbl. 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.

2 Tebb, Journ. of Physiol., 15; Rohmann, Ber. d. deutsch. chem. Gesellsch., 27;
Hamburger, Pfliiger's Arch., 60.

3 See Hammarsten, Maly's Jahresber., 1; Chittenden and Griswold, Amer. Chem.
Journ., 3; Langley, Journal of Physiol., 3; Nyl6n, 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; Schtesinger, Virchow's Arch.,
125; Schierbeck, Skand. Arch. f. Physiol., 3; Ebstein and C. Schulze, Virchow's Arch.
134; Kiibel, Pfliiger's Arch., 56.

4 Compt. rend. soc. biol., 62.

ACTION OF PTYALIN. 433

only inhibited because saliva made inactive in this manner can be react-
ivated by a small quantity of saliva or pancreatic juice. The experi-
ments recorded in support of this assumption are not conclusive enough
for such a view. That boiled starch (paste) is quickly, and unboiled
starch only slowly, converted into sugar is also of interest. Various
kinds of unboiled starch are converted with different degrees of rapidity.
Several series of investigations have been made upon the velocity
with which ptyalin acts, and as in testing enzyme action in general, the
experimenters have not made use of the different times required to pro-
duce equal chemical changes as a measure of the velocity, but have
taken the quantities of substance changed in equal times. Although
the results are somewhat divergent it is possible to deduce the following
from them. The velocity increases, at least under conditions otherwise
favorable, with the amount of enzyme and with an increasing temperature
to a little above 40° C. Foreign substances, such as metallic salts,1 have
different effects. Certain salts, even in small quantities, completely arrest
the action; for example, HgCl2 accomplishes this result completely by
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 addition of salts the dialyzed saliva becomes active again, espe-
cially on the addition of calcium or potassium chloride. ROGER 2 be-
lieves 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 accel-
erting action, may in large quantities have a retarding action. As an
example we can mention MgSO4, but unfortunately the opinions in
regard to this salt, as well as others, are widely divergent. The
presence of peptone has an accelerating action on the sugar formation
(CHITTENDEN and SMITH and others) . The accumulation of the products
of the amylolytic decomposition also checks the action of the saliva.
This has been showrn by special experiments made by SH. LEA.S He
made parallel experiments with digestions in test-tubes and in dialyzers,
and found on the removal of the products of the amylolytic decomposition
by dialysis that the formation of sugar took place sooner, but also that
considerably more maltose and less dextrin were formed.

To show the action of saliva or ptyalin on starch the three ordinary
tests for dextrose may be used, namely, MOORE'S or TROMMER'S test or

1 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.

2 Guyenot, Compt. rend. soc. biol., 63; Roger, ibid., 65.

3 Journ. of Physiol., 11.

434

DIGESTION.

the bismuth test (see Chapter XV). It is also necessary, as a control,
to first test the starch-paste and the saliva for the presence of dextrose.
The steps in the transformation of starch into amidulin, erythrodextrinr
and achroodextrin may be shown by testing with iodine.

Maltase occurs in saliva to only a slight extent. It converts maltose
into dextrose. According to STICKER l saliva also has the power of split-
ting sulphuretted hydrogen from the sulphur oils of radishes, onions,
and certain other vegetables.

The quantitative composition ot the mixed saliva must vary considerably,
not only because of individual differences, but also because under vary-
ing 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.

BJ

CS

.

H

55

m

p

13

I
B

5

i

II

wO

i

w

E

s

X
H

i a
ai u

11

«

*

£

H

K

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

2 2

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 (SO3) 63.8,
phosphoric anhydride (PA) 188.48, and chlorine 183.52.

The quantity of saliva secreted during twenty-fours 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,3
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,

1 Munch, med. Wochenschr., 43.

2 Zeitschr. f. physiol. Chem., 5. The other analyses are cited from Maly, Chemie
der Verdauungssafte, Hermann's Handbuch d. Physiol., 5, part II, 14.

3 Bidder and Schmidt, 1. c., 13; Tuczek, Zeitschr. f. Biologic, 12.

PHYSIOLOGICAL IMPORTANCE OF THE SALIVA. 435

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 secre-
tion under physiological conditions equals that of the salivary glands.
A remarkably 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 substances
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 *), iodine, and bromine compounds, but not others,
for example, iron compounds and dextrose. It is also noticeable 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 (HEIDEN-
HAIN, WERTHER, LANGLE Y and FLETCHER, Novi 2) .

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 substances.
The importance of the saliva in mastication is especially marked in
herbivora, and there is no question as to its importance in facilitating the
act of swallowing. The saliva containing mucin is especially important
in this regard, and PAWLOW'S school has shown that the secretion
also regulates itself in this regard. The saliva is also of importance, as it
serves in washing out the mouth and thereby acts as a protection against
destructive substances or bodies foreign to the mouth. The power of con-
verting starch into sugar is not inherent in the saliva of all animals, and
even when it possesses this property the intensity varies in different ani-
mals. 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

1 Virchow's Arch., 53.

2 Heidenhain, Pfliiger's Arch., 17; Werther, ibid., 38; Langley and Fletcher,
Proc. Roy. Sex;., 45, and especially Phil. Trans. Roy. Soc. London, 180; Novi, Arch,
f. (Anat. u.) Physiol., 1888.

436 DIGESTION.

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.

II. 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 coating
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.

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 pro-
teins; but their relation to coloring-matters seems to show that the
protein substances of both are not identical. The nucleus must con-
sist chiefly 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 HEIDENHAIN, inde-
pendent of the columnar epithelium of the excretory ducts they take no
part worthy of mention in the formation of mucus, which according to

SECRETION OF GASTRIC JUICE. 437

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 coat-
ing of the stomach.

LIEBERMANN J obtained an acid-reacting residue on digesting the mucosa
of the stomach with pepsin-hydrochloric acid, which strangely enough contained
no nuclein, but only a protein containing lecithin, called lecithalbumin. To
this lecithalbumin he^ ascribes a great importance in the secretion of hydrochloric
acid.

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 2
in 1842 on a dog. VERNEUIL performed the same on a man 1876 with
successful results. PAWLOW 3 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 digestion
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 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

1 Pfliiger's Arch., 50.

2 Helm, Zwei Krankengeschichten, Wien, 1803, cited from Hermann's. Handbuch,
5, part II, 39; Beaumont, "The Physiology of Digestion," 1833; Bassow, Bull, de
la soc. desnatur. de Moscou, 16, cited from Maly in Hermann's Handbuch, 5, 38; Ver-
neuil, see Ch. Richet, " Du sue gastrique chez 1'homme," etc. (Paris, 1878), 158.

3 Pawlow, Die Arbeit der Verdauungsdriisen (Wiesbaden, 1898), where the works
of his pupils are also mentioned. See also Er^ebnisse der Physiologic, 1, Abt. 1.

438 DIGESTION.

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 sensation
of satisfaction and pleasure on partaking it — and the chemical moment,
the action of certain chemical substances on the mucous membrane 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 continuous 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 certain unknown
extractive substances contained in meat and meat extracts, in impure
peptone, and also, it seems, in milk. According to HERZEN and RAD-
ZIKOWSKI l and others, alcohol is also a strong agent in producing a flowr
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 recent deter-
minations2 it would seem as if the concentration of the carbonate as well
as of sodium chloride exercises a certain influence, so that a weaker con-
centration is indifferent or retarding, while somewhat stronger concen-
tration has an accelerating action upon secretion, though investigators
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 (BoRissow, STRASHESKO 3) . 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 sub-
stances, such as egg-albumin, which act as chemical stimulants, cannot
be digested by the " psychical " secretion, but may perhaps cause a chem-
ical secretion by their decomposition products.

The quantity of juice secreted during digestion is proportional to the
quantity of food, and the secretion of gastric juice may also be influenced
by the kind of food. This action of various foods — meat, bread, and
milk — may be arranged in progressive series as follows:

Acidity.

Digestive Activity.

Duration of Secretion.

1.

Meat.

Bread.

Bread.

2.

Milk.

Meat.

Meat,

3.

Bread.

Milk.

Milk.

1 Pfliiger's Arch., 84, 513.

2 See Rozenblatt, Bioch. Zeitschr., 4; Mayeda, ibid., 2; Pimenow, Bioch. Centralbl.,
6; Lonquist, Maly Jahresb., 36.

3 Borissow, Arch. f. exp. Path. u. Pharm., 51; Strashesko, see Biochem. Centralbl.,
4, 148.

SECRETION OF GASTRIC JUICE. 439

The acidity is greatest with a meat diet and lowest with bread; the
quantity of enzyme is, on the contrary, highest with a bread diet and
lowest with milk.

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 exception
that in this case the retarding action is observed only in the first hours
of digestion. According to PIONTKOWSKI 1 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 secretion of
juice is stopped, as just at this time the soaps are being formed. Accord-
ing to FROUIN the food in the intestine produces a secretion of gastric
juice which continues after the action of the psychic moment has ceased.
LECONTE 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 KRYSHY-
SCHKOWSKY3 have 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. 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 peritoneal injection of an extract of the salivary glands
of dogs produces a secretion of gastric juice.

We know very little positively in regard to the gastric secretion in
man. According to the earlier authorities the irritants may be mechanical,
thermic, and chemical. Among the chemical excitants we include
alcohol and ether, which in too great a concentration bring about no

1 See Biochem. Centralbl., 3, 660.

2 Frouin, Compt. rend. soc. biol., 53; Leconte, La Cellule, 17.

3 Gross, Bioch. Centralbl., 5, 669; Kryshyschkowsky, Maly's Jahresb., 36, 403.

4 Edkins, Journ. of Physiol., 34; Hemmeter, Bioch. Zeitsclir., 11.

440 DIGESTION.

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 contra-
dictory.

The question as to how far the observations made by PAWLOW and
his school can be applied to man is of special interest. Many observa-
tions on this question have been collected 1 and they compare favorably
with the observations made upon dogs. Thus in man a psychic secre-
tion of gastric juice can also be brought about, and it has also been observed
that it can be stopped by emotions. As in dogs, so also in man, after
sham feeding, a secretion takes place after a pause, whose duration 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 SHULE, 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 proven without doubt, but we are not agreed 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
secretion of juice. On the other hand FROUiN3 observed that the intro-
duction of saliva into the large stomach of dogs acts favorably upon
the secretion in the small stomach (see p. 437), 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

1 Hornborg, 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.

2 The literature may be found in Umber's work, 1. c.

3 Compt. rend. soc. biol., 62.

GASTRIC JUICE. 441

from residues of the food or from mucus and saliva, is a clear, or only
very faintly cloudy, and nearly colorless fluid of an insipid, acid taste
and strong acid reaction. It contains, as form-elements, glandular cells
or their nuclei, mucus-corpuscles, and more or less changed columnar
epithelium.

The acid reaction of the gastric juice depends on the presence of free
acid, which, 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 1 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 acicl of the stomach is lactic
acid, according to GMELix.2 The quantity of free hydrochloric acid in_
the gastric juice is,. according to PAWLOW and his pupils, in dogs 5-6 p. m.r
and in cats an average of 5.20 p. m.'HCl. 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, SOMMERFELD 3) that the amount of hydro-
chloric 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 physical methods
are nearly identical with those obtained by titration (P. FRANCKEL 4).

As chief organic constituent, perfectly fresh gastric juice (of dogs)
contains a very complex substance (or perhaps a mixture of substances)
which coagulates on boiling and which separates on strongly cooling
the juice. This substance is considered, by certain experimenters (NENCKI
and SIEBER, and PAWLOW) as the conveyor of the several ferment actions
of the gastric juice, i.e., the pepsin as well as the rennin action. It con-
tains lecithin and chlorine, and yields nucleoprotein, proteose, purine
bases, and pentose as cleavage products (NENCKI and SIEBER 5).

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 Bidder and Schmidt, Die Verdauungssafte, etc., 44; Richet, 1. c.; Contejean, Con-
tributions a I'Stude de la physiol. de 1'estomac, Theses, Paris, 1892.

2 Pfliiger's Arch., 90 and 103.

3 See Richet, 1. c.; Contejean, 1. c.; Verhaegen, "La Cellule," 1896 and 1897; Som-
merfeld, Bioch. Zeitschr. 9, and also footnote 1, page 440, and the literature on the
estimation of hydrochloric acid in the gastric juice contents (p. 465).

4 Zeitschr. f. exp. Path. u. Therap., 1.

5 Zeitschr. f. physiol. Chem., 32.
"I.e.

442 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 quan-
tity 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 in a liter of gastric juice of a dog.

In the ash of human gastric juice after sham-feeding ALBu4 found
356.2 p. m. K2O; 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
nearly 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. Accord-
ing to KLUG and WROBLEWSKI 5 the pepsins found in man and various
higher animals are somewhat different, an observation which accord-
ing to the experience of HAMMARSTEN is very probable. 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 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

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.

5 Klug, Pfluger's Arch, 60; Wroblewski, Zeitschr. f. physiol. Chem., 21.

PEPSIN. 443

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 retard-
ing 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. SCHOUMOW-
SIMANOWSKI, NENCKI and SIEBER, and also PEKELHARING, have designated
as the true enzyme the substance containing chlorine, which they obtained
by strongly cooling the gastric juice. That this precipitate is .not a
chemical individual, and hence cannot be pepsin, follows from the investi-
gations 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. FRIED-
EN THAI, and MIYAMOTA 3 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 there-
with, 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
BIERNACKI 4 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 (GROBER5). Pepsin in acid solution is destroyed by heating
to 65° C. for five minutes. 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

1 Glaessner, Hofmeister's Beitrage, 1; Klug, Pfliiger's Arch., 92; Reach, Hofraeis-
ter's Beitrage, 4; Pekelharing, Arch, des scienc. hiolog., St. Petersbourg, 11; Pawlow-
Festband, 1904; Bergmann, Skand. Arch. f. PhysioL, 18.

2 Zeitschr. f. Biologic, 44.

3 Brucke, 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 Zeitschr. f. Biologic, 28.

5 Arch. f. exp. Path. u. Pharm., 51.

444 DIGESTION.

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 renriin action (HAM-
MARSTEN l) . 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 neutralized
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 reaction 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. HCJ). 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 transparent before
it dissolves. Unboiled fibrin swells up in a solution containing 1 p. m.
HC1, forming a gelatinous mass, and does not dissolve at ordinary tem-
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 per-
ceptibly changed by dilute acid (2-4 p. m. HC1) at the temperature of
the body in the course of several hours. . But the simultaneous presence

1 Zeitschr. f. physiol. Chem., 56. 2 Ibid., 55.

PEPSIN AND PEPSIN TESTS. 445

of pepsin causes 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, £, £, |, ^,
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> i> TB> of one series is digested in the same time as tests p = l, ^, i 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
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-

1 Griitzner, Pfliiger's Arch., 8 and 106. See also A. Korn, " Ueber Methoden Pepsin
quantitativ zu bestimmen," Inaug.-Dissert., Tubingen, 1902.

443 DIGESTION.

ing influence upon pepsin digestion, is to be tested, then the liquid must be first
properly diluted with 2-4 p. m. hydrochloric acid (NIERENSTEIN and SCHIFF *).

Objections have been raised against these methods from several sides, espe-
cially by GRUTZNER, but they can be recommended for practical purposes as
being simple and rather accurate. 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 precipitatipn 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.

JACQBY 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 (T^ 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 acids are unequal in their action ; hydrochloric acid shows
in slight concentration, 0.8-1.8 p. m., 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-
tory.6 Sulphuric acid, it seems, has a weaker action than the other

1 Mett, see Pawlow, I.e., 31; Nierenstein and Schiff, Berl. klin. Wochenschr., 40;
Jastrowitz, Bioch. Zeitschr., 2.

2 Huppert and Schiitz, Pfliiger's Arch., 80; J. Schiitz, 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.

8 See Wroblewski, Zeitschr. f. physiol. Chem., 21, and especially Pfleiderer, Pfliiger's
Arch., 66, which also gives references to other works; Larin, Biochem. Centrabl.,
1, 484; and A. Pick, Wien. Sitzungsber., M. N. Klasse, 112.

PEPSIN DIGESTION. 447

inorganic acids. The degree of acidity is also of the greatest importance.
With hydrochloric acid the degree of acidity is not the same for different
protein bodies. For fibrin it is 0.8-1 p. m., for myosin, casein, and vege-
table proteins about 1 p. m., for coagulated egg-albumin, on the con-
trary, about 2.5 p. m. The rapidity of the digestion increases, at least
to a certain point, with the quantity of pepsin present, unless the pepsin
added is contaminated by a large quantity of the products of digestion,
which may prevent its action.

According to E. ScnuTz,1 whose observations have been confirmed by
several others, the digestion products obtained in a certain time are,
within certain limits, proportional to the square root of the relative
amounts of pepsin (the ScnuTZ-BoRissow rule) while, as explained in
detail in Chapter II, under certain conditions another rule exists where
the quantity digested increases in proportion to the quantity of enzyme.
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 produc'ts of digestion has a retarding action on
digestion, 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 digestion takes place very slowly
at this temperature. With increasing temperature the rapidity of diges-
tion also increases until about 40° C., when the maximum is reached.
According to the investigations of FLAUM 3 it is probable that the rela-
tion between proteoses and peptones remains the same, irrespective
of whether the digestion takes place at a low or high temperature, so
long as the -digestion is continued for a long enough time. If the swelling
up of the protein is preve'nted, 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 (PFLEID-
ERER), retard digestion, while arsenous acid promotes it (CHITTENDEN),
and hydrocyanic acid is relatively indifferent. 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. ^ normal salt solutions), J. SCHUTZ 4

1 Zeitschr. f. physiol. Chem., 9. 3 Zeitschr. f. Biologie, 28.

2 Journ. of Physiol., 14. 4 Hofmeister's Beitrage, 5.

448 DIGESTION.

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 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 proteins insoluble or difficultly soluble combinations.
The alkaloids may also retard the pepsin digestion (CHITTENDEN and
ALLEN l) . A very large number of observations have been made in regard
to the action of foreign substances on artificial pepsin digestion, but as
these observations have not given any direct result in regard to the action
of these same substances on 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 dyspeptone by
MEISSNER. This name is therefore not only unnecessary but indeed
erroneous, as this residue does not consist of bodies related to the pep-
tones. In the digestion of proteins, substances similar to acid albumi-
nates, parapeptone (MEISSNER 2), antialbumate, and antiaibumid (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 dif-
ferent proteins, according to the researches of KLUG,3 yield on pepsin
digestion unequal quantities of the various digestive products. In the
digestion of unboiled fibrin an intermediate product may be obtained
in the earlier stages of the digestion — a globulin which coagulates at

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.

PEPSIN DIGESTION. 449

55° C. (HASEBROEK l). For information in regard to the different pro-
teoses 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
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 119). 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 boiling
with a mineral acid. Mucoids from tendons, cartilage, and bones dissolve,
according to POSNER and GiES,2 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 com-
bination of proteid with glucothionic acid (Chapters VII and VIII). The
solution contains primary and secondary mucoproteoses and mucopep-
tones. The former contain glucothionic acid, but the latter do not.
Elastin is dissolved more slowly and yields the above-mentioned sub-
stances (page 116). Keratin and the epidermal formations are insoluble.
The nucleins are dissolved with difficulty, and the cell nuclei, therefore,
remain in great part undissolved in the gastric juice. 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 membrane of the plant-cell is not dissolved. Oxy hemoglobin
is changed into hoimatin 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 fat, but, on
the contrary, dissolves the cell-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
proceed without the catalytes.

1 Zeitschr. f. physiol. Chem., 11. 2 Ainer. Journ. of Physiol., 11.

450 DIGESTION.

Chymosin (RENNIN) and Parachymosin. Besides the enzyme called
rennin (chymosin) which is found in the calf's stomach, there exists,
according to BANG/ another chymosin, the parachymosin, which is the
rennet enzyme of the pig and human stomach. This latter occurs in
the gastric juice of man under physiological conditions, but may be
absent under special pathological conditions (SCHUMBURG, BOAS, JOHN-
SON, KLEMPERER2). Chymosin 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 substance,
a rennin zymogen, occurs, which is converted into rennin by the action
of an acid (HAMMARSTEN). We have no knowledge as to whether the
rennet enzyme found in different animals is chymosin or parachymosin.
That the chymosin occurring in the calf's stomach is not identical with
that found in certain animals, for example the pike, follows without
question from the experience of HAMMARSTEN. Enzymes acting like
rennin are also found in the blood and several organs of higher animals,
as well as in invertebrates. Similar enzymes also occur widely diffused
in the plant kingdom, and numerous micro-organisms have the power
of producing rennin enzymes. Antibodies to the rennet enzymes, anti-
rennins, also occur in the animal kingdom, as shown by HAMMARSTEN
and RODEN in blood-serum, and may be produced in the animal body
by the introduction of rennin into the latter (MORGENROTH 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 concen-
tration. If an active and strong infusion of the gastric mucosa in water
containing 3 p. m. HC1 is heated to 40-45° C. for 48 hours, the rennin
is destroyed, while the pepsin remains. A pepsin solution free from
rennin can be obtained in this way. Rennin is characterized by its
physiological action, which consists in coagulating milk or a casein solu-
tion containing lime, if neutral or very faintly alkaline. The law of the
action of this enzyme is different from that of the action of pepsin. As

1 Deutsch. med. Wochenschr., 1899, and Pfliiger'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 See R6d<§n, Upsala Lakaref. Forh., 22; Morgenroth, Centrabl. f. Bakter., 26
and 27.

CHYMOSIN; PARACHYMOSIN. 451

specially shown by FuLD,1 within certain limits, the coagulation t,
multiplied by the quantity of rennin, I, equals a constant, k. As shown
by BANG, this law does not apply to parachymosin.

Parachymosin differs from chymosin by being much more resistant
toward acids, but is more readily destroyed by alkalies. Calcium
chloride accelerates the casein coagulation with parachymosin very
much more than with chymosin. GEWIN 2 has raised objections to these
points of difference between chymosin and parachymosin, although the
proofs that he puts forth are still not so conclusive that we have
sufficient ground to doubt the existence of parachymosin.

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 SAWJ ALOW3 and GEWIN, consider, on the contrary, that the coagula-
tion of milk is only a pepsin action and indeed as the first step in the
beginning of proteolygis, namely the beginning of peptic digestion of
casein.

The simultaneous occurrence in the animal and plant kingdoms of
enzymes having a proteolytic and rennet action indicates an identity
of both enzymes and enzyme actions and the parallelism of the pepsin
and rennet action. 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.

The pathological cases in man, if the observations are reliable, where
only one enzyme action occurs, seems to suggest the identity of the
action of both enzymes. In opposition, however, is 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 solutions can be made which coagulate milk but do not have
digestive action in acid reaction (H AMMARSTEN) . The observations of

1 Hofmeister's Beitrage 2; Bang, 1. c.

2 Zeitschr. f. physiol. Chem., 54.

3 The recent literature on this question can be found in Hammarsten, Zeitschr.
f. physiol. Chem., 56.

452 DIGESTION.

DuccHESCHi,1 that pepsin but no chymosin occurs in the stomach of the
Didelphys, also conflict with the identity of the two enzymes.

The views of NENCKT and SiEBER2 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 preparation of chymosin solutions free from pepsin
and of pepsin solution free from chymosin see the work of HAMMARSTEN.3

Plastein. As mentioned on page 134, D ANILE WSKY first showed the
power of rennin solutions to cause a partial coagulation of proteoses
and of converting them into so-called plastein. It is unknown whether
this action is due to pepsin, chymosin or to another enzyme.

Gastric Lipase (STOMACH STEAPSIN) . F. VOLHARD 4 made the discovery
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 great. 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 also,
or both, take part in the formation of free acid is disputed.5 There can
be no doubt that the hydrochloric acid of the gastric juice originates
in the chlorides of the blood, because, as is well known, a secietion
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,6 that in dogs after a sufficiently long common-salt starvation

1 Centrabl. f . Physiol., 22, 784.

2 Zeitschr. f. physiol. Chem., 32.

3 Zeitschr. f . physiol. Chem., 56.

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).

5 See Heidenhain, Pfliiger's Arch., 18 and 19, and Hermann's Handbuch, 5, part I,
" Absonderungsvorgange " ; Klemensiewicz, Wien. Sitzungsber., 71; Friinkel, Pfliiger's
Arch., 48 and 50; Contejean, I.e.; Kranenburg, Archives Teyler, Ser. II, Haarlem,
1901, and Mo. se,. Centralbl. f. Physiol., 17, 217.

8 Zeitschr. f . physiol. Chem., 10.

SECRETION PROCESSES. 453

the stomach secreted a gastric juice containing pepsin, but no free hydro-
chloric 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 WOHLGEMUTH, the quantity
of juice diminishes greatly, and on the introduction of NaCl the quantity
of juice secreted increases. According to PUGLIESE l the gastric juice
in starvation, after a certain time, has a neutral reaction, and the intro-
duction of NaCl does not now change its properties. In the secretion of
free acid it is assumed by PUGLIESE that the gland cells, which decom-
pose the chloride, have sufficient amounts of protein at their disposal.
On the introduction of alkali iodides or bromides, KULZ, NENCKI and
ScHOUMow-SiMAXowsKi 2 have shown that the hydrochloric acid of the
.gastric juice is replaced by HBr, and to a less extent by HI. According
to KOEPPE the seat of formation of hydrochloric acid is not the blood
or the glands, but the interior of the stomach in the immediate neigh-
borhood of the wall. The hydrochloric acid is assumed to be produced
from the chlorides of the food, as the semipermeable wall is not permeable
for the Cl ions, but is for the Na ions and for the II ions. As the Na ions
leave the stomach contents, an equivalent quantity of H ions wander
from the blood through the stomach wall into the interior of the stomach
and combine with the Cl ions. This theory is difficult to reconcile with
the fact that in dogs with sham feeding the empty stomach secretes
.abundant juice. Other objections have also been raised against this
by BENRATH and SACHS. The secretion of free hydrochloric acid from
the alkaline blood has been explained in other ways (MALY, BUNGE, L,
SCHWARZ), but as yet no satisfactory theory has been suggested.3

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. LANGLEY4 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 solution of
3 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

1 Wohlgemuth, Arbeiten aus d. pathol. Institute, Berlin, 1906; Pugliese, Maly's
Jahresb., 30, 394.

2 Kiilz, Zeitschr. f. Biologic, 23; Nencki and Schoumow, Arch, des sciences biol.
de St. Petersbourg, 3.

3 Koeppe, Pfliiger'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.

4 Schiff, Le';ons sur la physiol. de la digestion, 1867, 2; Langley and Edkins,
Journ. of Physiol., 7.

454 DIGESTION.

and possibly also of a steapsinogen, in the mucous coat has been men-
tioned above.

According to HERZEN and his collaborators l we must differentiate between
pepsinogenic and other bodies accelerating the flow of juice. To the first belong
mulin and glycogen, while alcohol belongs to the latter class of bodies. Dextrin not
only accelerates the flow of juice, but also acts as a pepsinogenic, especially as the
latter. Meat extract which has both actions is especially a flow-accelerator.
The pepsinogenic action consists in converting the zymogen into pepsin, and in
this way increases the quantity of pepsin ; the flow-accelerating substances increase
the quantity of secreted juice.

The question in what cells the two zymogens, especially the propepsin,
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.2

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. This secretion
is alkaline, viscous, jelly-like, rich in mucin, of a specific gravity of 1.009-
1.010, and containing 16.5-20.5 p. m. solids. It habitually contains
pepsin, which has been proven by HEIDENHAIN by observations on a
permanent pyloric fistula, and the amount may sometimes be considerable.
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 opera-
tion, because the stomach readily yields an alklaline juice instead of an
acid one under abnormal conditions. The reports of HEIDENHAIN and
KLEMENSIEWICZ have nevertheless been substantiated by AKERMANN,
KEESTEFF, SCHEMIAKINE and others.3

The secretion of gastric juice under different conditions ma}r vary
considerably. The statements concerning the quantity of gastric juice
secr'eted in a certain time are therefore unreliable. ROSEMANN 4 ob-
served on sham feeding in dogs a secretion of 917 cc. in the course of
3J hours — a considerable quantity.

The Chyme and the Digestion in the Stomach. By means of the
chemical stimulation caused by the food, a copious secretion of gastric

Pfliiger's Arch., 84.

See footnote 5, p. 452.

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.

Pfliiger's Arch., 118.

CHYME AND THE DIGESTION IX THE STOMACH. 455

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, such as HOFMEISTER
and SCHUTZ, MORITZ, CANNON, SCHEMIAKINE/ and others, on the move-
ments 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 by 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
contraction 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) GRUTZNER 2 later showed that when
the animals 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 laj^ers are so
arranged that the food first taken is found in direct contact with the
mucosa, while the food taken later is inclosed 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 oaly 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. The
fundus part is therefore less a digestion-organ than a storage-organ, and

1 Hofmeister and Schiitz, Arch. f. exp. Path. u. Pharm., 20; Moritz, Zeitschr. f.
Biologic, 32; Cannon, Amer. Journ. of Physiol., 1; Schemiakine, 1. c.

2 See Ellenberger, Pfliiger's Arch., 114, and Scheunert, ibid., 144; Griitzner, ibid.,
106.

456 DIGESTION.

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. Accord-
ing 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.

The fact that only that part of the ingesta lying on the mucous mem-
brane 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 DA.Y1 by
special experiments upon animals. The occurrence of sugar and dextrin
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 abundant digestion
of starch takes place in the various stomachs, due 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 oc 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.

This alternate action, according to CANNON 2 is due to the fact that
the acid in the pylorus which acts upon the sphincter and makes possible
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

1 Ellenberger and Hofmeister, Maly's Jahresb., 15 and 16; Cannon and Day, Amer.
Journ. of Physiol., 9.

2 Amer. Journ. of Physiol., 20.

DIGESTION IN THE STOMACH. 457

is neutralized the contractions of the sphincter cease and the passage
of new portions of the chyme occur. If the flow of bile and pancreatic
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 accord-
ing to conditions, and in consequence the reports of observers are
widely divergent. BEAUMONT l 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 impor-
tant, 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 cf its subdivision, and its action upon the
gastric secretion, upon the pyloric reflexes, etc.

The observations of BOLDYREFF 2 on the action of fats are con-
clusive 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 the1 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 intestinal juice migrate
from the intestine into the stomach so that the digestion 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
the researches of E. ZuNZ,3 LONDON 4 and his co-workers. LONDON,
POLOWZOWA and SAGELMANN 6 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. According to these

1 The Physiology of Digestion, 1833,

1 Pfliiger's Arch., 121. See also Abderhalden and Medigreceanu, Zeitschr. f. physiol.
€hem., 57.

3 E. Zunz, Hcfmeister's Eeitrage, 3; Annal de la soc. roy. des sceinc. med. Bruxelles,
12, 13- and Memoires publ. par 1'Acad. roy. Belg., 1906, 1907, and 1908.

4 The numerous works of London and co-workers will be found in Zeitschr. f .
physiol. Chem., 45-53, 55-57.

5 London with Polowzowa, Zeitschr. f. physiol. Chem., 49, with Sagelmann, ibid., 52.

458 DIGESTION.

authors the stomach has a sort of " selective capacity," but this is strongly
disputed by SCHEUNERT and GRIMMER. 1 Nevertheless the researches
of CANNON2 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.

The reason why different food-stuffs leave the stomach with unequal rapidity
is explained by CANNON by the above-mentioned action of the hydrochloric acid
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 there-
fore 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 facili-
tate 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 an abundant secretion
of gastric juice, and thereby facilitates digestion. The accelerating
action of alcohol upon the flow of gastric juice has already been men-
tioned on page 439.

In regard to the importance of the stomach we used to be of the gen-
eral opinion that an abundant peptonization of protein does not occur in
the stomach, and that the food rich in protein is more likely to be chiefly
prepared in the stomach for the real digestion in the intestine. That the

1 Scheunert, Zeitschr. f. physiol. Chem,, 51; Grimmer, Bioch. Zeitschr., 3.
3 Amer, Journ. of Physiol., 12 and 20.

DIGESTION IN THE STOMACH 459

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 new-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 overbur-
dening 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 individual animals of
the same species, depending upon the fineness or coarseness of the food,
upon the greater or less rapidity with which peptonization 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 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-workers 4 have shown
that in the stomach proteoses and peptones, besides so-called rest bodies,
are formed, but no amino-acids, or at least in any mentionable quan-
tity. In like manner we must agree in the belief that a part of the
protein always leaves the stomach undigested and that the chief mass,
about 80 per cent, passes into the intestine more or less digested.
LONDON and SANDBERG 5 indeed have from their experiments with
gliadin or with egg albumin (LONDON), suggested the proposition that
a certain percentage of the partaken protein is always digested, irre-

1 Tobler, 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 3 and 4, p. 457.

2 Rosenfeld, Ueber die Eiweissverdauung im Magen des Pferdes, Inaug.-Dissert.,
Dresden, 1908.

3 Lotsch, Zur Kenntnis der Verdauung von Fleisch im Magen und Diinndarm des
Schweines, Inaug.-Dissert. Freiburg i. Sa., 1908.

4 Abderhalden and London, with Kautsch, Zeitschr. f. physiol. Chem., 48, with
L. Baumann, ibid., 51, and with v. Korosy, ibid., 51.

5 Zeitschr. f. physiol. Chem., 56

460 DIGESTION.

spective of the quantity partaken of. We express this by the formula,
— = K, where q is the quantity of protein fed, Fr is the undi-
gested part and K a constant. For the division of the digested protein
into the three groups — proteoses, peptones and rest bodies, figures have
also been given. On comparing the results obtained by different investi-
gators one finds such great discrepancies that no positive conclusions
can be drawn. Still it seems to be certain that the rest-bodies occur
to a very small extent as compared with the proteoses and peptones.
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 con-
tradictory in regard to the absorption of the decomposition products of
the proteins in the stomach. While several investigators, like TOBLER,
LANG, COHNHEIM, 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, CAR-
VALLO and PACHON), and 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 possible to keep the animal alive, well
fed, and strong for a shorter or longer time. This is also true for human
beings.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 favorable circum-
stances, and according to STRAUSS and BIALOCOUR the limit of lactic*

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.) ; Grohe,
Arch. f. exp. Path. u. Pharm. 49; in regard to the case in man, see Schlatter in
Wroblewski, Centralbl. f. Physiol. 11, p. 665, and the surgical journals.

FERMENTATION IN THE STOMACH. 461

acid fermentation lies at 1.2 p. m. hydrochloric acid united to organic
bodies. The hydrochloric acid of the gastric juice has unquestionably
an antifermentative action, and also, like all dilute mineral acids, an
antiseptic action. This action is of importance, as many pathogenic
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 toxalbumins,
such as tetanotoxine and diphtheria toxine, is also of great interest
(XENCKI, SIEBER, and ScHouMowA1).

Because of this antifermentative and antitoxic action of gastric
juice it is considered that the chief importance of the gastric juice lies
in its antiseptic action. The fact that intestinal putrefaction is not
increased on the extirpation of the stomach, as derived from experiments
made on man and animal,2 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 generated 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 CO2, and
only a small quantity, 0.8-6.1 per cent, of oxygen. ScniERBECK3 has
shown that a part of the carbon dioxide is formed by the mucous mem-
brane of the stomach. The tension of the carbon dioxide in the stomach
corresponds, according to him, to 30-40 mm. Hg in the fasting condi-
tion. 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 SCHIERBECK also
found that the carbon-dioxide tension is considerably increased by pilo-
carpine, 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

1 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, Pfluger'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 handbooks of bacteriology.

2 See Carvallo and Pachon, 1. c., and Schlatter in Wroblewski, 1. c.

3 Planer, Wien. Sitzungsber., 42; Schierbeck, Skand. Arch. f. Physiol., 3 and 5.

462 DIGESTION.

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 gastric
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 can-
not be contradicted; but the reason is not to be found in the neutral-
ization of the acid. The investigations of FERMI and OTTE l show that
the blood circulation acts in an indirect manner by the normal nourish-
ment 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 the secretion of the antipepsins discovered
by DANILEWSKY, HANSEL, and WEINLAND, but this is hard to understand.
Undoubtedly bodies occur in the gastric mucosa which can inhibit the
action of pepsin, but whether these bodies are of an enzymotic nature
or not is undecided. WEINLAND'S antipepsin is related to the enzymes
because it is thermolabile, while the antipepsin of DANILEWSKY, HAN-
SEL, and O. SCHWARZ 2 is resistant toward heat and can hardly be con-
sidered as an enzymotic body. This is true for at least the thermo-
8tabile antipepsin of SCHWARZ, which does not give the biuret reaction.
Without mentioning the still unknown nature of these bodies, the natural
gastric juice, as well as an acid infusion of the mucosa, has such a strong
digestive action that the retarding action of the antipepsin can only be
shown under special conditions, and it is therefore difficult to conceive
how the antipepsin could have a protective action in life.

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 chymosin), 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 probable, 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 445, 446. 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

1 Pavy, 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.

2 See Hansel, Biochem. Centralbl., 1, p. 404, and 2, p. 326; Weinland, Zeitschr. f.
Biologic, 44; Schwartz, Hofmeister's Beitrage, 6.

INVESTIGATION OF THE GASTRIC CONTENTS. 453

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^OLIN 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-vanillin, and
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 proven. 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 oxidized into aldehyde and formic acid on careful oxida-
tion with sulphuric acid and manganese dioxide. The aldehyde is detected by

1 Bioch. Zeitschrift, 8.

464 DIGESTION.

its forming iodoform with an alkaline iodine solution or by its forming aldehyde-
mercury with NESSLER'S reagent. CRONER and CRONHEIM l 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 hydrochloric 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 that 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. ScntJTz.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 defined whether one wishes
to determine the actually free or the physiologically active hydro-
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 4 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
contains only free acid; if it has an ac;d 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.

1 Boas. Deutsch. med. Wochenschr., 1893, and Miinchener med. Wochenschr, 1893,
Croner and Cronheim, Berl. klin. Woschenschr., 1905. See also Thomas, Zeitschr. f.
physiol. Chem., 50.

2 Block. Zeitschr., 12.

3 Alb. Miiller, Deutsch. Arch. f. klin. Med., 88, and Pfluger's Arch., 116; J. Schiitz,
Wien. klin. Wochenschr., 20, and Wien. med. Wochenschr., 1906 (older literature).

'Centralbl. f. d. med. Wissensch., 1889, p. 481; Pfluger's Arch., 48, and Berlin,
klin. Wochenschr., 1905, p. 1491.

GLANDS AND SECRETION OF THE INTESTINE. 465

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 a previous chapter (see estimation of the alkalinity of the blood-serum,
page 264). For this determination physico-chemical methods are necessary,
but they have not been used to any great extent for clinical purposes on account
of the difficulty in their manipulation.

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
S.IOQUIST, 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 WEINTRAND and of SAHLI.
The same applies to the tests for volatiles fatty acids.

III. 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 PONOMAREW
finds that the secretion is inactive in alkaline reaction and contains 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. SCHEUNERT and GRIMMER l
indeed found diastatic enzyme in the duodenal glands of the horse, ox,
pig and rabbit, but no proteolytic or rennin enzyme.

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 BOLDYREFF 2,
in dogs with an empty stomach a scanty secretion lasting about 15 min-
utes occurs at regular intervals for about two hours. According to this
experimenter, during gastric digestion the juice is periodically but less
abundantly secreted as the time interval is much longer, namely three,
four to 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 DELEZENNE and FROTJIN

1 Griitzner, Pfliiger's Arch., 12; Glaessner, Hofmeister's Beitrage, 1; Ponomarew,
Biochem. Centralbi., 1, 351; Abderhalden and Rona, Zeitschr. f. physiol. Chem., 47;
Scheunert and Grimmer, cited in Bioch. Centralbl., 5, 673.

2Thiry, Wien. Sitz.-Ber., 50;. Vella, Molleschott's Untersuch., 13; Boldyreff,
Zeitschr. f. physiol. Chem., 50.

466 DIGESTION.

show without question that the passage of chyme into the intestine increases
the secretion of the intestinal juice. The acid causes a formation of secre-
tin (see below), and this produces, according to the above investigators,
a secretion of intestinal juice. As the secretin undoubtedly also increases
the secretion of pancreatic juice and as this latter, according to PAWLOW,
by its action upon the intestine excites a secretion of intestinal juice,
it is difficult to understand why, according to BOLDYREFF, the secretion
of intestinal juice should be so weak during the entire gastric diges-
tion, sometimes so weak that little if any juice is secreted. Soaps,
chloral, ether and on intravenous injection, also intestinal juice or an
extract of the intestinal mucosa (FROUIN), are chemical excitants of
intestinal juice. Several salts, NaCl, Na2SO4, and others, may cause
an abundant secretion of fluid into the intestine when injected intra-
venously or subcutaneously, 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 (GAMGEE *).

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 2 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.

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 gelatinous ;
in the lower part it is more fluid, with gelatinous lumps or flakes (Ron-
MANN). 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 Na2CO3, 4.8-5 and 4-5
p. m. respectively (GUMILEWSKI, R6HMANN3). The intestinal juice
of the lamb corresponded to an alkalinity of 4.54 p. m. Na2CO3. It

1 Delezenne and Frouin, Compt. rend. soc. biol., 56; Frouin, ibid., 56 and 58;
MacCallum, University of California Publications, 1, 1904; Gamgee, Pbysiol. Chem-
istry, 2, 410 (literature).

2 Journ. de Physiol. et d. path, gen., 1902 and 1904.

8 Gumilewski, Pfliiger's Arch., 39, Rohmann, ibid., 41.

INTESTINAL JUICE. 467

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 DEMANT, TURBY and MANNING, H.
HAMBURGER and HEKMA and NAGANO 1 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. NaCl. The
determination of the freezing-point was-0.62° (HAMBURGER and HEKMA).

The intestinal juice of the dog contains, according to BoLDYREFF,2
a lipase which acts especially upon emulsified fat (milk) , and is different
from pancreas lipase, in that its action is not accelerated by bile.
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 pep-
tones. It also possibly contains a nudease, and it also has a faint
amylolytic action. The juice, and to a high degree the mucous
coat, contains invertase and waltase, which fact has been substan-
tiated by the observations of PASCHUTIN, BROWN and HERON, BAS-
TIANELLI, and TEBB.S A lactose-inverting enzyme, a lactase, also occurs,
as shown by ROHMANN and LAPPE, PAUTZ and VOGEL, WEINLAND,
and ORBAN,4 in new-born infants and young animals, and also in
grown mammals which were fed upon a milk diet. 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 5) .

1 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.

2 Boldyreff, Archiv d. sciences biolog. de St. Pe'tersbourg, 11.

3 Paschutin, Centralbl. f. d. med. Wissensch., 1870, 561; Brown and Heron, Annal.
d. Chem. u. Pharm., 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.

4 Rohmann and Lappe, Ber. d. deutsch. chem. Gesellsch., 28; Pautz and Vogel,
Zeitschr. f. Biologic, 32, Weinland, ibid., 38; Orban, Maly's Jahresber., 29.

5 Frouin and Thomas, Arch, internat. de Physiol., 7; Omi, Das Verhalten des
Salizins im tierischen Organismus. Inaug.-Dissert. Breslau, 1907.

468 DIGESTION.

Besides erepsin and the other enzymes mentioned, the intestinal
mucosa also contains antienzymes, antipepsin and antitrypsin (DANILEW-
SKY and WEiNLAND1), also enterokinase or a mother-substance of the
Fame, and finally also the so-called prosecretin. These two last-men-
tioned bodies, which are closely connected with the secretion of pancreatic
juice, will be discussed in connection with this digestive fluid.

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 occurs
only in the lower parts. The kinase occurs only in the upper part of the
intestine (BOLDYREFF, BAYLISS and STARLING, DELEZENNF). Accord-
ing 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,
HEKMA, FALLOISE, and others, which, however, DELEZENNE says,2 is
formed in the leucocytes and PEYER'S glands.

BOTTAZZI 3 obtained a very complex protein from the intestinal mucosa,
which is readily soluble in water and alkali but is precipitated by acids. It
coagulates at 55° to 56° and probably also contains carbohydrate and considerable
iron. Intravenous injection of this protein brings about an abundant secretion
of saliva, pancreatic juice, bile, and intestinal juice, and promotes the peristalic
movements of the intestine.

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 polypep tides. 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

1 See footnote 2, p. 462.

2 Boldyreff, Arch. d. scienc. biolog. de St. Pe"tersbourg, 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.

3 See Biochem. Centralbl., 3. p. 65.

EREPSIN. PANCREAS. 469

(SALASKIN, KUTSCHER and SEEMAXX]). 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 nuclease, 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. Erepsin shows a great similarity to the
intracellular enzymes active in autolysis, and according to VERNON,
erepsins occur in the various tissues of invertebrates as well as vertebrates.
These tissue erepsins, which are closely related to the autolytic enzymes,
if they are not identical with them, behave somewhat differently from the
intestinal erepsin and are not identical therewith. Enzymes, haxing
an. action similar to erepsin, occur, VINES believes,2 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 certain of
the autolytic enzymes studied so far. The optimum of alkalinity is, accord-
ing to EuLER,3 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 consist
chiefly of mucus. Fistulas have also been introduced into these 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 anatomically
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 HE PATO PANCREAS. In man and in most vertebrates the for-
mation of bile, and of certain secretions, containing enzymes important
for digestion, is divided between the two organs, the liver and the pancreas.

'Cohnheim, Zeitschr. f. physiol. Chem., 33, 35, 36, and 47; Salaskin, ibid., 35;
Kutscher and Seemann, ibid., 35.

2 Bayliss and Starling, Journ. of Physiol., 30; Vernon, ibid., 30 and 33; F. Sachs,
Zeitschr. f. physiol. Chem., 46; Nakayama, ibid., 41; Vines, Annals of Botany, 18,
19. and 23

3 Zeitschr. f . physiol. Chem., 51.

470 DIGESTION.

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 homogeneous,
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 HEIDENHAIN l
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 homo-
geneous zone, which contains the repairing material, is then converted
into the granular substance. The so-called islands of LANGERHANS
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
of nudeoproteins, while the globulin and albumin occur only to a slight
extent as compared with the nucleoproteins. Among the compound
proteins is tjie substance studied and isolated by UMBER but previously
discovered by HAMMARSTEN3 and called a-proteid. This nucleoprotein
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 (#-proteid on boiling, which is much
richer in phosphorus than the nucleoprotein. 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.

1 Pfluger's Arch., 10.

2 See Diamare and Kuliabko, Centralbl. f. Physiol., 18 and 10; Rennie, ibid., 18;
Sauerbeck, Virchow's Arch., 177, Suppl.

3 Umber, Zeitschr. f. klin. Med., 40 and 43; Hammarsten, Zeitschr. f. physiol.
Chem., 19.

PANCREATIC JUICE. 471

Among the extractive bodies, which are probably in part formed by
post-mortem changes and chemical action, we must mention leucine
(butalanine) , tyrosine, purine bases in variable quantities,1 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 inves-
tigations of OIDTMANN the pancreas of an old woman contains 745.3
p. m. water, 245.7 p. m. organic and 9.5 p. m. inorganic substances.
GOSSMANN 2 found in a man 17.92 p. m. ash and 13.05 p. m. in a woman.

Besides the already-mentioned (Chapter VIII) relation to the trans-
formation of sugar in the animal 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.S

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.
During starvation the secretion almost stops, but commences again
after partaking of food and reaches its maximum, it is claimed by BERN-
.STEIN, HEIDENHAIN, and others, within the first three hours. Accord-
ing to PAWLOW and his school (WALTHER 4) this maximum is dependent
upon the character of the food. With milk diet it appears within three
to four hours, after bread diet at the end of the second hour, and with
a meat diet it arrives still sooner. The quality of the juice is also, accord-
ing to PAWLOW'S school, dependent upon the food, and the amount of
the three enzymes, diastase, trypsin, and steapsin, changes with the
variety of food. That the juice is secreted in varying amounts and
composition after various foods has been shown by many observers. On
the other hand its composition can undergo striking variation with
one and the same food (MAZURKIEWICZ), and it is difficult to find any
positive connection between the food and the composition of the juice.
In man WOHLGEMUTH observed a more abundant secretion after car-
bohydrates than after food rich in protein. GLAESSNER and POPPER 5

1 See Kossel, Zeitschr. f. physiol. Chem., 8.

2 Gossmann, Maly's Jahresber., 30; Oidtmann, cited from Gorup-Besanez, Lehr-
buch, 4th ed., 732.

3 Bernard, Lemons de Physiol., 2, 190; Ludwig, see Bernstein, Arbeiten a. d. physiol.
Anstalt zu Leipzig, 1869; Heidenhain, Pfliiger's Arch., 10, 604; Pawlow, Die Arbeit
derVerdauungsdriisen, Wiesbaden, 1898, and Ergebnisse der Physiologic, 1, Abt. 1.

4 Bernstein, 1. c., footnote 3, Walther, Arch, des sciences biol. de St. Petersbourg, 7.

5 Mazurkiewicz, Pfliiger's Arch., 121; Wohlgemuth, Berl. klin. Wochenschr., 44;
K. Glaessner and H. Popper, Deutsch. Arch. f. klin. Med., 94.

472 DIGESTION.

observed the reverse in man, that is, after carbohydrate feeding the
smallest amount of juice was secreted, and with a mixed diet about the
same amount as after the partaking of protein or fat. Objections have
also been raised in many quarters against PAWLOW'S teachings as to an
accommodation of the quantity of enzyme to the kind of food, and some
of the observations on which these teachings are based have been some-
what differently explained in the light of recent investigations on the
conditions necessary for the activation of the trypsinogen.

PAWLOW and his pupils, especially SCHEPOWALNIKOFF, have shown
that the above-mentioned (page 468) enterokinase activates the trypsino-
gen into trypsin. These observations were later confirmed by others,
by DELEZENNE and FROUIN, POPIELSKI, CAMUS and GLEY, BAYLISS and
STARLING, £UNZ, and have been further studied. The pure juice con-
tains, at least as a rule, on/y trypsinogen, and no trypsin. By mixing with
the intestinal juice, or by contact with the intestinal mucosa, the tryp-
sinogen is converted into trypsin by the k'nase. Enterokinase, which
itself has no action upon proteins, and therefore is not a proteolytic
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
PEYER'S patches and from the lymph-glands and leucocytes, hence
impure fibrin containing leucocytes acts as a kinase. These deductions
of DELEZENNE are disputed by BAYLISS and STARLING, HEKMA and others.

From the experience with enterokinase, the report as to the accom-
modation of the enzyme content of the juice to the kind of food has also
been explained by the fact that after different foods the proteolytic
power of the juice becomes unequally activated (LTNTWAREW and others).
For instance, a diet of bread and milk causes the secretion of a large
quantity of juice which is rich in trypsinogen but contains almost no
trypsin, while after a rich meat diet the secretion becomes scant and the
juice contains only trypsin but no trypsinogen. The theory based upon
the investigations of DELEZENNE, FROUIN, POPIELSKI, BAYLISS and STAR-
LING, PRYM, and others,1 that the pure juice collected under the influence
of the food contained only trypsinogen but no trypsin, opposes this

1 In regard to the literature on enterokinase, secretin, and secretion of pancreatic
juice, see O. Cohnheim, Bioehem. Centralbl., 1, 169, and S. Rosenberg, ibid., 2, 708;
Prym, Pfliiger's Arch., 104 and 107.

ACTIVATION OF THE PANCREATIC JUICE. 473

assumption. According to FROUIN l the influence of the various foods
consists in that the latter give to the juice a different activation. Accord-
ing to FROUIX, for the activation to maximum amount of trypsin the
meat juice requires 1/500—1/1000 of its volume of intestinal juice, and
bread-juice 1/20-1/10 of its volume. The question as to the influence
of the food upon the properties of the juice is still unsettled and requires
further study.

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 activation 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,
(SAWITSCH 2) .

The activation of £he 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
by the spleen has been repeatedly suggested by SCHIFF,S 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,* but he claims that this is due essentially to micro-
organisms.

The formation of lactase after the introduction of milk sugar into the intestine
as observed by WEINLAND and BAINBRIDGE, is to be considered as an intraglandular
enzyme formation in the pancreas. This is a special example of the general
rule based upon BROCARD'S researches, that the kind of food has a marked influence
upon the formation of hydrolytic ferments in the body: " c'est 1 'ailment qui fait
le ferment." This formation of lactase is denied by others such as BIERRY and

PLIMMER.5

1 Compt. rend. soc. biol., 63.

2 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.

3 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 pankre"as et la rate, Lausanne, 1901.

4 Pfliiger's Arch., 104 and 107.

5 Weinland, Zeitschr. f. Biologic, 38 and 40; Brocard, Journ. de physiol. et de
path, gen., 4; Bainbridge, Journ. of Physiol., 31. Contradictory views are held

474 DIGESTION.

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 trypsin
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 incorrect by HEKMA.1
Besides the enterokinase and the micro-organisms, there are other activa-
tors of the trypsinogen, namely liver-press-juice (WOHLGEMUTH 2) and
certain amino-acids and finally, as first shown by DELEZENNE and then
by ZUNZ by further investigations, especially the lime salts.3 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 neces-
sary 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. Accord-
ing 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 trypsino-
gen. This enzyme is .also activated by enterokinase.

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,4 the trypsin, on the
contrary, is a combination of the kinase and trypsinogen, analogous to
the haBmolysins, which according to EHRLICH'S side-chain theory are
combinations between a complement and an amboceptor.

The specific excitants for the secretion of pancreatic juice are, accord-
ing to PAWLOW and his collaborators, acids of various kinds — hydro-
chloric 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. Water, which causes a secre-
tion of acid gastric juice, likewise becomes, indirectly, a stimulant for the

by Bierry, Compt. rend., 140, and Compt. rend. soc. biolog., 58, and Plimmer, Journ.
of Physiol., 34.

1 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.

2 Bioch. Zeitschr., 2.

3 Delezenne, Compt. rend. soc. biol., 59, 60, 62, 63; Zunz, footnote 2, p. 473.

4 Bayliss and Starling, Journ. of Physiol., 30 and 32, which also cites the other
investigators. See also footnote 1, p. 472.

SECRETIN AND ACID ACTION. 475

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 intes-
tine, causing a secretion of juice. That a reflex action is here produced
is not contradicted by the investigations of POPIELSKI, WERTHEIMER
and LEPAGE, FLEIG,* and others. According to the researches of BAY-
LISS 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 hydro-
chloric-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 secretin, which accord-
ing to BAYLISS and STARLING 2 is the same in all vertebrates examined, is
not destroyed by heat; it is therefore not identical with enterokinase, and
is not considered an enzyme. It is formed from another substance,
prosecretin, by the action of acids. According to DELEZENNE and POZER-
SKi3 secretin occurs as such in the intestinal mucosa, and the acids act
only by the destruction 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. On the introduction of intestinal extract into-
the animal body the blood-pressure is suddenly and very strongly reduced,
the blood becomes uncoagulable and a secretion of saliva, tears, pan-
creatic juice, gastric juice and bile (not constantly) occurs. The same
action is also produced by the injection of an aqueous extract of other
organs, and the action is not due to choline, but to a special substance,
which is called vasodilatin, and which also occurs in WITTE'S peptone.
The proteoses and peptones are themselves without action and the view
of PICK and SPIRO (page 311), that their action upon the coagulation
of the blood is due to a contaminating substance, a peptozyme, seems to
be correct. POPIELSKI and PANEK have suggested another method of

1 Fleig, Centralbl. f. Physiol., 16, 681, and Compt. rend. soc. biol., 55. See also
foot-note 1, p. 472.

2 Journ. of Physiol., 29.

3Delezenne and Pozerski, Compt. rend. soc. biol., 56; Popielski, Centralbl. f.
Physiol., 19.

476 DIGESTION.

preparing vasodilatin.1 GIZELT disputes the occurrence of a specific
secretin and considers this body a peptone, v. FURTH and SCHWARZ 2
&lso 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 a secretin exciter.

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,
BABKiNE3), 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 4 found that by maceration of the mucosa
of the upper part of the duodenum with soap solution a substance goes
into solution which he calls sapokrinin and which when introduced into
the blood brings about a strong secretion of pancreatic juice. This
sapokrinin, 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 produced by a special secretin, chloral
secretin. The secretion of pancreatic juice can also be increased by
alcohol and FLEIG 5 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.

The estimation as to the quantity of pancreatic juice secreted in the
twenty-four hours differs very much. According to the -determina-
tions of PAVVLOW and his collaborators, KUWSCHINSKI, WASSILIEW, and
JABLONSKY,6 the average quantity (with normally acting juice) from a
permanent fistula in dogs is 21.8 cc. per kilo in the twenty -four hours.
GLAESSNER 7 found in man in one case 600-800 grams in the 24 hours.

The pancreatic juice of the dog is a clear, colorless, and odorless
alkaline fluid which when obtained from a temporary fistula is very

1 Popielski, Pfliiger's Arch., 128; Popielski and Panek, ibid., 128.

2 Gizelt, Pfliiger's Arch., 123; v. Fiirth and Schwarz, ibid., 124 (literature on
secretin).

3 Arch des scienc. biol. de St. Pe"tersbourg, 11, and Zeitschr. f. physiol. Chem., 56.

4 Compt. rend. soc. biol., 55, and Journ. de Physiol. et de Pathol. ge"n., 1904.

5 Wertheimer and Lepage, Compt. rend. soc. biol., 52; Fleig, ibid., 55; Falloise,
Bull. Acad. Roy. Belg., 1903.

6 Arch, des sciences de St. Pe"tersbourg, 2, 391. The previous claims of Keferstein
and Hallwachs, Bidder and Schmidt, and others may be found in Kiihne, Lehrbuch,
114.

7 Zeitschr. f . physiol. Chem., 40.

PANCREATIC JUICE. 477

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 KUHNE. Besides the above-
mentioned bodies the pancreatic juice invariably contains small quan-
tities of leucinCj 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 SAWITSCH/ 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 2 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 3 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.4 The secretion was clear, foamed readily,
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.

1 Mazurkiewicz, 1. c.; Babkine and Sawitsch, Zeitschr. f. physiol Chem., 56.

2 Cited from Maly in Hermann's Handbuch der Physiol., 5, Theil II, 189.

3 Journ. of Physiol., 31.

4 Zeitschr. f. physiol. Chem., 40. See also Ellinger and Kohn, ibid., 45, and the
investigations upon cystic fluids from the pancreas by Schumm, ibid., 36, and Murray
and Gies, American Medicine, 4, 1902.

478 DIGESTION.

Amylopsin or pancreatic diastase, which, according to KOROWIN
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 energetically
upon boiled starch, and according to KUHNE also upon unboiled starch,
especially at 37° to 40° C., and according to VERNON l best at 35° C. It
forms, similarly to the action of saliva, besides dextrin, chiefly isomaltose
and maltose, with only very little dextrose (MUSCULUS and v. MERING,
KULZ and VoGEL2). The dextrose 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 veiy small quantities of hydrochloric acid, but is diminished by larger
amounts. VERNON, GRUTZNER, and WACHSMANN4 find that the action
is indeed accelerated by very small quantities of hydrochloric 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.)

Steapsin or Fat-splitting Enzyme. The action of the pancreatic
juice on fats is twofold. First, the neutral fats are split into fatty acids
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 j uice 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 gly-
cerin 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.

1 Korowin, Maly's Jahresber., 3; Zweifel, footnote 3, p. 431; Kiihne, Lehrbuch,
117; Vernon, Journ. of Physiol., 27.

2 See footnote 1, p. 432.

3 See Tebb, Journ. -;f Physloi., 15; Bierry and Terroine, Compt. rend. soc. biolog.,
58; Bierry, ibid., G2.

4Rachfcrd, Amer. Journ. of Physiol., 2; Vernon, 1, c,; Grutzner, Pfliiger's
Arch., 91.

STEAPSIX. TRYPSIN. 479

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 accord-
ing to the following equation. C^L^O^.R^ (neutral fat)-r-3H2O =
C3H5.O3.H3 (£lycerin)+3(H.O.R) (fatty acid). This depends upon
a hydrolytic splitting, which was first positively proven by BERNARD and
BERTHELOT. The pancreas enzyme also decomposes other esters, just
as it does the neutral fats (NENCKI, BAAS, LOEVENHART l and others) .
The fat-splitting enzyme of the pancreas is, according to PAWLOW and
BRUNO and many others 2 aided in its action by the bile, and according
to EXCEL obeys ScnuTz-BoRissow's rule that the extent of cleavage
during a given time is proportional to the square root of the quantity of
ferment . The investigations of KANITZ 3 have led to the same
results.

POTTEVIN 4 found that the pancreas (free from water) could form
olein from oleic acid and glycerin. It is claimed that the gland can form
other esters from oleic acid or stearic acid with other alcohols (amyl
alcohol) if we operate only in the absence of water. In the presence of
considerable water the pancreas has a reverse saponifying action. (See
page 65).

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 proven by CORVISART.S 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 tryp-
sinogen. According to ALBERTONI 6 this zymogen is found in the gland
in the last third of the intra-uterine life. Enzymes more or less like tryp-
sin occur in other organs, and are very widely diffused in the vegetable

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;
Kalaboukoff and Terroine, Compt. rend. soc. biol., 63.

3 Engel, Hofmeister's Beitrage, 7; Kanitz, Zeitschr. f. physiol. Chem., 46.

4 Compt. rend., 138; Annal. Institute Pasteur, 20, and Bull. soc. Chem. (3), 35.

5 Gaz. hebdomadaire, 1857, Xos. 15, 16, 19, cited from Bunge, Lehrbuch, 4. Aufl.,
185.

6 See Maly's Jahresber., 8, 254.

480 DIGESTION.

kingdom,1 in yeast and in higher plants, and are also formed by various
bacteria. The enzymes similar to trypsin occurring in the plant king-
dom are, according to VINES, a mixture of peptases, which transform the
proteins into peptone and ireptases, which split the peptones 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. The
results as to the specificity of these antitrypsins in various animals, as well as
the possibility of producing antitrypsins by immunization, is still disputed.

.Trypsin, like other enzymes, has not been prepared in a pure condi-
tion. Nothing is positively known in regard to its nature, but as obtained
thus far it shows a variable behavior (KtJHNE, 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,
SCHWARZCHILD) . Trypsin dissolves in water and glycerin, while KUHNE'S-
trypsin is 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 solution
of 3-5 p. m. it is still more readily destroyed (BIERNACKI, VERNON 2).
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 (VERNON 3) .
Trypsinogen, according to the unanimous statements of several exper-
imenters, is more resistant toward alkalies than trypsin. Tiypsin is
gradually destroyed by gastric juice and even by digestive hydrochloric
acid alone.

The preparation of pure trypsin has been tried by various experimenters.
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 mak-
ing use of the combined salting out with NaCl and MgSO4. 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 extracting with
glycerin (HEIDENHAIN 4) . An impure but still very active infusion

1 In this connection see Vines, Annals of Botany, 16, 17, 18, 19, 22, and 23, and
Oppenheimer, Die Fennente, 1900.

3 Kiihne, 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.

3 Bayliss, Arch, des scienc. biolog. de St. Petersbourg, 11, Suppl.; Vernon, Journ.
of Physiol., 31.

4 Pfliiger's Arch., 10.

TRYPSIN AND TRYPSIN DIGESTION. 481

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 ($AL-
KOWSKI) at the temperature of the room. This infusion can be kept
very active for several years at the cellar temperature. For digestion,
experiments the active commercial trypsin preparations can be employed.

Like otner 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 com-
bined 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 l). According to the latter this erepsin
has a strong action upon peptone, and he believes that the peptone-
splitting action of a pancreas infusion is in great part due to the erepsin.
The pancreas besides these also contains a nuclease (see page 469), whosej
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
this enzyme glutinase. This glutinase is much more resistant toward acids
than trypsin, arid 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 Nsppi.2 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 putre-
faction a little thymol, chloroform, or toluene should be added to the

1 Bayliss and Starling, Journ. of Physiol., 30; Vernon, ibid., 30; and Zeitschr. f.
physiol. Chem., 50; Mays, ibid., 49 and 51.

2 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.

482 DIGESTION.

liquid. Tryptic digestion differs essentially from pepsin digestion, irre-
spective of the difference in the digestive products, in that the first takes
place in neutral or alkaline reaction and not, as is necessary 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 previously 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,1 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 filtrate 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-
LISS follows the digestion by the electrical conductivity, and HEDIN 2 determines
the quantity of nitrogen not precipitated by tannic acid.

Many circumstances exert a marked influence on the rapidity of the
trypsin digestion. With an increase in the quantity of enzyme present
the digestion is hastened, at least to a certain point. According to
PAWLOW and his school, the rule of SCHUTZ-BORISSOW is perfectly applicable
to trypsin, and the amount digested is proportional to the square root
of the quantity of ferment. This does not agree with the results of
BAYLISS, HEDIN and others, making use of other methods of determina-
tion. HEDiN,3 who has especially studied this question, found under cer-
tain conditions a direct proportion between the quantity of enzyme and
the intensity of digestion as explained in detail on pages 62, 63. Tryptic
digestion is also accelerated by an increase of temperature, at least to
about 40° C., at which temperature the protein is very rapidly dissolved
by the trypsin. The reaction is also of the greatest importance. Tryp-
sin acts energetically in neutral, or still better in alkaline, solutions, and
according to older statements, best in an alkalinity of 3-4 p. m. Na2CO3;

1 Arch. f. Hyg., 12 and 55.

2 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; Hedin, ibid., 32.

3 Pawlow, Die Arbeit der Verdauungsdriisen, Wiesbaden, 1898, p. 33; Bayliss, 1. c.;
Hedin, 1. c.; and Chapter II, pp. 62 and 63.

TRYPSIN DIGESTION. 483

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.1 The action of the alkali depends upon the number of hydroxyl
ions (DIETZE, KANITZ), and according to KAXITZ 2 the digestion pro-
ceeds 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 RACHFORD and SOUTH-
GATE, 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.3

Carbon dioxide, according to ScniERBECK,4 has a retarding action in
acid solutions, but it accelerates the tryptic digestion in faintly alkaline
liquids. Foreign bodies, such as potassium cyanide, may promote tryptic
digestion, while other bodies, such as salts of mercury, iron, and others
(CHITTENDEN and CUMMINS), or salicylic acid in large quantities, may
have a preventive action. According to WEISS 5 the halogen alkali
salts disturb tryptic digestion only slightly, and NaCl seems to have
the strongest action. The sulphates have a much stronger retarding
action than the chlorides. The nature of the proteins is also of impor-
tance. Unboiled fibrin is, relatively to most other proteins, dissolved
so very quickly that the digestion test with raw fibrin gives an incor-
rect 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. Na2CO3 (VERNON 6). The resist-
ance of certain native protein solutions, such as blood-serum and egg-

1 See Kudo, Bioch. Zeitschr., 15.

2 Kanitz, Zeitschr. f. physiol. Chem., 37, who also cites Dietze.

3 Chittenden 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.

4 Skand. Arch. f. Physiol., 3.

5 Weiss, Zeitschr. f. physiol. Chem., 40. See also Kudo, 1. c.
e Journ. of Physiol., 28.

484 DIGESTION.

white, against the action of trypsin is remarkable — a behavior which
can be explained by the occurrence of anti-bodies in these solutions
and which HEDIN 1 has carefully studied. An accumulation of the prod-
ucts of indigestion tends to hinder the trypsin digestion.

An interesting contrast to the inactivity of trypsin toward native egg-
'albumin and seralbumin is the enzyme papain, which, as the investigations of
DELEZENNE, MOUTON and POZERSKI and of JONESCU and SACHS 2 have shown,
is hindered in its action by an anti-body in the albumin which is destroyed by heat
or by the action of acid.

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
intermediate product (HERRMANN 3) . Besides this one obtains from
fibrin, as well as from other proteins, the products previously mentioned
in Chapter III. 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 diges-
tion, as shown by ABDERHALDEN and REINBOLD, using the protein edestin,
and by ABDERHALDEN and VoEGTLiN,4 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, phenylalanine, and gly-
^cocoll, stubbornly resist the cleavage action of the trypsin. The poly-
peptide-like bodies discovered by FISCHER and ABDERHALDEN, which
are produced in digestion, and which do not give the biuret reaction,
&re the atomic complexes which resist the action of trypsin. These
peptoids contain the pyrrolidine carboxylic acid and phenylalanine
groups of the protein, but also yield other monamino-acids such as leu-
cine, alanine, glutamic acid, and aspartic acid. Among the above-
mentioned products we find on the autodigestion of the gland other sub-
stances, such as oxyphenylethylamine (EMERSON), which is produced
from tyrosine by fermentive C02 cleavage, also uracil (LEVENE), guani-
dine (KUTSCHER and OTORI), the purine bases, which originate from the
nuclein bodies, and choline, which latter is formed from lecithin (KUT-
SCHER and LoHMANN5). If putrefaction is not completely prevented,

1 See Zeitschr. f. physiol. Chem., 50, where the works of Hedin are cited.

2 Delezenne, Mouton and Pozerski, Compt. rend., 142; Jonescu, Bioch. Zeitschr.,
2, Sachs, Zeitschr. f. physiol. Chem., 51.

3 Herrmann, Zeitschr. f. physiol. Chem., 11.

4 Abderhalden and Reinbold, Zeitschr. f. physiol. Chem., 44 and 46, with Voegtlin,
ibid. 53.

5 Fischer and Abderhalden, Zeitschr. f. physiol. Chem., 39; Emerson, Hofmeister's

ACTION OF TRYPSIN. 485

still other bodies occur which will be considered later in connection with
the putrefactive' processes in the intestine.

The Action of Trypsin upon other Bodies. The neudeoproteins and
nudeins are so digested that the protein complex is separated from the
nucleic acid and then digested. The nucleic acids may, nevertheless,
be somewhat changed (ARAKI), which is probably brought about by
another enzyme, the nudease (SACHS). A cleavage of nucleic acids with
the setting free of phosphoric acid and purine bases is, according to IWA-
NOFF,1 not brought about by trypsin. The splitting is first produced by
the action of nuclease or erepsin (see page 467). Gelatin is dissolved
and digested by pancreatic juice. A cleavage with the separation of
glycocoll and leucine does not occur (KUHNE and EWALD), or only to a
trivial extent (REICH-HERZBERGE 2) .

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 substance (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, con-
nective tissue, fat-corpuscles, and the remainder of the nervous tissue.
If the muscles are boiled, then the connective tissue is also dissolved.
Mucin is dissolved and split by trypsin, while chitin and horn substance
do not seem to be acted upon by the enzyme. Oxyho3moglobin is decom-
posed by trypsin with the splitting off of ha3matin. Trypsin lias 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
OULEWITSCH, GONNERMANN, and ScHWARZscHiLD,3 but the investigations
of FISCHER and of ABDERHALDEN and his co-workers,4 are much more
complete and important.

Beitrage, 1; Levene, Zeitschr. f. physiol. Chem., 37; Kutscher and Lohmann, ibid.,
-39; Kutscher and Otori, ibid., 43, and Centralbl. f. Physiol., 18.

1 Iwanoff, Zeitschr. f. physiol. Chem., 39, which also contains the literature; Sachs,
ibid., 46.

2 Kiihne and Ewald, Verb. d. naturh.-med. Vereins zu Heidelberg (N. F.), 1; Reich-
Herzberge, Zeitschr. f. physiol. Chem., 34.

3 Hofmeister's Beitrage, 4, where the other works are also cited.

4 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, o3, 54, 55, and 57.

486 DIGESTION.

As has been indicated (Chapter III) only those pepticles are split
hydrolytically by enzymes which are formed from the ammo-acids
occurring in nature. It has also been shown that with this limited
action of trypsin or the pancreatic juice only a certain number of pep-
tides, di-, tri-, as well as tetrapeptides are split, while it is without action
upon a number of others. The structure of these plays an important
role, as, for example, alanyl-glycine, CH3.CH(NH2).CO.NH.CH2.COOH,
is split, while its isomer glycl-alanine, NH2.CH2.CO.NH.CH(CH3).COOH,
is, on the contrary, not split. The nature of the amino-acids existing
in the peptide is also of importance. Those dipeptides which contain
alanine as acyl — for example, alanylglycine, alanyl-alanine, and alanyl-
leucine A — are readily hydrolyzed, while several dipeptides in which
a-aminobutyric acid or leucine functionates as acyl are very resistant.
The number of amino-acid groups is also of importance, as, for example,
triglycyl-glycine is not split, while tetraglycyl-glycine is. In those
peptides which are racemic bodies the hydrolysis takes place asym-
metrically, so that (see Chapter III) only one-half of the racemic body
is attacked, and those active amino-acids result as products which are
contained in the natural protein bodies.

The behavior of the polypeptides with trypsin, or nearly related
enzymes, is of the very greatest interest and in many regards very
important. 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, which are composed of amino-
acids, occurring in nature. By the aid of the polypeptide reaction ABDSR-
HALDEN 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.

By observations upon the optical behavior of a solution of a certain
optically active polypeptide on the addition of enzyme we can follow the
decomposition step by step, as many polypeptides have a stronger rota-
tion than their cleavage products, or as a change in the direction of the
rotation occurs during decomposition . In this manner we can determine
the enzyme hydrolysis in a certain time and in this way the interesting
investigations of ABDERHALDEN and GIGON l have shown that the hydrolysis
of glycyl-Z-tyrosine with yeast press-fluid is retarded by the addition ot
optically active amino-acids occurring in nature, while the optical anti-
podes of the same amino-acids is without action or at least have only
a very weak retarding action. An analogous condition in digestion

1 Zeitschr. f. physiol., Chem. 53.

PANCREATIC REXMX. 487

explains in part why the decomposition of proteins in vitro is slower than
in the digestive tract, where the decomposition products are removed
by absorption. On following the changes in the optical behavior during
the enzymotic cleavage of polypeptides one can, as ABDERHALDEN with
KOELKER and BRAHM l have shown, determine at which point the enzyme
first applies its action in the cleavage of a tripeptide. We cannot enter
into further detail as regard this and the other related behaviors.

Pancreatic rennin is an enzyme found in the gland and in the juice,
which coagulates neutral or alkaline milk (KfJHNE 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 enterokinase 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 XIV),
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.

The property of pancreatic juice of giving plastein precipitates is just as
inexplicable as in the case of the gastric juice and other enzyme solutions.

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.

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 collabora-
tors, occurs chiefly 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

1 Abderhaiden with Koelker, Zeitschr. f. physiol. Chem., 54 and 55; with Brahm,
ibid., 57.

2 Kiihne 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.

3 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.

488 DIGESTION.

refer the reader to what has been previously stated on this subject in
Chapter VIII, pages 385 and 386.

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;1 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 reactiva-
tion 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 pancreatic juice. MAR-
TIN, WILLIAMS, PAWLOW, and BRUNO 3 have observed a beneficial action
of the bile on the diastatic action of the pancreas infusion. To this
may be added that the organized ferments 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 dextrose
in the intestine. Cane-sugar is inverted in the intestine, and, at least
in certain animals, also lactose. There does not seem to be any doubt
that cellulose, especially the fine and tender varieties, is in part dissolved
in the intestine. LOHRISCH 4 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 cellulose undergoes
a fermentation in the intestine by the action of micro-organisms, produc-
ing 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.5

1 See Wroblewski and collaborators, Hofmeister's Beitrage, 1.

2 Compt. rend. soc. biol., 62.

3 Martin and Williams, Proceed, of Roy. Soc., 45 and 48; Bruno, footnote 2,
p. 479.

4 Cited from Bioch. Centralbl., 8, 334.

5 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,

CHEMICAL PROCESSES IN THE INTESTINE. 489

The bile has, as shown by MOORE and ROCKWOOD l and then especially
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 absorption
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,2 it accelerates the fat-splitting action of the steapsin.
According to v. FURTH and SCHUTZ 3 the bile-salts are the active con-
stituents of the bile in this cleavage, and the fatty acids set free can com-
bine 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 4) . 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 452).

Bile completely prevents peptic zymolysis in artificial 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,S to have no dis-
turbing action on gastric digestion. According to BOLDIREFF,G after con-
tinuous starvation, on feeding fat and food rich in fat, as well as after

11; Weiske, Zeitschr. f. Biologic, 22, 373; Tappeiner, ibid., 20 and 24; Mallevre,
Pfliiger's Arch., 49; Omeliansky, Arch. d. scienc. biol. de St. Pe"tersbourg, 7; E. Miiller,
Pfliiger's Arch., 83; Lohrisch, Zeitschr. f. physiol. Chem., 47 (literature); and 1. c.,
footnote 4, p. 488.

1 Proceedings of Roy. Soc., 60, and Journ. of Physiol., 21. In regard to Pfliiger's
work see Absorption.

2 Nencki, Arch. f. exp. Path. u. Pharm., 20; Rachford, Journal of Physiol., 12.

3 Centralbl. f . Physiol., 20.

4 Briicke, Wien. Sitzungsber., 61, Abt. 2; Gad, Arch. f. (Anat. u.) Physiol., 1878;
Loewenthal, ibid., 1897.

5 Oddi, in Centralbl. f. Physiol., 1, 312; Dastre, Arch, de Physiol. (5), 2, 316.
8 Centralbl. f. Physiol., 18, 457, and Pfliiger's Arch., 121.

490 DIGESTION.

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 diges-
tion 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. On the
contrary, the bile does not disturb the digestion of proteins by the pan-
creatic juice in the intestine. The action of these digestive 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 tryp-
sin is accelerated by the bile. In a dog killed while digestion is going
on, the faintly acid, bile-containing material of the intestine shows regu-
larly a strong digestive action on proteins.

The precipitate 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 hydrochloric 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 precipitation 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-
organisms. These are less intense in the upper parts of the intestine,
but 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.
MACFADYEN, M. NENCKI, and N. SEEBER l 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 con-
tents of the intestine after it had been exposed to the action of the mucous
membrane of the entire small intestine. The mass was yellow or yellowish

1 Arch. f. exp. Path. u. Pharm., 28.

PROTEIN DIGESTION IN THE INTESTINE. 491

brown, due to bilirubin, and had an acid reaction which, on a mixed
but chiefly 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 resemb-
ling that of indol. The essential acid present was acetic acid, accom-
panied by fermentation and paralactic acid, volatile fatty acids, succinic
acid, and bile-acids. Coagulable proteins, peptone, mucin, dextrin, dex-
trose, 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 l 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 2 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 caBcum 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 3
reobtained 73 per cent of the coagulable protein from a fistula in the
ileum (2-3 cm. in front of the caecum). KUTSCHER and SEEMANN,
ABDERHALDEN, LONDON and collaborators4 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,

1 Jakowsky, Arch, des scienc. biol. de St. Pe"tersbourg, 1; Ad. Schmidt, Arch. f.
Verdauunskr., 4.

2 The works of London and collaborators cannot be cited in detail, but may be
found in Zeitschr. f. physiol. Chem., 46-57.

3 Zeitschr. f. physiol. Chem.. 46.

4 Kutscher and Seemann, ibid., 34; Abderhalden and London, with Kautzsch,
ibid., with L. Baumann, ibid., 51, with v. Korosy, ibid., 53.

492 DIGESTION.

it seems, according to ABDERHALDEN, LONDON, OPPLER and REEMLiN,1
is similar to the artificial digestion with trypsin, namely, that the destruc-
tion takes place step-wise, that certain ammo-acids, such as tyrosine,
are split off earlier than others.

The decomposition products of the proteins formed by the action of
gastric juice can, according to LONDON,2 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 (LEViTES3) may be so completely
split in the stomach and small .intestine that their absorption is com-
plete before the contents pass into the caecum. According to LONDON
and POLOWZOWA 4 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 Jie intestine, though the cleavage also
goes on in the other parts, .namely in the jejunum and the upper part
3f the ileum.

As above remarked, ordinarily no putrefaction takes place in the
small intestine, but occurs generally only in the large intestine. This
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-
acids, and ammonia) indol, skatol, paracresol, phenol, phenylpropionic
acid, and phenylacetic acid, also paraoxyphenylacetic acid and hydro-
paracoumaric acid (besides paracresol, produced in the putrefaction of
tyrosin), volatile fatty acids, carbon dioxide, hydrogen, marsh-gas, methyl-
m,ercaptan, and sulphuretted 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 oxy acids, 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 XV). The quantity of these bodies in the urine also varies
with the extent of the putrefactive processes in the intestine; at least

1 Zeitschr. f. physiol. Chem., 55 and 58. 3 Zeitschr. f. physiol. Chem., 49 and 53.

2 Ibid., 49. 4 Ibid., 56.

INTESTINAL GASES. 493

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 l 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 461, 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
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. Hydro-
gen occurs in largest quantities after a milk diet, and in smallest quan-
tities 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 circum-
stances. There is no doubt that the methylmercaptan and sulphuretted
hydrogen which occur normally in the intestine originate from the pro-
teins. The marsh-gas undoubtedly originates in the putrefaction of
proteins. As proof of this HUGE 2 found 26.45 per cent marsh-gas in
the human intestine after a meat diet. He found a still greater quan-
tity of this gas after a vegetable (leguminous) diet ; this coincides with the
observation that marsh-gas may be produced by a fermentation of car-
bohydrates, but especially of cellulose (TAPPEiNER3). 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 decomposi-
tion of lecithin (HASEBROEK4).

1 Baumann, Zeitschr. f. physiol. Chem., 10; Harley and Goodbody, Brit. Med.
Journ., 1899.

2 Wien. Sitzungsber., 44.

3 Zeitschr. f. Biologie, 20 and 24. 4 Zeitschr. f. physiol. Chem., 12.

494 DIGESTION.

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 urobilin 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 excrement
of certain animals, while taurocholic acid is so completely decomposed
that it is entirely absent in the feces. In the fetus, on the contrary,
in whose intestinal tract no putrefaction processes occur, undecomposed
bile-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 at 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 follows
from the fact that putrefaction may also continue during complete fasting.
From the observations of M TILLER* upon CETTI it was found that the
elimination of indican during starvation rapidly decreased 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 elimination of indican
during starvation is considerable, but the phe"nol elimination 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, peptones, poly-
peptides and amino-acids is concerned. The question has indeed been
asked (PASTEUR), is digestion possible without micro-organisms? NUTT Al-
and THIERFELDER have shown that guinea-pigs, removed from the uterus
of the mother by CaBsarian 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 conditions, kept in sterile rooms and

1 Berlin, klin. Wochenschr., 1887.

2 Nuttal and Thierfelder, Zeitschr. f. physiol. Chem., 21 and 22, Schottelius, Arch,
f. Hygiene, 34, 42, and 67.

PUTREFACTIVE PROCESSES IN THE INTESTINE. 495

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 variety of bacteria from hen feces,
they gained weight again and recovered.

The bacterial action in the intestinal canal is, at least in certain cases,
necessary, and it acts in the interest of the organism. This action may,
by the formation of further cleavage products, involve a loss of valuable
material to the organism, and it is therefore 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 putrescent odor. Also the odor of the con-
tents 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.

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 proven
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 (HIRSCHLER *). 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, LINDBER-
GER3). There is no question that the free bile-acids have a strong
preventive action on putrefaction outside of the organism, and it is there-
fore difficult to deny such an action in the intestine. Notwithstanding
this, the anti-putrid action of the bile in the intestine is not considered
by certain investigators (VoiT, ROHMANN, HIRSCHLER and TERRAY,
LANDAUER and ROSENBERG 4) as of great importance.

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, Centr-albl. f. d. med. Wiss., 1893, 467, and Seelig, Virchow's Arch., 126
(literature) .

3 Maly and Emich, Monatshefte f. Chem., 4; Lindberger, footnote 3, p. 483.

4 Voit, Beitr. zur Biologic, Jubilaumschrift, Stuttgart, 1882; Rohmann, Pfliiger's

496 DIGESTION.

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 have a light-gray or pale color.
The extent of deviation from the normal after the operation is essentially
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 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 quantity of pro-
teins and fats be increased, then the latter, which can be only incom-
pletely 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 putrefaction.
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 carbohydrates, it may
remain quite normal, and the leading off of the bile does not cause any
increased putrefaction. The. carbohydrates may be uninterrupedly
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 preventive action
on putrefaction.

To this conclusion the objection may be made that the carbohydrates,
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,2 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

Arch. 29; Hirschler and Terray, Maly's Jahresber., 26; Landauer, Math. u. Naturw.
Ber aus Ungarn, 15; Rosenberg, Arch. f. (Anat. u.) Physiol., 1901.

1 Schwann, Muller's Arch. f. Anat. u. Physiol., 1844; Blondlot, cited from Bidder
and Schmidt, Verdauungssafte, etc., 98.

2 See Hirschler and Terray, 1. c.

PUTREFACTIVE PROCESSES IN THE INTESTINE. 497

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 its
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-
MAXX, WASBUTZKI, BIERNACKI and MESTER had proven that an increased
putrefaction in the intestine occurred when the quantity of hydrochloric
acid in the gastric juice was diminished or deficient, SCHMITZ l has lately
shown in man that on the administration of hydrochloric acid, produc-
ing a hyperacidity of the gastric juice, the putrefaction 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 hydro-
chloric acid, but chiefly to organic acids, acid salts, and free carbon
dioxide, 'there are several observations as to the reaction of the intes-
inal contents, by MOORE and ROCKWOOD, MOORE and BERGIN, MATTHES
and MARQUARDSEX, I. MUXK, NEXCKT 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 presence
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 NaHCO3 and free CO2, 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 BIEXSTOCK 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 putrefactive pro-
ducers or retard their development. According to HOROWITZ an unequal

1 Zeitschr. f. physiol. Chem., 19, 401, which includes all the pertinent literature.

2 Moore and Rockwood, Journ. cf 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.

498 DIGESTION.

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. Perhaps, also, agreeing
with the .experience of CONRADI and KuRpjuwEiT,1 the toxines pro-
duced by the intestinal bacteria may, by their antiseptic action, keep
the putrefactive processes in the intestine within bounds.

Feces. It is evident that the residue which remains after com-
plete digestion and absorption in the intestine must be different, both
qualitatively 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,2 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 has 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 in protein.
The individuality also plays an important role in the utility of the food
and the formation of feces (SCHIERBECK 3) . 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 fibres, 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 contains
indigestible bodies, such as the remains of plants, keratin substances,
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,*
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. Occasionally, also,
parasites of different kinds occur; and lastly, the excrement contains
micro-organisms of various species.

1 Bienstock, Arch. f. Hygiene, 39; Horowitz, Zeitschr. f. physiol. Chem., 52; Conradi
and Kurpjuweit, Munch, med. Wochenschr., 1905.

2 Zeitschr. f . Biologic, 25, 264.

3 Arch. f. Hygiene, 51.

4 In regard to the purine bases in feces, see Hall, Journ. of Path, and Bacteriol., 9;
Schittenhelm, Arch. f. klin. Med., 81; Schittenhelm and Kriiger, Zeitschr. f. physioh
Chem., 45.

FECES. 499

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,1 that a clean, isolated loop
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 (Chapter
VIII). 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, nearly entirely, of intestinal secretions. Many
foods, therefore, produce a large quantity of feces chiefly by causing an
abundant secretion.2

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 calomel 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. On the contrary, there is
found stercobilin (MASIUS and VANLAIR), which is identical with urobilin
(JAFFE 3) . Bilirubin may occur in pathological cases in the feces of
mature persons. It has been observed in a crystallized state (as hffimatoi-
din) 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

1 Hermann, Pfliiger's Arch., 46. See also Ehrenthal, ibid., 48; Berenstein, ibid.,
53; Klecki, Centralbl. f. PhysioL, 7; 736, and F. Voit, Zeitschr. f. Biologic, 29; v.
Moraczewski, Zeitschr. f. physiol. Chem., 25; F. Lippich, Prager med. Wochenschr., 32.

2 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.

3 See bile-pigments, Chapter VIII, and urobilin, Chapter XV.

500 DIGESTION.

cases a large quantity of crystals has been observed which consist chiefly
of magnesium soaps or sodium soaps. Hemmorrhage in the upper parts
of the digestive tract yields, when it is not very abundant, a dark-brown
excrement, due to ha?matin.

EXCRETIN, so named by MARCET,* 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 disease 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 brown-
ish gray, of variable size, consisting of concentric layers and containing
chiefly ammonium-magnesium phosphate and calcium phosphate, besides
a small quantity of fat or pigment. The nucleus ordinarily consists of
some foreign body, such as the stone of a fruit, a fragment of bone,
or something similar. In those countries where bread made from oat-

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.

5 See Schmidt and Strassburger, 1. c.

INTESTINAL CONCREMEXTS. 501

bran is an important food, we often find in the large intestine balls similar
to the so-called hair-balls (see below). Such calculi contain calcium
and magnesium phosphate (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-fibres, and termed hair-balls. The so-called
"jBGAGROPiL-ae," which occur in the ANTILOPUS RUPICAPRA, belong to this group,
and are generally considered as nothing else than the hair-balls of cattle.

The so-called oriental bezoar-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 LITHOFELLTC
ACID, C2oH36O4, 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 CUH6O3,
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 problem of digestion consists in part in separating the valuable
constituents of the food from the useless ones and dissolving or trans-
forming them into forms which are adapted for the processes of absorp-
tion. 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.

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 protein as are
introduced intravenously, subcutaneously, or into a body -cavity, i.e.,
evading the intestinal canal, or, as OPPENHEIMER calls it, parenteral.

1 Ber. d. d. chem. Gesellsch., 36.

502 DIGESTION.

Since the first investigations of ZUNTZ and v. MERING on this sub-
ject several experimenters, such as NEUMEISTER, FRIEDENTHAL and
LEWANDOWSKY, MUNK and LEWANDOWSKY, OPPENHEIMER, MENDEL,
and ROCKWOOD, HEILNER, CRAMER and PRINGLE, MICHAELIS and RONA
and others,1 have shown, without any doubt, that the animal body can
more or less completely utilize different, parenterally introduced pro-
teins, although different varieties of animals show a difference in this
regard. Still we do not know where and how these foreign proteins are
changed and assimilated, but CRAMER ascribes great importance to the
leucocytes in this regard.

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 LATSCHENBERGER,
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. MEN-
DEL and ROCKWOOD, on the contrary, in experiments with casein and
edestin in the living intestinal loop, could prove only the slightest absorp-
tion on excluding digestion as completely as possible, while the corre-
sponding proteoses were abundantly absorbed.

It is difficult to decide in these experiments as to how far the proteins
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 have shown the
passage of non-modified protein into the blood and lymph. Based upon
many investigations on this subject we can consider it possible that under
certain circumstances, as on flooding the intestinal canal with protein,

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; Munkand 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. Wcchenschr., 49; Cramer,
Journ. of Physiol., 37, with Pringle, ibid.; Rona and Michaelis, Pfluger's Arch., 123
and 124.

2 Briicke, Wien. Sitzungsber., 59; Bauer and Voit, Zeitschr. f. Biologie, 5; Eich-
horst, Pfluger'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.

ABSORPTION OF PROTEINS. 503

with a greater permeability of the intestinal wall, as in new-born and
sucking animals, and with a diminished modification by the gastric juice,
a passage of non-modified protein may take place in the blood-vessels, but
that under normal conditions this is not the case, or at least does not take
place to any mentionable degree. As a rule, the absorption of protein
follows a modification of it, and the next question is whether the pro-
teins are chiefly absorbed as proteoses 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 1 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 contents
of the intestine. The amino-acids occur to a less extent than the pro-
teoses 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 previous
cleavage into amino-acids.

NOLF and HONORE found, what was later substantiated by ZuNZ,2
that the proteoses 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
large quantities in the intestine pass in small amounts into the blood.
Another proof is the findings of BoRCHARDT3 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 HoFMEisTER4 the walls of the

1 Schmidt-Miilheim, Arch. f. (Anat. u.) Physiol., 1877; Munk and Rosenstein,
Virchow's Arch., 123.

2 Nolf and Honore", Arch, internat. de Physiol., 1905; Nolf, Journ. de Physiol. et
Pathol. gen., 1907; Zunz, Me"moires cour., etc., Acad. Roy. Med., Belg., 20, Fasc. 1.

3 In regard to the literature on proteoses in the blood see Chapter VI, footnotes 3
and 4, p. 255, and 1, p. 256.

4 Zeitschr. f . physiol. Chem., 6, and Arch. f. exp. Path. u. Pharm., 19, 20, 22.

504 DIGESTION.

stomach and intestine are the only parts of the body in which proteoses
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 pages 255 and 256 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 proteoses in the blood,
or at least in the blood plasma, has not been positively shown under
physiological conditions.1 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 quantity 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. KOROSY).2

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.3 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
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

1 See foot-note 3, p. 503.

2 Kutscher and Seemann, Zeitschr. f. physiol. Chem., 34; v. Korosy, ibid., 57.

3 Arch. f. (Anat. u.) Physiol., 1880, Supplbd. See also Cathcart and Leathes,
'Journ. of Physiol., 33.

FATE OF THE ANIMO-ACIDS AND PROTEOSES. 505

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,1 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 ZALESKi2 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 CRAMER 4 urged the theory of the importance of the
leucocytes, and the observations of INAGAKI 6 also show, the possibility
of the leucocytes taking up the proteosee 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 ammo-acids

1 Bioch. Zeitschr., 12.

'Arch, des scienc. biol. de St. Petersbourg, 4; Arch. f. exp. Path. u. Pharm., 37;
see also Salaskin, Zeitschr. f. physiol. Chem., 25.

3 Zeitschr. f. physiol. Chem., 59.

4 Hofmeister, 1. c.; Heidenhain, Pfliiger's Arch., 43; Pringle and Cramer, Journ,
of Physiol., 37.

'Zeitschr. f. physiol. Chem., 50.

506 DIGESTION.

the coagulable proteins are constructed by synthesis. In support
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, HEN-
RIQUES and HANSEN, and especially by ABDERHALDEN and his co-workers 1
on dogs, mice and mts, 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. Espe-
cially important are certain experiments of ABDERHALDEN and RON A
and ABDERHALDEN with OLINGER, MESSNER and WINDRATH with com-
pletely decomposed meat.

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. It
is nevertheless true that a nitrogen retention is not synonymous with
a new formation of protein, and we must not overlook the fact that
LiJTHjE2 observed a nitrogen retention on feeding with one amino-
acid and abundance of carbohydrate, a condition where we can hardly
speak of a synthesis of protein. Still these experiments do not seem
to be conclusive enough to discard the above-mentioned experiments
and for the present, as above stated, we must admit that these experi-
ments give proof as to the synthesis of protein. With this we naturally
do not explain the occurrence or the extent of such a synthesis under
normal conditions.

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 3 made
an experiment on a dog with an ECK fistula (see page 376), 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 equibrilium but also in nitrogen retention. According to
them the liver has no special function in the protein synthesis and the
theory is more reasonable that the protein synthesis takes place in
the intestine.

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, 4", and
52; Henriques and Hansen, ibid., 4,3, 49; Henriques, ibid., 54; Abderhalden with
Olinger, ibid., 57, with Messner and Windrath, ibid., 59.

2 Pfliiger's Arch., 113.

3 Zeitschr. f. physiol. Chem., 54.

PROTEIN SYNTHESIS. 507

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 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 wre can also add that according to the investigations of FREUND
and v. KOROSY l the blood coming from the intestine during digestion
is richer in coagulable protein than other blood, and also that this pro-
tein 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 decomposed
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 rel'a-
tively greater quantity of coagulable protein should occur in the rnucosa
of the digesting intestine as compared with the non-digesting intestine.
PRINGLE and CRAMER, by a method * which requires confirmation,
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 intes-
tine, are richer in non-coagulable protein than the starving animal, a
condition which is related to the role of the leucocytes in the protein
assimilation. This question of the absorption of proteins in the intes-
tine 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 vege-
table source. As proof of this the following observations are given:
In his experiments on the utilization of certain foods in the intestinal
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 condi-
tions are quite different with vegetable food, as shown by the researches
of MEYER, RUBNER, HULTGREN and LANDERGREN, who made experi-
ments with various kinds of rye bread and found that the loss of nitro-

1 v. 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, Jounu
of Physiol., 37.

508 DIGESTION.

gen through the feces amounted to 22-48 per cent. Experiments with
other vegetable foods, and also the investigations of SCHUSTER, CRAMER,
MEINERT, MoRi,1 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 gen-
eral that the nitrogen deficit by the feces increases with a larger quan-
tity 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
hy 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 substances
seem to be indigestible, and because of the difficultly digestible vegetable
food, a large quantity of digestion fluids containing nitrogen are 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
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
digestion 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.2
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 diges-

1 Rubner, Zeitschr. f. Biologie, 15; Meyer, ibid., 7; Hultgren and Landergren,
Nord. med. Arch., 21; Schuster, in Voit's "Untersuch. d. Kost," etc., 142; Cramer,
Zeitschr. f. physiol. Chem., 6; Meinert, "Ueber Massennahrung," Berlin, 1885; Kell-
ner and Mori, Zeitschr. f . Biologie, 25.

2 Abelmann, " Ueber die Ausniitzung der Nahrungsstoffe nach Pankreasexstirpa-
tion" (Inaug. -Dissert. Dorpat, 1890), cited from Maly's Jahresber., 20; Sandmeyer,
Zeitschr. f. Biologie, 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.

CARBOHYDRATE ABSORPTION. 509

tion fluids act substitutingly. In this regard Zuxz and MAYER J 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.
Dextrose, levulose, 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 dextrose and levulose.
Lactose is also, at least in certain animals, inverted in the intestine.
In other mature animals, 011 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 undergo fer-
mentation, or, as ROHMANN and NAGANO 2 assumed, if it is not trans-
formed in the intestinal mucosa in some unknown way. An absorption
of non-inverted carbohydrates is not improbable, and according to OTTO
and v. MERIXG the portal blood contains, besides dextrose, a dextrin-like
carbohydrate after a carbohydrate diet. MOSCATI 3 believes that when
homogeneous starch solutions are injected intravenously or subcutaneously
the starch is taken up by the organs, namely the spleen, liver and lungs,
and is utilized as the starch is changed into glycogen. A part of the car-
bohydrates is destroyed by fermentation in the intestine, with the forma-
tion 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 LEPINE, ROHMANN and NA-
GANO 4). 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, ALBERTONI, WAYMOUTH
REID, ROHMANN and NAGANO). There seems* to be no doubt that lac-
tose is absorbed more slowly than the two other disaccharides. Accord-
ing to the extensive experiments of ROHMANN and NAGANO, saccharose
is absorbed more quickly than maltose. NAGANO 5 contends that the
pentoses are absorbed more slowly than hexoses.

1 Mem. de 1'acad. roy. de m£dic. de Belg., 18.

2 Voit and Lusk, Zeitschr. f. Biologie, 28; Rohmann and Nagano, Pfliiger's Arch.,
95, which contains the references to the literature.

3 Otto, see Maly's Jahresber., 17; v. Mering, Arch. f. (Anat. u.) Physiol., 1877;
Moscati, Zeitschr. f. physiol. Chem., 50.

4 Lannois and Lepine, Arch.de physiol. (3), 1; Rohmann, Pfliiger's Arch., 41; see
also footnote 2.

5 In regard to the literature on the absorption of sugars, see footnote 2.

510 DIGESTION.

On the introduction of starch even in very considerable quantities
into the intestinal tract no dextrose 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 substance
in order to produce an alimentary glycosuria gives, according to HOF-
MEiSTER,1 the assimilation limit for that same sugar. This limit is dif-
ferent 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 circumstances.
In general it can be said that in regard to the ordinary varieties of sugarr
such as dextrose, levulose, galactose, saccharose, maltose, and lactose,
the assimilation limit is highest for dextrose 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 ROHMANN and
NAGANO. It is, therefore, not remarkable that disaccharides, as well,
have been found in the urine in cases of alimentary glycosuria.2

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 measureable 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 observations
of I. MUNK and ROSENSTEIN 3 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 HEIDEN-
HAIN,4 to be found in the anatomical conditions, in the arrangement of
the capillaries close under the layer of epithelium. Ordinarily these
capillaries find the necessary time for the removal of the water and the
soJids 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,

1 Arch. f. exp. Path. u. Pharm., 25 and 26.

2 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 2, p. 375. See also Blumenthal, Zur Lehre von der Assimilationsgrenze der
Zuckerarten, Inaug.-Dissert. 1903, Strassburg and Brasch, Zeitschr. f. Biol., 50.

3 v. Mering, Arch. f. (Anat. u.) Physiol., 1877; Munk and Rosenstein, Virchow's
Arch., 123.

4 Pfliiger's Arch., 43, Suppl.

CARBOHYDRATE ABSORPTION. 511

and in these cases a part of the dissolved bodies passes into the chylous
vessels and the thoracic duct (GINSBERG and RdHMANN1).

The passage of sugar into the urine when at one time large quantities 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 observa-
tions of FILIPPI 2 upon dogs with ECK fistula, it seems as if the role 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 somewhat 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,
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,3 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 question
how these bodies are absorbed after the extirpation of the pancreas.
As on the absorption of proteins, so also on the absorption of starch,
the observations have given variable results. In certain cases the absorp-
tion 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 (ROSEN-
BERG, CAVAZZAXI 4) .

1 Ginsberg, Pfliiger's Arch., 44; Rohmann, ibid., 41.

2 Zeitschr. f. Biol., 49 and 50.

3 Rubner, Zeitschr. f. Biologic, 15 and 19; Constantinidi, ibid., 23; Hultgren and
Landergren, 1. c.

4Cavazzani, Centralbl. f. PhysioL, 7. See footnote 2, p. 508; also Lombroso,
Hofmeister's Beitrage, 8.

512 DIGESTION.

Emulsification used to be considered as of the greatest importance in
the absorption of fats, and this emulsion occurs in the chyle on the intro-
duction 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 l) .

The assumption that the fat is absorbed chiefly as an emulsion is
partly based on the abundance of emulsified fat in the chyle after feeding
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 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 2 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 KUHNE and recently
shown by MOORE and KRUMBHOLZ,3 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.4

1 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, 30;
Moore, see Biochem. Centralbl., 1, 741; Frank and Ritter, Zeitschr. f. Biologie, 47.

2 Zeitschr. f . Biologie, 36.

3 Kiihne, Lehr. der physiol. Chem., 122; Moore and Krumbholz, Journ. of Physiol.,
22.

4 In regard to the recent literature on fat absorption, see the works of Pfliiger,
Pfliiger's Arch., 80, 81, 82, 85, 88, 89, and 90, where the work of other investigators
is cited and discussed.

FAT ABSORPTION. 513

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
of soaps.

The importance of the bile, the soaps, and the alkali carbonates has.
been closely studied, chiefly 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 con-
clusion 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 forma-
tion of an emulsion is, according to this view, that the fat in this con-
dition 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. It
is the opinion of the author that it is still too early to give a positive
verdict as to how these conditions in the intestine are brought about
and the conclusion must be left for further investigation.

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 l on dogsx
it seems that only a small part of the fats, or at least of the fatty acids
fed, pass into the chylous vessels; but these observations can hardly
be applied to the absorption of neutral fats, or to the absorption irx
man under normal circumstances. MUNK and RosENSTEiN,2 in their
investigations on a girl with a lymph fistula, found 60 per cent of the

1 Walther, Arch. f. (Anat. u.) Physiol., 1890; Frank, ibid., 1892.

2 Virchow's Arch., 123.

514 DIGESTION.

fat ingested in the chyle, and of the total quantity of fat in 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
ishe 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,1
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
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 RuBNER2 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 produce
an absorption of the emulsified fat without the pancreatic juice. DASTRE 3
lias 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 ancf pancreatic
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, ROHM ANN, FR. MULLER, I. MuNK,4 and

1 Munk, Virchow's Arch., 80 and 95; Arnschink, Zeitschr. f. Biologie, 26.

2 Voi.t, Zeitschr. f. Biologie, 9; Rubner, ibid., 15.

3 Arch, de Physiol. (5), 2

4 F. Miiller, Sitzungsber. der phys.-med. Gesellsch. zu Wiirzburg, 1885; I. Munk,
Virchow's Arch., 122. See also footnotes 4, p. 495 and 1, p. 496.

FAT ABSORPTION. 515

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 oi 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 ROHM ANN 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 feces consists of fatty acid, while under normal conditions the
feces contain 1 part neutral fat to about 2-2 J 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, HEDON 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 other results, and HARLEY
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

1 Miiller, "Unters. iiber den Icterus," Zeitschr. f. klin. Med., 12; He"don 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 footnote 2,
p. 508.

616 DIGESTION.

Ho 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 secre-
tion 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 x) . This investigator is also of the opinion that the
pancreas, independent of the external secretion in any way (by endo-
crinic 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 pan-
creatic juice from the intestine acts upon the other factors of the diges-
tion, 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 509), indicates that such a reverse action is possible,
tinder 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 in 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 pan-
creatic juice has been shown by the investigations of HEDON and VILLE

and CUNNINGHAM.2

The reason for the fact that the fat absorption i i diminished in the
absence of bile from the intestine must be sought for in the above-men-
tioned role 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), chiefly 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 pancreas
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

1 Lombroso, see Bioch. Centralbl., 3, 67 and 566, and 4, 738; also Compt. rend.
BOC. biol., 57; Hofmeister's Beitrage, 8, 11; Pfliiger's Arch., 112; and Arch. f. exp.
Path. u. Pharm., 56 and 60; Niemann, 1. c.

2 He\ion and Ville, 1. c.; Cunningham, Journ. of Physiol., 23.

ABSORPTION. 517

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 favorable.

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 enzymes may also be absorbed like other soluble constituents of
the digestive secretions, as is demonstrated by the passage of pepsin into
urine. The occurrence of urobilin in urine attests the absorption of the
bile-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 proven
by other observations. TAPPEINER l 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 investiga-
tors, and more recently by PREVOST and BINET, and specially by STADEL-
MANN and his pupils.2 After the introduction of f6reign 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 (fivefold). 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.

1 Wien. Sitzungsber., 77.

' Schiff, Pfliiger's Arch., 3; Prevost and Binet, Compt. rend., 106; Stadelmann,
see footnote 1, p. 394.

3 Proceed. Roy. Soc., 64.

518 DIGESTION.

ERLANGER and HEWLETT 1 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.

After the exclusion of the colon in rabbits, BERGMANN and HULT-
GREN 2 could find no definite action upon the availability of the cellulose
nor could any diminution in the utility of the other constituents of the
food be observed. ZUNTZ and USTJANZEW 3 also found that the removal
of the caecum had no influence on the utilization of nitrogen; but in
respect of other factors they arrived at different results. They found,
namely, that the caecum of the rodent is of great importance for the
digestion of crude fibre and the pentosans. On feeding hay and wheat
to rabbits after the removal of the caecum, the digestion coefficient for
crude fibre 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 answered. It is certain that thus far the laws
of diffusion and osmosis alone are not sufficient to explain absorption,
although some claim that it is. With all these facts in view, and as
it is not within the scope of this book to enter more in detail upon the
numerous investigations of this subject, we must refer to larger works 4
and to text-books on physiology for further information.

1 Arner. Journ. of Physiol., 6.

2 Skand. Arch. f. Physiol., 14.

3 Verhandl. d. physiol. Gesellsch. zu Berlin, 1904-1905.

4 See Hober, Physikalische Chemie der Zelle, Leipzig, 1906, and I. Munk, Ergeb-
nisse der Physiologic, I, Abt. 1; Hamburger, Osmotischer Druck und lonenlehre,
Bd. 2, Weisbaden, 1904.

CHAPTER X.
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 serglobulin
and seralbumin, which occur in the parenchymatous fluid (LoEBiscn1).

The connective tissue also often contains fibres or formations con-
sisting of elastin, sometimes in such great quantities that the connective
tissue is transformed into elastic tissue. A third variety of fibres, the
reticular fibres, also occurs, and according to SIEGFRIED these consist
of reticiilin.

If finely divided tendons are extracted in cold water or NaCl solutions,
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 precipitated 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 miicin, 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 2) .

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

1 Zeitschr. f. physiol. Chem., 10.

2 Levene, ibid., 31 and 39; Cutter and Gies, Amer. Journ. of Physiol., 6; Chittenden
and Gies, Journ. of Exp. Med., 1.

519

520 TISSUES OF THE CONNECTIVE SUBSTANCE.

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 115
to 120.

The tissues described under the names mucous or gelatinous tissues
are characterized more by their physical than by their chemical properties,
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 Acalephae, 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 has been described on page 166. C. TH. MORNER J
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 myxoedema,
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 nuchae (VAN DE-
GRIFT and GIES 3) :

Achilles tendon. Ligament.

Calf. Ox. Calf. Ox.'

Water.... 675.1p.m. 628.7p.m. 651.0p.m. 575.7p.m.

Solids 324.9 "• 371.3

Organic bodies 318.4 366.6

Inorganic bodies ... 6.1 " 4.7

Fat 10.4

Proteid 2.2

Mucoid 12.83

Elastin 16.33

Collagen 315.88

Extractives, etc 8 . 96

394.0 ' 424.3

342.4 " 419.6 '

6.6 " 4.7 '

11.2 '

6.16'

5.25'

316.70'

72.30'

7.99'

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

1 Zeitschr. f. physiol. Chem., 18, 250.

2 Mucin in Myxoedema: Further Analyses. King's College Collected Papers No. 1,
1893.

3 Buerger and Gies, Amer. Journ. of Physiol., 6; Vandegrift and Gies, ibid., 5.

CARTILAGE. 521

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 Fe203, 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 recent investigations of FRAUENBERGER l who
found, in Wharton's jelly, about one-twentieth 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 fibres 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 collagen,
so-called chondrigen. The recent investigations of MOROCHOWETZ and
others, but especially those of C. M6RNER,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
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

1 Schulz, Pfliiger's Arch., 84 and 89; Frauenberger, Zeitschr. f. physiol. Chem., 57.

2 Morochowetz, Verhandl. d. naturh. med. Vereins zu Heidelberg, 1, Heft 5; Morner,
Skand. Arch. f. Physiol., 1.

522 TISSUES OF THE CONNECTIVE SUBSTANCE.

quantities of mineral acids. The precipitation may be retarded by
neutral salts or by chondroitin-sulphuric acid. The solution containing
NaCl and acidified with HC1 is not precipitated by potassium ferro-
cyanide. 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 MILLON'S
and ADAMKIEWICZ'S 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 varie-
ties of cartilage and also in the tunica intima of the aorta and as traces
in the bone substance. K. MORNER 1 has also found it in the ox-kidney
and in human urine as a regular constituent. Its occurrence in amyloid
as mentioned on page 168 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 Ci8H27NSOi7, according
to ScHMiEDEBERG.3 As primary products this acid yields, on cleavage,
sulphuric acid and a nitrogenous substance, chondroitin, according 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
more strongly than dextrose. 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

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.

CHONDROITIN-SULPHURIC ACID. 523

acid and glucosamine. This assumption does not seem to have sufficient
foundation. According to ORGLER and XEUBERG, 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 C6Hi3O6N
and also a hexosamine acid or tetraoxyaminocaproic acid. In opposition
to this S. FRANKEL l 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 C6HnNO6, which he calls aminoglucuronic
acid, which gives the above .tests and also reduces.

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 solu-
tion 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 acid, sugar of lead, mercuric 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 chondroit in-
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,
the excess of this acid removed with sugar of lead, and the lead removed
from the filtrate by H2S. 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.

SCHIMIEDEBERG prepared the acid from the septum narium of the pig
according to the following method: The finely divided cartilage is
first exposed to artificial peptic digestion, then carefully washed with
water and the insoluble residue treated with 2-3 per cent hydrochloric
acid. This cloudy liquid containing hydrochloric acid is precipitated with
alcohol (about one-quarter vol.) and the clear filtrate treated with absolute
alcohol and some ether. The precipitate, consisting chiefly of a com-
bination or a mixture of chondroitin-sulphuric acid and gelatin pep-
tone (peptochondrin), is first washed with alcohol and then with water.

1 Orgler and Neuberg, Zeitschr. f. physiol. Chem., 37; Frankel, Annal. d. Chem. u.
Pharm., 351.

524 TISSUES OF THE CONNECTIVE SUBSTANCE.

It is then dissolved in alkaline water and the basic alkali compound
precipitated from this solution by the addition of alcohol, whereby the
gelatin -peptone alkali remains in solution. The precipitate is purified
by repeated solution in alkaline water and precipitated by alcohol. To
obtain chondroitin-sulphuric acid entirely free from chondroitin it is
more advantageous to prepare the potassium-copper compound of the
acid, from the alkaline solution, by the alternate addition of copper acetate
and caustic potash and precipitation with alcohol. The reader is referred
to the original article for more details and also tor ODDI'S method.

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 following 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 r
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,! 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 Pfluger's in Pfliiger Arch., 92; Handel, ibid.

CORNEA. 525

extent in all matrices, and of these it is richest in the cartilage. Ten-
dons, ligament-urn nuchae, 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 chondroi tin-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 substance.
The cartilage is richest in sodium of all the tissues of the body, and accord-
ing 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
NaoO 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 XV). 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 2 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 167), which contains 14.77 per cent N and 0.90
per cent S.

In the cornea of oxen His3 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 Zeitschr. f. physiol. Chem., 18.

3 Cited from Gamgee, Physiol. Chem., 1880, 451.

526 TISSUES OF THE CONNECTIVE SUBSTANCE.

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 is not usually present in the bony structure
(HERBERT SMITH *).

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 GIES 2 have shown, mucoid and albuminoid.
After the removal of the lime-salts by hydrochloric acid of 2-5 p. m.
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 following
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 11.75 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. Na2C(>3, 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 25.68J GIES

The inorganic constituents of the bony structure, the so-called bone-
earths, which after the complete calcination of the organic substance
remain as a white brittle mass, consist chiefly of calcium and phosphoric

1 Zeitschr. f. Biologie, 19. 2 Amer. Journ. of Physiol., 5 and 7.

BONE. 527

acid, but also contain carbonic acid and, in smaller amounts, magnesium,
chlorine, and fluorine. Alkali sulphate and iron, which have been found
in bone-ash, do 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 MOR-
NER,1 from the chondroitin-sulphuric acid. According to GABRIEL,
potassium and sodium are essential constituents of bone-earth, and this
has been substantiated by ARON.-2

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 in apatite (CaCl2,3Ca3P2O8) .
If we eliminate the magnesium, the chlorine, and the fluorine, the
last, GABRIEL claims, occurring only as traces, the remaining mineral
bodies form the combination 3(Ca3P2Os)CaCO3. In his opinion the
simplest expression for the composition of the ash of bones and teeth
is (Ca3(PO4)2 + Ca5HP3Oi3 + Aq), in which 2-3 per cent of the lime is
replaced by magnesia, potash, and soda, and 4-6 per cent of t]\e phos-
phoric acid by carbonic acid, chlorine, and fluorine.

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.3 The figures represent parts per thousand :

Man. Ox. Tortoise. Guinea-pig.

Calcium phosphate, Ca3P2O8 838 . 9 860 . 9 859 .8 873 . 8

Magnesium phosphate, Mg3P2O8 10.4 10.2 13.6 10.5

Calcium combined with CO2, Fl, and Cl. .. 76.5 73.6 63.2 70.3

CO2 57.3 62.0 52.7

Chlorine 1.8 2.0 1.3

Fluorine4 2.3 3.0 2.0

Some of the C02 is always lost on calcining, so that the bone-ash does not
contain the entire CO2 of the bony substance.

AD. CARNOT 5 found the following composition for the bone-ash of
man, ox, and elephant:

Calcium phosphate

Man.
Femur Femur
(body). (head).

874 5 878 7

Ox.
Femur

857 2

Elephant.
Femur.

900 3

Magnesium phosphate

15 7 17 5

15 3

19 6

Calcium fluoride

35 37

4 5

4 7

Calcium chloride

23 30

3 0

2 0

Calcium carbonate

101 8 92 3

119 6

72 7

Iron oxide .

10 13

1 3

1 5

1 Zeitschr. f. physiol. Chem., 23.

2 Gabriel, ibid., 18, which also contains the pertinent literature; Aron, Pfliiger's
Arch., 106.

3 Hoppe-Seyler, Med.-chem. Untersuch., p. 19.

4 The reports as to the quantity of fluorine disagree ; see Harms, Zeitschr. f .
Biologic, 38; Jodlbauer, ibid., 41.

5 Compt. rend., 114.

528 TISSUES OF THE CONNECTIVE SUBSTANCE.

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 com-
pact 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-SEYLER1 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.

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,2 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 235. The protein consists of a globulin coagulating
at 47-60° C. (FORREST) and a nucleoprotein with 1.6 per cent phos-
phorus (HALLIBURTON3) besides fibrinogen (P. MiiLLER4), traces of
albumin and proteose. In the extractives are formed 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 5).

The diverse quantitative composition of the various bones of the
skeleton depends probably on the varying quantities of other tissues,

1 Physiol. Chem., 102-104.

2 Bioch. Zeitschr., 10.

3 Forrest, Journ. of Physiol., 17; Halliburton, ibid., 18.

4 See footnote 1, p. 245.

5 Nerking, 1. c.; Hutchinson and Macleod, Journ. of Anat. and Physiol., 36.

BONE. 529

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. SCHRODT l has made comparative analyses of different
parts of the skeleton of the same animal (dog) and has found an essential
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 vertebrae 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 2).

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 3 has found in rabbits, 6J-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 ©f 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.4

1 Cited from Maly's Jahresber., 6.

2 Killer, cited from Maly's Jahresber., 14; During, Zeitschr. f. physiol. Chem., 23.
•Volt, Zeitschr. f. Biologic, 16; Brubacher, ibid., 27; Graffenberger in Maly's

Jahresber., 21.

4 See H. Weiske, Zeitschr. f. Biologie, 31, and W. Stoeltzner, Pfliiger's Arch., 122,
and H. Stoeltzner, Bioch. Zeitschr., 12.

530 TISSUES OF THE CONNECTIVE SUBSTANCE.

On feeding sufficient calcium and phosphorus in the food ARON l 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 metab-
olism 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. Otherwise pathological conditions seem to affect
chiefly the quantitative composition of the bones, and especially the
relation between the organic and the inorganic substance. In exos-
tosis and osteosclerosis the quantity of organic substance is generally
increased. In rachitis and osteomalacia the quantity of bone-earths is
considerably decreased. 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 ERWIN VOIT, ARON and SEBAUER and others2
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, B AGINSKY 3) . WEISKE, on the contrary, has shown,
by administering dilute sulphuric acid or monosodium phosphate with
the food (presupposing that the food gave no alkaline ash) to sheep and
rabbits, that the quantity of mineral bodies in the bones might be dimin-
ished. On feeding continuously for a long time with a feod which
yielded an acid ash (cereal grains), WEISKE observed a diminution in
the mineral substances of the bones in full-grown herbivora.4 A few
investigators are of the opinion that in rachitis, as in osteomalacia,
a solution of the lime-salts by means of lactic acid takes place. This
was suggested by the fact that O. WEBER and C. SCHMIDT 5 found lactic
acid in the cyst-like, altered bony substance in osteomalacia.

Well-known investigators have disputed the possibility of the lime-

1 Pfliiger's Arch., 10G.

2 Zeitschr. f. Biologie, 16; Aron and Sebauer, Bioch. Zeitschr., 8; A. Baginsky,
Arch. f. (Anat. u.) Physiol., 1881.

3 Heitzmarm, Maly's Jahresber., 3, 229; Heiss, Zeitschr. f. Biologie, 12; Baginsky,
Virchow's Arch., 87.

4 See Maly's Jahresber., 22; also Weiske, Zeitschr. f. physiol. Chem., 20, and
Zeitschr. f. Biologie, 31.

5 Cited from v. Gorup-Besanez, Lehrb. de. physiol. Chm., 4. Aufl.

TOOTH STRUCTURE. 531

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 can be easily proven.
The investigations of LEVY 1 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 decalcification in which one
molecule of phosphate-carbonate after the other is removed.

In rachitis the quantity of organic matter has been found to vary between 664
and 811 p. m. The quantity of inorganic substance was 189-336 p. m. These
figures refer to the dried substance. According to BRUBACHER, rachitic bones
are richer in water than the bones of healthy children, and poorer in mineral
bodies, especially calcium phosphate. 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 2 found a larger quantity of magne-
sium than calcium in a bone. The ash contained 417 p. m. phosphoric acid, 222
Lm. lime, 269 p. m. magnesia, and 86 p. m. carbon dioxide. Other investigators
ve on the contrary found considerably 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 contains
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

1 Zeitschr. f . physiol. Chem., 19.

2 Chabrie, Les ph6nomenes chim. de 1'ossification, Paris, 1895, 65.

532 TISSUES OF THE CONNECTIVE SUBSTANCES.

in bone-earths. The quantity of chlorine according to him is remarka-
bly high, 0.3-0.5 per cent, while BERTZ l found that the ash of enamel
was free from chlorine and that dentin was very poor in chlorine.

CARNOT,2 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.

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.3 The same investigator found that the amount of phos-
phates is strikingly small in the enamel, and in the teeth considerable
lime is replaced by magnesia. This coincides with BERTZ'S findings,
that dentin contains twice as much magnesia as the enamel.

According to GASSMANN 4 the teeth among themselves have different
composition, and in man the wisdom teeth are poorer in organic substance
and richer in lime than the canine teeth. The great tendency of the first
to caries is probably explained by this fact. The reason for the degene-
ration of the teeth is considered by C. ROSE 5 to be a lack of earthy salts
and according to him one finds the best teeth in localities where the
drinking water has strong 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 cellular 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 REINECKE 6 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 chiefly consists of triglycerides of
stearic, palmitic, and oleic acids. Besides these, especially in the less

1 See Maly's Jahresber., 30. 4 Zeitschr. f. physiol. Chem., 55.

2 Compt. rend., 114. 5 Deutsch. Monatsh. f. Zahnheilk., 1908.
* Sec footnote 4, p. 527. « Annal. d. Chem. u. Pharm., 142.

FATTY TISSUE. 533

solid kinds of fats, there are glycerides of other fatty acids (see Chapter
V). In all animal fats there are besides these, as Fn. HOFMANN 1 has
shown, also free, non-volatile tatty 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.2 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.S 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 properties of fats in general, and the three most important varieties
of fat in particular, have already 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
tat 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
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

1 Ludwig-Festschrift, 1874, Leipzig.

2 See Jaeckle, Zeitschr. f. physiol. Chem., 36 (literature).

3 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.

534 TISSUES OF THE CONNECTIVE SUBSTANCES.

been derived from the fat of the food. PETTENKOFER and Vorr 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 transforned by synthesis with gly-
cerin 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 by ROSENFELD. CORONEDI and
MARCHETTI and in particular WINTERNITZ l have recently shown that
the 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
Iv. 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
poisoning, that we cannot consider the formation of fat as conclusively
proven. Recent investigations of ATHANASIU, TAYLOR, SCHWALBE,
and others, especially of RosENFELD,2 have shown that probably no new

1 Coronedi arid 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.

2 Bauer, Zeitschr. f. Biologic, 7; Leo, Zeitschr. f. physiol. Chem., 9; Polimanti,
Pfluger'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 1,
p. 368.

FORMATION OF FAT FROM PROTEINS. 535

formation of fat from protein took place, but rather a fat migration
(ROSENFELD).

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 1 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.

WEINLAND 2 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 investi-
gations of PETTENKOFER and VOIT are often quoted. These investi-
gators 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-
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
glycogen. On recalculation of the figures, using these coefficients, PFLUGER

1 See Rosenfeld, Fettbildung, Ergebnisse der Physiologic, 1, Abt. 1.

2 Zeitschr. f. Biol., 51.

536 TISSUES OF THE CONNECTIVE SUBSTANCES.

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
proven. KAUFMANN has recently substantiated this view by a method
which will be spoken of in detail in Chapter XVIII, 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 proven.

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
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 combated for some time, and until
lately it was the general opinion that a direct formation of fat from
carbohydrates not only had not been proven, but also that it was
improbable. The undoubtedly great influence of the carbohydrates on
the formation of fat as observed and proven 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

1 See 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 FROM CARBOHYDRATES. 537

with foods especially rich in carbohydrates LAWES and GILBERT, SOXH-

LET, TSCHERWINSKY, MEISSL, and STROMER (on pigs), B. SCHULTZE,

CHANIEWSKI, E. VOIT and C. LEHMANN (on geese), I. MUNK and RUBNER
and LUMMERT l (on dogs) apparently prove 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 complicated 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 rela-
tion between the inspired oxygen and the expired carbon dioxide, i.e., the

CO

respiratory quotient -W^, was found greater than 1 in certain cases (HAN-
RIOT and RICHET, BLEIBTREU, KAUFMANN, LAULANIE 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
iipase is of importance here, as LOEVENHARTS has found that all over
the body where fat is deposited in large amounts Iipase 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
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.

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. Biologie, 20; Voit and Lehmann, see C. v. Voit, Sitz-
ungsber. d. k. bayer. Akad. d. Wissensch., 1885; I. Munk, Virchow's Arch., 101;
Rubner, Zeitschr. f. Biologie, 22; Lummert, Pfliiger's Arch., 71.

2 Hanriot and Richet, Annal. de Chim. et de Phys. (6), 22; Bleibtreu, Pfluger's
Arch., 56 and 85; Kaufmann, Arch, de Physiol. (5), 8; Laulanie, ibid., 791.

3 Amer. Journ. of Physiol., 6.

CHAPTER XI.
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 bo
satisfied at the present time with a few microchemical reactions in the
investigation of the chemical composition of the muscular fibres.

Each muscle-tube and each muscle-fibre 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 posesses the power of contractility, has in the inactive
muscle an alkaline reaction, or, more correctly speaking, an amphoteric
reaction 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 monophosphate chiefly. The dead muscle has an acid reaction, or,
more correctly, 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 ROHMANN 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. If the
muscular fibres are treated with reagents which dissolve proteins,

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.

538

PROTEINS OF THE MUSCLES. 539

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 shrink-
ing, the fibres split longitudinally into fibrils; and this behavior shows
that several chemically different substances of various solubilities enter
into the construction of the muscular fibres.

The protein myosin is generally considered as the chief 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 D ANILE WSKY, con-
firmed by J. HOLMGREN,1 myosin may be completely extracted from
the muscle \\lthout changing its structure, by means of a 5-per cent
solution of ammonium chloride, which fact conflicts with the above view.
D ANILE WSKY claims that another protein-like substance, insoluble in
ammonium chloride and only swelling up therein, enters essentially
into the structure of the muscles. The proteins, which form the chief
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 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 thoroughly frozen 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

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. Forth, 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.

540 MUSCLES.

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 alka-
line reaction. It varies in different animals. Muscle-plasma from the
frog spontaneously coagulates slowly at a little above 0° C., but more
quickly at the temperature of the body. Muscle-plasma from mammals
coagulates slowly, according to v. 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
proteins, 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 separately discuss 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 chief mass of contractile protoplasm. The findings as to the
occurrence 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,1 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

1 Zeitschr. f. physiol. Chem., 7.

2 Studies from the Physiol. Chem. Laboratory of Yale College, New Haven, 3, 115.

MYOSIN, MUSCULIN. 541

myosin separates as fibres, 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. It is insoluble in water, but
soluble in dilute saline solutions as well as in dilute acids or alkalies,
which readily convert it into albuminates. 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
from 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 magnesium-
sulphate solution. Tne filtered extract is then treated with magnesium
sulphate in substance until 100 cc. of the liquid contain about 50 grams
of the salt. The so-called paramyosinogen or musculin separates. The
filtered liquid is then treated with magnesium sulphate until each 100 cc.
of the liquid hold 94 grams of the salt in solution. The rnyosin which
now separates is filtered off, dissolved in water by aid of the retained
salt, precipitated by diluting with water, and, when necessary, purified
by redissolving in dilute salt solution and precipitating with water.

The older and perhaps the usual method of preparation consists,
according to DANILEWSKY/ 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 MgSC>4 (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

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).

542 MUSCLES.

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.

Myogldbulin. After the separation of the musculin and the myosin from the
salt extract of the muscle by means of MgSO4, the myoglobulin may be precipitated
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 fibre the
lt muscle-stroma." According to DANILEWSKY the amount of such stroma
substance is connected with the muscle activity. He maintains that
the muscles contain a greater amount of this substance, compared with
the myosin present, when the muscles are quickly contracted and
relaxed, the correctness of which report has recently been disputed by

SAXL.1

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 coaguable proteins,
and dissolves in dilute alkalies, forming an albuminate. The elementary
composition of this substance is nearly 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 contains 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

1 Hofmeister's Beitrage, 9. 2 See footnote 1, p. 539.

PROTEINS OF THE MUSCLE PLASMA. 543

the muscle nuclei. According to BOTTAZZI and DUCCESCHI l 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'S 2 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. 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 solu-
tion (5 per cent MgSO4), 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 warm-
ing or by the addition of myosin ferment, is, according to HALLIBURTON,
a process analogous to the coagulation of the muscle-plasma. Accord-
ing to this same investigator, myosin when dissolved 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

1 Pekelharing, Zeitschr. f. physiol. Chem., 22; Bottazzi and Ducceschi, Centrabl.
f . Physiol., 12.

2 Arch. f. exp. Pathol. u. Pharm., 53.

544 MUSCLES.

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. HALLI-
BURTON'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
musculm 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
this way from the rest of the musculin. The myogen exists in the new
filtrate 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 filtrate 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 myosin-
ogen-f-myoglobulin. 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 fibrin + myogen
fibrin, and corresponds, it seems, also to myosin mixed with paramyo-
sinogen (HALLIBURTON). STEWART and SOLLMANN differ from HALLI-
BURTON in considering that paramyosinogen 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.

For these reasons the author is not sure whether he has understood and correctly
given the work of the different investigators.

EXTRACTIVE BODIES OF THE MUSCLES 545

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 investiga-
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 J 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 hsemo-
chromogen, and called myohcematin by him, has not been substantiated,
at least for muscles of higher animals (LEVY and MORNER 2) . 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.

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. The disputed glycolytic
enzyme (Chapter VIII), whose nature is unknown, probably belongs to
the oxidases. An amylolytic and a proteolytic enzyme (HEDIN and
ROWLAND3) have also been found, and the hydrolytic and oxidizing
enzymes (Chapter XV) active in the formation and destruction of uric
acid are also present.

Extractive Bodies of the Muscles.

The nitrogenous extractives consist chiefly of creatine, on an average
of 1-5 p. m. in the fresh muscles containing water, also the purine bases,
hypoxanthine and xanthine, besides guanine and carnine, but chiefly
hypoxanthine. The purine bases probably do not occur as such, but as
complex combinations. The quantity of nitrogen as purine bases amounts,
according to BURIAN and HALL, in the fresh flesh of the horse, ox, and calf
to 0.55, 0.63, and 0.71 p. m. respectively, or 1.3-1.7 p. m. calculated as

1 Przibram, Hofmeister's Beitrage, 2; Steyrer, ibid., 4.

2 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.

3 Zeitschr. f. physiol. Chem., 32.

546 MUSCLES.

hypoxanthine. In the embryonic ox-muscles, KOSSEL l found more
guanine than hypoxanthine. The purine bases are produced in the
muscles themselves, and their production, which also takes place while
at rest, is greatly increased during work (BuRiAN2).

Among the apparently habitually occurring nitrogenous extractives,
we should also mention phosphocarnic acid as well as inosinic add, which
is perhaps allied to it, carnosine, carnitine, and perhaps also other bodies
which have recently been found in meat extract and which will be men-
tioned later.

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. THESEN in fish-flesh is nothing but impure creatinine,
according to POULSSON, SCHMIDT and KoRNDORFER.3 Uric acid, urea, taurine,
and leucine are found as traces in the muscles, in certain cases only in a few species
of animals. In regard to the amounts of these different extractives in the muscles,
KRUKENBERG and WAGNER 4 have shown that they vary greatly in different
animals. A large quantity of urea is found in the muscles of the shark and ray;
uric acid is found in alligators; taurine in cephalopoda; glycocoll in gasteropoda,
and creatinine especially in fishes. 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,S
urea is a regular constituent of the muscles, although M. NENCKI and KOWARSKI
dispute this.

The purine bases, with the exception of carnine, have been treated
on pages 184-188, and therefore among the extractive bodies we will
first consider the creatine.

/NH2

Creatine, C-iHgNsC^, C^=NH , or methyl -guanidine-

\N(CH3).CH2COOH

acetic acid, occurs in the muscles of vertebrate animals in variable amounts,
1.4-5 p. m., in different species; the largest quantity is found in birds.
It is also found in the brain, blood, transudates, amniotic fluid, and some-
times also in the urine. Creatine may be prepared synthetically from
cyanamide and sarcosine (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 investiga-

1 Burian and Hall, Zeitschr. f. physiol. Chem., 38; Kossel, ibid., 8, 408.

2 Ibid., 43.

3 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.

4 Zeitschr. f. Biologie, 21; see also M. Henze, Zeitschr. f. physiol. Chem., 43;
Mendel, Hofmeister's Beitrage, 5; Kelly, ibid., 5.

5 Kaufmann, Arch, de Physiol. (5), 6; Schondorff, Pfliiger's Arch., 62; Nencki
and Kowarski, Arch. f. exp. Path. u. Pharm., 36; Brunton-Blaikie, Journ. of Physiol.,
23, Supplement.

CREATINE. 547

tors 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, C4H7N3O, which
is retransformed into creatine by the action of alkali.

The question as to the relation of creatine to creatinine within the
animal body is disputed, and is intimately related to the question as
to the role of the creatinine in protein metabolism. As this question
is best discussed in connection with creatinine and its elimination in
the urine (Chapter XV), we will here only discuss the direct relation of
creatine to the muscles and its metabolism.

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-dialyzabla
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. SEEM ANN 1
indeed 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 autolytic 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
STANGASSINGER2 with the kidneys and livers of dogs not only point to
the ability of these organs to decompose creatine, but also for a re-for-
mation of creatine in the liver. Further investigations are still very
necessary.

Opinions are not unanimous in regard to the organ where creatine
is formed. From recent investigations it is concluded that the liver
plays an important part therein. Other organs, especially the muscles,

1 Urano, Hofmeister's Beitrage, 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.

2 Zeitschr. f. physiol. Chem., 55.

548 MUSCLES.

are also included. According to MELLANBY the creatinine is probably
formed in the liver and transformed into creatine in the muscles and there
deposited as such.

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-
tion of creatine in water is not precipitated by basic lead acetate, but
gives a white, flaky precipitate with mercurous nitrate if the acivd 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 (JAFFE1).

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 H2S, 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. The quantitative estimation of creatine is performed
by transforming it into creatinine (see Chapter XV) .

Carnine, C7H8N4O3 + H2O, 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, which may be transformed into hypoxanthine
by oxidation is, according to HAISER and WENZEL,3 probably only an
equimolecular mixture of hypoxanthine and a pentoside, CioH^NjO-,
called inosine, which is crystalline, and which readily splits' into
hypoxanthine and pentose by the action of acid.

Carnine has been obtained as a white crystalline mass. It dissolves

1 Ber. d. d. Chem. Gesellsch., 35.

3 Zeitschr. f. analyt. Chem., 2 and 6.

3 Weidel, Annal. d. Chem. u. Pharm., 158; Krukenberg and Wagner, Sitznngsber.
d. Wiirzb. phys.-med. Gesellsch., 1883; Pouchet, cited from Neubauer-Huppert,
Analyse des Harnes, 10. Aufl., 335; Haiser and Wenzel, Monatsch. f. Chem., 29.

CARNIXE, CARXOSIXE, CARNITINE. 549

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. Carnine does not give the so-called WEIDEL'S xanthine
reaction. Its watery solution is precipitated by basic lead acetate;
but the lead compound may be dissolved on boiling.

Carnine is prepared by the following method: The meat extract diluted with
water is completely precipitated by baryta-water. The filtrate is precipitated
by basic lead acetate, the lead precipitate boiled with water, filtered while hot,
and sulphuretted hydrogen passed through the filtrate. Remove the lead sul-
phide from the filtrate and concentrate strongly. The concentrated solution
is now completely precipitated with silver nitrate, the precipitate washed free
from silver chloride by ammonia, and the carnine silver oxide suspended in
water and treated with sulphuretted hydrogen.

Carnosine, C9H14N4O3, has been isolated by GULEWITSCH and AMIRADZIBI
from meat extracts. It is a base which is readily soluble in water, crystallizing
in flat needles. It is precipitated by phosphotungstic acid and by silver nitrate
in the presence of an excess of barium hydrate, and forms a copper compound
which crystallizes in hexagonal plates. Carnosine, which also occurs, according
to KRIMBERG, in fresh meat to the extent of 1.3 p. m., is probably a histidine
derivative (GULEWITSCH) which is identical with ignotine, isolated by KUTSCHER
from meat extract. According to KUTSCHER * these extractive bodies are more
likely isomeric bodies.

Carnitine, C7H15NO3, is another base isolated by GULEWITSCH and KRIMBERG
from meat extracts, having a strong alkaline reaction, and is very readily soluble
in water, and which KRIMBERG also found in fresh meat. Carnitine according to
KRIMBERG 2 is a trimethylamine derivative and probably trimethyloxybutyro-

/O— —CO

betaine with the formula (CH3)3N<f | . According to him

\CK2-CH.OH-CH2

it is also very probably identical with novaine prepared by KUTSCHER from meat
extracts, which is also a trimethylamine derivative. It gives crystalline double
compounds with platinum, gold and mercuric chlorides, among which the follow-
ing, C7H15NO32HgCl2, with a melting-point of 196-197° C., is especially used in the
isolation of the base.

From LEIBIG'S extract of beef KUTSCHER has isolated besides the above-
mentioned ignotine and novaine, several other bodies, neosine, C8H17N02, which
according to KUTSCHER and ACKERMANN is a homologue of choline, vitiatine
(as gold salt, C6Hl4N6.2HC1.2AuCl3), carnomuscarine, methylguanidine (also found
by GULEWITSCH), oblitine, Gi8H38N2O5, which probably contains two novaine
groups, which corresponds well with KRIMBERG 's 3 view, and also choline and
neurine. ZUNZ * has been able to obtain from fresh meat the three so-called hexone

:

1 Gulewitsch and Amiradzibi, Zeitschr. f. physiol. Chem., 30; Gulewitsch, ibid.,
50, 51, and 52; Krimberg, ibid., 48; Kutscher, ibid., 50 and 51.

2 Gulewitsch and Krimberg, Zeitschr. f. physiol. Chem., 45; Krimberg, ibid., 49,
50, 53, 56.

3 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.

4Zunz, reference in Centralbl. f. Physiol., 18, 852.

550 MUSCLES.

bases besides leucine, aspartic and.glutamic acids, and MicKo1 found in meat extracts
small quantities of alanine, glutamic acid, taurine and inosite, but no dipeptides.
In fish-flesh SUWA 2 found creatine, creatinine and methylguanidine, but not
the numerous bodies found by KUTSCHER in meat extract. In crab extract
KUTSCHEK and AcKERMANN'3 found no creatine and creatinine, but among others
betaine and two new bases, crangitine, C,3H20N2O4 and crangonine, C13H26N2O3.

The base musculamine, isolated by ETARD and VILA on the hydrolysis of veal,
is nothing but cadaverine, according to POSTERN AK.4

Inosinic acid has been discussed on page 179. 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, xantko-
creatinine, C5H10H4O, crusocreatinine, C5H8N4O, amphicreatine, Q,H19N704, and
pseudoxanthine, C4H5N5O.

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,8 for details of which the reader is referred to the original article.

Phosphocarnic acid 7 is a complicated substance, first isolated by SIEGFRIED
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,
torni/emne, from the extract of the muscles free from proteins. The quantity
of phosphocarnic acid, calculated as carnic acid, can be determined by multiply-
ing the quantity 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 MANIC ARDI found, in the plant kingdom. Phos-
phocarnic 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.

The non-nitrogeneous extractive bodies of the muscles are inosite, glyco-
gen, sugar, and lactic acid.

1 Zeitschr. f. physiol. Chem., 56.

2 Centralbl. f. Physiol., 22, 307.

3 Zeitschr. f. Unters. d. Nahrungs-u. Genussmittel, 13 and 14:

4 Etard and Vila, Compt. rend., 135; Posternak, ibid., 135.

5 See Maly's Jahresber., 16, 523.

6 Ibid., 22, 335.

7 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
Ide, ibid., 21, and Balke, ibid., 22; Macleod, ibid., 28; E. Cavazzani, Centralbl. f.
Physiol., 18, 666; Panella, Maly's Jahresber., 34.

INOSITE. 551

Inosite, C6H1206 + H20 = C6HG(OH)6 + H20. This body, discovered
by SCHERER, is not a carbohydrate, but belongs to the hydroaromatic
compounds, and is a hexahydroxy benzene (MAQUEXNE 1). That it stands
in certain relation to the carbohydrates follows from the fact that NEUBERG
obtained some furfurol from inosite by distillation with phosphoric
anhydride and also that P. MEYER2 found fermentation lactic acid in
the urine of rabbits after the introduction of inosite p3r os. It has been
known for some time that inosite undergoes lactic acid fermentation.
The acid formed thereby is sarcolactic acid according to HILGER and
fermentation lactic acid according to VOHL.S

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. ROSENBERGER attempted to show
that in certain animals (rabbits) and organs (muscles) the inosite did
not occur free,but as an inositogen, but this claim has not sufficient founda-
tion and it is denied by STARKENSTEiN.4 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.
According to WINTERSTEIN a phosphorized compound occurs in the
vegetable kingdom which yields inosite as a cleavage product and whose
Mg and Ca compound is called phytin. POSTERNAK considers this
body as an anhydroxymethylenediphosphoric acid. From the cleavage
experiments of WINTERSTEIN as well as the observations of SUZUKI,
YOSHIMURA and TAKAISHI 5 on the occurrence in rice-bran of a special
enzyme, phytase, which splits phytin into inosite and phosphoric acid,
it seems as if this body is more likely an inosite-phosphoric acid. Ino-
site is found in plants, especially in the developing organs (MEILLERE),
and according to STARKENSTEIN 6 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 decomposition product of
metabolism, but rather a body necessary for the development of the cells.

Inosite, which nearly 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

1 Bull. soc. chem. (2), 47 and 48; Compt. rend., 104.

2 Neuberg, Bioch. Zeitschr., 9; P. Mayer, ibid., 9.

3 Hilger, Annal. d. Chem. u. Pharm., 160; Vohl, Ber. d. d. Chem. Gesellsch., 9.

4 Rosenberger, Zeitschr. f. physiol. Chem., 56, 57, and 58; Starkenstein, ibid., 58.

5 Winterstein, Ber. d. d. chem. Gesellsch., 30, and Zeitschr. f. physiol. chem., 58;
Posternak, Contribution a 1'etude chim. de 1'assimilation chlorophyllienne. Revue
generate botanique, Tome 12 (1900), and Compt. rend., 137; Suzuki, Yoshimura and
Takaishi, Bull, agric. Univers. Tokio, 7.

6 Meillere Jour. d. Chim, et Pharm. (6) 28; Starkenstein, Zeitschr. f, Exp. Path. u.
Therap., 5.

552 MUSCLES.

groups of fine crystals similar to cauliflower. It loses its water of crys-
tallizatio'n 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 wat-er 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 not 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
add, and the following reaction depends upon this.

If inosite is evaporated to dryness on platinum-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 (G ALLOTS'
inosite test). New inosite reactions have been suggested by DENICES.*

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
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 H2S. 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 obtained from the alcoholic
solution directly, are recrystallized by redissolving in very little boiling
water and adding 2-4 vols. of alcohol. MEILLERE 2 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 .Ton. MULLER,S
and which was found long ago in the kidneys, liver and spleen of Plagiostomata.
Scyllite crystallizes in shining prisms, is soluble in water 1 : 100 at 180° C., is similar
to inosite in its reactions, but has a much higher melting-point, namely about
360° C.

1 Compt. rend. soc. biol., 62.

2 Compt. rend. soc. biol., 60, and Journ. d. Chim. et Pharm., (6) 24; see also
Starkenstein, Zeitschr. f. exp. Path. u. Ther., 5.

3 Ber. d. d. chem. Gesellsch., 40.

GLYCOGEN, MUSCLE SUGAR, LACTIC ACIDS. 553

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. 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 SCHONDORFF 1 has found a max-
imum 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 dif-
ference 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, JENSEN2). 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-
ing of food. As stated in Chapter VIII, 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 muscle,
and which is probably formed after the death of the muscle from the
muscle-glycogen, is, according to the investigations of PANORMOFF,
in part dextrose, but consists chiefly of maltose (OSBORNE and ZoBEL3)
with some dextrin.

Lactic Acids. Of the oxypropionic acids with the formula C3H003
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 three physical isomers, is of importance. These three
ethylidene lactic acids are the ordinary, optically inactive FERMENTA-
TION LACTIC ACID, the dextrorotatory PARALACTIC or SARCOLACTIC ACID,
and the LEVOLACTIC ACID obtained by SCHARDINGER by the fermenta-
tion of cane-sugar by means of a special bacillus. This levolactic acid,
which has also been detected by BLACHSTEIN in the culture of GAFFKY'S
typhoid bacillus in a solution of sugar and peptone, and which is formed
by various vibriones, need not be described here.4

1 Bohm, Pfliiger's Arch., 23, 44; Schondorff, ibid., 99; Moscati, Hofmeister's
Beitrage, 10.

2 Boruttau, Zeitschr. f. physiol. Chem., 18; Jensen, ibid., 35.

3 Panormoff, Zeitschr. f. physiol. Chem., 17; Osborne and Zobel, Journ. of Physiol.,
29.

4 See Schardinger, Monatshefte f. Chem., 11; Blachstein, Arch, des sciences biol.
de St. Pe"tersbourg, 1, 199; Kuprianow, Arch. f. Hygiene, 19, and Gosio, ibid., 21;
Herzog anc Horth, Zeitschr. f. physiol. Chem., 60.

554 MUSCLES.

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
has recently been disputed by MomYA.1 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, lym-
phatic glands, thymus, thyroid gland, blood, bile, pathological transudates,
osteomalacious bones, in perspiration in puerperal fever, in the urine
after fatiguing marches, in acute yellow atrophy of the liver, in poison-
ing 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 fasting
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 dextrose and also often
albumin) takes place through the urine, an observation which has been
confirmed by SAITO and KATSUYAMA.2 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 part
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

1 Heintz, Annal. d. Chem. u. Pharm., 157, and Gscheidlen, Pfliiger's Arch., 8,
171; Moriya, Zeitschrift f. physiol. Chem., 43.

2 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.

ORIGIN OF THE LACTIC ACID. 555

the liver and muscles was always diminished. He also calls attention
to the fact that dextrolactic acid may be formed from glycogen, as directly
observed by EKUNINA/ and also to the numerous observations on the
formation of lactic acid and the consumption of glycogen in muscular
activity. 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. HOPPE-SEYLER 2 also positively defends the view as to
the formation of lactic acid from carbohydrates. He was of the opinion
that lactic acid is produced from the carbohydrates by the cleavage of
the sugar only with lack of oxygen, while with sufficient oxygen the sugar
is burned into carbon dioxide and water. The formation of lactic acid
in the absence of free oxygen and in the presence of glycogen or dextrose
is, according to HOPPE-SEYLER, very probably a function of all living
protoplasm. In the anaerobic metabolism of the animal cells, accord-
ing to the recent investigations on alcoholic fermentation in the tissues
(see Chapter VIII), carbon dioxide and alcohol are formed from the
sugar, with lactic acid as an intermediary step; but even if this view
be correct and when the cells, as STOKLASA 3 and his collaborators have
shown, contain a lactic-acid-forming enzyme, it is not known what
kind of lactic acid is here produced. MORISHIMA believes that an increase
in the lactic acid in the liver occurs after death, probably from the
liver glycogen, but this acid is chiefly fermentation lactic acid, and the
fact must not be overlooked that INOUYE and KONDO 4 found dextro-
rotatory lactic acid on the autolysis of muscles.

ASHER and JACKSON 5 experimented by transfusing blood (with and
without the addition of sugar) through the lower extremities of dogs,
and neither in these experiments nor in those where the larger organs
(liver and abdominal viscera) were excluded from the circulation could
they detect any increase of lactic acid due to the sugar. Although
these last-mentioned investigations do not show any formation of lactic
acid from carbohydrates, still, on the other hand, we have recent
investigations that make such an origin for lactic acid very probable.
Thus EMBDEN 6 found, on percolating blood through a surviving liver
rich in glycogen, that lactic acid was formed, and also that this acid
was produced in abundance when blood rich in sugar was transfused

1 Journ. f. prakt, Chem. (N. F.), 21.

2 Virchow's Festschrift, also Ber. d. deutsch. chem. Gesellsch., 25, Referatb., 685.
3Simacek, Centralbl. f. Physiol., 17; Stoklasa, Jelinek, and Cerny, ibid., 16.
4Morishima, Arch. f. exp. Path. u. Pharm., 43; Inouye and Kondo, Zeitschr. f.

Physiol. Chem., 54.

5 Zeitschr. f. Biologic, 41.

6 Centralbl. f. Physiol., 18, 832.

556 MUSCLES.

through a glycogen-free liver, while, on the contrary, blood poor in sugar
led to only a very inconsiderable formation of lactic acid. The investi-
gations of A. R. MANDEL and LUSK 1 also indicate a formation of lactic
acid from carbohydrates, in the animal body. They have shown that
in dogs, after phosphorus poisoning, an abundance of lactic acid occurs
in the blood and urine, and that this disappears from these fluids on bring-
ing about a phlorhizin diabetes in the animal. Phosphorus intoxication
caused no lactic-acid formation in a phlorhizin-diabetic dog. Although
it is difficult to give a satibfactory explanation of the results of these
experiments, still it seems probable that by elimination of the sugar in
phlorhizin diabetes a mother-substance of the lactic acid is lost.

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.
In a previous chapter (VIII) we mentioned the formation of lactic acid
in the animal body by a deamination of alanine, and this gives us an
indication of a lactic-acid formation from protein. Phosphocarnic acid
is considered by SIEGFRIED as another source of sarcolactic acid.

The lactic acids are amorphous. They have the appearance of color-
less or faintly yellowish, acid-reacting syrups which mix in all propor-
tions 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 their different optical properties — paralactic acid being dex-
trogyrate, while fermentation lactic acid is optically inactive — also by
their different solubilities and the different amounts of water of crystalliza-
tion of 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(C3H5O3)2
+ 3H2O. The zinc salt of paralatic 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 micro-
scopic 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 estimation of
the lactic acids. The lithium salt contains 7.29 per cent Li. For fur-
ther information as to the salts and specific rotation of the lactic acids
see' HOPPE-SEYLER-THIERFELDER'S Handbuch, 8. Aufl., 1909.2

1 Amer. Journ. of Physiol., 1(>.

2 See also E. Jungfleisch, Compt. rend., 139, 140, and 142.

FAT AND PHOSPHATIDES. 557

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 an I 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 nitra-
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
shaken with ether to remove the fat. The residue is dissolved in water
and phosphoric acid 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, tne 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 and to the work

Of JERUSALEM.1

Fat is never absent in the muscles. Some fat is always found in the
intermuscular connective tissue; but the muscle-fibres 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
considerable quantity of fat in the muscle-fibres is found only in fatty
degeneration. 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 sub-
stance itself and is richer in free fatty acids, standing, according to
ZUXTZ and BoGDANOw,2 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
extraction and which is rich in fatty acids depends in part on a decom-
position of the lecithin and the phosphatides. ERLANDSEN has shown,
that phosphatides of various kinds occur in the muscles and indeed different
quantities in different muscles. According to him the ox-heart muscle
is richer in phosphatides than the muscle of the thigh, and RUBOW 3
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.

1 Bioch. Zeitschr., 12.

2 Arch. f. (Anat. u.) Physiol., 1897. e

3 Erlandsen, Zeitschr. f. physiol. Chem 51; Rubow, Arch. f. exp. Path. u. Pharm.,
52.

558 MUSCLES.

Tlie 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
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. SCHMEY l found
variations between 0.0129 p. m. (rabbits) and 0.0793 p. m. (human),
calculated on the fresh muscle substance. The heart-muscle was com-
paratively richer in iron, 0.06-0.109 p. m.

URANO 2 has removed the salts of the intermediary fluid (blood, lymph)
from frogs' muscle by treating them with an isotonic cane-sugar solution
(of 6 per cent) and in this manner found that 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 cal-
culated 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 more recent investiga-
tions of URANO the possibility of a disturbance in the osmotic properties
of the muscle fibres by the sugar solution is not entirely excluded, and the
question whether the muscle fibres are free from sodium or not has there-
fore not been positively decided. FAHR'S 3 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 studied by several experimenters (LOEB, LINGLE,
HOWELL, OVERTON, LANGENDORFF and HuECK, and others 4) . Further
proof as to the previously discussed ion action of the electroytes and the
antagonism of various ions has been given by many very interesting
investigations. These researches also indicate that each of the ions
Na, Ca, and K plays a certain part in the maintenance of the excitability,

1 Macallum, Journ. of Physiol., 32; Schmey, Zeitschr. f. physiol. Chem., 39.

2 Zeitschr. f. Biol., 50.

3 Urano, ibid., 51; Fahr, ibid., 52.

4 Loeb, Amer. Journ. of Physiol., 3, and Pfliiger's Arch., 80, 91; Lingle, Amer.
Journ. of Physiol., 4 (also references to literature); Overton, Pfliiger's Arch., 92 and
105; Langendorff and Hueck, ibid., 96.

PERMEABILITY OF THE MUSCLES. 559

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-
tained about 7 p. m. NaCl, besides small amounts of CaCl2 (0.2 p. m.),
KC1 (0.1 p. in.), 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-fibres, 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-fibres 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-fibres, 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-fibres 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-fibres
take place. The permeability changes essentially on the death of the
muscle.

The living muscle-fibres 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 belong to those bodies to which the muscle-fibres (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 organism

1 Pfliiger's Arch., 92. See also Hober, ibid., 106, and Hamburger, Osmotischer
Druck und lonenlehre, Bd. 3.

560 MUSCLES.

in part makes use of a similar artifice in order to regulate the concentra-
tion of the nutritive bodies within the protoplasm.

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 appear-
ing under these circumstances is called the spontaneous or the fermentative
rigor, because it seems to depend in part on the action of an enzyme.
A muscle may also become stiff for 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. The heat-rigor depends upon the coagula-
tion of certain proteins, and its occurrence at lower temperatures in
cold-blooded as compared with warm-blooded animals is due, accord-
ing to v. Ft)RTH,,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 l 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 this probably
depends upon the fact that the reaction of the muscle changes on heating
with the formation of lactic acid. 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. The rigor which is produced by means of acids
or other agents which, like alcohol, coagulate proteins must be considered
as produced by entirely different processes from those causing spontaneous
rigor.

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 reports in regard to the presence or absence
of free lactic acid in the rigor-mortis muscle are contradictory.2 Besides

1 Zeitschr. f. Biol., 48. See also E. B. Meigs, Amer. Journ. of Physiol., 24.

2 It is impossible to enter into the details of the disputed theories as to
the reaction of the muscles, etc. We shall only refer to the works of Rohmann,
Pfliiger's Arch!, 50 and 55, and Heffter, Arch. f. exp. Path. u. Pharm., 31 and 38

RIGOR MORTIS. £61

the formation of acid, the chemical processes which take place in rigor
of the muscles are the following: By the coagulation of the plasma a
myosin-clot is produced which is the cause of the hardening and of the
diminished transparency of the muscle; but this view must be changed
on account of the researches of v. FURTH, which have shown that the
clot consists of myogen fibrin and myosin fibrin. The appearance of this
clot may be hastened by the simultaneous occurrence of lactic acid.
Carbon dioxide is also formed, which does not seem to be a direct oxida-
tion product, but a product of the cleavage processes. HERMANN l
claims that carbon dioxide is produced in the removed muscle, even in
the absence of oxygen, when it passes into rigor mortis. In connec-
tion with this view we must call attention to FOLIN'S 2 observations
that no protein coagulation took place in rigor under special con-
ditions.

As many investigators admit of an increased formation of lactic acid on
the appearance of rigor mortis, the question arises, from what con-
stituents of the muscle is this acid derived? The most probable explana-
tion 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. On the other side,
BOHM 3 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.
It is therefore possible that the consumption of glycogen and the forma-
tion 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 lactic acid of the muscle may be considered as a decomposition prod-
uct of protein. The origin of the carbon dioxide is also not to be
sought for in the decomposition ot the glycogen or dextrose. PFLUGER
and STINTZING 4 found that in the muscle a substance occurs which
evolves large quantities of carbon dioxide on boiling with water, and it
is probably this substance which is decomposed with the formation of
carbon dioxide in tetanus as well as in rigor. In this connection we
call attention to the fact that phosphocarnic acid yields lactic acid as
well as carbon dioxide as cleavage products.

After the muscles have been rigid for some time they relax again and

These works contain also the researches of the earlier investigators more or less com-
pletely.

1 Untersuchungen iiber den StofTwechsel der Muskeln, etc., Berlin, 1867.

2 Amer. Journ. of Physiol., 9.

3 Nasse, Beitr. z. Physiol. der kontrakt. Substanz, Pfliiger's Arch., 2; Werther,
ibid., 46; Bohm, ibid., 23 and 46; Moscati, Hofmeister's Beitrage, 10.

4 Pfliiger's Arch., 18.

562 MUSCLES.

become softer. This is in part produced by the strong acid dissolving
the myosin-clot and in part by autolytic processes (VoGEL).1

Metabolism in the Inactive and Active Muscles. It is admitted
by a number of prominent investigators, PFLUGER and COLASANTI,
ZUNTZ and Ro'HRiG,2 and others, that the metabolism in the muscles is
regulated by the nervous system. When at rest, when there is no mechan-
ical 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 decid-
ing 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 procedure have
been adopted. The same active and inactive muscles have been compared
after removal, also the arterial and venous muscle-blood in rest and activ-
ity, and lastly the total exchange of material, the receipts and expend-
itures 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.
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

1 R. Vogel, Unters. iiber Muskelsaft, Deutsch. Arch. f. klin. Med., 1902.

2 See the works of Pfluger and his pupils in Pfliiger'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.

REST AND ACTIVITY. 563

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 several
investigators that the quantity of glycogen is increased and its correspond-
ing 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 consumption of glyco-
gen is increased, and it has been positively proven by the researches of
several investigators (NASSE, WEISS, KULZ, MARCUSE, MANCHE, MORAT
and DUFOUR 3) that the quantity of glycogen in the muscles in activity
decreases quickly and freely. As shown by the researches of CHAUVEAU
and KAUFMANN, QUINQUAUD, MORAT and DUFOUR, CAVAZZANI, and
especially those of SEEGEN,4 the sugar is removed from the blood and
consumed during activity. 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 aniphoteric reaction of the inactive muscles is changed dur-
ing 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,G more acid dur-
ing activity than the more slowly contracting red muscles. The acid
reaction appearing during activity was formerly considered to be due
to the formation of lactic acid, a view which has been contradicted by
ASTASCHEWSKY, PFLUGER, and WARREN, who found less lactic acid in
the tetanized muscle than when at rest. MONARI also found a decrease
in the quantity of lactic acid during activity, and according to HEFFTER

1 1. c. In regard to gas exchange in removed muscles, see aso J. Tissot, Arch, de
Physiol. (5), 6 and 7, and Compt, rend., 120.

2Chandelon, Pfliiger's Arch., 13; Vay, Arch, f. exp. Path. u. Pharm., 34, which
also contains the pertinent literature.

3 Nasse, Pfluger's Arch., 2; Weiss, Wien. Sitzungsber., 64; Kiilz, in Ludwig's
Festschrift, Marburg, 1890; Marcuse, Pfliiger's Arch., 39; Handle", Zeitschr. f. Biolo-
gic, 25; Morat 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; Pfliiger's Arch., 50.

5 Joh. Miiller, Zeitschr. f. allgem. Physiol., 3; Camis, ibid., 8; Locke and Rosen-
heim, Journ. of Physiol., 36.

6 Pfluger's Arch., 41.

564 MUSCLES.

the quantity of lactic acid in the muscle is diminished in tetanus produced
by poison. Contrary to the results of these investigations, MARCUSE and
WERTHER have been able to prove the formation of lactic acid during
activity; still the reports are discordant. Other observations indicate
a formation of lactic acid during activity. Thus SPIRO found an increase
in the quantity of lactic acid in the blood during work. COLASANTI
and MOSCATELLI found small quantities of lactic acid in human urine
after strenuous marches, and WERTHER observed an abundance of lactic
acid in the urine of frogs after tetanization. According to HOPPE-
SEYLER, on the contrary, in agreement with his view in regard to the
formation of lactic acid, it is not produced regularly during work, but
only when insufficient oxygen is supplied. ZILLESEN 1 has also found
that on artificially cutting off the oxygen from the muscles during life
more lactic acid was formed than under normal conditions.

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. Of importance in considering the
formation of lactic acid in the muscle and the conflicting opinions on
this subject is the work of FLETCHER and HopKiNS.2 They find that in
the preparation of the muscle and its preparation for the investigation
for lactic acid several sources of error are possible. Thus mechanical
irritation such as warming or treating the muscle with alcohol, which
is not ice-cold, brings about the formation of lactic acid. It was also
shown that the absence of oxygen is favorable to the formation or
accumulation of lactic acid, while abundant oxygen supply acts reversely.
The question as to the formation of lactic acid under different physiolog-
ical conditions requires further study.

According to SIEGFRIED the amount of phosphocarnic acid is dimin-
ished during activity. MACLEOD claims that this is true only for intense
muscular activity, while with ordinary work the organic phosphorus

1 Astaschewsky, Zeitschr. f. physiol. Chem., 4; Warren, Pfiiiger'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
and Moscatelli, Maly's Jahresber., 17, 212; Hoppe-Seyler, 1. c., and Zeitschr. f. physiol.
Chem., 19; Zillesen, ibid., 15.

2 Journ. of Physiol., 35.

REST AND ACTIVITY. 565

not present as nucleons is diminished ard the quantity of phosphates
is increased. This stands in accord with WEYL and ZEITLER'S l observa-
tions that the active muscle contains more phosphoric acid than the
inactive muscle. As in the dead muscle, so in the active muscle, the
somewhat stronger acid reaction is in part due to a greater quantity of
monophosphate.

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 M.ONARI 2 the total quanticy 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. In excessive activity MONARI also found xantho-
creatmine in the muscle, and the quantity was one-tenth that of the
creatinine. The recent investigations of GRAHAM BROWN and CATH-
CART 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 wrork. 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. The
purine bases are, according to BiiRiAN,4 increased during work, due to
a greater formation (see above, page 546). It seems to have been posi-
tively shown that the active muscle contains a smaller quantity of bodies
soluble in water and a larger quantity of bodies soluble in alcohol than the
resting muscle. (HELMHOLTZ 5).

Attempts have been made to solve the question relative to the
behavior of the nitrogenized constituents of the muscle at rest and during
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

1 Siegfried, Zeitschr. f. physiol. Chem., 21; Macleod, ibid., 28; Weyl and Zeitler,
ibid., 6.

2 Maly's Jahresber., 19, 296.

3Cathcart and Graham Brown, Journ. of Physiol., 37; Weber, Arch. f. exp. Path,
u. Pharm., 58.

1 Zeitschr. f . physiol. Chem., 43.
5 Arch. f. (Anat. u.) Physiol., 1845.

566 MUSCLES.

by other investigators, especially I. MUNK and HiRSCHFELD,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 therefore
also a measure of the protein catabolism. The earlier researches of ENGEL-
MANN, FLINT, and PAVY, as well as the more recent ones of BECK and
BENEDICT,3 and DUNLOP and his collaborators, show an increased elimina-
tion of sulphur during or after work, and this indicates an increased pro-
tein 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/ that a retention of nitrogen
and a exposition of protein occur during work. The discordant
observations 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
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.

Recently STEYRER 5 has found that the muscle juice of a continuously tetani zed
muscle was somewhat poorer in musculin and correspondingly richer in myogen
than the juice from a similar non-tetanized muscle. We cannot draw any con-

1 Voit, Untersuchungen iiber den Einfluss des Kochsalzes, des Kaffees und der
Muskelbewegungen auf den Stoffwechsel (Mimchen, 1860), and Zeitschr. f. Biologie, 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. Funke, Kellner, cited from Voit, Hermann's Handb., 86, 197; Dunlop
Noel-Paton, Stockman, and Maccadam, Journ. of Physiol., 22; Krummacher, Zeitschr.
f. Biologie, 33; Pfluger, Pfluger's Arch., 50; Zuntz, Arch. f. (Anat. u.) Physiol., 1894.

3 Engelmann, Arch. f. (Anat. u.) Physiol., 1871; Beck and Benedict, Pfluger's Arch.,
54, and also footnote 2.

4Caspari, Pfliiger's Arch., 83; Bornstein, ibid.; Kaup, Zeitschr. f. Biologie, 43;
Wait, U. S. Depart. Agricult. Bulletin, 89 (1901); Atwater and Benedict, ibid., Bull.,
69 (1899); Loewy, Arch. f. (Anat. u.) Physiol., 1901.

5 Hofmeister's Beitrage, 4.

MUSCLE WORK. 567

elusions from this experiment, but it seems to show that the proteins are not
consumed in 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,1 the fat belong-
ing to the muscle-fibres, which is difficultly extracted, takes part in
work. Besides these there are several researches by VOIT, PETTEN-
XOFER and VOIT, J. FRENTZEL,2 and others which make an increased
destruction of fat during work probable or proven.

If the results of the investigations thus far made of the chemical
processes going on in the active and inactive muscle 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 quotient,

, is found to be regularly raised during work ; yet this rise, which will

C02

O

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 consumption
of carbohydrates, glycogen, and sugar takes place. The acid reaction
of the muscle becomes greater with work. In regard to the extent of
a re-formation of lactic acid opinion is divided. An increased consump-
tion of fat has occasionally been observed. The quantity of organic
phosphorus decreases, and an increase in the nitrogenous extractives
of the creatinine group seems also to occur. Protein metabolism has
been found increased in certain series of experiments and not in others;
but an increased elimination of nitrogen as a direct consequence of mus-
cular exertion has thus far not been positively proven.

In close connection with the above-mentioned facts there is the
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 consisted
of a catabolism of the protein bodies; to-day another generally accepted
view prevails. FICK and WiSLiCENUS3 climbed the Faulhorn and calcu-
lated 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

1 Arch. f. (Anat. u.) Physiol., 1897.

2 Pfliiger's Arch., 68.

3 Vierteljahrsschr. d. Zurich, naturf. Gesellsch., 10, cited from Centralbl. f. d.
med. Wiss., 1866, 309.

568 MUSCLES.

urine, and found that the work really performed was not by any moans
compensated by the consumption of protein. It was therefore proven
by this that proteins alone cannot be the source of muscular activity,
and that this depends in great measure on the metabolism of non-nitro-
genous 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 generally considered as
positively proven 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 PFLUGER1 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-
nutriments, and according to SEEGEN, CHAUVEAU, and LAULANiii2 the
sugar is indeed the only direct source of muscular force. The last-
mentioned 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 investigations

1 Pfliiger's Arch., 50.

2 See Seegen, footnote 4, page 563. The works of Chauveau and his collaborators
are found in Compt. rend., 121, 122, and 123; Laulanie, Arch, de Physiol. (5), 8.

COMPOSITION OF THE MUSCLES. 569

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 BENEDICT 1 have also led to similar results as
to the fats being a source of muscular energy. The law of the substitu-
tion 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 substitu-
tion of the body material by the bodies supplied as food does not take
place in the muscular activity in the ordinary nutritive condition. Accord-
ing to JOHAXSSOX and KoRAEN2 the CO2 excretion produced by cer-.
tain 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 3 researches, phosphocarnic acid, which
yields on cleavage, among other bodies, carbon dioxide, occurs in part preformed
m 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 pur-
poses, in order to determine the nutritive value of different varieties
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 anahses are incomplete or of little value.

To give the reader some idea of the variable composition of muscle-
substance the following summary is presented, chiefly obtained from
K. B. HoFMANN's4 book, although it does not correspond to the present
demands. The figures are parts per 1000.

1 Loeb, Arch. f. (Anat. u.) Physiol., 1894; Heinemann, Pfliiger's Arch., 83; Frentzel
and Reach, ibid. ; Atwater and Benedict, U. S. Dept. of Agric., Bull. 136, and Ergeb-
nisee der Physiologie, 3.

2 Skand. Arch. f. Physiol., 13.

3 Zeitschr. f. physiol. Chem., 22.
4Lehrbuch d. Zoochemie (Wien, 1876), 104.

570 MUSCLES.

Muscles of Muscles of

Mammals. Birds. Anials

Solids 217-255 225-282 200

Water 745-783 717-773 800

Organic bodies 208-245 217-263 180-190

Inorganic bodies 9-10 10-19 10-20

Myosin 35-106 29.8-111 29.7-87

Stroma substance (D ANILE WSKY). ... 78-161 88.0-184 70.0-121

Creatine 2-4 4.9 2.3

Xanthine bodies 1.3-1.7 0.7-1.3

Inosinic acid (barium salt) 0.1 0. 1-0 . 3

Protic acid 7.0

Taurine 0.7 (horse) 1 . 1

Inosite 0 . 03

Glycogen 4-37 3-5

Lactic acid 0 . 4-0 . 7 —

Phosphoric acid 3 . 4-4 . 8

Potash 3.0-4.0

Soda 0.3

Lime 0.2

Magnesia 0.4

Sodium chloride 0. 04-0. 1

Iron oxide 0.04-0.1

In this table, which has little viilue because of the variation in the
composition of the muscles, no results are given as to the estimates of
fat. Owing to the variable quantity of fat in meat and the incomplete-
ness of the older methods of estimation, it is hardly possible to quote
a positive average for this substance. After most caretul 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 SxEiL.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 extremities
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 respec-
tively 100, 164, and 329 p. m. fat.2

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.

COMPOSITION OF THE MUSCLES. 571

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. Work and .rest also influence
the quantity of water, for the active muscle contains more water than
the inactive. The uninterruptedly active heart should therefore be the
muscle richest in water. That the quantity of water may vary inde-
pendently 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 strikingly the
different amounts of water (independent of the quantity of fat) in the
flesh of different animals. According to the analysis of ALMEN 1 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 quantity of nitrogen we generally accept VOIT'S figure, namely,
3.4 per cent, as an average for fresh lean meat. According to NOWAK
and HuppERT2 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 follows:
C 49.6; H 6.9; N 15.3; O + 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;
O + S 22.3 per cent, which are very similar results. This investigator
also made similar analyses of the flesh of various animals and deter-
mined the calorific value of the ash- and fat -free dried meat substance.
This value was, per gram of substance, 5599-5677 cal. The relation of
the carbon to nitrogen, which ARGUTINSKY calls the " flssh 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

1 Nova Act. Reg. Soc. Sclent. Upsal., Vol. extr. ord., 1877; also Maly's Jahresber., 7.

2 Voit, Zeitschr. f. Biologie, 1; Huppert, ibid , 7; Nowak, Wien. Sitzungsber., 64,
Abt. 2.

572 MUSCLES.

variation 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 proteins, and 12.52 per cent other soluble bodies.
FRENTZEL and SCHREUER 1 find that about 7.74 per cent of the total
nitrogen belongs to the nitrogeous extractives.

There exist complete investigations by KATz-2 as to the quantity
of mineral constituents of the muscles from man and animals. The
variation in the different elements is considerable. Pork is much richer
in sodium as compared with potassium than other kinds of meat. The
quantity of magnesium is greater, and often considerably greater, than
calcium in all kinds of flesh investigated, with the exception of the
haddock, the eel, and the pike. Beef is very poor in calcium. Potassium
and phosphoric acid are the most abundant mineral constituents of all
flesh.

Non-striated Muscles.

The smooth muscles have a neutral or alkaline reaction (DuBois-
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 KuHNE,3
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 HELWic;4 but they were first carefully
studied according to newer methods by MUNK and VELICHI.S These
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

1 Argutinsky, Pfliiger'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.

2 Pfluger's Arch., 63. See also Schmey, Zeitschr. f. physiol. Chem., 39.

3 Du Bois-Reymond in Nasse, Hermann's Handb., 1, 339; Bernstein, ibid., Heiden-
hain, ibid., 340, with Hellwig, ibid., 339; Kiihne, Lehrbuch, 331.

4 Heidenhain in Nasse, Hermann's Handb., 1, 340, with Hellwig, ibid., 339; Kiihne,
Lehrbuch, 331.

6 Munk and Velichi, Centralbl. f . Physiol., 12.

NON-STRIATED MUSCLES. 573

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/ 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. FiJRTH,2 some on th« 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.
Haemoglobin occurs in the smooth muscles of certain animals, but is absent
in others. In the smooth muscles (in certain varieties of animals)
creatinc, creatinine, hypoxanthine, taurine, inosite, glycogen, and lactic
acid have been found. The mineral constituents show the remarkable
fact that the sodium compounds exceed the potassium compounds.
According to SAIKI 3 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/ occurs invthe 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 vetebrates, and are nearly twice
as rich in sulphur as these.

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 Journ. of Biol. Chem., 4.

4 Henze, ibid., 43; Fremy and Valenciennes, cited from Kiihne's Lehrbuch, p. 333.

CHAPTER XII.
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,
chiefly 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 composition
of the brain and nerves has been somewhat extended by the investiga-
tions of modern times, still it must be admitted that this subject 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 chiefly 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 at 55-60°. LEVENE
obtained adenine and guanine but no hypoxanthine as cleavage
products. According to HALLIBURTON there are two globulins, namely,
the neuroglobulin a, which coagulates at 47°, or at, in the case of
birds 50-53° and the neuroglobulin /?, whose coagulation temperature is
70-75°; but which varies somewhat in different animals. In the frog
stili another protein body occurs, which coagulates at a still lower tem-
perature, about 40°. It must be remarked that the coagulation tempera-
ture of tt-globulin corresponds with the temperature of the first heat
contraction of the nerves of different classes of animals (HALLIBURTON).

There does not seem to be any doubt that the proteins chieflj belong
to the gray substance of the brain and to the axis-cylinders. The same

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).

574

CONSTITUENTS OF 1HE NERVOUS SYSTEM. 575

remark also applies to the nuclein, which v. JACKSCH l found in large
quantities in the gray substance. Neurokeratin, which was first detected
by KIHNE, and which partly forms the neuralgia, and as a double sheath
envelops the outside of the nerve-medulla under SCHWANN'S sheatn and
the inner axis-cylinders, occurs in the nerves, but chiefly, or according
to KOCH entirely, in the white substance (KUHNE and CHITTENDEN,
BAUMSTARK 2) .

The so-cailed 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 the non-phosphorized cerebron or with
a mixture of cerebrosides (see below). There does not seem to be any
doubt that these last, especially the cerebron as well as lecithin and cephalin,
occur preformed in the brain and nerves. From the investigations
made thus far it is difficult to state with positiveness whether the last
two mentioned bodies (which have been discussed in Chapter V) belong
to the gray or white substance. According to KOCH they occur much
more abundantly in the white substance. FALKS prepared a phos-
phatide from human brain, which behaves essentially like cephalin
and also yields cephalinic acid, which is difficult of characterization.
The strikingly high nitrogen content is remarkable, namely in one prepara-
tion 2.905 per cent and 2.76 per cent in another. The phosphorus
content was 3.28 and 3.16 per cent. The relation P:N is correspond-
ingly about 1:2. The isolated substance is a diamido-phosphatide
while the cephalin thus far investigated is a monamido-phosphatide.
Under these circumstances, irrespective of the purity and the chemical
identity of the isolated substance, we are not warranted in calling this
substance cephalin, as was done by FALK. On the other hand it is also
questionable whether we are warranted in including cephalin among the
lecithins as done in the text on page 234. The observations of KOCH 4
show that at least the human and ox brain contain jecorin, and that
the lipoid sulphur occurs, much more abundantly, in the white than in
the gray substance. The same is true for the numerous brain phos-
phatides described by TnuDicnuM5 under different names, and whose
chemical individuality has not been sufficiently established; cholesterin
chiefly occurs in the white substance. Fatty acids and neutral fats may

1 Pfliiger's Arch., 13.

2 Koch, Amer. Journ. of Physiol., 11; Kiihne and Chittenden, Zeitschr. f. Biologic,
26; Baumstark, Zeitschr. f. physiol. Chem., 9.

3 Bioch. Zeitschr., 10.

4 Zeitschr. f. physiol. Chem., 53.

5 Thudichum, Die chemische Konstitution des Gehirns des Menschen und der Tiere,
Tubingen, 1901.

57G BRAIN AND NERVES.

be prepared from the brain and nerves; but as these may be readily
derived from a decomposition 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 fibres, 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.

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, paral-actic acid (MORIYA), phosphocarnic acid, uric
acid, and the diamine neuridine, C5Hi4N2, discovered by BRIEGER *
and which is most interesting because of its appearance in the putrefac-
tion of animal tissues or in cultures of the typhoid bacillus. Under
pathological conditions leucine and urea have been found in the brain.
Urea is also a physiological constituent of the brain of cartilaginous
fishes.

Of the above-mentioned constituents of the nerve-substance pro-
tagon and the cerebrins or cerebrosides must be specially described.

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 for carbon and phosphorus, namely 66.25 and 0.97 per cent,
found by KOSSEL and FREYTAG, correspond well with these figures while
the result for the nitrogen, 3.25 per cent, is too high. They also found,
as shown previously by RUPPEL, that protagon contains sulphur, namely
0.51 per cent. CRAMER also found that the protagon contained sulphur,
but obtained about the same figures as GAMGEE and BLANKENHORN.
Recently WILSON and CRAMER2 have reported newer analyses and they
find for protagon, recrystallized 4-5 times, nearly the same figures as
GAMGEE and BLANKENHORN, namely C 66.53, H 10.97, P 0.95 and S
0.73 per cent. They consider protagon as a unit substance.

1 Brieger, Ueber Ptomaine, Berlin, 1885 and 1886.

2 Lieberich, 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.

PROTAGOX. 577

GIES, POSNER and ROSENHEIM and TEBB 1 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, hav-
ing variable composition, especially different P and N contents. They
are, therefore, as are LESEM, THUDICHUM, WORNER and TniERFELDER,2
and others, of the opinion that protagon does not exist as a chemical indi-
vidual, but as a mixture of cerebrosides and phosphatides. It is not easy to
come to any decision on this disputed question. The above-mentioned
investigations, and especially the recent ones of ROSENHEIM and TEBB,
imply the absence of unity of protagon but 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 crystal -
lizable bodies can be so easily crystallized and yield a product, which
with proper care, can be recrystallized repeatedly without changing
its composition. According to ROSENHEIM and TEBB if the proper
quantity is used in solution a crystalline product can be obtained from
the decomposition products of protagon, which has the same specific
rotation as protagon and can be repeatedly recrystallized without
changing its composition or its optical activity.3 If this is the case it
would be better to investigate the cleavage products than to analyze the
various fractions in order to get information on the disputed question.

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 indeed found three
cerebrosides, namely, CEREBRIN, KERASIN (homocerebrin), and ENCEPH-
ALIN. According to KOCH 4 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 THIERFELDER 5 that protagon dissolved in methyl alcohol containing
chloroform, deposits crusts of cerebron (not pure) after a time at

1 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.

2 Lesem, 1. c.; Thudichum, 1. c.; Worner and Thierfelder, Zeitschr. f. physioK
Chem., 30.

3 Journ. of Physiol., 37; Proc. physiol. Soc., January, 1908, p. 3.

4 Zeitschr. f. physiol. Chem., 53.

5 Kitagawa and Thierfelder, ibid., 49; Rosenheim and Tebb, Journ. of Physiol., 37,
341 and 348.

578 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, accord-
ing to ROSENHEIM and TEBB it is probably a diamido-phosphatide,
called sphingomyelin by THUDICHUM. On boiling protagon with dilute
mineral acids it yields a reducing sugar (galactose) due to the decompo-
sition 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. It decomposes on heating even below 100° C. 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 cerebroside. Pro-
tagon is soluble in pyridine at 30° C., yielding a clear solution, and this
solution has a specific rotation (a')D= +6.8° ( WILSON and CRAMER). On
warming or cooling according to ROSENHEIM and TEBB, the rotation
changes with the separation of sphingomyelin so that it first diminishes
in rotation, 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 levo-
rotation 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. On fusion with saltpeter and soda it yields alkali phosphate.

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 burnt 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.

Cerebrosides or Cerebrins.

On decomposing protagon (or the protagons) 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 a reducing variety of sugar (galactose) on boiling with dilute mineral

CEREBRIX, KERASIN, ENCEPHALIN. 579

acids. The cerebrosides isolated from the brain are cerebrin, kerasinr
encephalin, and cerebron, but it must be remarked that there is ro doubt
that sometimes the same body of varying purity has received different
names. The bodies isolated by KOSSEL and FREYTAG from pus, and called
pyosin and pyogenin, also belong to the cerebrosides (see Chapter VII).
Cerebrin. Under this name W. MULLER 1 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
baryta-water. Following a method essentially the same, but differing,
slightly, GEOGHEGAN 2 prepared,, from the brain, a cerebrin with the
same properties as MULLER'S, but containing less nitrogen. According
to P ARGUS 3 the cerebrin isolated by GEOGHEGAN, as well as by MULLER,
consists of a mixture of three bodies, " cerebrin," " homocerebrin,"
and " encephalin." KOSSEL and FREYTAG isolated two cerebrosides
from protagon which were identical with the cerebrin and homocerebrin
of P ARGUS. According to these investigators, the two bodies phrenosin
and kerasin, as described by THUDICHUM, seem to be identical with cere-
brin and homocerebrin.

Cerebrin, according to PARCUS, has the following composition: C 69.08,
H 11.47, N 2.13, O 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 burnt
fat, and burns with a luminous flame. 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 consist of a mass of globules
or grains on microscopical examination. Cerebrin forms a compound
with baryta, which is insoluble in water and is decomposed by the action
of carbon dioxide. Cerebrin dissolves in concentrated sulphuric acid,
and on warming the solution it becomes blood-red. The variety of
sugar split off on boiling with mineral acids — the so-called brain-sugar
— is, in THIERFELDER'S 4 opinion, galactose.

Kerasin. (THUDICHUM), or homocerebrin (PARCUS), has the following
composition: C 70.06, H 11.60, N 2.23, and O 16.11 per cent. Enceph-
alin has the composition C 68.40, H 11.60, N 3.09, and O 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

1 Annal. d. Chem. u. Pharm., 105.

2 Zeitschr. f. physiol. Chem., 3.

3 Parcus, Ueber einige neue Gehrinstoffe, Inaug.-Diss. Leipzig, 1881.

4 Zeitschr. f. physiol. Chem., 14.

580 BRAIN AND NERVES.

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. Like cerebrin and kerasin, it yields a reducing substance (prob-
ably galactose) on boiling with dilute acid.

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.

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,1 identical with cerebron. Cerebron can be prepared
directly from the brain without saponification with baryta, by treatment
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 C^HgsNOg; 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 solution in methyl alcohol
(containing 75 per cent chloroform) is (a)D=+7.6° (KITAGAWA and
THIERFELDER). According to THIERFELDER it yields as cleavage prod-
ucts, galactose, cerebronic acid (THUDICHTJM " neurostearic acid ") and
a mixture of two bases, which have not been closely studied, of which one
is crystalline, gives a beautiful crystalline compound with HC1 and
has the probable composition CigH39NO2. The relation of these bases
to THUDICHUM'S base sphingosin has not been explained. Cerebronic
acid is crystalline, melts at 99-100° C. and gives a crystalline methyl
ester melting at 65° 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; Gamgee, Text-book of Physiol. Chem., London,
1880; Thudichum, 1. c.; Gies, Journ. of Biol. Chem., 1 and 2.

COMPOSITION OF THE BRAIN. 581

chloroform (see page 577), 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.

The two monamido-monophosphatides, lecithin and cephalin, have
already been discussed in Chapter V.

BETHE l prepared the following decomposition products from the brain
of the horse after treatment with CuCl2 and alkali: Aminocerebrinic-acid glucoside,
C44H8108N, which on boiling with hydrochloric acid yields cerebrinic acid, amino-
cerebrinic-acid chloride, and a hexose (galactose ?) ; phrenin, perhaps identical
with THUDICHUM'S krinosin; cerebrinic-phosphoric acid, and a stearic acid, differ-
ing somewhat from the ordinary one.

Neuridine, C5Hi4N2, 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
§79_926 p. m. water. The observations of WEISBACH 2 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 Years.

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

722.0

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

801.7

•Cerebellum

788 . 3

794.9

778

7

789.0

787.9

784

5

803

4

797.9

Pons Varolii. .

. 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

Quantitative analyses of human brains at different ages, namely
6 weeks, 2 and 19 years, have been made by KOCH and MANN.2 These
analyses show that with increasing age the water, proteins, extractives
and salts diminish relatively, while the phosphatides, cerebrosides and
especially cholesterin strikingly increase. The sulphur of the lipoids is

1 Arch, f . exp. Path. u. Pharm., 48.

2 Cited from K. B. Hoffmann's Lehrbuch d. Zooch., Wien, 1877, p. 121.

3 Journ. of Physiol., 36, Proc. physiol. Soc., 1907.

5S2 BRAIN AND NERVES.

increased until the second year, but from this age to the nineteenth
year the quantity was the same.

The older analyses of PETROWSKY and of BAUMANN l are not sufficient
for present conditions, but they substantiate in substance what has been
stated (page 575) in regard to the unequal division of the organic con-
stituents between the gray and white substance.

BAUMSTARK claims to have found that a part of the cholesterin in
the brain occurs in a combined state, perhaps as ester; this view has
been found to be incorrect by the recent investigation^ 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 2 has also found only free cholesterin.

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 estimation 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

Nuerokeratin • 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

As the cerebrosides chiefly occur in the myelin sheath, KOCH, starting from
the amount in the investigated part of the brain, attempts to calculate the amount
of the analyzed cortical substance in the white nerves, and on the basis of these
calculations, he finds the following values for the pure gray substance, free from
nerve-fibres, and compares them with the corpus callosum. The results are in
1000 parts of the dry substance:

Gray Substance

Corpus (free from

Callosum. white substance).

Protein 100.0 217.0

Nucleoproteins 115.6 96 . 6

Neurokeratin 84 . 0

Extractives 47 . 5 59 . 2

Lecithins 162.2 76.7

Cephalin and myelin 109 . 1

Phrenosin and kerasin 142 . 9

Cholesterin 152.0

Sulphurized substance 43 . 7 54 . 3

1 Petrowsky, Pfluger's Arch., 7; Baumann, Zeitschr. f. physiol. Chem., 9.

2 Biinz, Zeitschr. f. physiol. Chem., 46; Tebb, Journ. of Physiol., 34.

3 Amer. Journ. of Physiol., 11.

BRAIN AND NERVES. 583

The above assumption has not sufficient foundation and is less prob-
able, as FALK l has found cerebrosides in the medullary nerve fibres as
well as in 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 fibres Medullary fibres in

in p. m. of the total p. m. of the total

extract extract

Cholesterin 470 250

Cephalin 237 124

Cerebrosides 60 182

Lecithins 98 29

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 2 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 prsecox (5 cases), KOCH 3
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.4
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
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,5 neurochitin replaces neurokeratin in insects and crus-
tacea, the quantity of the first being 10.6-12 p. m.

1 Falk, Bioch. Zeitschr., 13.

2 Noll, Zeitschr. f. physiol. Chem., 27; Mott and Halliburton, Philos. Transactions,
Ser. B, 191 (1899), and 194 (1901).

3 Arch, of Neurology, 3.

4 Zeitschr. f . Biologie, 26.

5 Compt. rend., 115.

584 BRAIN AND NERVES.

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 CO3:
0.102-0.220 SO4; 0.01-0.098 Fe2(PO4)2; 0.005-0.022 Ca; 0.016-0.072
Mg; 0.58-1. 778 K; 0.450-1.1 14 Na. The gray substance yields an alkaline
ash, the white an acid ash.

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 CAHN1). The mineral bodies
consist of 422 p. m. Na2HPO4 and 352 p. m. NaCl.

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, erythropsin, or VISUAL RED, is
the pigment of the rods. BoLL,2 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 3 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 these
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 chiefly occurs in
the rods and only in their outer parts. In animals whose retina has no
rods the visual purple is absent, and is also necessarily absent in the macula
lutea. In a variety of bat (Rhinolophus hipposideros) , in hens, pigeons
and newborn 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, which 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 corre-

1 Zeitschr. f. physiol. Chem., 5.

2 Monatsschr. d. Kgl. Preuss. Akad., 12. Nov., 1876.

3 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.

VISUAL PURPLE. 585

spending 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 l 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 regeneration 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.

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

1 Centralbl. f. Physiol., 9; also Maly's Jahresber , 25, 351.

586 BRAIN AND NERVES.

in cholates is precipitated by saturating with ina.mnesium sulphate,
ing the precipitate with a sat united solution of uia^iifsiuiii sulphate,
and then dissolving in water by the aid of the cholates simultaneously
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 concc.nl raird 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 XVI.

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 shown 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 so) ids
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 342.

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 membranin
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 hydro-
chloric acid or ADAMKIEWICZ'S reagent. They are dissolved with great
difficulty by pepsin-hydrochloric acid or trypsin solution, but are soluble
in dilute acids and alkalies in the warmth. Membranin of the capsule

1 Kiihne, Zeitschr. f. Biologie, 32.

2 Kiihne, Die nichtbestandigen Earben der Netzhaut, Untersuch. aus dem physigl.
Institut Heidelberg, 1, 341.

3 Kiihne, 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. Biologie, 31. A
complete review of the literature will also be found here.

CRYSTAL! JNK LENS, ,>S7

of the lens contains 14.10 per cent N and 0.83 per cent S, and is a little
less soluble than that from DKSCKMKT'S membrane.

The chief mass of the solids of the crystalline lens consists of proteins,
whose nature has been investigated by C. MORNER.* Some of these pro-
teins dissolve in dilute salt solution, while others remain insoluble in
this solvent.

The Insoluble Protein. The lens fibres consist of a protein substance
which is insoluble in water and in salt solution and to which Mo,
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
1o an alkali-albuminate solution, but coagulates at about 50° C. on nearly
complete neutralization and the addition of 8 per cent NaCl. Albu-
moid has the following composition: C 53.12, H 6.8, N 16.62, and S
0.79 per cent. The lens fibres 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 album okl in the entire
lens amounts on an average to about IS 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 fi-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; /9-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, /?-crystallin is precipitated from a salt-free solution
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 sulphate in sub-
stance 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;
/J-crystallin, on the contrary, from within outward.

A. BECHAMP distinguishes the two following protein bodies in the watery
extract of the crystalline lens: phacozymase, which coagulates at 55° C., con-
tains a diastatic enzyme, and has a specific rotatory power of («)/= —41°,
and ihc cryvtalbumin, with a specific rotatory power of (a)/ = —80.3°. From
the residue of the lens, which was insoluble in water, BECHAMP extracted,
by means of hydrochloric acid, a protein body having a specific rotatory power
of (a)/= -80.2°, which he called crystdfibrin.

The lens does, not seem to contain any protein bodies which coagulate
spontaneously like fibrinogen. That cloudiness which appears after
death depends, according to KUHNE, upon the unequal changing of the
concentration of the contents of the lens-tubes. This change is produced

1 Zeitschr. f. physiol. Chem., 18. This contains also the pertinent literature.

588 BRAIN AND NERVES.

by the altered ratio of diffusion. A cloudiness of the lens may also be
produced in life by a rapid removal of water, as, for example, when a
frog is plunged into a salt or sugar solution. The appearance of cloudi-
ness in diabetes has been attributed by some to the removal of water.
Opinions on this subject are, however, conflicting.

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 3 :

Albumoid (lens fibres) 170 p. m.

/?-Oystallin 110 "

a-Crystallin 68 "

Albumin 2 "

The corneal tissue has been previously considered (page 525). Th&
sclerotic has not been closely investigated, and the choroid coat is chiefly
of interest because of the coloring-matter (melanin) it contains (see
Chapter XVI).

TEARS consist of a water-clear, alkaline fluid of a salty taste. Accord-
ing to the analyses of LERCH 4 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 I.e.

* Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 401.

CHAPTER XIII.
ORGANS OF GENERATION.

(a) Male Generative Secretions.

The testes have been little investigated chemically. We find in the
testes of animals protein bodies of different kinds — seralbumin, alkali
albuminate (?), and an albuminous body related to ROVIDA'S hyaline
substance', also leucine, 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, leuciner
aminovaleric acid, aminobutyric 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,S 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
nucleoproteins, traces of mucin, albumin, and a substance similar to pro-
teose (found earlier by POSNER). According to CAVAZZANi4 semen
contains relatively considerable nucleon, more than any organ. The
mineral bodies consist chiefly of calcium phosphate and considerable
NaCl. Potassium occurs only in smaller amounts.

1 Compt. rend., 74.

2 Amer. Journ. of Physiol., 11.

3 Zeitschr. f . physiol. Chem., 35.

4 Posner, BerL.klin. Wochenschr., 1888, No. 21, and Centralbl. f. d. med. Wissensch.,
1890; Cavazzani, Biochem. Centralbl., 1, 502, and Centralbl. f. Physiol., 19.

589

590 ORGANS OF GENERATION.

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 1),
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, C2H5N. This combination has
been called BOTTCHER'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 leucamia (Tn. COHN, B. LEWY2). They are,
according to SCHREINER,S as above stated, a combination of phosphoric
acid with a base, spermine, C^H^N, 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.

CAMUS and GLEY 6 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.

1 Iversen, Nord. med. Ark., 6; also Maly's Jahresber., 4, 358; Stern, Biochem.
Centralbl., 1, 748.

2 Th. Cohn, Centralbl. f. allg. Path. u. path. Anat., 10 (1899), and Zeitschr. f. Urolog.,
1908; B. Lewy, Centralbl. f. d. med. Wissensch., 1899, 479.

3 Annal. d. Chem. u. Pharm., 194.

4 Ladenburg 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.

5 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.

6 Compt. rend, de soc. biolog., 48, 49.

SPERMATOZOA. 591

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 rr.ovements, 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 alter 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.1 .

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 composition
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 hist ones. 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 MIESCHER 2
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
6.5-7.5 p. m. inorganic bodies. The last consist chiefly 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
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

1 See G. Giinther, Pfliiger's Arch., 118.

2 See Miescher, " Die histochemischen und physiologischen Arbeiten von Friedrich
Miescher, gesammelt und herausgegeben von seinen Freunden," Leipzig, 1897.

592 ORGANS OF GENERATION.

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 development
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 (!VERSEN 2). The other kind is larger, sometimes the size
of the head of a pin, and 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, paralbumin
or metalbumin, which are found in certain pathological ovarial fluids,
but seems to be a serous liquid. The corpora lutea are colored yellow
by an amorphous pigment called lutrin. Besides this another coloring-
matter sometimes occurs which is not soluble in alkali; it is crystalline,
but not identical with bilirubin or hsematoidin ; but it may be identified
as a lutein by its spectroscopic behavior (PICCOLO and LIEBEN, KUHNE
and EwALD3).

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 (HYDRO PS 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.

1 Zeitschr. f. physiol. Chem., 23.

2 Nord. med. Ark., 6.

3 See Chapter VI, p. 290.

OVARIAL CYSTS. 593

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 between 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 GLUGE'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. 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 1 such a colloid is designated /3-pseudomucin. Some-
times a colloid is found which, when treated with a very dilute alkali,

1 Arch. f. Gynak., 38.

594 ORGANS OF GENERATION.

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 J 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 ScnERER2 gave to a protein substance
found by him in an ovarial fluid. The met albumin was considered by
SCHERER to be an albuminous body, but it belongs to the mucin group,
and it is for this reason called pseudomucin by HAMMARSTEN.S

Pseudomucin. 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.

MITJUKOFF 4 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 solution
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-

1 Panzer, Zeitschr. f. physiol. Chem., 28; Wiirtz, see Lebert, Beitr. zur Kenntnis
des Gallertkrebses, Virchow's Arch., 4.

2 Verb. d. physik.-med. Gesellsch. in Wurzburg, 2, and Sitzungsber. der physik.-
med. Gesellsch. in Wurzburg fur 1864-1865; Wurzburg med. Zeitschr., 7, No. 6.

3 Zeitschr. f. physiol. Chem., 6.

4 K. Mitjukoff, Arch. f. Gynakol., 49.

COLLOID AND PSEUDOMUCIN. 595

cially shown by FR. MULLER, NEUBERG and HEYMANN.1 From pseudo-
mucin ZANGERLE 2 obtained 30 per cent glucosamine, and NEUBERG and
HEYMANN have shown that the glucosamirie 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 3
obtained on the hydrohsis 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 pseudo-
mucin, 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
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 fat is ordinarily 2-4 p. m. The remaining

1 Miiller, Verb. 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.

2 Munch, med. Wochenschr., 1900.

3 Otori, Zeitschr. f. physiol. Chem., 42 and 43; Ptegl, ibid., 58.

596 ORGANS OF GENERATION.

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 LIGAMENTA 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 quantitative composition of the fluid from ovarial
cysts we refer the reader to the work of OERUM.1

E. LUDWIG and R. v. 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/ page 232.

The colloid from a uterine fibroma analyzed by STOLLMANN 3 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.

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 4) . The occur-

1 Kemiske Studier over Ovariecystevsedsker, etc., Koebenhavn, 1884. See also
Maly's Jahresber., 14, 459.

2 Ludwig and v. Zeynek, Zeitschr. f. physiol. Chem., 23; Ameseder, ibid., 52.

3 Amer. Gynecology, 1903.

4 Pfliiger's Arch., 43.

OVUM. 597

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 (BRIEGER 1); purine bases (MESERNITZKI 2),
glucose in very small quantities, and mineral bodies. The occurrence of
cerebrin and of granules similar to starch (DARESTE 3) has not been posi-
tively proven.

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 lipoiytic, and a chromolytic (?) enzyme

(WOHLGEMUTH4) .

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 seeds 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
that the lecithin is chemically united with the vitellin (HOPPE-SEYLER 5).
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 [NH412SO4), 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 coagulating
at 76-77° C. in a solution containing salt, and PLIMMER 6 found a protein

1 Ueber Ptomaine, Berlin, 1885.

2 Mesernitzki, Biochem. Centralbl., 1, 739.

3 Compt. rend., 72.

4Miiller and Masuyama, Zeitschr. f. Biologic, 39; Stepanek, Centralbl. f. Physiol.,
18, 188; Wohlgemuth in Salkowski's Festschrift and Zeitschr. f. physiol. Chem., 44.

5 Med. chem. Untersuch., 216.

6 Osborne and Campbell, Connecticut Agric. Exp. Station, 23d Ann. Report, New

598 ORGANS OF GENERATION.

which he calls livetin which only contained 0. 1 per cent phosphorus 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. BUNGE l prepared a pseudonuclein by digesting the yolk
with gastric juice, and his pseudonuclein, he claims, is of great
importance in the formation of the blood, and on these grounds he called
it hcematogen. This hsematogen 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. NEUBERG 2
has also split off glucosamine from the yolk and has identified it as nori-
sosaccharic acid. It is difficult to state whether this glucosamine was
derived from the vitellin or from some other constituent of the yolk.

The chief 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 carbohydrate on boiling with sulphuric acid. Ichthulin has

Haven, 1900; Gross, Zur Kenntn. d. Ovovitellins, Inaug.-Diss. Strassburg, 1899;
Plimmer, Journ. Chem. Soc. London, 93.

1 Zeitschr. f. physiol. Chem., 9, 49. See also Hugounenq and Morel, Compt. rend.,
140 and 141.

2 Ber. d. d. chem. Gesellsch., 34.

YOLK. 599

the following composition: C 53.42, H 7.63, N 15.63, O 22.19, S 0.41, P 0.43 per
<;ent. It also contains iron. The ichthulin investigated by LEVENE from codfish
eggs had the composition C 52.44, H 7.45, N 15.96, S 0.92, P 0.65, Fe + O 22.58
per cent, and yielded no reducing substances on boiling with acids. The pure
vitellin isolated by HAMMARSTEN 1 from pereh 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.

The yolk also contains albumin, besides vitellin and the above-men-
tioned proteins.

The fat of the yolk of the egg, LIEBERMANN 2 claims, Is a mixture
of a solid and a liquid fat. The solid fat consists chiefly 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 dig] yce rides, or upon a quantity of fatty acid deficient in carbon
(LIEBERMANN). The composition of yolk fat is dependent upon the
food, as HENRIQUES 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 ot these,
which was soluble in alcohol -ether, behaved like lecithin. The second
was difficultly soluble in alcohol but readily soluble in ether, contained
1.37 per cent N and 3.96 per cent P. The third was a diamido phos-
phatide 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 4 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 triamido-monophosphatide having the formula C^H^NsPO^.
BARBIERI has obtained a sulphurized phosphatide called ovin, con-
taining 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. Yellow or orange-red amorphous coloring-matters 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. These coloring-

1 Walter, Zeitschr. f. physiol. Chem., 15; Levene, ibid., 32; Hammarsten, Skand.
Arch. f. Physiol., 17.

2 Pfluger's Arch., 43.

3 Skand. Arch. f. Physiol., 14.

4Thierfelder and Stern, Zeitschr. f. physiol. Chem., 53; Frankel and Bolaffio,
Bioch. Zeitschr., 9; Barbieri, Compt. rend., 145.

COO ORGANS OF GENERATION.

matters, which also occur in the vegetable kingdom (THUDICHUM), and
whose relation to the vegetable pigments, the xanthophyll group, has
recently been shown by SCHUNCK;I have been called luteins or lipo-
chromes.

The luteins, which among themselves show somewhat different proper-
ties, are all soluble in alcohol, ether, and chloroform. 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, and, lastly, they ordinarily show an
absorption-spectrum of two bands, of which one covers the line F and
the other lies between the lines F and G. LEWIN, MIETHE and STENGER 2
have given exact reports as to the absorption behavior of the lutein
from egg-yolk in various solvents. " The luteins withstand the action of
alkalies so that they are not changed when we remove the fats present
by means of saponification.

Lutein has not been prepared pure. MALY 3 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 beautifully green by concentrated
sulphuric acid. The absorption-bands, especially of the vitellolutein, correspond
very nearly to those of ovolutein.

The mineral bodies of the yolk of the egg consist, according to PoLE£K,4
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 preceding analyses.

The yolk of the hen's egg weighs about 12-18 grams. The quantity
of water and solids amounts, according to PARKE,S to 471.9 p. m. and
528.1 p. m. respectively. Among the solids he tound 156.3 p. m. protein,
3.53 p. m. soluble and 6.12 p. m. insoluble salts. The quantity of fat,
according to PARKE, is 228.4 p. m.; the lecithin, calculated from the

1 Thudichum, Centralbl. f. d. med. Wissensch., 1869; Schunck, see Chem. Cen-
tralbl., 1903, 2, 1195.

2 Pfliiger's Arch., 124.

3 Monatshefte f. Chem. 2.

4 Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 740.

5 Hoppe-Seyler, Med. chem. Untersuch., Heft 2, 209.

OVOGLOBULIN. 601

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 tramework 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
consists, seems to be a body closely related to horn substances (LIEBER-
MANN) .

The white of the egg has a specific gravity of 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. Among the extractive
bodies LEHMANN found a fermentable variety of sugar which amounted
to 5 p. m. or, according to MEISSNER, 80 p. m. of the solids.1 Besides
these one finds in the white of the egg traces of fats, soaps, lecithin and
cholesterin.

The white of the egg of the Insessores becomes transparent on boiling and acts
in many respects like alkali albuminate. This albumin TARCHANOFF 2 called
" tatalbumin."

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 proven nor is it probable that the glucosamine belongs to the pro-
tein molecule. According to the solution and precipitation properties
they are similar to the globulins, albumins or proteases. The representa-
tives of the first two groups, which until recently were considered as
true proteins, 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-

1 Cited "from v. Gorup-Besanez, Lehrbuch, 4. Aufl., 739.

2 Pfliiger's Arch., 31, 33, and 39.

C02 ORGANS OF GENERATION.

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, GATJTIER, 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 chief fractions;
the cry st alii z able 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
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 3 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 the following: The specific rota-
tion 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 ABDER-

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., 1-1, 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.

3 Zeitschr. f . physiol. Chem., 31.

OVOMUCOID. 603

HALDEN and PREGL l on the hydrolysis of ovalbumin do not show any-
thing 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 prepared
partly free from phosphorus and partly with a variable phosphorus
content of 0.1-3.06 per cent (KAAS, WILLCOCK and HARDY2).

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 nitrate
to slowly evaporate 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
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 602, foot-note 2, for the HOPKINS and PINKUS method.) WILL-
COCK 3 has recently found that magnesium sulphate can also be used in
the crystallization of ovalbumin.

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 4 found a fibrinogen-like substance in the white of the 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,5 a mucoid with 12.65 per cent nitro-
gen and 2.20 per cent sulphur. Ovomucoid exists in hen's eggs to the
extent of about 10 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 nolj precipitated by an

1 Zeitschr. f. physiol. Chem., 46.

2 Kaas, Monatsh. f. Chem.. 27; Willcock and Hardy, cited from Chem. Centralbl.,
1907, 2, 821.

3 Journ. of Physiol., 37.

4 Compt. rend., 135.

5 R. Neumeister, Zeitschr. f. Biologic, 27; Salkowski, Centralbl. f. d. med. Wis-
sensch., 1893, 513 and 706; C. Morner, Zeitschr. f. physiol. Chem., 18. See also Lang-
stein, Hofmeister'p Beitrage, 3 (literature).

604 ORGANS OF GENERATION.

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.1

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
ducks, contain a special protein in the egg-white, which is not identical with
that of the hen's egg. WORM 2 has prepared a crystalline albumin from 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 the egg have been analyzed by
POLECK and WEBER.S They found in 1000 parts of the ash: 276.6-
284.5 grams potash, 235.6-329.3 soda, 17.4-29 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 grams carbon
dioxide. Traces of fluorine have also been found (NiCKLES4). 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 consists,
as above stated (page 112), of a keratin substance. The shell contains
very little organic substance, 36-65 p. m. The chief 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 bird's eggs is due to several different coloring-matters.
Among these we find a red or reddish-brown pigment called " oorodein " by SORBY,S
which is perhaps identical with hsematoporphyrin. The green or blue coloring-
matter, SORBY'S oocyan, seems, according to LIEBERMANN 8 and KRUKENBERG,T
to be partly biliverdin and partly a blue derivative of the bile-pigments.

1 Zanetti, Chem. Centralbl., 1898, 1; Seemann, cited from Langstein, Ergebnisse
der Physiol., 1, Abt. 1, 86.

2 Panormow, see Bioch. Centralbl., 5; Worm, cited from Chem. Centralbl., 1906,
2, 1508.

3 Cited from Hoppe-Seyler, Physiol. Chem., 778.

4 Compt. rend., 43.

5 Cited from Krukenberg, Verh. d. phys.-chem. Gesellsch. in Wiirzburg, 17.
* Ber. d. deutsch. chem. Gesellsch., 11.

7 I.e.

FISH EGGS. 605

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 (HiJFNER).1

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 2) .

The white of the egg of cartilaginous and bony fishes contains only traces of
true albumin, but consist 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 3 claims, mucin in the envelope in the
unripe state and only mucinogen in the ripe state. The crystalline formations
(yolk-spherules, or dotterplattcheri) 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 chiefly of phos-
phoglycoproteins. The klupeovin obtained by HUGOUNENQ 4 from the herring's
eggs and from which he obtained the three so-called hexone bases and abundant
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 cyano-
crystallin, becomes red on boiling in water.

C. MORNER 5 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.

In fossil eggs (of APETNODYTES, PELECANUS, and HALL^US) in old guano
deposits, a yellowish white, silky, laminated compound has been found which
is called guanovulit, (NH4)2SO4 + 2K2SO4+3KHSO4+4H2O, 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. The occurrence of glycogen is
doubtful, and the carbohydrates are perhaps represented by a very small
amount of sugar and glycoproteins. On the contrary, the egg contains a
large proportion of fat, which doubtless is important as a source of supply
for nourishment and in maintaining respiration for the embryo. The

1 Arch, f . (Anat. u.) Physiol., 1892.

2 Zeitschr. f. Biol. 43.

3 Giacosa, Zeitschr. f. physiol. Chem., 7; Hammarsten, Skand. Arch. f. Physiol., 17.

4 Valenciennes and Fremy, cited from Hoppe-Seyler, Physiol. Chem., p. 77;
Hugounenq, Bull. soc. chim. (3), 33, and Compt. rend., 143.

5 Zeitschr. f. physiol. Chem., 40

606 ORGANS OF GENERATION.

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 598) is formed, is doubtless, as
BUNGE claims, of great importance in the formation of the hemoglobin
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, chiefly due 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 l 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 incubation ;
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 fcetus. 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 2 has also shown that the fertilized hen's egg not only gives off
nitrogen the first five or six hours of incubation, but a] so 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. The weight of the shell and the quantity
of lime-salts contained therein does 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.

Very complete and careful chemical investigation on the development

1 Tangl and v. Mituch, Pfliiger's Arch., 121; Liebermann, ibid., 43.

2 Bohr and Hasselbalch, Maly's Jahresber., 29; Hasselbalch, Skand. Arch. f.
Physiol., 13.

3 Tangl with Hammerschlag, Pfliiger's Arch., 121.

DEVELOPMENT OF THE EGG. 607

of the embryo of the hen have been made by LiEBERMANN.1 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 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 chondrin.
A body similar to mucin occurs in the embryo when about six days old,
but then disappears. The quantity of hemoglobin shows a continual
increase compared with the weight of the body. LIEBERMANN found
that the relation of the hemoglobin to the body weight was 1:728 on
the eleventh day and 1 : 421 on the twenty-first day.

By means of BERTHELOT'S thermometric methods TANGL2 has deter-
mined 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 3 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-

1 1. c. 2 Pfluger's Arch., 93 and 121. 3 Pfliiger's Arch., 104.

608 ORGANS OF GENERATION.

tilization, development and artificial parthogenesis of the eggs of sea
animals have already been discussed in connection with the physico-
chemical processes and action of ions (Chapter II). Here it will be
sufficient to call attention to the fact that LOEB believes that a synthesis
of the nuclear substance, i.e., of nucleoproteins from the constituents
of the yolk, is the means of stimulating development, and that oxidation
processes direct the proper path of the nuclear syntheses. The setting
free of the nuclear syntheses and these oxidation processes also depend
upon a change in the peripheral layers of the egg, which is in general
followed by a formation of a membrane, and this latter seems to be brought
about by zytolytically acting bodies and the liquefaction of a lipoid.
The impulse for the development of the egg is according to this assump-
tion the liquefaction of a lipoid on the surface of the egg, and from this
follows another assumption that the head of the spermatozoa contain
a lipoid-liquefying substance.

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 nucleoprotein, found by others, is
not clear. The protein found by SAVARE contained 0.45 per cent phos-
phorus. The nucleic acid studied by KIKKOJI/ which is very similar to
the thymus nucleic acid, originates from this nucleoprotein. 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
CRAMER2 the quantity of glycogen in the placenta is not increased by
food rich in carbohydrate. In the foetus (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 (AscoLi,
RAINERI, BERGELL and LIEPMANN, SAVARE 3) . 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.4

1 Bottazzi and Delfino, Centralbl. f. Physiol., 18, 114; Savare", Hofmeister's Beitrage,
11; Kikkoji, Zetischr. f. physiol. Chem., 53.

2 Moscati, Zeitschr. f. physiol. Chem., 53; Lochhead and Cramer, Proc. Roy. Soc.,
80 B. (1908).

3 Ascoli, Centralbl. f. Physiol., 16; Raineri, Bioch. Centralbl., 4, 428; Bergell and
Liepmann, Munch, med. Wochenschr., 1905; Savar£, Hofmeister's Beitrage, 9.

4 See Etti, Maly's Jahresber., 2, 287, and Preyer, Die Blutkristalle, Jena, 1871.

AMNIOTIC FLUID. 609

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
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.
protein 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 hardly 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 levulose. The human amniotic fluid also con-
tains some urea, uric acid, and allantoin. 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. Creatine
and lactates are doubtful constituents of the amniotic fluid. The quantity
of urea in the amniotic fluid, is, according to PROCHOWNICK, 0.16 p. m.
In the fluid in hydramnion PROCHOWNICK and HARNACK found respectively
0.34 and 0.48 p. m. urea. The chief mass of the solids consists of salts.
The quantity of chlorides (NaCl) is 5.7-6.6 p. m. The molecular con-
centration of the amniotic fluid is somewhat lower than that of the blood,
which is no doubt due to a dilution by the foetal urine (ZANGEMEISTER
and MEISSL J) .

1Weyl, 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 Griinbaum, ibid., 1904.

CHAPTER XIV.
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,
investigated by ODENIUS, contains as an average the following: 17.28
per cent N, 0.89 per cent S, and 0.277 per cent P. MANDEL has made
an analysis of the hydrolytic cleavage products of the nucleoprotein of the
mammary glands, carefully prepared according to HAMMARSTEN'S method,
and finds the ammo-acids in nearly the same quantitative proportions as
they occur in casein, as determined by FISCHER and ABDERHALDEN and by
HART. Besides this compound proteid we have at least one other, as
MANDEL and LEVENE and LOEBISCH 1 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 pen-
tose reactions and yielded abundance of levulmic acid. Besides this
nucleic acid, MANDEL and LEVENE 2 isolated from the glands a gluco-
thionic acid with 2.65 percent S and 4.38 per cent N. We cannot state
what relation these substances bear to that constituent of the gland
found by BERT, which on boiling with dilute mineral acids yielded a
reducing substance. A similar substance, which acts perhaps as a step
toward the formation of lactose, has also been observed by THIERFELDER.
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
mammary glands have been little investigated, but among them are
found considerable amounts of purine bases. The mammary glands

1 Odenius, Maly's Jahresber., 30; Mandel, Bioch. Zeitschr., 22; Mandel and Levene,
Zeitschr. f. physiol. Chem., 46; Loebisch, Hofmeister's Beitrage, 8.

2 Zeitschr. f . physiol. Chem., 45.

610

COW'S MILK. 611

also contain a proteolytic enzyme which, according to HiLDEBRANDT,1
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 chiefly 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
re-action 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
sulphuric 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 reation 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,
FoA4 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 transformation
of the milk-sugar into lactic acid, caused by micro-organisms.

1 Bert, Compt. rend., 98; Thierfelder, Pfluger'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 .

3 Thorner, Maly's Jahresber., 22; Sebelien, ibid.', Courant, Pfluger's Arch., 50.

4 Compt. rend. soc. biolog. (58), 59, 51.

612 MILK.

Perfectly fresh amphoteric milk does not coagulate on boiling, but
forms a pellicle consisting of coagulated casein and lime-salts, which
rapidly reforms after being removed. Even after passing a current of
carbon dioxide through the fresh milk it does not coagulate on boiling.
In proportion as the formation of lactic acid advances this behavior
changes, and soon a stage is reached when the milk, which has previously
had carbon dioxide passed through it, coagulates on boiling. At a second
stage it coagulates alone on heating; then it coagulates by passing carbon
dioxide alone without 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, especially 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 oonverted
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 some time, 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

1 On the Conditions Influencing the Number and Size of Fat-globules in Cow's
Milk, Wisconsin Exp. Station, 0, 1892.

FAT GLOBULES. 613

whose diameter is 0.0024-0.0046 mm. 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.

The observations of ASCHERSON 1 show that drops of fat, when
dropped in an alkaline protein solution, are covered with a fine albuminous
•coat, a so-called haptog en-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 albuminous membrane for the fat-globule has been
generally abandoned. The observations 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 sur-
rounded by a stratum of casein solution held by molecular attraction,
and this prevents the globules from uniting with each other. Every-
thing 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.
DROOP-RICHMOND and BONNEMA,S on the other hand, present several
deductions conflicting with STORCH'S theory. If STORCH'S observation

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;
Droop-Richmond, see Chem. Centralbl., 1904, 2, 356; Bonnema, ibid., 1243.

614 MILK.

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.1 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 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
chiefly of olein and palmitin. Besides these it contains, as triglycerides,
myristic acid, stearic acid, small amounts of lauric acid, arachidic acid,
and dioxyslearic 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 butty ic acid and
30-33 p. m. is caproic acid. The non-volatile fat consists of A-TV
olein, and the remainder is chiefly palmitin. The composition of butter
is not constant, but varies considerably under different circumstances.2
According to LEMUS 3 the small fat-globules contain more olein and
less volatile acids than the large globules.

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 two carbohydrates, of
which the one, lactose, is of great importance. It also contains extract-
ive bodies, traces of urea, creatine, creatinine, or otic acid, hypoxanthine (?),
cholesterin, citric acid (SOXHLET and HENKEL4), and lastly also mineral
bodies and gases.

1 Zeitschr. f. physiol. Chem., 59.

2 Riegel, Maly's Jahresber., 34; Duclaux, Compt. rend., 104. Various statements
as to the composition of milk-fat can be found in Koefoed, Bull. d. 1'Acad. Roy.
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.

3 See Maly's Jahresber., 34.

4 Cited from Soldner, Die Salze der Milch, etc., Landwirthsch. Versuchsstation,.
35, Separatabzug, 18.

CASEIN. 615

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 the following com-
position: 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 (a)D=— 80°; its faintly alkaline solution has a
stronger rotation, namely, —97.8 to —111.8°, in a solution of N/10 — N/5
NaOH (LONG x). 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 CSOKAS 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 SLYKES). It
dissolves readily in water with the aid of alkali or alkaline earths, also
calcium carbonate, from which it expels carbon dioxide. If casein is
dissolved in lime-water and this solution carefully treated with very
dilute phosphoric acid until it is neutral in reaction, 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

1 Hoppe-Seyler, Handb. d. physiol. u. pathol. chem. Analyse, 7. Aufl., 368; Long,
Journ. Amer. Chem. Soc., 27.

2 Pfliiger's Arch., 121.

3 Laqueur and Sackur, Hofmeister's Beitrage, 3; M. Arthus, Theses presentees
a la faculte des sciences de Paris, 1893; Robertson, Journ. of biol. Chem., 2; L. and
D. van Slyke, Amer. Chem. Journ., 38.

616 MILK.

the white color of the milk is. due partly to the casein and calcium phos-
phate. SOLDNER has prepared two calcium compounds of casein with
1.55 and 2.36 per cent CaO, and these compounds are designated di- and
tricalcium casein by COURANT.

Besides the rather earlier investigations on the salts of casein by SOLD-
NER, COURANT, ROHMANN, LAQUETJR, RAUDNITZ l and others we have
the recent observations and theoretical discussion of ROBERTSON2 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 presen.ce 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 easily 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 coagu-
lates with rennet in the presence of a sufficiently large amount of lime-
salts. In solutions free from lime-salts the casein does not coagulate
with rennet, but it is changed so that the solution (even if the enzyme
is destroyed by heating) yields a coagulated mass, having the properties
of a curd, if lime-salts are added. The rennet enzyme, rennin, has there-
fore an action on casein even in the absence of lime-salts. These last

1 Soldner, Die Salze d. Milch, etc., and Maly's Jahresber., 25; Courant 1. c.;
Rohmann, Berl. klin. Wochenschr., 1895; Laqueur, 1. c., and Hofmeister's Beitrage,
7; Raudnitz, Ergebn. d. Physiol., 2, Abt. 1.

2 Journ. of physik. 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, Milchwirkch. 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.

CASEIN. 617

are only necessary 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 proven 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 coagulation 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 DAM2 claims
that it is the quantity of lime combined with the casein which is of
importance in the coagulation process. The role of the lime-salts in
coagulation is not clear, and this follows from the chemical procedure
in rennin coagulation. •—*-*•. *i

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 coagu-
lation with rennin is considered as a cleavage process, in which the chief
mass of the casein, sometimes more than 90 per cent, is split off as para-
casein,4 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 Kaseins 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 Me"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
3eitrage, 6 and 7, with Reichel, ibid., 7 and 8; Laqueur, ibid., 7.

2 Zeitschr. f . physiol. Chem., 58.

3 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 accqunt the author calls the curd paracasein, according to
Schulze and Rose (Landwirthsch. Versuchsstat., 31). A summary of the literature on

618 MILK.

amounts of lime-salts the paracasein-lime precipitates out while the
proteose-like substance (whey protein) remains in solution.

The paracasein is very similar to casein, but cannot be recoagulated
by rennin. As solution of alkali-paracaseinate is much more readily
precipitated by CaCl2 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 transfor-
mation has been found in many cases (PETRY, SLOWTZOFF, v. HERWER-
DEN l). This is explained by the presence of another proteolytic enzyme
in the (impure) rennin preparation. This assumption seems to be plaus-
ible, and we are here probably dealing only with a secondary process
which has nothing whatever to do with the true formation of para-
casein. Whey protein is also formed after the very short action of rennin,
and the continued cleavage occurs with varying speed. Thus SCHMIDT-
NEILSEN 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 investiga-
tions all 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 proven.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 LAXA, coagulates with rennin the same as a solution of casein lactate,
which indicates, he believes,2 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 proven.

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, MORACZEWSKI, SEBELIEN,
and ZAiTSCHEK.4 The amount of phosphorus in the pseud onucleins

the casein coagulation may be found in E. Fuld, Ergebnisse der Physiol., 1; Raudnitz,
ibid., 2; and Laqueur, Biochem. Centralbl., 4, 344.

1 Petry, Hofmeister's Beitrage, 8; Slowtzoff, ibid., 9; v. Herwerden, Zeitschr. f.
physiol. Chein., 52.

2 See also Werncken, Zeitschr. f. Biol., 52.

3 Laxa, 1. c.

4Salkowski, Zeitschr. f. physiol. Chera., 27; Salkowski and Hahn, Pfliiger's Arch.,

DIGESTION OF CASEIN. 619

obtained also varies considerably. SALKOWSKI considers that the quar-
tity of pseudonuclein split off is dependent upon the relation between
the casein and the digestion fluid, e.g., the quantity of the pseudonu-
cleins diminishes as the pepsin-hydrochloric acid increases. In the presence
of 500 grams of pepsin-hydrochloric acid to 1 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 digestion
progresses. Another part of the phosphorus is retained in organic com-
bination in the proteoses as well as in the true peptones (SALKOWSKI,
BIFFI, ALEXANDER, ADERS PLIMMER and BAYLISS 1).

From the products of peptic digestion of casein, after the separation
of the pseudonuclein, SALKOWSKI 2 has isolated an acid rich in phos-
phorus. He considers this a paranucleic acid. It is soluble in water,
insoluble m alcohol, levorotatory, and contained N 13.25-13.55, and P
4.05-4.31 per cent. The acid differs from the nucleic acids in that it
gives the biuret test and a faint xanthoproteic reaction. Presupposing
its purity, it is not an acid comparable with the nucleic acids. A still
richer product in phosphorus, with 6.9 per cent P, has been isolated
as a uranium compound by REH 3 from the peptic digestive products
of casein. He calls this body polypeptid phosphoric acid. On hydrolysis
this product gave 18.7 per cent nitrogen as diamino-acids, 56.7 per cent
as monamino-acids and the remarkably high result 23.8 per cent amido-
nitrogen. This pseudonucleic acid also gave the biuret and the xantho-
proteic tests and MILLON'S reaction, and behaved like a protein rich in
phosphorus.

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

59; Salkowski, ibid., 63; v. Moraczewski, Zeitschr. f. physiol. Chem., 20; Sebelien
ibid., 20; Zaitschek, Pfliiger's Arch., 104.

1 Salkowski, 1. c.; Biffi, Virchow's Arch., 152; Alexander, Zeitschr. f. physiol.
Chem., 25; Pliramer and Bayliss, Journ. of Physiol., 33.

2 Zeitschr. f. physiol. Chem., 32.

3 Hofmeister's Beitrage, 11.

620 MILK.

it had the properties of serglobulin; the globulin isolated by TIEMANN l
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-
MANN 2 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:

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 MgSO4 in substance and the filtrate treated as previously
stated (page 255).

The occurrence of other proteins, such as proteases and peptones, in milk has
not been positively proven. 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 lacloprotein, which is a mixture of a little casein with
changed albumin, and proteose 3 which is formed by chemical action. In regard
to opalisin, see Human Milk, p. 628.

Milk also contains, SIEGFRIED 4 claims, a nucleon related to phos-
phocarnic acid, which yields fermentation lactic acid (instead of para-
lactic acid) and a special carnic acid, orylic add (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 mention
catalases, oxidases, peroxidases, and reductases, 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.
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 fermentation enzyme which in the absence of micro-organisms
decomposes the lactose into lactic acid, alcohol, and CC>2, occurs, according
to STOKLASA 5 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 prop-
erty at least of acting upon monobutyrin. BABCOCK and RUSSEL have

1 Zeitschr. f . physiol. Chem., 25.

2 Sebelien, Zeitschr. f. physiol. Chem., 9; Wichmann, ibid., 27.

3 See Hammarsten, Maly's Jahresber., 6, 13.

4 Zeitschr. f . physiol. Chem., 21 and 22.

5 See Chem. Centralbl., 1905, 1, 107.

LACTOSE. 621

found in these two kinds of milk, as well as certain others, a proteolytic
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, CgHnNaO^HX), 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, C^H^On + H^O. This sugar, on hydrolysis,
can be split into two hexoses, dextrose 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, of which, according to TANRET,S there are three modifica-
tions, occurs ordinarily as colorless rhombic crystals with 1 molecule 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 water; 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)D=+52.5°. 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 4 found that the milk-sugar is first split into dextrose
and galactose by an enzyme, lactase, existing in the yeast. The prep-
aration of milk-wine, " kumyss," from mare's milk and " kephir " from
cow's milk is based upon this fact. Other micro-organisms also take
part in this change, causing a lactic-acid fermentation of the milk-sugar.

1 Babcock 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.

3 Bull. soc. chim. (3), 13. See also Hudson, Journ. Amer. Chem. Soc., 30.

4 Ber. d. d. Chem. Gesellsch., 27.

622 MILK.

Lactose responds to the reactions of dextrose, 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
lactbsazone, 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 dextrose 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 centimetres long (NEUBERG 2).

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, SEBELIEN 3 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. (SEBLIEN 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. LANDWEHR considers
this as animal gum, and BECHAMP 4 as dextrin.

1 The well-known beautiful red color, which milk produces after the addition of
alkali, at the room temperature and to which recently attention has been called 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.

3 Sebelien, Hammarsten's Festschrift, 1906; with Sunde, Zeitschr. f. angew.
Chem., 21.

4 Ritthausen, Journ. f. prakt. Chem. (N. F.), 15; Landwehr, Zeitschr. f. physiol.
Chem. 8, 9; Bechamp, Bull. Soc. chim. (3)., 6.

MINERAL BODIES. 623

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 they cannot all be treated here, we will give the chief 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 asbestus. 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
precautions 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 RITTHATJSEN'S
method is employed, namely, the precipitation of the milk with copper
sulphate according to the modification suggested by MuNK.1 He pre-
cipitates all the proteins by means of cupric hydoxide 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 lactalbumin 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. SiMON2 found that the precipitation with tannic acid, also with phospho-
tungstic acid, is the simplest and most accurate. The objection to this and
other methods in which the proteins 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 ^ of the total nitrogen belongs to the
extractives in cow's milk, and rr in woman's milk. CAMERER and" SOLDNER
determine the nitrogen in the filtrate from the tannic-acid precipitate by KJEL-
DAHL'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,4

1 Ritthausen, Journ. f. prakt. Chem. (X. F.), 15; I. Murik, Virchow's Arch., 134.

2 Sebelien, Zeitschr. f. physiol. Chem., 13; Simon, ibid., 33.

3 Zeitschr. f . Biologie, 33 and 36.

4 Hoppe-Seyler, Med. chem. Untersuch., 272.

624 MILK.

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 precipitate 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 lactalbumin may also be precipitated by tannic acid from
the filtrate from the casein precipitate containing MgSO4, after dilut-
ing with water, the nitrogen determined by KJELDAHL'S method and the
result multiplied by 6.37.

SCHLOSSMANN l 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 nitrate 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 weigh-
ing 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 numerous other methods
for estimating milk-fat, but they cannot be considered here.

In determining the milk-sugar the proteins are first removed. For
this purpose 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 XV). 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 com-
plete works.

Instead of these volumetric determinations other methods of estima-
tion, such as ALLIHN'S method, the polariscope method, and others,, may
be used* In calculating 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. Casein. Albumin. Fats. Sugar. Salts.

871.7 128.3 30.2 5.3 36.9 48.8 7.1

35.5

1 Zeitschr. f. physiol. Chem., 22.

2 Chemie der menschlichen Nahrungs- und Genussmittel, 4. Aufl.

COLOSTRUM. 625

The quantity of mineral bodies in 1000 parts of cow's milk is, according
to the analyses of SOLDNER, as follows: K20 1.72, Na20 0.51, CaO 1.98,
MgO 0.20, P2O5 1.82 (after correction for the pseudonuc.lein) , Cl 0.98
grams. BUNGE l found 0.0035 gram Fe2O3. 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
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. 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 chiefly 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
diameterwith abundant fat-granules and fat -globules. Thefat of colostrum
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 difference between
it and ordinary milk is that colostrum coagulates on heating to boiling
because of the absolutely and relatively greater quantities of globulin and
albumin that it contains.4 The composition of colostrum varies consider-
ably. 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.

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

1 Zeitschr. f . Biologic, 10. 2 Pfliiger's Arch., 2. 3 See Maly's Jahresber., 21.

4 See Sebelien, Maly's Jahresber., 18, and Tiemann, Zeitschr. f. physiol. Chem.,
135. See also Simon, ibid., 33; Winterstein and Strickler, ibid., 47.

626 MILK.

KUMYSS and KEPHIR are obtained, as above stated, by the alcoholic and lactic-
acid fermentation of the milk-sugar, the former from mare's milk and the latter
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 another,
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 1 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
investigations 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-hydro-
chloric acid without leaving a residue. 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 ELLENBERGER 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 pseudonuclein on pepsin digestion, which agrees
well with the above-mentioned investigations 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,2 by being very rich in fat, 144.6-197.3 p. m., and casein, 80.6-
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:3

Milk of the
Dog

Water.
. . . 754 . 4

Solids.
245.6

Proteins.
99 . 1

Fat.
95.7

Sugar.
31.9

Salts.

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
Ass

. . . 900.6
. . . 900.0

99.4
100.0

18.9
21.0

10.9
13.0

66.5
63.0

3.1
3.0

Pig

. . . 823.7

167.3

60.9

64.4

40.4

10.6

Elephant

678 5

321 5

30 9

195 7

SS 5

6 5

Dolphin

486 7

513 3

437 6

4 6

Whale*.

. . . 698.0

302.0

94.3

194.0

traces

9.9

1 Studien iiber die Eiweissstoffe des Kumys und Kefirs, St. Petersburg, 1886 (Ricker).
2Zaitschek, 1. c.; Schlossmann, Zeitschr. f. physiol. Chem., 22; Ellenberger, Arch,
f. (Anat. u.) Physiol., 1899 and 1902; Werenskiold, Maly's Jahresber., 25.

3 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.

4 Scheibe, cited in Bioch. Centralbl., 7, 553.

HUMAN MILK. 627

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 nevertheless
a lower absolute reaction for alkalinity as well as for acidity. He found
between the tenth day and the fourteenth month after confinement prac-
tically 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 elect rometrically is, according to FoA,1 still
nearly neutral, like the other kinds of milk.

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 PARMENTIER 2 constant
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, 3 relatively poor in volatile fatty
acids. The non-volatile fatty acids consist of one-half oleic acid, 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 investigators4 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.5 It may be precipitated by gastric juice, but

1 Compt. rend. soc. biolog., 58.

2 See Maly's Jahresber., 34.

3 Ruppel, Zeitschr. f. Biologic, 31; Laves, Zeitschr. f. physiol. Chem., 19.

4 See Biedert, Untersuchungen iiber die chemischen Unterschiede der Menschen-
und Kuhmilch (Stuttgart), 1884; Langgaard, Virchow's Arch., 65; Makris, Studien
iiber die Eiweisskorper der Frauen- und Kuhmilch, Inaug.-Diss. Strassburg, 1876.

5 See among others Bienenfeld, Bioch. Zeitschr., 7, and Fuld and Wohlgemuth,
ibid., 8.

628 MILK.

dissolves completely and easily in an excess of juice; the casein precipi-
tate 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. We are not clear
as to this difference between the digestibility of the cow's casein and
human casein, as the first seems to be utilized in the intestinal tract of
the infant to the same extent as human casein (P. MULLER, RUBNER and
HEUBNER 1).

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 been decided as yet. According to SZONTAGH
and ZAITSCHEK and also WROBLEWSKY, the casein from human milk
does not yield any pseudonuclein on peptic digestion, and hence it cannot
be a nucleoalbumin. WROBLEWSKY found the following for the com-
position 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 KOBRAK 2 woman's casein yields some pseu-
donuclein, 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 nucleo-
albumin and a basic protein.

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
precipitation, so that the casein masses become larger to a certain extent
and the precipitation becomes easier. ENGEL 3 recommends dilution with

1 Miiller, Zeitschr. f. Biologic, 39; Rubner and Heubner, ibid., 37.

2Szontagh, Maly's Jahresber., 22; Zaitschek, 1. c.; Wroblewsky, Beitrage zur
Kenntniss des Frauenkaseins, Inaug.-Diss. Bern, 1894, and Ein neuer eiweissartiger
Bestandteil der Milch, Anzeiger der Akad. d. Wiss. in Krakau, 1898; Kobrak,
Pfliiger's Arch., 80; Langstein and Bergell, cited in Bioch. Centrabl., 8, 323.

8 Fuld and Wohlgemuth, Bioch. Zeitschr., 5; Engel, ibid., 14.

.HUMAN MILK. 629

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/ 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.

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 rela-
tively 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
percentage 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. 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 com-

1 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.

630 MILK.

bination. This does not agree with the results of SIKES who found on
an average of only 42 per cent of the total P2O5 in organic combina-
tion. Because of the large amount of casein (and calcium phosphate)
cow's milk is much richer in phosphorus than human milk. The relation
1*205 : N, according to SCHLOSSMANN/ is equal to 1:5.4 in human milk
and 1:2.7 in cow's mi-Ik. 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
(SCHEIBE 2), 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 (SIEBER 3).
This reaction consists in treating 5 cc. of woman's milk with 2.5 cc. ammonia
(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 4 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).5 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 (^4), 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:

K2O... . 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

P2O5 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

1 Burow, Zeitschr. f. physiol. Chem., 30; Koch, ibid., 47; Wittmaack, ibid., 22;
Siegfried, ibid., 22; Nerking and Haensel, Bioch. Zeitschr., 13; Valenti, Biochem.
Centralbl., 4; Schlossmann, Arch. f. Kinderheilkunde, 40; Sikes, Journ. of Physiol., 34.

2 Maly's Jahresber., 21.

3 Zeitschr. f. physiol. Chem., 30.

4Rubner, Zeitschr. f. Biologic, 36; Camerer and Soldner, ibid., 39; Schondorff,
Pfliiger's Arch., 81.

5 Bunge, Zeitschr. f. Biologic, 10; Camerer and Soldner, ibid., 39 and 44.

HUMAN COLOSTRUM. C31

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 l
find about the same amount, namely, 10-20 milligrams Fe2O3 = 3.5-7
milligrams iron in 1000 grams human milk.

The gases of woman's milk have been investigated by KiiLZ.2 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 dif-
ference in the protein bodies of these two kinds of milk has been completely
studied.

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 3 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 lactation,
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, CAM-
ERER 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 BRUNNER.* 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

1 De Lange, Maly's Jahresber., 27; Jolles and Friedjung, Arch. f. exp. Path. u.
Pharm., 46; Camerer and Soldner, Zeitschr. f. Biologic, 46>

2 Zeitschr. f . Biologic, 32.

3 See Hoppe-Seyler, Physiol. Chem., 734.

4 Sourdat, Compt. rend., 71; Brunner, Pfliiger's Arch., 7.

632 MILK.

contains less casein than that of brunettes, a difference which TOLMATSCHEFF 1
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 slightly diminish the milk-sugar and
to considerably increase 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 stand-
point 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. GENSER,2 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.

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 chief groups of organic nutritive substances — proteins, carbohy-
drates, 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, also some lecithin an .1 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 3 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) :

A B

K20 114.2 149.8

Na20 106.4 ' 88.0

CaO 295.2 272.4

MgO 18.2 15.4

Fe2O3 7.2 1.2

P2O5 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 in soda relatively decreases. In regard to the amount

1 1'Heritier, cited from Hoppe-Seyler, Physiol. Chem., 738; Vernois and Bec-
querel, Du lait chez la femme dans 1'etat de sante", etc. (Paris, 1853); Tolmatscheff,
Hoppe-Seyler, Med.-chem. Untersuch., 272.

2 Schlossberger and Hauff, Annal. d. Chem. u. Pharm., 96; Gubler and Quevenne,
cited from Hoppe-Seyler 's Physiol. Chem., 723; v. Genser, ibid.

3 Zeitschr. f. physiol. Chem., 13.

MINERAL BODIES OF THE MILK AND THE NURSLING. 633

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 l
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.

A B

K20 78 314

Na2O 91 119

CaO 361 164

MgO 9 26

Fe2O3 8 6

P2O3 389 . 135

Cl 77 200

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 2 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 ABDERHALDEN,S 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.

1 Hugounenq, Compt. rend., 128; de Lange, Zeitschr. f. Biologie, 40; Camerer
and Soldner, ibid., 39, 40, and 44.

2 Bunge, "Die zunehmende Unfahigkeit der Frauen ihre Kinder zu stillen," Miin-
chen, 1900, cited by Camerer, Zeitschr. f. Biologie, 40.

3 Proscher, Zeitschr. f. physiol. Chem., 24; Abderhalden, ibid., 27; Pages, Arch*
de Physiol. (5), 7.

634 MILK.

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 DECAISNE l
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 others 2 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 seems
to cause no constant, direct action on the quantity of the milk constituents.3
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 CoMMAiLLE4
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.5

Chemistry of Milk-secretion. That the constituents which occur actu-
ally dissolved in milk pass into the secretion and not alone by filtration
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 them-
selves. A further proof lies in the fact that the lactalbumin is not identical
with seralbumin; and lastly, as BUNGE G has shown, the mineral bodies

1 Cited from Hoppe-Seyler, 1. c., 739.

2 See Maly's Jahresber., 26. See also Basch, Ergebnisse der Physiologic, 2, Abt. 1.

3 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, 37, and Morgen, Beger and Fingerling, Landw.
Versuchsst., 61, and Raudnitz, Monatschr. f. Kinderheilk.

4 Cited from Konig, 2, 235.

5 See Beck, Maly's Jahresber., 25.

8 Lehrbuch d. physiol. und pathol. Chem., 3. Aufl., 93.

CHEMISTRY OF MILK SECRETION. 635

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. There does not seem to be any doubt that the pro-
toplasm of the cells takes part in the secretion in such a manner that it
becomes itself a constituent of the secretion, and this also agrees with
HEIDENHAIN'S l views. 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 HiLDEBRANDT2 upon
the proteolytic enzyme of the mammary gland, and the autolysis of the
gland have not given any clue as to the mode of formation of casein.
There is no doubt that it is formed in the mammary glands by a synthesis.
The findings of MANDEL 3 .that the hydrolytic cleavage products of the
nucleoprotein from the mammary glands occur approximately quan-
tatively 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. JANTZEN has shown that after feeding
iodized casein, the milk-fat of goats contained a little iodine, which
indicates that the iodized milk-fat could also have a different origin.
As a contamination of the casein fed with iodized fat was not excluded
in these experiments, they do not seem to modify the proof of the investi-
gations of WINTERNITZ and others (CASPARI, PARASCHTSCHUK4). 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

1 Hermann's Handbuch, 5, Teil 1, 380.

2 Basch, Jahrb. f. Kinderheilkunde, 1898; Hildebrandt, Hofmeister's Beitrage, 5;
Ldbisch, ibid., 8.

3 Bioch. Zeitschr., 22.

4 Winternitz, Zeitschr. f. physiol. Chem., 24; Jantzen, Centralbl. f. Physiol., 15 ;
Caspar!, Arch. f. (Anat. u.) Physiol., 1899, Supplbd. and Zeitschr. f. Biologic, 46;
Paraschtschuk, Chem. Centralbl., 1903, 1.

636 MILK.

containing iodine was unchanged iodized fat of the food. The investi-
gations of SPAMPANI and DADDI, PARASCHTSCHUK, GOGITIDSE 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 uncertain 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-fat in the glandular
cells is possible. The results of the experiments of GOGITIDSE 1 with
soaps also indicate that the mammary glands have the property of form-
ing 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 proven, 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 as food, and this proves that at least
a part of the fat secreted by the milk is produced from proteins or carbo-
hydrates, 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 dextrose 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 TniERFELDER2 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 dextrose introduced as food or
formed in the body. Certain observations indicate such an origin, among
others those of PORCHER, who found that dextrose appeared in the urine
after delivery when the mammary glands of the goat had previously been

1 Spampani and Daddi, Maly's Jahresber., 26; Henriques and Hansen, ibid., 29;
Gogitidse, Zeitschr. f. Biologie, 45, 46, and 47. See also Basch, Ergebnisse d. Physiol.,
2, Abt. 1.

2 Miintz, Compt. rend., 102; Bert and Thierfelder, footnote 1, p. 611.

CHEMISTRY OF MILK SECRETION. 637

extirpated. This glycosuria is explained simply by the. fact that the
lactose-forming action of the gland was removed at the time of delivery,
when large amounts of dextrose were produced, but it must not be for-
gotten that MARSHALL and KIRKNESS found with similar experiments
upon guinea-pigs no passage of sugar into the urine. The experiments
of KAUFMANN and MAGNE 1 upon cows indicate a formation of lactose
from dextrose. They found that during secretion the glands took sugar
from the blood, so that the venous gland -blood was poorer in sugar than
otherwise.

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 nursing
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
may also pass into the milk, but probably not in such quantities as to
have any direct action on the nursing child.2 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 3 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 4 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 the glands finally, instead of yielding milk,
gave only blood-serum or a serous fluid. HUSSON 5 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 Porcher, Compt. rend., 138 and 141; Marshall and Kirkness, Bioch. Journ., 2;
Kaufmann and Magne, Compt. rend., 143.

2 See Klingemann, Virchow's Arch., 126, and Rosemann, Pfliiger's Arch., 78.

3 Schlossberger, Annal. d. Chem. u. Pharm., 96; Joly and Filhol, cited from
v. Gorup-Besanez, Lehrb., 4. Aufl., 438.

4 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.

5 Compt. rend., 73.

CHAPTER XV.
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 veiy 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
kidneys also contain, according to LONNBERG, a mucin-like substance.

638

PHYSICAL PROPERTIES OF THE URINE. 639

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.
The nucleic acid obtained by MANDEL and LEVENE from beef kidneys
yielded guanine, adenine, thymine, and cytosine on cleavage. Accord-
ing to MORNER l chondroitin-sulphuric add occurs as traces. MANDEL
and LEVENE 2 have also obtained glucothionic acid from the kidneys.
Fat occurs only in very small amounts in the cells of the convoluted
tubules. FRANKEL and NOGUEIRA 3 found a cephalin-like substance,
a triamido-diphosphatide and a diamino-monophosphatide. Among
the extractive bodies of the kidneys one finds purine bases, also 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. OIDTMANN 4 found 810.94 p. m. water, 179.16
p. m. organic and 0.99 p. m. inorganic substance in the kidney of an
old woman.

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 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
atrophied; 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
carnivora, 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,

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, Zeitschr. f.
physiol. Chem., 47; Morner, Skand. Arch. f. Physiol., 6.

2 Zeitschr. f . physiol. Chem., 45. See also Mandel and Neuberg, Bioch. Zeitschr., 13.

3 Bioch. Zeitschr., 16. See also Dunham, Zeitschr. f. physiol. Chem., 64.

4 Cited from v. Gorup-Besanez, Lehrb., 4. Aufl., 732.

640 URINE.

yellowish-brown, rose-colored, or often brick-red precipitate (sedimentum
lateritium) 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; t*he 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 carnivora, 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 flesh, as then the urine
voided is acid.

The urine of a healthy man on a mixed diet has an acid reaction,
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
oxy acids, oxyproteic acids l 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 See St. Kozlowski, Bull. Acad. d. scien. de Cracovie, Jan., 1909, 37.

ACIDITY OF THE URINE. 641

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 con-
taining hydrochloric acid is secreted, the urine may be neutral or even
alkaline.1 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 HOFFMANN, RINGSTEDT, ODDI, and TARULLI and VOZARIK;
muscular work raises the degree of acidity, but ADUCCO 2 claims that it
decreases it. Abundant perspiration 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 burnt up with difficulty ar§
ingested in large quantities. When the supply of carbonates of the
fixed alkalies stored up in the organism for this purpose is not sufficient
to combine with °the excess of acid, then ammonia is split off from the
proteins or their decomposition products, and this excess of acid combines
therewith, forming ammonium salts, which pass into the urine. In her-
bivora such a combination of the excess of acid with ammonia seems
not to take place, or not to the same extent, and therefore herbivoia
soon die wrhen acids are given. This is true at least for rabbits, while
according to BAER 3 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 l>e caused, he says, from a different amount of
protein in the food which yields ammonia. One can by food rich
in protein make herbivora (also rabbits) resistant toward the intro-
duction of acid, while dogs with food poor in protein behave like
rabbits. The question as to the action of acids upon the elimination
of fixed alkalies by the urine and the removal of these from the tissues
seems to be rather complicated as, according to STAAL, in rabbits the
quantity of sodium in the subcutaneous connective tissue is not diminished
after the continuous introduction of acid for several days, but is rather

1 Contradictory statements are found in Linossier, Maly's Jahresber., 27.

2 Hoffmann, see Maly's Jasresber., 14; Ringstedt, ibid., 20; Oddi and Tarulli,
ibid., 24; Aducco, ibid., 17; Vozdrik, Pfluger's Arch., 111.

3 See Winterberg, Zeitschr. f. physiol. Chem., 25, and J. Baer, Arch. f. exp. Path, m
Pharm., 54.

642 URINE.

increased. It must not be overlooked that, as A. LOEWY l has found,
the sensitiveness of different individuals toward the action of acid
varies considerably.

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 burnt 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 determina-
tions. We now determine the acidity simply by acidimetric methods,
titrating with N/10 caustic alkali, using phenol phthalein 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
disturbing 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 Er]en-
meyer flask (about 200 cc. capacity), treated with 1-2 drops of -^-per cent
phenolphthalein solution, and shaken with 15—20 grams of powdered

1 Eppinger, Zeitschr. f. exp. Path. u. Therap., 3; with Tedesko, Bioch. Zeitschr.,
16; Staal, Zeitschr. f. physiol. Chem., 58; A. Loewy, Centralbl. f. Physiol., 20, 337.

OSMOTIC PRESSURE OF THE URINE. 643

potassium oxalate and immediately titrated with N/10 caustic soda

with constant shaking until a pronounced pale-rose color appears.

VOZARIK 1 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 264),
the ion acidity cannot be determined by titration, wrhile 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.2 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, 100X1CT7, and 49 X 10 ~7, respectively. On an average the
urine therefore contains 30-50 grams of hydrogen ions in 10 million
liters, and as in the same quantity of purest water there is contained in
round numbers 1 gram of hydrogen ions, the urine contains, therefore,
30-50 times as many hydrogen ions as the water. From HOBER'S
investigations it also follows that no direct relation exists between
the titration acidity and the ion acidity, 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 J= 0.87-2. 71° C.3
After partaking of considerable water it may be markedly lower, and on
diminished supply of water it may be considerably higher.

According to BUGARSKY a certain relation exists between the freezing-

£

point depression and the specific gravity, namely, — r = constant = 75. This

5 — 1

equation, where s represents the specific gravity, has no general application, and
according to STEYRER 4 is only approximate for normal urines. The validity
of the relation found by BUGARSKY between the electrical conductivity and
the ash content of the urine, seems also to require further proof.

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; Vozd-rik, 1. c.; de Jager, Zeitschr. f. physiol. Chem., 55, and Ringer, ibid., 60.

2 v. Rhorer, Pfliiger's Arch., 86; Hober, 1. c. See also Jolles, Bioch. Zeitschr., 13.

3 See Strauss, Zeitschr. f. klin. Med., 47.

4Bugarsky, Pfluger's Arch., 68; Steyrer, Hofmeister's Beitrage, 2.

644 URINE.

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 alter
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 dif-
ferent 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-
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

UREA. 645

by LoHNSTEiN.1 JoLLES2 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.

;il. ORGANIC PHYSIOLOGICAL CONSTITUENTS OF URINE.

+ /NH2

Urea, Ur, CON2H4=CO<f , has been synthetically prepared in sev-

XNH2

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 others.

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. Urea is also found in
certain tissues and organs of mammals, especially 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.

1 Pfluger's Arch., 59; Chem. Centralbl., 1895, 1, and 1896, 2.

2 Wien. med. Presse, 1897, No. 8.

3 Pfluger's Arch., 117.

4 v. Schroeder, Zeitschr. f. physiol. Chem., 14.

5 Hammarsten, ibid., 24.

646 URINE.

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
different 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, such as BORLAND, E. SCHULTZE,
CAMERER, VOCES, MORNER and SJOQVIST, GUMLICH, BODTKER, FoLiN,1
and others, on the relation of the different nitrogenous constituents to
each other in the normal urine of adults. 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 nitrogeneous substances (extractives) . . . 7-12 7.3-14.7

The variable relation between uric acid, ammonia, and urea
nitrogen in children and adults is remarkable, since the urine of chil-
dren is considerably richer in uric acid and ammonia, and considerably
poorer in urea, than the urine of adults. A much larger number of anal-
yses 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' nitrogen-
ous substances may be markedly changed, and a decrease in the quan-
tity of urea and an increase in the quantity of ammonia have been

1 Pfluger and Bohland, Pfiiiger'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, 1982), 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.

FORMATION OF UREA. 617

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 1 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 divi-
sion of the nitrogen of the urine. We determine a, the total nitrogen;
b, the nitrogen of the phosphotungstate precipitate; and c, the nitrogen
in the nitrate from the phosphotungstate precipitate. This last con-
tains the urea, hippuric acid, oxyproteic acids, and other bodies whose
nitrogen is ordinarily designated as monamino-acid nitrogen. The
urea nitrogen is especially determined. The bodies precipitated by
phosphotungstic acid are not all known; but uric acid and purine
bases, ammonia, creatinine, pigments, diamino-acids, diarnines and
ptomaines (if they occur), sulphocyanides, 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-
tion. According to v. JACKSCH2 normal human urine contains from 1.5
to 3 per cent of the total nitrogen as amino-acid nitrogen and 5.16 to 8.5
per cent as ammonia and purine bodies. Other experimenters have
obtained different results, and our knowledge on this subject is not suffi-
cient. Very great variations seem to occur not only in the healthy
individual, but also and to a greater degree in diseased conditions.3

Formation of Urea in the Organism. The experiments to produce
urea directly from proteins by oxidation have led to the formation of
some guanidine, bat urea has not been obtained positively. On the
hydrolysis of proteins arginine has been 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, KOSSEL, see Chapter III). 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

1 Zeitschr. f. physiol. Chem., 30.

2 Zeitschr. f. klin. Med., 50.

3 See Satta, Hofmeister's Beitrage, 6, which also gives the literature, and Erben,
Zeitschr. f. Heilkunde, 25.

648 URINE.

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 l has recently
given a direct proof for the formation of urea from arginine. The
introduction of arginine into the body of a dog either per os or sub-
cutaneously 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. In
these cases, without mentioning that the arginine seemed to raise the
nitrogen catabolism, probably also urea was formed from the ornithine.
This can be explained by a deamidation of the ornithine and formation
of urea from the ammonia and carbon dioxide split off.

By the action of alkalies, as above mentioned (Chapter XI), urea
may be formed from creatinine; still such an orgin of urea in the animal
body has not thus far been proven.

The amino-acids are considered as special mother-substances of urea.
By the researches of SCHULTZEN and NENCKI and SALKOWSKI with
leucine and glycocoll, those of STOLTE with several amino-acids, and
those of v. KNIERIEM with asparagin, it has been shown that the amino-
acids are in part converted into urea in the animal organism. 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 substance. The researches
of LOEWI with the " urea-forming " enzyme of the liver, discovered by
RICHET, on glycocoll or leucine, as also the researches of AscoLLi,2
have led to similar results, but it must be remarked that we have no
proof as to the identity of the newly formed substance with urea. The
formation of urea*from amino-acids is considered as proven, and, like
the amino-acids, the polypeptides are also decomposed into urea in
the animal organism, as shown by ABDERHALDEN with TERUUCHI and
BABKIN and with ScniTTENHELM.3

Nothing positive can be said in regard to the manner in which this

1 Kossel and Dakin, Zeitschr. f. physiol. Chem., 41; Thompson, Journ. of Physiol..
32 and 33.

2 Schultzen and Nencki, Zeitschr. f. Biologie, 8; v. Knieriem, ibid., 10; Salkowski,
Zeitschr. f. physiol. Chem., 4; Salaskin, ibid., 25; Loewi, ibid., 25; Stolte, Hofmeister's
Beitrage, 5; Richet, Compt. rend., 118, and Compt. rend. Soc. biol., 49; Ascoli,
Pfliiger's Arch., 72.

3 Abderhalden with Teruuchi and with Babkin, Zeitschr. f. physiol. Chem., 47,
with Schittenhelm, ibid., 51.

FORMATION OF UREA. 649

formation of urea occurs ; but there is no doubt that a formation of
ammonia is here of great importance.

The possibility of a formation of urea from ammonia has been pos-
itively shown. Thus the researches of v. KNIERIEM, SALKOWSKI, FEDER,

I. MUNK, CORANDA, SCHMIEDEBERG and FR. WALTER, HALLERVORDEN,

and POHL and MUNZER/ on the behavior of ammonium salts in the animal
body and the elimination of the ammonia under various conditions,
have shown that not only ammonium carbonate, but also those ammonium
salts which are burnt into carbonate in the organism, are transformed
into urea by carnivora as well as herbivora. v. ScHROEDER,2 by irrigat-
ing the surviving dog's liver with blood treated with ammonium car-
bonate or ammonium formate, has shown that the formation of urea
takes place, at least in part, in this organ. NENCKI, PAWLOW, ZALESKI
and SALASKIN 3 have also found that, in dogs, 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 proven fact, and the urea formation from ammonium
carbonate is to be considered as a synthesis with the elimination of
water.

The assumption of a splitting off of ammonia from amino-acids is
not difficult of conception, as now, especially from the investigations
mentioned in Chapter VIII, we know with positiveness that deamidation
of amino-acids does take 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,4 on the equilibrium of car-
bonate 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,5 namely, that the amino-acids are
transformed into urea with carbamic acid as an intermediate step.
DRECHSEL has shown that the amino-acids yield carbamic acid by oxida-
tion in alkaline fluid outside of the organism, and he obtained urea from
ammonium carbamate by passing an alternating electric current through
its solution, i.e., by alternate oxidation and reduction. DRECHSEL has

1 v. 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.

2 Arch. f. exp. Path. u. Pharm., 15. See also Solomon, Virchow's Arch., 97.

3 Arch, des sciencs biol. de St. Pe"tersbourg, 4; see also Chapter VI, p. 317.

4 Nolf, Zeitschr. f. physiol. Chem., 23; Macleod and Haskins, Journ. of biol. Chem., 1.

5 Zeitschr. f. Biologie, 8.

650 URINE.

also been able to detect small quantities of carbamates in blood, and
later in conjunction with ABEL he detected carbamic acid in alkaline
horse's urine. DRECHSEL therefore accepts the theory of the formation
of urea from ammonium carbamate, and believes that the alternating
oxidation and reduction take place in the following way:

H4N.O.CO.NH2 + 0 = H2N.O.CO.NH2 + H2O

Ammonium carbamate

H2N.O.CO.NH2 + H2 = H2N.CO.NH2 + H20.

Urea

ABEL and MUIRHEAD 1 have later observed an abundant elimination
of carbamic acid in human and dog's urine after the administration of
large quantities of milk of lime, and the probability of the regular appear-
ance of this acid in normal acid-reacting human and dog's urine has been
demonstrated by M. NENCKI and HAHN 2. These last-mentioned inves-
tigators have also given very important support to the theory of the
formation of urea from ammonium carbamate by observations on dogs
with ECK'S fistula. NENCKI and HAHN observed violent symptoms
of poisoning, in dogs fed on meat and operated upon by PAWLOW and
MASSEN, and these symptoms were quite identical with those obtained
on introducing carbamate into the blood. These symptom's also appear
after the introduction of carbamate into the stomach, while the intro-
duction of carbamate into the stomach of a normal dog had no action.
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-transformation
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.

The experience of ROTHBERGER and WINTERBERG was that the
phenomena with meat feeding and with carbamic acid intoxication are
not the same, and HAWKS has also arrived at other results. He
observed violent toxic symptoms on abandoning meat feeding with such
fistular dogs, but not always. In the latter cases the symptoms
appeared on the simultaneous feeding of LIEBIG'S extract of beef, while
this was without effect with a meat-free diet. The administration of
sodium carbamate or intravenous injection of this salt in dogs with

1 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.

2 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. Pe"tersbourg, 1, No. 4, 1892.

3 Rothberger and Winterberg, Zeitschr. f. exp. Path. u. Therap., 1; Hawk, Amer.
Journ. of PhysioL, 21.

FORMATION OF UREA. 05 1

Ecx-fistiila did not, on the contrary, produce the same toxic symptoms
as occurred where meat and extract were fed, and these observations
on these fistular animals do not allow of any positive conclusions as
to the relation of the carbamates to the formation of urea. On the
other hand neither do they contradict the assumption of such a mode
of formation of the urea.

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 l 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 CON"H2,
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;2 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
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 by NENCKI and HAHN, SLOSSE, LIEBLEIN, NENCKI
and PAWLOW, SALASKIN and ZALESKIS 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

1 Arch. f. exp. Path. u. Pharm., 37.

2 In regard to the investigations of Prevost and Dumas, Meissner, Voit, Gre"hant,
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.

3 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 scienc. biol. de St. Pe"ters-
bourg, 5. See also v. Meister, Maly's Jahresber., 25; Salaskin and Zaleski, Zeitschr. f .
physiol. Chem., 29.

652 URINE.

which, irrespective of the pronounced atrophy, an abundant formation
of urea occurs, and no appreciable, 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, KAUFMANN 1 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 2 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 elimina-
tion of ammonia may, as shown by MUNZER in the case of acute phos-
phorus 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 accord-
ing to the law of elimination of ammonia, which will be given later.
That an abnormal formation of acid occurs after the cutting out of the
liver has been especially shown by SALASKIN and ZALESKI.S

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 dissolves

1 Compt. rend. soc. biol., 46, and Arch, de Physiol., (5), 6.

2 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;. Miinzer, ibid., 52; Frankel, Berlin, klin. Wochenschr., 1878; Richter, ibid.>
1896; Morner and Sjoqvist, Skand. Arch. f. Fhysiol., 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.

3 Zeitschr. f. physiol. Chem., 29.

PROPERTIES OF UREA. 653

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 anhydrous
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 ajkali (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 hypobromite
decomposes urea into nitrogen, carbon dioxide, and water according
to the equation

CON2H4 + SNaOBr = 3NaBr + C02 + 2H2O + N2.

With a concentrated solution of furfurol 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 HUPPERT 1 the test is best performed by taking 2 cc. of a
concentrated furfurol 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.HNO3. On crystallizing quickly this
compound forms thin rhombic or six-sided overlapping tiles, or colorless
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.

1 Huppert-Neubauer, Analyse des Hams, 10. Aufl., 296.

654 URINE.

UREA OXALATE, 2.CO(NH2)2.H2C2O4. 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 cry stalliz able 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 difficultly
soluble and whose composition is somewhat disputed.1 With phenylhydrazine,
urea in strong acetic acid gives a colorless crystalline compound of phenyl-
semicarbazid, C6H6NH.NH.CONH2, which is soluble with difficulty in cold water
and melts at 172° C. (JAFFE 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 anima) charcoal, and filter while warm. The urea which crystallizes
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-

'See Tollens and his pupils, Ber. d. deutsch. chem. Gesellsch., 29, 2751; Gokl-
schmidt, ibid., 29, and Chem. Centralbl., 1897, 1, 33; Thorns, ibid., 2, 144 and 737.
2 Zeitschr. f . physiol. Chem., 22.

DETERMINATION OF UREA. 655

saturating with alkali and the ammonia collected in standard sulphuric
acid. The following reagents are necessary:

1. Sulphuric Acid. Either a mixture of equal volumes of pure
concentrated and fuming sulphuric acid or else a solution of 200 grams
phosphoric anhydride in 1 liter ot 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 its 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 colorless.
On cooling the contents are transferred to a voluminous distil ling-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.

Among the methods suggested for the special estimation of urea,
that of MORNER-SJOQVIST, in combination with FOLIN'S method, is per-
haps the most trustworthy and readily performed. For this reason
only this method wrill be given in detail, while we must refer to special
works for the other methods, such as BUNSEN'S method with its many
modifications as suggested by PFLUGER, BORLAND and BLEiBTREU.1

Principle of Morner-Sjdqvist's Method? According to this method
the nitrogenous constituents of the urine, with the exception of urea,

1 Pfluger's Arch., 38, 43, and 44.

2 Skand. Arch. f. Physiol., 2, and Morner, ibid., 14, where the recent literature
may also be found.

£56 URINE.

ammonia, hippuric acid, creatinine, and traces of allantoin,1 are precipi-
tated by a mixture of alcohol and ether after the addition of a solution
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 KJEDLDAHL'S nitrogen estima-
tion. Because of the slight error due to the presence of hippuric acid
and creatinine, several modifications have been suggested by SALASKIN
and ZALESKI and by BRAUNSTEiN.2 These errors are best prevented,
according to MORNER, by the use of FOLIN'S method.

Principle of Folin's Method.3 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 Urea by the Morner-Sjoqvist and Folin Method.4
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.
After cooling it is diluted to about J 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 CO2 and cooling, the acid is retitrated. Corrections must be
made for the ammonia of the urine and for that contained in the magnes-
ium chloride.

If a special determination of the preformed ammonia has been made,
then a direct treatment of the urine, according to FOLIN (nevertheless
after the evaporation of the urine with hydrochloric acid), gives good
results. In the presence of sugar the treatment of the urine with barium
hydroxide is absolutely necessary in MORNER'S opinion, otherwise the
humin substances produced from the sugar take up and retain nitrogen.

1 According to Wiechowski, Hofmeister's Beitriige, 11, the quantity of allantoin
is so great in urine that it must be considered in this method.

2 Braunstein, Zeitschr. f. physiol. Chem., 31; Salaskin and Zaleski, ibid., 28.

3 Ibid., 32, 36, and 37.

4 See Morner, Skand. Arch. f. Physiol., 14.

UREIN, CARBAMIC ACID. 657

HASKINS has changed FOLIN'S method, as he precipitates the urine
first with phosphomolybdic acid and after a further preparation proceeds
according to FOLIN. According to GLAESSNER 1 the MORNZR-SJOQVIST
method cannot be used in the presence of large quantities of amino-acids,
as they remain in part in the alcohol-ether solution.

KNOP-HUFNER'S method2 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 653). This method
is less accurate than the preceding ones, and therefore in scientific work it is dis-
carded. v 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.

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). See
also HOPPE-SEYLER-THIERFELDER'S Handbuch, 8. Aufl. SALKOWSKI 3
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 MOOR/ 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.5

/NH2

Carbamic Acid, CH3NO2 = CO^ . This acid is not known in the free

\OH

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 Na,CO3 to a

1 Haskins, Journ. of biol. Chem., 2; Glaessner, Zeitschr. f. exp. Path. u. Therap., 4.

2 Knop, Zeitschr. f. analyt. Chem., 9; Hufner, Journ. f. prakt. Chem. (N. F.), 3.
In regard to the extensive literature, see Huppert-Neubauer, 10. Aufl., 304, and follow-
ing.

3 Arbeiten aus dem pathol. Institute, Berlin, 1906.

4 O. Moor, Bull. Acad. de St. Petersbourg, 14 (also Maly's Jahresber., 31, 415),
and Zeitschr. f . Biologie, 44 and 45, and Zeitschr. f . physiol. Chem., 40 and 48.

5 See Kubiabko, 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.

658 URINE.

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.1

Carbamic-acid ethylester (urethane), as shown by JAFFE,2 may pass, by the
mutual action of alcohol and urea, into the alcoholic extract of urine when one
is working with large quantities.

FOLIN 3 claims that all human urine contains a body which is probably methylurea*

NH - CO

Creatinine, C^yNsO, or NH : C^ | , is generally considered as

>N(CH3).CH2

the anhydride of creatine (see page 546) found in the muscles. It occurs
in human urine and in that of certain mammalia. It has also been
found in ox-blood, milk, though in very small amounts, and in the flesh
of certain fishes.

JOHNSON'S statement that the creatinine of the urine is different from that
produced by the action of acids on creatine is incorrect according to TOPPELIUS

and POMMEREHNE, WOERNER and THELEN.4

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-
BAUER), or on an average 1 gram. JOHNSON 5 found 1.7-2.1 grams per
day, and similar results have been obtained by v. HOOGENHUYZE and VER-
PLOEGH.6 The quantity of creatinine with a diet free from meat is,
FOLIN 7 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 per cent, as
determined by several experimenters.

As the two bodies, creatine and creatinine, can easily be transformed

1 Nolf, Zeitschr. f. physiol. Chem., 23; Macleod and Haskins, Amer. Journ. of
Physiol., 12, and Journ. of biol. Chem., 1.

2 Zeitschr. f . phygi^i 6&em., 14.

4 S.^-cording to WiecH. Roy. Soc., 42, 43; Chem. News, 55; Toppelius and Pom-
merehne,*, in urine tharm., 234; Woerner, Arch. f. (Anat. u.) Physiol., 1898.

5 Huppeitein, Zeauer, Harnanalyse, 10. Aufl., 387.

6 Zeitschr. i36hysiol. Chem., 46.

7 Amer. Journ. of Physiol. 13; v. Klercker, Hofmeister's Beitrage, 8.

CREATININE. 659

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, partly based upon autolytic
experiments (see Chapter XI). According to v. HOOGENHUYZE and
VERPLOEGH 2 a part of the creatine formed in the body is oxidized and a
part changed into creatinine.

The proteins, or rather the guanidine groups therein, are considered
as the piobable mother substance of these two bodies. JAFFE has
indeed shown, which has been substantiated later by DORNER,S that in
rabbits glycocyamine (guanidine acetic acid) is in part changed into
creatine with the annexation of methyl. Guanidine occurs in the pro-
teins as arginine, but the observations of OTORI 4 show that it is not
improbable that the proteins also contain other guanidine groups. JAFFE
considers it improbable that the arginine is the mother substance of the
creatine, but rather another guanidine group. Under the circumstances
the proteins can be considered as the mother substance of the creatine
and creatinine, and in a previous chapter (XI) the experiments of SEEMAN 5
showed the abundant formation of creatine from protein by autolysis.

If then the creatinine (creatine) originates from the protein, it is
evident that we must differentiate between food protein and body
protein. The quantity 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,6 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. On the contrary, its extent,
as shown by other conditions, is dependent upon the intensity of the
metabolism in the cells, and the creatinine, according to FOLIN, is a
product of the endogenous protein metabolism.

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 547, and v. Hoogenhuyze and Verploegh, Zeitschr. f. physiol.
€hem., 59.

3 Jaffe, Zeitschr. f. physiol. Chem., 48; Dorner, ibid., 52.

4 Zeitschr. f. physiol. Chem., 42, 43.
5 1. c., footnote 1, page 547.

6 Besides the works cited above see also Closson, Amer. Journ. of Physiol., 16.

660 URINE.

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 l also finds an absolute increase in the elimination of
creatinine only in starving dogs.

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 2). Little is known
about the behavior of creatinine in disease nor are the observations in
accord. In anemia and cachexia the elimination of creatinine is dimin-
ished, 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.3 In diseases of the liver
a diminished elimination of creatinine may occur and in cases of car-
cinoma of the liver considerable creatine has been found in the urine
(v. HOOGENHUYZE ana VERPLOEGH, MELLANBY).

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.4 It is more soluble
in warm alcohol and nearly insoluble in ether. In alkaline solution
cieatinine 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 5).
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(C4H7N3O.HCl.HgO)3HgCl2 separates on stand-
ing some time (JOHNSON). Among the compounds of creatinine, that

1 Arch. f. exp. Path. u. Pharm., 58. Further literature may be found in v. Hoog-
enhuyze and Verploegh, Zeitschr. f. physiol. Chem., 46.

2 v. Hoogenhuyze and Verploegh, Zeitschr. f. physiol. Chem., 57; Cathcart, Bioch.
Zeitsehr., 6; Benedict and Myers, Amer. Journ. of Physiol., 18; Jaffe, 1. c.

3 See O. v. Klercker, Zeitschr. f. klin. Med., 68.

4 See Huppert-Neubauer, 10. AufL, and Hoppe-Seyler-Thierf elder's Handbuch,
8. Aufl.

5 Kerner, Pfliiger's Arch., 2; Hofmeister, Zeitschr. f. physiol. Chem., 5.

CREATININE. 661

with zinc chloride, creatinine-zinc chloride, (C-jH^NaO^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-
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 solu-
tion 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'S 1 reaction) . An alkaline bismuth solution (see Sugar Tests)
is not reduced by creatinine.

An aqueous solution 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 water (or mine) 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's2 reac-
tion). Acetone gives a more reddish-yellow color. Dextrose gives
with this reagent a red coloration only after heating.

If we add a few drops of a freshly prepared very dilute sodium-nitro-
prusside 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,

1 Zeitschr. f. analyt. Chem., 17.

2 Zeitschr. f. physiol. Chem., 10.

3 Ber. d. deutsch. chem. Gesellsch., 11.

662 URINE.

a crystalline precipitate of a nitroso-compound (C4H6N402) of creatinine
separates on stirring (KRAMM *) . If, on the contrary, the yellow solu-
tion is treated with an excess of acetic acid and heated, the solution
becomes first green and then blue (SALKOWSKI 2) ; 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.3 This reaction is not due to creati-
nine. 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 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 absorption band as well as in that the
creatinine reaction requires more sodium nitroprusside.

In preparing creatinine from urine the creatinine-zinc chloride is first
prepared according to NEUBAUER'S 4 method. Creatinine may also be
prepared from urine by precipitating with a mere uric -chloride solution
according to either MALY'S or JOHNSON'S 5 process.

The best method for preparing creatinine is the following, suggested
by FoLiN.6 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
KHCO3 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.

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's7 modification of this method. As this method
is now seldom used we refer the reader to other hand-books.

FOLIN 8 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\or five

1 Centralbl. f. d. med. Wissensch., 1897.

2 Zeitschr. f. physiol. Chem., 4.

3 Arnold, Zeitschr. f. physiol. Chem., 49; Holobut, ibid., 56.

4 Ann. d. Chem. u. Pharm., 119.

5 Maly, Annal. d. Chem. u. Pharm., 159; Johnson, Proceed. Roy. Soc., 43.

6 Zeitschr. f. physiol. Chem., 41.
''Ibid., 10 and 14.

8 Ibid., 41.

URIC ACID. 663

minutes it is diluted with water to 500 cc. and mixed. This solution
is now compared in a DUBOSCQ colorimeter with a 1 /2 normal potassium-
dichromate 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

O 1

= ~X10, or 11.25 milligrams. This method has been tried by many

authorities and found to be trustworthy.

The same method is used in the determination of creatine, which
for this purpose is first converted into creatinine by warming 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 VERPLOEGH, GOTTLIEB and STANGASSINGER.

In regard to other methods, see the works of KOLISCH and GREGOR.1

Xanthocreatinine, C5H10N40. This bodv, 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.S

HN-CO

Uric Acid, Ur, Cgl^N^; 2, 6, 8-trioxypurine, OC C-NHV , has

I II >0

HN-C-NHX

been piepaied synthetically by HORBACZEWSKI by fusing urea and
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

1 Kolisch, Centralbl. f. innere Med., 1895; Gregor, Zeitschr. f. physiol. Chem., 31.

2 Gautier, Bull, de 1'acad. de med. (2), 15, and Bull, de la soc. chim. (2), 48: Monari,
Maly's Jahresber., 17; Colasanti, Arch. ital. d. Biologic, 15, Fasc. 3; Stadthagen,
Zeitschr. f. klin. Med., 15.

3 Achelis, Centralbl. f. Physiol., 20, 455, and Zeitschr. f. physiol. Chem., 50; Kut-
scher and Lohmann, ibid., 49; Engeland, ibid., 57.

664 URINE.

acid (E. FISCHER and TULLNER) and finally E. FISCHER and ACH l have
prepared uric acid fiom 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,
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, C5H4N403 +
O + H2O = C4H2N204+ (NH2)2CO. On warming with nitric acid, alloxan
yields carbon dioxide and oxalyl urea, or parabanic acid, C3H2N2O3.
By the addition of water the parabanic acid passes into oxaluric acid,
C3H4N2O4, 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,
C5H8N4O6, which may then be changed into oxonic acid, C4H.5N3O4.2
Uric acid may, as F. and L. SESTINI as well as GERARD have shown,
undergo bacterial fermentation with the formation of urea. According
to ULPIANI and CiNGOLANi,3 uiic acid is quantitatively split into urea
and carbon dioxide, according to the equation

C5H4N4O3 + 2H2O + 3O = 3C02 + 2CO (NH2)2.

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

1 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.

2 See Sundwik, Zeitschr. f . physiol. Chem., 20 and 41; also Behrend, Annal. d. Chem.
u. Pharm., 333.

3 See Chem. Centralbl., 1903, where the other investigators are cited, and Centralbl.
f. Physiol., 19.

ELIMINATION OF URIC ACID. 665

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 *).

The amount of uric acid eliminated with human urine is subject to
considerable individual variation, but amounts on an average to 0.7
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,2 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.3

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 purine-free
diet is considerably greater than in starvation. HIRSCHSTEIN explains this
by the formation of secretions containing purines due to the digestion, an
explanation which BRUGSCH and SCHITTENHELM 4 do not accept. 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 5 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 Philos. Trans. Roy. Soc., 186, B, 661.

2 See the extensive review of the literature in Wiener, " Die Harnsaure," in Ergeb-
nisse der Physiologic, 1, Abt. 1, 1902.

3 J. Ranke, Beobachtungen und Versuche iiber die Ausscheidung der Hafnsaure,
etc. (Munchen, 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.

4 Zeitschr. f. exp. Path. u. Therap., 4; Hirschstein, Arch. f. exp. Path. u. Pharm., 57 »

5 Siven, Skand. Arch. f. Physiol., 11; Leathes, Journ. of Physiol, 35.

666 URINE.

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
abundant destruction of nucleated cells as in pneumonia, after the
crisis, and in leucaemia. In leucaemia in most cases not only is the elimina-
tion to the urea increased absolutely, but a] so 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 with a purine-free diet (BRUGSCH and SCHITTENHELM) . Accord-
ing to the extensive investigations of BRUGSCH and SCHITTENHELM 5 the
endogenous uric acid elimination (see below) is not higher in chronic gout,
but rather lower than normally, and gout is more probably characterized
by a diminished enzymotic formation of uric acid as well as by a decreased
enzymotic destruction of uric acid. This last moment is the cause for
the appearance of uric acid in the blood and its accumulation in certain
tissues.

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 and purine bases (alloxuric bases 6) . The original view of
HORBACZEWSKI, that the nucleins do not directly cause an increased
elimination of uric acid, but indirectly by causing a leucocytosis with
a consequent destruction of leucocytes, has been generally discarded.

1 See Schondorff, Pfluger's Arch., 46, which contains the pertinent literature.

2 See Clar, Centralbl. f. d. med. Wissensch., 1888; Haig, Joura. 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.

5 Zeitschr. f . exp. Path. u. Therap., 4.

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
der Physiol., 1, Abt. 1, 1902.

FORMATION OF URIC ACID. 667

At present it is considered that a direct formation of uric acid from the
nucleins takes place by the transformation of the purine bases of the
nucleins into uric acid.

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 SCHUR l 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 SIVEN, RocKwooD,2 and many others have also
led to the same results. Other investigators have arrived at different
results, or they draw different deductions from their observations; still
this does not change the essential fact 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.

In man and other mammalia the greatest amount if not all of the uric
acid originates from the nucleins or the purine bases. This formation
of uric acid seems 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
PARTRIDGE,4 by more careful investigations have shown that enzymes
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

1 Pfliiger's Arch., 80, 87, and 94.

2 Siven, 1. c.; Rockwood, Amer. Journ. of Physiol., 12.

3 See footnote 6, page 666.

4 Schittenhelm, Zeitschr. f. physiol. Chem., 42, 43, 45, 46, 57, with Schmid, ibid.,
50 and Zeitschr. f. exp. Path. u. Therap., 4; Burian, Zeitschr. f. physiol. Chem., 43;
Jones and Partridge, ibid., 43; Jones with Winternitz, ibid., 44 and 60; Jones, ibid.,
45, with Austrian, ibid,., 48.

668 URINE.

his collaborators.1 The distribution is nob the same in all animals and
the reports regarding it are conflicting. SCHITTENHELM indeed claims
that it has not been proven that guanase and adenase are two different
enzymes. Still we have other grounds for the non-identity of the two
amidases.

In birds the conditions are different, v. MACH2 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 tormed
in birds by synthesis.

The formation of uric acid in birds is increased by the admin-
istration 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 correspond-
ing 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;
although, as SALASKIN and ZALESKI and LANG have shown, after the
extirpation of the liver, an 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
KOWALEWSKI and SALASKIN 3 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.4

The possibility of a formation of uric acid from lactic acid has been
shown in another manner by WiENER,5 namely, by feeding birds with
urea and lactic acid and different non-nitrogenous substances, oxy-,

1 See footnote 4> p. 667. See also Mendel and Mitchell, Amer. Journ. of Physiol., 20.

2 Arch. f. exp. Path. u. Pharm., 24.

3v. Schroder, Zeitschr. f. physiol. Chem., 2; Meyer and Jaffe", Ber. d. d. Chem.
Gesellsch., 10; Minkowski, Arch. f. exp. Path. u. Pharm., 21 and 31; Salaskin and
Zaleski, Zeitschr. f. physiol. Chem., 29; Lang, ibid., 32; Kowalewski and Salaskin,
ibid., 33.

4 Zeitschr. f . Biologic, 13.

5 Hofmeister's Beitrage, 2. See also Arch, f . exp. Path. u. Pharm., 42, and Ergeb-
nisse d. Physiol., 1, Abt. 1, 1902.

FORMATION OF URIC ACID. 669

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
attachment of a second urea residue.

Among the substances tested, only tartronic acid and its ureide, diamric 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).COOIJ, which by the attachment

xNH— COv
of a urea residue forms dial uric acid, COS >CHOH, and from this, by

\NH— CO/
the attachment of a second urea residue, uric acid is formed.

In opposition to the above-mentioned observations and opinions we must remark
that recently FRIEDMANN and H. MANDELA by transfusion experiments with
geese livers, have come to other conclusions. They found an increase in the uric
acid content in the transfused blood without adding any substance, but found no
increase in the quantity of uric acid when the transfused blood was treated with
urea alone or with urea and sodium lactate or malonate. Further research
must explain this contradiction.

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 2 could find no increase in the elimina-
tion 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 BURIAN 3 we have for the present no
proof of a synthetical formation of uric acid in the mammalian liver.
Dialuric acid and tartronic acid, according to him, do not cause any
marked uric-acid formation with extracts of the ox-liver in the absence
of purine bases; on the contrary they accelerate the enzymotic oxida-
tion of purine bases and hence, according to BURIAN, this explains, per-
haps, the increase in uric-acid elimination.

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 4

1 Arch. f. exp. Path. u. Pharm., 1908; Suppl. Schmiedeberg's Festschrift.

2 Hofmeister's Beitrage, 10.

3 Zeitschr. f. physiol. Chem., 43.
41. c.

670 URINE.

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 investigations. The liver, spleen, and muscles
are considered as the most important organs in the oxidative formation
of uric-acid from nucleins and purine bases, but it must not be for-
gotten that these organs in different animals behave dissimilarly 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
by several experimenters,1 in great part destroyed and more or less com-
pletely changed into urea. In rabbits, according to WIENER, the uric
acid is destroyed with the formation of glycocoll as an intermediate step.
HIRSCHSTEIN has recently accepted this view, but WIECHOWSKI, SAMUELY,
BRUGSCH and SCHITTENHELM 2 dispute it. Still we must consider the
earlier opinion of WOHLER and FRERICHS as probably correct, that allan-
toiri is the chief intermediary product. This is based upon the work of
SALKOWSKI, MENDEL and BROWN, MENDEL and WHITE, and WIECHOW-
SKI, but it is denied by WIENER, POHL and PODUSCHKA. The inter-
mediary transformation of uric acid into allantoin occurs, in WIECHOW-
SKi's3 opinion, only in the mammals investigated, but not in man.

The demolition of uric acid seems to be possible, according to the
numerous researches of CHASSEVANT and RICHET, ASCOLI, JACOBY,
WIENER, SCHITTENHELM, BURIAN, ALMAGIA, PFEIFFER, and WIECHOWSKI,
in several organs, such as the liver, kidneys, muscle, and bone-marrow,
although this behavior differs in various animals. This destruction of
uric acid, which is called uricolysis, is an enzymotic process. WIECHOW-
SKI and WIENER 4 have made careful investigations on the active

1 Wohler and Frerichs, Annal. d. Chem. u. Pharm., 65. See also Wiener, Ergeb-
nisse der Physiologic, 1, Abt. 1.

2 Hirschstein, Zeitschr. f. exp. Path. u. Ther., 4, and Arch. f. exp. Path. u. Pharm.,
59; Wiechowski, Hofmeister's Beitrage, 9; Samuely, Zeitschr. f. exp. Path. u. Ther.,
4; Brugsch and Schittenhelm, ibid., 4.

3 Wiener, Arch, f . exp. Path. u. Pharm., 40 and 42, and Ergebnisse der Physiologic,
1, Abt. 1; Pohl, Arch. f. exp. Path. u. Pharm., 48; Poduschka, ibid., 44; Salkowski,
Zeitschr. f. physiol. Chem., 35, and Ber. d. d. Chem. Gesellsch-, 9; Mendel and Brown,
Amer. Journ. of Physiol., 3; Mendel and White, ibid., 12; Wiechowski, Arch. f. exp.
Path. u. Pharm., 60, with Wiener, Hofmeister's Beitrage, 9. Conflicting reports in
Croftan, Pfluger's Arch., 121.

4 Chassevant and Richet, Comp. rend. soc. biolog., 49; Ascoli, Pfluger'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 and 45; Burian, ibid.,
43; Almagia, Hofmeister's Beitrage, 7; Pfeiffer, ibid., 7; Wiechowski and Wiener >
ibid., 9.

DESTRUCTION OF URIC ACID. 671

enzyme of the ox-kidney and dog-liver and find that it is an oxidase
only active in faintly alkaline or neutral reaction. WIECHOWSKI has
also found that this enzyme transforms the uric acid into allantoin
almost entirely. ASCOLI and IZAR 1 have also made the remarkable
observation that if an extract of liver which has completely destroyed
a known quantity of uric acid (air or oxygen being passed through) is
allowed to stand for some time in the thermostat, with the exclusion
of air, the destroyed uric acid is gradually reformed again. It has not
been determined from what transformation products this is produced.
Stil] it is very suggestive in connection with WIECHOWSKI'S reports on
the formation of allantoin that the allantoin is inactive in this regenera-
tion of the uric acid. Further enlightenment on this point would be
of the greatest interest.

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. Bum AN and ScnuR2 have indeed
suggested a tactor, 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. Accord-
ing to them, carnivora eliminate unchanged about -gV - 3^ of the uric
acid introduced into the circulation, rabbits about £, and man \. Such
calculations are necessarily very uncertain, and for man, in whom accord-
ing to WIECHOWSKI practically no intermediary destruction of uric acid
occurs, they 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

1 Zeitschr. f. physiol. Chem., 58. 2 Pfluger's Arch., 87.

672 URINE .

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
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, but SMALE claims that the monophosphate has only a slight
solvent action. RUDEL 1 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 car-
bonates, but also by several organic bases, such as ethylamine and propyl-
amine, piperidine and piperazine. Uric acid dissolves, without decom-
posing, in concentrated sulphuric acid. It is completely precipitated
from the urine by picric acid (JAFFE2). Uric acid gives a chocolate-
brown precipitate with phosphotungstic acid in the presence of hydro-
chloric acid.3

Uric acid is dibasic and consequently forms two series of salts,
neutral 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
lateritium) 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 grams sodium, and 0.4141 grams ammonium urate,
and at 37° C. 2.7002, 1.5043. and 0.7413 grams of the respective urates.
The salts of the alkaline earths are soluble with great difficulty. The
above solubilities apply only, in GUDZENT'S 4 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-foim).

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.

2 Zeitschr. f. pyhsiol. Chem., 10.

3 In regard to the combinations of formaldehyde and uric acid, see Nicolaier, Deutsch.
Arch. f. klin. Med., 89 (1906).

4 Zeitschr. f. physiol. Chem., 56 and 60.

PROPERTIES OF URIC ACID. 673

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.

If uric acid is converted into alloxan by the careful action of nitric
acid and the excess of acid carefully expelled, on treating this with a few
drops of concentrated sulphuric acid and commercial benzene (contain-
ing thiophene) a beautiful blue coloration is obtained (DENIGES' reaction1).

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 dextrose 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 reaction2).

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 SEOS we are here dealing with
combinations of 1 molecule nucleic acid and 2 molecules uric acid, which.
protects the uric acid within the body against destruction or transforma-
tion into allantoin.

1 Journ. de Pharm. et de Chim., 18. Cited from Mary's Jahresber., 18.

2 Cited f. Bioch. Cenetralbl., 8, 250.

3 Goto, Zeitschr f. physiol. Chem., 30; Seo, Arch. f. exp. Path. u. Pharm., 58.

674 URINE.

Preparation of Uric Acid 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 Unne. As the older
method suggested by HEINTZ, even after recent modifications, gives
inaccurate results, it will not be considered here.

SALKOWSKI and LUDWIG'S l 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 AMMONIAC AL SILVER-NITRATE SOLUTION, which contains in 1 liter 26
grams of silver nitrate and a quantity of ammonia sufficient to redissolve com-
pletely the preciptate 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 is col-
lected on a filter, washed with ammoniacal water, and then returned to
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

1 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. 675

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.

HOPKINS' method 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.1 The last
named converts the uric acid into urea by oxidation with potassium
permanganate in sulphuric-acid solution and then determines the quan-
tity of this by sodium hypobromite. 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
filter b}^ 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.

1 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.

676 URINE.

Purine Bases (ALLOXURIC BASES) . The purine bases found in human
urine are xanthine, guanine, hypoxanthine, adenine, paraxanthine, heteroxan-
thine, episarkine, epiguanine, l-methylxanthine, and carnine. The occurrence
of guanine and carnine (POUCHET) is, according to KRUGER and SALOMON/
not positively shown. The quantity of these bodies in the urine is
extremely small and varies in different individuals. FLATOW and REIT-
ZENSTEiN2 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 the investigations
of ALBANESE, BONDZYNSKI and GOTTLIEB, E. FISCHER, M. KRUGER
and G. SALOMON, and SCHMIDT and KOTAKE 3 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,4 and the same factors apply as for the uric acid,
viz., the endogenous purine formation represents a value which is some-
what variable for different individuals and relatively constant for the
same individual. According to SiVEN,5 with purine-fiee diet the elimina-
tion of purines is lowest at night and highest in the morning hours. Rest
and work do not show any positive difference. As the four true nuclein
bases and carnine have been treated in Chapters III and XI, it only
remains to describe the special urinary purine bodies.

HN— CO
Heteroxanthine, C9H9N402, 7-monomethylxanthine, OC C.N.CH3, was first

1 Zeitschr. f. physiol. Chem., 24; Pouchet, " Contributions & la connaissarice des
matieres extractives de 1'urine." These Paris, 1880. Cited from Huppert-Neubauer,
333 and 335.

2 Deutsch. med. Wochenschr., 1897.

3Albanese, 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; K.
Fischer, ibid., 30, 2405; Kruger 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.

4 See Burian and Schur, footnote 1, page 667, and Kaufmann and Mohr, Deutsch.
Arch. f. klin. Med., 74.

5 Skand. Arch. f. Physiol., 18.

PURINE BASES. 677

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 NEUBERG * 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 WEIDEL'S reaction, according to FISCHER (see Chapter III).

CH3.N— CO

I I
i-Methylxanthine, C6H6N4O2, OC C.NH , was first isolated from the

urine and studied by KRUGER, and then by KRUGER and SALOMON.2 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.

CH3.N— CO

oi

Paraxanthine, C7H8N402, . 1.7-dimethylxanthine, OC C.NCH3, urotheo-

I II ^CH

HN— C.N-/

bromine (THUDICHUM), was first isolated from the urine by THUDICHUM and
SALOMON.3 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 4 in pigs' and dogs' urine,

1 Salkowski's Festschrift, Berlin, 1904.

2 Kruger, Arch. f. (Anat. u.) Physiol., 1894; Kruger and Salomon, Zeitschr. f.
physiol. Chem., 24.

3Thudichum, " Grundzuge d. anal. med. klin. Chemie " (Berlin, 1886); Salomon,
Arch. f. (Anat. u.) Physiol., 1882, and Ber. d. deutsch. chem. Gesellsch., 1(> and 18.

4 Balke, " Zur Kenntnios dcr Xanthinkorper " (Ina,ug.-Diss., Leipzig, 1893); Salo-
mon, Zeitschr. f. physiol. Chem., 18.

678 URINE.

as well as in urine in leucaemia. BALKE gives C4H6N30 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 compound. The
silver compound is difficultly soluble in nitric acid. Episarkine is possibly
identical with epiguanine.

HN— CO

Epiguanine, C6H7N5O, 7-methylguanine, H2N.C C.N.CH3, was first pre-

L,

pared from the urine by KRUGER.1 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. 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.2

Quantitative Estimation of Purine Bases according to SALKOWSKI.S
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 675. 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-

1 Arch. f. (Anat. u.) Physiol., 1894; Kriiger and Salomon, Zeitschr. f. physioL
Chem., 24 and 26.

2 Zeitschr. f. physiol. Chem., 26, and also Hoppe-Seyler-Thierfelder's Handbuch,
8. Aim1., 188.-

3 Pfliiger's Arch., 69.

ESTIMATION OF PURINE BASES. 679

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 of 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.1

MALFATTI 2 determines the nitrogen of the purine bases in the hydrochloric-
acid filtrate from the separated uric acid. This nitrate 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, ARNSTEIN 3). SALKOWSKI has raised the objection to
this procedure that it is not possible to remove all the ammonia from the silver
precipitate by washing. According to ARNSTEIN 4 this can readily be done by
boiling the precipitate in water with some magnesia, and under these circum-
stances this method is quite serviceable. The nitrogen is estimated by KJEL-
DAHL'S method. The uric-acid nitrogen multiplied by 3 gives the quantity of
uric acid. As the mixture of purine bases in the urine is but little known, the
quantity of nitrogen of the purine bases is always calculated as a certain purine
base, for example xanthine (CAMERER), and the quantity so found used as a
measure for the purine bases.

According to a new method of KRUGER and SCHMID 5 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.

We cannot discuss the other methods, such as those of DENIGES and NIEMI-
LOWICZ, and the method suggested by HALL 6 for clinical purposes.

Oxaluric Acid, C3H4N2O4= (CON,H3).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 oxaluric retained by the charcoal may be obtained by
boiling with alcohol.

COOH

Oxalic Acid, C2H2O4, or . , occurs under physiological conditions

COOH

in very small amounts in the urine, about 0.02 gram in twenty-four hours
(FfJRBRiNGER7). According to the generally accepted view it exists in

1 In regard to the details we refer the reader to the original paper.

2 Centralbl. f. innere Med., 1897.

3 Camerer, Zeitschr. f. Biologic, 26 and 28; Arnstein, Zeitschr. f. physiol. Chem., 23.

4 Salkowski, 1. c.; Arnstein, Centralbl. f. d. med. Wissensch., 1898.

5 Zeitschr. f. physiol. Chem., 45 and Hoppe-Seyler-Thierf elder's Handbuch, 8.
Aufl., 590.

8 Niemilowicz, Zeitschr. f. physiol. Chem., 35; Gittelmacher-Wilenko, ibid., 36;
Hall, Wien. klin. Wochenschr., 16.

7 Deutsch. Arch. f. klin. Med., 18. See also Dunlop, Journ. Path, and Bacteriol., 3.

68 J URINE.

the urine as calcium oxalate, which is kept in solution by the acid phos-
phates present. Calcium oxalate is a frequent constituent of urinary
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;1 and as many vegetables and fruits, such as cabbage, spinach,
asparagus, sorrel, apples, grapes, etc., contain oxalic acid, it is possible
that a part oi 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,2 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, according
to SALKOWSKI,S 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 (SALKOWSKI). Gelatin and
gelatin-yielding tissues seem to increase the excretion of oxalic acid,
which stands in accord with the observations oi KUTSCHER and ScHENCK4
that on the oxidation of gelatin oxamic acid is produced from the glyco-
coll and this then readily decomposes into ammonia and oxalic aciJ.
After feeding nucleins no constant increase in the elimination of oxalic
acid has been observed.5 The production of oxalic acid due to an incom-
plete combustion of the carbohydrates has also been suggested. The
work of HILDEBRANDT and P. MAYER seems to indicate this under abnor-
mal conditions. In alimentary glycosuria or diabetes LUZZATO 6 could
not observe any rise in the elimination of oxalic acid. According to
CAKIN/ in rabbits an increased elimination of oxalic acid occurs after
the introduction of glycollic or glyoxylic acids, and the oxalic acid seems
to be an intermediary product of metabolism, which is further burnt.
We cannot exclude the possibility of the formation of oxalic acid in

1 In regard to the behavior of oxalic acid in the animal body, see page 733.

2 Mills, Virchow's Arch., 99; Liithje, Zeitschr. f. klin. Med., 35.

3Reale and Boeri, Wien. med. Wochenschr., 1895; Terray, Pfliiger's Arc'i., 65;
Salkowski, Berl. klin. Wochenschr., 1900.

4 Zeitschr. f. physiol. Chem., 43.

5 See Stradomsky, Virchow's Arch., 163; Mohr and Solomon, Deutsch. Arch. f.
klin. Med., 70; Salkowski, 1. c.

6 Hildebrandt, Zeitschr. f. physiol. Chem., 35; P. Mayer, Zeitschr. f. klin. Med., 47;
Luzzato, Salkowski's Festschrift, 1904.

7 Journ. of Biol. Chem., 3, 57.

ALLAXTOIN. G81

the oxidation of uric acid in the animal body, yet we have no positive proof
of such a formation.1

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. The method suggested by AUTEN-
RIETH and EARTH is as follows:

The twenty-four-hour urine is precipitated by CaCl2 and ammonia
in excess. After 18-20 hours the precipitate is collected (the filtrate
must be clear) and dissolved in a little hydrochloric acid and shaken
out 4-5 times with 150-200 cc. ether (containing 3 per cent absolute
alcohol) . The united ethereal extracts are filtered through a dry filter
and distilled after the addition of about 5 cc. of water. The liquid, if
necessary, is decolorized with animal charcoal and precipitated with
CaCl2 and ammonia, made acid after a certain time with acetic acid,
and finally the oxalate is collected, washed, burned to CaO, and weighed.
MACLEAN 2 finds that this method yields too low results, as some calcium
oxalate always remains in the filtrate and the original method as suggested
by SALKOWSKI is more trustworthy.

/NH.CH.HN.CO.NH2,
Allantoin (GLYOXYLDIUREIDE), C4H6N4O3, OC<(

XNH.CO

occurs 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 mine
of adults, if it contains any allantoin at all, has only traces, and he could
not detect any in the urine of nurslings or in the ammotic 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 animals. It is also found, as first shown by VAUQUELIN
and L-ASSAiGNE,3 in the allantoic fluid of the cow (hence the name) . That
allantoin is formed from the uric acid in mammalia but not in human
beings (WIECHOWSKI) is almost certain, and the investigations on which
this is based have already been given in discussing the decomposition
of uric acid. The allantoin thus originates from the purine bodies, and

1 See Wiener, Ergebnisse der Physiol., 1, Abt. 1.

2 Salkowski, Zeitschr. f. physiol. Chem., 29; Autenrieth and Earth, ibid., 35;
Mac Lean, ibid., 60.

3 Ziegler and Hermann, see Gusserow, Arch, f . Gynakol, 3 — both cited from Huppert-
Neubauer, Harn- Analyse, 10. Aim., 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.

682 URINE.

consequently the excretion of allantoin is considerably increased,
according to MINKOWSKI, COHN, SALKOWSKI, and MENDEL and BROWN,*
after feeding thymus or pancreas. A strong allantoin excretion is also
found in dogs after poisoning with hydrazine (Bomssow), hydroxylamine,
semicarbazide, and aminoguanidine (POHL), and this increase in the
excretion of allantoin is connected with the nuclein metabolism. POHL 2
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 enzymoytic uric acid formation with a
subsequent uricolysis with the formation of allantoin. According to
PODUSCHKA and MiNKOwsKi,3 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 burnt.

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 allan-
toin solution gives no precipitate with silver nitrate alone, but by the
careful addition of ammonia a white flocculent precipitate is formed,
C4H5AgN4O3, 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 FEELING'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. In preparing allantoin from urine, proceed according
to LOEWI'S method, which consists of the following: The faintly acidified
urine is precipitated with a mercurous-nitrate solution, the filtrate treated
with H^S, and the new filtrate precipitated by magnesium oxide and
silver nitrate after the removal of the H2S. The precipitate is filtered
off and washed with warm water and decomposed with H2S, and the

1 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.

2 Borissow, Zeitschr. f. physiol. Chem., 19; Pohl, Arch. f. exp. Path. u. Pharm.,
46; Poduschka, ibid., 44. According to Underfill! 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.

3 Poduschka, Arch. f. exp. Path. u. Pharm., 44; Minkowski, ibid., 41.

HIPPURIC ACID. 683

filtrate evaporated to dryness. The residue is extracted with hot water
and then the solution is precipitated with mercuric nitrate. The pre-
cipitate is collected and decomposed by H2S. From the evaporated
filtrate the allantoin crystallizes out. This method, DAKIN claims, is
not suited for the quantitative estimation of allantoin. For this pur-
pose we make use of the method suggested by WiEcnowsKi,1 which
consists in precipitating the allantoin by a dilute mercuric acetate solu-
tion in the presence of concentrated sodium acetate solution. For
details we refer to the original publications.

Glyoxylic Acid, C2H404, • , is produced on boiling allantoin as well as

COOH

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,2 as it is readily destroyed in the body, and
its passage into the urine is very improbable, or at least only occurs seldom.

OC.C3H5
Hippuric Acid (BENZO YL-AMINO ACETIC ACID) , CgHgNOs, •

'HN.CH2.COOH.

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 =
C^H5.CO.NH.CH2.COOH + H2O. This acid may be synthetically pre-
pared from benzamide and monochloracetic acid, C6H5.CO.NH2 + CH2C1.
COOH = C6H5.CO.XH.CH2.COOH + HC1, 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 hippuric
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

1 Loewi, ibid., 44; Wiechowski, Hofmeister's Beitrage, 11, and Arch. f. exp. Path.
u. Pharm., 60; Dakin, Journ. of biol. Chem., 3, 73.

2 The literature on the occurrence and detection of glyoxylic acid in the urine can
be found in Granstrom, Hofmeister's Beitrage, 11.

684 URINE.

acid, hydrocinnamic acid) or by reduction (quinic acid) are converted
into benzoic acid. The question of the origin of hippuric acid is there-
tore 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.

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 SHEPHERD, SALKOWSKI, and others l). 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.CH2.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 aminophenylpropionic acid (phenylalanine), which is derived
from several protein substances. The supposition that the phenyl-
propionic 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 disin-
fecting the intestine of dogs with calomel the hippuric acid disappears,
from the urine (BAUMANN 3) .

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,
originates 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

1 Salkowski, Ber. d. deutsch. chem. Gesellsch., 11; Meissner and Shepard, Unter-
such. iiber das Entstehen der Hippursaure im thierischen Organismus. Hanover, 1866.

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.

FORMATION OF HIPPURIC ACID. 685

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 (HuPFER1).

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 benzole 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 2 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, accord-
ing to BRUGSCH and R. HIRSCH, FEIGEN and BRUGSCH, as here there is
no more glycocoll available for hippuric acid formation than is split off
from the proteins on hydrolysis. LEWINSKI 3 believes that, nevertheless,
in man after abundance of benzoic acid about 34 per cent of the total
nitrogen may be excreted as hippuric acid, but BRUGSCH claims that
these observations are incorrect. The abundant production of hippuric
acid in herbivora induced ABDERHALDEN, 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 glycocoll from complexes rich in
carbon, and it is quite possible that the benzoic acid combines with higher
amino-acids and that the hippuric acid is then formed from this combina-
tion by oxidation. The investigations of MAGNUS-LEVY 4 to prove this

1 Weiss, Zeitschr. f. physiol. Chem., 25, 27, 38; Lewin, Zeitschr. f. klin. Med., 42;
Hupfer, Zeitschr. f. physiol. Chem., 37. See also Wiener, " Die Harnsaure," Ergeb-
nisse der Physiol., 1, Abt. 1.

2 Wiechowski, Hofmeister's Beitrage, 7 (literature); A. Magnus-Levy, Munch, med.
Wochenschr., 1905.

3 Brugsch and Hirsch, Zeitschr. f. exp. Path. u. Therap., 3; Brugsch, Maly's
Jahresber., 37, 621, and Bioch. Centralbl., 8, 336; Feigen, Maly's Jahresber., 36, 631;
Lewinski, Arch. f. exp. Path. u. Pharm., 58.

4 Abderhalden, Gigon and Strauss, Zeitschr. f. physiol. Chem., 51; Magnus-Levy,
Bioch, Zeitschr., 6.

686 URINE.

assumption, where he used benzoylated higher amino-acids, have not
given support to this assumption, and the question how the abundant
formation and elimination of glycocoll take place is still unexplained.

The kidneys may be considered in dogs as special organs for the syn-
thesis of hippuric acid (SCHMIEDEBERG and BUNGE l). 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 decom-
poses, producing a red mass and a sublimate of benzoic acid, with the
generation, first, of a peculiar pleasant odor of hay and then an odor
of hydrocyanic 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
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 2) :

1 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.
* 2 Arch. f. exp. Path. u. Pharm., 6. In regard to other methods, such as Blumen-

ETHEREAL SULPHURIC ACIDS. 687

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 separately funnel, then evaporated at a medium temperature
and the dry residue treated repeatedly 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. The mother-liquor is repeatedly
shaken with acetic ether. This last is removed and evaporated; the
residue is added to the above crystals on the filter, dried and weighed.

Phenaceturic Acid, C10HnN03 = C6H5.CH2.CO.NH.CH2.COOII. This acid,
which is produced in the animal body by a combination of glycocoll with the phenyl-
acetic acid, CeH5.CH2.COOH, formed in the putrefaction of the proteins, has
been prepared from horse's urine by SALKOWSKi,1 but it probably also occurs in
human urine. According to VASILIU 2 it is just as important a constituent of the
urine of herbivora as hippuric acid is.

Benzoic Acid, C7H,,O2 or C6H5.COOH, is found in rabbit's urine and sometimes,
though in small amounts, in dog's urine (\VEYL 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 decomposition
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, accord-
ing to SCHMIEDEBERG and MiNKOWSKi,3 contain a special enzyme, SCHMIEDE-
BERG'S histozym, which splits the hippuric acid with the seoaration of benzoic
acid.

Ethereal Sulphuric Acids. In the putrefaction of proteins in the
intestine, phenols, — whose mother-substance is considered to be tyrosine,
— indol and skatol are produced. These phenols directly, and the
two last-named bodies after they have been oxidized respectively into
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

thai as well as Pfeiffer, Bloch and Riecke, see Maly's Jahresber., 30 and 32. See also
Wiechowski, 1. c.

1 Zeitschr. f. physiol. Chem., 9.

2 Mitteil. d. landw. Inst., Breslau, 4.

3 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.

688 URINE.

not been isolated. The ethereal sulphuric acids of the urine were dis-
covered and specially studied by BAUMANN.J 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. 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 putrefac-
tion 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; stiU 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 betwreen 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
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 acid 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, Cells. O.SO2. OH and

XO.SO2.OH
C^H^ . These acids are found as alkali salts in human urine,

XCH3
in which also orthocresol has been detected. The quantity of cresol-

1 Pfl tiger's Arch., 12 and 13.

2 v. d. Velden, Virchow's Arch., 70; 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 P-CRESOL SULPHURIC ACID. 689

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 ac'ds
of the urine amounts to 17-51 milligrams in the twenty-four hours
(MUNK). The methods for the quantitative estimation used heretofore
give, according to RUMPF, as well as KOSSLER and PENNY/ such inac-
curate results that new determinations are very desirable. After a
vegetable diet the quantity of these ethereal -sulphuric acids is greater
than after a mixed diet. 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 pheno], it forms phenyl-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.

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.4

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 these ethereal-
sulphuric acids was usually made by weighing the phenol which was
separated from the urine as tribromphenol. At the present time the
following method is employed:

1 Munk, Pfliiger's Arch., 12; Rumpf, Zeitschr. f. physiol. Chem., 16; Kossler and
Penny, ibid., 17.

2 See Baumann, Pfluger's Arch., 12 and 13, and Baumann and Preusse, Zeitschr.
f. physiol. Chem., 3, 156.

3 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 14.

4 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.

690 URINE.

KOSSLER and PENNY'S Method with NEUBERGV 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:

C6H5OH + 3NaIO =C6H2I3.OH + 3NaOH.

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). For
details, see NEUBERG, 1. c., and HOPPE-SEYLER-THIERFELDER'S Hand-
buch, 8. Aufl.

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 protocateehuic 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 2).

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 3 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 ultimately black in the presence of alkali and
the oxgyen 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 acteate. 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

1 Kossler and Penny, Zeitschr. f. physiol. Chem., 17; Neuberg, ibid., 27.

2 Baumann and Herter, Zeitschr. f. physiol. Chem., 1; Preusse, ibid., 2; Baumann,
ibid., 3.

3Ebstein and Muller, Virchow's Arch., 62; Bodeker, Zeitschr. f. rat. Med. (3), 7.

INDOXYL-SULPHURIC ACID. 691

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 pyroeatechin 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,1 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 pyroeatechin, it easily reduces
metallic oxides. It acts like pyroeatechin with alkalies, but is not precipitated
by lead acetate. It is oxidized into quinone by ferric chloride and other oxidiz-
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-
sulphuric acid.

C.O.SO2.OH
Indoxyl-sulphuric Acid, C8H7NS04, C6H4/\CH , also called

NH

URINE INDICAN, formerly called UROXANTHINE (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,2 for man is 5-20 milligrams per
twenty-four hours. 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 492),
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-
SEYLER 3) . 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

1 Virchow's Arch., 92.

2 Pfliiger's Arch., 3.

3 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 Physiol., 7.

692 URINE.

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.1 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 indicanr
while an obstruction in the large intestine showed no such action.

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 starvation
and also after phlorhizin poisoning; but these statements are vehemently
opposed by other investigators, such as P. MAYER, SCHOLZ, and ELLINGER,
and are improbable. The indol, it seems, is not formed from the trypto-
phane (indolaminopropionic acid) as intermediary step in the demoli-
tion of the proteins in the animal body, but rather from the putrefac-
tion of the tryptophane in the intestine. GENTZEN,2 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 bacterial
decomposition in the large intestine. The reports as to the elimination
of indican after oxalic-acid poisoning are conflicting. After poisoning
with oxalic acid HARNACK and v. LEYEN found an increased indican elimi-
nation, and MORACZEWSKI believes he has proven a certain parallelism
between the quantity of indican and the quantity of oxalic acid in
diabetes. ScnoLZ,3 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. Inclol-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

1 Jaffe1, Virchow's Arch., 70; Ellinger and Prutz, Zeitschr. f. physiol. Chem., 38.

2 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., 39; Gentzen, " Ueber die Vor-
stufen des Indols beider Eiweissfaulnis im Thierkorper," Inaug.-Dissert. Konigsberg,
1904.

3 Harnack, Zeitschr. f. physiol. chemie, 29; Scholz, 1. c., Moraczewski, Centralbl.
f. innere Med., 1903.

INDOXYL-SULPHURIC ACID. 693

analogous blue or green pigments are produced only from those derivatives

CH CH

of indol which, like n-methyl indol C6H4<^\CH, a-naphtmdol,C10H8<^\CH or

N.CH, NH

N.CH,
CH2

n-methyl indolin, C6H4<T >CH2, do not have the hydrogen atoms of the two

N.CH3

methine groups substituted by alkyl. From those derivatives in which one or two
hydrogen atoms are substituted by alkyl, such as a-methyl indol, dimethyl indol,
C.CH3 CH

C8H4<^Sc.CH3, and bz. 3, pr. 2-dimethyl indol, CH3. CeH§/\C.CH3, red pigments

NH NH

are produced, a behavior which PORCHER and HERVIEUX * 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 a 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 split 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

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 Jaffe, 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.

694 URINE.

latter suggested by PORCHER and HERVIEUX1). 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
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 HERVIEUX2).

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 3), 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 4 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-

1 Jaffa", Pfliiger's Arch., 3; Obermayer, Wien. klin. Wochenschr., 1890; Salkowski,
Zeitschr. f. physiol. Chem., 57; Porcher and Hervieux, Zeitschr. f. physiol. Chem., 39.

2 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. Centralb., 8, 54.

3 Krauss, Zeitschr. f. physiol. Chem., 18; Adrian, ibid., 19; Wang, ibid., 25; Sal-
kowski, ibid., 42.

4 Bouma, Zeitschr. f. physiol. Chem., 32; Oerum, ibid., 45.

SKATOXYL-SULPHURIC ACID. 695

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 l
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 2 have recently observed such cases.

C.CHg

Skatoxyl-sulphuric Acid, C9H9NSO4, C6H4^Sc.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-
BERG3), 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 (MES-
TER4). 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. 5

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

1 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.

2 Grober, Munch, med. Wochenschr., J904; Wang, Salkowski's Festschrift, 1904.

3 Otto, Pfluger's Arch., 33; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29.

4 Brieger, Ber. d. deutsch. chem. Gesellsch., 12, and Zeitschr. f. physiol. Chem.,
4, 414; Mester, ibid., 12.

5 Journ. of biol. Chem., 4.

696 URINE.

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 JAFPE'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 1 is of the opinion that no
skatol chromogen exists in human urine, and that the observations made
heretofore were due to a confusion of skatol red with indigo red or urorosein.
It cannot be disputed that derivatives of skatol sometimes occur in
human urine, and to prevent confusion with indigo red it must be borne
in mind that indigo red is soluble in chloroform as well as in ether, while
skatol red is inso]uble 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 A = 577-550 (PORCHER
and HERViEUX2).

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. Accord-
ing to HERTER 3 urorosein is not identical with skatol red, although the
investigations of STAAL, GROSSER, PORCHER and HERVIEUX 4 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), C10H9N02,

NH

This acid, whose occurrence in the urine was first shown by SALKOWSKT, 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 5 it occurs (more
correctly as urorosein) as traces in normal urine, abundantly in horse urine,
and in especially large quantities in cow urine. When introduced into the animal
body it appears unchanged in the urine.

1 Rosin, Virchow's Arch., 123.

2 Zeitschr. f. physiol. Chem., 45.

3 Journ. of biol. Chem., 4.

4Staal, Zeitschr. f. physiol. Chem., 46; Grosser, ibid., 44; Porcher and Hervieux,
ibid., 45; Compt. rend., 138, and Journ. de Physiol., 7.

5 Salkowski, Zeitschr. f. physiol. Chem., 9; Wechselmann, cited in Bioch. Centralbl.,
5, 784.

AROMATIC OXYACIDS. 697

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, ^,nd 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 NENCKI l to a red pigment which occurs in
the urine under the conditions mentioned under indol acetic acid. This pig-
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. According to HERTER this does not occur in perfectly fresh urine,
but only after a formation of nitrite by bacterial action which acts in the reaction
as an oxidizing agent. Urorosein differs from indirubin essentially by the same
properties as skatol, with which, according to some, it is identical (see above).

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, oxymandelic acid, found by SCHULTZEN and
RIESS in urine in acute atrophy of the liver, oxyhydroparacoumaric acid,
found by BLENDERMANN in the urine on feeding rabbits with tyrosine,
gallic acid, which, according to BAUMANN,2 sometimes appears in horse's
urine, and kynurenic 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.

/Oil

Paraoxyphenylacetic Acid, C8H8O3, C6H4\ , and p-Oxyphenylpropionic

\CH2.COOH

/OH
Acid (Hydroparacoumaric Acid), C9H1003, C8TI4\ , are crystalline

\CH2.CH,COOH

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 MIL-
LON'S reagent.

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

1 Nencki and Sieber, Journ. f. prakt. Chem., (N. F.), 26.

2 Schultzen and Riess, Chem. Centralbl., 1869; Blendermann, Zeitschr. f. physioK
Chem., 6, 267; Baumann, ibid., 6, 193.

698 URINE.

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), C8H4O4 =
/OH(1)

;— OH(4) . This acid, which was discovered by MARSHALL1 and

called 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 alcap-
tonuric urine in this case were due to this acid. This acid has later
been found in many cases of alcaptonuria by EMBDEN, GARNIER and VOIRIN,
OGDEN, GARROD, and many others. Glycosuric acid, 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
ingestion of tyrosine by persons with alcaptonuria, WOLKOW and BAU-
MANN and EMBDEN observed a greater quantity of homogentisic acid
in the urine. Since LANGSTEIN and E. MEYER showed in a case of alcap-
tonuria that the quantity of tyrosine in the protein, even when calculated
to a maximum, was not sufficient to account for the quantity of homo-
gentisic 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 homogentisic 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 phenylalanine in the form of polypeptides, dipeptides as
well as tripeptides. The p-tyrosine and phenylalanine are quantitatively
converted into homogentisic acid, in alcaptonuria (FALTA). The m-

1 The Medical News, Philadelphia, January 8, 1887.

2 Wolkow and Baumann, Zeitschr. f. physiol. Chem., 15; Embden, ibid., 17 and
18; Gamier and Voirin, Arch, de Physiol. (5), 4; Ogden, Zeitschr. f. physiol. Chem.,
20; Geyger, cited from Embden, 1. c., 18. The literature can be found in Fromherz,
Ueber Alkaptonurie, Inaug.-Dist. Freiburg, 1908.

3 Langstein and Meyer, Deutsch. Arch. f. klin. Med., 78; Falta and Langstein, Zeit-
schr. f. physiol. Chem., 37; Falta, Der Eiweiss-Stoffwechsel bei der Alkaptonurie,
Habilitationsschrift, Naumburg a. S., 1904.

4 Zeitschr. f. physiol. Chem., 52.

HOMOGENTISIC ACID.

and o-tyrosine, on the contrary, are not converted, according to BLUM,1
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 BAUMANN 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 ABDERHALDEN, BLOCK and RoNA2 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 NH2, OH
or H in the a-position to the carboxyl group and not in the /?-posi-
tion, p-tyrosine, phenylalanine, phenyl-a-lactic acid and phenyl-pyro-
racemic acid are such acids. It can be admitted with FALTA that
the phenylalanine 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)2C6H3.CH2.CHOH.COOH, is formed, and then from this by oxida-
tion dioxyphenylacetic acid (homogentisic acid), (OH)2C6H3.CH2.COOH,
is produced. Tyrosine is also supposed to undergo an analogous
transformation whereby a removal of OH groups in the para position
must be admitted.

According to NEUBAUER,4 on the contrary, the tyrosine, as well as

1 Arch. f. exp. Path. u. Pharm., 59.

2 Zeitschr. f. physiol. Chem., 52.

3 Ibid., 42; see also footnote 3, page 698, and Fromherz, 1. c.

4 Cited from Centralbl. f. Physiol., 23, 76.

700 URINE.

the other amino-acids, is first transformed into the corresponding keto-
acid, p-oxyphenyl pyroracemic acid, OH.CGH4.CH2.CO.COOH, which
is then oxidized into the corresponding chiiiol and transformed into
hydroquinone pyroracemic acid, (OH)2C6H3.CH2.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<f J>OH (homo-

GHaCOOH

gentisic acid) and ^ y (tyrosine) do not exist now, since we have

CH2CHNH2COOH
learnt of other analogous processes. For example, the oxidation, by KUMAGAI and

\VoLFFENSTEiN,1 cf paracresol H|OC yOH with potassium persulphate i

acid solution. In this manner the expected 3.4 dioxytoluene 3^^

was not obtained, but instead homohydroquinone HO\ /OH, and hence a

CH3

transference of the alkyl group must have occurred.

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

1 Ber. d. d. chem. Gesellsch., 41.

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.

HOMOGENTISIC ACID. 701

acid has also been prepared synthetically by BAUMANN and FRANKEL,
starting with gentisic aldehyde and by NEUBAUER and FLATOW 1 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 optically inactive and non-fermentable. Its watery solution has the
properties of so-called alcaptonuric mine. 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. It 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 H2S. 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 and MITTELBACH, GARROD and HURTLEY. DENIGES 3 has suggested
another method.

Uroleucic acid, C9H10O5, is, according to HUPPERT, probably a dioxyphenyl-
lactic acid, C6H3(OH)2.CH2.CH(OH).COOH. This acid was first prepared by KIRK
from the urine of children with alcaptoriuria, which also contained homogentisic
acid. LANGSTEIN and MEYER 4 have also found a small amount of this acid in

1 Baumann and Frankel, Zeitschr. f. physiol. Chem., 20; Neubauer and Flatow,
ibid., 52.

2 Orton and Garrod, Joura. of Physiol., 27; Garrod, ibid., 23.

3 Mittelbach, Deutsch. Arch. f. klin. Med., 71 (which contains the work of Bffumann
and Morner); Garrod and Hurtley, Journ. of Physiol., 33; Deniges Chem. Cenralbl.
1897, 1, 338.

4 Huppert, Zeitschr. f. physiol. Chem., 23; Kirk, Brit. Med. Journ., 1886 and 188*8;
Langstein and Meyer, 1. c.

702 URINE.

a case of alcaptonuria studied by them. It melts at 130-133° C. Otherwise,
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 * 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.

Oxymandelic acid, C8H8O4, paraoxyphenylglycolic acid, HO.C6H4.CH(OH)COOH
is, as above stated, found in the urine in acute atrophy of the liver. The acid
crystallizes in silky needles. It melts at 162° C., dissolves readily in hot water,
less in cold water, and readily in alcohol and ether, but not in hot benzene. It
is precipitated by basic lead acetate, but not by lead acetate.

CH COTI

HC C C.COOH
Kynurenic acid (^-oxy-/?-quiriolincarboxylic acid) , C10H7NO3 , |

' HC C CH

has only been found thus far in dogs' urine, but not always ; its quantity is increased
by meat feeding. It does not occur in the urine of cats. According to the obser-
vations of GLAESSNER and LANGSTEIN the mother-substance seems to be con-
tained among the products of pancreatic digestion which are soluble in alcohol
and precipitable by acetone. ELLINGER 2 has been able to show positively that
tryptophane is the mother-substance of this acid. By the introduction of tryp-
tophane in the organism he has shown the formation of a l<ynurenic 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 CO2 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,

1 Journ. of Physiol., 36.

2 Glaessner and Langstein, Hofmeister's Beitrage, 1; Ellinger, Ber. d. cl. 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.

0 3 Zeitschr. f. physiol. Chem., 7. In regard to kynurenic acid, see also Huppert-
Neubauer, 10. AuflL, and Mendel and Jackson, Amer. Journ. of Physiol., 2; Mendel
and Schneider, ibid., 5; Camps, Zeitschr. f. physiol. Chem., 33.

UROCHROME. 703

•

by the action of light (SAILLET) and air (JAFFE, DISQUE l and others).
Besides this chromogen, urine contains various other bodies from which
coloring matters may be produced by the action of chemical agents.
Humin substances (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 substances may sometimes originate from the rea-
gents used, as from impure amyl alcohol (v. UDRANSZKY2). 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
iiiomdanins and other bodies described by different investigators (PLOSZ,
YHUDICHUM, SCHUNCKS). Indigo blue (uroglaucin of HELLER, urocyanin,
cyanurin, and other coloring matters of earlier investigators4) 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 5) 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 (VI) 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 GARROD to the yellow pigment of
the urine. THUDICHUM 6 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
questionable whether anybody has ever, had this pigment in a pure
form. Urochrome is free from iron, but contains nitrogen. DOMBROW-
SKY found 11.15 per cent nitrogen, HOHLWEG found 9.89 per cent nitrogen,
and KLEMPERER found only 4.2 per cent nitrogen. According to
DOMBROWSKY urochrome contains about 5 per cent sulphur, while
other investigators like HOHLWEG, SALOMONSEN, and MANCINI found
that is was free from sulphur.7 According to GARROD it stands in

1 Jaffe, Centralbl. f. d. med. Wissensch., 1868 and 1869, and Virchow's Arch., 47;
Disque, Zeitschr. f. physiol. Chem., 2; Saillet, Revue de medecine, 17, 1897.

2 v. Udranszky, Zeitschr. f. physiol. Chem., 11, 12, and 13.

3 Plosz, Zeitschr. f. physiol. Chem., 8; Thudichum, Brit. Med. Journ., 201, and
Journ. f. prakt. Chem., 104; Schunck, cited from Huppert-Neubauer, 10. Aufl., 509.

4 See Huppert-Neubauer, 161.

5 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.

7 Dombrowsky, Zeitschr. f. physiol. Chem., 54; Hohlweg, Bioch. Zeitschr., 13;
Salomonsen, ibid., 13; Mancini, ibid., 13; Klemperer, Berl. klin. Wochenschr., 40.

704 URINE.

•

9

close relation to urobilin and is transformed into urobilin by the action
of "active" acetaldehyde, while RIVA 1 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 DOMBROWSKY. Never-
theless it is known that urochrome gives the pyrrol reactions under
certain conditions. SALOMONSEN and especially MANCINI have prepared
a bromine derivative of urochrome whose mother-substance is called
uropyrryl by MANCINI, and has the approximate formula C34H47N7Oi2.

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 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 solution. 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 (see GARROD,
O. BoccHi2). 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, SALOMONSEN and MANCINI also remove the pigment from
the urine, which iias previously been precipitated by calcium or barium
salts, by means of animal charcoal. DOMBROWSKY 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.

DOMBROWSKY, BROWINSKI and DOMBROWSKY 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 used 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

1 Garrod, Journ. of Physiol., 21 and 29; Riva, cited from Huppert-Neubauer, 524.

2 Garrod, 1. c.; Bocchi, Hofmeister's Beitrage, 11.

3 Dombrowsky, Zeitschr. f. physiol. Chem., 54, with Browinski, Bull. Acad. d.
d. scien. Cracovie, 1908; Klemperer, 1. c.

UROBILIN. 705

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.

Urobilin is the pigment first isolated from the urine by JAFFE,1 and
which is characterized by its strong fluorescence and by its absorption-
spectrum. Various investigators have prepared, from the urine, by different
methods, pigments which differed slightly from each other but behaved
essentially like JAFFE'S urobilin. Thus different urobilins have been
suggested, such as normal, febrile, physiological, and pathological uro-
bilins.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. According
to SAILLET 3 no urobilin exists originally in human urine, but only the
mother-substance of the same, urobilinogen, from which the urobilin
is formed in the excreted urine by the influence of light.

Urobilin-like bodies, so-called urobilinoids, have been prepared from
bile-pigments as well as blood-pigments, and indeed by oxidation as well
as by reduction. MALY obtained his hydrobilirubin by the reduction of
bilirubin with sodium amalgam, and DISQUE obtained a product which is
still more similar to urobilin, 'while STOKVIS prepared, by the oxidation
of cholecyanin, with a little lead peroxide, a choletelin, which acted very
much like urobilin. HOPPE-SEYLER, LE NOBEL, NENCKI and SIEBER
have obtained urobilinoid bodies by the reduction of hsematin and ha3ma-
toporphyrin with tin or zinc and hydrochloric acid, while MAcMuNN*
obtained, by the oxidation of ha3matin with hydrogen peroxide in alcohol
containing sulphuric acid a pigment, which seemed to be identical with
urinary urobilin. It is apparent that all these urobilins cannot be identical.

Many investigators declare that urobilin is identical with hydrobilirubin>
but according to the researches of HOPKINS and GARROD 5 this view is not
correct, because, irrespective of other small differences, each body has an
essentially distinct composition. Hydrobilirubin contains C 64.68, H 6.93,

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 m&iecme, 17, 1897.

4Maly, Ann. d. Chem. u. Pharm., 163; Bisque", 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 Journ. of Physiol., 22.

706 URINE.

N 9.22 (MALY), while urinary urobilin, on the contrary, contains C 63.46,
H 7.67, N 4.09 per cent. The urobilin from feces, stercobilin, has the same
composition as urinary urobiiin with 4.17 per cent nitrogen.

Urinary urobilin may not be identical with hydrobilirubin, but this
does not exclude the possibility that urobilin, according to the generally
admitted view, is derived from bilirubin (although not by simple reduction
and taking up of water) in the intestine. Several physiological as well as
c.inical observations 1 point to the truth of this theory, among which we
must mention the regular appearance in the intestinal tract of stercobilin,
undoubtedly derived from the bile-pigments and having the same composi-
tion as urinary urobilin, the absence of urobilin in the urine of new-born
infants as well as on the complete exclusion of bile from the intestine, and
also the increased elimination of urobilin with strong intestinal putrefac-
tion. On the other hand there are investigators who, basing their opinion
on clinical observations, deny the intestinal origin of urobilin and claim
that the urobilin is derived from a transformation of the bilirubin else-
where than in the intestine, by an oxidation of the bile-pigment or by a
ti ansf ormation of the blood-pigments.2 The possibility of a different
mode of formation of urinary urobilin in disease is not to be denied; but
there is no doubt that this pigment is formed from the bile-pigments in
the intestine under physiological conditions.

Urobilin does not occur in the urine of all animals, and according to
FROMHOLDT it is absent in the urine as well as in the feces of rabbits.
BIFFI 3 finds abundance of urobilin or urobilinogen in the blood of human
cadavers, while it is normally absent in the blood during life. In cases
with inflammation of the lungs it occurs according to BIFFI in the blood
during life.

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 in
disease, especially by JAFFE, DISQUE, GERHARDT, G. HoppE-SEYLER,4

1 See Fr. Miiller, Schles. Gesellsch. f. vaterl. Kultur, 1892; D. Gerhardt, " Ueber
Hydrobilirubin und seine Bezieh. zurn Ikterus " (Inaug.-Diss., Berlin, 1889); Beck,
Wien. klin, Wochenschr., 1895; Harley, Brit. Med. Journ., 1896; Fischler, Zeitschr.
f physiol. Chem., 48.

2 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.

3Fromholdt, Zeitschr. f. physiol. Chem., 59; Biffi, Folia hsematol, 4, and 1. c.
Boll., 78.

4 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.

UROBILIN. 707

and others. The quantity is increased in hemorrhage and in diseases
where the blood-corpuscles are destroyed, as is the case after the action of
certain blood-poisons, such as antifebrin and antipyrine. 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 sul-
phate, especially after the addition of sulphuric acid (MEHu1). 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 neutralor 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.2

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, y, between b and F, which borders on Fr
or in greater concentration extends over F. The alkaline solutions are
brownish yellow, yellow, or (the ammoniacal) yellowish green, according
to concentration. If some zinc-chloride solution is added to an ammoni-
acal solution of the pigment it becomes red and shows a beautiful green
fluorescence. This solution, as also that made alkaline with fixed alkalies,
shows a darker and more sharply defined band, d, between b and F,
almost midway between E and F. If a sufficiently concentrated solution
of urobilin alkali is carefully acidified with sulphuric acid it becomes
cloudy and shows a s'econd band exactly at E and connected with ?• by a
shadow (GARROD and HOPKINS, SAILLETS).

Urobilinogen is colorless or is only slightly colored. Like urobilin it

1 Journ. de Pharm. et Chim., 1878, cited from Maly's Jahresber., 8.

2 See Salkowski, Berlin, klin. Wochenschr., 1897, and Stokvis, Zeitschr. f. Biologie,
34.

3 Garrod and Hopkins, Journ. of Physiol., 20; Saillet, 1. c.

708 URINE.

is precipitated from the urine by saturating with ammonium sulphate.
According to SAILLET it may be extracted by acetic ether from urine
acidified with acetic acid. It also dissolves in chloroform, ethyl-ether
and amyl-alcohol. It shows no absorption-bands and is readily converted
into urobilin by the influence of sunlight and oxygen, and, according to
NEUBAUER and BAUER, 1 gives the EHRLICH reaction with dimethylamido-
benzaldehyde and hydrochloric acid (see below).

In preparing urobilin from normal urine, precipitate the urine with
basic lead acetate (JAFFE), wash the precipitate with water, dry at the
ordinary temperature, then boil it with alcohol, and decompose it when
cold with alcohol containing sulphuric acid. The filteied alcoholic
solution is diluted with water, saturated with ammonia, and then treated
with zinc-chloride solution. This new precipitate is washed free from
chlorine with water, boiled with alcohol, dried, dissolved in ammonia,
and this solution precipitated with sugar of lead. This precipitate,
which. is washed with water and boiled with alcohol, is decomposed by
alcohol containing sulphuric acid, the filtered alcoholic solution is mixed
with ^ vol. chloroform, diluted with water, and shaken repeatedly, but
not too energetically. The urobilin is taken up by the chloroform. This
last is washed once or twice with a little water and then distilled, leaving
the urobilin. The pigment may be precipitated directly from the urine
rich in urobilin by ammonia and zinc chloride, and the precipitate treated
as above described (JAFFE).

The method suggested by MEHU (precipitation with ammonium sul-
phate) has been modified by GARROD and HOPKINS in that they first
remove the uric acid by saturating with ammonium chloride and then
saturating the filtrate with ammonium sulphate. In regard to small
details, and to a second method suggested by these experimenters, we
refer to the original work.2

SAILLET extracts the urobilinogen from the urine by shaking with
acetic ether, using a kerosene-oil light. In regard to this and other
methods we must refer to other hand-books.

The color of the acid or alkaline solution, the beautiful fluorescence
of the ammoniacal solution treated with zinc chloride, and the absorp-
tion-bands of the spectrum, all serve as means of detecting urobilin.
In fever-urines the urobilin may be detected, directly, or after the addi-
tion of ammonia and zinc chloride, by its spectrum. It may also some-
times be detected in normal urine, either directly or after the urine has
stood exposed to the air until the chromogen has been converted into
urobilin. If it cannot be detected by means of the spectroscope, then
the urine may be treated with a mineral acid and shaken with ether or,
still better, with amyl alcohol. The amyl-alcohol solution is, either
directly or after addition of a strongly ammoniacal alcoholic solution
of zinc chloride, tested spectroscopically. According to SCHLESINGER 3
it can be readily detected if the urine is precipitated by an equal volume

1 Neubauer, cited from Centralbl. f. PhysioL, 19, 145; Bauer, cited from Biochem.
Centralbl., 4, 390.

2 Journ. of PhysioL, 20.
3Deutsch. med. Wochenschr., 1903.

UROERYTHRIN. 709

of a 10 per cent solution of zinc acetate in absolute alcohol. Disturb-
ing bodies are here precipitated and the filtrate gives the fluorescence
directly, and also the spectrum. GRIMBERT 1 has given another com-
paratively simple method.

The detection of urobilin in feces can be accomplished in various
ways and very simply by the aid of alcoholic extracts as suggested by

SALKOWSKI.2

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 evapora'ted, then
dried and weighed. According to this method G. HOPPE-SEYLER found
0.08-0.14 gram of urobilin in one day's urine of a healthy person, or an
average of 0.123 gram.

Urobilin may also be determined spectrophotometrically according to FR.
MULLER or SAiLLET.4 SAILLET found that the limit for the perceptibility of
the absorption-bands of an acid-urobilin solution lies in a concentration of 1
milligram of urobilin in 22 cc. of solution when the thickness of the layer of fluid
is 15 mm. In a quantitative estimation the urobilin solution is diluted to this
limit and then the quantity of urobilin calculated from the extent of dilution.
The freshly voided urine, shielded from light, is acidified with acetic acid, com-
pletely extracted in kerosene-oil light with acetic ether, and the dissolved uro-
bilinogen oxidized to urobilin with nitric acid. On the addition of ammonia
and shaking with water the urobilin passes into the watery solution. This is
acidified with hydrochloric acid and diluted until the above limit is reached.

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,5 has a pink color, is amorphous, and is very quickly destroyed
by light, especially when in solution. The best solvent is amyl alcohol;

1 See Chem. Centralbl., 1904, 1, 1623.

2 Arbeit, aus. d. pathol. Inst. Berlin. Festschr., 1906.

3 Virchow's Arch., 124.

4 FT Miiller, see Huppert-Neubauer, 861; Saillet, 1. c.

5 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., 1" and 21.

710 URINE.

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 solutions grass
green, and often a play of colors from pink to purple and blue is observed.
PORCHER and HERVIEUX l 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 hrematoporphyrin 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 acids, such as formic acid, acetic acid, and perhaps also butyric
acid, occur under normal conditions in human urine (v. JAKSCH), also in that of
dogs and herbivora (SCHOTTEN). The acids poorest in carbon, such as formic
acid and acetic acid, are more constant 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 grain. 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
normal urine (SALKOWSKI 2) . Non-volatile fatty acids have been detected as
normal constituents of urine by K. MORNER and HYBBINETTE.S

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, arid this probably
explains the occurrence of lactic acid in the urine after epileptic attacks (!NOUYE
and SAIKI) . MINKOWSKI 4 has shown that lactic acid occurs in the urine in large
quantities on the extirpation of the liver of birds.

1 Journ. de Physiol., 7.

2 v. Jaksch, Zeitschr. f. physiol. Chem., 10; Schotten, ibid., 7; Rokitansky, Wien.
med. Jahrbuch, 1887; Salkowski, Zeitschr. f. physiol. Chem., 13; Magnus-Levy, Sal-
kowski's Festschrift, 1904; Rosenfeld, Deutsch. med. Wochenschr., 29.

3 Skand. Arch. f. Physiol., 7.

4 Colasanti and Moscatelli, Moleschott's Untersuch., 14; Schultzen and Riess,

CARBOHYDRATES AND REDUCING SUBSTANCES. 711

Glycerophosphoric acid occurs as traces in the urine,1 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 dextrose, as traces, in normal urine is highly probable, as the investiga-
tions of BRUCKE, ABELES, and v. UDRANSZKY 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 BAU-
MANN and WEDENSKI, and especially by BAISCH. Besides dextrose normal
urine contains, according to BAISCH, another not well-studied variety
of sugar; according to LEMAIRE, probably isomaltose is present, and
besides this a dextrin-like carbodydrate (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 not sufficiently
trustworthy, varies considerably between 1.5 and 5.09 giams.2

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 3 claims, a nitrogenous carbohydrate which has strong reduc-
ing 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 partly conjugated compounds of glucuronic
acid, C6H10O7, which is closely allied to dextrose. The reducing power of
normal urine corresponds, according to various investigators, to 1.5-5.96
p. m. dextrose. That portion of the reduction belonging to dextrose
alone is equal to 0.1-0.6 p. m. LAVESON4 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.

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; Schutz, Zeitschr. f. physiol. Chem., 19; Inouye and
Saiki, ibid., 37; Minkowski, Arch. f. exp. Path. u. Pharm., 21 and 31.

1 See Pasqualis, Maly's Jahresber., 24.

2 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.

3 Berlin, klin. Wochenschr., 1905.

4 Fluckiger, Zeitschr. f. physiol. Chem., 9; Laveson, Bioch. Zeitschr., 4; see also
Huppert-Neubauer, page 72.

712 URINE.

Several new methods for the determination of the reducing power of the urine
have been suggested.1

Conjugated glucuronates occur, as indicated by FLLCKIGER and first
positive^ shown by MAYER and NEUBERG,2 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. Besides these conjugated glucuionates perhaps
the urine sometimes contains the urea glucuronic acid, the ureidoglucu-
ronic acid prepared synthetically by NEUBERG and NEIMANN.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 dyspncea, in diabetes mellitus, and by the direct
introduction of large amounts of dextrose. According to P. MAYER,
as stated on page 215, in the oxidation of dextrose a part of it forms
glucuronic acid, hence it is to be expected that the glucuronic acid can
in part be derived from the dextrose. 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 glyco-
suria a corresponding reduction of the glucose elimination would take
place with the increased excretion of conjugated glucuronates. In order
to prove this possibility O. LOEWI 4 fed dogs with camphor during phlor-
hizin 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.S
ilabbits normally convert almost all the camphor introduced into
conjugated 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 dextrose while

1 See Rosin, Munch, med. Wochenschr., 46; Niemilowicz, Zeitschr. f. physiol. Chem.,
3>j; Niemilowicz with Gittelmacher-Wilenko, ibid., 36, and Helier, Compt. rend., 129.

2 Fliickiger, 1. c.; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29.

3 Zeitschr. f. physiol. Chem., 44.

4 Arch. f. exp. Path. u. Pharm., 47.

5 Zeitschr. f. klin. Med., 47,

CONJUGATED GLUCORONIC ACIDS. 713

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.
HILDEBRANDT 1 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^ 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,S by a combination taking
place first between the conjugator and the dextrose by means of the alde-
hyde group, and then the end alcohol group, CH2OH, 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:* Based upon their cleavage
(as far as they have been investigated) by kephir lactase and emulsin,
but not by yeast lactase (NEUBERG and WOHLGEMUTHS), 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) .6

According to the body with which they are conjugated the glucuronates
vary in behavior. On taking up water they split into glucuronic acid
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 othei 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.

1 Arch. f. exp. Path. u. Pharm., 44.

2 See Maly's Jahresber., 34.

3 E. Sundwik, Akademische Abhandlung Helsingfors, 1886; see also Maly's
Jahresber., 16, 76; Fischer and Piloty, Ber. d. d. chem. Gesellsch., 24.

4 Zeitschr. f. physiol. Chem., 44.

5 See Neuberg, Ergebnisse der Physiologic, Bd. 3, Abt. 1, 444.

6 Jaffe, Zeitschr. f. physiol. Chem., 43; Magnus-Lavy, Bioch., Zeitschr., 6.

714 URINE.

•

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, chrondroitin-
sulphuric acid and protein bodies, but in greater part are made up of antoxy-
proteic 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 differentiate it
from the " acid " sulphur of the sulphate and ethereal-sulphuric acid
(SALKOWSKI 2) . The neutral sulphur in normal urine as determined by
SALKOWSKI is 15 per cent, by STADTHAGEN 13.3-14.5 per cent, and by
LEPINE 20 per cent, and HARNACK and KLEINE 3 19-24 per cent of the
total sulphur. In starvation, according to FR. MULLER, with insufficient
supply of oxygen (REALE and BOERI, HARNACK and KLEINE), as in
chloroform narcosis (KAST and MESTER), as also after the introduction of
sulphur (PRESCH and YVON 4), the quantity of neutral sulphur is increased.
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 upon the extent of the sulphuric-acid excretion.
According to HARNACK and KLEINE,S the relation of the oxidized
sulphur to the total sulphur changes always in the same way as the
relation of the nitrogen o^f 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 6 show that it is probable that the difficultly oxidizable
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-

1 Gscheidlen, Pfliiger's Arch., 14; Munk, Virchow's Arch., 69.

2 Ibid., 58, and Zeitschr. f. physiol. Chem., 9.

3Stadthagen, Virchow's Arch., 100; Lepine, Compt. rend., 91 and 97: Harnack
and Kleine, Zeitschr. f. Biologic, 37.

4 Fr. Muller, Berl. klin. Wochenschr., 1887; Reale and Boeri, Maly's Jahresber., 24;
Harnack and Kleine, 1. c.; Presch, Virchow's Arch., 119; Yvon, Arch, de Physiol.
(5), 10.

5 Benedict, Zeitschr. f. klin. Med., 36; Harnack and Kleine, 1. c.; Folin, Amer.
Journ. of Physiol., 13.

6 Lepine, 1. c.; Smith, Zeitschr. f. physiol. Chem., 17.

OXYPROTEIC ACID. 715

tion with nitric acid.1 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 2).

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 (FR. MULLER, SALKOWSKI 3). 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.4 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 5 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 EHRLICH'S diazo reaction (see below).
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 6
to a nitrogenous acid containing sulphur and which they prepared from
human urine, which has recently been further studied by BONDZYNSKI,
DOMBROWSKI and PANEK. This acid contained C 39.62, H 5.64, N 18.08,
S 1.12, and O 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
diazo 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 PnvJGER-BoHLAND's
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.

1 See Abderhalden and Funk, Zeitschr. f. physiol. Chem., 58 and 59, which also
cites other methods. S3e Folin, Journ. of biol. Chem., 1.

2 Jerome, Pfluger's Arch., 60.

3 Fr. Miiller, Berlin, klin. Wochenscbr., 1887; Salkowski, ibid., 1888.

4 Heffter, Pfluger's Arch., 38; Salkowski, ibid., 39; Presch, Virchow's Arch., 119.
13 Zeitschr. f. physiol. Chem., 4(J.

3 Centralbl. f. d. med. Wissensch., 1897, No. 33.

716 URINE.

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 ant oxyproteic 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 l
from the urine.

Alloxyproteic acid is a thiid acid related to the above, which was first
isolated by BONDZYNSKI and PANEK2 from the urine and then carefully
studied with DOMBROWSKI. The composition is: C 41.33, H 5.70, N 13.55,
S 2.19, and O 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 LIEBERMANN 3 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 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 fil-
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 4 for details,

Uroferric acid is an acid isolated by THIELB 5 from the urine, according
to SIEGFRIED'S method for preparing pure peptone. It also contains
sulphur, 3.46 per cent, and has the formula C35H56N8SOi9. 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. (a)D= —32.5°. On cleavage
it yields melanine substances, sulphuric acid, aspartic acid, but no hexone

%

1 Cloetta, Arch. f. exp. Path. u. Pharm., 40; Pregl, Pfluger's Arch., 75.

2 Ber. d. d. chem. Gesellsch., 35.

3 Zeitschr. f. physiol. Chem., 52.

4 Ibid-., 46.

5 Ibid., 37.

POLYPEPTIDES AND AMINO- ACIDS. 717

bases. The existence of this acid is disputed by BONDZYNSKI, DOM-
BROWSKI 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 1. According to
their determinations in man with a mixed diet the nitrogen of the oxypro-
teic 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 2 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.

Non-dialyzable substances, the so-called adialyzable bodies, or bodies that dialyze
with difficulty, also occur in the urine. They consist in part of chrondroitin-
sulphuric acid whose daily amount, according to PONS, is 0.08-0.09 gram, and
also of nucleic acid, mucoids 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.

Amino-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 SAMUELY * in rabbits. EMBDEN and REESE, FORSSNER,
ABDERHALDEN and SC^TTENHELM, SAMUELY, EMBDEN and MARX 5 were able,

1 Gawinski, Zeitschr. f. physiol. Chem., 58. ; Ginsberg, Hofmeister's Beitrage, 10.

2 Zeitschr. f. physiol. Chem., 46.

3 Pons, Hofmeister's Beitrage, 9; Sasaki, ibid., 9; Savare, ibid., 9 and 11; Ebbecke,
Bioch. Zeitschr., 13.

4 R. Hirsch, Zeitschr. f. exp. Path. u. Therap., 1; Plaut and Reese, Hofmeister's
Beitrage, 7; Abderhalden and Samuely, Zeitschr. f. physiol. Chem., 47.

5 Forssner, Zeitschr. f. physiol. Chem., 47; Abderhalden and Schittenhelm, ibid.,
47; Samuely, ibid., 47; Embden and Reese, Hofmeister's Beitrage, 7, with Marx,

718 URINE.

by means of the naphthalene sulphoehloride method to detect gtycoooll in normal
human urine, and this glycoeoll must occur in the urine in a combination which
is readily split by alkali. Although there have been numerous investigations,
no amino-acids besides glycoeoll could be detected in normal human urine, while,
on the contrary, in pathological conditions other amino-acids have been found
several times. The ammo-acid fraction of the urine seems to be increased in starva-
tion 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. For
the quantitative estimation of the amino-acids we can, according to HENRIQUES,S
make use to advantage of the method suggested by SORENSEN, mentioned on
page 162.

Organic combinations containing phosphorus such as glycerophosphoric acid, phos-
phocarnic acid (ROCKWOOD), etc., which yield phosphoric acid on fusing with salt-
peter and caustic alkali, are also found in urine (LEPINE and EYMONNET, OERTEL).
With a total elimination of about 2.0 grams total P2O5, OERTEL found on an
average about 0.05 gram P2O6 as phosphorus in organic combination. According
to SYMMERS 4 the organic combined phosphoric acid may in many pathological
conditions be 25-50 per cent of the total phosphoric acid. In lymphatic 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 (BRUCKE and others), which, according to MATTHES,
undoubtedly originates from the stomach, and a diastatic enzyme (COHNHEIM and
others) and trypsin.5

Mucin. The nubecula consists, as shown by K. MORNER,' 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 nucleoalburnins 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 subtances, occur in normal urine, as shown
by earlier investigations (POUCHET, BOUCHARD, ADUCCO and others7) and also
by recent researches of KUTSCHER, LOHMANN and ENGELAND. The trimethyl-
amine, which originates from the phosphatides, and first detected by DE FILIPPI
and later by K. BAUER belong to the leucomaines 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 C7H17NO2, gynesin, C19H23N3O3 (from female urine), mingin,
C13Hi8N2O2, vitiatin (Chapter XI) and methylpyridine chloride, which is not a

ibid., 11, which also cites the rather conflicting deductions of Neuberg and Wolgemuth
and of Hirschstein.

1 Deutsch. med. Wochenschr., 1905.

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 Zeitschr. f. physiol. Chem., 60.

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 Jahresber., 31;
Symmers, Journ. of Path, and Bact., 10.

5 In regard to the literature on enzymes in the urine, see Huppert-Neubauer, 599;
Matthes, Arch. f. exp. Path. u. Pharm., 49; Wilenko, Berl. klin. Wochenschr., 45.

6 Skand. Arch. f. Physiol., 6.

7 A complete bibliography on the ptomaines and leucomaines of the urine is found
in Huppert-Neubauer, 403.

CHLORIDES. 719

leucomaine, but is probably derived from smoking tooacco or from drinking coffee.
The imidazole derivatives histidine and imidazolamino-acetic acid found by KUT-
SCHER and ENGELAND l also belong to this group.

Under pathological conditions the quantity of lucomaines and other bodies
may be increased (BOUCHARD, LEPINE and GUERIN, 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

BAUMANN and v. UDRANSZKY have shown that ptomaines may occur in the
urine under pathological conditions. They demonstrated the presence of the
two ptomaines discovered and first isolated by BRIEGER — putrescine, C4H12N2
(tetramethylenediamine), and cadaverine, C5H14N2 (pentamethylenediamine) — in
the urine of a patient suffering from cystinuria and catarrh of the bladder. Cadav-
erine has later been found by STADTHAGEN and BRIEGER in the urine in two cases
of cystinuria. BRIEGER, v. UDRANSZKY and BAUMANN, and STADTHAGEN have
shown that neither these nor other diamines occur under physiological condi-
tions, while DoMBROWSKi,3 on the contrary, found cadaverine in normal urine.

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,
also found in dog's urine and which seems to stand in some relation to the
purine bases ; damaluric acid and damolic acid (according to SCHOTTEN, 4 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.

III. 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 com-
bination 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 BAUMGARTEN 5 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.

2 See footnote 7, page 718.

3 Baumann and Udranszky, Zeitschr. f. physiol. Chem., 13; Stadthagen and Brieger,
Virchow's Arch., 115; Dombrowski, Arch, polonais. d. sciences biol., 1903.

4 Zeitschr. f. physiol. Chem., 7.

5 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.

720 URINE.

mixed diet islO-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, KAST *) .

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 corresponding
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 indicate^ 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 first be
destroyed. For this purpose evaporate to dryness 5-10 cc. of the urine,
after the addition of 1 gram of chlorine-free soda and 1-2 grams chlorine-
free saltpeter, and carefully fuse. The mass is dissolved in water, acidified
faintly with nitric acid, and then neutralized exactly with pure calcium
carbonate. This neutral solution is used for the titration.

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 AgNO3 in 1 liter.

1 Zeller, 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.

CHLORINE DETERMINATION. 721

FREUND and TOEPFER, as well as BODTKER/ have suggested modifica-
tions of MOHK'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 AgNO3 solution and correct the sul-
phocyanide solution by the careful addition of water until 20 cc. exactly corre-
sponds 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
ver\ simple. For example, if 4.6. cc. of the sulphocyanide solution
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-

1 Freund and Toepfer, see Maly's Jahresber., 22; Bodtker, Zeitschr. f. physio! .
Chem., 20.

722 URINE.

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 AgNO3 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

If it is necessary to destroy the organic urinary constituents before titration,
this can be best performed, according to DEHN,* 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 '& 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.; (&) 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 (shaking
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,2 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, M2HPO4, phosphates, both
of which may be found in acid urines at the same time. OTT 3 found that
on an average 60 per cent of the total phosphoric acid was di- and
40 per cent was mono- hydrogen phosphate. This proportion may
vary, and in many cases the urine seems to contain only dihydrogen
phosphate and sometimes indeed only a small quantity of phosphoric
acid. The total quantity of phosphoric acid varies and depends on
the character and the quantity of food. The average quantity of ?2O5
is in round numbers 2.5 grams, with a variation of 1-5 grams per

1 Zeitschr. f. physiol. Chem., 44.

2Ekehorn, Hygiea, Stockholm, 1906; Morner, Upsala Lakaref. Forh. (-N. F.), 11.

3 Zeitschr. f. physiol. Chem., 10.

PHOSPHATES. 723

day. A small part of the phosphoric acid of the urine originates from the
burning of organic compounds, such as nuclein, protagon, and lecithin,
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 quantity
of phosphoric acid eliminated is greater when the food is rich in alkali
phosphates in proportion to the quantity of lime and magnesium phos-
phates. If the food contains much lime and magnesia, large quantities
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 experiments
about one-half of the phosphoric acid taken as CaHPO4 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 subcutane-
ously 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 VoiT3 in dogs, when the nitrogen and phosphoric acid
(P2Or) of the food exactly reappeared in the urine and feces, the relation
was 8.1:1. In starvation, as shown by the compilation of R. TIGER-
STEDT,4 the phosphorized constituents of the body are destroyed to a

1 See A. Gumlich, Zeitschr. f. physiol. Chem., 18; Roos, ibid., 21; Weintraud,
Arch. f. (Anat. u.) Physiol., 1895; Milroy and Malcolm, Journ. of Physiol., 23; Rohmann
and Steinitz, Pfluger's Arch., 72; Loewi, Arch. f. exp. Path. u. Pharm., 44 and 45.

2 Ehrstrorn, Skand. Arch. f. Physiol., 14; Bergmann, Arch. f. exp. Path. u. Pharm.,
47.

3 Physiologic des allgemeinen Stoffwechsels und der Ernahrung in L. Hermann's
Handbuch, 6, Thl. 1, 79.

4 Skand. Arch. f. Physiol., 16.

724 URINE.

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,%and I. MUNK l 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 alloxuric
bodies was increased the phosphoric acid would also be augumented.
This is not the case, and indeed we have observed cases with an increased
elimination of alloxuric 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 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 diminished
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 2) .

Quantitative Estimation of the Total Phosphoric Acid 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
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 ?2O5 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.

1 Preysz, see Maly's Jahresber., 21; Olsavszky and Klug, Pfliiger's Arch., 54; Munk
3, Arch. f. (Anat. u.) Physiol., 1895.

2 Panek, see Maly's Jahresber., 30, 112; Iwanoff, Biochem. Centralbl., 1, 710;
Soetber and Krieger, Eteutsch. Arch. f. klin. Med., 72; Campani, Biochem. Centralbl.,
3, 616; Tobler, Arch. f. exp. Path. u. Pharm., 52,

PHOSPHATE DETERMINATION. 725

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 titrating
with a solution of sodium phosphate of known strength (10.085 grams crystallized
salt in 1 liter, which corresponds to 0.100 gram P2O3 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 corre-
sponds 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 filtei, 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 determinations
gives the quantity of phosphoric acid combined with the alkalies. The
results obtained are not quite accurate, as a partial transformation 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
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 H2SO4 per day.

726 URINE.

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 par-
allelism can hardly be expected, as in the first place a part of the sulphur
is always eliminated as neutral sulphur, and secondly because the small
proportion of sulphur in different protein bodies undergoes greater varia-
tion 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 5-sulphuric acid.

The quantity of total sulphuric acid is determined in the following
waj/, but at the same time 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 boil-
ing precipitate with 2 cc. of a saturated BaCl2 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 BaCl2 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 SALKOwsKi1 :

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.

FOLIN 2 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 unlike 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

1 Baumann, Zeitschr. f. physiol. Chem., 1; Salkowski, Virchow's Arch., 79.

2 Journ. of Biol. Chem., 1, and Arner. Journ. of PhysioL, 13.

POTASSIUM AND SODIUM. AMMONIA. 727

and CITRON, l 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,2 3-4 grams K2O and 5-6 grams Na2O, 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.3

Ammonia. Some ammonia is habitually found in human urine and
in that of carnivora. As above stated (page 694), on the foimation 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 alka-
lies. This view is confirmed by the observations of CORANDA, who found
that the elimination of ammonia was smaller on a vegetable diet and
larger on a rich meat diet .than on a mixed diet. On a mixed diet the
average amount of ammonia eliminated by the urine is about 0.7 gram
NHs per day (NEUBAUER), corresponding to 4.6-5.6 per cent of the total
nitrogen of the urine according to CAMERER, Jn As above stated, all
the ammonia of the urine is not represented by the residue which has
eluded synthesis into urea by neutralization with acids, because, as shown
by STADELMANN and BECKMANN,4 ammonia is eliminated by the urine
even during the continuous administration of fixed alkalies.

Ammonia exists on an average of about 0.90 milligram in 100 cc. of
human blood, and in different amounts in all the tissues thus far investi-
gated.5 According to NENCKI and ZALESKI 6 it is abundantly formed in

1 Schonbein, Journ. f. prakt. Chem., 92; Weyl, Virchow's Arch., 96, with Citron,
ibid., 101.

2 Ibid., 53.

3 Pribram and Gregor, Zeitschr. f. analyt. Chem., 38; Autenreith and Bernheim,
Zeitschr. f. physiol. Chem., 37.

4 Coranda, Arch. f. exp. Path. u. Pharm., 12; Stadelmann (and Beckmann), Ein-
fluss der Alkalien auf den Stoffwechsel, etc. Stuttgart, 1890; Camerer, Zeitschr. f.
Biologic, 43.

5 See Salaskin, Zeitschr. f. physiol. Chem., 25, 449, and footnote 6, page 317.

8 Arch, des science biol. de St. Petersbourg, 4, and Salaskin, 1. c. See also Nencki
and Zaleski, Arch, f . exp. Path. u. Pharm., 37.

728 URINE.

the cells of the digestive glands, the stomach, the pancreas, and the in-
testinal mucosa (of dogs) as the time when protein foods are being digested
and transported to the liver. As the ammonia introduced into the liver
is transformed into urea (see above), we can therefore expect that in
certain diseases of the liver an increased elimination of ammonia and a
decreased excretion of urea will occur. In how far this is true has already
been stated (page 651), and we refer to the researches of the various
authors there cited.

In man and certain animals the elimination of ammonia is increased
by the introduction of mineral acids; and, as shown by JoLiN,1 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. This dissimilar
tendency of different animals toward acidosis has been discussed in the
previous pages.

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 — ^-oxybutyric acid and acetoacetic acid — are pro-
duced, which pass into the urine combined with ammonia.2 Other
observations also indicate that the elimination of ammonia by the urine
is increased on insufficient or diminished supply of alkalies or alkaline
earths.

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.*

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,

1 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, 34; Kowalcwski and
Markewicz, Bioch. Zeitschr., 4, and the works cited on page 641.

2 On the elimination of ammonia in disease, see the works of Rumpf, Virchow's
Arch., 143; Hallervorden, ibid.

3Pfluger's Arch., 43, 32.

CALCIUM AND MAGNESIUM. 729

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 l 25 cc. of the urine are placed in a distil] ation-flask with about
10 grams of XaCl and 1 gram of Xa2CO3, 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. SCHAFFER'S method is practically the same STEEL arid GIES
have raised objections to FOLIN' s method (passing air through the urine),
as they found low results in the presence of large quantities of magnesium
phosphate in the urine. MALFATTI 2 has recently suggested a method
based upon an entirely different principle.

Calcium and magnesium occur in the urine chiefly as phosphates.
The quantity of earthy phosphates eliminated daily is somewhat more
than 1 gram, and of this amount f is magnesium and J calcium phos-
phate. This statement, as found by RENWALL and GROSS,3 is not correct,
or at least is not valid in general, as they found more calcium than mag-
nesium in the urine. 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 insolubleris particularly augmented
by the presence in the urine of dihydrogen alkali phosphates and sodium,
chloride (On4). The quantity of alkaline earths in the urine depends
on the composition 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. According to GRANSTROM 5 starvation in rabbits or the intro-
duction of food which yields an acid ash and yields an acid urine produces
the same effect as the introduction of acid. Nothing is known with
certainty in regard to the constant and regular change in the elimina-
tion of calcium and magnesium salts in disease, and in these conditions
the excretion is chiefly dependent upon the diet and the formation and
introduction of acid.6

1 Zeitschr. f. physiol. Chem,, 39; Schaffer, Amer. Journ. of Physiol., 8, which con-
tains the literature.

2 Steel and Gies, Journ of biol. Chem., 5; Malfatti, Zeitscjir. f. anal. Chem., 47.

3 Renwall, Skand. Arch. f. Physiol., 16; Gross, Biochem. CentralbL, 4, 189.

4 Zeitschr. f . physiol. Chem., 10.
*IUd., 58.

B See Albu and Neuberg, 1. c., and Zak, Wien. klin. Wochenschr., 21.

730 URINE.

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, as it seems from the
investigations of KUNKEL, GIACOSA, ROBERT and his pupils, it does not exist
as a salt, but as an organic combination — in part as pigment or chromogen. The
reports in regard to the iron present seem to show that the quantity ranges from
1 to 11 milligrams per liter of urine (MAGNIER, GOTTLIEB, ROBERT and his pupils).
JOLLES found as an average for twelve persons 8 milligrams of iron in twenty-
four hours, while HOFFMANN, NEUMANN and MAYER 1 found lower results — an
average of 1.09 and 0.983 milligrams. The quantity of silicic acid is ordinarily
stated to amount to about 0.3 p. m. 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 gi eater 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 ordinarily cal-

1 Kunkel, cited from Maly's Jahresber., 11; Ciacosa, 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; Hoffmann,
Zeitschr. f. anal. Chem., 40; Neumann and Mayer, Zeitschr. f. physiol. Chem., 37.

QUANTITY AND COMPOSITION OF THE URINE. 731

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 houis 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 approximate 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 eliminated in twenty-four hours; therefore the quantity of

solidsexcretedwas2lX2.33 = 48.9and =51.35 grams. LONG 1

-LUL/U

has made a new determination of the coefficient for a 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 melli-
tus) , 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 diarrhcea, 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 notwith-
standing the oliguria, and be poor in solids and light in color.

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 0.7 and 1 ; as a rule, it amounts on an average to 0.87, but changes

1 Journ. Amer. Chem. Soc., 25.

732 URINE.

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, and others J) .

It is difficult to give a tabular view of the composition of urine on
account of its variation. For certain purposes the following table may be
of some value, but it must not be overlooked that the results are not
given for 1000 parts of urine, but only approximate figures for the quan-
tities 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 carbodydrate
per day, and to a man of average weight.

Daily quantity of solids = 60 grams.

Organic constituents = 35 grams. Inorganic constituents =25 grams.

Urea 30.0 grams. Sodium chloride (NaCl). . . 15.0 grams.

Uric acid 0.7 " Sulphuric acid (H2SO4). ... 2.5 "

Creatinine 1.5 " Phosphoric acid (P2O5) 2.5 "

Plippuric acid 0.7 Potash (K2O) 3.3 "

Remaining organic bodies . . 2.1 " Ammonia (NH3) 0.7 "

Magnesia (MgO) \ n Q

Lime (CaO) / a*

Remaining inorganic bodies 0.2 "

Urine contains on an average 40 p. m. solids. The quantity ot 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 deter-
minations, 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 of the common salt and others; or attempts 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, the views are unfortunately
still too divergent.

1 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.

CASUAL URINARY CONSTITUENTS. 733

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
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 belong-
ing to this series, which are otherwise decomposed into water and carbon-
ates 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 2)-, but as to oxalic acid authorities
disagree. In birds, according to GAGLIO and GIUNTI, it is not oxi-
dized. In mammals it is in great part oxidized, according to GIUNTI,
while GAGLIO and POHL claim that it is not destroyed. MARFORI and
GIUNTI claim that in human beings oxalic acid is in great part oxidized.
The recent investigations of SALKOWSKI, PIERALLINI, STRADOMSKY,
KLEMPERER and TRITSCHLER and especially those of HILDEBRANDT and
of DAKIN,S where oxalates were introduced subcutaneously, show that
oxalic acid, when introduced in medium quantities, is in great part
oxidized in the animal body. Tartaric acids act differently, according

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.

2 Schotten, Zeitschr. f. physiol. Chem., 7; Grehant and Quinquaud, Compt. rend.,
104.

3 Gaglio, Arch. f. exp. Path. u. Pharm'., 22; Giunti, Chem. Centralbl., 1897, 2;
Marfori, Maly's Jahresber., 20 and 2"; 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, Joiirn. of biol. Chem., 3.

734 URINE.

to BRION 1 ; thus in dogs the levotartaric acid is almost entirely consumed,,
while a little more than 70 per cent of dextrotartaric acid is burnt. Race-
mic acid is oxidized to a still less extent in the animal body. Succinic
and malic acids are completely combustible, according to PoHL.2 Ex-
amples of the different behavior of stereoisomeric substances have already
bee"n given on page 199.

The acid amides do not appear to be altered in the body (SCHULTZEN and
NENCKI 3) . The amino-acids may, indeed, when introduced into the body
in large quantities, be in part eliminated unchanged by the urine; but
otherwise, as stated above (page 648) for leucine, glycocoll, and aspartic
acid, they are decomposed within the body, and may therefore cause an
increased excretion of urea. That in the demolition of the amino-acids
a deamidation takes place is shown by alanine yielding lactic acid and
diaminopropionic acid, yielding gly eerie acid, as mentioned in a previous
chapter (VIII). The amino-acids give an instructive example of the
unequal behavior of stereometric substances in the animal body, as the
racemic acids are so changed and transformed that the component foreign
to the body is burnt with more or less difficulty, while the component
occurring in the body protein is burnt, on the contrary, with ease and
more completely. ABDERHALDEN 4 and his collaborators have shown that
the polypeptides when introduced into the body are decomposed with
the formation of urea similar to the amino-acids, and this probably occurs
by a previous decomposition into their simplest fractions, the amino-
acids.

We do not know exactly how the amino-acids are split in the animal body*
DAKIN has shown that in the oxidation of the amino-acids by hydrogen peroxide
outside of the body under certain conditions the reaction proceeds with the
formation of ammonia, carbon dioxide and an aldehyde, which latter is then further
oxidized with the formation of the corresponding acid and other products.
For example, leucine first yielded isovaleric aldehyde, then isovaleric acid,
(CH3)2.CH.CH2.COOH, from which, DAKIN claims, acetone is derived (see under
acetone bodies, page 774), with /?-oxyisovaleric acid, (CH3)2.C(OH).CH2.COOH,
probably as intermediary step. In a series of investigations DAKIN 5 calls atten-
tion to the correspondence which exists between the artificial oxidations and
several known decompositions in the animal body, which indicate a similar
decomposition of amino-acids in the living organism. According to NEUBAUER°
the destruction of the amino-acids occurs, in that the corresponding keto acid,
R.CO.COOH is first formed by an oxidative deamidization, and not, as generally
admitted, the corresponding oxyacid. The keto acids are by oxidation with the
splitting off of CO2 then transformed into the next lower fatty acid.

1 Zeitschr. f. physiol. Chem., 25.

2 Pohl, Arch. f. exp. Path. u. Pharm., 37, which also contains reports on the
intermediary products formed in the oxidation of the fatty bodies.

3 Zeitschr. f. Biol., 8.

4 Zeitschr. f. physiol. Chem., 39, 46, 47, 48, 52, 55, 57.

5 Dakin's work can be found in Journ. of biol. Chem., 1, 2, 3, 4.

6 Cited in Physiol. Centralbl., 23, 76.

CASUAL URINARY CONSTITUENTS. 735

By substituting one of the hydrogen atoms in the NH2 group of
norma] « -ammo-acids by an alkyl radical (methyl) the combustion of the
acids of the series C2 and €4 is considerably retarded and almost entirely
prevented in the members of the C5 and C6 series (FRIEDMANN). 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 acid, NH2.CO.N(CH3).CH2.COOH (ScnuLTZEN1), is an example of this
kind. The substitution of both hydrogen atoms of the NH2 group by
methyl radicals does not seem to make the demolition of the amino-acids
more difficult (FRIEDMANX). The a -amino-acids with branched carbon
chains behave differently in the body from the n-amino-acids, and the
formation of /?-oxybutyric acid and acetoacetic acid, which will be dis-
cussed on page 775, is an example. No general rule for the mode of
demolition of the amino-acids can be given (FRIEDMANN 2) .

The nitrites, including hydrocyanic acid, pass, according to LANG, into
sulphocyanide combinations, and this sulphocyanide apparently originates
from the non-oxidized sulphur of the proteins, which is readily split off.
PASCHELES' observations indicate that, in an alkaline reaction and at the
temperature of the body, this sulphur can readily convert the alkali-
cyanides into sulphocyanides. The alkali sulphocyanides when ingested
are almost quantitatively eliminated in the urine, according to POLLAK.S

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 alhedydes 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.4

By conjugation with sulphuric acid, the alcohols which are otherwise
readily oxidizable may be guarded against combustion, and conse-
quently the alkali salt of ethylsulphuric acid is not burnt in the body
(SALKOWSKI 5) .

The organic combinations containing sulphur act differently. Taurine,

1 Ber. d. deutsch. chem. Gesellsch., 5. See also Baumann and v. Mering, ibid., 8,
584, and E. Salkowski, Zeitschr. f. physiol. Chem., 4, 107.

2 Hofmeister's Beitrage, 11.

3 Lang, Arch. f. exp. Path. u. Pharm., 34; Pascheles, ibid.; Pollak, Hofmeister's
Beitrage, 2.

4 See Harnack and Griindler, Berlin, klin. Wochenschr., 1883; Zeller, Zeitschr. f
physiol. Chem., 8; Kast, ibid., 11; Binz, Arch. f. exp. Path. u. Pharm., 28; Zeehuisen,
Maly's Jahresber., 23.

5 Pfluger's Arch., 4.

736 URINE.

whose behavior varies in different animals (SALKOWSKI 1), passes in
human beings, at least in part, into the corresponding uramino-acid,
taurocarbamic acid, NH2.CO.NH.C2H4.SO2.OH. A part of the taurine
also appears as such in the urine. In rabbits, when taurine is introduced
into the stomach, nearly all its sulphur appears in the urine as sulphuric
and hyposulphurous acids. After subcutaneous injection the taurine
appears again in great part unchanged in the urine. In dogs a great part
of the sulphur of cystine appears in the urine as sulphate- (also as thiosul-
phate) (BLUM, ABDERHALDEN and SAMUELY2).

According to W. SMITH the sulphur of the thio-acids, like thioglycolic
acid, CH2.SH.COOH, is in part oxidized to sulphuric acid, and according
to GOLDMANN the same result occurs with aminothiolactic acid (cysteine)
and the sulphur of the thio-alcohols (ethyl mercaptans). On the con-
trary, ethylsulphide, sulphonic and sulpho acids in general (SALKOWSKI,
SMITH 3) are not changed into sulphuric acid. Oxyethylsulphonic acid,
HO.C2H4.SO2.OH, which is in part oxidized to sulphuric acid, is an
exception (SALKOWSKI).

Conjugation with glucuronic acid occurs, according to the investiga-
tions of SUNDVIK. and especially of O. NEUBAUER, in many substituted
as well as non-substituted alcohols, aldehydes, and ketones. Chloral
hydrate, C2C13OH + H20, passes, after it has been converted into tri-
chlorethyl-alcohol by a reduction, into a levogyrate reducing acid, uro-
chloralic acid or trichlorethylglucuronic acid, C2C13H2.C6H9O7 (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
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. That the
benzene ring is destroyed in the body in certain cases is very probable.

1 Ber. d. deutsch. chem. Gesellsch., 6, and Virchow's Arch., 58.

2 Blum, Hofmeister's Beitrage, 5; Abderhalden and Samuely, Zeitschr. f. physiol.
Chem., 46.

3 Smith, Pfluger's Arch., 53, 55, 57, and Zeitschr. f. physiol. Chem., 17; Salkowski,
Virchow's Arch., 66; Pfluger's Arch., 39; Goldmann, Zeitschr. f. physiol. Chem., 9;
also Baumann and Kast, ibid., 14.

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, Pfluger's
Arch., 28 and 33; O. Neubauer, Arch. f. exp. Path. u. Pharm., 46.

CASUAL URINARY CONSTITUENTS. 737

The fact that benzene may be oxidized outside of the body into caibon
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. As the benzene nucleus can protect a substance
belonging to the fatty series from destruction when conjugated with it,
which is the case with the glycocoll of hippuric acid, it seems that the
aromatic nucleus itself may likewise be protected from oxidation in the
organism by synthesis with other bodies. The aromatic ethereal-sul-
phuric acids are examples of this kind.

The difficulty in deciding whether the benzene ring itself is destroyed
in the body lies in the face that we do not know all the different aromatic
transformation products which may be produced by the introduction of
any such substance into the organism, and which must be sought for in
the urine. On this account it is also impossible to learn by exact quanti-
tative determinations whether or not an aromatic substance ingested or
absorbed appears again unchanged in the urine. Certain observations
render it probable that the benzene ring, as above mentioned, is at least
in certain cases destroyed in the body. SCHOTTEN, BAUMANN, and others
have found that certain amino-acids, such as phenylamino-propionic
acid, amino-cinnamic acid, and tyrosine, when introduced into the body
cause no increase in the quantity of known aromatic substances in the
urine; this makes a destruction of these amino-acids in the animal body
seem probable. According to F. KNOOP 1 phenyl-a-lactic acid and
phenyl-a-ketopropionic acid (phenyl pyroracemic acid) have a similar
behavior. According to Ju YALTA 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
readily destroyed than the corresponding meta- and para-compounds.
The claims of .Tu YALTA and PORCHER are unfortunately disputed by
PRIBRAM and PoHL.3

1 Schotten, Zeitschr. f. physiol. Chem., 7 and 8; Baumann, ibid., 10, 130. In regard
to the behavior of tyrosine, see especially Blendermann, ibid., 6; Schotten, ibid., 7;
Bass, ibid., 11: and R. Cohn, ibid., 14; F. Knoop, Der Abbau aromatischer Fett-
sauren ini Tierkorper, Habilit.-Schrift, Freiburg, 1904.

2 Zeitschr. f. physiol. Chem., 17.

3 Juvalta, Zeitschr. f. physiol. Chem., 13; Pribram, Arch. f. exp. Path. u. Pharm.,
51; Porcher, Bioch. Zeitschr., 14; Pohl, ibid., 16.

738 URINE.

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, C6H5.NH2, passes into paraminophenol, which latter passes into
the urine as its ethereal-sulphuric acid, H2N.C6H4.O.SO2.OH (F. MULLER).
Acetanilid is in part converted into acetyl paraminophenol (JAFFE and
HILBERT, K. MORNER), and carbazol into oxycarbazol (KLINGENBERG).1

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, C6H5.CH3 (SCHULTZEN and NAUNYN), ethyl-
benzene, C6H5.C2H5, and propylbenzene, C6H5.C3H7 (NENCKI and GiACosA),2
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 the side chain has several members,
the behavior is somewhat different. Phenylacetic acid, C6H5.CH2.COOH,
in which only one carbon atom exists between the benzene nucleus and
the carboxyl, is not oxidized, but is eliminated after conjugation with
glycocoll as phenaceturic acid (SALKOWSKI 3) . Phenylaminoacetic acid,
C6H5.CHNH2.COOH is in part converted into mandelic acid (phenyl-
glycollic acid), C6H5.CHOH.COOH, and in great part is eliminated as
such (SCHOTTEN, KNOOP4). Phenylpropionic acid C6H5.CH2.CH2.COOH,
with three carbon atoms in the side chain, is, on the contrary, oxidized
into benzoic acid, and H. and E. SALKOWSKI 5 have proposed the rule that
the homologues of the benzoic acids are converted into benzoic acid when
the side chain contains more than two carbon atoms.

KNOOP 6 has shown by experiments with several acids, that this rule

1 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; Jaff^ and Hilbert, Zeitschr. f. physiol. Chem., 12; Morner, ibid., 13; Klingen-
berg, " Studien iiber die Oxydation aromatischer Substanzen," etc., Inaug.-Diss.
Rostock, 1891. In regard to formanilid, which acts essentially as acetanilid, see
Kleine, Zeitschr. f. physiol. Chem., 22.

2 Zeitschr. f. physiol. Chem., 4.

3 Ibid., 7 and 9.

4 Ibid., 8.

5 Ibid., 7.

6 Hofmeister's Beitrage, 6, and Habilit.-Schrift, Freiburg, 1904.

CASUAL URINARY CONSTITUENTS. 739

does not hold good. He found that the aromatic fatty acids with straight
carbon chains such as phenyl butyric acid and phenyl caproic acid, are
changed into phenyl acetic acid, which is then conjugated with glycocolJ,
forming phenylaceturic acid, while the acids with uneven carbon side-
chains, such as phenyl propionic and phenylvalerianic acids, yield benzoic
acid and are eliminated as hippuric acid. This behavior is explained
if, as is made probable by KNOOP, in the demolition those aromatic fatty
acids an oxidation takes place in those groups which are in the /^-position
to the carboxyl group. FRIEDMANN has nevertheless raised objections
to this view, but DA.KIN,1 on the other hand, has reported observations
which support it strongly. This author found that in the dog phenyl
propionic acid was eliminated as hippuric acid, and also a part as
phenyl-9-oxypropionic acid, C6H5.CH(OH).CH2COOH and acetophenone,
C6H5.CO.CH3, which presupposes an oxidation in the /9-position. As
the last two mentioned bodies can be converted into benzoic acid in the
body, DAKIN suggests that the demolition of phenylprop ionic acid at
least in part occurs with phenyl-/?-oxypropionic acid and acetophenone
as mtemediary steps, and he also undoubtedly finds that in the demolition
of the saturated fatty acids that the first stage is the oxidation of the
hydrogen combined to a carbon atom in the /^-position. KNOOP'S rule
does not apply to the propionic acids substituted in the a-position, i.e.,
phenylalanine, phenyl-a-lactic acid, and phenyl-a-ketopropionic acidr
which, like tyrosine and a-amino-cinnamic acid, are burnt in the body.
SCHOTTEN'S rule, according to which all acids having three carbon atoms
in the side chain of which the middle one has a NH2 group attached, are
almost completely burnt in the organism, has been extended by these
exceptions.

If several side chains are present in the benzene nucleus, then only one
is always oxidized into carboxyl. Thus xylene, C6H4(CH3)2, is oxidized
into toluic acid, CoH4(CH3)COOH (SCHULTZEN and NAUNYN) ; mesitylene,
CbH3(CH3)3, into mesitylenic acid, C6H3(CH3)2.COOH (L. NENCKI);
cymene, (CH3)2CH.C6H4.CH3, into cumic acid (M. NENCKI and ZiEGLER2) ;

XOCH3

and vanillin, OH.C6H3<^ , into vanillinic acid (Y. KOTAKES).

X5HO

Reductions may also occur and examples of this kind are the conver-
sion, as observed by E. MEYER,4 of nitrobenzene, C6H5NO2, or of nitro-
phenol, HO.06H4.NO2 into aminophenol, HO.C6H4.NH2, and also the
behavior of w-nitrobenzaldehyde in the animal body as mentioned below.

1 Friedmann, Hofmeister's Beitrage, 11; Dakin, Journ. of biol. Chem., 4, 419.

2 L. Nencki, Arch. f. exp. Path. u. Pharm., 1; Nencki and Ziegler, Ber. d. deutsch.
chem., Gesellsch., 5. See also O. Jacobsen, ibid., 12.

3 Zeitschr. f. physiol. Chem., 45.

4 Ibid., 46.

740 URINE.

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 l more cor-
rectly to KELLER and URE. All the numerous aromatic substances which
are converted into benzoic 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,2 benzoic acid does not pass into hippuric acid
in birds, but into another nitrogenous acid, ornithuric acid, CigH^o^O^
This acid yields as splitting products, besides benzoic acid, ornithine, a body
which has been spoken of on page 159. Not only are the oxybenzoic acids
and the substituted benzoic acids conjugated with glycocoll, forming corre-
sponding 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 conju-
gation with glycocoll has been shown only with salicylic and p-oxybenzoic
acid (BERTAGNINI, and others), while BAUMANN and HERTERS find it
only very probable for ra-oxybenzoic acid. According to BALDONI 4
in dogs the salicylic acid does not pass into salicyluric acid, and he indeed
found two acids which he calls ursalicylic acid, Ci5Ni4O8 and uramin-
salicylic acid, C16Hi6NO8. The oxybenzoic acids are also in part elimi-
nated as conjugated sulphuric acids, which is especially true for m-oxy-
benzoic 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,S that
in rabbits m-aminobenzoic acid passes in part into uraminobenzoic acid,
H2N.CO.ftN.C6H4.COOH. It is also in part eliminated as aminohip-
puric 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-bromtoluene is completely changed to hippuric acid, the m- and
2>-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.

1 Die Ausscheidung korperfremder Substanzen im Hani, Ergebnisse der Physiol.,
4, 252.

2 Ber d. d. chem. Gesellsch., 10 and 11.

3 Zeitschr. f . physiol. Chem., 1, where Bertagnini's work is also cited. See also
Dautzenberg, Maly's Jahresber., 11, 231.

4 Arch. f. exp. Path. u. Pharm., 1908, Suppl. Bd. (Schmiedeberg's Festschrift).
5Salkowski, Zeitschr. f. pyhsiol. Chem., 7; Cohn, ibid., 17; Hildebrandt, Hof-

meister's Beitrage, 3.

CASUAL URINARY CONSTITUENTS. 741

The substituted aldehydes are of special interest as substances which
may undergo conjugation with gl) cocoll. According to the investigations
of R. COHN 1 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 SMIRNOW 2 m-nitrobenzaldehyde passes in dogs into m-nitro-
hippuric acid, and according to COHN into urea-ra-nitrohippurate, 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 nitro-
group 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.CO.NH.CeH^COOH. This process is
analogous to the action of furfurol, and the reduction does not take
place in the intestine, but in the tissues. The p-nitrobenzaldehyde acts
in rabbits in part like the m-aldehyde and passes in part into p-acetyl-
aminobenzoic acid. 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.3

To those substances which undergo a conjugation with glycocoll
belongs also furfurol (the aldehyde of pyromucic acid), which, when intro-
duced into rabbits and dogs, as shown by JAFFE and CoHN,4 is first oxidized
into pyromucic acid and then, after conjugation with glycocoll, elimin-
ated as pyromucuric acid, C7H7NO4. In birds this behavior is different,
namely, the acid is conjugated with another substance, ornithine,
C.5Hi2N2O2, which is a diaminovaleric acid, forming pyromucinornithuric
acid.

Furfurol in mammals also undergoes conjugation with glycocoll in
other forms. Thus JAFFE and COHN found that it is in part combined with
acetic acid, forming furfuracrylic acid, C4H3O.CH:CH.COOH, which passes
into the urine coupled writh glycocoll as furfuracryluric acid.

It has not been proven how thiophene, C4H4S, behaves in the animal
body. Of methylthiophene (thiotolene), C4H3S.CH3, a very small part is
oxidized to thiophenic acid, C4H3S.COOH (LEVY). This acid, as shown

1 Zeitschr. f. physiol. Chem., 17.

2 Monatshefte, f. Chem., 8.

3 In regard to the extensive literature on glycocoll conjugations we refer the reader
to O. Kiihling, Ueber Stoffwechselprodukte aromatischer Korper. Inaug.-Diss.,
Berlin, 1887.

4 Ber. d. d. Chem. Gesellsch., 20 and 21.

742 URINE.

by JAFFE and LEVY,1 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.2

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,3 the quantity of ethereal-sulphuric acid in the urine, while
ROST asserts, contrary to earlier claims, that the same occurs with gallic
acid (trioxybenzoic acid) and tannic acid*

While acetophenone (phenylmethylketone) , C6H5.CO.CH3, as shown
by M. NENCKI, is oxidized to benzoic acid and eliminated as hippuric acid,
the aromatic oxyketones with hydroxyl groups, such as resacetophenoner

C6H3(OH) (OH) (CO.CH3), paraoxypropiophenone, C6H4(OH) (COCH2.CH3),

and gallacetophenone, C6H2(OH)(OH)(OH)(CO.CH3), pass into the urine
without previous oxidation as ethereal-sulphuric acids and in part after
conjugation with glucuronic acid (NENCKI and REKOWSKI 5). Euxanthon,
which is also an aromatic oxyketone, passes into the urine as euxanthic
acid after the conjugation with glucuronic acid previously mentioned.

A conjugation of other aromatic substances with glucuronic acid, which
last is protected from combustion, occurs rather often. The phenols, as
above stated (page 689), 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 HILDEBRANDT and FROMM
and CLEMENS 6 have shown that the cyclic terpenes and camphors, by oxida-
tion or hydration, or in certain cases by both, are converted into hydroxyl
derivatives when the body in question is not previously hydroxylized, and

1 Levy, Ueber das Verhalten einiger Thiophenderivate, etc., Inaug.-Diss., Konigs-
berg, 1889; Jafte and Levy, Ber. d. d. chem. Gesellsch., 21.

2 In regard to the literature, see O. Ktihling, 1. c.

3 Zeitschr. f. physiol. Chem., 21.

4 In regard to the action 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.
Cesellsch. zur Beford. d. ges. Naturwiss. zu Marburg, 1898.

5 Arch. d. scienc. biol. de St. Pdtersbourg, 3, and Ber. d. deutsch. chem.
Gesellsch., 27.

8 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.

CASUAL URINARY CONSTITUENTS 743

that these hydroxyl derivatives are eliminated as conjugated ghicuronic
acids. Conjugated glucuronic acids are detected in the urine after the
introduction of various substances into the organism, e.g., therapeutic
agents, such as terpenes, borneol, menthol, camphor (camphoglucuronic
acid was first observed by SCHMIEDEBERG), naphthalene, oil of turpentine,
oxyquinolines, antipyrine, and many other bodies.1 Orthonitrotoluene in
dogs passes first into o-nitrobenzyl alcohol and then into a conjugated
glucuronic acid, uronitrotoluolic acid (JAFFE2). The glucuronic acid split
off from this conjugated acid is levogyrate and hence is not identical, but
only isomeric, with the ordinary glucuronic acid. Dimethylaminobenzal-
dehyde, according to JAFFE, is converted in part into dimethylaminoben-
zoglucuronic acid in rabbits. The same conjugated glucuronic acid is also
produced, according to HILDEBRANDT, 3 from p-dimethyltoluidine, which
is first changed into p-dimethylaminobenzoic acid. Indol and skatol seem,
as above stated (page 695), to be eliminated in the urine partly as con-
jugated glucuronic acids.

A synthesis in which compounds containing sulphur, mercapturic acids,
are formed and eliminated after conjugation wTith glucuronic acid, occurs
when chlorine and bromine derivatives of benzene are introduced into the
organism of dogs (BAUMANN and PREUSSE, JAFFE). Thus chlorbenzene
combines with cysteine, forming chlorphenylmercapturic acid, CiiH12ClSXO3.
The important investigations of FRIEDMANN 4 show that the phenylthio-
lactic acid which forms the foundation of the mercapturic acids belongs
to the /9-series, and in this way the direct chemical connection of this body
with the protein-cystine (a-amino-/?-thiolactic acid) is established. FRIED-
MANN has also been able to convert cysteine into bromphenylmercapturic
acid.

Pyridine, C5H5N, which does not combine either with glucuronic acid
or with sulphuric acid after previous oxidation, shows a special behavior.
It takes up a methyl group as found by His and later confirmed by COHN,
and forms an ammonium combination, methylpyridylammonium hydroxide,
HO.CH3.NC5H5, while in rabbits it occurs unchanged in the urine, accord-
ing to ABDERHALDEN, BRAHM and SCHITTENHELM.S

1 See O. Kiihling, 1. c., which gives the literature up to 1887; also E. Kiilz, Zeitschr,
f. Biologie, 27; the works of Hildebrandt, Fromm and Clemens, see footnote 6,
page 742; Brahm, Zeitschr. f. physiol. Chem., 28; Fenyvessy, ibid., 30; Bonanni.
Hofmeister's Beitrage, 1; Lawrow, Ber. d. d. chem. Gesellsch., 33.

2 Zeitschr. f . physiol. Chem., 2.

3 Jaffe, Zeitschr. f . physiol. Chem., 43; Hildebrandt, Hofmeister's Beitrage, 7.

4 Baumann and Preusse, Zeitschr. f. physiol. Chem., 5; Jaffe", Ber. d. deutsch.
chem. Gesellsch., 12; Friedmann, Hofmeister's Beitrage, 4.

5 His, Arch. f. exp. Path. u. Pharm., 22; Cohn, Zeitschr. f. physiol. Chem., 18;
Abderhalden and collaborators, ibid., 59.

744 URINE.

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, chrysophinic 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

Eroducts of hydroquinone and humin substances. After naphthalene the urine
as 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 disgusting odor which is probably due to methylmercaptan, according
to M. NENCKi.1 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 repeatedly observed by many investigators, such as POSNER
PLOSZ, v. NOORDEN, LEUBE, and others. According to K. MORNER*
proteid regularly occurs as a normal urinary constituent to the extent
of 22-78 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, or of both. The monohydrogen calcium phosphate decom-
poses on boiling, and the normal phosphate may separate out. The

1 Arch. f. exp. Path. u. Pharm., 28. 2 Skand. Arch. f. Physiol., 6 (literature).

PROTEIDS. 745

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.

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 wine
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 98) : The urine is very
carefully floated on the surface of nitric acid in a test-tube. The presence
of proteid is shown by a white ring between the two liquids. With this
test a red or reddish-violet transparent ring is always obtained with normal
urine; it depends upon the indigo coloring-matters and can hardly be
mistaken for the white or whitish proteid ring, and this last must not be
mistaken for the ring produced by bile-pigments. 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 separa-
tion of urea nitrate may also appear. This ring consists of shining

746 URINE.

crystals, and it does not appear in urine previously diluted. A con-
fusion 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 nucleoalbumin. In this
case proceed as described below for the detection 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
second one, such as the heat test. In performing this test the (primary)
proteoses are also precipitated.

The reaction with metaphosphoric acid (see page 98) 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 ft 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
quantities 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 1 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.

ROCK'S Test. 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 pre-
cipitate the uric acid or the resin acids.2

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

JSpiegler, Wien. klin. Wochenschr., 1892, and Centralbl. f. d. klin. Med., 1893;
Jolles, Zeitschr. f. physiol. Chem., 21.

2 Pharmaceut. Centralbl., 1889, and Zeitschr. f . physiol. Chem., 29.

PROTEIDS. 747

ordinary cases HELLER'S test is sufficiently delicate. If no reaction is
obtained with this test within 2^ 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, SCHWEISSINGER *).

The different color reactions cannot be directly used, especially 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
of proteid, the precipitate obtained in the boiling test may be filtered, washed,
and then tested with MILLON'S reagent. The precipitate may also be dissolved in
dilute alkali and the biuret test applied to the solution. The presence of proteoses
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 proven 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 riot appear immediately, but only after
a certain time, and it must not be mistaken for the globulin precipitate.
In detecting seralbumiii heat the filtrate from the globulin precipitate
to boiling-point, or add about 1 per cent acetic acid to it at the ordinary
temperature.

1 Jolles, Zeitschr. f. anal. Chem., 29; Gnitzner, Chem. Centralbl., 1901, 1; Schweis-
singer, ibid.

748 URINE.

For the detection and also for the quantitative estimation of the various
globulins (fibringlobulin, euglobulin, and pseudoglobulin) OSWALD l has proposed
the fractional precipitation with ammonium sulphate.

Proteases 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-
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 addition
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 3) . The following modification by BANG of DEVOTO'S 4 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 £ 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 filtrate 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 recently somewhat modified by v. ALDOR and 6ERNY.5 In regard to other
more complicated methods we refer to HUPPERT-NEUBAUER.

MORAWITZ and DIETSCHY 6 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.

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 im Harn, Sputum und Eiter (Inaug.-Diss., Dorpat, 1891); E. Hirsch-
feldt, Ein Beitrag zur Frage der Peptonurie (Inaug.-Diss., Dorpat, 1892); and espe-
cially Stadelmann, Untersuchungen iiber die Peptonurie. Wiesbaden, 1894; Ehrstrom,
Bidrag till kannedomen om Albumosurien, Helsingfors, 1900; Ito, Deutsch. Arch,
f. klin. Med., 71.

3Salkowski, Berlin, klin. Wochenschr., 1897; Stokvis, Zeitschr. f. Biologic, 34.

4 Devoto, Zeitschr. f. physiol. Chem., 15; Bang, Detusch. med. Wochenschr., 1898

5 Salkowski, Centralbl. f. d. med. Wissensch., 1894; v. Aldor, Berl. klin. Wochenschr. r
36; Cerny, Zeitschr. f. analyt. Chem., 40.

6 Arch. f. exp. Path. u. Pharm., 54.

PROTEIDS. 749

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 III. 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 protoproteose the urine gives HELLER'S test, is precipitated even
in neutral solution on saturating with NaCl, but does not coagulate on boiling.
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 temperature
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
in the spinal marrow. It gives a precipitate on heating to 40-60° C.r
which on further heating to boiling dissolves again more or less completely,
depending upon the reaction and upon the amount of salt present. It
does not separate on dialysis, but can be precipitated 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). 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
hydroiytic cleavage products as the other proteins (ABDERHALDEN and
ROSTOSKI) . l

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 generally 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 writh MgSO4 added to saturation (HAMMAR-

1 Magnus-Levy, Zeitschr. f . physiol. Chem., 30 (literature) ; Grutterink and de
Graaff, ibid., 34 and 40; Moitessier, Compt. rend. soc. biolog., 57; Ville and Derrien,
ibid., 62; Abderhal-.V:i :irnl Rostoski, Zeitschr. f. physiol. Chem.. 46.

750 URINE.

STEN), or simply by adding an equal volume of a saturated neutral solution of
ammonium sulphate (HOFMEISTER and POHL *). 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.

ESBACH'S 2 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 TsuscHUA3 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, 4 of ROBERTS and STOLNI-
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.

Nudeoalbumin 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, 5 is of an entirely different kind.

All urine, according to MORNER, contains a little proteid and in
addition substances precipitating proteid. If the urine freed from salts
by dialysis is shaken with choloroform after the addition of 1-2 p. m.
acetic acid, a precipitate is obtained which acts like a nucleoalbumin.

1 Hammarsten, Pfliiger's Arch., 17; Hofmeister and Pohl, Arch. f. exp. Path. u.
Pharm., 20.

2 In regard to the literature on this method and the numerous experiments to deter-
mine its value, see Huppert-Neubauer, 10 Aufl., 853.

3 Christensen, Virchow's Arch., 115; Tsuschija, Centralbl. f. Med., 1908.

4 Detusch. med. Wochenschr., 1908.

5 Skand. Arch. f. Physiol., 6.

NUCLEO ALBUMINS. 751

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 chrondroitin-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
by MORNER to be a combination of proteid chiefly with chrondroitin-
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 Nudeoalbumins. 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 nitrate from the nucleoalbumin can be used for the ordinary proteid
tests.

Nudeohistone. In a case of pseudoleucsemia A. JOLLES found a phosphorized
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 Bum AN.1

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.

752 URINE.

Blood and Blood-coloring Matters. The urine may contain blood from
hemorrhage in the kidneys or other parts of the urinary passages (H.EMA-
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,
methaemoglobin (H^MOGLOBINURIA). 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 haemoglo-
binuria with fever. In hsemoglobinuria 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 VI.

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, wliich 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 beautiful
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 1 this reaction
is brought about by the blood pigments acting as catalyst upon the
organic peroxides existing in the turpentine, accelerating the decomposi-

1 Pfluger's Arch... 104.

BLOOD AND BLOOD COLORING MATTERS 753

tion of these and the active oxygen taken up by the guaiaconic acid
which is oxidized to guaiac blue (guaiaconic acid ozonide). Urine con-
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 J). 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 shouid be kepr,
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

HELLER-TEICHMAXN'S Test. If a neutral or faintly acid urine con-
taining blood is heated to boiling, one always obtains a mottled pre-
cipitate consisting of proteid and haBmatin. If caustic soda is added
to the boiling hot test, the liquid becomes clear and turns green when
examined in thin layers (due to hffimatin alkali), and a red precipitate,
appearing green by reflected light, re-forms, consisting of earthy phos-
phates and haBmatin. 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 hffimin test (see page 286). 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 h^min crystals.
If, on the contrary, the amount of phosphates is very small, then first
add a little MgCl2 solution to the urine, heat to boiling, and add simulta-
neously 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 ha3min crystals
(STRUVE 3) .

O. and R. ABLER 4 have recommended leucomalachite green or benzidine in
the presence of peroxide arid acetic acid as especially sensitive reagents for blood,

Haematoporphyrin. Since the occurrence of haBinatoporphyrin 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

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 haBmatoporphyrin in the urine may be
found in R. Zoja, Su qualche pigmento di alcune urine, etc., in Arch. Ital. di. clin.
Med., 1893.

754 URINE.

in the urine after sulfonal intoxication. It was first isolated in a pure
crystalline state by HAMMARSTEN l from the urine of insane women after
sulfonal intoxication. According to GARROD and SAiLLET2 traces of
hsematoporphyrin (SAILLET'S urospectrin) regularly occur in normal
urines. It is also found in the urine during different diseases, although
it occurs only in small quantities. It has been found in considerable
quantities in the urine after the lengthy use of sulfonal.

Urine containing hsematoporphyrin 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 hsematoporphyrin, but upon other red or
reddish-brown pigments which have not been sufficiently studied.

In the detection of small- quantities of hsematoporphyrin 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 by 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,S with a barium-acetate solution. The
washed precipitate, wrjich contains the hsematoporphyrin, 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 hsematoporphyrin in acid solution and gives
the spectrum of alkaline hsematoporphyrin 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 hsematoporphyrin, while the
upper layer of alcohol and water contains the other pigments besides
some hsematoporphyrin.

Other methods which have no advantage over this one of GARROD have been
suggested by RIVA and ZOJA as well as SAILLET. 4

BAUMSTARK 5 found in a case of leprosy two characteristic coloring-matters
in the urine, " urorubrohaematin " and " urofuscohsematin," which, as their names
indicate, seem to stand in close relation to the blood-coloring matters. Uro-
rubrohcematin, C68H94N8Fe2O2e, contains iron and shows in acid solution an absorp-

1 Salkowski, Zeitschr. f. physiol. Chem., 15; Hammarsten, Skand. Arch. f. Physiol., 3

2 Garrod, Journ. of Physiol., 13 (contains review of literature) and 17; Saillet,
Revue de M6decine, 16.

3 Salkowski, 1. c.; Hammarsten, 1. c.

4 Riva and Zoja, Maly's Jahresber., 24; Saillet, 1. c. See also Nebelthau, Zeitschr.
f. physiol. Chem., 27.

5 Pfliiger's Arch., 9.

PUS. BILE ACIDS. 755

tion-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 bro\vnish-red
non-dichroic liquid with alkalies. Urofuscohcematin, C68H106N8026, 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) haematoporphyrin.

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 XVI). 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. Urine containing melanin or melanogen is colored black by a ferric-chloride
solution (v. JAKSCH *).

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 DONNE'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 recognizeol 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 MAC-
KAY and v. UDRANSZKY and K. MORNER 2 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.

1 K. Morner, Zeitschr. f. physiol. Chem., 11; v. Jaksch, ibid., 13.

2 Cited from Huppert-Neubauer, Hani-Analyse, 10. Aufl. 229.

756 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 evapo-
ration 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, pre-
cipitate consisting of the alkali salts of the biliary acids is used in perform-
ing PETTENKOFER'S test.

HAYCRAFT has suggested a reaction for clinical purposes which consists in
sprinkling flowers of sulphur upon the urine. In icteric urine the powder quickly
sinks to the bottom, while in normal urine is remains on the surface. The value
of this test is still questioned.

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. In regard to the occurrence of
urobilin in icteric urine see p. 707.

Detection of Bile-coloring Matters in Urine. Many tests have been
proposed for the detection of these substances. Ordinarily we obtain
the best results either with GMELIN'S or with HUPPERT'S test.

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 CaCl2 solution,
and then with a solution of sodium or ammonium carbonate. The precipi-
tate which contains the bile-coloring matter is filtered, washed, dissolved
in alcohol which contains 5 cc. of concentrated hydrochloric acid in 100

BILE PIGMENTS. 757

cc. (I. MUNK), and heated to boiling when the solution becomes green
or bluish green. According to XAKAYAMA 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 408), 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 wiien 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 BaCl2 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,
wrhich 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.

BOUMA 2 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. 3

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
& test so that it showrs 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 notation test of OBERMAYER and POPPER 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.

SUGAR IN URINE.

The occurrence of traces of dextrose in the urine of perfectly healthy
persons has been, as above stated (page 711), 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-

1 Munk, Arch. f. (Anat. u.) Physiol., 1898; Nakayama, Zeitschr. f. physiol. Chem.,
36.

2 Deutsch. med. Wochenschr., 1902 and 1904.

3 Wien. med. Wochenschr., 1902 and 1904.

758 URINE.

tioned, and the reader is referred to Chapters VIII and IX for the essential
facts in regard to the appearance of sugar in the urine.

In man the appearance of dextrose 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 an elimination of 1 kilogram or even more of dextrose 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.

The properties and reactions of dextrose have been considered in a
previous chapter, and it remains but to mention the methods for the detec-
tion and quantitative determination of dextrose 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
page 207. This test may lead to very great mistakes in urines 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

SUGAR. 759

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 MULLER.* 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 per-
fectly 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 de-
scribed on page 208. 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.

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 3 on properly performing the test. 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, more-
over, 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

1 In regard to this test see Pfliiger, Pfliiger's Arch., 105 and 106; Hammarsten,
ibid., 116, and .Zeitschr. f . phyisol. Chem., 50.

2 Schondorff, Pfluger's Arch., 121; Bohmansson, Bioch. Zeitschr., 19.

3 Bechhold, Zeitschr. f. physiol. Chem., 46; Zeidlitz, Upsala Lakaref. Forh. (N. F.),
11 (Hammarsten Festschr).

760 TJRINE.

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. Like TROMMER'S
test it is a reduction test, and shows also certain other reducing bodies
besides the sugar. These bodies are certain conjugated glucuronic
acids which may appear in the urine. After the use of certain thera-
peutic 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, especially when the
reduction is not very great.

According to BOHMANSSON and BANG l this test is perfectly reliable
if about 10 cc. of the urine is treated with one-fifth volume of 25 per
cent HC1 and about 1 vol. moist animal charcoal (or J vol. dry) and shaken
for about one minute and then filtered. The filtrate on neutralization
with a few cubic centimeters of caustic soda is used for the ALMEN test.
The disturbing reducing substances are removed by the animal charcoal,
but the sugar is not.

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
sulphuric or hydrochloric acid, is treated with compressed yeast, or yeast
which has previously been washed by decantation with water. Pour
this urine to which the yeast has been added into a SCHR OTTER'S gas-
burette or a LOHNSTEIN'S saccharimeter (see below). As the fermenta-
tion proceeds, the carbon dioxide collects in the upper part of the tube,
while a corresponding quantity of liquid is expelled below. As a con-
trol 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 solution and yeast to determine the activity of the yeast.
According to VICTOROW 2 the fermentation is complete after six hours
at a temperature 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 car-
bon 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

1 Bioch. Zeitschr., 19. 2 Pfluger's Arch., 118.

SUGAR. 761

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 a little sulphuric acid, with yeast whose activity has been
tested by a special test on a sugar solution, and allow it to stand 24-30
hours at about 30°. 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, unfermentable
substances.

In performing the fermentation test care should be takea that the urine
be acid before as well as after fermentation. If the reaction becomes
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. According to v. JAKSCH this test is performed in
the following way : Add in a test-tube containing 6-8 cc. of the urine two
knife-pointfuls of phenyihydrazine hydrochloride and three knife-point fills
of sodium acetate, and when the salts do not dissolve on warming add
more water. The test-tube is placed in boiling water and warmed on
the water-bath. It is then placed in a beaker of cold water. If the
quantity of sugar present is not too small, a yellow crystalline precipitate
is now obtained. If the precipitate appears amorphous, there are found,
on looking at it under the miscroscope, yellow needles singly and in groups.
If very little sugar is present, pour the test into a conical glass and examine
the sediment. In this case at least a few phenylglucosazone crystals
are found, while the occurrence of larger and smaller yellow plates or
highly refractive brown globules does not show the presence of sugar.
This reaction is very reliable, and by it the presence of 0.03 per cent
sugar can be detected (ROSENFELD, GEYER 2) . In doubtful cases where
certainty is desired, prepare the crystals from a large quantity of urine,
dissolve them on the filter by pouring over them 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 levulose
gives the same osazone as dextrose, and that a further investigation is
necessary in certain cases.

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

1 On the performance of the fermentation test and certain sources of error, see
Salkowski, Berlin, klin. Wochenschr., 1905 (Ewald-Festnummer), and Pfliiger, Pfluger's
Arch., 105 and 111.

2 Rosenfeld, Deutsch. med. Wochenschr., 1888; Geyer, cited from Roos, Zeitschr.
f. physiol. Chem., 15.

762 URINE.

acid (30-per cent) saturated with sodium acetate, 2 drops of pure phenylhydrazine
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 beau-
tifully 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 reaction. SALKOWSKI * 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 HIRSCHL,
not to be apprehended when the test is heated in the water-bath for a
long time (one hour). KISTERMANN 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 HAM-
MARSTEN'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 cc.). Too great a delicacy of 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 dextrose 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 levo rotatory, may
contain 0.2 per cent sugar 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 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 of the same may be prepared according to BAUMANN'S msthod. 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 tho
odor of benzoyl chloride has disappeared. After standing sufficiently long the
ester is collected, finely divided, and saponified with an alcoholic solution of sodium

1 Neumann, Arch. f. (Anat. u.) Physiol., 1899, Suppl. See also Margulies, Berlin,
klin. Wochenschr., 1900; Salkowski, Arbeiter aus dem pathol. Inst., Berlin, 1906.

2 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 Pfluger's Arch., 91.

DETERMINATION OF SUGAR. 763

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 sus-
pended 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
negative 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 recommended 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,S 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 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 con-
stituents, 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. dextrose (see page 711). In such cases the titra-
tion procedure must be employed in connection with the fermentation
method, which will be described later.

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

1 Zeitschr. f. physiol. Chem., 19.

2 Pfliiger's Arch., 121, which cites the work of Patein and Dufau.

3 See Roos and Treupel, Zeitschr. f . physiol. Chem., 15 and 16.

764 URINE.

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 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 (b) a solution containing 6.55 grams hydrox-
ylamine sulphate in 2 liters.

The copper solution is prepared in the following manner : 500 grams potassium
carbonate, 400 grams potassium thiocyanate and 100 grams potassium bicar-
bonate are dissolved in 1200 cc. water in a graduated flask and if necessary
warmed to 50-60° C. On cooling to the ordinary temperature add very slowly
150 cc. of a cool, aqueous solution of cupric sulphate which contains 25 grams
cupric sulphate (CuSO4-h5H20) in 150 cc.; then add water up to 2 liters. After
standing at least 24 hours filter, and this solution can be kept indefinitely. The
hydroxylamine solution is prepared by dissolving 200 grams potassium thiocyan-
ate 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 also 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 cop-
per 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. If more than 3 per cent sugar is
suspected in the urine the latter must be diluted with a known amount of
water. In the estimation 10 cc. of the fluid containing sugar is always
used. If the urine contains less than 0.6 per cent sugar, then 10 cc. are
used; otherwise, according to the amount of sugar, 5-2 cc. of the urine
are diluted with water to 10 cc. and this used in the determination.

Performance of the determination. 10 cc. of the sugar fluid are placed
in a g]ass 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-
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.
This yellow coloration may disturb the end reaction somewhat, so that
with inexperienced workers an error of 0.5 cc. hydroxylamine solution
(corresponding to 0.5 milligram sugar) may be the result. If necessary
the urine can be decolorized, according to ANDERSEN, by mercuric nitrate.

1 Journ. f. prakt. Chem., (N. F.), 21.

2 Pavy, The Physiology of the Carbohydrates, London, 1894; Kumagawa and Suto,
Salkowski's Festschr., 1904; Sahli, Deutsch. med. Wochenschr., 1905.

3 Bioch. Zeitschr., 2 and 11. See also Funk, Zeitschr. f. physiol. chem., 56; Jessen-
Hansen, Bioch. Zeitschr., 10 and Andersen, ibid., 15.

DETERMINATION OF SUGAR.

765

The sugar in milligrams is directly obtained from the amount of hydroxyl-
amine solution used by referring to the following reduction table:1

Hydroxyl-
amine
solution
used.

Milligrams
sugar

Hydro xyl-
amine
solution
u^ed.

Milligrams
sugar.

Hydroxyl-
amine
solution
u.,ed

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.

For exact determinations of sugar the method as suggested by ALLIHN
and modified by PFLUGER2 is the best suited.

The TITRATION ACCORDING TO KNAP? depends on the fact that mercuric
cyanide in alkaline solution is reduced to metallic mercury by dextrose. 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 ec. of this solution should correspond to exactly 0.05 gram of dextrose.
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 preliminary
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 KNAP.P'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 arid allow the diluted
urine to flow gradually into the hot solution, at first 2 cc., then 1 cc., then 0.5 cc.,

1 This table is given with the permission of the publisher, Julius Springer, Berlin.

2 Pfliiger's Arch., 66.

766 URINE.

then 0.2 cc., and lastly 0.1 cc. After each addition let it boil £ 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 (OTTO J)- The calculations are just as simple as for the
previous method.

This titration, unlike the previous one, may be performed equally well by
daylight and by artificial light. KNAPP'S method has the following advantages
over FEHLING'S method: It is applicable even when the quantity of sugar in
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 hydro-
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 24 hours the fermentation is ordinarily ended, but th'is 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 pyknometer sup-
plied with a thermometer and an expansion-tube, this method, when the
quantity of sugar is not less than 4-5 p. m., gives, according to WORM-
MULLER, very exact results, but this has been disputed by BUDDE.S

1 Journal f. prakt. Chem., 26.

2 Pfliiger's Arch., 16 and 23.

3 Roberts, Edinburgh Med. Journ., 1861, and The Lancet, 1, 1862; Worm-Miiller,

DETERMINATION OF SUGAR. 767

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.

^'hen the quantity of sugar is less than 5 p. m. these methods cannot
be used. Such small amounts cannot, as already mentioned, be deter-
mined by titration directly, because the reducing power of normal urine
corresponds to 4-5 p. m. of sugar. In such cases, according to WORM-
MULLER, it is better to first determine the reducing power of the urine
by titration with KNAPP'S solution, then ferment the urine with the
addition of yeast and titrate again with KNAPP'S solution. The dif-
ference 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 CO2 can be estimated or the volume
of the gas measured. For this last purpose LOHNSTEIN 1 has constructed
a special fermentation saccharometer, and his " precision saccharometer "
is to be recommended. Based upon LOHNSTEIN'S instrument, WAG-
NER2 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 dextrose. The
urine may contain several levo rotator}' substances such as proteids, /?-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 levulose is detected in a
special manner (see below), and the dextrorotatory milk-sugar differs
from dextrose in its not fermenting readily. By using a delicate instru-
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.

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

Pfliiger's Arch., 33 and 37; Budde, ibid., 40, and Zeitschr. f. physiol. Chem., 13. See
also Huppert-Neubauer, 10. Aufl., and Lohnstein, Pfliiger's Arch., 62.

1 Berlin, klin. Wochenschr., 35, and Allg. med. Central-Ztg., 1899; Goldman, Chem.
Centralbl., 1907, 1, 1149.

2 Miinch. med. Wochensch., 1905.

768 URINE.

have been found where levulose exists in the urine besides dextrose.
Reports on this subject do not agree however.1

Levulose 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 phenyiglucosazone. With
methylphenyJhydrazine it gives the characteristic levulose 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
give the reaction (see page 211 and the works of ROSIN and UMBER2).
After heating and cooling it can be neutralized with soda and shaken
out with amyl alcohol, or with acetic ether (BORCHARDT). The amyi
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 levulose 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, for one minute (BORCHARDT). In
order to remove other disturbing pigments MALFATTI 3 suggests the
oxidation of the urine with a little hydrochloric acid and potassium
permanganate.

Maltose sometimes occurs in the urine according to Lepine and Boulud, and
to Geelmuyden 4 and the latter has suggested a method of detecting maltose in
the presence of dextrose by means of the fractional precipitation of the osazones.

Laiose is a substance named by HUPPERT and found by LEO 5 in diabetic urines
in certain cases, and which he considers 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.

Lactose. The appearance of lactose in the urine of pregnant women
was first 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 IX on absorption). LANGSTEIN and STEINITZ have
observed the passage of lactose and also of galactose 6 into the urine of
nurslings with diseases of the stomach. The passage of lactose into the
urine is called lactosuria.

1 See Borchardt, Zeitschr. f. physiol. Chem., 55, W. Voit, ibid., 58.

2 Umber, Salkowski's Festschrift, Berlin, 1904; Rosin, ibid., and Zeitschr. f. physioL
Chem., 38.

3 Rosin, 1. c.; Borchardt, 1. c.; Malfatti, Zeitschr. f. physiol. Chem.,- 58.

4 Zeitschr. f. klin. Med., 63; Lepine and Boulud, Compt. rend., 132.

5 Virchow's Arch., 107.

6 Hofmeister, Zeitschr. f. physiol. Chem., 1, which also contains the pertinent
literature. See also Lemaire, ibid., 21 ; Laiigstein and Steinitz, Hofmeister's Beitrage, 7»

PENTOSES. 769

The positive detection of this sugar in the urine is difficult, because
it is, like dextrose, 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/ best performed by using pure cultivated
yeact (saccharomyces apiculatus). This yeast only ferments the dex-
trose, while it does not decompose the milk-sugar. VOIT claims that if
RUBNER'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.2

R. BAUER 3 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 phenyl hydrazine
reaction directly, but only after boiling with an acid. The reason of this is not
known, but in a case examined by SMOLENSK! 4 the reaction to all appearances
was due to cane-sugar.

Pentoses. SALKOWSKI and JASTROWITZ 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
KfJLZ and VOGEL small amounts of pentose also occur in the urine of
diabetics, as also in the urine of dogs with pancreatic or phlorhizin diabetes.5

The pentose isolated by NEUBERG from the urine in chronic pentosuria
was i-arabinose. LUZZATTO has studied a case of pentosuria and found
/-arabinose (see page 203). 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.G

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 dextrose small amounts of pentose may also undergo fermentation.
The preparation of the osazone serves in the detection of pentoses;

1 Carl Voit, Ueber Die Glycogenbildung nach Aufnahme verschiedener Zuckeraten,
Zeitsehr. f. Biologie, 28.

. 2 Hofmeister Zeitsehr. f. physiol. Chem., 1, which also contains the pertinent
literature.

3 Zeitsehr. f. physiol. Chem., 51.

4 Ibid., 60.

5 In regard to the literature, see footnote 1, page 201. See also Blumenthal,
" Die Pentosurie," Deutsche Klinik, 1902.

6 Blumenthal, Deutsche Klinik, 1902; v. Jaksch, Centralbl. f. innere Medizin, 1906.

770 URINE.

this compound when pure melts at 166-168° C., but when obtained from
the urine has a melting-point of 156-160° C. The phloroglucin or orcin
tests can also be employed (see page 202). 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 is 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 * 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 pen-
tose the liquid turns a beautiful green. The usefulness 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 reac-
tion we refer to page 203. The same for the quantitative estimation
of pentoses (page 202) .

ROSENBERGER '* believes he has detected a heptose in the urine in a case of
diabetes. According to him and to GEELMTJYDEN 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 dextrose and levulose can be pre-
vented by the fermentation test. They may also be distinguished from
dextrose 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

^eutsch. med. Wochenschr., 1903; see also footnote 3, page 203.

2 Zeitschr. f . physiol. Chem., 49.

3 Rosenberger, Centralbl. f. inn. Med., 28; Geelmuyden, Zeitschr. f. klin. Med.,
58 and 63.

CONJUGATED GLUCURONIC ACIDS. 771

be isolated and identified by their osazones. The occurrence of conjugated
glucuronic acid 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. hydrochloric acid (sp.gr. 1.19), boil for
one minute, allow to stand four minutes, 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 216.

A further proof is that suggested by v. ALFTHAN;2 500 cc. of the
urine is benzoylated and the ester obtained saponified with sodium
ethylate. The free and conjugated glucuronic acid is thus obtained as
sodium compounds, insoluble in alcohol, while the pentoses, if present,
remain in the alcoholic filtrate. We have no sufficient experience as to
the value of this method.

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 216) with p-bromphenylhydrazine hydrochloride and sodium
acetate. HERVIEUX 3 has slightly modified this method.

Inosite seems to be a normal urinary constituent, although it occurs
only in very small quantities (HOPPE-SEYLER, STARKENSTEIN 4) . 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 552,
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 especially
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).

1 Ber. d. d. chem. Gesellsch., 41, 1788, and C. Tollens, Zeitschr. f. physiol. Chem.,
56. See also Mandel and Neuberg, Bioch. Zeitschr., 13.

2 Arch. f. exp. Path. u. Pharm., 47.

3 Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Hervieux, Compt. rend. soe.
biol., 63.

4 Starkenstein, Zeitschr. f . exp. Path. u. Therap., 5, which contains the literature..

5 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

772 URI:;E.

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
and acetoacetic acid during inanition (v. JAKSCH, FR. MULLER*). 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 product
from gelatin and protein (BLUMENTHAL and NEUBERG, ORGLER2).
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, BLUM, BORCHARDT and
L.ANGE,4 has showp. jbhat there can be no doubt that leucine in par-
ticular is a strong acetone former, and consequently that acetone can be
formed from protein. Prot amines and hist ones 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

Stoffwechsels. Berlin, 1906, and for recent work, Magnus-Levy, Die Azetonkorper,
Ergbn. d. inn. Med. u. Kinderheilk., I.

1 v. Jaksch, Ueber Acetonurie und Diaceturie. Berlin, 1885; Fr. Midler, Bericht '
iiber die Ergebnisse des an Cetti ausgefiihrten Hungerversuches. Berlin, klin. Woclien-
schr., 1887.

2 Blumenthal and Neuberg. Deutsch. med. Wochenschr., 1901; Orgler, Hofmeis-
ter's Beitrage, 1.

3 Hofmeister's Beitrage, 8.

4Erabden, ibid., 11, with Marx, I^ngel, Lattes and Michaud, ibid., 11; Baer and
Blum, Arch. f. exp. Path. u. Pharm.. 55 and 56; Borchardt, ibid., 53, with Lange,
Hofmeister's Beitrage, 9.

ACETONE BODIES. 773

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
quantity of protein, and an increase in the latter above the average causes
a diminution in the elimination of acetone (ROSEXFELD, HIRSCHFELD,
FR. VoiT1). The carbohydrates cannot be considered as material' for
the formation 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), tartaric
acid, lactic acid and citric acid (SATTA),alanine and asparagin (BORCHARDT
and LANGE 2) . Certain bodies like glycerin, lactic acid, alanine, asparagin,
which cause a sugar formation or increased elimination 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 (M ARUM 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 (/9-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
the body fat is consumed, and in several cases a certain relation has

1 Hirschfeld, Zeitsclir. f. klin. Med., 28; Geelmuyden, see Maly's Jahresber., 26,
and Zcitschr. f. physiol. Chem., 23 and 26; Rosenfeld, Centralbl. f. innere Med., 16;
Voit, Deutsch. Arch. f. klin. Med., 60.

2 1. c. Hofmeister's Beitriige, 9, which also cites the other works.

3 See footnote 1.

4 Hofmeister's Beitriige, 10.

774 URINE.

been found between the fat consumed and the acetone bodies eliminated.
Certain investigators (GEELMUYDEN, SCHWARZ, WALDVOGEL x) have
also observed an increase in the acetonuria on partaking of fatty food,
and at the present time 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 /9-oxybutyric acid, and this last is considered
as the mother-substance of the other two. If /?-oxybutyric acid,
CH3.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, CH3.CO.CH2.COOH. This acid can also be burnt,
but if large quantities are introduced it appears in part in the urine and
readily splits into acetone, CH3.CO.CH3, 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 /?-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 /?-oxybutyric acid (acidosis).

That acetone bodies can be formed from proteins is shown by the
perfusion experiments of EMBDEN and ENGEL with livers and leucine,
where acetone is formed in the liver with acetoacetic acid as intermediary
body. It is also probable, in correspondence with the observations of
BAER and BLUM,2 that in diabetics leucine and isovalerianic acid cause an
increase in the elimination of /9-oxybutyric acid, and that /?-oxybutyric acid

CH3X
is formed from leucine, with isovaleric acid, )>CH.CH2COOH, as

CH/

intermediary step. In the formation of acetone from tyrosine and
phenylalanine, acetoacetic acid is formed as an intermediary, and probably
also /?-oxy butyric acid (EMBDEN and ENGEL).

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 MARX3 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

1 Magnus-Levy, Arch. f. exp. Path. u. Pharm., 42; Geelmuyden, 1. c., and Norsk,
Magazin for Laegevidenskaben, 1900, see also Zeitschr. f. physiol. Chem., 41, Schwarz,
Deutsch. Arch. f. klin. Med., 1903; Waldvogel, Centralbl. f. innere Med., 20.

2 Arch. f. exp. Path. u. Pharm., 55 and 56; Embden and Engel, Hofmeister's Bei-
tra-e, 11.

3 Hofmeister's Beitrage. 11.

ACETONE. 775

are without action in this regard. This is true at least for the acids from
?i-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 ^-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 which take part in the formation of
/?-oxybutyric acid. A synthetical formation of /?-oxybutyric acid has
been accomplished by GEELMUYDEN and others, but especially by MAG-
NUS-LEVY, starting with acetaldehyde, according to the hypothesis of
SPIRO. It is also interesting that FRIEDMANN 1 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.COII
= CH3.CH(OH).CH2.COH, and that ^-oxybutyric 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,2 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.

Acetone, C3H6O, dimethylketone, CH3.CO.CH-.5, is a thin, water-
clear liquid, boiling at 56.3° and possessing a pleasant odor of fruit,
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 al)
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-

1 Geelmuyden, Zeitschr. f. physiol. Chem., 23 and 26; Magnus-Levy, Arch. f. exp.
Path. u. Pharm., 42; Friedmann, Hofmeister's Beitrage, 11.

2Embden and Lattes, Hofmeister's Beitrage, 11; Embden and Michaud, ibid., 11.

776 URINE.

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.

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 section, 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 l 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
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.

BELA v. BITTO'S 2 reaction is based on the fact that on adding a solution of
metadinitrobenzene, made alkaline with caustic potash, to acetone, a violet-red
color is produced which becomes cherry-red on acidifying with an organic acid or
metaphosphoric acid. Aldehyde gives a similar violet-red color which becomes
yellowish-red on acidification. Creatinine does not give this reaction. FROM-
MER 3 has suggested the following method for detecting acetone: Treat 10 cc.
of the urine with 1 gram potassium hydroxide and add 10-20 drops of an alkaline
solution of salicyl-aldehyde. On warming a purple-red coloration is obtained in
the presence of acetone.

Acetoacetic acid, C4H6O3, acetylacetic acid, diacetic acid, CH3.CO.
CH2-COOH, is a. colorless, strongly acid liquid which mixes with water,

1 Journ. of Physiol., 87.

2 Annal. d. Chem. u. Pharm., 269.

3 Berlin, klin. Wochenschr., 1905.

ACETOACETIC ACID. 777

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 account of the decom-
position 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. HC1 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 0.0 1 p.m. acetoacetic acid. Large
amounts of acetone (but not the quantity occurring in urines) give this
reaction according to ALLARD.1

BONDI and ScnwARz's2 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 irritating vapors of iodo-acetone will
attack the eyes. Acetone does not give this reaction.

Detection of Acetone and Acetoacetic Acid 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

1 Arnold, Wicn. klin. Wochenschr., 1899, and Centralbl. f. innere Mod., 1900;
Lipliawsky, Deutsch. med. Wochenschr., 1901; Allard, Berl. klin. Wochenschr., 1901.

2 Wien. klin. Wochenschr., 1906.

778 URINE.

acid make the urine slightly alkaline and shake in a separatory funnel
with ether free from alcohol and acetone. Remove the ether arid 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 PENZOLDT'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 thy 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 distilled
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 dropwise 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 to3; EMBDEN, and
SCHLIEP and FOLIN 4 have suggested methods for determining the quan-
tity of acetone and acetoacetic acid separately.

/2-Oxybutyric Acid, C4H8O3, 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)D= —24.12°
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 71-72a
C., and water, CH3.CH(OH).CH2.COOH = H2O+CH3.CH:CH.COOH,
It yields acetone on oxidation with a chromic-acid mixture.

1 See also Salkowski, Pfliiger's Arch., 56.

2 Hofmeister's Beitrage, 8.

3 Hoppe-Seyler, Thierf elder, 8. Aufl., 617 and 618.

*Embden and Schliep, Centralbl. f. d. ges. Phys. u. Path. d. Stoffwechsel, 1907;
Folin, Journ. of biol. Chein., 3.

/5-OXYBUTYRIC ACID 779

Detection of ft-Qxybuiyric 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 with ether, evaporate,
and test the melting-point of the residue which has been washed with
water. According to MINKOWSKI the acid may be isolated as a silver
salt.1

The quantitative estimation is done by complete extraction of the
3-oxybutyric acid by ether and determining the specific rotation. The
extraction can be done according to MAGNUS-LEVY 2 or according to
BERGELL.3 Other methods of estimating /?-oxybutyric acid have been
suggested by DARMSTADTER, BOEKELMAN and BouMA.4

EHRLicn's5 Urine Test. Mix 250 cc. of a solution which contains 50 cc. of
HC1 and 1 gram of sulphanilic acid is 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
certain time give red azo bodies which color the upper layer of the phos-
phate sediment). In pathological urines there sometimes occurs (and this
is the characteristic diazo reaction) a primary yellow coloration, with a 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 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 reaction are the aromatic oxyacids, antoxyproteic acid and the imidazole
derivative found by ENGELAND (see page 719).

Another urine test suggested by EHRLICH consists in adding hydrochloric
acid containing 2 per cent dimethylaminobenzaldehyde to the urine ; normal urines
arc colored faintly red, while certain pathological urines become cherry-red.
The cause of this reaction is not sufficiently known; according to NEUBAUER it
appears to be connected with the urobilinogen. HERTER" found that it was
increased by a meat diet.

1 Arch. f. exp. Path. u. Pharm., 18, 35; Zeitschr. f. anal. Chem., 24, 153.

2 See Hoppe-Seyler, Thierf elder's Handbuch, 8. Aufl., 619, and Geelmuyden, Ham-
marsten's Festschr., 1906.

3 Zeitschr. f. physiol. Chem., 33.

4 Darmstadter, ibid., 37; Boekelman and Bouma, see Mary's Jahresber , 31.

5 Ehrlich, Zeitschr. f. klin. Med., 5. See also Clemens, Deutsch. Arch. f. klin.
Med., 63 (literature). Kutscher and Engeland, footnote 1, page 719.

8 See Proscher, Zeitschr. f. physiol. Chem., 31, and Clemens, Deutsch. Arch, f
klin. Med., 71; Neubauer, Centralbl. f. Physiol., 19, 145; HERTER, Journ. of biol.
Chem.. 4.

780 URINE.

ROSEN BACH'S urine test, which consists in adding nitric acid drop by
to the boiling-hot urine and obtaining a claret-red coloration and a bluish-iv 1
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.

Amino-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 717), but also
in pathological urines.2

Cystine (see page 146). BAUMANN and GOLDMANNS 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 phosphorus. Cystine itself is only found with pos-
itiveness, and £ven then very rarely, in urinary calculi and in pathologi-
cal 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 (pentamethylen-
diamine) and putrescine (tetramethylendiamine) , which are produced
in the putrefaction of proteins. These two diamines were also found
in the contents of the intestine in cystinuria, while under normal condi-
tions they are not present. HAMMARSTEN therefore considers that per-
haps some connection exists between the formation of diamines in the
intestine, by the peculiar putrefaction in cystinuria, and cystinuria
itself. This is less probable, and cystinuria is, as generally admitted,
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

1 See Rosin, Virchow's Arch., 123.

2 Ignatowski, Zeitschr. f. physiol. Chem., 42; Abderhalden and Schittenhelm, ibid.,.
45; Abderhalden and Barker, ibid., 42. See also footnote 5, page 717.

3 Baumann, Zeitschr. f. physiol. Chem., 8. In regard to the literature on cystinuria
see Brenzinger, ibid., 16; Baumann and Coldmann, ibid., 12; Baumann and v. Udran-
szky, ibid., 13; Stadthagen and Brieger, Berlin, klin. Wochenschr., 1889; Cammidge
and Carrod, 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.

CYSTINE. 781

cystinurics, at least sometimes, such cystine introduced is quantitatively
transformed.1 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 CAMMIDOE and CARROD 2 in one case, that the
diamines occur only from time to time in the feces. The properties
and reactions of cystine have been given on pages 147 and 148.

Cystine is easLy prepared from cystine calculi by dissolving them
in ti.kaii 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 GOLDMAN.
For the detection and estimation of cystine we can proceed to advantage
in the following manner, suggested by GASKELLS: 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 wrhich precipitate are dissolved in ammonia and then purified
by reprecipitation with acetone.

VII. 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 constituent^. 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.

As previously mentioned (page 639), the urine of healthy individuals
may sometimes, even on voiding, be cloudy on account of the phosphates
present, or become so alter a little while because of the separation of
urates. As a rule, urine just voided is clear, and after cooling shows

1 See Wolf and Schaffer, Journ. of biol. Chem., 4.
2 1. c. footnote 2, page 780.
3 Journ. of Physiol., 36:

782 URINE.

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 writh the mates of the urine. Monohydrogen
phosphates besides acid urates 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 ureae, bacterium urea?, and
other bacteria. Muscunjs2 has isolated an enzyme from the micro-
coccus urese which decomposes urea, is soluble in water and is called
urease. 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.

•
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

1 See Huppert-Neubauer, 10. Aufl., and A. Ritter, Zeitschr. f. Biologic, 35.

2 Musculus, Pfliiger's Arch., 12.

3 Salkowski, Zeitschr. f. physiol. Chem., 13.

NON-ORGANIZED SEDIMENTS. 783

with a drop of hydrochloric acid small crystals of uric acid are obtained
which can be easily seen under the microscope.

Acid Urates. 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 thornapple. 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 oxalati 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 782), 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.

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.

784 URIXE.

It is not yellow or brown like ammonium urate, and does not give the
murexid test.

Calcium Phosphate. The CALCIUM TRIPHOSPHATE, Ca3(PO4)2, 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, CaHPO4+2H2O, occurs in neutral
or only in veiy faintly acid urine.1 It is found sometimes as a thin
fiim 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 rnurexid 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.

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, Mg3(PO4)2, occurs with calcium triphosphate
in urines rendered alkaline by a fixed alkali. Crystalline magnesium
phosphate, Mg3(PO4)2H-22H2O, has been observed in a few cases in
human urine (also in horse's urine) as strongly refractive, long rhombic
plates.

Kyestein is the film which appears after a little while on the surface of the urine.
This coating, which was formerly considered as characteristic of urine in preg-
nancy, contains various elements, such as fungi, vibriones, epithelium-cells, etc.
It often contains earthy phosphates and triple-phosphate crystals.

As more rare sediments we find cystine, tyrosine, hippuric acid, xanthine, h&ma-
toidine. In alkaline urine blue crytsals of indigo may also occur, due to a decom-
position of indoxyl-glucuronic acid.

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. EBSTEiN2 considers the essential difference between an

1 C. Th. Morner, Zeitschr. f. physiol. Cliem., 58.

2 Die Natur und Behandlung der Harnsteine. Wiesbaden, 1884.

URINARY CALCULI. 7S5

amorphous and crystalline sediment in the urine on 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 a calculus and its further development take place
in an undecomposed 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 cystine; 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.

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.

786 URINE.

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 a pale powder. Thev
give the murexid test and develop much ammonia with caustic soda.

Calcium-oxalate 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-
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-

URINARY CALCULI. 787

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 tost.

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,* 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
-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.

1 Chem. Untersuch. z. wissensch. Med., 2. Cited from Maly's Jahresber., 19, 422.
3 Zeitschr. f. physiol. Chem., 18.

788

URINE.

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CHAPTER XVI.
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 x three different
substances can be detected in the horn substance, designated by him A,
B and C keratin.

The .d-keratin, which forms the envelope of the horn and hair cells arid 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 5-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
different substances and for this reason it is unnecessary to give the
older elementary analyses of the various epidermoidal structures.

The quantity of sulphur and of mineral bodies is of certain interest.
The sulphur and cystine content of these structures can be found on

1 Monatsch. f. prakt. Dermat., 44.

789

790 THE SKIN AND ITS SECRETIONS.

pages 112, 113 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 also 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 3 claims that at least a part of the silicic acid exists in the
feathers in organic combination as an ester.

According to GAUTIER and BERTRAND 4 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 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
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 dextrose, as shown first by FRANCHIMONT 5 and later confirmed by WIN-

TERSTEIN.

1 Journ. of Biol. Chem., 3.

2 Lehr. d. physiol. Chem., 4. Aufl., 660, 661; Baudrimont, ibid.

3 Centralbl. f. Physiol., 11, 361.

4 Gautier, Compt. rend., 129, 130, 131; Bertrand, ibid., 134.

5 Ambronn, Maly's Jahresber., 20; Berthelot, Annal. de Chim. et Phys., 56, Compt.

CHITIN. 791

Chitin is not found in vertebrates. In invertebrates chit in is alleged
to occur in several classes of animals; but it can be positively asserted
that true, typical chitin is found only in cephalopods and in articulated
animals especially, in which it forms the chief organic constituent of
the shell, etc. KRAWKOW 1 found that chitin of the shell, etc., does
not seem to occur free, but in combination with another substance, prob-
ably a protein-like body. In GILSON and WINTERSTEIN'S 2 investigations
chitin occurred in certain fungi as well.

According to SUNDVIK the formula of chitin is probably CeoHiooNgOas
+ n(H2O), where n may vary between 1 and 4. ARAKI claims that it
has on the contrary the composition Ci8H3oN2Oi2. KRAWKOW found
that the chitins of different origin show different action with iodine,
and he therefore concludes that there must exist quite a group of
chitins, which seem to be amino derivatives of different carbohydrates,
such as dextrose, glycogen, dextrins, etc. According to ZANDER 3 only
two chitins exist, one of which turns violet with iodine and zinc
chloride, and the other brown.

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. Chitin
can hardly be considered as a simple acetyl glucosamine, but probably
has a much more complicated constitution, which is not known at the
present. FRANKEL and KELLY as well as OFFER4 have obtained acetyl
glucosamine, C6HiiO5NH(COCH3) and acetyldiglucosamine (C^H^^Og)
COCH3 as characteristic cleavage products.

The acetyldiglucosamine has the same empirical formula, Ci4H26N2Oio,
as the chitosan prepared by ARAKI, but it is not identical therewith.
The chitosan, which v. FURTH and Russo 5 obtained as a crystalline
hydrochloric acid combination, has a different composition, and accord-
ing to them, is more likely a multiple of C^H^N^O^. On heating
with acetic anhydride chitosan is converted into a chitin-like substance
which is not identical with chitin. Chitosan is insoluble in water and
alkali, but dissolves in dilute acids. It splits into acetic acid and gluco-
samine by the action of hydrochloric acid. According to v. FURTH and

rend., 47; Winterstein, Zeitschr. f. physiol. Chem., 18; Franchimont, Ber. d. deutsch
chem. Gesellsch., 12.

1 Zeitschr. f . Biologic, 29.

2 Gilson, Compt. rend., 120; Winterstein, Ber. d. deutsch. chem. Gesellsch., 27 and 28.

3 Sundvik, Zeitschr. f. physiol. Chem., 5; Araki, ibid., 20; Zander, Pfluger's Arch., 66.

4 Ledderhose, Zeitschr. f. physiol. Chem., 2 and 4; Araki, 1. c.; Frankel and Kelly,
Monatshefte f. Chem., 23; Offer, Bioch. Zeitschr., 7.

5 Hofmeister's Beitrage, 8.

792 THE SKIN AND ITS SECRETIONS.

Russo on acid cleavage it yields 25 per cent acetic acid and 60 per cent
glucosamine.

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 chit ins 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.

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 Li JCKE 1 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.

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, epithelium-
cells of the retina, in sepia, in certain pathological formations, and in the
blood and urine in disease. Of these pigments there are a few, such as

1 Virchow's Arch., 19.

MELANINS. 793

'

SCHMIEDEBERG'S sarcomelanin, and that from the melanotic sarcomata
of horses, the hippomelanin (NENCKI, SIEBER, and BERDEZ), which are
soluble with difficulty in alkalies, while others, such as the coloring mat-
ter of certain pathological swellings in man, the phymatorhusin (NENCKI
and BERDEZ), are readily soluble, in alkalies. The humus-like products,
called melanoidic acids by SCHMIEDEBERG, obtained on boiling proteins
with mineral acids, are rather easily soluble in alkalies.

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 and of horse-hair,
are rather rich in sulphur (2-4 per cent), while the phymatorhusin found
in certain 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 deriv-
ative 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 sarco-
melanin (from a sarcomatous 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 sarcomelanin investigated by ZDAREK
and v. ZEYNEK also contained 0.4 per cent iron. Recently WOLFF. L
prepared two pigments from a melanotic liver, of which one was no
doubt modified. The other, which was soluble in a soda solution, con-
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 contrary, 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 product could not be obtained from the
pigment of the hair (SPIEGLER) nor from hippomelanin (v. FURTH and

JERUSALEM2).

1 Zdarek and v. Zeynek, Zeitschr. f. physiol. Chem., 36; Wolff, Hofmeister's Beitragc,
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 Beitrage, 4, and especially v. Fiirth,
Centralbl. f. allg. Path. u. Path. Anat., 15, 1907, 617.

2 Wolff , Hofmeister's Ec'ir".-^, 5; Spiegler, ibid., 10; v. Fiirth and Jerusalem,
ibid.. TO.

794 THE SKIN AND ITS SECRETIONS.

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 be of secondary importance only.

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
hsemopyrrol according to SPIEGLER. Several melanins — and this is also
true of the melanoids produced from proteins on cleavage with acids
(SAMUELY l) — 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 last two 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 others 3 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 4 has
also prepared an extract from the melanotic metastases of a primary
adrenal tumor which formed a dark-brown pigment from adrenalin
and p-oxyphenylethylamine, but not from tyrosine. As indicated

1 Hofmeister's Beitrage, 2.

2 Compt. rend., 122.

3 The literature can be found in v. Fiirth and Jerusalem, Hofmeister's Beitrage, 10.

4 Virchow's Arch., 192.

TETRONERYTHRIN, CARMINIC ACID. 795

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 oxyha3moglobin. It must be remarked that according to LAIDLAW l
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 2 found a large number of coloring matters in birds' 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 inver-
tebrates (HALLIBURTON, DE MEREJKOWSKI, MACMUNN). Besides tetronerythrin
MACMUNN found in the shells of crabs and lobsters a blue coloring matter cyano-
crystallin, which turns red with acids and by boiling water. Hoematoporphyrin,
according to MAcMuNN,3 also occurs in the integuments of certain of the lower
animals.

In certain butterflies (the pieridinse) the white pigment of the wings consists,
as shown by HOPKINS/ 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,S 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,6 cochenillw acid, C10H8O7, and coccinic acid.
G9H8O5, the first being the tri-carboxylic acid, and the other the di-carboxylic
acid, of w-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. Its chemical nature has not been investigated.

Among the remaining coloring matters found in invertebrates may be men-
tioned blue stentorin, actiniochrom, bonellin, polyperyihrin, pentacrinin, antedonin,
crustaceorubin, janihinin, 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

1 Journ. of Physiol., 31.

2 Vergleichende physiol. Studien, Abth. 5, and (2. Reihe) Abth. 1, 151, Abth. 2, 1,
and Abth. 3, 128.

3 \Yurm, cited from Maly's Jahresber., 1; Halliburton, Journ. of Physiol., 6; Merej-
kowski, Compt. rend., 93; MacMunn, Proc. Roy. Soc., 1883, and Journ. of Phyisol., 7.

t4 Phil. Trans., 186.

5 Pfliiger's Arch., 98.

6 Ber. d. deutsch. chem. Gesellsch., 30.

796 THE SKIN AND ITS SECRETIONS.

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 LINSER claim that true fat occurs only to
a very slight extent. On saponification the sebum gives an oil, dermolein,
which combines readily with iodine, and another body, dermocerin,
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
aermocerin 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 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. .

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,
LIEBREICH 3 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 role 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 aim SUNDVIK 4
found psylla-alcohol, C33H68O, which exists there as an ester in combination with
psyllic acid, C32H65COOH. This alcohol has also been found in the wax of the
humble-bee.

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; Riippel,
Zeitschr. f. physiol. Chem., 21; Ameseder, ibid., 52.

2 Monatsch. f. prakt. Dermat., 45. »

3 Virchow's Arch., 121.

4 Zeitschr. f . physiol. Chem., 17, 25, 32, 53 and 54.

CERUMEN, VARIOUS SECRETIONS. 797

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.1

The preputial secretion, smegma prceputii, contains chiefly fa*t, 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 2) .

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, C30H6o04, and lanopatmitic add, C16H32O3.
Isocholesterin, oxycholesterin and carnaubyl alcohol, C24H49OH, are besides the
two last-mentioned acids, substances that are characteristic of wool-fat. Accord-
ing to ROHMANN * wool-fat contains a body lanoc&rin, 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, C18H38O, 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 isorners of lauric and
myristic acid. The fatty acids are in great part combined with the oetadecylic
acid, and this is probably formed by the reduction of stearic acid or oleic 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 CASAU), bufotalin and the disputed bodies bufonin and bufotenin
(FAUST, BERTRAND and PHISALIX 4). Tkalassin is the crystalline body discovered
by RICHET 5 which is the poisonous constituent of the feelers of the sea nettle.

1 See Lamois and Martz, Maly's Jahresber., 27, 40.

2 Aldrich, Journ. of Exp. Med., 1; Beckmann, Maly's Jahresber., 26, 566.

3 Darmstadter 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., 234.

4De 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.

5 Pfliiger's Arch., 108.

798 THE SKIN AND ITS SECRETIONS.

The Perspiration. Of the secretions of the skin, whose quantity
amounts to about -^ 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 perspiration 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 1 showed that horse's sweat is strongly alkaline.

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. nu
water. The solids are 4.4-22.6 p. m. The molecular concenti ation
also varies widely and the freezing-point depression depends essentially
upon the content of NaCl. ARDIN-DELTEIL found J= —0.08-0.46°,
average-0.237°. BRIEGER and DISSELHORST found with perspiration

1 Triimpy and Luchsinger, Pfliiger's Arch., 18; Heuss, Maly's Jahresber., 22; Cam-
erer, Zeitschr. f. Biologic, 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.

PERSPIRATION. 799

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. The organic bodies are neutral fats, cholesterin,
volatile fatty acids, traces of protein (according to LECLERC and SMITH
always in horses, and according to GAUBE regularly in man, while LEUBE 2
claims only occasionally after hot baths, in BRIGHT'S disease, and after
the use of pilocarpin), creatinine (CAPRANICA), aromatic oxyacids, ethereal-
sulphuric acids of phenol and skatoxyl (KAST 3), sometimes also of indoxyl,
and lastly urea. The quantity of urea has been determined by ARGUTIN-
SKY. In two steam-bath experiments, in which in the course of 4
and J 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 ARGUNTINSKY'S experiments, and also from
those of CRAMER,4 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,5 KAST). The relation, according to
KAST, is as follows:

Chlorine

In perspiration 1

In urine . . . . 1

Phosphate
0.0015
0.1320

Sulphate
0.009
0.397

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 XV).
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

1 Ardin-Delteil, Maly's Jahresber., 30; Brieger and Disselhorst, Deutsch. med.
Wochenschr., 29; Tarugi and Tomasinelli, cited in Physiol. CentralbL, 22, 748.

2 Leclerc, Compt. rend., 107; Gaube, Maly's Jahresber., 22; Leube, Virchow's Arch.,
48 and 50, and Arch. f. klin. Med., 7.

3 Capranica, Maly's Jahresber., 12; Kast, Zeitschr. f. physiol. Chem., 11.

4 Argutinsky, Pfliiger's Arch., 46; Cramer, Arch. f. Hygiene, 10.

5 Compt. rend., 35. and Arch, gener. de Med. (5), 2.

800 THE SKIN AND ITS SECRETIONS.

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 l). 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 REGNAULT
and REISET, is small, and according to ZUELZER amounts under the
most favorable circumstances to T-J-{r 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 toWiLLEBRAND beginning at 33°) .2 It especially
increases with hyperaemia 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-
BRAND3 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,4 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 1\ 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, "l. c.

4 Arch. f. (Anat. u.), Physiol., 1894, and Maly's Jahresber., 24.

CHAPTER XVII.
CHEMISTRY OF RESPIRATION.

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.

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. LUDWIG and 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
experiments, calculated for 0° C. and 760 mm. pressure. The nitrogen

801

802 CHEMISTRY OF RESPIRATION.

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, a different temperature, alkalinity, rest and activity
cause a change. In regard to the gases they contain, the greatest dif-
ference 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. LOEWY 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

Oxygen is absorbed only to a small extent by the plasma, ' whose
absorbability for oxygen is 97.5 per cent of that of water, according to

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, 33^13, 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.

GASES OF THE BLOOD. 803

BOHR. The greater part or nearly all of the oxygen is loosely combined
with the haemoglobin. The quantity of 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 exchange 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,1 ZuNTz,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. BOHR 4 claims
that about 30 mm. may be considered as the average pressure of the
carbon dioxide in the organsim, 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 CC>2, theie-
fore about 5 per cent of the total carbon dioxide is simply dissolved.
Under the assumption that the blood corpuscles make up about J of
the volume of the blood, of the physically dissolved CO2, 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. CC>2, only a small part of its CO2 is physi-
cally dissolved. The chief mass of the CO2 is loosely combined and the
constituent of these cells which unites with the CO2 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
alkali diphosphate takes place. It is generally admitted that the blood-
coloring matters, especially the oxyhaemoglobin, which can expel carbon

1 Ber. d. k. sachs. Gesellsch. d. Wissensch. math.-phys. Klasse, 1867.

2 Centralbl. 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 Handbucb
der Physiologic des Menschen, Bd. 1.

804 CHEMISTRY OF RESPIRATION.

dioxide from sodium carbonate in vacuo, acts like acids; and as the glob-
ulins 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 Tonur,2 have shown, be
able to hold the carbon dioxide loosely combined even in the absence
of alkali. BOHR has also found that the disassociation 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 jn
'the organism, the combination of the carbon dioxide in the blood
corpuscles 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
oil m a vacuum, while a smaller part can be removed only after the
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.

1 Centralbl. f. d. med. Wissensch., 1877. See also Zuntz in Hermann's Handbuch,
76.

« 3 Zuntz, 1. c., 76; Bohr, Maly's Jahresber., 17; Torup, ibid.
3 Centralbl. f. d. med. Wissenssh., 1867.

CARBON DIOXIDE OF THE BLOOD. 805

Absorption experiments with blood-serum have shown us further
that the carbon dioxide which can be pumped out is in great part loosely
chemically combined, and fiom 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
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 tlie bicarbonate,
in the serum from which the carbon dioxide can be set free by simple

1 E. Pfliiger's Ueber die Kolfcnsaure des Blutes, Bonn, 1864, 11. Cited from Zuntz
in Hermann's Handbuch, 65.

806 CHEMISTRY OF RESPIRATION.

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 ^rom 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 ammo bodies. SIEGFRIED has found that amino-acids com-
bine with carbon dioxide, thereby being converted into carbamino-

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

1 Hoppe-Seyler, Med. chem. Untersuch., 350.

2 Zeitschr. f. physiol. Chem., 44 and 46.

GASES OF THE LYMPH. 807

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, /?-oxybutyric acid
(STADELMANN, MINKOWSKI), and MINKOWSKI l 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 HENSEN2 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 These gases contained traces of
oxygen and consisted of 37.4—53.1 per cent CO2 and 1.6 per cent N at 0°
C. and 760 mm. Hg 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
animal, is explained by ZUNTZ 4 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.

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-

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.

3 Ber. d. k. sachs. Gesellsch. d. Wissensch., math.-phys. Klasse, 23.

4 Buchner, Arbeiten aus der physiol. Anstalt zu Leipzig, 1876; Zuntz, 1. c., 85.

808 CHEMISTRY OF RESPIRATION.

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 l
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 foi the
total quantity of carbon dioxide in milk and urine are 10 and 18.1-19.7
per cent respectively.

EWALD 2 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
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 the introduction (Chapter I, p. 3) 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

1 Pfliiger, Pfliiger's Arch., 1 and 2; Kiilz, Zeitschr. f. Biologie 23. It seems as if Kiilz's
results were not calculated at 760 millimeters Hg, but rather at 1 meter.

2 C. A. Ewald, Arch. f. (Anat. u.) Physiol., 1873 and 1876.

EXTERNAL AND INTERNAL RESPIRATION. 800

tissues. It IB nevertheless true that oxidations take place in the blood,
although 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 l the lungs take a variable but
always an 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 secretory 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.

Oxygen occurs in the blood in a disproportionately large part as
oxy haemoglobin, and the law of the dissociation of oxyhaemoglobin 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 a
pure solution of hemoglobin, and HtiFNER2 has made very careful and
important determinations on such solutions. Recent investigations
of BoHR3 and his pupils, as well as of LOEWY and ZuNTZ,4 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

1 Centralbl. f . Physiol., 6, and Maly's Jahresber., 27.

2 Arch. f. (Anat. u.) Physiol., 1890 and 1894.

3 See Nagel's Handbuch, and Krogh, Skand. Arch. f. Physiol., 16.

4 Arch. f. (Anat. u.) Physiol., 1904.

810 CHEMISTRY OF RESPIRATION.

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 l 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 con-
centration of the haemoglobin manifests itself in that in dilute solutions
the oxygen is more firmly combined (HUFNER, LOEWY and ZUNTZ, BOHR)
and that consequently blood made laky with water has a lower dissocia-
tion tension and a firmer binding of the oxygen than undiluted blood.

Of especial interest is the finding of BOHR, HASSELBALCH and KROGH 2
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 3 upon horse's blood at 38° and a con-
stant carbon dioxide tension will be given below. In calculating the
results in column 4 the quantity of oxygen chemically combined at
150 mm. oxygen pressure is equal to 100.

In 100 cc. blood Oxygen taken up

Chemically Oxygen Per oont

. ~* ffiS* *;—

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.

1 Arch. f. (Anat. u.) Physiol., 1904.

2 Centralbl. f. Physiol., 17, and Skand. Arch. f. Physiol., 16.

3 Skand. Arch, f. Physiol., 16.

OXYGEN TENSION AND PARTIAL PRESSURE. 811

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 hemoglobin.

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 l 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 mentionable decrease observed. A. LOEWY 2 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 wTith 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 oxyhaBmo-
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,3 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, FAL-
LOISE), and by means of the lung-catheter (see below) equal to 25.5-27
mm. (WOLFBERG, NUSSBAUM). For human venous blood LOEWY and

1 Ueber die Wirkungen der verdiinnten Luft auf den Organismus., Berlin, 1883.

2 A. Loewy, Untersuch. iiber die Respiration und Zirculation, etc., Berlin, 1895;
also Centralbl. f. Physiol., 13, 449, and Arch. f. (Anat, u.) Physiol., 1900.

3 Bert, La pression barometrique, Paris, 1878; Herter, Zeitschr. f. physiol. Chem.,
3; Hiifner, 1. c.; Fr&lericq, Centralbl. f. Physiol., 7, and Travaux der laborat. de 1'inst.
de physiol. de Liege, 5, 1896.

812 CHEMISTRY OF RESPIRATION.

v. SCHROTTER 1 found an average of 37.68 mm. These results do not
coincide with the investigations of BoHR,2 who in many cases obtained
essentially higher figures for the oxygen tension in arterial blood.

He 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 h?emataerometer. 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
KROGH 3 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 FREDERicQ4 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
detends his experiments, but also finds errors in the experiments of his
opponents, while HALDANE and SMITH'S 5 experiments, making use of

1 Strassburg, Pfliiger's Arch., 6; Falloise, Bull. Acad. Roy. Belg., 1902; Wolfberg,
Pfliiger's Arch., 4 and 6; Nussbaum, ibid., 7; Loewy and v. Schro'tter, cited by Loewy
in Oppenheimer's Handb., 4, 76.

2 Skand. Arch. f. Physiol., 2, and Nagel's Handbuch der Physiologie.

3 Skand. Arch. f. Physiol., 20.

4 Hiifner, Arch. f. (Anat. u.) Physiol. 1890; Fre'dericq, Centralbl. f. Physiol., 7, and
Traveaux du laboratorie de Finstitute de physiologie de Liege, 5, 1896.

5 Haldane, Journ. of Physiol., 18; Haldane and Smith, ibid., 20.

OXYGEN TENSION. 813

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 oxgyen 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.

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.

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 :

/~i_ Nitrogen Carbon

Oxygen *

(and agon) Dioxide

Atomspheric 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 exphed 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 xesidue
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

1 See Zuntz, 1. c.. Hermann's Handbuch, 105 and 106.

814 CHEMISTRY OF RESPIRATION.

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 oxygen 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
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 specific activity of the lungs, and according to
him a secretory activity of the lungs also exists besides diffusion.

As opinions on the taking up of oxygen are disputed so also are those
on the giving up of caibon 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.1

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
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 carbon
dioxide of the gas mixture corresponds as closely as possible, in the beginning, to

1 Wolffberg, Pfliiger's Arch., 6; Strassburg, ibid.; Nussbaum, ibid., 7.

CARBON DIOXIDE TENSION. 815

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 alveoli 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
by 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 CC>2 in the air taken
out with the catheter. NUSSBAUM also determined the carbon-dioxide
tension in the blood Irom 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
place. The method of catheterizing the lungs is, as shown by LOEWY
and v. ScHROTTER,2 also applicable to man, and they found that the
carbon dioxide tension of human venous blood was equal to 6 per cent

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.

2 1. c., footnote 1, page 812.

816 CHEMISTRY OF RESPIRATION.

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
alveoli 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 812), 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, form 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 alveolar air is richer in carbon dioxide than the bifui-
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,1 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,2 in FREDERICQ'S laboratory has made
experiments with animals which respiied 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. In oppo-
sition to these investigations BOHR has presented strong objections; he
has demonstrated the principles for the construction of the tonometer, and
claims that the earlier experiments with the tonometer are not conclu-
sive, 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
in WERIGO. Still ZUNTZ has presented weighty objections to WERIGO'S
experiments, and BOHR 3 has later also shown that we have no positive
basis for the above assumption.

1 See footnote 3, page 811.

2 Centralbl. f. Physiol. 10, 482; Falloise, see Maly's Jahresber, 32.

3 Holmgren. Wien. Sitzungsber., 48 Werigo, Pfliiger's Arch., 51 and 52; Zuntz, ibid.,
52; Bohr, see Nagel's Handbuch der Physiologic.

SECRETION OF CARBON DIOXIDE. 817

The conditions as to the elimination of carbon dioxide in the lungs
are not quite clear, and from the above we see that in regard to the gas
exchange in the lungs we have two opposing theories. According to the
older view suggested by the PFLUGER school the exchange of gas follows
simple physical "laws and is on the whole a diffusion process. . Accord-
ing to BOHR'S opinion diffusion does take place, but the lung is a gland
which has the powei of secreting gases, and the gas exchange in the lungs is
essentially a secretory process.

The theory that a secretion of carbon dioxide takes place in the lungs
has been further supported by recent, investigations by Bonn.1

One group of experiments includes those carried out, essentially,
according to an earlier principle, but using the microtonometer of KROGH.
The other group includes those experiments where each lung breathed
by itself, the one atmospheric air and the other a gas mixture containing
a considerable quantity of carbon dioxide, namely, about 8 per cent.
The first group of experiments gave new support for the secretion
theory, but the second group were of still greater interest. In these
experiments — in which the alveolar air of both lungs had a different
quantity of carbon dioxide, for example in one about 3 per cent CC>2
and the other about 9 per cent, while the circulating blood was the same
in the two sepaiated respiring lungs (right heart blood) — the carbon
dioxide tension in the venous blood was always lower than in the air
of the CC>2 respiring lung, and nevertheless CO2 was eliminated in this
lung. This cannot be explained by the diffusion theory; and in the
opinion of HAMMARSTEN it cannot be disputed that the investigations
thus far reported seem to support BOHR'S theory strongly, and this is
also corroborated by the secretion ol gases detected in certain animals.

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. MOHEAIT 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
hore a secretion and not a diffusion of oxygen. Recently JAEGER 2 has given a
further explanation as to the secretory activity of the swimming-bladder.

1 Centralbl. f. Physiol., 21, 367.

2 Biot, 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.

818 CHEMISTRY OF RESPIRATION.

From what has been said above (page 809) 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 PrLUGER,1 and the oxygen tension in
blood on entering the capillaries must be higher. The oxygen tension
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
regulating and varying the oxygen tension, and such a means is the car-
bon dioxide produced in the tissue which, according to BOHR, HASSEL-
BACH, and KROGH,2 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 dissocia-
tion 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 combination to the quantity of iron of the blood
or the hemoglobin solution.

As the haemoglobin prepared from different blood portions does not, according
to BOHR, always take up the same quantity of oxygen for each gram, so the
haemoglobin within the blood-corpuscle may show a similar behavior. He calls
the quantity of oxygen (measured at 0° C. and 760 mm. Hg) which is taken up
by 1 gram of haemoglobin of the blood at 15° C. and an oxygen pressure of 150 mm.
the specific oxygen capacity.3 This quantity, he claims, may vary not only in
different individuals, but also in the different vascular systems of the same
animal, and it may also be changed experimentally by bleeding, breathing air
deficient in oxygen, or poisoning. It is now evident that one and the same quan-
tity of oxygen in the blood, other things being equal, must have a different ten-
sion according as the specific oxygen capacity is greater or smaller. The tension
of the oxygen, BOHR says, may be changed without changing the quantity of
oxygen, and the animal body must have means of varying the tension of the oxygen

1 Estor and Saint Pierre with Pfliiger in Pfliiger's Arch., 1.
'Centralbl. f. Physiol., 17, and Skand. Arch. f. Physiol., 16.
'Centralbl. f. Physiol., 4, and Nagel's Handb. d. Physiol.

RESPIRATORY EXCHANGE OF GAS. 819

in the tissues in a short time without changing the quantity of oxygen contained
in the blood. The importance for respiration of such a property of the tissues
is evident; but it is perhaps too early to give a positive opinion on BOHR'S state-
ments and experiments.

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 1 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 HEGNAULT
and REISET and of PETTENKOFER, determine the total gas exchange,
and indeed for a long time, while the three other methods determine the
respiratory gas exchange alone, and this only for a short time.

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 by
exactly measured quantities. This method, which also makes possible a direct
determination of the oxgyen used as well as the carbon dioxide produced, has since
been modified by other investigators, such as PFLUGER and his pupils, SEEGETNT
and NOWAK, and HOPPE-SEYLER, ROSENTHAL and OPPENHEIMER and especially
by ATWATER and BENEDICT.2

PKTTENKOFER'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

1 Pfliiger's Arch., 6.

2 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 Bene-
dict, Bull, Dept. of Agriculture, Washington, 69, 109, and 136. See also Krogh, Wien.
Sitz. Ber., 115, III., and Skand. Arch. f. Physiol., 18.

820 CHEMISTRY OF RESPIRATION.

method. The large respiration apparatus of iSoxDEN and TTGERSTEDT as well a.s
of ATWATER and ROSA * are based upon this principle.

SPECK'S Method.2 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 4 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
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 5 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 (FR.

MlJLLER6).

The lungs have a strong reducing property, which BOHR explains by
the extensive oxidation processes in the lungs. According to N. SIEBER

1 Pettenkofer's method; see Zuntz, 1. c.; Sonde"n and Tigerstedt, Skand. Arch. f.
Physiol., 6; Atwater and Rosa, Bull, of Dept. of Agriculture, 63. Washington.

2 Speck, Physiologie des menschlichen Atmens. Leipzig, 1892.

3 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.

4Compt. rend., 104.

5 Cited from Maly's Jahresber., 18, 248.

6 Jacoby, Zeitschr. f. physiol. Chem., 33; Miiller, Verhandl. d. Kongress. f. inn.
Medizin, 1902.

LUNGS AND THEIR EXPECTORATIONS. 821

they also have the ability to decompose neutral fats, while, RIEHL 1
says, they do not have the ability to invert milk sugar.

The blank 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, a slightly dextrorotatory carbohydrate
differing from glycogen, found by POUCHET in consumptives, and finally
also cellulose, which, according to FREUND,2 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, P2O5 485, SO3 8, and sand 134 grams. According to
OiDTMANN,3 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.

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
products 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, SIMON 4),
volatile fatty acids, glycogen, CH ARGOT'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 5 is not always constant.

- N. Sieber, Zeitschr. f. physiol. Chem., 55; Riehl, Zeitschr. f. Biol., 48.

2 Pouchet, Compt. rend., 96; Freund, cited from Maly's Jahresber, 16, 471.

3 Schmidt, cited from v. Gorup-Besanez, Lehrbuch, 4. Aufl., 727; Oidtmann, ibid.,
732.

4 Wanner, Deutsch. Arch. f. klin. Med., 75; Simon, Arch. f. exp. Path. u. Pharm., 49.

5 Gerhartz and Strigel, Beitr. z. kb'n. d. Tuberkulose, 10, which also cites Zickgraf.

CHAPTER XVIII.

METABOLISM WITH VARIOUS FOODS, AND THEIR NECESSITY

TO MAN.

I. EXCHANGE OF MATTER AND FORCE IN GENERAL WITH METHODS OF STUDY.

THE conversion of chemical energy into heat and mechanical work
which characterizes animal life, leads, as previously stated in Chapter I,
to the formation of relatively simple compounds — carbon dioxide, urea,
€tc. — which leave the organism, and which, moreover, being very poor in
energy, are for this reason of little or no value to the body. It is there-
fore 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 that which has been
•exhausted. This is accomplished 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 wrhich 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 foodstuffs 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.

Such knowledge can be attained only by a series of systematic and
thorough observations, in wrhich the quantity of nutritive material, rela-

822

EXCRETA OF THE BODY. 823

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 AT WATER.

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 com-
position 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 ejec-
tion 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, perspira-
tion, and epidermal formations — wrhich 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 constituents of the animal fluids or tissues, cannot be considered 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 difficulty,
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 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 individu-
alities, different modes of living, different foods, etc. Only approximate values can
therefore be given for the constant excreta of the human body.

824 METABOLISM.

The following figures represent the quantity of excreta for twenty -
four hours from a grown man, weighing 60-70 kilos, on a mixed diet.
The numbers are compiled from the results of different investigators :

Grams.

Water 2500-3500

Salts (with the urine). . 20-30

Carbon dioxide 750-900

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 (^ = 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 as
stated in Chapter IX consists in pare of non-absorbed remnants of the
food, but in chief part and sometimes entirely of constituents of the epi-
thelium and the secretions. 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 fluids. It may not only vary in different
individuals, 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

ELIMINATION OF NITROGEN AND SULPHUR. 825

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 J), 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 norma^ 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 neverthe-
less 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 OppENHEiHER.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 metab-
olism 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
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

1 Rieder, Zeitschr. f. Biologie, 20; Rubner, ibid., 15; Tsuboi, ibid., 35.

3 Berlin, klin. Wochenschr., 1887.

2 See Regnault and Reiset, Annal. d. chem. et phys. (3), 26, and Annal. d. Chenu
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, in Hermann's Handbuch, 6, Thl. 1; Griiber, Zeitschr. f.
Biologie, 16 and 19.

826 METABOLISM.

from the sulphates of the food, makes nearly 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, H2S04, produced by their combustion is in the ratio 5.2:1, or about
the same as in the urine (see page 726) . 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. A determination of the nitrogen alone is 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
f eces 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 sulphur of the catabolized proteins is more quickly eliminated,
according to v. WENDT, HAMALAINEN and HELME l than the nitrogen, and
this behavior of sulphur gives a more positive picture of the temporal catab-
olism of protein than the nitrogen. This is of importance, as the elim-
ination 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
vith varying rapidity, and the same is true, according to HAMALAINEN
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 (GuMLicn,
SANDMEYER, MARCUSE, ROHMANN, and STEINITZ, LoEwi,2 and others).

1 Wendt, Skand. Arch. f. PhysioL, 17; Hamalainen and Helme, ibid., 19; Falta,
Deutsch. Arch. f. klin. Med., 86.

2 In regard to the investigations on the metabolism of phosphorus and the methods
used therein, see Steinitz, Pfliiger's Arch., 72; Zadik, ibid., 77; Leipziger, ibid., 78;
Oertel, Zeitschr., f. physiol. Chem., 26; Mandel and Oertel, Bull. Med. Sciences, N. Y.
Univ., 1, and Ehrlich, Inaug.-Diss., Breslau, 1000; Loewi, Arch, f. exp. Path. u. Pharm.,
45; Slowtzoff, Hofmeister; Beitrage 8. On the absorption of casein, see Poda, Praus-
nitz, Micko, and P. Miiller, Zeitschr. f . Biologic, 39. The literature on the phosphorus

CALCULATION OF THE EXCHANGE OF MATERIAL. 827

On the other hand, the phosphorized protein substances, lecithins and
phosphatides, 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 718). For these reasons the phosphorus is of great
importance in certain investigations on metabolism.

If 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 oi 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 6.25.1 Usually, according to
YOIT'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 thai
lean meat contains 3.4 per cent nitrogen is arbitrary, and the relation of N : G
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 XI).

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 formerly
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. This
does not seem to be trustworthy, as this relation varies and depends,
according to TANGL and PFLUGER, LANGSTEIN, and STEiNixz,2 upon the
kind of food. TANGL has shown that the richer the food is in carbohydrates
the more carbon and hence the more heat of combustion per gram of
nitrogen does the urine contain. He found the following for 1 gram of
nitrogen in the urine: With diet rich in fab 0.747 gram C and 9.22 calories;
for carbohydrate-rich diet he found 0.936 gram G and 11.67 calories.

The extent of the gas exchange can be determined by any of the methods
given on pages 819, 820. By multiplying the quantity of carbon dioxide
found by 0.273 one obtains the quantity of carbon eliminated as CC>2.

metabolism can be found In Albu and Neuberg, Physiol. u. Pathol. des Mineralstoff-
\vechsels, Berlin, 1906.

1 In calculating the protein catabohsm from the nitrogen of the urine it must not
i e forgotten that the food often contains nitrogenous extractives whose nitrogen cannot
1 e calculated as protein and for which a special correction must be made, if necessary.

2Tangl, Arch. f. (Anat. u.) Physiol., 1899, Supplbd.; Pfliiger in Pfluger's Arch., 79;
Lungstein and Steinitz, Centralbl. f . Physiol., 19.

828 METABOLISM.

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 coriesponding loss of body
substance.

The nature of the substances here deposited or lost, whether they consist
of proteins, fats, or carbodydrates, is learned from the total quantity of the nitrogen
of the excretions. The corresponding quantity of proteins may be calculated 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 decomposed 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 — (3.3 X 10) = 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 assump-
tion must be made that the person experimented upon has used only fat and pro-
teins. As animal fat contains on an average 76.5 per cent carbon, the quantity

100

of transformed fat may be calculated by multiplying the carbon by =^ = 1.3.

t o.o

In the case of the above example, the person experimented upon would have used
62.5 grams of proteins and 167 X 1.3 = 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
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

CALORIFIC VALUE OF THE FOODSTUFFS. 829

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-
ILFWSKI, 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

Dextrose 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.

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 crganism than outside of it, and they
must therefore be specially determined. For this purpose RUBNER 2
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 corre-
sponded 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-

1 See Rubner, Zeitschr. f . Biologie, 21, which also cites the works of Frankland and
Danilewski; see also Berthelot, Compt. rend., 102, 104, and 110; Stohmann, Zeitschr.
f. Biologie, 31; Benedict and Osborne (vegetable proteins), Journ. of biol. Chem., 3.

2 Zeitschr. f. Biologie, 21.

830 METABOLISM.

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:

i 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 the expired air we learn, on measuring
them when dry and at the same temperature and pressure, that the volume
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

PO
volume of the inspired oxygen, and the relation — -, 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

RESPIRATORY QUOTIENT. 831

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.

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 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,
the same number, 5047 calories, are produced. In an analogous manner
the average calorific value of fat for 1 liter of oxygen, 4.686 calories, and
for 1 liter carbon dioxide, 6629 calories, can be calculated.

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

52.3Sg.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 41.50; 4.40; 7.69; O0~; OXX

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

1 Zuntz, Loewy, Miiller and Caspari, Hohenklima und Bergwanderungen, Berlin,
1006, pages 102, 103

832 METABOLISM.

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

431 63
of nitrogen eliminated in the urine (16.28 gram.) there is produced -, -5- =26.51

lO.Jo
QA AQ

calories; the corresponding quantity of oxygen is jg'7Kr = 5.91 liter O and the

77 39
corresponding quanity of CO2 produced is = ^.75 liters CO2. The calorific

c\n r-i

value for 1 liter of oxygen consumed is therefore - ' = 4.485 calories, and for

1 liter of carbon dioxide produced -r-'=^ = 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 O = 4.66 and for 1 liter CO2 = 5.87 calories. If we
take the average of these calculations we obtain a calorific value for the com-
bustion of protein, which for 1 liter of oxygen amounts to 4.573 calories and for
the carbon dioxide 5.725 calories.

According to these calculations the combustion of proteins in the
animal body has a calorific value for 1 liter of oxygen of, in round numbers,
4.57 calories, and for 1 liter CO2, 5.73 calories. We therefore have the
following calorific values for the three foodstuffs:

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

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
CO2 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 considering
the respiratory quotient. For this purpose ZUNTZ and SCHUMBURG have

1 Voit, Zeitschr. f . Biol., 44; Kummacher, ibid.

CALORIFIC VALUE OF OXYGEN. 833

constructed a table, an abstract of which we give below, taken from the

\VOrk Of MAGNUS-LEVY.1

R. Q Calories value Division in per cent.

per 1 liter 0. 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 820, 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 REGNAULT-REISET principle are only
useful in experiments over a longer period.

KAUFMANN 2 incloses the individual to be experimented upon in a capacious
sheet-iron room, which serves both as a respiration-chamber and a calorimeter,
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 formulas 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, KAUF-
MANN attempts to explain the various decomposition processes in the body under
different nutritive conditions.

As will be shown, the fats and carbohydrates may decrease the metab-
olism of proteins in the body, while, on the other hand, the quantity
of proteins in the body or in the food acts on the metabolism of fat in
the body. In physiological combustion the various foods may replace
one another to a certain extent, and it is therefore important to know the
ratio of replacement. The investigations made by RUBNER have taught

1 A. Magnus-Levy in v. Noorden's Handb. d. Pathol. des Stoffwechsels, Bd. 1. (1906).

2 Arch. d. Physiologie (5), 8.

834 METABOLISM.

that this, if it relates to the force and heat production in the animal body,
Is a proportion that corresponds with the figures of the heat value of the
same. 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:

100 grams fat are equal to or isodynamic with

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

Dextrose 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 grams of
protein and carbohydrate are equal to or isodynamic \vith 100 grams of
fat in regard to source of energy, because each yields 930 calories on com-
bustion in the body.

By means of recent very important calorimetric investigations, RCJBNER 1
has shown that the heat produced in an animal in several series of experi-
ments extending over forty-five days corresponded to within 0.47 per cent
of the physiological heat of combustion calculated from the decomposed
body and foods. ATWATER and his collaborators 2 have made some
very thorough investigations on this subject on men. In their experiments
they made use of a large respiration calorimeter, which not only exactly
determined the excreta, but also made a calorimetric determination of
the heat given out by the person experimented upon, i.e., the work per-
formed. 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 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,

1 Zeitschr. f . Biologic, 30.

2 Bull, of Dept. of Agnc., Washington, 44, 63, 69, and 109 and Ergebnisse des
Physiologic, 3.

PHYSIOLOGICAL AVAILABILITY. 835

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.1 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.

F , Loss in per cent. Total loss Availability

In Urine. In the Feces. in per cent, in per cent.

Cow's milk 5.13 5.07 10.20 89.8

Mixed diet (rich in fat) 3.87 5.73 9.60 90.4

Mixed diet (poor in fat) 4.70 6.00 10.70 89. 3

Potatoes 2.00 5.60 7.60 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 exists, besides
the above-mentioned standard figures for the physiological calorific value of the
organic foodstuffs, also for the carbon of the carbon dioxide, and for the oxygen
other standard factors. 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 *
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 *
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.

Instead of the direct determination the heat of combustion can also be deter-
mined from the elementary composition acording to the following principle as
suggested by E. VoiT.4 If we designate the heat of combustion for 1 gram of the
substance by calories and the quantity of oxygen necessary for the complete com-
bustion of 1 gram of the substance ( = oxygen capacity of the substance) by O,,

then — ~~jr\ — ~=K, which is the combustion value for 1 gram of oxygen. The

oxygen capacity can be calculated from the elementary composition, and when
the value of K is known, the combustion heat of a chemical compound or a known
mixture can be readily determined. The value K is almost constant for substances
of the same groups ; but also different groups show among themselves only slight
deviation for this value. VOIT obtained the following values for a few of the food-
stuffs:

K (in kg. Calories.) O Capacity.

Plant protein 3 .298 1 .740

Animal protein 3 . 273 1 . 741

Fat 3.271 2.863

Carbohydrate 3 . 525 1 . 156

These methods of calculation are, according to VOIT and KRUMMACHER,
admissible for practical purposes.

1 Zeitschr. f . Biologic, 42.

2 Zeitschr. f. physiol. Chem., 31.

3 The works of Frentzel and Schreuer may be found in Arch. f. (Anat. u.) Physiol.,,
1901, 1902, and 1903.

4 Zeitschr. f . Biologic, 44. See also Krummacher, ibid.

83G METABOLISM.

II. METALBOLISM 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 CHOSSAT/ 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,2 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 can live without food
from four to eight weeks, birds five to twenty days, snakes and frogs more
than half a year or 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
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 the metabolism is referred to the unit of the weight of the
body, namely, 1 kilo, it appears that this quantity remains almost
unchanged during starvation. The investigations of ZUNTZ, LEHMANN,
and others3 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

1 Cited from Voit in Hermann's Handbuch, 6, Thl. 1, 100.

2 See Luciani, Das Hungern. Hamburg u. Leipzig, 1890.

3 Berlin. Win. Wochenschr., 1887.

PROTEIN METABOLISM IN STARVATION. 837

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.1 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 VoiT2 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.

Grams of Urea Eliminated in Twenty-four Hours.
Day of Starvation. ger j ger n Ser m

First 60.1 26.5 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

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,
TIGERSTEDT, LANDERGREN,3 as follows: At the commencement of star-
vation the protein metabolism is reduced by the glycogeri 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

1 See also Tigerstedt and collaborators in Skand. Arch. f. Physiol., 7.

2 See Hermann's Handbuch. 6, Thl. 1, 89.

3 Prausnitz, Zeitschr. f. Biologic, 29; Tigerstedt and collaborators, 1. c.; Landergren,
Undersokningar ofver menniskans agghviteomsat tiling, Inaug.-Diss. Stockholm,
1902.

838 METABOLISM.

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 fat 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 collaborators l).

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-tour 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,2 who had been poorly nourished.

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
constant, 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-

1 Zeitschr. f. Biologie, 41, 167 and 502. See also Kaufmann, ibid., and-iV. Schulz,
ibid., and Pfluger's Arch., 76, with Mangold, Stiibel and Hempel, ibid., 114.

2 Berl. kliii. Wochenschr., 1887.

LOSS OF WEIGHT IN STARVATION. 839

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. Starving animals, as a rule, do not partake of
water.

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. If we bear, these conditions in mind, then, it seems, according to BOHT-
LiNGK,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 destruc
tion 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 conditions,
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.

MUNK observed, in CETTI'S case, an increase in the elimination of
phosphoric acid in relation to the Ar-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 WELLMANNS 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-
lion of this point we give the following results of CHOSSAT'S experiments
on pigeons, and those of VOIT 4 on a male cat. The results are percentages
of weight lost from the original weight of the organ.

1 Arch, des sciences biol. de St. Pe"tersbourg, 5.

2 Bdhtlingk, 1. c.; Katsuyama, Zeitschr. f. physiol. Chem., 26.

3 Munk, Berl. klin. Wochenschr., 1887; Wellmann, Pfliiger's Arch., 121.

4 Cited from Voit in Hermann's Handbuch, 6, Part 1, 96 and 97.

8-iO

METABOLISM.

Pigeon (CHOSSVT). 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 14

Nervous system 2 3

The total quantity of blood, as well as the quantity of solids contained
therein, decreases, as PANUM and others 1 have shown, in the same pro-
portion as the weight of the body. Concerning the loss of water by
different organs authorities disagree, LUKJANOW 2 claiming that the
various organs differ from each other in this respect.

The above-tabulated results cannot serve as a measure of the metabo-
lism m 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,
but it depends more likety upon the disturbance in the nutrition of a few
less vitally important organs (E. VOIT 3) .

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. VoiT4 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 values 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

1 Panum, Virchow's Arch., 29; London, Arch. d. scienc. biol. de St. Pe"tersbourg, 4.

2 Zeitschr. f. physiol. Chem., 13.

3 Zeitschr. f. Biologic, 41.

4 Ibid., 46.

BASAL REQUIREMENT. 841

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/ 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,
CATHCART 2) .

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 3) . 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
determine the gas exchange in a person lying down with as perfect
complete muscular rest as possible, or sleeping in the early morning and
at least twelve hours after a light meal not rich in carbohydrates. This
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,4 and by
JOHANSSON 5 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 gram CO2 per hour and 24.8
gram CO2 in the ordinary resting. According to MAGNUS-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.

The food may be quantitatively insufficient, and the final result of

1 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.

2 Zeitschr. f. klin. Med., 36; Cathcart, 1. c.

3 Magnus-Levy in v. Noorden's Handbuch, and Loewy in Oppenheimer's Handbuch
d. Blochemie, Bd. 4.

4 The literature can be found in the works of Magnus-Levy and Loewy.

5 Skand. Arch. f. Physiol., 7, $, 21, and Nord. Med. Arch. Festband, 1897; see also
Magnus-Levy.

842 METABOLISM.

this is absolute inanition. The food may also be qualitatively insufficient
or, as we say, inadequate. This occurs when any of the necessary nutri-
tive 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.1 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 from
lack of water than from complete inanition (LANDAUER, NOTHWANG).

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 SpiEGLER,2 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 (VoLKMANN3),
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

1 See Voit, in Hermann's Handbuch, 6, part 1, 345.

2 Landauer, Maly's Jahresber., 24; Nothwang, Arch. f. Hyg., 1892; Straub, Zeitschr.
f. Biol., 37 and 38; Spiegler, ibid., 41.

3 See Hermann's Handbuch., 6, pt. 1. 353.

LACK OF MINERAL SUBSTANCES IN THE FOOD. 843

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 J).

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 be so or not has not, especially in man, been
sufficiently investigated; but generally we consider the need of mineral
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 FORSTER. 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 2 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 cleath in 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, BUXGE and LUNIN 3 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

1 Forster, Zeitschr. f. Biologie, 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.

2 University of California Publications, Pathol., 1.

3 Bunge, Lehrbuch der physiol. Chem., 4. Aufl., 97; Lunin, Zeitschr. f. physiol.
Chem., 5.

844 METABOLISM.

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 IX) 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 combinations 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 hi NaCl, which therefore must be taken in greater amounts
from the outside (BUNGE) . This occurs continuously in herbivora, and in
man with vegetable food rich in potash. For human beings, and espe-
cially for the poorer classes of people who chiefly live on potatoes and
foods rich in potash, common salt is, not only a condiment, but a
necessary addition to the food (BUNGE 1). 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 investiga-
tions, which may be found in ALBU and NEUBERG'S work, previously
cited.

Lack of Alkali Carbonates or Bases in the Food. The chemical processes
in the organism are dependent upon the presence of tissue-fluids of a cer-
tain reaction, and this action, 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 of great importance, not
only as a solvent for certain protein bodies and as constituents of cer-
tain 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

1 Zeitschr. f. Biologic, 9.

LACK OF PHOSPHATES AND EARTHS. 845

as well as the unequal resistance of man and other animals toward this
action of acids has already been discussed in Chapter XV.

Lack of Phosphates and Earths. With the exception of the value of
the alkaline earths as carbonates and more especialty 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 earths in adults, and no average results can
be given. The same is true for the need of phosphates or phosphoric acid,
whose vame 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 EHRSTROM/ 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 Chap-
ter X 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 ROHM ANN 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 MIESCHER, the develop-
ment of 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 recent investiga-
tions of HART, MACCOLLUM and FULLER,S who found that pigs with food
,poor in phosphorus develop just as well with inorganic phosphates as

1 Skand. Arch. f. Physiol., 14.

2 See Marcuse, Pfliiger's Arch., 67, and also footnote 2, page 826.

3 Hart, MacCollum and Fuller, Amer. Journ. of Physiol., 23.

846 METABOLISM.

with organic phosphorus compounds, also indicate such a formation.
Other investigators, such as v. WENDT/ also admit of a synthesis of phos-
phorized protein substances by the aid of inorganic phosphates.

Lack of Iron. As iron is an integral constituent of haemoglobin,
absolutely necessary for the supply of oxygen, hence it is an indispen-
sable 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 on 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. According to the doctrine of C. VOIT, which has been defended
by recent investigations of E. VOIT and KoRKUNOFF,2 the protein metab-
olism is never so low under these conditions as in starvation. Accord-
ing to several investigators, such as HIRSCHFELD, KUMAGAWA, KLEM-
PERER, SIVEN, LANDERGREN, and others, the protein metabolism may
indeed, with exclusively fat and carbohydrate diet, be smaller than in
complete starvation. Thus LANDERGREN has observed on an adult,
healthy man in nitrogen starvation but with sufficient supply of energy
(about 40 calories per kilo as carbohydrates and fat) on the fourth
starvation day that the nitrogen excretion was not more than 4 grams.
On the seventh day, with only carbohydrates, the nitrogen excretion
sank to 3.34 grams, which corresponded to 0.047 gram N per kilo of body
weight and to 0.29 gram protein. The recent investigations of MICHAUD 3
on dogs also show that in protein starvation, as by exclusive feeding
with fat and carbohydrate, lower results are obtained for the nitrogen
elimination than in complete starvation.

The absence of fats and carbohydrates in the food affects carnivora and
herbivora somewhat differently. It is not known whether carnivora
can be kept alive for any length of time by food entirely free from fat

1 Skand. Arch., f. PhysioL, 17.

2 Zeitschr. f. Biologie, 32.

3 Hirschfeld, Virchow's Arch., 114; Kumagawa, ibid., 116; Klemperer, Zeitschr.
f. klin. Med., 16; Sive"n, Skand. Arch. f. Physiol., 10 and 11; Landergren, l.c-., 11; foot-
note 3, page 837, also Maly's Jahresber., 32; Michaud, Zeitschr. f . physiol. Chem., 59.

ARTIFICIAL FOODS. 847

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 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.

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. This was not possible in the experiments of
BUNGE and LUNIN, cited above. Later investigators, such as HALL and
STEINITZ, FALTA and NOEGGERATH, KNAPP and JACOB have come to the
same conclusion, although they obtained somewhat better results. ROH-
MANN 3 obtained much better results, and he showed that the selection of
the protein is of the greatest importance. " If mice are fed with a mixture
of 7.2 parts vitellin, 14 parts casein, 4 egg albumin, 27 margarine, 120
wheat starch, 60 potato starch, 10 dextrose, 4 salts (a mixture of 10 parts
calcium phosphate, 40 acid potassium phosphate, 20 sodium chloride,
15 sodium citrate, 8 magnesium citrate, 8 calcium lactate) it is not only
possible to keep all the mice alive for some time but it is also possible to
raise young mice by artificial feeding of the mother and then the small
animals themselves (RoHMANN4). They become mature and deliver
active offspring who in turn become mature. The young that these
deliver cannot be raised to maturity." If the food only contained one
of the above-mentioned proteins the adult mice lost weight and succumbed
after a certain time. If the casein is replaced by egg albumin or the egg
albumin was replaced by casein the weight of all the mice remained con-
stant for some time. Young mice had their development retarded in a
remarkable manner. They remained at the same body weight for some
time and then succumbed.

These experiments show that the various proteins have different
importance in nutrition and especially for the develpoment of the young.

1 See Horbaczewski, Maly's Jahresber., 31, 715.

2 Pfliiger's Arch., 50.

3 Hall, Arch. f. (Anat. u.) PhysioL, 1896; Steinitz, Ueber Versuche mit kiinstlicher
Ernalirung, Inaug.-Diss., Breslau, 1900; Falta and Noeggerath, Hofmeister's Beitrage,
7; Knapp, Zeitschr. f. exp. Path. u. Therap., 5; Jacob, Zeitschr. f. Biol., 48; Rohmann,
Klin, therap. Wochenschr., No. 4, 1902 and Allg. med. Zentral.-Zeitg., 1908, No. 9.

4 Allg. med. Zentral-Zeitg., 1908, No. 9.

848 METABOLISM

III. 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, or 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 ohe 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 nitrogeneous
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 which is in nitrogenous equilibrium, hav-
'ing been fed on large quantities of meat, is suddenly given a small
quantity of meat" per day, then it uses up its own body proteins, the
amount decreasing from day to day. The elimination of nitrogen and
the catabolism of proteins decrease constantly, and the animal may in
this case also pass into nitrogeneous equilibrium, or almost into this
condition. These relations are illustrated by the following table (Vorr) l :

Grams of Meat in the Food per Day.

Before the Test. During the Test.

1 500 1500

2 1500 1000

Grams of Flesh Metabolized in Body per Day.

1222 1310 1390 1410 1440 1450 1500

1153 1086 1088 1080 1027

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
] 000 grams meat, decreased considerably, and on the fifth day an almost

1 Hermann's Handbuch, 6, Part I, 110.

EXCLUSIVE PROTEIN FEEDING. 849

nitrogeneous 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 nitrogeneous 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 1 found in man but not, or at least not to the same extent,
in clogs, 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 HELME 2 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 protein.
From the unequal rate at which the. different proteins are decom-
posed it follows chat 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
cf 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, as detailed in Chapter X, that there may even take place a
formation of fat under these circumstances. The objections presented by
PFLUGER to these experiments, as well as the proofs of the formation of
fat from proteins, are also given in the above-mentioned chapter.

According to PFLUGER'S doctrine, the protein can influence the forma-
tion of fat only in an indirect way, namely, in that it is consumed instead
of the non-nitrogeneous bodies and hence the fat and fat-forming carbo-
hydrates are spared. If sufficient protein is introduced with the food
to satisfy the total nutritive requirements, then the decomposition of
fat stops; and if non-nitrogenous food is taken at the same time, this is

1 Deutsch. Arch. f. kiln. Med., 86. * Skand. Arch, f . Physiol., 19.

850 METABOLISM.

not consumed, but is stored up in the animal body, the fats as such,
and the carbohydrates at least in great part as fat.

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 J (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 property,
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 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-
proteins, are ordinarily catabolized to only a small extent, about 1 per
cent daily (VOIT). By an increased supply of proteins the activity of
the cells md their ability to decompose nutritive proteins is also increased
to a certain degree. When nitrogenous equilibrium is obtained after an

1 See Schondorff, Pfliiger's Arch., 71.

TISSUE AND CIRCULATING PROTEINS. 851

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, arid 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
especially by PFLUGER. PFLUGER'S belief, based on an investigation
made by one of his pupils, ScHONDORFF,1 is that the extent of protein
destruction 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 PFLL'GER. VOIT 2 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 been taken up
by the cells from the fluids washing them. The point of VOIT'S theory
is that all proteins are not destroyed in the body with the same degree of
ease. The organized protein, which is fixed by the cells and has become
a part of them, is destroyed less readily than the protein taken up by the
cells from the nutritive fluid, which serves as material for the chemical con-
struction of the much more complicated organized proteins. This nutritive
protein, which circulates with the fluids before it is taken up by the cells,
and which can exist in store in the cells as well as in the fluids, agreeably
to VOIT'S view, has been called circulating protein or store protein by
him. It is clear that these names may lead to misunderstanding, and
therefore too much stress should not be put upon them. The most
essential part of VOIT'S theory is the supposition that the food protein
of the cells is more easily destroyed than the organized, real protoplasmic
protein, and this assertion can hardly, for the present, be considered as.
refuted or exactly proven.

The investigations in recent years, especially those of FOLIN, 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 differen-

1 Pfliiger, Pfliiger's Arch., 54; Schondorff, ibid., 54.

2 Zeitschr. f . Biologic, 11.

852 METABOLISM.

tiate bstween endogenous and exogenous protein metabolism. The
experience on protein feeding and the endeavor of the body, as observed
by ScHREUER,1 on going to an ordinary diet after abundant protein feed-
ing, to remain at the old state previous to the overfeeding of protein,
point to the fact that protein retained by the body is not quite the same
as the other body protein.

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
investigations 'of PANUM, FALCK, ASHER and HAAS and others2 on the
transitory progress of the elimination of nitrogen after a meal rich in
proteins throws light on this question. From experiments upon a dog
it was found that the elimination of nitrogen increases almost immediately
after, a meal rich in proteins, and that it reaches its maximum in about
six hours, when about one-half pf the quantity of nitrogen corresponding
to the administered proteins is' eliminated. If we also recollect
according to an experiment of ScHMiDT-MiJLf1!™3 uPon a do%> about
37 per cent of the given proteins are absorbed in theT&rst.two hours *
the meal and about 59 per cent in the course of the first s£&nours> li ma^
then be inferred that the increased elimination of nitrogen a . e r a n
is due to a catabolization of the digested and assimilated proteii/? (
food not previously organized. If it is admitted that the catablG
protein must have been organized, then the greatly increased eliminatiS11
of nitrogen after a meal rich in proteins supposes a far more rapid and1*
comprehensive destruction and reconstruction of the tissues than has '
been generally assumed.

The extensive cleavage of the proteins in digestion and the repeatedly
observed deamidation of amino-acids in the animal body make it prob-
able that the abundant elimination of nitrogen after a diet rich in pro-
teins is in great part due to a progressive demolition of the food protein
in digestion, whereby certain atomic complexes are more readily split
than others. The abundant elimination of nitrogen by the urine after
partaking of considerable protein may also depend in great part upon those
nitrogenous atomic complexes which are split off and whose nitrogen is
split off as ammonia and therefore cannot be used by the body. The
abundant formation of ammonia in the cells of the digestive apparatus

1 Folin, Amer. Journ. of Physiol., 13; Schreuer, Pfliiger's Arch., 110.

2 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, Pfliiger's Arch., 65, and Veraguth, Journ. of Physiol., 21;. Schlosse,
Maly's Jahresber., 31.

3 Arch, f. (Anat. u.) Physiol., 1879.

NUTRITIVE VALUE OF GELATIN. 853

after food rich in proteins, as observed by NENCKI and ZALESKI/ seems to
speak in favor of this view.

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 Vorr's experiments upon a dog:

Food per Day. Flesh.

Moat Gelatin. Fat. Sugar. Cataholizerl. On the Body.

400 0 200 0 450 -50 *

400 0 0 250 43Q -39

400 200 0 0 356 +44

I. MuNK3 has later arrived at similar results by means of more deci-
sive experiments. He found that in dogs on a mixed diet which con-
tained 3.7 grams protein per kilo of body, of which hardly 3.6 grams were
catabolized, nearly five-sixths could be replaced by gelatin. The same dog
catabolized on the second day of starvation three times as much protein
as with the gelatin feeding. MUNK also states that gelatin has a much
greater sparing action on proteins than the fats or the carbohydrates.

This ability of gelatin to spare the proteins is explained by VOIT by
the fact that the gelatin is decomposed instead of a part of the circulat-
ing proteins, whereby a part of this last may be organized.

The recent investigations of KRUMMACHER and KIRCHMANN show the
extent of the sparing action of gelatin upon proteins. The extent of
protein destruction during gelatin feeding was compared 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. KAUFMANN,
who experimented upon dogs, found that one-filth of the protein nitrogen
could be readily replaced by gelatin nitrogen, while in an experiment upon

1 Arch, des scienc. biol. de St. Petersbourg, 4; Salaskin, Zeitschr. f. physiol. Chem.,
25; Nencki and Zaleski, Arch. f. exp. Path. u. Pharm., 37.

2 Voit, 1. c., 123; Panum and Oerum. Nord. Med. Arkiv., 11.

3 Pfliiger's Arch., 58.

854 METABOLISM.

himself with 93 per cent gelatin nitrogen, 4 per cent tyrosine nitrogen,
2 per cent cystin nitrogen, and 1 per cent tryptophane nitrogen, he found
instead of the equal quantity of protein nitrogen in the periods before
and after, that the gelatin replaced by amino acids had almost the same
physiological value as the proteins. The correctness of this action of the
amino-acids has unfortunately not been confirmed by others. RONA and
MiCHAELis,1 who also proved that one-fifth of the protein metabolism
could be replaced by gelatin, found that no mentionable increase in the
the nutritive value of gelatin on the addition of tyrosin or tryptophane
occurred.

The value of gelatin has been found by MURLIN 2 to be dependent to
a high degree upon the protein condition ot 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
value, and when two-thirds of the total calories partaken of were
supplied by carbohydrates, 63 per cent of the total nitrogen could be
replaced by gelatin nitrogen.

Gelatin may also somewhat decrease the consumption of fat, although
it is of less value in this respect than the carbohydrates.

The nutritive value of proteoses (and peptones) stands in close
relation to the nutritive value of the proteins and gelatin. The early
investigations made by MALY, PLOSZ and GYERGYAY, and ADAMKIEWICZ
have led to the conclusion that with food which contains no proteins
besides peptones (proteoses) an animal may not only preserve its nitrog-
enous equilibrium, but its protein condition may even increase.
According to recent and more exact investigations by POLLITZER, ZUNTZ,
and MUNK the proteoses have the same nutritive value as proteins, at
least in short experiments. POLLITZER claims that this is true for dif-
ferent proteoses as well as for true peptone; but this does not agree
with the experience of ELLINGER,S who finds that the true antipeptone
(gland peptone) is not able to replace proteins entirely or to prevent
the loss of protein in the animal body. On the contrary, he claims
it has, like gelatin, the property of sparing proteins. VOIT long ago
expressed a similar view. He believes that the proteoses and peptones
may indeed replace the proteins for a short time, but not permanently;

1 Krummacher, Zeitschr. f. Biologie, 42; Kirchmann, ibid., 40; Kaufmann, Pfliiger's
Arch., 109; Rona and Michaelis, Zeitschr. f. physiol. Chem., 50.

2 Amer. Journ. of Physiol., 19.

3 Maly, Pfliiger's Arch., 9; Plosz and Gyergyay, ibid., 10; Adamkiewicz, " Die Natur
und der Nahrwerth des Peptons " (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. Biologie, 33 (literature).

PROTEOSES, ASPARAGIN, MIXED DIETS. 855

"they can spare the proteins, but cannot be converted into proteins.
According to the researches of BLUM the different proteoses have various
nutritive values. In his experiments the heteroproteose from fibrin
could not replace the proteins of the food, while casein protoproteose
had this property. In the researches of HENRIQUES and HANSEN 1
on white mice it was shown that the heteroproteose as well as the dys-
proteose (from WITTE peptone) had the ability of protecting the body
from loss of nitrogen and that they deposited nitrogen. If the animal
body has the ability of again constructing protein from simple fractions
then there is nothing strange in these reports on the nutritive value of
proteoses. The proteoses and peptones are formed by cleavages, and
perhaps certain atomic complexes are absent which occur in the mixture
of cleavage products and which are necessary for a regeneration of special
protein bodies.

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
dependent 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.2 If, as is
generally 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. The most important facts in connection with the
relation of the amino-acids to the protein metabolism and protein syn-
theses have been mentioned in a previous Chapter (IX).

Metabolism on a Diet consisting of Protein, with Fat or Carbohydrates.
Fat cannot arrest or prevent the catabolism of proteins; but it can decrease
it, and so spare the proteins. This is apparent from the following table
of VoiT.3 A is the average for three days, and B for six days.

Food. Flesh.

A

Meat.
1500

Fat.

o

Metabolized.
1512

On the Bodv.
-12

B

1500

150

1474

+ 26

1 Blum, Zeitschr. f. physiol. Chem., 30; Voit, 1. c., 394; Henriques and Hansen,
Zeitschr. f. physiol. Chem., 48.

2 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-
ber., 27, and Zeitschr. f. Biologic, 39; Pfliiger's Arch., 113; Kellner and Kohler, Chem.
Centralbl., 1, 1906. Voltz, Pfliiger's Arch., 107, 117, with Yakuwa, ibid., 121; v.
Strusiewicz, Zeitschr. f. Biol., 47; Rosenfeld and Lehmann, Pfliiger's Arch., 112;
Lehmann, ibid., 115; M. Miiller, ibid., 117; Henriques and Hansen, Zeitschr. f. physiol.
Chem., 54.

3 Voit in Hermann's Handbuch, 6, 130.

856 METABOLISM.

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.

Because of the sparing action of fats an animal to whose food fat is
added may, as is apparent from the table, increase its store of protein
with a quantity of meat which is insufficient to preserve nitrogenous
equilibrium.

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 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.

600 250 ... ... 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 administered protein without a pre-
vious 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 (VoiT) .

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

1 Voit, ibid., page 143.

2 See Ergebnisse der Physiologic, 3.

3 1. c., Inaug.-Diss., and Skand. Arch. f. Physiol., 14.

FAT AND CARBOHYDRATES AS PROTEIN SPARERS. 857

carbohydrates have double the protein-sparing action as compared with
an isodynamic quantity of fat.

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. TALLQUIST l made a series of experiments on this
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,2 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 carbo-
hydrates.

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; arid when the dail} 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 experiments upon man as well as upon

1 Finska Lakaresallskapets Handl., 1901. See also Arch. f. Hygiene, 41.
21. c., Inaug.-Diss. See also Skand. Arch. f. Physiol., 14. .unfits tn9vi8 £

J09I

858 METABOLISM.

dogS by HlRSCHFELD, KUMAGAWA, KLEMPERER, MUNK, ROSENHEIM,1

and others. It follows from these experiments that the lower limit of
protein needed for human beings, for a week or less, is about 30^0 grams
or 0.4-0.6 gram per kilo with a body of average weight, v. NOORDEN 2
considers 0.6 gram protein (absorbed protein) per kilo and per day as the
lower limit. The above-mentioned figures are valid only for short series
of experiments; still there exists the observation of E. VOIT and CON-
STANTINIDI 3 on 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 protein per kilo. CASPARI 4 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 com-
plete nitrogenous 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 CONST ANTINIDI'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 entirety 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
the 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 nitro-
gen 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 5 also, in an experiment on himself, could not attain com-

1 See footnote 3, page 846; also Munk, Arch. f. (Anat. u.) PhysioL, 1891 and 1896;
Rosenheim, ibid., 1891; Pfliiger's Arch., 54.

2 Grundriss einer Methodik der Stoff wechseluntersuchungen. Berlin, 1892.

3 Zeitschr. f . Biologe, 25.

4 Physiologische Studien iiber Vegetarismus, Bonn, 1905.

BSiven, Skand. Arch. f. PhysioL, 10 and 11; Caspari, Arch. f. (Anat. u.) PhysioL,
1901.

WEAR AND TEAK QUOTA, SPECIFIC DYNAMIC ACTION. 859

plete 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
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 per cent of
the total calorific needs. The energy supply of the food is under these
conditions entirely assumed by the non-nitrogenous foodstuffs.

All proteins do not have the same value in replacing the protein
minimum. MiCHAUD2 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 a foreign protein, like
gliadin and edestin.

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
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.S 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.

1 Rubner, Theorie d. Ernahrung nach Vollendung des Wachstums, Arch. f. Hyg.,
6(V 1-80 and Ernahrungsvorgange beim Wachstum des Kindes, ibid., 66, 81-126.

2 Zeitschr. f. physiol. Chem., 59.

3 Rubner, 1. c., and Gesetze des Energieverbrauches, 70.

860

METABOLISM.

In close relation to the second purpose, the betterment of the condition
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 porportion 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:

.Food.

Number of
Days of Ex-
perimentation .

Total
Deposit of
Flesh.

Daily
Deposit of
Flesh.

Nitrogenous
Equilibrium.

Meat, Grams.

Fat, Grams.

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
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)
are shown by the series of experiments of KRUG, BORNSTEIN, SCHREUER,
HENNEBERG, PFEIFFER and KALB,1 and there is no doubt that such a

1Krug, Cited by v. Noorden, Lerb. der Path, des Stoffwechsel, 1. Aufl., p. 120;
Bornstein, Berl. klin. Wochenschr., 1898, and Pfliiger's Arch.. 83 and 106; Bornstein

FLESH AND FAT DEPOSITION. 861

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. Different theories as to the value of this
protein fattening are entertained. According to a prevailing opinion this
enrichment of protein is not continuous, but may easily be retrograde,
and the question whether we are here dealing chiefly with a taking up of
proteins by the cells (LUTHJE) or with a new formation of living tissues,
requires further solution.

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.1

The experiences of graziers show that in food-animals a flesh deposit
does not occur, or at least is only inconsiderable, on overfeeding. The
individuality and the race of the animal are of importance for flesh deposi-
tion.

The conditions in young, growing individuals are different than 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 X), respecting 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.

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

and Schreuer, Pfliiger's Arch., 110; Henneberg and Pfeiffer, see Maly's Jahresb.,
20; Pfeiffer and Kalb, ibid., 22.

1 See also Svenson, Zeitschr. f . klin. Med., 43.

862 METABOLISM.

increased elimination of urine causes in fasting animals (VoiT, FORSTER),
but not to any appreciable degree in animals taking food (SEEGEN, SAL-
KOWSKI and MUNK, MAYER, DuBELiR1), 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 HEiLNER2 has advocated VOIT'S
view. The introduction of water in a starving animal is, HEILNER
believes, to a certain extent an excessive supply of a foodstuff; hence
the metabolism is increased and the water acts under these conditions
in a sort of specific dynamic manner.

In this connection it must be mentioned that, according to HEILNER,S urea,
when introduced subcutaneously in physiological salt solution, causes an increased
action upon the protein catabolism, a fact which he mentions as an example that
the products of metabolism probably have an influence upon the same processes
which produce them.

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, STRAUS 4) . 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.

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.
Investigations of STRAUB and ROST 5 show that the action of salts stands
in close relation to their power of abstracting water. Small amounts of

1 Voit. Untersuch. iiber den Einfluss des Kochsalzes, etc. (Munchen, I860); Forster,
cited from Voit in Hermann's Handbuch, 6, 153; Seegen, Wien. Sitzungsber., 63; Sal-
kowslci and Munk, Virehow's Arch., 71; Mayer, Zeitschr. f. klin. Med., 2; Dubelir,
Zeitschr. f. Biologic, 28.

2 See R. Neumann, Arch, f . Hygiene, 36; Heilner, Zeitschr. f. Biologic, 47 and 49;
Hawk, University of Pennsylvania Med. Bull., xviii.

3 Zeitschr. f . BioL, 52.

4 Landauer, Maly's Jahresber., 24; Straub, Zeitschr. f. Biologie, 37.

5 W. Straub, Zeitschr. f. Biologie, 37 and 38; Rost, Arbeiten aus d. Kaiserliche
Gesundheitsamte, 18 (literature). See also Griiber, Maly's Jahresber., 30, 612.

ACTION OF SALTS AND ALCOHOL. 863

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
protein 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 J). As the alcohol has a high calorific value (1 gram = 7
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,
ZTJNTZ and GEPPERT, BJERRE, CLOPATT, NEUMANN, OFFER, RosEMANN,2 and
others, seem to show positively that, in man, alcohol can diminish the con-
sumption not only of fat and carbohydrates, but also the proteins, although
at first, due to its poisonous properties, it may increase the protein
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-
rious 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.

1 Arch, f . (Anat. u.) Physiol., 1896, which contains the literature.

2 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 ge-
samten wissenschaftlichen Literature iiber den Alcohol und den Alcoholismus," Berlin
and Wien, 1904. See also Rosemann in Oppenheirner's Handb. d. Bioch., Bd., 4. 1.

864 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/ only 10-20 per cent of the total
calories of the basal requirement belongs to the circulation and
respiration 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
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 2 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 the result 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 cannot

1 Cited from v. Noorden's Handbuch. 2. Aufl, page 97.

3 Beitrage zur Ernahrung im Knabenalter, etc. Berlin, 1902.

SURFACE OF BODY AND METABOLISM, 865

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 determines 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 ol 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,1 and others show,
that they vary only slightly from a certain average in individuals of
different weights.

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 homoio-
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).
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 RUB-
NER,2 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 diet, work 1399

Suckling 1221

Child with medium diet 1447

Aged men and women 1099

Women 1004

The variation in the calorific values 3 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

1 Rubner, Zeitschr. f. Biologic, 19 and 21; Richet, Arch, de Physiol., 5., (2).

2 Rubner, Ernahrung im Knabenalter, page 45; E. Voit, Zeitschr. f. Biologie, 41;
Oppenheimer, ibid., 42.

3 See Magnus-Levy, Pfliiger's Arch., 55; Slowtzoff (u. Zuntz), ibid., 95.

866 METABOLISM.

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 l are of value:

A

Hyc

3
5
7
9
12}
15

ge. Weight of Body in Ki
ars 10 80

Urea in Grams.
los- Per Day. Per Kilo.
12.10 1.35
11.10 0.90
12.37 0.76
14.05 0.75
17.27 0.69
17.79 0.54
17.78 0.50

13 30

16 20

18 80

25 10

32 60

. 35.70

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.

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
FALK2 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, FORSTER 3) .

In old age the metabolism is very much reduced ; and even when calcu-

1 Zeitschr. f. Biologic, 16 and 20.

2 Tigerstedt and Sond6n, 1. c.; Rubner, 1. c.; and Arch. f. Hygiene, 66; Magnus-
Levy, Arch. f. (Anat. u.) Physiol., 1899, Suppl.

3 Cited by A. Loewy in Oppenheimer's Handb., Bd. 4, 189.

SEX, REST AND WORK. 867

lated upon the square meter of surface of body it is lower than in an indi-
vidual 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 two 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.1

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 (XI), 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 2 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,3 thirst, and
drinking of water, can also influence the excretion of nitrogen. The

1 Tigerstedt and Sond6n, Skand. Arch. f. Physiol., 6; Magnus-Levy and Talk,
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 med. de Belgique,
1897 and 1901, and Maly's Jahresber., 30 and 31). See also Magnus-Levy in v. Noor-
den's Handb. d. Pathol. d. Stoffwechsels.

2 Virchow's Arch., 67 and 71. In regard to disputed views see C. Voit, Zeitschr*
f. Biol., 49 and Frankel, ibid., 50.

3 See Zuntz and Schumburg, Arch. f. (Anat. u.) Physiol., 1895.

868 METABOLISM.

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 elimina-
tion 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 1 have made investiga-
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. CO2 Eliminated. O Consumed.

-P ,. / Rest 79 209 ... 716 761

nng- • • • ^ Work 75 380 . 1187 1071

Mixed diet /Rest 137 72 352 912 831

' { Work 137 173 352 1209 980

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 pupils2 have made important investigations into
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 3 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.

1 Zeitschr. f . Biologie, 2.

2 See the works of Zuntz and Lehmann, Maly's Jahresber., 19; Katzenstetn, Pfliiger's
Arch., 49; Loawy, ibid.', Zuntz, ibid., 68, Zuntz and Slowtzoff, ibid., 95; and especially
the large work " Untersuch. uber den Stoffwechsel des Pferdes bei Ruhe und Arbeit,"
Zuntz and Hagemann, Berlin, 1898; Hohenklima und Berg wand erungen by Zuntz,
Loewy, Miiller and Caspan, which also contains a bibliography.

3 Nord. Med. Arkiv. Festband, 1897; also Maly's Jahresber., 27; Speck, " Physiol.
des menschl. Atmens," Leipzig, 1892.

METABOLISM AND WORK. 869

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 chemical processes going on during work, as we have a series
of experiments made by ZUNTZ and his collaborators, LEHMANN, KAT-
ZENSTEIN and HAGEMANN/ in which the respiratory quotient remained
almost wholly unchanged in spite of work. According to LoEWY2 the
combustion processes in the animal body go on in the same way in work
as in rest, arid a raising of the respiratory quotient (irrespective of the
transient change in the respirator} 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
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 wdth 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).

The 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.3 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-

1 See footnoted, page 808. 2 Pfliiger's Arch., 49. 3 Arch, de Physiol. (5), 8, 572.

870 METABOLISM.

SON 1 upon human beings, teach us that if the muscular work is elim-
inated the metabolism during waking 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,2 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.3

Mental activity does not seem to have any influence on metabolism
according to the means at hand for studying this influence.

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. In this case
we must differentiate between cold-blooded and warm-blooded animals.
In the first the metabolism rises with an increase in the surrounding
temperature, while in the second group the conditions are different.
The experiments of SPECK, LOEWY and JOHANSSON4 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. A chemical heat regulation, i.e., a rise in metabolism
without noticable 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 pro-
duction 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. On the contrary in several cases a
small rise in the metabolism has been observed on raising the tem-
perature above the zone within which the fundamental catabolism of
the individual showed its minimum (Vorr, RUBNER 5). The investiga-
tions of EYKMAN 6 upon inhabitants of the tropics also show the same
result, namely, that in human beings no appreciable chemical heat regula-
tion occurs.

In animals, a rise in the external temperature causes to a certain extent

1 Rubner, Ludwig-Festschr., 1887; Loewy, Berl. klin. Wochenschr., 1891, 434;
Johansson, Skand. Arch. f. Physiol., &.

2 Speck, 1. c.; Loeb, Pfliiger's Arch., 42; Ewald, Journ. of Physiol., 13.

3 See larger handbooks for the literature on this question.

4 Speck, 1. c.; Loewy, Pfliiger's Arch., 46; Johansson, Skand. Arch. f. Physiol., 7.

5 C. Voit, Zeitschr. f. Biol., 14; Rubner, Arch. f. Hyg., 38.

6 Virchow's Arch., 133, and Pfliiger's Arch., 64.

HEAT REGULATION. 871

a rise in the metabolism; the conditions on lowering the external tem-
perature are rather more complicated, and a chemical heat regula-
tion in the strict sense is difficult of exclusion, especially in small animals.
If the natural muscular activity is eliminated by poisoning with curare
or by section of the spinal cord, then, as shown by PFLUGER l and his
pupils, the warm-blooded animal behaves 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 met-
abolism. On sufficiently cooling the body the temperature sinks
irrespective of the increased metabolism, and at a certain limit of tem-
perature the catabolism of substance is still lower with decreasing tem-
perature.

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. CASPARi.2

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 and also by
HEiLNER.3 It follows from these investigations that this rise in metabo-
lism, 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 COHNHEIM 4 has shown
that in sham feeding an increased catabolism of non-nitrogenous body

1 See footnote 2, page 562.

2 Hohenklima und Bergwanderungen in ihrer Wirkung auf den Menschen, Berlin,
1906.

3Zuntz and v. Mering, Pfliiger's Arch., 15: Zuntz, Naturw. Rundschau. 21 (1906),
with Hagemann, 1. c., with Magnus-Levy, Pfliiger's Arch., 49; Magnus-Levy, ibid.,
55, and v. Noorden's Handbuch; Voit, Hermann's Handbuch, 6; Rubner, Zeitsehr.
f. BioL, 19 and 21, and Arch. f. Hyg., 66; Johansson, Skand. Arch. f. Physiol., 21,
with Koraen, ibid., 13; Heilner, Zeitsehr. f . BioL, 48 and 50.

4 Arch. f. Hyg., 57.

S72 METABOLISM.

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 consumption of more
than 50 per cent of the total energy content of the raw fodder.

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 cause a specific dynamic
action seems to be generally accepted. It is difficult to decide how
far the fats have such an action while, from the investigations of
JOHANSSON,1 there is no doubt that the carbohydrates have a specific
dynamic action.

JOHANSSON, who has studied the rise in the CO2 elimination after the intro-
duction of carbohydrates, found that this rise was proportional to the sugar
supply up to a maximal limit, but that this rise was considerably less, or indeed
absent when the store of glycogen was diminished. This behavior, as well as the
circumstance that levulose increased the C02 elimination nearly twice that of
glucose, cannot be explained by digestion work, which indicates a specific
dynamic action of carbohydrates.

The investigations of JOHANSSON, HELLGREN and GIGON 2 have also
shown that the foodstuffs, at least in the first hours after the taking of food,
act for each other in the metabolism, but not according to their iso-
dynamic values, and that they probably first pass into the various depots
of the body and then are transformed by the various tissues. JOHANSSON
and KORAEN have in a similar manner found that the muscles in their
work do not probably take the carbohydrate directly from the intestine,
but first, after their transformation into glycogen, from the supply.

1 Skand. Arch. f. Physiol., 21.

2 Johansson with Hellgren, Hammarsten's Festschrift, 1906; Gigon, Skand. Arch.
f. Physiol., 21.

NECESSITY OF FOOD BY MAN. 873

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 RTJBNER,
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 ri'ch in undigestible cellulose, a much larger quantity must be subtracted.

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.

874

METABOLISM.

I

Soldier during peace. . .
Soldier light service. . . .
Soldier in field . . .

'roteins.

119
117
146

Fat.

40
35
46

Carbohy-
drates.

529
447
504

Calories

2784
2424

2852

i. Authority.

PLAYFAIR.1
HlLDESHEIM.
HlLDESHEIM

Laborer. ... ...

130

40

550

2903

MoLESCHOTT

Laborer at rest

137

72

352

2458

PETTENKOFER and VOIT

Cabinetmaker (40 years)
Young physician
Young physician

131
127
134

68
89
102

494
362
292

2835
2602
2476

FORSTER.2
FORSTER.

FORSTER

Laborer (36 years)

133

95

422

2902

FoRSTER

English smith

176

71

666

3780

PLAYFAIR

English pugilist

288

88

93

2189

PLAYFAIR

Bavarian wood-chopper.
Laborer in Silesia

135
80

208
16

876
552

5589
2518

LIEBIG.
MEINERT 3

Seamstress in London . .
Swedish laborer
Japanese student
Japanese shopmUn. .

54
134

83
55

29
79
14
6

292
485
622
394

1688
3019
2779
1744

PLAYFAIR.
HULTGREN and LANDERGREN.*

ElJKMAN.5
TAWARA.5

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 of 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:

Proteins.
For men 118 grams

Fat.
56 grams

Carbod yd rates. Calories.
500 grams 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.

1 In regard to the older 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 Untersuchung 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.

NECESSITY OF FOOD BY MAN. 875

The quantity of food required by a woman engaged in moderate work
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 these 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 BORLAND 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 page 858) that an almost
complete or indeed a complete nitrogenous equilibrium may be attained
by the sufficient administration of non-nitrogenous 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

1 Bohland, Pfliiger's Arch., 36; Bleibtreu, ibid., 38.

876 METABOLISM.

of climate, it is not remarkable that a person accustomed to a mixed
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 l experimented on him-
self during 764 days in three series of experiments, and his diet consisted
of 7.42 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
CniTTENDEN2 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 consisted of careful
observations upon the manner of living, food taken, nitrogen elimina-
tion, 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
HAMMARSTEN also includes the figures for VOIT'S diet:

Group 1
Group 2
Group 3
Volt's figures

Urine Nitrogen.
Min. Max.

5.69 8.99
7.03 8.39
7.47 11.06
16

Catabolized Protein.
Min. Max.

35.6 » 56.19
43.9 52.44
46.7 69.10
100

Protein per Kilo.
Min. Max.
0.61 0.86
0.74 0.87
0.75 0.92
1.43

1 Arch, f . Hygiene, 45.

2 R. H. Chittenden, Physiological Economy in Nutrtition, New York, 1904.

PROTEIN REQUIREMENT. 877

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 remained in perfect health and were not only able to perform the
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 1) 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
LAPICQUE 2 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 874 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., 83; Grindley, Sammis, and others, ibid., 91.

2 Hultgren and Landergren, 1. c.; Lapicque, Arch, de Physiol. (5), 6.

878 METABOLISM.

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 containing 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.

2 Sonden and Tigerstedt, Skand. Arch, f . Physiol., 6; Johansson, ibid., 7; Tigerstedt,
Nord. Med. Arkiv. Festband, 1897.

WORK AND FOOD REQUIREMENT. 879

the following taoles give the rations of soldiers in peace and in the field
and the average figures from the detailed data of various countries.1

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 VOIT'S figures for a soldier in manceuver 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 874 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).

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.

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.

2 See footnote 1, page 877.

880 METABOLISM.

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.Z

Woman in poorhouse. ... 80 49 266 1725 FORSTER.

The figures given by VOIT are, he says, the lowest reported for a non-
working prisoner. 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 also are 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.

1 See Voit, Unters. der Kost, Munchen, 1877, page 142. See also Hirschfeld,
Maly's Jahresber., 30.

2 Voit, Unters. der Kost, page 186.

ANIMAL FOODS.
TABLE I.— FOODS.1

881

i. Animal Foodstuffs.

a. MEAT WITHOUT BONES.

1000 Parts contain

11

Fat beef 2 183 166

Beef (average fat *) 196 98

Beef 2 190 120

Corned beef (average fat) 218 115

Veal 190 80

Horse, salted and smoked 313 65

Smoked ham 255 365

Pork, salted and smoked 3 100 660

Meat from hare , . . . 233 11

" chicken 195

" partridge 253 14

" wild duck 246 31

6. MEAT WITH BONES.

Fat beef 2. . 156 141

Fcef (average fat *) 167 83

Eeef, slightly corned ! 175 93

Feef, thoroughly corned 190 100

Mutton, very fat 135 332

" average fat 160 ICO

Pork, fresh, fat 100 4CO

" corned, fat 120 540

Smoked ham 200 300

c. FISHES.

River 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 (rideX salted 200 108

Kabeljau (salted haddock) 246

Codfish (dried ling) 532 5

(dried torsk) 665 10

Fish-meal from variety of GADUSJ 736

II

X: ej

11

18

18

117

13

125

100

40

12

11

14

12

9
15

85

100

8

10

5

eo

70

10

11
11

100

107

132

178

106

59

87

640
G88
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

Relation of
1:2:3.

100
100
100
100

100
100
100
ICO
100
100
100
100

100
100
100
ICO
ICO
100
ICO
100
100

100
100
100
100
100
100
100

roo

100
100
100
100
100
100

90

50

C3

53

42

20

143

6CO

5

48

6

13

90

49

53

53

246

100

4CO

450

150

246

56

31

9

2

1

1

100

37

54

1

1

1

1

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.

2 Meat such as is ordinarily sold in the markets in Sweden.

3 Pork, chiefly from the breast and belly, such as occurs in the rations of Swedish soldiers.

882

FOOD TABLES.
TABLE I.— FOODS— (Continued).

i. Animal Foodstuffs.

1000 Parts contain

Relation of
1:2:3.

Proteids and
Extractives.

2

2

Carbohy-
drates.

4
A

5
c

V

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
10G
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
10
9
7
6

0
0
0
0
0
0

0

0
0
100
0

143
143
93
95
17
15
512

4r

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 (av-
erage results)

e. OTHER ANIMAL FOODS.
Variety of pork-sausage (Mett-
wurst)

Same for frying

Butter

Lard

Meat extract . . .

Cow's milk (full)

" " (skimmed)
Buttermilk

Cream

Cheese (fat)

" (poor)

Whey cheese (poor)
Hen's egg, entire

" without shell
Yolk of egg

White of egg

2. Vegetable Foodstuffs.

Wheat (Drains)

Wheat-flour (fine)
" " (very fine)

Wheat-bran
WTheat-bread (fresh)
Macaroni

Rye (grains)

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

VEGETABLE FOODS AND LIQUORS.
TABLE I— FOODS— (Continued}.

8C3

2. Vegetable Foodstuffs.

1000 Parts contain

Relation of
1:2:3

1

11
11

!•

2

1

3

>> .
fj

•SS
g«

4

4

10
7
10
8
12
6
19
10
4
7
9
61
3
6
29
50

5

tj

O>

1

6

1

1

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

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

I

Carbon
Dioxide.

Alcohol.

Extract.

Proteids.

i

02

Dextrin.

d

3

e

<5

Glycerine.

4
3

Porter

871

2

54

76

7

13

3 0

4

Beer (Swedish). . . .

887

28

15

. —
g

5

5

" (Swedish export) . .

885

32

7

7

3

Q

Draught-beer

911

2

35

55

8

10

31

2 0

2

2

Lager-beer

903

2

40

58

4

7

47

1 5

2

2

Bock-beer

881

2

47

72

6

13

1 7

3

Weiss-beer

916

3

?•>

59

5

4 o

2

Swedish ' ' S vagdricka "

945

22

7

^-^^
2

1 1^.'

Q

Q

884

FOOD TABLES.

TABLE III.— WINES AND OTHER ALCOHOLIC LIQUORS.

1000 Parts by Weight contain

1

Alcohol, Vol.
Per Cent.

Extract.

02

Acid and
Potassium
Bitartrate.

Glycerin.

x

8|J

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)
Tokay . ....

801

808

94
120

105
72

87
51

6.0
7 0

1.0
9.0

2.0
30

(• 60-70

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

4.0

Swedish punch

479

263

332

Brandy

460

_

French cognac

550

=

_

Liqueurs

442-590

260-475

886 INDEX TO SPECTRUM PLATE.

SPECTRUM PLATE.

1. Absorption spectrum of a solution of oxyhcemoglobin.

2. Absorption spectrum of a solution of haemoglobin, obtained by the action of an

ammoniacal ferro-tartrate solution on an oxyhaemoglobin 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 hcemochromogen, obtained by the

action of an ammoniacal ferro-tartrate solution on an alkaline-hsematin solution.

7 Absorption spectrum of an acid solution of urobilin.

8. Absorption spectrum of an alkaline solution of urobilin after the addition of a zinc-
chloride solution.

9 Absorption spectrum of a solution of lutein (ethereal extract of the egg-yolk).

INDEX OF AUTHORS

The numbers bearing a * refer to footnotes only .

Abderhalden, cleavage of polypeptides, 67;
action of yeast-press juice, cleavage of
polypeptides, 68; hydrolytic cleavage,
80, protein cleavage, 83*, 88; histone,
106*, 107, 108; ichthylepidin, 114*, 121;
peptones, 124,* 127; polypeptide-like
bodies, 131, 132; leucine, 140; glutamic
acid, 145; cystine, 146, 147; trypto-
phane, 154, 155; histidine, 157; diam-
inotrioxydodecanoic acid, 162; seral-
bumin, 163*, 254 ; enzymes in blood
serum, 256*, 259; blood serum analysis,
262, 263; stromata, 267; haemoglobin,
270; globin, 281; composition of blood
corpuscles, 292; blood corpuscles, 293;
tables of blood analyses, 314; haemo-
globin in blood, 316, 321; blood cor-
puscles, 32*1*, 322*, 323; adrenalin, 358,
359; cholesterin, 420, 421; digestion,
457*, 459; secretion of Brunner's
glands, 465; trypsin digestion, 484;
action of trypsin on acid-amides and
polypeptides, 485; hydrolysis of
glycyl-Z-tyrosine, 486; cleavage of
polypeptides, 487; putrefaction of pro-
teins, 491 ; artificial digestion with
trypsin, 492; protein synthesis, 506;
plasma protein, 507; ovalbumin, 602;
nucleoprotein of mammary gland, 610;
milk fat-globules, 614; human milk,
626*, 633; urea, 648; hippuric acid,
685; homogentisic acid, 698, 699;
polypeptides in urine, r-alanine and
glycocoll in urine, 517*, 717; fate of
polypeptides in body, 734; elimination
of cystine in urine, 736; pyridine elim-
ination, 743; protein, 749, 780*

Abel, epinephrin, 358; spermine, 590;
carbonic acid elimination, 650

Abeles, uric acid hi blood, 260; dextrose
in urine, 711

Abelmann, pancreas extirpation, 508;
fat absorption, 515; splitting of fats,
516

Abelous, oxidases, 11, 12; oxidases, 14*,
15; adrenalin, 359

Abelsdorf, visual purple, 585

Abt, 7*, 298

Ach, uric acid, 664

Achard, 259*

Achelis, methylguanidine, 663 ; ptomaines
in urine, 718, 719*

Ackermann, viridinine, 24; blood cor-
puscles, 267; vitiatine, 549; crab ex-
tract constituents, 550

Acree, proteid detection, 99

Adamkiewicz, proteid detection, 100;
nutritive value of proteoses and pep-
tones, 854

Adamson, osmotic pressure, 39

Aders, casein, 118*, 619

Adler, protoproteose, 134

Adler, O., pentose reactions, 203; detect-
ing levulose, 211; blood test, 753

Adler, R., pentose reactions, 203; detect-
ing levulose, 211; blood test, 753

Adrian, indican, 694

Adriance, J., human milk analysis, 631

Adriance, V., human milk analysis, 629,
631

Aducco, ptomaines in urine, 641*, 718

Afanassiew, bile pigments, 419

Akermann, pyloric secretion, 454

Albanese, purine bases, 676

Albert, 9*

Albertoni, solids in bile, 393 ; trypsinogen,
479; sugar absorption, 509

Albrecht, blood corpuscles, 265

Albro, trypsin digestion, 483

Albu, gastric juice, 442; leucomaines in
urine, 719; results of lack of chlorides,
720*, 729*, 827*, 843*, 844

von Aldor, detecting proteins, 748

Aldrich, sphygmogenin, 358; anal secre-
tion, 797

Alexander, proteoses, 132; casein, 619

Alexandroff, proline detection, 152

von Alfthan, conjugated glucuronic acids
in urine, 711*, 771

Allard, sugar formation from fat, 391 ;
acetoacetic acid test, 777

Allen, pepsin digestion, 448

Allers, tryptophane, 154; relation of lime
to other bases in blood, 297

Allihn, sugar estimation, 765

Almagia, carbohydrate formation, 390;
uric acid, 670

Almen, xanthine, 165; sugar test, 208;
fat in muscle, 570; water in muscle,
571; sugar test, 759

887

888

INDEX OF AUTHORS

Aloy, oxidases, 14*, 15

Alsberg, proteid reactions, 104; nucleic

acid, heminucleic acid, thymic acid,

177; nucleotin, 178
Altmann, nucleic acid, 176*, 181
Amberg, action of HF1 on enzymotic

cleavage, 74
Ambronn, 790

Amerman, digestion products, 447*
Amiradzibi, carnosine, 549
Amseder, ethal, 232; dermoid cysts, 596;

dermocerin, 796

Amthor, oleic acids, 230 ; animal fat, 533
Andersen, estimating sugar, 764
Anderson, A. C., 152*, 356*
Andersson, N., blood serum, 257; sugar

in blood, 317

Andrlik, glutamic acid, 145
von Anrep, carbon-monoxide methaemo-

globin, 279; benzoic acid, 687
Anselm, iron in bile, 411
Ansiaux, fibrinogen, 96, 245
Anthon, solubility of dextrose, 206
Applcyard, adsorption, 48, 49
Araki, I., methaemoglobin, 277; action' of

trypsin on nucleic acids, 279*, 381*, 485;

paralactic acid, 554, 710; lactic acid,

556; chitin, 711*, 791
Ardin-Delteil, perspiration, 798
Argiris, protagon, 112*, 114*, 576 .
Argutinski, flesh analysis, " flesh quotient,"

571; perspiration, 572*, 799; nitrogen-
carbon relation, 827
Arinkin, 17*
Armstrong, emulsin, Kefir lactose, 62*,

63*, 65; inversion of cane sugar, in-

vertin action, 68
Arnheim, 387*
Arnold neutral hsematin, 280; adrenal

chromogen, 357; creatinine reaction,

662
Arnold-Lipliawsky, acetoacetic acid test,

777

Arnschink, fat absorption, 514
Arnstein, purine bases, estimation, 679
Aron, methaemoglobin, 274; bone earth,

527; bone development, 530
Arrhenius, osmotic pressure, 29; catalysis,

54, 55; rule of enzyme action, 59*, 63*,

64; Ehrlich's theory, 72
Arronet, blood corpuscles, 314
Arteaga, 379*
Arthus, blood coagulation, 242, 243; fibrin,

247; glycolytic enzyme, 258; buty-

rinase, 259; coagulation of blood, 300,

301; calcium salts in blood coagulation,

303, 304; fluoride plasma, 305; action

of tissue extracts, 307; glycogen, 377;

casein, 615, 617
Artmann, colloids, 44
Ascher, isoserine, 144
Ascherson, haptogen membrane, 613
Ascoli, plasminic acid, 176*, 181; uracil,

190; glutinase, 481; trypsin, 482;

protein assimilation, 502; enzymes in

placenta, 608; urea, 648; uric acid,

670, 671
Asher, sugar in blood serum, 257; lymph,

327, 331-333; spleen, 353; submaxillary

saliva, 360*, 427; nitrogen eliminated

in urine, 505; lactic acid, 555; protein

metabolism, 852
Aso, oxidase, 13; 96

Astaschevvsky, muscle reaction, 563; 564*
Athanasiu, substance retarding blood

coagulation, 311; fat formation, 363*,

534
At water, muscle energy, 566, 569;

respiratory gas exchange, 819, 820;

metabolism, 823; alcohol in metabolism,

863; food requirements, 874; food

demand for working individuals, 877*,

879; calorimetric investigations of foods.

834
Aubert, carbon dioxide eliminated by the

skin, 800

Austin, glycogen estimation, 371
Austrian, 667*
Autenrieth, oxalic acid, 681; potassium

and sodium in urine, 727
Ayres, 584

Baas, steapsin, 479; hippuric acid, 684

Babcock, milk, 620

Babkine, pancreatic secretion activator,.

107*, 476; solids in pancreatic juice,

477; urea, 648
Bach, autooxidation 7, 8; oxidases, 11,

12; peroxidases, 13; 14*
Baer, cystine, 146; thiolactic acid, 148*

urine, 149*, 641; acetone bodies in

urine, 772, 774
Baeyer, A., sunlight synthesis, 1; animal

oxidations, 6; assimilation hypothesis,

205

Baginsky, mucin in bile, 413; bone, 530
Bainbridge, lymph, 333; lactase forma-
tion in intestine, 473
Baisch, carbohydrates in urine, 711 ; sugar

test, 763
Baker, 219*
Baldi, jecorin, 364
Baldoni, pancreatic calculi, 487; fate of

salicylic acid, 740
Balean, haemoglobin, 289
Balke, purine bases, 184; adenine, 188;

phosphocarnic acid, 550; episarkine,

677
Bang, I., nucleic acid, 175; guanylic acid,

178, 179, 180; nucleohistone, 295;

lymphatic glands, 317*, 347, 348;

thymus, 348, 349; glycogen estimation,

371; hyperglycaemia, 382; glycosuria,

381, parachymosin, 450; law of rennin

action, 451; modified Devoto method,

637*, 748; sugar test, 759, 760; method

estimating sugar, 764
Barbera, lymph, 327, 331, .332; lymph

secretion, 393
Barbieri, phenylalanine, 150; ovin, 599

INDEX OF AUTHORS

889

Bardachzi, 397*

Barendrecht, inversion of cane sugar, 68

Barker, proteolytic enzymes, 295, 780*

Barral, sugar in blood, 258

Barratt, carbon dioxide eliminated by
the skin, 800

Barszcewski, arabinose, 203

Barth, oxalic acid, 681

Bartoschewski, 688*

Basch, milk secretion, 633, 634*, 636*

Basedow's disease, 355

Baserin, bile pigments, 418

Bashford, 686*

Baskoff, jecorin, 364

Bassow, gastric juice, 437

Bass, 737*

Bastiannelli, intestinal juices, 467

Battelli, 386*

Baudrimont, iron in hair, 790

Bauer, keratin, 112; atmidkeratose, 113;
histidine, 157; protein assimilation, 502;
fat formation, 534; urobilinogen, 708;
trimethyl amine in urine, 718, 719*

Bauer, Fr., inosinic acid, 179

Bauer, R., detecting galactose and lactose
in urine, 769

Baum, skatosine, 156

Baumann, L., cystine, thiolactic acid, 24*,
148; tryptophane, 154; sugar test,
209, iodothyrin, 281*, 356; protein in
sugar formation, 390, 459; putrefaction
of proteins, 491*, 492; intestinal putre-
faction, 493; brain composition, 582;
hippuric acid, 684; ethereal sulphuric
acid, 688; phenolsulphuric acids, 689;
pyrocatechin sulphuric acid, 690; hydro-
quinone indican elimination, 691;
potassium indoxylsulphonate, 693 ; oxy-
acids in urine, 697 ; homogentisic acid,
699,701; carbohydrates in urine, 711;
ptomaines in urine, 719; determining
sulphuric acid in urine, 726, 735* ; fate
of amino-acids in the body, 736*, 737;
oxidation of benzene, 738; elimination
of ethereal sulphuric acid, 740, 742;
mercapturic acids, 743; sugar testing,
762; cystine in urine, 780; detecting
cystine, 781

Baumgarten, cerebrospinal fluid, 342;
diabetes, 382; chlorine in urine, 719

Baumstark, neurokeratin, protagon, 575;
creatine in brain, 576; cholesterin in
brain, 582; urorubrohsematin, urofusco-
haematin, 754

Bayer, plasteinogen, 135

Bayliss, kinase and secretin in intestine,
enterokinase, 61*, 468, 472; erepsin,
469; action of trypsin on trypsinogen,
474; pancreatic juice excitants, pro-
secretin, 475; trypsin, 480; enzyme
in pancreas, 481 ; trypsin estimation,
speed of trypsin digestion, 482, 619

Beatty, polypeptides, 89; 106*

Beaumont/ gastric juice, 437; gastric
digestion, 457

Beccari, iron in bile, 411

Bechamp, A., phacozyme, crystalbumin,
crystalfibrin, 587; ovalbumin, 602;
milk carbohydrates, 622

Bechhold, colloids, 39, 40, 45; colloid
filtration, 51 ; sugar test, 759

Beck, sulphur elimination, 566; 634*, 706*

Beckmann, determining osmotic pressure,
29 ; ammonia in urine, 727 ; anal secre-
tion, 797

Becquerel, fibrin in blood, 320; human
milk, 631

Beebe, mineral substances in cells, 22

Beger, 634

Behrend, uric acid, 663; 664*

von Behring, anti-bodies, 70

Beier, bile acids, 416

Beil, milk, 626

Beilter, 153

Bela v. Bitto, acetone test, 776

Bellamy, activation of trypsinogen, 473

Belloni, orotic acid, 621

van Bemmelen, adsorption, 50; gels, 51

Bence, blood corpuscles, 298

Bendix, pentoses, 201, 202; glycogen
formation, 373

Benedicenti, indican, 126*, 692; alcohol
in metabolism, 863

Benedict, sugar test, 208, sulphur elim-
ination, 566; muscle energy, 569;
creatinine, 660; sulphur compounds
in urine, 714; respiratory gas exchange,
819; calorific value of foods, 829;
sulphur excretion in starvation, 841;
alcohol in metabolism, 863

Benedikt, R., 228*

Benrath, gastric secretion, 453

Berard, globulins, ovalbumin, 96*, 602

Berdez, hippomelanin, phymatorhusin,
793

Berenstein, 499*

Bergell, protein cleavage, 89; preparing
lecithin, 238; seralbumin, 254; sphyg-
mogenin, 358; enzymes in placenta,
485*, 608; human milk/ 628

Berger, ptyalin, 431

Bergh, elastin, 115, 116

Bergholz, pancreatic calculi, 487

Bergin, reaction of intestinal contents, 497

Bergmann, amino-acids in blood serum,
260; bile acids, 356*, 417; pepsin, 443;
effect of removing colon, 518; phos-
phates in urine, 723

Berlinerblau, lactic acid in blood, 318

Berlioz, chlorine in urine, 719

Bernard, Claude, sugar in blood, 258;
316; glycogen in liver, 368, 377; piqure,
380; pancreatic juice, 381*, 383*,471 ;
steapsin, action of pancreatic juice on
proteins, 479; fat absorption, 514; meta-
bolism in muscles, 563

von Berneek, catalysis, 57

Bernert, ascitic fluid 82*, '226*, 340

Bernheim, ascitic fluid, 340; potassium
and sodium in urine, 727

890

INDEX OF AUTHORS

Bernstein, muscle reaction, 572

Bert, P., origin of milk sugar, 611*, 636;
blood gases, 802; oxygen in blood, 811

Bertagnini, 740

Berthelot, colloidal precipitation, 48;
steapsin, 479; tunicin, 790; calorific
value of foods, 829

Bertin-Sans, methaemoglobin, 277; car-
bon monoxide methaemoglobin, 279

Bertrand, oxidases, 11, 12, 13; xylose,
22*, 204; thyroid arsenic, 354; arsenic
in skin, 790; bufonin, bufotenin, 797

Bertz, chlorine in teeth, 532

Berzelius, catalysis, 3*, 54

Bethe, brain decomposition products, 581

Bial, M., test for pentoses, 201*, 203;
diastases in blood, 258; lymph; 328;
glucosamine as a glycogen former, 375;
glycogen, 377; test for pentoses, 378*,
770

Bialcour, lactic acid fermentation, 460;
461*

Bialobrzeski, 285

Biarnes, oxidases, 11, 12

von Bibra, liver constituents, 367

Bickel, gastric juice, 440*, 441

Bidder, buccal mucus, 428; influence of
bile on digestion, 434*, 441*, 476*, 496;
fat absorption, 514

Biedermann, tyrosinases, 12

Biedert, 627*

Biedl, ammonia in blood, 318

Biel, 629

Bielfeld, iron in liver, 366

Bienenfeld, 627*

Bienstock, influence of bacteria in intes-
tines, 497, 498

Biernacki, blood corpuscles and serum,
297*, 313; pepsin, 443; trypsin, 480;
putrefaction in intestine, 497, 688

Bierry, enzymotic processes, 73; lactase
formation in intestine, 473; maltase
in pancreatic juice, 478

Biffi, pigments in blood serum, 260; casein
619, urobilin, 706

Billitzer, colloidal precipitation, 47

Biltz, molecular movements, 42 ; colloids,
44; adsorption, 49, 73

Bing, lecithin sugar, 237; blood serum,
257; jecorin, 364; 378*

Binet, bile absorption, 517

Bingel, amino-acids in blood serum, 260

Binz, 735

Biot, gas secretion in fish, 817

Biscaro, orotic acid, 621

Bizzio, glycogen, 369; chromhydrosis, 800

Bizzozero, blood-plates, 295; blood coag-
ulation, 301

Bjerre, alcohol in metabolism, 863

Blachstein, levolactic acid, 553

Blankenhorn, protagon, 576

Blanksma, galactosamine, 164, 215; esti-
mating acetone, 778

Bleibtreu, fat formation, 537; estimating
urea, 655; protein metabolism, 875

Bleibtreu, L., blood corpuscles, 312, 314*

Bleibtreu, M., blood corpuscles, 312, 314*

Bliele, sugar in blood, 316

Blendermann, protein in sugar formation,
390; oxyacids in urine, 697; 737*

Bliss, 126

Blix, haematocrit, 313

Bloch, homogentisic acid, 687*, 698, 699

Blondlot, influence of bile on digestion,
496

Blum, autolysis, 19; putrefactive prod-
ucts, 81; protogen, 98*, 126; adrenalin
glycosuria, 384; homogentisic acid, 699;
elimination of cystine in urine, 736;
acetone bodies in urine, 772, 774;
nutritive value of proteose, 855

Blumenthal, protein oxidation products,
82; pentose, 201; glycogen, 362*, 373;
indican elimination, 510*, 686*, 692;
pentoses in urine, 769; acetone bodies
in urine, 772

Boas, parachymosin, 450; reagent for
free HC1 in gastric juice, reagent for
lactic acid in gastric juice, 463

Bocarius, sperm reaction, 590

Bock, carbon - monoxide haemoglobins,
278*, 279; glycosuria, 379, 380*, 381*

Bodeker, alcapton, 690

Bodlander, alcohol in metabolism, 863

Bodon, transudates, 337

Bodong, fluoride plasma, 305, 310*

Bodtker, urine nitrogen, 646 ; determining
chlorine in urine, 721

Boechi, O., urochrome, 704

Boehm, 360*

Boeckelman, estimating betaoxybutyric
acid, 779

Boeri, oxalic acid, 680; sulphur com-
pounds in urine, 714

Bogdanow, fatty acids, 557; muscle work,
567

Bogdanow-Beresowski, 430

Bogen, 440*

Bogomoloff, 705*

Bohland, urine nitrogen, 646; estimating
urea, 655; ammonia in urine, 666*,
728; protein metabolism, 875

Bohm, glycosuria, 381; glycogen, 553;
rigor mortis, 561

Bohmansson, sugar test, 759, 760

Bohr, C., hsemochrom, 268; haemoglobin,
270; oxyhaemoglobin, 271, 272; car-
bon-dioxide haemoglobin, 275*, 280

Bohr, hemoglobin, erythrocites, 323;
egg development, 606; blood gases,
801-804; respiration, 809, 810; oxygen-
ation of blood, 812; respiratory air, 814;
carbon dioxide tension in blood, 816,
817; gas secretion in fish, 817; varying
oxygen tension of blood, specific
oxygen capacity of blood, 818; reducing
property of lungs, 820

Bohtlingk, increased elimination of nihu
eral substances during starvation
839

INDEX OF AUTHORS

891

Du Bois-Reymond, R., muscle reaction,
299, 563, 572

Bokorny, active proteins, 4*, 5; sugar
synthesis, 206

Bolaffio, neottin, 233, 599

Boland, 347*

Boldryeff, gastric digestion, 457; secre-
tion of Lieberkuhn's glands, 465; intes-
tinal juice, 466, 467; enzymes in intes-
tinal juice, 468; digestion, 489

Bolin, J., lucerne-laccase, 13

Boll, visual red, 584

Boltazzi, protein in placenta, 608

Bonamartini, 95*

Bonanni, molecular concentration of
bile, 412, 743*

Bondet, serolin, 256

Bondi, fibroin, sericin, 122*, 123; relation
of lime to other bases in blood, 297;
synthetic glycocholic acid, 397, 398;
synthetic taurochoiic acid, 399; cholic
acid, 402; amniotic fluid, 609

Bondi-Schwarz, acetoacetic acid test, 777

Bondzynski, koprosterin, hippokoproste-
rin, 82*, 423; ovalbumin, 602; purine
bases, 676; ahtoxyproteic acid, 715;
oxyproteic acid, alloxyproteic, 716;
uro ferric acid, 717

Bonnema, milk fat-globules, 613

Boos, 429*

Borchardt, glycogen, 377; absorption of
proteose, 503; estimating sugar, 768;
acetone bodies in urine, 772; acetone
elimination, 773; acetoacetic acid test,
778

Bordet, immune bodies, 72; precipitins
in blood, 259; blood coagulation, 301;
prothrombin, 305

Borissow, gastric juice, 438; allantoin, 682

Borkel, 135*

Bornstein, muscle work, 566; protein
fattening, 860

Boruttau, glycogen, 553

Bosshard, leucine, 141

Bottazzi, osmotic pressure, 35; nucleated
erythrocytes, 293; liver fat, 363; glyco-
gen, 369; nucleoprotein, 370*, 543;
nucleon in muscle, 573

Bottcher, spermine crystals, 590

Bottger-Alme'n, sugar test, 208

Bouchard, autointoxication, 25; glycogen
formation, 372; ptomaines in urine,
718; leucomaines in urine, 719

Boulud, glucuronic acid, 215; pentose,
in blood serum, 257; glucuronic acids,
258, maltose in urine, 768

Bouma, indican, 694; test for bile pig-
ments, 757; estimating betaoxybutyric
acid, 779

Bourcet, iodine and arsenic in blood
serum, 261, menstrual blood, 320

Bourquelot, oxidases, 11; glycogen forma-
tion, 375

Bouveault, isoleucine, 143

Bovet, putrefactive products, 81

Bowman, " disks," 539

Boyle-Mariott, law of gas pressure, 27

Bradley, 22*

Brahm, cleavage of polypeptides, 487;
pyridine elimination, 743

Brahn, inosinic acid, 179, 180; xylose, 204

Brand, bile secretion, 392; solids in bile,
molecular concentration of bile, 412;
sulphur, in bile, 413

Brantlecht, plant proteins, 77

Brandenburg, alkalies in blood, 297;
estimating proteid, 750

Brandl, phymatorhusin, 793

Brasch, isoleucine, 143

Brat, test for pentoses, 203

Brauer, bile, 415

Braunstein, estimating urea, 387*, 656

Bredig, metallic sols., 37; precipitation of
colloids, 46; catalysis, 54, 57, 58

Brieger, H., ptomaines, 24; putrefaction
of proteins, 492; neuridine, 576, 581;
egg yolk, 597; indican elimination, 691;
potassium indoxylsulphonate, 693; ska-
toxylsulphuric acid, 695; putrescine
and cadaverine in urine, 719; perspira-
tion, 780*, 798

Briggs, 381*

Brion, fate of tartaric acids in body, 734

Brocard, influence of food on enzyme
formation, 473

Brodie, sugar formation in kidney, 248*,
379; action of pancreatic juice on
casein, 487

Brook, putrefactive products, 81

Brooks, sugar formation from glycogen, 378

Browinski, proteic acids in serum, 260;
urochrome, 704

Brown, ptyalin, 61*, 62*, 219*, 223*, 236*,
432; intestinal juice, 467; uric acid,
670; allantoin, 682

Brown, A. J., inversion of cane sugar, 68

Brown, Graham, muscle work, 565

Brown, R., molecular movement, 41

Browne, 614*

Brubacher, bone analysis, 529; bone, 531

Brucke, peptone, 129; blood coagulation,
300; fibrin ash, 303; glycogen prepa-
ation, 371; pepsin, 443, 444; pepsin
test, 445; emulsifying action of fatty
acids, 489; protein assimilation, 502;
dextrose in urine, 711; pepsin in urine,
718

Briicke-Kiilz, glycogen estimation, 371

Brugsch, uric acid, 665, 666, 670; hip-
puric acid, 685; urea loss in starvation,
718*, 841

Bruhns, 189*

Brunner, 57*

Brunner, human milk, 631

Bruno, steapsin, 479; tryptic digestion,
483; action of bile on diastatic action
of pancreas infusion, 488; 719*

Brunton-Blaikie, urea in muscle, 546

Bryant, food demand for working indi-
viduals, 877*3 879

892

INDEX OF AUTHORS

Buchanan, thrombin, 248

Buchner, E., ferment, 9, 10; enzymes,
59, 60; sugar fermentation, lactacidase,
zymase, 200; lymph gases, 207*, 807

Buchtala, keratin cleavage products, 113;
cystine, 114*; 146

Budde, estimating sugar, 766

Buerger, analysis of achilles tendon, 520

Bufalini, haemoglobin, 321

Bugarsky, proteids, 94; molecular con-
centration of sera, 263; urine, 643

Bull, doeglic acid, 230

Bulnheim, cholic acid, 401

Biilow, 94*, 221*

Bunge, blood serum analysis, 263; blood
corpuscles, 293; estimating blood cor-
puscles and blood fluid, 314; iron in
blood, 322; blood corpuscles, 323;
liver constituents, 366; HC1 secretion,
453; cartilage, 461*, 525; pseudonu-
clein, 598; egg constituents, 606; min-
eral bodies of milk, 625; human milk,

630, 633; chemistry of milk secretion,
634; hippuric acid, 686; death from
lack of mineral substances, 843 ; results
of lack of chlorides, 844; metabolism,
847

Buntzen, blood corpuscles, 321

Biinz, cholesterin in brain, 582.

Buraczewski, ruemopyrrol, 288

Burckhardt, globulins in blood serum, 262

Burian, xanthine oxidase, 12, 13; purine
bases, 183, 184; xanthine oxidase, 189*,
351; spleen, 354; muscle extractions,
545; muscle work, 565; uric acid, 665;
uric acid, xanthine oxidase, 667; uric
acid, 669, 670, 671; histone in urine,
751; 676*

Burow, human milk, 629, 630*

Biirker, blood coagulation, 301

Busch, lymph, 331*, 332

Butlerow, sugar synthesis, 205

Bywaters, seromucoid, 252, 256

Cade, 440*

Cahn, gastric secretion, 452; retina con-
stituents, 584

Camerer, determining proteins in milk,
623; determining lactose in milk, 624;
human milk analysis, 629; human milk,

631, 632, 633; urine nitrogen, 646;
purine bases estimation, 665*, 679;
ammonia in urine, 727; perspira-
tion, 798; metabolism, 864; urea
elimination, 866

Cammidge, sugar reaction, 769; cystin-
uria, 780, 781

Campani, 724*

Campbell, bilicyanin, 408, 410; globu-
lins, 601; ovalbumin, 602; ovovitellin,
597, 598

Camps, 702*

Camus, antitryptic action, 68; muscle
metabolism, 563; prostate fluid, vesicu-
lase, 590; enterokinase, 472; pan-

creatic secretion, 473; pancreatic ex-
citants, 475

Cannon, gastric digestion, 455, 456, 458

Cappelli, nucleated erythrocytes, 293;
nucleon in muscle, 573

Capranicka, guanine, 186; creatinine in
perspiration, 799

Carlier, blood coagulation, 300, 329*

Carlson, lymph, 330

Carnot, bone ash, 527; dentin, 532

Carvallo, substances retarding blood
coagulation, 311; digestion, 460, 461*

Casali, bufidin, 797

Caspari, W., muscle work, 323*, 566;
milk fat formation, 635; catabolism
limits, 831*, 858; effect of altitude on
metabolism, 868*, 871

Castoro, 158*

Castro, 224*

Cathcart, tryptic digestion, 69; glucosa-
mine as a glycogen former, 375; muscle
work, 376*, 504*, 565; creatinine, 660;
urea loss in starvation, 841

Cavazzani, E., cerebrospinal fluid, 342;
sugar formation from glycogen, 378;
starch utilization, 511; nucleon, 550;
nucleon in semen, 589; muscle metab-
olism, 563

Cetti, putrefaction of proteins, 494;
nitrogen excretion, 825; urea, excre-
tion in starvation, 838

Cerny, detecting proteids, 555*, 748

riuibbas, glucose in vitreous humor, 586

Chabrie, bone, 531

Chaniewski, fat formation, 537

Chauveau, sugar formation from fat, 391;
fat formation, 536; muscle metabolism,
563; muscle work, 568

Chandelon, metabolism muscles, 563

Chassevant, uric acid, 670

Cherry, 72*

Chigin, gastric juice, 437

Chittenden, elastin, 115; protoelastose,
deuteroelastose, 1.16; gelatin, 119;
proteoses, 128*, 129; protoproteoses,
134, 137*; buccal saliva, 430; ptya-
lin, 431*, 432; saliva, 433; digestion
products, 447; pepsin digestion, 448;
trypsin digestion, 483; mucoids, 519;
myosin, 540; neurokeratin, 451*, 575,
583; protein requirements, 876

Chodat, animal oxidation, 7; oxidases,
11, 12; peroxidases, 13, 14*

Chossat, tissue loss in starvation, 839;
starvation, 836

Christenn, 629*

Christensen, estimating proteid, 750

Ciamician, indol test, 156

Cingolani, uric acid, 664

Citron, nitrates in urine, 727

Clapp, hydrolytic cleavage, 80; poly-
peptides, 89; 107*

Claus, 387*

Clar, 666*

Clarke, sulphsemoglobin, 279

INDEX OF AUTHORS

893

Clemens, elimination of camphors and
terpenes, 742, 743*, 779*

Clemm, human milk analysis, 631

Clerc, 259*

Cleve, cholic acid, 401

Cloetta, haimatin, 283; hseniin, 286;
uroproteic acid, 716

€loez, bile acids, 416

Clopatt, alcohol in metabolism, 863

Closson, hyperglyc83mia, 380, 659*

Cohn, R., hydrolytic cleavage, 80; leu-
cinimide, 142; carbohydrate metabol-
ism, 390; lactic-acid fermentation, 430*,
460, 461*; allantoin, 682; fate of
benzene derivatives in the body, 737;
fate of furfurol and orthonitrobenz-
aldehyde in the body, 740*, 741; pyri-
dine elimination, 743

Cohn, Th., crystals in semen, 590

Cohnheim, intestinal absorption, 21;
proteids, 76*, 80*, 94; alkalinity of
blood, 297; blood corpuscles, haemo-
globin, 320; lymph formation, 334;
glycolysis, glycolytic enzyme in pan-
creas, 386*, 387; ptyalin, 431; diges-
tion, 459; gastric absorption, 460;
erepsin, 467, 468; deamidation of
digestive products, 469*, 472*, 505;
diastatic enzyme in urine, 718; effect
of food in digestion on metabolism, 871

Colasanti, metabolism in muscles, 562;
lactic acid in muscles, 564; xantho-
creatinine, 663; paralactic acid, 710

Cole, proteid detection, 99, 100; trypto-
phane, 106*, 153, 154, 155

Colenbrander, 258*

Collmaim, chromhidrosis, 800

Commaille, milk constituents, 634

Connstein, lipolysis, 259

Constantinidi, sugar in intestines, 511;
catabolism limits, 858

Conradi, autolysis, 19; bodies retarding
coagulation, 311; action of autotoxines
in intestines, 498; fat absorption, 534

Contejean, substances retarding blood
coagulation, 311; gastric juice, 379*.
441 ; pyloric secretion, 452*, 454

Copeman, blood test, 753

Coranda, urea, 649; ammonia in urine,
727

Cordua, hsematoidin, 290

Corin, fibrinogen, 96*, 245; globulins,
ovalbumin, 602

Corvisart, action of pancreatic juice on
proteins, 479

Le Count, 114*

Courant, milk, 611; casein, 616, 617;
human milk, 627

Cousin, lei, chins, 234

Couvreur glycogen formation, 372

Cramer, thrombosin, 122*, 245*, 303;
protein absorption, 502; influence of
leucocytes on proteoses, 505; influence
of leucocytes on protein assimilation,
507; utilization of foods, 508; pro-

tagon, 576, 578; glycogen in placenta,

608; perspiration, 686*, 799
Cremer, yeast-press juice, 65; pentoses,

201; glycogen in plants, 258*, 369;

glycogen, 371*, 372; glycogen formers,

375; sugar formation from glycerin,

379*, 390; fat formation, 536
Croftan, bile acids in blood, 316, 670*
Croft-Hill, catalysis, 64
Crookewitt, 122*
Cummins, trypsin digestion, 483; myosin,

540, 541*

Cunningham, fat absorption, 516
Curtius, cholic acid, 85*, 401
Cutter, tendon mucin, 165; submaxillary

saliva, 427; tendon mucin, mucoids,

519

Csokas, casein, 615
Cybulski, adrenal pressure raising action,

357
Czerny, glycogen, 295; digestion, 386*,

460; protein assimilation, 502

Daddi, fat in blood, 321; milk fat forma-
tion, 636

Daenhardt, lymph gases, 807

Dakin, hydrolysis by liver-press juice,
67; protein cleavage, 90; protamines,
99*, 109*, 110; valine, 139; serine, 143;
proline, 152; arginine, 156*, 158;
lysine, 160; urea, 648; oxalic acid,
680; allantoin, 683; fate of oxalates
in body, 733; fate of amino-acids in
body, 734 ; oxidation of fatty acids, 739

Daland, estimating blood corpuscles, 313

van Dam, casein, 617

Danilewsky, W., sulphur in proteins, 79;
antialbumid, 134; plastein, 236*, 452;
auto digestion, 462; antienzymes in
intestinal juice, 468; myosin, 539, 540;
myosin, 541; muscle-stroma, 542;
milk globules, 613; calorific values of
food, 829

Danoise, 614*

Dareste, testes constituents, 589; egg
yolk, 797

Darmstadter, estimating betaoxybutyric
acid, 424*, 779; wool-fat, 797

Dastre, fibrinogen, 244; fibrin, fibrin-
olysis, 247; glycogen, 295; blood
coagulation, 301; substances retarding
coagulation, 311; glycogen, 328; liver
pigments, glycogen, liver globulin, 362;
iron in liver, 366; glycogen formers,
375; sugar formation from glycogen,
377*, 378; bile secretion, 392; bili-
prasin, 410; iron in bile, 411; eritero-
kinase, 472; pepsinogen conversion,
474; action of bile on gastric action,
489; fat absorption, 514

Dauwe, 59*

Day, gastric digestion, 456

Dean, protein synthesis, 222, 506

Decaisne, influence of food on milk com-
position, 634

894

INDEX OF AUTHORS

Dehn, determining chlorine in urine, 722

Dekhuisen, 35*

Delezenne, tryptic digestion, 68; blood
Coagulation, 242, 300; action of tissue
extracts on blood coagulation, 305, 307;
substances retarding blood coagulation,
311, 312; lymphagogues, 333; secre-
tion of Lieberkuhn's glands, 465; intes-
tinal secretion, 466; enterokinase, 468,
472; trypsinogen activators, 474; pan-
creatic juice excitants, prosecretin,
475; tryptic digestion, 484; pancreatic
rennin, 487

Delfino, protein in placenta, 608

Demant, intestinal juice, 467

Deniges, proline test, 152; inosite tests,
552; uric acid reaction, 673; purine
bases estimation, 679; homogentisic
acid, 701

Derrien, methaemoglobin, 274, 749*

Desgrez, glycogen formation, 372

Deucher, pancreatic duct closure, 408;
fat absorption, 515; splitting of fats,
516

Devillard, hydrocele fluid, 341

Devoto, detecting proteoses, 748

Dewitz, melanins, 794

Diaconow, lecithin, 235, 236; preparing
lecithin, 238

Diamare, 470*

Diels, cholesterin, 420, 421

Dietschy, detecting proteids, 748

Dietz, synthetic action of pancreas, 62*,
65; reversible reaction, 66; splitting of
fats, 227

Dietze, trypsin, 483

Dillner, globulins, 601, 602*

Disqu6, urobilinogen, 703; urobilin, 705

Disselhorst, perspiration, 798

Ditthorn, compound protein, 164*, 170;
galactosamine, 215

Dittrich, methsemoglobin, 277

Dock, 381*

Dohrn, internal secretion in pancreas, 385

Dombrowski, urochrome, 703, 704; auto-
oxyproteic acid, 715; alloxyproteic
acid, 716; uroferricacid, 717; ptomaines
in urine, 718; cadaverine in urine, 719

Domenicis, glycogen, 279*, 383

Donath, cerebrospinal fluid, 342

Donne, pus test, 755

Dony-Henault, oxidase action, 13

Doree, koprosterin, hippokoprosterin, 423

Dorner, creatine, 659

Dorpinghaus, cleavage products of keratin,
113; valine, 139; leucine, 140; seral-
bumin, 254

Dagon, fibrinogen, 244, 245; glycolytic
enzyme, 258; butyrin, 259; fibrinogen
in Iblood, 319; olive oil as a cholagogue,
393; cholagogues, 394; biliverdin, 410;
cholesterin in bile, 414

Dragendorff, bile acids, 755

Drechsel, oxidations, 14; hydrolytic
cleavage 79, 80; gelatin, 93*, 118;

porgonin cleavage products, 123; diam-
inoacetic acid, 159; lysine, lysuric acid,
160; lysatine 161; purine bodies, 184;
thyroids, 356; jecorin, 364; urea, 647,
649, 650 ; silicic acid in feathers, 790

Dresser, ion-action, 35*, 74

Droop-Richmond, milk fat-globules, 613

Drosdoff, haemoglobin in blood, 319

Dubelir, effect of water on metabolism,
862

Duecheschi, digestion, 452

Duclaux, volatile fatty acids in butter,
96, 614

Ducleau, 61*

Dufau, sugar estimation, 763

Dufour, glycogen in muscles, 563

Dufourt, olive oil as a cholagogue, 393;
cholagogues, 394; cholesterin in bile..
414

Duggan, 96*

Dull, 223*

Dumas, 651*

Dunham, carnaubic acid, 232, 639*

Dunlop, sulphur elimination, 566, 697*

During, bone constituents, 529

Ebbecke, adialyzable bodies in urine, 717
Ebstein, pentoses, 114*, 201; pentose

determination, 202; ptyalin, 432; pyro-

catechin, 690; calculi, 784
Eck, fistula operation, 376
Eckhard, chorda-saliva, 427
Edie, E., preparing lecithin, 238
Edkins, gastric juice, 439, 453*, 487*
Ehrenfeld, protein oxidation products,

protein reaction products, 80*, 82;

leucine, 141; lecithin, 237
Ehrenreich, enzymes, 59, 481*
Ehrenthal, 499*
Ehrlich, Felix, leucine, isoleucine, 141,

142
Ehrlich, P., antibodies, 69; measuring.

toxin value, 70; side-chain theory,

toxoid, immune bodies, 71; glucosa-

mine, 214; precipitins in blood, 259;

bilirubin test, 407; urine test, 779*,

826*

Ehrmann, glycosuria, 384
Ehrstrom, lotahistone, 108; phosphates in

urine, 723 ; results of lack of phosphates-

and earths, 748*, 845
Eicholz, globulins, 262, 602*, 705*
Eichler, glycosuria, 384
Eichorst, protein assimilation, 502
Eijkman, 874
Einhorn, 156*

Ekborn, desoxycholic acid, 403
Ekehorn,determmingchlorinein urine, 722
van Ekenstein, Alberda, carbohydrates,

164*, 196; galactosamine, 215; esti-
mating acetone, 778
Ekholm, diet required, 878
Ekunina, glycogen, 555
Ellenberger, gastric digestion, 455, 456;

milk, 626

INDEX OF AUTHORS

895

Ellmger, isoserine, 144; tryptophane, 153,
154, 155; ornithine, 159; lysine, 160;
substances retarding blood coagulation ,
311; lymph formation, 333*, 334; indi-
can elimination, 692; indican, indigo
red, 694; kynurenic acid, 702; nutritive
value of antipeptone, 654

Elvove, 14*

Ely, buccal saliva, 430, 432*

Embden, hydrolytic cleavage, 78; sys-
tine, 79*, 146; liver, 360; carbohydrate
formation, 387*, 390; lactic acid, 555;
homogentisic acid, 698; glycocoll in
urine, 717; acetone bodies in urine, 772;
acetone formation, 774; acetone bodies,
775; estimating acetone, 778

Emerson, autodigestion of pancreas, 484

Emich, glycocholic acid, 398; bile acids
as preventive of putrefaction in intes-
tine, 495

Emmerling, maltose synthesis, amygdalin
reconstruction, 65; cleavage products
of keratin, 113; isomaltose, 219

Engel, H., steapsin, 452*, 479; human
casein, 628; acetone formation, 772*,
774

Engeland, methyl guanidine, 663; pto-
maines in urine, 7 18-719 ; diazo-reaction,
779

Engelmann, sulphur elimination, 566

Engler, animal oxidations, 6; auto-
oxidation, 7

Eppinger, hsematin, 283; haemin, 286;
adrenalin glycosuria, 335*, 355*, 384;
urine, 641, 642*

Erb, proteids, 94

Erben, leucsemic blood, 226*, 295, 323*,
324; 328*, 647*, 657*

Erlandsen, cuorin, 233; phosphatides,
lecithins, 234; preparing lecithin, 238;
cuorin, 241; phosphatides, 557

Erlanger, effect of removing jejunum and
ileum, 518

Erlenmeyer, leucine, 140

Erlenmeyer, Jr., serine, 144; cysteinic
acid, 147; phenylalanine, tyrosine, 150

d'Errico, 370*

Esbach, method of estimating proteid in
urine, 750

Estor, quantity of oxygen in blood, 818

Etard, musculamine, 550

Etti, 608*

Eulenberg, glycogen, 377; determining
acidity of gastric juice, 465

Euler, lucerne-laccase, 7*, 13; enzymes,

' 60; cane sugar inversion, 62*, 63;
enzyme action, 73; erepsin, 469

Eves, 432*

Ewald, hsematoidin, 119*, 290; gelatin
cleavage, 485; lutein, 584*, 592;
gases in pathological transudates, 808;
influence of light on metabolism, 870

Eykman, blood corpuscles and serum,
313; influence of temperature on
metabolism, 870

Eymonnet, phosphorus compound in
urine, 718

Fabian, glucosamine as a glycogen former,
375, 376

Fahr, muscle salts, 558

Fajans, catalysis, 58

Falck, protein metabolism, 852

Falk, phosphatides, 575; composition of
medullary fibers, 583, 586; metabolism
in old and young, 877

Falloise, bile secretion, 394; enzymes
in intestine, enterokinase, 452*, 468;
chloral secretin, 476; oxygen tension
in blood, 811; carbon dioxide tension
in blood, 816

Falta, thyroid, 355; adrenalin glycosuria,
383*, 384; pancreas diabetes, 385; sugar
formation from fat, 388*, 391; homo-
gentisic acid, 698, 699; sulphur excre-
tion, 826; metabolism, 847; protein
catabolism, 849

Fano, blood coagulation, 243

Farkas, blood alkalinity 264; egg devel-
opment, 607

Farwick, fat in muscle, 570

Faust, sepsine 24; bufonin, bufotenin,
samandarin, 117*, 797

Favre, perspiration, 787

Fawitzki, 652*

Fedeli, 689*

Feder, urea, 649

Fehrsen, 321*

Feigen, hippuric acid, 685

Feinschmidt, 387*

Fenaroli, cholesterin, 421

Fenyvessy, glucuronic acid, 713, 743*

Fermi, fibrin, 247; .autodigestion, 482;
trypsin, 482

Fick, muscle work, 567

Filehne, blood pigments in bile, 415

Filhol, human milk, 637

De Filippi, glycogen formation, 376; pas-
sage of sugar into urine, 511; trimeth-
ylamine in urine, 718, 719*

Finkler, 3*

Fingerling, 634*

Fischer, Ch., glycocoll, 138

Fischer E., kefir-lactose, 65; cleavage of
amygdalin and polypeptides, 67; cleav-
age of polypeptides, 68; hydrolytic cleav-
age, 80; protein cleavage products, 84,
89; cleavage products of keratin, 113;
gelatin, 118; fibroin, 123; polypeptide-
like bodies, 124*, 131, 132; polypeptides,
135; glycocoll, 138; d-alanine, 139;
leucine, 140, 141; leucine leucinimide,
142; serine, 143, 144; cystine, 146, 147;
phenyl alanine, 150; tyrosine, 151;
proline, 152; oxyproline, 153; histidine
reaction, 157; ornithine, 159; lysine,
160 ; diaminotrioxydodecanoic acid, 162;
nuclein bases, purine bases, 181-182;
modified Weidel reaction, 184*, 185;
guanine, 186; hypoxanthine, 187; ura-

896

INDEX OF AUTHORS

cil-thymine, 190; carbohydrates, 193,
194, 195; osimines, 197; glucosides, 198;
carbohydrate synthesis, 205; galactose,
208*, 210; d- glucosamine, 213*, 214;
glucuronic acid, 215; isomaltose, 219;
trypsin digestion, 484; action of trypsin
on acid-amides and polypeptides, 485;
micleoprotein of mammary glands, 610;
lactose, 621 ; uric acid, 664; purine bases,
676; methylxanthine, 677; glucuronic
acid, 706*, 713

Fischer, Hans, tryptophane, 154; purine
bases, 184

Fischer, Martin, glycosuria, 379, 380*

Flacher, 358*

Flamand, tryptophane, 153, 154, 155

Flatow, purine bases, 676; homogentisic
acid, 701 ; uroleucic acid, 702

Flaum, digestion products, 447

Fleckseder, saliva, 430

Fleig, bile secretion, 394; pancreatic juice
excitants, 475; ethyl seretin, sapokrinin,
prosapokrinin, 476

Fleischer, piperidine-glycosuria, 381

Fleischl, hsemometer, 292; bile acids, 416

Fleischmann, 614*

Fleigmann, sulphur in proteins, 79

Fleroff, parahistone, 109

Fletcher, saliva, 435; lactic acid in muscles,
564

Flint, protein metabolism, sulphur elimina-
tion, 566

Florence, sperm reaction, 590

Floresco, substances retarding blood coag-
ulation, 311; liver pigments, 362; iron
in liver 366; biliprasin, 410

Fliickiger, glucuronates, 711*, 712

Floa, 430*

Folin, O., mucin, 165; rigor mortis, 297*,
561; urine nitrogen, 643*, 646; estimat-
ing urea, 656; methylurea, 658; creati-
nine, 659, 662, 663; sulphur compounds
in urine, 665*, 714, 715*; determining
sulphate-sulphuric acid in urine, 726;
estimating ammonia in urine, 729;.
estimating acetone, 778; protein metab-
olism, 851

Folin-Schaeffer, estimating uric acid, 675

Fordos, pus, 347

Forrest, bone marrow, 528

Forschbach, glucosamine in glycogen
formation, 376

Forssner, glycocoll in urine, 717

Forster, blood corpuscles, 325; mineral
bodies in tissues, 326*, 843; effect of
water on metabolism, 862; metabolism
in nurslings, 866, 874, 880

Foster, 156*

Frank, absorption of fats, 512; passage
of fats into chylous vessels, 513, 562*

FranKel, S., catalysis, 55; hydrolytic
cleavage, 78, 79*; protein cleavage
products, 83; proteid test, 101; pep-
tone precipitants, 130; proteoses and
peptones, 137; thiolactic acid, 149;

histidine reaction, 157; albamine, 214;
neottin, 233; blood alkalinity, 264;
thyroid, 356; glucosamine as a glyco-
gen former, 375; gastric juice, 376*,
44 1 ; chondrosin , amino-glucuronic acid ,
452*, 523; neottin, 599; kidney constit-
uents, 639; homogentisic acid, 652*,
701; chitin, 791; oxygen pressure in
blood, 811; influence of rest and work
on metabolism, 867

Framm, 207*, 258*

Franchimont, tunicin, 790, 794*

Frankland, calorific value of foods, 829

Franz, hirudin, 243

Frauenberger, connective tissue, 521

Fredericq, L., serglobulin, 96*, 252;
haemocyanin, 292; blood coagulation,
300; blood gases, 803; oxygen in
blood, 811 ; oxygen tension in blood, 812;
carbon dioxide tension in blood, 816

Fremy, creatine in muscle, 573; emydin,
ichthin, ichthidin, ichthulin, 605

Frenkel-Heiden, cerebrospinal fluid, 341;

342

Frentzel, glycogen, 369, 372; muscle work,
567, 569; meat nitrogen, 572; calorific
food values, 835

Frerichs, synovial fluid, 344; composition
of bile, 412; uric acid, 670

Freud berg, alkalinity of blood, 297

Freund, E., scrglobulins, 251, haema-
tinogen, 256*, 289; blood coagulation,
300; coagulable protein in blood, 507;
determining chlorine in urine, 721;
bodies in pathological lungs, 821 ; urine-
nitrogen loss in starvation, 840; urea
loss in starvation, 841

Freund, O., urine-nitrogen loss in starva-
tion, 840; urea loss in starvation, 841

Freundlich, colloids, 43; adsorption, 49,
50

Freytag, pus cells, 346; pyosin, pyo-
genin, 579; brain constituents, 580,
protagon, 576, 577

von Frey, M., blood coagulation, 301*

Friedenthal, alkalinity of blood, 264;
pepsin, 443 ; protein metabolism, 502

Fried jung, human milk, 631

Friedlander, protein assimilation, 502

Friedmann, hydrolytic cleavage, 78; kera-
tin, 79*, 113; protoproteoses, 134;
isoleucine, 143; cystine, 146, 147;
thiolactic acid, 148; taurine, 149;
sphygmogenin, 358; bile acids, 417;
uric acid, 669; fate of amino-acids in
the body, 735; oxidation of fatty acids,
739; mercapturic acids, 743; acetone
bodies in urine, 775

Friend, stromata, 267; pericardial fluid,
338

Fromherz, 698*

Fromholdt, urobilin, 706

Fromm, elimination of terpenes and
camphors, 742, 743*

Fromme, A., 452*

INDEX OF AUTHORS

897

Frommer, acetone test, 776
Frouin, gastric secretion, 439, 440; secre-
tion of Lieberkuhn's glands, 465;
intestinal juice, 466; enzyme in intes-
tinal juice, 467; enterokinase, 472;
pancreatic juice, 473
Frugoni, adrenalin glycosuria, 384
Fubini, carbon dioxide eliminated by

the skin, 800
Fuchs, 29*, 114*

Fuld, E., rennin, 63; thrombin, 248;
fluoride plasma, 305; plasmozyme,
cytozyme and holozyme, fluidity of
blood, retarding blood coagulation,
306 ; alpha-thrombin, beta-thrombin,
neozym metazym, beta-prothrombin,
307 ;1 substances retarding blood coagula-
tion, 311; pepsin test, 446; law of
rennin action, 450, 451; casein, 617;
human casein, 618*, 627*, 628
Fuller, influence of phosphates, 845
Funk, glutamic acid, 145, 715*, 764*
von Funke, protein metabolism, 566
Furbringer, oxalic acid, 679; proteid test,

747

von Furth, tyrosinases, 12; peroxypro-
teic acid, 81; xanthomelanin, 82;
nucleic acids, 177, choline, 239, 179*;
iodothyrine, 354*, 356; suprarenin,
358; secretin, 476; bile salts, 479*489;
muscle plasma, 539, 540; myosin, 541;
myosin fibrin, 542; musculin, 543;
myosin ferment, 544; myoproteid, 545;
rigor mortis, 560; muscle albumin, 572;
nuclein in muscle, 573; chitin, 791;
melanin, 793, 794

Gabriel, cystine, 147; bone earth, 527;
fluorine in teeth, 532, 602*, 855*

Cachet, activation of trypsinogen, 473

Gad, emulsifying action of fatty acids,
489

Gaglio, lactic acid in blood, 318; para-
lactic acid, 554; fate of oxalic acid hi
the body, 735

Galdi, 335*

Galeotti, 95*

Gallimard, keratin, 113

Galli, pigments in blood serum, 260

Gallois, inosite test, 552

Gamgee, nucleoproteins, 172; nucleic
acids, 176; haemoglobin, 248*, 273;
oxyhaemoglobin, 274; intestinal secre-
tion, 466; protagon, 525*, 576; pseu-
docerebrin, 580

Ganassini, saliva, 431; uric acid reaction,
673

Gansser, haemoglobin, 270; oxyhaemoglo-/
bin, 271

Gardner, koprosterin, hippokoprosterin,
423

Gamier, homogentisic acidrf 698

Garrod, hsematoporphyrin, 287; alcapton-
uria, 700; homoffentisic acid, 701;
uroleucic acid, 702; urochrome, 703,

704; urobilin, 708; uroerythrin, 709,
710, 754; haematoporphyrin, 780*

Gartner, 394*

Gaskel, detecting cystine, 781

Gassmann, tooth composition, 532

Gatin-Gruzewska, submicrons, 41; glyco-
gen, 368; glycogen, 369; index of
rotation of glycogen, 370

Gaube, perspiration, 799

Gaunt, ferments, 10

Gautier, A., ptomaines, 22*, 24; leuco-
maines, 25; fibriiiogen, 244; menstrual
blood, 320; thymus, 349; thyroid
arsenic, 354; glycosuria, 384; fat
formation, 536; meat extractives, 550;
ovalbumin, 602; egg white, 603;
xanthocreatinine, 622*, 663; cystin-
uria, 781; arsenic in skin, 790

Gautier, Cl., iodine in blood serum, 261

Gawinski, uroferric acid, 717

Gay-Lussac, law of gas pressure, 27

Gebhard, 562*

Geelmuyden, maltose in urine, 768;
sugars in urine, 770; acetone elimina-
tion, 773, 774; acetone bodies in urine,
775, 779*

Geiger, serine, 144

Geissler, albumin test paper, 747

Generali, thyroid, 355

Gengou, blood coagulation, 301; pro-
thrombin, 305

von Genser, human milk, 632

Gentzen, indican elimination, 692

Geoghegan, cerebrin, 579; brain constit-
uents, 580; mineral constituents of
brain, 584

Gephart, F., lecithin, 237

Geppert, oxygen pressure in blood, 297*,
811; determining respiratory gas ex-
change, 820 ; alcohol in metabolism, 863

Gerard, cholesterin, 15*, 422; uric acid,
664

Gerber, 629*

Gerhardt, urobilin, 706; acetoacetic acid
test, 777

Gerhartz, lung concretions, 821

Gerngros, thymine, 190

Gessard, melanins, 794

Geyer, phenylhydrazine test, 761

Geyger, glycosuric acid, 698

Gewin, chymosin and parachymosin, 451

Giacosa, mucins, 164; haematin, 290;
egg white, 605; iron in urine, 730;
oxidation of ethylene, 738

Giaja, enzymotic processes, 73

Giertz, pseud onucleins, 104, 173

Gies, elastin, elastoses, 115, 116, 117;
tendon mucin, 161*, 165; mucin, 166,
167; lymph, 331*, 332; pepsinhydro-
chloric acid digestion, 449; tendon
mucin, mucoids, 519; analysis of
achilles tendon and ligamentum nuchae,
520; bone matrix, 526; protagon, 577,
578; phrenosin, 580; estimating am-
monia in urine, 657*, 729

898

INDEX OF AUTHORS

Gigon, action of yeast press juice, 68;
hydrolysis of glycyl-M-yrosine, 486;
hippuric acid, 685; influence of food on
metabolism, 872

Gilbert, fat formation, 537

Gilkin, 226*

Gilson, preparing lecithin, 234*, 238;
chitin, 791

Ginsberg, effect of sugar in intestines,
511; uroferric acid, 717

Githens, globulins in blood serum, 262

Gittelmacher-Wilenko, 679*

Giunti, fate of oxalic acid in body, 733

Gizelt, secretin, 476

Glaessner, K., liver, 360; pseudopepsin,
442; secretion of Brunner's glands,
443*, 465; pancreatic juice, 471; quan-
tity of pancreatic juice, 476; pan-
creatic secretion, 477; pancreatic ren-
nin, 487; estimating urea, 657; kynu-
renic acid, 702

Gleiss,x muscle reaction, 563

Gley, antitryptic action, 69; iodine in
blood serum, 261; substances retarding
blood coagulation, 311, 312; lympha-
gogues, 333; thyroid, 335; enterokin-
ase, 472; pancreatic secretion, 473;
pancreatic excitants, 475; prostate
fluid, vesiculase, 590

Gliddinning, 61*

Glikin, lecithin, 235, 237; liver autolysis,
365

Gmelin, bile-pigment reactions, 407;
gastric juice, 441; detecting biliary
pigments, 756

Gogitidse, milk fat formation, 636

Goldmann, cystine, 148; hsemopyrrol,
288; cystine in urine, 356*, 736*, 767*,
780; detecting cystine, 781

Goldschmidt, F., proteose cleavage, 55*,
132; ptyalin, 431, 654*

Gompel, tryptic digestion, 68

Gonnermann, glycocoll, 138; action of
trypsin in acid-amides and polypep-
tides, 485

Goodbody, intestinal putrefaction, 493

Goodwin, 129*

Gorodeski, bile pigments, 418

von Gorup-Besanez, pericardial fluid,
314*, 338; composition of bile, 367*, 412;
silicic acid in feathers, 426*, 530*, 790

Gosio, 553*

Goto, protamines, 110, 111; uric acid, 673

Gottlieb, urea in blood, 317; iron in bile,
411; creatine, 547; creatinine, 659,
663; purine bases, 676; oxyproteic
acid, 715, 716; iron in urine, 730

Gourlay, thyroid proteins, 354

de Graff, proteoses, 749

Graebe, ellagic acid, 501

Graffenberger, bone analysis, 529

Graham, colloids, crystalloids, hydrosol,
hydrogel, dialysis, 36

Granstrom, calcium and magnesium in
urine, 365*, 683*, 729

Grawitz, blood corpuscles, 323

Green, globulins in fibrin, 121*, 247;
calcium salts in blood coagulation, 303;
lymph, 330

Gregor, creatinine, 663; potassium and
sodium in urine, 727

Grehant, urea in blood, 317, 319; fate of
volatile fatty acids in body, 733

Griffiths, neurochitin, 583; leucomaines in
blood, 719

Grimbert, urobilin, 709

Grimm, 706*

Grimmer, gastric digestion, 458; diastatic
enzyme in Brunner's glands, 465

Grindley, 877*

Griswold, 432*

Grober, pepsin, 443, 695*

Groll, haemoglobin, 321

Grosjean, substances retarding blood
coagulation, 311

Gross, ascitic fluid, 340; gastric juice,
439; pepsin test, 446; trvpsin estima-
tion, 482; ovovitellin, 588*, 597, 598;
calcium and magnesium in urine, 727

Grosser, skatol red, 696

Grossmann, pancreatic juice, 471, 762*

Grossenbacher, spleen, 353

Grouven, fat in muscle, 570

Grube, K., glycogen formation, 375

Gruber, nitrogen equilibrium, 223*, 825,
862*

Griibler, 93*

Gruenhagen, aqueous humor, 342

Griinbaum, amniotic fluid, 609

Grund, pentoses, 201; pentose determina-
tion, 202

Griindler, 735*

Grutterink, proteoses, 749

Griitzner, pepsin, 445*, 446; gastric
digestion, 455, 456; secretion of Brun-
ner's glands, 465; amylopsin, 478, 747

Gruzewska, seralbumins, 253

Gryns, plasmolysis, 31

Gscheidlen, saliva, 431; lactic acid in
brain, 554; sulphur combinations in
urine, 651*, 714

Gubler, lymph, 329; human milk, 632

Gudzent, uric acid, 672

Guerin, leucomaines in urine, 719

Gugenot, saliva, 433

Guggenheim, adrenalin, 359

Guillemonat, spleen, 352; liver con-
stituents, 366

Guinochet, ascitic fluid, 340

Guldberg, catalysis, 53

Gulewitsch, arginine, 158; thymine, 191;
choline, 240; action of trypsin on acid
amides and polypeptides, 485; carno-
sine, carnotine, oblitine, 549

Gullbring, taurocholeic acid, 400

Giimbel, 77*

Gumilewski, intestinal secretion, 466

Gumlich, A., urine nitrogen, 646; metab-
olism, 723*, 826

Gundermann, 81*

INDEX OF AUTHORS

899

Gunning, acetone test, 775

Gunther, G., 591*

Gimzberg, reagent for free HC1 in gastric

juice, 463
Giirber, plasmolysis, 33; seralbumins, 253;

seralbumin, 255; alkali in blood, 265*,

298; bile, 415; amniotic fluid, 609
Gusserow, allantoin, 681
Guth, 226*
Gyergyay, nutritive value of proteoses

and peptones, 854

Haas, haBmopyrrol 288; nitrogen elimin-
ated in urine, 505; protein metabolism,
852

Habermann, hydrolytic cleavage, 80;
protein oxidation products, 82; protein
reaction products, 83; leucine, 141;
glutamic acid, 145; tyrosine, 151

Haensel, human milk, 629, 630*

Hafner, 226*

Hagemann, carbon dioxide eliminated by
the skin, 800 ; blood gases, 802 ; respir-
atory quotient during work, 868*, 869,
871*

Hagen, 189*

Hahn, M., 9*

Harm, antitryptic action, 68; casein, 618;
carbamic acid elimination, 650; urea
elimination, 651

Haig, 666*

IJaiser, inosinic acid, 179, 180; carnine,
548

Haldane, methsemoglobin, 276; cyan-
methsemoglobin, 277; blood, 291*, 325;
oxygen tension of blood, 812

Hall, Walker, muscle extractives, 189*,
498*, 545; metabolism, 679*, 718*, 847

Hallauer, bile, 415

Halle, adrenalin, 359

Hallervorden, urea, 649, 652*, 728*

Hallwachs, 476*

Halliburton, choline, 96*, 223*, 239; seral-
bumin, 248*, 254 ; blood-serum analysis,
262; stromata, 267; tetronerythrin,
292; globulin in cells, 294; intra-
vascular coagulation, 310; cerebro-
spinal fluid, 338*, 341; liver globulin,
361; glycogen precipitant s, 370; action
of pancreatic juice on casein, 487; con-
nective tissue, 520; bone marrow, 528;
muscle plasma, 539, 540; myosin,
paramyosinogen, 541; myoglobulin,
.seralbumin a, 542; myosin ferment,
paramyosinogen, myosinogen, 543; nu-
cleoprotein in brain, 574 ; protagon, 583 ;
kidney constituents, 638; tetronery-
thrin, 795

Halpern, cerebrospinal fluid. 342

Halsey, tyrosine, 150

Ham, haemoglobin, 289

Hamalainen, sulphur excretion, 826;
protein catabolism, 849

Hamburger, plasmolysis, 31, 33; diastases
in blood, 35*, 258; blood serum, 261;

dissociation of blood serum, 263; stro-
ma, 265; erythrocytes, 293; blood
corpuscles, alkali in blood, 297*, 298;
lymph, 332, 333; ascitic fluid, 340;
iron in bile, 411; ptyalin, 432; intes-
tinal secretion, 466; intestinal juice,
467; enterokinase, 472; 518*, 559*
Hammarsten, ferments, 10; antitryptic
action, 68; nucleoalbumin, 102*, 105;
mucins, 163*, 164, 165, 167; helico-
proteid, 170; nucleotides, 175; pen-
tose, 201; pnosphatides, 232; thrombin,
233*, 245*, 249; fibrin-globulin, 250;
globulin, 251, 252; serglobulin, 253,
254, 255*; pigments in blood serum,
260; blood-plasma analysis, 261, 262;
pseud ohsemoglobin, 276; thrombin,
fibrinogen transformation, calcium salts
in blood coagulation, 289*, 303; fibrin,
304; gases in lymph, 328; transudates,
336; pericardial fluid, 338; pleural
fluid, 339; ascitic fluid, 340; hydrocele
and spermatocele fluids, 341; synovia!
fluid,. 343; alcoholic fermentation in
animal tissues, 386; sugar formation in
diabetes, 388; sugar formation from
protein, 392; bile secretion, 392; bile
constituents, 395; glycocholic acid, 398;
hyoglycocholic acid, 399; taurocholic
and taurocholeic acids, 400; cholic
acid, 401, 402; desoxycholic acid, 403;
glycocholic acid, ursocholic acid, 404;
bilirubin reaction, 408; cholohaBmatin,
410; bile constituents, 411; solids in
bile, 412; sulphur in bile, 413; glyco-
cholic acid in bile, phosphatides in
bile, 414; pepsin, 432*, 442, 443;
rennin enzyme, chymosin, antirennin,
450; protein digestion, 451; chymosin,
452; a-proteid, 470; preparing trypsin,
480; pseudomucin, 594; vitellin, 599;
egg-white, 605; casein, 616*, 617,
620*, 645*; estimating globulin? and
albumins, 749; hsematoporphyrin, 750*,
754; test for bile pigments, 757; sugar
test, 759; phenylhydrazine test, 762;
cystinuria, 780; lymph gases, 807;
carbon dioxide secretion, 817; influence
of food on metabolism, 872

Hammerbacher, ash from saliva, 434

Hammerl, 499*

Hammerschlag, sp.gr. of blood, 296

Handel, cartilage, 524

Hanriot, lipase in blood, 258; lipolysis,
259; respiratory quotient in diabetes,
382; fat formation, 537; determining
respiratory gas exchange, 820

Hansel, autodigestion, antipepsin, 462

Hansen, W., lecithins, 162*, 226*, 234;
protein synthesis, 506; human fat, 533;
yolk fat, 599; milk fat formation, 636;
nutritive value of heteroproteose, 855

Hanssen, O., amyloid, 168, 169, 170;
chondroitin-sulphuric acid, 522

Harden, enzymes, 60

900

1JSDEX OF AUTHORS

von Hardt-Stremeyer, 77*

Hardy, colloids, 37; cataphoresis, 42;
colloids, 43; precipitation theory of
colloids, 46; gels, 51; ovalbumin, 603

Harley, V., sugar formation in liver, 378;
intestinal putrefaction, 493; pancreas
extirpation, 508; fat absorption, 515;
splitting of fats, 516; effect of removing
sections of intestine on absorption, 517;
urohsematin, 703, 706*

Harms, 527*

Harmack, E., iodospongin, 94*, 122;
acid haemoglobin, 278; sulpha)moglobin,
279; sulphur compounds in urine, 714;
amniotic fluid, 735*, 742*, 609; indican
elimination, 692

Harries, protein oxidation products, 82

Harriot, monobutyrin synthesis, 65

Harris, nucleic acid, 77*, 175; plant
nucleic acid, 180, 181

Hart, E., elastin, 77*, 106*, 115; pro-
toelastose, deuteroelastose, 116; hetero-
proteose, 133; protoproteose, 134;
nucleoprotein of mammary gland, 157*,
161*, 610; influence of phosphates, 845

Hartogh, sugar formation from fat, 391

Hartung, iron content of egg, 605

Hartwell, 129*

Hasebroek, lecithin, 237; pericardial
fluid, 338; intestinal decomposition,
493

Haskins, carbamate formation, 646*, 649;
estimating urea, 657; carbamic acid,
658

Haslam, proteids, 94; protoproteoses,
34; separating hetero- and proto-
proteoses, 137; globulins, 251; ser-
globulins, 253

Hasselbalch, egg development, 606; res-
piration, 810 ; varying oxygen tension of
blood, 818

Hatcher, glycogen formation, 376

Hauff, human milk, 632

Hausermann, 322*

Hausmann, gelatin, 77*, 118; kopros-
terin, 423; cholesterin, 424

Hawk, bone matrix, 526; urea, 650;
sulphur in hair, 790

Hay, ascitic fluid, 340

Haycraft, hirudin, 96*, 243; blood coagu-
lation, 300; biliverdin, 382*, 409; bile
acids, 756

Hayem, hsematoblasts, 295; haemoglobin,
324

Heckel, leucine, 140

Hedenius, J., keratin, 115*; bilirubm test,
411

Hedin, autolysis, 17*, 18; plasmolysis,
32, 33; enzymes, 59, 61; trypsin, 62*,
63; enzymes, 64; tryptic digestion,
68; adsorption, 69; trypsin, 73; elastin,
115, 116; gelatin, 118; histidine, 157;
argenine, 158; lysatine, 161; hsemato-
crit, estimating blood corpuscles, blood
corpuscles and serum, 313; spleen, 351;

trypsin estimation, speed of trypsin
digestion, 482; tryptic digestion, 484;
muscle enzymes, 545

He"don, internal secretion in pancreas,
385; sugar absorption, 509; fat absorp-
tion, 515, 516

Heffter, enzymes, 15; muscle reaction,
363*, 538*, 560*, 563; sulphuretted
hydrogen in urine, 564*, 715; hippuric

acid formation, 733*, 740

Heger, liver, 360

Heidenhain, lymphagogues, 35; lymph,
100*, 331, 332, 333; capillary endothe-
lium, 335; bile secretion, 393; sublin-
gual saliva, 428; saliva, 435; pyloric
glands, 436; gastric juice, 437; 452*;
pepsin formation, 454; pyloric secre-
tion, 455; pancreatic juice, 470, 471;
trypsinogen transformation into sugar,
474; preparing trypsin, 480; action
of leucocytes on proteoses, 505; muscle
reaction, 572; milk secretion, 635

Heilner, protein absorption, 502; effect
of water on metabolism, 862; effect of
food ingestion on metabolism, 871;
influence of food on metabolism, 872

Heinemann, muscle work, 569

Heinsheimer, 452*

Heinsius, bilicyanin, 110, 408

Heintz, stearin, 228; palmitic acid, 229;
lactic acid in muscles, 554; uric acid,
665; estimating uric acid, 673

Henze, M., 546*

Heiss, bone, 530

Heitzmann, bone, 530

Hekma, intestinal secretion, 466; kinase
in intestine, enterokinase, 468, 472;
action of trypsin on trysinogen, 474

Hele, 700*

Heller, proteid test, 98; uroxanthine,
691; urophain, uroglaucin, urorhodin,
703; proteid te t, 745; blood test, 75 j;
scheme for investigating calculi, 788

Hellgren, influence of food on metabolism,
872

Helm, gastric juice, 437

Helme, sulphur excretion, 826; protein
catabolism, 849

Helmholtz, muscle work, 565

Helwig, muscle proteins, 572

Hemmeter, gastric juice, 439; saliva,
440; reaction of intestinal contents,
497

Hempel, 838*

Henderson, L., reaction of blood, 77*, 297;
protein synthesis, 506

Henkel, milk plasma, 614

Henneberg, protein fattening, 860

Henninger, 137*

Henocque, hsematoscope, 292

Henri, inversion of cane sugar, tryptic
digestion, 68; enzymotic processes, 73;
oxyheemoglobin, 272

Henriques, lecithins, 232*, 234; blood
serum, 257; sugar in blood, 317; pro-

INDEX OF AUTHORS

901

tein synthesis, 506; human fat, 533;
yolk fat, 599; milk fat formation, 636;
amino-acids in urine, 718; blood gases,
802; respiration, 809; nutritive value
of heteroproteose, 855

Henry, law of absorption, 48

Hensen, lymph, 330; lymph gases, 807

Henze, gorgonin cleavage products, 123;
aspartic acid, 144; nucleoproteins, 171;
hsemocyanin, 292 ; copper in liver, 367 ;
spongosterin, 424; taurine in muscle,
573

Heptner, 413*

L'Heritier, human milk, 631

Herlant, nucleic acids, 180

Herlitzka, glycosuria, 7*, 384

Hermann, A., 665*, 666*

Hermann, L., muscle work, 15; haemo-
globin, 22*, 321; tryptic digestion,
484; feces, 499; rigor mortis, 561;
metabolism in muscles, 563; allantoin,
681

Heron, ptyalin, 223*, 432; intestinal
juice, 467

Herter, ethereal sulphuric acid, 688;
urorosein, 690*, 697; indol and skatol
formation, 695; urorosein, indolacetic
acid, 696; ethereal sulphuric acid
elimination, 740, 742; diazo-reaction,
779; oxygen in blood, 811

Herter, C. A., 156*

Herth, deuteroproteose, 129, 137*

Hervieux, indol in blood serum, 260;
indican elimination, 691*, 692; indol,
693; indican, 694; indoxylsulphuric
acid, 695; skatol red, 696; uroerythrin,
710; detecting conjugated glucuronic
acids, 771

von Herwerden, paracasein, 618

Herzen, spleen, 353; gastric secretion,
438, 454; activation of trypsinogen,
473; pancreatic juice excitants, 475

Herzfeld, hydrazones, 199

Herzog, ferments, 10; histidine reaction,
157, 553*

Hess, autolysis, 18

Hetper, hsemin, 285; hsemopyrrol, 288

Heubner, mytolin, 250*, 543; human
milk, 628

Heuss, perspiration, 798

Hewlett, 96*

Hewson, blood coagulation, 300

Heyl, nucleic acid, 174; plant nucleic
acids, 181

Heymann, pseudomucin, 595

Heynsius, protein precipitants, 128

Hiestand, lecithins, 237

Hilbert, oxidation of acetanilid, 738

Hildebrandt, antienzyme, 69; proteolytic
enzyme in mammary glands, 611;
casein formation in milk, 635; oxalic
acid, 680; glucuronic acid, 706*, 713;
fate of oxalates in the body, 733; fate
of aminobenzoic acids in the body, 740;
elimination of terpenes and camphors,

742 ; paradimethylaminobenzoic acid.
743

Hilclesheim, 874

Hilger, inosite fermentation, 167*, 551

Hill, isomaltose, 219

Hiller, mineral bodies in bone, 529

Hirsch, R., alimentary glycosuria, 385;
hippuric acid, 685; amino-acids in
urine, 717

Hirschberg, 295

Hirschfeld, muscle work, 461*, 566;
uric acid, 665; acetone elimination,
773; protein metabolism, 846; catab-
olism limits, 858; nitrogenous equilib-
rium^; 880*

Hirschfeldt, 748*

Hirschl, phenylhydrazine test, 762

Hirschler, putrefaction of carbohydrates,
495, 496*, 688*

Hirschstein, uric acid, 665, 670

His, corneal constituents, 189*, 525;
uric acid, 672; pyridine elimination,
743

Hlasiwetz, hydrolytic cleavage, 80; glu-
tamic acid, 145

Hober, R., osmotic pressure, 35; colloids,
37, 46; blood alkalinity, 264; viscosity
of blood, 298; urine, 643, 518

Hoernes, 78*

Hofbauer, 43*

Hofmann, koilin, 115; tyrosine test, 124*,
152; fats, fat absorption, 533; fat
formation, 535

van't Hoff, dissociation of oxygen mole-
cule, 6; osmotic pressure, 27, 28;
catalysis, 54, 55

Hoffmann, A., 686*

Hoffmann, F., transudates, 336; edema-
tous fluid, 339, 343; sugar formation
in liver, 378; glycosuria, 379; urine,
380*, 381*, 641; iron in urine, 730

Hofmeister, F., enzymes, 19; solidifica-
tion and melting-point of gelatin, 52;
putrefactive products, 77*, 80*, 81;

frotein cleavage products, 83; collagen,
17; gelatin, 119; synproteose, 133;
peptones, 136; serglobulin, 251, 253;
pus cells, 346; gastric digestion, 455,
456; intestinal absorption, 503; fate
of proteins, 504; action of leucocytes
on proteoses, 505; assimilation limit,
510; ovalbumin, 602, 603; urea, 651;
creatinine, 660; estimating globulins
and albumins, 750; lactose in urine,
768; detecting lactose in urine, 769

Hohlweg, blood serum, 260; urochrome,
703, 704

Holde, 226*

Hollinger, sugar in blood, 315

Holmgren, E., mucinogen, 427; phenyl-
hydrazine test, 762

Holmgren, Fr., blood gases, 802; carbon
dioxide elimination, 816

Holmgren, J., mucinogen, 427; myosin,
539; muscle stroma, 542

902

INDEX OF AUTHORS

Holobut, creatinine, 662

von Hoist, serosamucin, 336; synovia-
mucin, 343

Hone, bile acid, 755

Honore, absorption of proteoses and
peptones, 503

Hoogenhuyze, creatine, 547; creatinine,
547, 658, 660, 663

Hooker, lymph, 333

Hopkins, putrefactive products, 81; pro-
teid detection, 99; tryptophane, 106*,
153, 154, 155; lactic acid in muscles,
564; ovalbumin, 602, 603; uric acid,
665; estimating uric acid, 675; urobilin,
675*, 705, 707, 708

Hoppe-Seyler, F., cell activity, 3*, 5;
lecithalbumin, 104; nuclein, 163*, 172;
xanthine, 185; lecithin, 214*, 235, 236;
preparing lecithin, 238; blood plasma
analysis, 261; blood corpuscles, 267;
blood coloring matter, phlebin, 268;
haemoglobin, 270; oxyhaemoglobin, 274;
methaemoglobin, 277, 278*; sulphaemo-
globin, 279; estimating blood corpuscles
and blood fluid, 314; chyle, 329; peri-
cardial fluid, 338; pus serum, 345;
pus cells, 346; stroma cystica, 354;
glycogen, 368; bile composition, 412;
bile secretion, 415; cartilage, 525;
bone substance, 527*, 528; bone earths,
531; lactic acids, 555, 556; lactic acid
in muscles, 564; retina constituents,
584; ovovitellin, 597; casein, 600*,
615; determining casein and albumin
in milk, 623; indol, 631*, 634*, 695;
urobilinoids, 705; paralactic acid, 710;
test for bile acids, 756; inosite in urine,
771; chitin, 779*, 791; sebum, 796;
respiratory gas exchange, 797*, 819

Hoppe-Seyler, G., haemochromogen, 281,
282; haematin, 283; haematoporphyrin,
287, 289; haemoglobin, 290; composi-
tion of red blood corpuscles, 292; in-
dican elimination, 689*, 691; urobilin,
693, 706, 709

Horbaczewski, elastin, hemielastin, elas-
tinpeptone, 112*, 116; xanthine, 184;
guanine, 185; spleen, 353; uric acid,
663, 664*, 666, 667; urostealith, 787,
847*

Hornborg, gastric juice, 440, 441

Home, blood coagulation, 243

Horodjuski, ammonia in blood, 317

Horowitz, influence of bacteria in intes-
tines, 497, 498*

Horth, 553*

Hougardy, seralbumin, 254

Ho well, mineral bodies of muscles, 558;
amino-acids in blood serum, 260

Huber, blood coagulation, 243; fibrin,
247: glycogen, 377

von Hiibl^ iodine number, 231

Hiibner, 156*

Hudson, enzymes, 63, 621*

Hueck, mineral bodies of muscles, 558

Hiifner, leucine, 140; haemoglobin, 270;
oxyhaemoglobin, 271, 272, 275; met-
haemoglobin, 276, 277; carbon-mon-
oxide haemoglobin, 278; haemochromo-
gen, 282; estimating blood pigments,
291; bile, 398; gas in egg, 605; res-
piration, 809, 810; oxygen in blood,
811; oxygen tension in blood, 812, 817*

Hugounenq, biliverdin, 410; klupeovin,
598, 605; human milk, 633

Huiskamp, fibrinogen, 245; thrombin,
248; fibrin-globulin, nuceloprotein, 250;
action of tissue extracts, 307; thymus,
348, 349

Hulett, adsorption, 49

Hultgren; nitrogen loss, 507; utilization
of carbohydrates, 508*, 511; effect of
removing the color, 518; protein
requirements, 874*, 877

Humnicki, koprosterin, hippokoprosterin,
423

Hundeshagen, spongin, 122, 234*

Hunter, proteoses, 106*, 111; primary
proteoses, 133

Hupfer, hippuric acid, 685

Huppert, rule of enzyme action, 64;
proteoses, 132; glycogen, 295; index
of rotation of glycogen, 370; bilirubin
reaction, 408; pepsin test, 446; nitrogen
in muscle, 571; urea test, 653; uro-
leucic acid, 701; estimating proteid,
750; test for biliary pigments, 756;
laiose in urine, 768

Huppert-Messinger, estimating acetone,
778

Huppert-Neubauer, estimating uric acid,
675; detecting proteids, 748, 750*,.
767*, 782*

Hiirthle, cholesterin in blood serum, 256

Hurtley, sulphaemoglobin, 279; homo-
gentisic acid, 701; uroleucic acid, 702

Husson, milk, 637

Hutchinson, bone marrow, 528

Hybinette, non-volatile fatty acids in
urine, 710

Ide, phosphocarnic acid, 550

Ignatowski, 780*

Inagaki, seralbumin, 254"; erythrocytes,
262, 323; influence of leucocytes on
proteoses, 505; rigor mortis, 560

Inoko, haemoglobin, 270

Inouye, lactic acid, 176*, 452*, 555;
paralactic acid, 710, 711*

Irisawa, lactic acid in blood, 318, 711*

Iscovesco, thrombin, 250

Ito, proteoses and peptones in urine, 748

Iversen, prostatic concrements, 590*, 592

Iwanoff, nucleases, 176; cleavage of nu-
cleic acids, 485; phosphates in urine,
724

Jaarsveld, benzoic acid, 687
Jablonsky, quantity of pancreatic secre-
tion, 476

INDEX OF AUTHORS

903

Jackson, lactic acid, 555, 702*

Jacob, metabolism, 847

Jacobs> serine, 144; guanylic acid, ino-
sinicacid, 179, 180; guanosin adenosin,
181; xyloses, 204; spleen, 351

Jacobsen^ blood serum, 257; solids in bile,
412; taurocholic acid in bile, 413, 739*

Jacoby, oxidases, 11, 12; autodigestion,
17; fibrinogen, 245; fibrinolysis, 245*,
247; pepsin test, 446; trypsin esti-
mation, 482; uric acid, 670; autolysis
of lungs, 820

Jaderholm, methaemoglobin, 276, 277;
hsemochromogen, 282

Jaeckle, human fat, 226*, 228*, 533

Jaeger, gas secretion in fish, 817

Jaffa, food demand for working individ-
uals, 877*, 879

Jaffe, ornithine, 159; bilirubin, 408; urobi-
linin feces, 499; creatine, 548; urethane,
658; creatine, 659; creatinine, 661, 662;
uric acid, 668, 672; indican elimina-
tion, 691, 692, 694*; urobilinogen, 703;
urobilin, 705, 706, 708; glucuronates,
713; oxidation of acetanilide, 738;
hippuric acid formation, 740; fate of
furfurol in the body, 741; thiophenuric
acid, 742; uronitrotoluic acid, mer-
capturic acid, 743

Jaffe-Obermeyer, indican test, 693

de Jager, 643*

Jakowsky, putrefaction of proteins, 491

von Jaksch, alkali in blood, 297; urea in
blood, 317; determining acidity of
gastric juice, 465; nuclein in brain, 575;
urine nitrogen, 647; volatile fatty acids
in urine, 710; melanin, melanogen, 755;
phenylhydrazine test, 761; pentoses
in urine, 769; acetone bodies in urine,
771*, 772

Jalowetz, 219*

Jamieson, iodogorgonic acid, 123

Jantzen, milk fat formation, 635

Jappelli, submaxillary saliva, 427

Jaquet, oxidases, 11; haemoglobin, 270

Jastrowitz, pentoses, 201; pentoses in
urine, 446*, 718* 769

Jelinek, 386, 555*

Jensen, glycogen, 553

Jerome, 715*

Jerusalem, nucleic acids, 177; detection
and quantitative estimation of lactic
, acid, 179*, 557; melanin, 793, 794

Jesner, glucose in vitreous humor, 586

Jessen-Hansen, 764*

Joachim, serglobulins, 251; serosamucin,
336; ascitic fluid, 340

Jochmann, 295

Jodlbauer, enzymes, 59, 527*

Johansson, preparing seralbumin, 255;
muscle aciti vity , 569 ; determining basal
requirements in metabolism, 841; in-
fluence of rest on metabolism, 868; in-
fluence of sleep on metabolism, 869;
influence of external temperature on

metabolism, 870; effect of food in-
gestion on metabolism, 871; carbon
dioxide elimination, 872; metabolism
during sleep and rest, 878

Johnson, nucleic acid, 174; plant nucleic
acids, 181; cytosine, 189; uracil, 190;
parachymosin, 450; creatinine, 658, 660,
662

Jolin, thyroid, 357; glycocholic acid, 399;
ammonia in urine, 728

Jolles, A., protein oxidation products, 82;
pentose reactions, 202, 203; haemo-
globin, 290; bilirubin, 407; human
milk, 631; estimating uric acid, 643,*
675; iron in urine, 705,* 730; proteid
test, 746; nucleohistone in urine, 751

Joly, human milk, 637

Jolyet, blood gases, 802

Jones, W., enzymes, 17; nucleoproteins,
172; nucleic acid, 175; guanylic acid,
178; purine bases, 183; thymine, 191;
spleen, 351, 354; uric acid, 667

Jonescu, tryptic digestion, 484

De Jonge, coccygeal secretion, 797

Jorissen, 6*

Jornara, bufidin, 797

Josephsohn, 702*

Jowett, sphygmogenin, 358

Junger, 404*

Jungfleisch, colloidal precipitation, 48.
556*

Justus, mineral substances in cells, 22

Juvalta, fate of phthalic acid in the body,
737

Kaas, ovalbumin, 78*, 603

Kalaboukoff, 479*

Kalb, protein fattening, 860

Kalberlah, acetone bodies in urine, 772

Kanitz, steapsin, 479; trypsin, 483

Kareff, fibrinogen, 245

Kast, putrefaction in intestine, 497; sul-
phur compounds in urine, 714 ; chlorides
in urine, 720; skatoxjl in perspiration,
736*,799

Kastle, fat synthesis, 7*, 14*, 63*, 65

Katsuyama, lactic acid in blood, 318; lac-
tic acid elimination, 554

Katz, A., mineral constituents of meat,
572, 706*

Katzenstein, respiratory quotient dur-
ing work, 868*, 869

Kauder, serglobulin, 253, 254

Kaufmann, sugar formation in liver, 377*,
378; sugar formation from fat, 391;
fat formation, 536, 537; urea in muscle,
546; muscle metabolism, 563; milk,
milk, 637; urea, 652; determining cal-
orific oxygen value, 676*, 833; gelatin
as a protein sparer, 853, 838*, 854*

Kaup, muscle work, 566

Kausch, 375*

Kautzsch, 491*

Kaznelson, 440*

Keferstein, 476*

904

INDEX OF AUTHORS

Kempe, tryptophane, 154, 155

Keller, hippuric acid formation, 718,*
740

Kellner, protein metabolism, 508*, 566,
855*, 874*

Kelly, chitin, 791

Kermauner, 499*

Kerner, creatinine, 660

Keyes, hemolysis, 266

Kiesel, glycerophosphoric acid, 240

Kikkoji, nucleic acid in placenta, 608

Kiliani, carbohydrates, 193

Kirchmann, gelatin as protein sparer, 853,
854

Kirk, uroleucic acid, 701

Kirkness, milk, 637

Kistermann, phenylhydrazine test, 762

Kitagawa, protagon, 577; cerebron, 580

Kjeldahl, determining nitrogen, 654

Klages, 404*

Klatte, enzymes, 60

von Klaveren, katha3moglobin, 280

Klecki, 499*

Kleine, sulphur compounds in urine, 714,
728*

Kleiner, 682*

Kleinschmitt, 107*

Klemensiewiez, pyloric secretion, 452*,
454

Klemperer, parachymosin, 450; uro-
chromc, 703, 704; fate of oxalates in the
body, 733; protein metabolism, 846;
catabolism limits, 858; nitrogenous
equilibrium, 875

von Klercker, O., creatinine, 658*, 659,
660*

Klingemann, 637*

Klingenberg, oxidation of hydrocarbon,
and carbazol, 738

Klug, pepsins, 153*, 442; pepsin diges-
tion, 443*, 448; trypsin, 480; phos-
phates in urine, 724

von Knaffl-Lenz, glycogen, 368

Knapp, sugar test, 208; method of esti-
mating sugar, 765; metabolism, 847

von Knieriem, urea, 648, 649; uric acid, 668

Knoop F., histidine and its reaction test,
157, 158; methylimidazole, 197; fate
of amino-acids in the body, 737; elim-
ination of benzoic acids, 738; oxida-
tion of fatty acids, 739

Knop-Hiifner, estimating urea, 657

Knopfelmacher, human fat, 226*, 533

Kobert, H. U., hsemochrom, 268

Kobert, R., hemolysis, 266; cyan-
methsemoglobin, 277; iron in urine,
278*, 730, 793*

Kobner, bile acid, 416

Kobrak, human milk, 628

Koch, W., lecithin, 236*, 237; determi-
nation of lecithin and cephalin, 238;
neurokeratin, lecithin, jecorin, and
cephalin in brain, 575; protagon, 577;
brain composition 581 ; analysis of
brain, 582; degenerated nerves, 583

Kocher, thyroid, 356

Kochs, liver, 360, 686*

Koefed, 614*

Koelker, cleavage of polypeptides, 487

Koelichen, catalysis, 56

Koettgen, visual purple, 585

Kohler, flesh analysis, " flesh quotient,"
571; calorific food values, 572*, 835,
855*

Kolisch, creatinine, 317*, 663; histone
in urine, 751

Kolle, determining acidity of gastric
juice, 465

Kondo, indol test, 156; dextrorotatory
lactic acid, 555

Konig, B., fat in muscle, 570; composition
of milk, 624; composition of colostrum,
625; composition of milk of various ani-
mals, 626, 634*

Koppe, plasmolysis, 32, 33; blood cor-
puscles, 265; hemolysis, stromata, 266;
estimating blood corpuscles, 293, 313;
BC1 in gastric juice, 453

Koraen, muscle activity, 569; metabolism,
871, 872

von Korangi, osmotic pressure, 36; blood
corpuscles, 298; lymph formation, 333

Korkunoff, results of absence of proteins
from food, 846

Korn, A., 445*

Korndorfer, isocreatinine, 546

Korner, 114*

von Korosy, amino-acids and proteoses
in blood, 459*, 504; coagulable pro-
tein in blood, 507

Korowin, amylopsin, 478

Kossel, primar}' cell substances, 20;
hydrolytic cleavage, 77*, 80; protein
cleavage, 90; histones, 107, 108;
histone peptone, protamines, 109, 110,
111; gelatin, 118, 122*, 124*; scombrin,
136; valine, 139; serine, 143; proline,
152; histidine, 157; arginine, 158;
lysine, 160; lysatine, 161; nucleo-
proteins, 171; thymonucleic acids, 176;
thymic acid, 177; plasminic acid,
yeast nucleic acid, nuclein bases, 181;
purine bases, 182, 183; guanine, 185;
hypoxanthine, adenine, 187, 188; cyto-
sine, 189; uracil, thymine, 190; nucleo-
histone, 270*, 295;' blood-plates, 296;
pus cells, 343 ; thymus, 348, 349; purine
bases in liver, 364; muscle extractive,
471*, 546; protagon, 576, 577; pyosin,
pyogenin, 579; brain constituents^, 580;
protamine nucleate, 592; ichthulin,
598; urea, 647, 648

Kossler, ethereal sulphuric acids, 689, 690

Koster, casein, 617

Kostin, 279*

Kostytschew, thymonucleic acids, 177,
180

Kotake, Y., purine bases, 176*, 676; oxi-
dation of vanillin into vanillinic acid,
739

INDEX OF AUTHORS

905

Kotliar, E., 360*

Kowarski, urea in muscle, 546

Kowalewski, uric acid, 131*, 668, 728*

Kramm, creatinine, reaction, 662

Kranenburg, 452*

Krasnosselsky, spleen, 350

Kratter, adipocere, 534

Kraus, F., carbohydrate metabolism, 363*.
390

Krauss, indican, 694

Krawkow, amyloid, 168, 169, 170;
chitin, 791, 792

Krehl, histone in urine, 751

Kreis, 226*

Kresteff, pyloric secretion, 454

Kreusler, hydrolytic cleavage, 80

Kreuzhage, protein metabolism, 566

Krieger, proteids, 94; seralbumins, 253;
phosphates in urine, 255*, 724

Krimberg, carnosine, carnotine, 549

Krober, pentose determination, 202

Krogh, respiration, 810; microtonometer,
812; carbon dioxide secretion, 817;
varying oxygen tension of blood, 818;
nitrogen deficit, 819*, 825

Kronecker, benzoic acid, 687

Ivrouig, toxicity of mercury, 23; ion-
action, 74

Krug, protein fattening, 860

Kriiger, Fr., gelatin, 96*, 119, 120, 135*

Kriiger, M., sulphur in proteins, 79;
nucleiii bases, 181; purine bases, 184;
adenine, 188; hemoglobin in blood,
223*, 273*, 294*, 318, 319; spleen,
352; iron and calcium oxide in liver,
367; saliva, 430; muscle energy, 498*,
569; purine bases, 622*, 676; methyl-
xanthine, 677; epiguanine, 678; purine
bases estimation, 679; ammonia in
urine, 728; estimating ammonia in
urine, 729

Krukenberg, atmidkeratin, 112; skeletins,
113*, 121; cornicrystallin, 122*, 123,
hyalogens, 167; pigments in blood
serum, 260; haemerythrin, 292; muscle
extractives, 546; carnine, 548; oocyan,
604; urostealith, 748; pigments, in
feathers, 795

Krumbholz, preserving action of proteins
on fat emulsions, 512

Krummacher, protein metabolism, 566;
oxygen heat value, 832; gelatin as a
protein, sparer, 835*, 853, 854*

Kryshyschkowsky, gastric juice, 439

Kiibel, 432*, 433*

Kudo, 483*

Kiihling, O., 741*, 742*, 743*

Kuhn, glucose in vitreous humor, 586

Kiihne, enzymes, 9; keratins, 112; pep-
tones, 119*; proteoses, peptones, 129;
tryptic digestion, 131; protoproteoses,
134; antipeptones, 135; separating
proteoses and peptones, 137; para-
globulin, 250; haematoidin, 290; glyco-
gen, 368; antialbumid, true peptone,

448; rennin, 461*, 476*, 477; amy-
lopsin, 478; trypsin, 479, 480; gelatin
cleavage, 485; pancreatic rennin, 487;
preserving action of proteins on fat
emulsions, 512; muscle-plasma, myo-
sin, 539, 540; muscle reaction, muscle
proteins, 572; neurokeratin in brain,
575; neurokeratin, 583; visual purple,
584, 585; chlorophan, xanthophan,
rhodophan, fuscin, 586; changes in
crystalline lens, 587; lutein, 592

Kuliabko, 470*, 657*

Kulz, C., pericardial fluid, 337

Kiilz, E., cystine, 146; pentoses, 201;
isomaltose, 219; methaBmoglobin, 276;
glycogen, 369; glycogen formation, 372,
373, 376; levulose in glycogen formation,
379*, 382; parotid saliva, 429; ptyalin,
432; gastric juice, 451; amylopsin, 478;
glycogen in muscles, 563; human milk,
631; pentoses in urine, 736*, 743*, 769;
detecting betaoxybutyric acid, 779;
lymph gases, 807

Kumagawa, estimating fat, 231; fat
formation, 536; protein metabolism,
846; catabolism limits, 858; nitroge-
nous equilibrium, 875

Kumagawa-Suto, sugar test, 764

Kunkel, iron in milk, 279*, 322*, 411;
iron in bilirubin, 418; iron in urine, 730

Kuprianow, 553*

Kurajeff, putrefactive products, 81; anti-
albumid, 109*, 134, 153*

Kurbatoff, gadoleic acid, 230

Kurpjuweit, influence of autotoxines in
intestines, 498

Kusmine, lymphagogues, 333

Kusomoto, koprosterin, 423; cholesterin,
424

Kiister, absorption, 49; hsemochromogen,
282; hsematinic acid, haemin, 283, 284;
dehydrochloride haemin, 286; hsemo-
pyrrol, 288; bilirubin, 405, 406, 407;
bilirubin, biliverdin, 409; choleprasin,
410

Kutscher, protein oxidation products,
77*, 82; histones, 106*, 107; para-
histone, 109; gelatin, 118; pancreatic
autodigestion, 122*, 124*, 131; glu-
tamic acid, 145; histidine, 157; argi-
nine, 158, 159; lysine, 160; lysatine,
161; oxidation products of nucleic
acid, martamic acid, 178; cytosine,
189; thymus, 350; erepsin, 469; auto-
digestion of pancreas, 484; putrefac-
tion of proteins, 491; amino-acids and
proteoses in blood, 504; ignotine. 549;
meat constituents, 550; methylguani-
dine, 663; oxalic acid, 680; ptomaines
in urine, 718, 719

Kuwschinski, quantity of pancreatic
secretion, 476

Kyes, P., 236*

Laache, haemoglobin, 324

906

INDEX OF AUTHORS

von Laar, 112*

Lailenburg, spermine, 590

Laidlaw, haematin, 289; turacin, 795

Liiitinen, 297*

Lamois, 797*

Lumpel, 78*

Lariceraux, internal secretion in pancreas,
385

Landau, cerebrospinal fluid, 342

Landauer, antiputrid action of bile, 495;
death from lack of water, 496*, 842;
effect of water on metabolism, 862

Landergren, nitrogen loss, 507; utili-
zation of carbohydrates, 508*, 511; pro-
tein metabolism, 846; carbohydrates
as protein sparers, 856, 857; protein
requirements, 877

Landois, stroma fibrin, 268: quantity of
blood, 325

Landolt, melanins, 793

Landsteiner, antitryptic action, tryptic
digestion, 69; adsorption, 73

Landwehr, mucin, 165; milk carbohydrate,
622; dextrin in urine, 711

Lane-Claypton, liver autolysis, 18

Lang, digestion, 459; gastric absorption,
460; uric acid, 668; fate of nitriles in
the body. 735; estimating proteids, 750

Lang, J., taurine, 149; lymph, 330

de Lange, human milk, 631, 633; acetone
bodies in urine, 772; acetone elimina-
tion, 773

Langendorff, mineral bodies of muscles,
558

Langer, palmitin, 228

Langerhans, internal secretion of pancreas,
386; islands of , 470

Langgaard, 627*

Langhans, haematoidin, 20

Langheld, protein oxidation products,
82; desoxycholic acid, 403

Langley, ptyalin, 432; saliva, 435; pro-
pepsin, 453; pepsin formation, 454

Lailgstein, L., protein cleavage products,
83; peptic digestion, 131; skatosine,
156; fibrinogen, 164*, 214,* 244; globu-
lin, 245*, 252; glucosamine, 254; blood
serum, 260; albumin and globulin in
blood serum, 262; feeding experiments
with alanine, carbohydrate meta-
bolism, 390; globulin, 601; globulins,
602; human milk, 628; homogentisic
acid, 698, 699; uroleucic acid, 701;
kynurenic acid , 702 ; urine, 732; lactose
in urine, 768; nitrogen-carbon rela-
tion, 827

Lapicque, spleen, 352; iron in liver, 366,
367; protein requirement, 877

Lappe, lactase in intestinal juice, 467

Laptschinsky, composition of crystalline
lens, 588

Laqueur, casein, 452,* 615 616, 617;
paracasein, 618

Larin, 446*

Lassaigne allantoin, 681

Lassar, alkalinity of blood, 297

Lassar-Cohn, cholic acid, 401; 402, cholic
acids, 404; bile constituents, 411

Latarjet, 440*

Latchenberger, blood coagulation, 242;
bile pigments, 416, 418; protein assi-
milation, 502

Latchinoff, cholic acid, 401; cholecam-
pheric acid, choleic acid, 402; desoxy-
cholic acid, 403

Latham, 4

Lattes, acetone bodies, 775

Laulanie, fat formation, 537; muscle
work, 568; respiratory quotient during
work, 869

Laves, respiratory quotient in diabetes,
376*, 382; glycogen, 383; human milk,
627

Laveson, sugar in urine, 711

Lavoisier, influence of work on metabol-
ism, 868

Lawes, fat formation, 537

Lawrow, histones, 107; peptic digestion,
131; antialbumid, 134; coaguloses, 135;
decomposition of blood pigments, 157*,
281, 743*

Laxa, paracasein, 616*, 618

Lazarus-Barlow, lymph, 333

Lannois, sugar absorption, 509

Lankester, chlorocruorin, 292

Lea, Sh., saliva, 433

Leathes, spleen, 35*, 351 ; uric acid, 504*.
595*, 665

Leaven worth, plant proteins. 77; gly-
cogen, 368

Lebedeff, liver fat, 363; fat absorption,
533

Leclerc, perspiration, 799

Leconte, 439*

Ledderhose, d-glucosamine, 214; chitin,
791

Ledoux, substances retarding blood coagu-
lation, 311

Leers, 278*

van Leersum, test for pentoses, 203

Lefevre, glucuronic acid, 217

Lefmann, creatinine, 659

Legal, indol reaction, 156; acetone test,
776

Lehmann, egg white, 601; carbon dioxide
eliminated by the skin, 800; starvation
experiments, 836; respiratory quotient
during work, 855,.* 868*, 869"

Lehmann, C., fat formation. 537

Lehmann, K. B., hsemorrhodin, 280;
adipocere, 534

Lehndorff , cerebrospinal fluid, 342

Leichtenstern, haemoglobin, 320; blood
corpuscles, haemoglobin, 321

Leick, human fat, 533

Leipziger, 826*

Lemaire, isomaltose in urine, 711, 768*

Lemus, milk fat-globules, 614

Leo, liver fat, 363; respiratory quotient
in diabetes, 382; acid phosphates in

INDEX OF AUTHORS

907

gastric juice, 464; fat formation, 534;
laiose in urine, 768, 825*

Lepage, pancreatic juice excitants, 475;
chloral secretin, 476

Lepine, glucuronic acid, 215; pentose in
blood serum, 257; glucuronic acids,
sugar in blood, 258; chyle, 328; glyco-
gen formation, 372; glycolytic property
of blood, 386; sugar absorption, 509;
sulphur compounds in urine, 714;
phosphorus compounds, 718; leuco-
maines in urine, 767; maltose in urine,
767

Lepinois, chlorine in urine, 719

Lesem, protagon, 577

Lesmik, oxidation of naphthalene, 738

Lesser, lymph, 331

Letsche, cholesterin in blood serum, 256;
blood serum constituents, 259; uric
acid and amino-acid in blood serum,
260

Leube, proteid in urine, 744; perspira-
tion, 799

Leuchs, serine, 143, 144; oxyproline, 153;
osamines, 197; glucosamine, 214

Levene, enzymes, 17; polypeptide. 89;
proteid reactions, 104; gelatin, 106*;
118*, 120; protoproteose, 134; gluta-
mic acid, 145; tendon mucin, 154*, 165;
nucleic acid, 174; nucleotides, poly-
nucleotides, 175; thymonucleic acids,
176, 177; glycophosphothymic acid,
mononucleotides, 178; guanylic acid,
inosinic acid, 179; thymonucleic acid,
yeast nucleic acid, 180; guanosin,
adenosin, tubercle-bacilli nucleic acid,
181: purine bases, 183; guanine, 186;
thymine, 190; xyloses, 204; pus cor-
puscles, 295; spleen, 346*, 350; liver
cleavage products, 362; liver consti-
tuents, 365; trypsin, 379*, 480; auto-
digestion of pancreas, 484; tendon
mucin, 519; chondroitin-sulphurie acid,
522; nucleoprotein in brain, 574; testes
constituents, 589; ichthulin, 599; pro-
teid of mammary gland, 610; kidney
constituents, 639

Levison, pepsin test, 446

Levites, proteins, 78; cholesterin, 424;
splitting of carbohydrates and fats,
452*, 492

Levy, A., 'sp.gr. of blood, 296; phosphate
in bone, 530; inyoha?matin, 547; thio-
, phenuric acid, 742

Lewandowsky, protein absorption, 502

Lewin, methaemoglobin, 277; carbon-
monoxide hemoglobin, 279; haemover-
din,4SO; hsemochromogen, 282; lutein,
600; hippuric acid, 685; indican elimi-
nation, 692

Lewin, L., oxyhaemoglobin, 274

Lewinsky, proteins in blood plasma, blood
serum' 262 ; hippuric acid, 685

Lewis, 430*

Lewy, B., crystals in semen, 590

von Leyden, indican elimination, 692

Lieben, haematoidin, 290; lutein, 292;
acetone test, 775

Liebermann, proteids, 94; proteid detec-
tion, 99, 100; proteid, 105; nucleins,
pseudonucleins, 172, 173; protein cleav-
age, 162*, 163; gastric juice, 437; egg
yolk, 596; yolk fat, 599; yolk con-
stituents, 600; egg white, 601; oocyan,
604; egg development, 606, 607; kid-
ney constituents, 638; alloxyproteic
acid, 716; guaiac blood test, 752;
carminic acid, 795

Liebermann-Burchard, cholesterin reac-
tion, 422

Liebermeister, C. G., nucleoprotein, 250

Liebig, mineral substances in cells, 22;
inosinic acid, 179; fat formation, 536;
muscle work, 565, 567; determining
urea, 654, 874*

Lieblein, blister fluid, 343; urea elimina-
tion, 651

Liebrecht, putrefaction products, 81

Liebreich, protagon, 576; plant waxes,
796

Liepmann, enzymes in placenta, 608

von Lier, plasmolysis, 33

Lifschiitz, oleic acid, 230; cholesterin
reaction. 423; cholesterin, 424; wool-
fat, 797

Likhatscheff, gentisic acid, 742

Lilienfeld, nucleohistone, 107, 248*, 295;
blood plates, nuclein plates, 296; blood
coagulation, 301, 302, 303; thrombin,
303; thymus, 348, 350

Lillie, osmotic pressure, 39; osmotic
tension, 52

von Limbeck, alkali in blood, 297*, 298

Limpricht, protic acid, 546

Lindberger, trypsin digestion, 483; bile
acids as preventive of putrefaction in
intestine, 495

Lindemann, 202*

Linder, colloids, 44; colloidal precipitation,
47

Lindvall, 112*

Ling, 219*

Lingle, mineral bodies of muscles, 558

Linn, beta-cholestanol, 421

Linossier, 641*

Linser, sebum, 796

Lintner, 223*

Lintwarew, pancreatic juice, 472

Lipp, tyrosine, 150

Lippich, F., leucine, 142; 499*, 657*

von Lippmann, tyrosine, 151

Lister, blood coagulation, 300

Ljungclahl, glycosuria, 381, 382*

Ljubarsky, physetolic acid, 232

Lloyd-Jones, sp.gr. of blood, 296

Lobisch, milk secretion, 635

Lobry de Bruyn, carbohydrates, 40*,
196; osamines, 197; amino-sugars, 213

Lochhead, glycogen in placenta, 60S

Locke, muscle metabolism, 563

908

INDEX OF AUTHORS

Lockemann, 295

Locquin, isoleucine, 143

Loeb, J., cell life, 21*, 23; muscle exper-
iments, 33; artificial fertilization, 74;
blood coagulation, 248; influence of
light on metabolism, 305*, 306; min-
eral bodies of muscles, 558; muscle
work, 569; egg development, 607

Loeb, L., action of tissue extracts, blood
coagulation, 307; blood corpuscles, 308

Loeb, W., sunlight synthesis, 1

Loebisch, bilipurpurin, 410; form ele-
ments, 519; proteid of mammary
gland, 610

Loeschke, glycogen, 371

Loevenhart, ' fat synthesis, 7*, 63*, 65;
action of NaFl on enzymotic cleavage,
74; steapsin, 479; fat deposition, 537

Loew, O., active proteins,- 4; catalases,
7; oxidations, 8; putrefactive products,
77*, 81; sugar synthesis, 84*, 96*, 205

Loewenthal, emulsifying action of fatty
acids, 489

Loewi, O., protein synthesis, 379*, 391*,
506; urea, 648; preparing allantoin,
682; glucuronates, 712; metabolism,
723*, 826

Loewy, A., ornithine, 159: lysine, 160;
alkali in blood, 296; 297*, 298: liver
autolysis, 323*, 365; muscle work, 566;
urine, 642; amino-acids in urine, 718;
blood gases, 780*, 802; respiration, 809,
810; oxygen pressure in blood, 811;
alveolar air, 814 ; carbon dioxide tension
in blood, 815; maintenance value in
metabolism, 831*, 841 ; combustion proc-
esses, 866*, 869*, 869; influence of
external temperature on metabolism,
870; effect of altitude on metabolism,
871

Lohlein, trypsin estimation, 482

Lohmann, lysine, 160; choline, 239;
adrenals, 357; autodigestion of pan-
creas, 484; methylguanidine, 663;
ptomaines, in urine, 718

Lohnstein, urine, 645; estimating sugar,
767

Lohrisch, cellulose and hemicellulose
digestion, 488, 489*

Lombardi, 95*

Lombroso, influence of pancreatic juice
on protein absorption, 508; food
absorption, 511*, 516

London, rapidity of digestion, 452*, 457;
extent of digestion, 459; gastric
absorption, 460; putrefaction of pro-
teins, 491 ; artificial digestion with
trypsin, cleavage of starch, dextrins
and disaccharides, 492 ; protein synthe-
sis, 506

Long, lecithin, 236, 237; casein, 615, 616*

Lorinberg, cartilage, 524; kidney con-
stituents, 638

Ldnquist, 438*

Lorcher, 450*

Losev, adsorption, 50

Lessen, protein oxidation products, 82;
slow coagualtion of blood, 312

Lotsch, digestion, 459

Lottermoser, colloids, 44

Lowit, plasmoschisis, 301

Luchsinger, glycogen formation, 372, 374;
perspiration, 798

Luciani, haemoglobin, 321, 836*

Liicke, A., pus, 167*, 347; hyalin, 792

Luckhard, lymph, 330

Liidecke, lecithin, 235; glycerophosphoric
acid, 235

Ludwig, C., ethal, 232; pseudohsemoglo-
bin, 270*, 276; bile acids, 371*, 416;
digestion, 460; pancreatic juice, 471;
protein assimilation, 503; fate of pep-
tone in intestine, 504; sugar entrance
into blood stream, 510; blood gases,
801, 802; rheometer, 812

Ludwig, E., dermoid cysts, 596; esti-
mating uric acid, 674, 675

Lukjanow, solids in bile, 393; loss of
water in starvation, 840

Lukomnik, J., 135*

Lummert, fat formation, 363*, 537

Lunin, results of lack of mineral sub-
stances, 843; metabolism, 847

Lusk, sugar formation in liver, 379;
lactic acid and diabetes, 387; sugar
formation from fat, 391; lactase for-
mation, 509; lactic acid, 556; lactose
in urine, 769

Lussana, glycogen, 377

Luther, sugar test, 763

Liithje, origin of sugar in diabetes, 388,
389; sugar formation from glycerin,
390; protein synthesis, 506; ^oxalic
acid, 680; protein fattening, 861

Luzzato, R., arabinose, 203; oxalic acid,
680; Z-arabinose in urine, 769

Maas, alkali albuminose, 126

Macallum, potassium hi cells, 22; ash of
muscles, 322*, 558

Maccadam, 566*

MacCallum, secretion of intestinal juice,
466; influence of phosphates, 845

Macfadyen, putrefaction in intestines,
9*, 490

von Mach, uric acid, 668

Mackay, bile acids, 755

MacLean, phosphatide, 240*, 233; oxalic
acid, 681

Macleod, glycosuria, 3S1; bone marrow,
528; phosphocarnic acid, 550* 564;
carbamate formation, 565*, 649; car-
bamic acid, 658

MacMunn, hsematoporphyrin, 287; echin-
ochrom, 292; cholohaematin, 410; myo-
hsematin, 545; urobilin, 705; blood test,
753; tetronerythrin, cyanocrystallin, 795

Madsen, enzymes, 59; pyocyaneus pro-
tease, 63; anti-bodies, 69;" Ehrlich's
theory, 72; cholesterin, 424

INDEX OF AUTHORS

90$

Magnanini, indol test, 156

Magne, milk, 637

Magnier, iron in urine, 730

Magnus, R., enzymes, 60

Magnus-Levy, liver cells, 356*, 361;
volatile fatty acids in urine, 391*, 710;
glucuronates, 713; hippuric acid, 685;
proteoses, 749: acetone bodies in urine,
772*, 773; 774*, 775*; estimating
betaoxybutyric acid, 779; oxygen heat
value, 820*, 833; basal requirement in
metabolism, productive increase, 841;
metabolism in old and young, 865*,
866, 867; effect of food ingestion on
metabolism, 871

Maillard, toxicity of copper salts, 23;
ion-action, 74; indican, hemi-indigotin,
694

Makris, 627*

Majert, spermine, 590

Malcolm, 723*

Malengreau, thymus, 348, 349

Maleniick, protamines, 109*, 110, 111

Malfatti, peptic digestion, 131; purine
bases estimation, 679; estimating am-
monia in urine, 729; estimating sugar,
767

Mall, reticulin, 120

Mallevre, 489*

Maly, oxyprotosulphonic acid. 82; biliru-
bin, 137*, 406, 407; choletelin, 408;
HC1 secretion, 434*, 453; bile acids
as preventive of putrefaction in intes-
tine, 495; lutein, 600; creatinine, 662;
urobilinoids, 705; hydrobilirubin, 706;
nutritive value of proteoses and pep-
tones, 854

Manasse, jecorin, 364

Manche, glycogen in muscles, 563

Manchot, oxygen dissociation, 6, 7;
oxidases, 13

Mancini. pentose, 201; urochrome, uro-
pyrryl, 703, 704

Mandel, glutamic acid, 145; nucleic acid,
174; nucleotides, polynucleotides, 175;
thymonucleic acids, 176, 177; glyco-
phosphothymic acid, mononucleotides,
178; guanylic acid, 179; thymine, 190;
glucuronic corpuscles, 211*, 216; spleen,
346*, 350; liver cleavage products, 362;
liver constituents, 365; nucleoprotein
of mammary gland, 610; nucleoprotein
cleavage products, 635; kidney con-
stituents, 639, 718*, 771*, 826*

Mandel, A. R., lactic acid and diabetes,
387; lactic acid, 556

Mandel, H., uric acid, 669

Mandelstamm, olive oil as a cholagogue, 393

Mangold, glycogen, 369, 838*

Manicardi, nucleon, 550

Mann, brain composition, 76*, 80*, 581

Manning, intestinal juice, 467

Mansfield, G.. lipolysis, 259, 342*

Maquenne, starch, 220; dextrin, 223;
cellulose, 224; inosite, 551

Marcet, xanthine, 184; excretin, excreto-
licacid, 500

Marchetti, fat absorption, 534

Marchlewski, blood pigment, hsemopyrrol,
288, 269; htemin, 285; hsematopor-
phyrin 287; phyllocyanin, cholohse-
matin, phylloerythrin, 410

Marcus, serglobulins, 251

Marcuse, glycogen in muscle, 563; lactic
acid in muscles, 564; metabolism, 826,
845*

Margulies, 762*

Mark, 364*

Markewicz, 728*

Marquardsen, reaction of intestinal con-
tents, 497

Marshall, milk, 637; glycosuric acid, 698

Martin, enzymes, 63; thrombin, 248;
snake poison causing intravascular
coagulation, 310; action of bile on
diastatic action of pancreas infusion, 488

Martz, 797*

Marum, acidosis, 773

Marx, glycocoll in urine, 717; acetone
formation, 772*, 774

Marxer, internal secretion in pancreas,
386

Maschke, creatinine reaction, 93*, 661

Masius, bilirubin, 406; stereobilin in
feces, 499

Massen, carbamate elimination, 650

Masuyama, enzymes in egg yolk, 597

Mathews, arbacin, 108; nucleic acid, 109*,
160*, 177; fibrinogen, 244; protamine
nucleate, 245*, 592

Mathieu, blood gases, 802

Matthes, enzymes in urine, 718; reaction
of intestinal contents, 497; histone in
urine, 751

Mauthner, cholesterin, 420, 855*

Maximowitsch, seralbumin, 253, 254 t

May, enzyme in pancreas, 202*, 481

Mayeda, 438*

Mayer, E. W., beta-cholestanol, 421

Mayer, P., lecithalbumin, 105; isoserine,
144; cystine, 146, 147; stone-cystine,
148; sugar transformations, 197; sugar
fermentations, 199; glucuronic acid,
215,216; lecithin, 236: fibrinogen, 244;
glucuronic acids, 245*, 257*, 258; albu-
min and globulin in blood serum, 262;
meat digestion, 364*, 509; food absorp-
tion, 516; oxalic acid, 680; indican
elimination, 692; glucuronates, 712,
713; iron in urine, 730; detecting
conjugated glucuronic acids, 771; effect
of water on metabolism, 862

Mayo-Robson, bile secretion, 392

Mays, trypsin, 480 ; fuscin, 586

Maze, sugar fermentation, 200

Mazurkiewicz, pancreatic juice, 471 ; solids
in pancreatic juice, 477

McGuigan, sugar formation from glvcogen,
378

Meara, 129*

910

INDEX OF AUTHORS

Meddelelser, 138*

Medigreceanu, haemoglobin, 270, 457*

Medwedew, oxidases, 11

Mehu, pleura! fluid, 339; urobilin, 707, 708

Meigs, E. B., 560*

Meillere, inosite, 551; detecting and
estimating inosite, 552; inosite in
urine, 719*, 771

Meinert, utilization of foods, 508, 874

Meinertz, 364*

Meisenheimer, J., ferments, 10

Meissenheimer, sugar fermentation,
zymase, lactacidase, 200, 207*

Meissl, fat formation, 537; amniotic fluid,
609

Meissner, dyspeptone, parapeptone, 448;
allantoin, 651*, 681; hippuric acid, 684

von Meister, 651*

Mellanby, creatine, 250*, 547, 548; crea-
tinine, 659, 660

Mendel, lymph, 333; glycogen, 368;
activation of trypsinogen, 429*, 473;
protein absorption, 502; uric acid,
546*, 668*, 670; allantoin, 682, 702*

Mendes de Leon, 629*

Menozzi, bombicesterin, 424

Menschutkin, catalysis, 58

Menzies, methsemoglobin, 277

de Merejkowski, tetronerythrin, 795

von Mering, glucuronic acid, 215; dex-
trose in blood, 257*, 319; glycogen for-
mation, 373; phlorhizin diabetes, 379;
glycogen, 383; ptyalin, 431; amy lop-
sin, 432*, 478; protein absorption, 502;
carbohydrate absorption, 509; sugar
entrance into blood stream, 510; uro-
chloralic acid, 735*, 736; effect of food
ingestion on metabolism, digestion
work, 871

Merunowicz, haemin compounds, 287

Mesernitzki, egg yolk, 597

Messner, protein synthesis, 506

Mester, putrefaction in intestine, 497;
skatoxylsulphuric acid, 695; sulphur
compounds in urine, 714

Mett, pepsin test, 445; trypsin estimation,
482

Meyer, E., homogentisic acid, 698, 699;
conversion of nitrobenzene and nitro-
phenol into aminophenol, 739

Meyer, H., blood serum, 260

Meyer, Kurt, glucosamine in glycogen
formation, 376

Meyer, P.,' colloids, 44; glucuronic acid,
215; iron in liver, 367; inosite, 551;
blood pigments in bile, 415; nitrogen
loss, 507; uric acid, 508*, 668; uro-
leucicacid, 701

de Meyer, activating substance in pan-
creas, 387

Michaelis, colloids, 45; enzymes, 71*,
59; proteids, 96; proteicf test, 101;
proteoses, 133; sugar in blood serum,
257; lipolysis, 259; sugar in blood
corpuscles, 314; permeability of blood

corpuscles, 316; protein absorption,
317*, 502; gelatin as potein sparer,
854

Michaud, acetone bodies, 772*, 775; pro-
tein metabolism, 846; protein mini-
mum, 859

Michel, seralbumins, 253, 254, 255

Micheli, F., 135*

Micko, meat extracts, 499*, 550, 826*

Middendorff, haemoglobin in blood, 319

Miescher, F., protamines, 109, 110, 111;
nuclein, 172; thymonucleic acids, 176;
pus corpuscles, 345; pus, 346*, 347;
karyogen, 591*, 592; influence of
phosphates, 845

Miethe, oxyhaemoglobin, 274; methsemo-
globin, 277; carbon-monoxide haemo-
globin, 279; haemochromogen, 282;
lutein, 600

Milchner, mucin, 166

Millon, proteid test, 98

Mills, oxalic acid, 680

Milroy, nucleins, 172; haemochromogen,
282, 723*

Minkowski, ascitic fluid, 297*, 340;
phlorhizin diabetes, 376*, 379; glycogen,
382, 383; glycosuria, 384; internal
secretion in pancreas, 385; bile pig-
ments, 417, 418, 419; pancreas extir-
pation, 508; fat absorption, 512*, 515;
paralactic acid, 554, 710; uric acid, 668,
669; allantoin, 682; benzoic acid, 687;
detecting beta-oxy butyric acid, 711*,
779; blood gases, 807

Mitchell, 668*

Mitjukoff, ovarial colloid, paramucin, 594

Mittelbach, fibrinogen, 245; homogentisic
acid, 701; estimating proteid, 750

von Mituch, 606*

Miura, 372*, 467*

Miyamota, pepsin, 443

Modrakowski, choline, 240

Modrzejewski, amyloid, 170

Moeller, 499*

Mohr, sulphur in keratin, 112; pancreas
diabetes, 378*, 385; sugar formation
from fat, 391; reagent for free HC1 in
gastric juice, 463, 676*, 680*

Moitessier, carbon-monoxide methaemo-
globin, 279; proteid, 719*, 749

Molescbott, 874

Molinari, cholesterin, 421

Molisch, sugar test, 209

Moll, proteids, 103

Mollenberg, hemoglobin, 320

Moller, estimating acetone, 778

Monod, 622*

Monari, muscle reaction, 563; muscle
work, 564*, 565; xanthocreatinine, 663

Moor, Ovid, urein, 657

Moore, cell electrolysis, 21; adsorpates,
34; osmotic pressure, 37, 38, 39;
adsorption, 50; dextrose test, 207;
action of bile on fatty acids; 489: reac-
tion of intestinal contents, 497; fatty

INDEX OF AUTHORS

911

acids converted into neutral fats, pre-
serving action of proteins on fate mul-
sion, 512; solvent action of bile on
fatty acids, action of bile on soap solu-
tions, 513

Moraczewki, casein, 499*, 618; indican
elimination. 619*, 692

Moral, glycogen in muscles, 563

Morawitz, fibrinogen, 244; blood coagu-
lation, 245*, 262*, 301; hirudin, 305;
fluoride plasma, thrombokinase, throm-
bogen, retarding blood coagulation, 306;
non-coagulability of blood, 312; serum
reactivation, beta-thrombin, alpha-
thrombin, beta-prothrombin, action of
tissue extracts, 307; substances retard-
ing blood coagulation, 311; slow-
coagulation of blood, 312; detecting
proteids. 748

Morax, 688*

Moreau, dextrin, 223; gas secretion in
fish, 817

Morel, fibrinogen, 244, 245; glycolytic
enzyme, 258; butyrinase, 259, 479*,
598, 622*

Moreschi, bombicesterin, 424

Morgen, 634*

Morgenroth, antienzyme, 69; antitoxin,
72 ; antirennins, 450

Mori, utilization of foods, 508, 874*

Moriggia, perspiration, 798

Morishima, liver cells, 361 ; lactic acid, 555

Moritz, exudates, 335; gastric digestion,
379*, 455

Moriya, lactic acid in organs, 554; para-
lactic acid in brain, 576

Morkovvin, 109*

Morner, C. Th., albuminoid, 114; gelatin,
117,119; reticulin, 121; gorgonin, 122;
cornicry stalli n, 123; levulose, 167*,
210, 211; mucoid in vitreous humor,
520; cartilage, chondromucoid, 521;
chondroitinsulphuric acid, 522; pre-
paring chondromucoid, 523; cartilage
chondroitin balls, 524: membranin,
525: bone ash, 527; vitreous humor,
hyalomucoid, crystalline lens, 586;
solids of crystalline lens, albumoid, 587;
proteins in crystalline lens, 588; ovo-
mucoid, 603; 'percaglobulin, 605; kid-
ney constituents, 639; urine nitrogen,
646; estimating urea, 652*, 656; homo-
gentisic acid, 701; determining chlo-
rine in urea, 722; nucleoalbumin in
urine, 742*, 751, 784*

Morner, K., hydrolytic cleavage, 78,
80; sulphur in proteins, 79; keratin
cleavage products, 106*, 113; albumin-
ates, 126; cystine, 146, 147; thiolactic
acid, cy stein, 148; thiolactic acid, 149;
globulin, 251, 252; seralbumin, 254;
h£i>min, 285, 286; blister fluid, 343;
determining acidity of gastric juice,
465; chondroitinsulphuric acid, 522;
muscle syntonin, 543; muscle haemo-

globin, 545; nonvolatile fatty acids in
urine, 710; mucin in urine, 718; oxida-
tion of acetanilide, 738; proteid in
urine, 744; nucleoalbumin and mucin
in urine, 750: melanin, 755; bile acids,
755; phymatorhusin, 793

Morner-Sjoqvist, estimating urea, 655

Morochowetz, cartilage, 521

Morris, 9*, 219*, 223*

Moruzzi, 240*

Mosse, 378*

Miihler, 135*

Miihsam, fat in blood, 318

Muirhead, carbamic acid elimination, 650*

Mulder, 112*

Miiller, synthetic glycocholic acid, 36*r
397; synthetic taurocholic acid, 399;
cholic acid, 402; putrefaction of pro-
teins, 423*, 494; brain constituents,
499*, 572*, 580; enzymes in egg yolk,
597; nitrogen excretion, 825, 831*, 868*

Miiller, A., colloids, 37, 44; HC1 in gastric
juice, 464

Miiller, E., 295, 489*

Miiller, Franz, adrenalin, 323*, 358

Miiller, Fr., heterolysis, 18; proteins,
82; mucins, 135*, 164, 165, 167; pus
cells, 346; fat absorption, 514,' 515;
pseudomucin, 595; ethereal sulphuric
acid, 688; urobilin, 706*, 709; sulphur
compounds in urine, 714; sulphuretted
hydrogen in urine; 715; oxidation of
aniline, 738; acetone bodies in urine,
772; autolysis of lungs, 820; effect of
altitude on metabolism, 871

Miiller, Joh., scyllite, 552; muscle metab-
olism, 563; pyrocatechin, 690

Miiller, M., carnic acid, 550; " flesh
quotient," 571, 855*

Miiller, P., fibrinogen, 244; fibrinogen in
blood plasma, 245*, 262; bone marrow,
528; human milk, 628, 826*

Miiller, W., cerebrin, 579

Munk, I., chyle, 328; fat in chyle, 329;
chyle, 331; buccal saliva, 430; reac-
tion of intestinal contents, 497 ; protein
absorption, 502. 503; sugar absorption,
510; conversion of fatty acids into
neutral fats, 512; passage of fat into
chyle, 513; fat absorption, 514, 515;
518*; fat absorption, 533; fat forma-
tion, 537; muscle work, 566; muscle
proteins, 572; nucleon in muscle, 573;
proteins in milk, 623; urea, 649; ethe-
real sulphuric acid, 689; sulphur com-
binations in urine, 714; sulphates in
urine, 724; test for bile pigments, 757;
urea excretion in starvation, 838;
elimination of phosphoric acid in
starvation, 839; gelatin as a protein
sparer, 813; nutritive value of pro-
teoses, 854; catabolism limits, 855*,
858; effect of water on metabolism, 860

Miintz, origin of milk sugar, 636
Miinzer, urea, 649, 652

912

INDEX OF AUTHORS

Murlin, gelatin value, 854

Musculus, ptyalin, 223*, 431; amylopsin,

432*, 478; urochloralic acid, 736; urine

fermentation, 782
Miither, xyloses, 204
Myers, creatinine, 660
Mygge, estimating proteid, 750
Mylius, modified Pettenkofer test, 221*,

397; cholic acid, 401, 402; desoxycho-

lic acid, 403

Naegeli, 643*

von Nageli, plasmolysis, 30; starch, 220

Nagano, intestinal juice, 467; lactase
formation, 509; sugar formation, 509,
510

Nakaseko, 372*

Nakayama, nucleoprpteins, 176; erepsin,
469; test for bile pigments, 757

van Name, collagen, 117*, 120

Nasse, O., animal oxidations, 6; protein
oxidation, 14; muscle experiments,
33; proteid detection, 77*, 98; gelatin,
119; sp.gr. of blood during pregnancy,
fibrin in blood, 223*, 258*, 320; lymph,
331; spleen constituents, 352 ; glycogen,
368, 370; ptyalin, 431; musculin,
432*, 433*, 541*, 543; rigor mortis,
561; glycogen in muscles, 563

Naunyn, glycogen formation, 373; bile
pigments, 417, 418*, 419; oxidation of
benzene and toluene, 738; oxidation
xylene into toluic acid, 739

Nawratzki, cerebrospinal fluid, 342

Nebelthau, glycogen formation, 372, 754*

Necker, 748*

Neff, 207*

Neilson, catalysis, 57, 429*, 430*

Neimann, glucuronic acids, 215, 216;
liver constituents, 365; glucuronates,
366*, 712; glucuronic acids, 713

Nelson, protamines, 109*, 110

Nencki, L., oxidation of mesitylene into
mesitylenic acid, 739

Nencki, M., putrefactive products, 14*,
78*, 81; tryptophane, 153; indol, 156;
blood pigments, haemopyrrol, 269;
oxyhsemoglobin, 273; haemin, 285, 286;
hsematoporphyrin, 287; mesoporphyrin,
phyllocyanin, 288; diabetes, 382; bile
pigments, 418; gastric juice, 441, 442;
pepsin, 443; pepsin digestion, 452;
gastric juice, 453; action of gastric
juice on toxalbumins, 461; steapsin,
479; action of bile on steapsinogen,
489; putrefaction in intestines, 490;
putrefaction of proteins, 492; reaction
of intestinal contents, 497; ammonia
formation in digestive apparatus, 505;
urea in muscle, 546; urea, 648, 649;
carbamic acid elimination, 650; urea
elimination, 651; urorosein, 697, 703;
urobilinoids, 705; ammonia in urine,
727, 728; fate of ammo-acids in body,
734; oxidation of naphthalene and

ethylbenzene, 738; oxidation of cymene
into cumic acid, 739; acetophenone
elimination, 742; odor of urine after
asparagus ingestion, 744; hippomelanin,
phymatorhusin, 93; protein metabolism,
853

Neppi, glutinase, 481 ; trypsin, 482

Nerking, lecithin, 235; bone marrow,
528; human milk, 629, 638*

Nernst, membrane permeability, 21;
adsorption, 48*, 73

Neubauer, O., proteid detection, 100;
creatine, 548; creatinine, 658, (>(>2;
homogentisic acid, 699, 701; uroleucic
acid, 702; urobilinogen, 708; ammonia
in urine, 727; fate of amino-acids in
body, 734; glucuronic acid conjuga-
tion, 736; diazo-reaction, 779

Neubauer-Huppert, 548*

Neuberg, protein oxidation products, 81*,
82; isoserine, 144; oxyaminosuccinic
acid cystine, 146; stone-cystine, 148;
tryptophane, 154; ornithine, 159; ly-
sine, 160; mucin, 166; amyloid, 168,
16.9; inosinic acid, 179, 180; carbo-
hydrates, 196, 197; sugar fermenta-
tion, 199; pentoses, 201; test for
pentoses, arabinose, 203, 204; sugar
test, 208; galactose, levulose, 2JO; de-
tecting levulose, 211; amino sugars, 212;
glucosamine, 214 glucuronic acid, 215,
216; lysine in blood serum, 260; spleen,
337*, 351; adrenalin, 359; liver auto-
lysis, 365; glycogen, 372; feeding experi-
ments with alanine, carbohydrate met-
abolism, 390; cholesterin, 421; chon-
drosin, 523; inosite, 551; pseudomucin,
595; egg yolk, 598; lactose test, 621;
heteroxanthine, 639*, 677; phenolsul-
phuric acid, 690; skatoxylsulphuric
acid, 695; glucuronates, 712, 713;
phenylhydrazine test, 720*, 729*, 761;
pentoses in urine, 769; detecting con-
jugated glucuronic acids, 771; acetone
bodies in urine, 772, 780*; melanins,
794; results of lack of chloride s, 827*,

843*, 844

Neuberg-Rauchwerger, cholesterin reac-
tion, 422

Neumann, A., antipeptones, 135; protein
cleavage, 162*, 163; thymonucleic
acids, 176; thymic acid, 177; thymo-
nucleic acids, 180; cytosine, 189; thy-
mine, 190; pentose tests, 203; iron in
urine, 418*, 730; phenylhydrazine tests,
761 ; alcohol in metabolism, 762*, 863

Neumann, O., protein requirements, 863*,
876

Neumeister, R., keratins, 112; soluble
proteoses, 128*, 129; glycogen pre-
cipitants, 153*, 223*, 370; protein
absorption, 502; ovomucoid, 603

Neusser, blood test, 753

Nickles, fluorine in egg white, 604

Nicloux, glycerin in blood serum, 62*, 256

INDEX OF AUTHORS

9:3

Nicolaier, 672*

Memann, influence of pancreatic juice
on protein absorption, 508; food
absorption, 516

Niemilowicz, purine bases estimation, 679

Nierenstein, pepsin test, 446

Nilson, lichenin, 222; cholostrum, 625

Le Nobel, haematoporphyrin, 287; uro-
bilinoids, 705; blood test, 753

Noeggerath, metabolism, 847

Noel-Pa ton, lymph, 331; liver fat, phos-
phatides, 363; sugar formation from
glycogen, 378; bile secretion, 392;
bile pigment, 413, 566*

Noguchi, cholesterin, 424

Nogueira, kidney constituents, 639

Nolf, fibrinogen, 35*, 244, 245; fibrin-
olysis, 247; thrombin, 250; prothrom-
bin, 305; retarding blood coagulation,
306; blood coagulation, coagulation of
plasma, 308; antithrombin, thrombo-
plastic agents, fibrinolysis, fibrin for-
mation, 309; • substances retarding
blood coagulation, 311; thrombo-
kinase secreted by endothelial cells,
312; chorda-saliva, 427; absorption of
proteoses and peptones, 503; carbam-
ate formation, 649; carbamic acid, 658

Noll, nucleic acids, 17.7; protagon, 583

von Noorden, sugar formation from fat,
291*, 380*, 382*, 383*, 391; ethereal
sulphuric acid, 652*, 688; proteid in
urine, 744; catabolism limits, 858;
flesh deposition, 861

Nothwang, death from lack of water, 842

Notkin, thyroid protein, 356

Novi, iron in liver, 366; iron in bile, 411;
saliva, 435

Novy, 126*

Nowak, 825*; nitrogen in muscle, 571;
respiratory gas exchange, 819

Niirenberg, iodothyreoglobulin, 357

Nussbaum, oxygen tension in blood, 811;
carbon dioxide tension in blood, 814,
815

Niittal, digestion, 494

Nylander, sugar test, 208, 759

Nylen, ptyalin, 432

Obermayer, proteid test, 101; tests for

bile pigments, 694*, 757
Obermiiller, cholesterin, 420; cholesterin

test, 423

Odenius, protein in mammary gland, 610*
Oddi, action of bile on gastric digestion,

489 ; preparing chondroitin-sulphuric

acid, 524; urine, 641
Oertel, phosphorus compounds in urine,

718, 826*
Oertmanri, 3*
Oerum, quantity of blood, 325; sulphur

in bile, 413; cystic fluid, 596; indican

test, 694; gelatin as a protein sparer,

853
Offer, pentosamine, 213; glucosamine as

a glycogen former, 375; chitin, 376*,
791; alcohol in metabolism, 863

Ofner, detecting ketoses, 211; detecting
levulose, 212

Ogata, digestion, 460

Ogden, homogentisic acid, 698

Oidtmann, inguinal glands, 348; thymus,
350; spleen, 352; thyroid, 354; salivary
glands, 426; pancreas, 471; kidney
analysis, 639

Oker-Blom, adrenals, 357

Okunew, antialbumid, 134

Oliver, adrenal pressure raising action,
357

Ollendorff, hydrazones, 199

Ollinger, protein synthesis, 506

Olsavszky, phosphates in urine, 724

Omeliansky, 489

Omi, enzyme in intestinal juice, 467

van Oondt, 380*

Opie, proteolytic enzymes, 295

Oppenheimer, C., seralbumin, 162*, 210*,
254; artificial digestion with trypsin,
480*, 492; protein absorption, 502;
respiratory gas exchange, 819; nitrogen
deficit, 825; metabolism, 865

Oppler, sugar in blood, 316, 317*

Orban, lactose in intestinal juice, 467

Orgler, protein oxidation products, 82;
adrenals, 357; chondrosin, 523; uric
acid, 665; acetone bodies in urine, 772

Orglmeister, arginine, 159

Orndorff, bilirubin, 405, 406

Orton, homogentisic acid, 701

Osborne, plant proteins, 77; sulphur in
proteins, 79; hydrolytic cleavage, 80;
polypeptides, 89; globan, 102*, 103;
edestan, 107*, 108; nucleic acid, 174,
175; plant nucleic acids, 180, 181;
sugar of muscles, 553; ovovitellin, 597,
598; globulins, 601; ovalbumin, 602;
calorific value of foods, 829

Ost, 219*

Osterberg, 646*

Ostertag, 626*

Ostwald, Wo., colloids, 37; adsorption,
49; gelatin solutions, 52; catalysis, 54,
55; ion-action, 75

Ostwald-Luther, 29*

Oswald, thyroid proteins, 81*, 354;
thyroid protein, 356; iodothyreoglo-
bulin, 357; detecting and determining
globulins, 748

Otori, mucin, 165; transudates and exu-
dates, 337; autodigestion of pancreas,
484; pseudomucin, 595; guanidine,
659

Ott, phosphates in urine, 722, 729

Otto, J., blood serum, 257; methsemo-
globin, 270*, 276, 277; estimating
blood corpuscles and blood fluid, 277,
291*, 314; dextrose in blood, 319;
haemoglobin, 320, 321; autodigestion,
462 ; carbohydrate absorption, 509

Overton, osmosis, 21; plasmolysis, 31;

914

INDEX OF AUTHORS

muscle experiments, cell permeability,
33, 34; mineral bodies of muscles, 558;
permeability of muscles, 559
Owen-Rees, chyle, 328, 329*

Paal, C., gelatin, 77*, 120; lysalbinic and
protalbinic acids, 126, 137*

Paderi, 258*

Pachon, substances retarding blood co-
agulation, 311, 312; digestion, 460;
activation of trypsinogen, 461*, 473

Pages, blood coagulation, 243; calcium
salts in blood coagulation, 303, 304;
casein, 617; human milk, 633

Paijkull, transudates, 335, 336; pleural
fluid, 339; ascitic fluid, 340; bile con-
stituents, 395

Painter, 129*, 433*

Palladin, 386*

Panek, vasodilation, 475, 476*; ant-
oxyproteic acid, 715; oxyproteic acid,
alloxyproteic acid, 716; uroferric acid,
717; phosphates in urine, 724

Panella, phosphocarnic acid in blood
serum, 260; nucleon in muscle, 550*,
573

Panormoff, sugar of muscles, 553; oval-
bumin, 602; egg-white protein, 604

Pantanelli, 143*

Panum, serum casein, 250; blood con-
stituents, 321; blood corpuscles, 322,
325; quantity of blood, 325; blood
decrease in starvation, 326*, 840; pro-
tein metabolism, 852; gelatin as
protein sparer, 853

Panzer, protein reaction products, 83;
cerebrospinal fluid, 328*, 341; ovarian
colloid, 594; pseudomucin, 595

Parastschuk, protein digestion, 451 ; milk
fat formation, 635, 636

Parcus, cerebrin, homocerebrin, 579;
encephalin, 580

Parke, yolk fat, 600

Parker, action of bile on soap solutions,
513

Parmentier, human milk, 627

Partridge, purine bases, 183; spleen, 351;
uric acid, 667

Pascheles, fate of sulphur in body, 735

Pasehutin, lymph, 331; intestinal juice,
467

Pascucci, blood corpuscles, 265; hemoly-
sis, 266; stromata, 267

Pasqualis, 711*

Pasteur, organized ferments, 8; diges-
tion, 494

Patein, sugar estimation, 763

Patten, hydrolytic cleavage, 78; cysteine,
79*, 148; histidine, 157; lysatine,
151, 443*

Paul, toxicity of mercury, etc., 23; ion-
action, 74; uric acid, 672

Pauli, cataphoresis, 42; colloids, 46; solidi-
fication and melting-point of gelatin,
52; proteids, 94*, 96; gelatin, 119

Pauly, H., histidine, 81*, 157; histidine
reaction, 158; sphygmogenin, 358

Pautz, aqueous humor, 342; lactase in
intestinal juice, 467; paralactic acid in
vitreous humor, 586

Pavy, protein cleavage products, 83;
isomaltose in blood serum, 257; glyco-
gen estimation, 371; carbohydrate
groups in ovalbumin, 375; glycogen;
377; sugar formation in liver, 378,
phlorhizin diabetes, 379; autodigestion,
461 ; protein metabolism, sulphur elimi-
nation, 462*, 566; sugar test, 764

Pawlow, trypsin digestion, 64; bile
secretion, 392; parotid saliva, 429; sali-
va, 430, 435; gastric juice, 437, 439,
441; pepsin, 443*, 444; protein diges-
tion, 451; gastric digestion, 456; secre-
tion of Lieberkuhn's glands, 465; intes-
tinal juice, 466; pancreatic juice, 471;
enterokinase, 472; trypsinogen conver-
sion, 474; quantity of pancreatic
secretion, 476; steapsin, 479: speed of
trypsin digestion, 482; action of bile
on diastatic action of pancreas infusion,
488; putrefaction of proteids, 491;
urea, 649; carbamate elimination, 650;
urea elimination, 651

Peiper, alkalinity of blood, 297

Pekelharing, tryptophane, 131; thrombin,
248, 249; nucleoprotein, 250, 542;
fibrinogen transformation, thrombin,,
303; prothrombin and thrombin, 304;
action of tissue extracts, 307; pepsin,
442, 443, 444

Pemsel, proteids, 94

Penny, ethereal sulphuric acids, 689, 699

Penzoldt, piperidine-glycosuria, 381

Pernow, spleen, 352

Perrin, colloids, 37

Peskind, hemolysis, 266

Petersen, fat in muscle, 570

Petit, 719*

Petrone, blood coagulation, 301

Petrowsky, brain composition, 582

Petry, paracasein, 618

Pettenkofer, bile test, 230, 396; fat
absorption, 534; fat formation, 535;
muscle work, 565, 567, 568; test for
bile acids, 755; determining respiratory
gas exchange, 819; metabolism, 823;
nitrogen equilibrium, 825; fat metab-
olism, 849; influence of work on
metabolism, 868, 874

Pfaff, bile secretion, 392

Pfannenstiel, /9-pseudomucin, 593

Pfaundler, peptic digestion, 131; pro-
teoses, 132; method of precipitating
urine, 647

Pfeffer, copper-ferrocyanide membrane,
27; determining gas pressure, 28

Pfeiffer, human milk analysis, 629, 631;
uric acid, 669, 670; protein fattening,
687*, 860

Pfeiffer, L., phymatorhusin, 793

INDEX OF AUTHORS

915

Pfleiderer, digestion, 446*, 447

Pfliiger, animal oxidases, 3; living pro-
teins, 4; carbon dioxide in lymph, 328;
liver, 360; glycogen, 369, 370; glycogen
estimation, 371; glycogen formation,
372, 373, 374; pancreas diabetes, 383;
glycosuria, 384; internal secretion in
pancreas, 385; sugar formation from
fat, 388*, 391; chorda-saliva, 427;
action of bile on fatty acids, 489; fat
absorption, 512; importance of bile,
soaps and alkali carbonates, 513;
cartilage, 524; fat formation, 534, 535,
536; rigor mortis, 561; metabolism in
muscles, 562; muscle reaction, 563;
protein metabolism, 566; muscle work,
568; gases of milk, 625; urine nitrogen,
646; estimating urea, 655; sugar
estimation, 759*, 761*, 765; blood
gases, 801, 802, 805; lymph gases, 807;
animal oxidations, 808; carbon dioxide
tension in blood, 814, 815, 816, 817;
quantity of oxygen in blood, 818; res-
piratory gas exchange, 819; nitrogen-
carbon relation, 827 ; results of absence
of fat from food, 847; protein metab-
olism, 849; nutritive requirements,
850; metabolism, 851; influence of
external temperature on metabolism,
871; protein metabolism, 875

Pfliiger-Nerking, glycogen, 371

Phisalix, bufonin, bufotenin, 797

Picard, urea in vitreous humor, 586

Piccard, 109*

Piccolo, haematoidin, 290 ; lutein, 592

Pick, proteoses, 130, 132; glucoproteose,
protoproteose, 133; separating pro-
teoses and peptones, 137; serglobulins,
251; protozym as a cause of coagu-
lation, 311; glycogen, 377; adrenalin
glycosuria, 385; blood coagulation, 475

Pick, A., 446*

Pickering, intravascular coagulation, 85*,
310

Pickhardt, cartilage, 337*, 525

Picton, colloids, 44; colloidal precipita-
tion, 47

Pierallini, fate of oxalates in body, 733

Pierce, action of NaFl on enzymotic
cleavage, 74

Piettre, stromata, oxyhaemoglobin, 267;
haemin, 285

Pigeand, transudates, 336

Piloty, glucuronic acid, 215, 713; haBmatin.
284

Pilz, O., protokyrins, 136

Pilzecker, bile, 415

Pimenow, 438*

Pincussohn, colloids, 45

Pineles, adrenalin glycosuria, 385

Pinkus, putrefactive products, 81; oval-
bumin, 94*, 602, 603

Piontkowski, gastric juice, 439

Piria, tyrosine test, 151

Planer, gases of stomach, 461

Plaut, r-alanine in urine, 717

Plattner, crystallized bile, 396

Playfair, 874

Plimmer, nucleoproteins, 171; lactase
formation in intestine, 473; ovovitellin,
597; casein, 598*, 619

Plosz, blood corpuscles, 267; liver pro-
teins, 361, 362; uromelanins, urorubrin,
703; proteid in urine, 744; nutritive
value of proteoses and peptones, 854

Poda, 499*, 826*

Poduschka, uric acid, 670; allantoin, 682

Poehl, putrefaction, 495; spermine, 590

Pohl, oxidases, 11; serglobulin, 223*, 253;
liver "organ plasma," 361; urea, 649;
uric acid, 670; allantoin, 682; fate of
oxalic acid in the body, 733; fate of
succinic and malic acids in body, 734,
fate of benzene derivatives in the body,
737; estimating globulins and albu-
mins, 750

Poleck, mineral bodies of yolk, 600;
mineral bodies of egg white, 604

Polimanti, fat formation, 534

Politis, 855*

Pollak, glutinase, 481; elimination of
sulphocyanide in urine, 735

Pollitzer, nutritive value of proteoses, 854

Polowzowa, rapidity of digestion, 457;
cleavage of starch, dextrins, and disac-
charides, 492

Pommerehne, creatinine, 658

Ponfick, quantity of blood, 325, 326*

Ponomarew, secretion of Brunner's glands,
465

Pons, chondroitinsulphuric acid in urine,
717

Popel, 321*

Popielski, protozym as a cause of coagu-
lation, 311; parotid saliva, 429; saliva,
430; enterokinase, 472; pancreatic
juice excitants, secretin, vasodilation,
475

Popper, H., pancreatic secretion, 471 ;
pancreatic rennin, 487; tests for bile
pigments, 757

Popowsky, tryptophane, 154, 155*

Porcher, indican elimination, 637*, 691*,
692; indols, 693; indican, 694; indoxyl-
sulphuric acid, 695; skatol red, 696;
uroerythrin, 710; fate of phthalic acid
in the body, 737

Porges, serglobulins, 251

Porteret, glycogen formation, 372

Portier, lactase formation, 386*, 509

Posner, mucin, 166, 167; pepsin-hydro-
chloric acid digestion, 449; protagon,
577; semen constituents, 589; proteid
in urine, 590*, 744

Posselt, 122*

Posternak, musculamine, 550; phytin, 551

Pottevin, mono- and triolein synthesis,
65; splitting of fats, 219*, 227; pan-
creas action, 479

Pouchet, carmine, 548; purine bases, 676;

916

INDEX OF AUTHORS

ptomaines in urine, 718; bodies in path-
ological lungs, 821

Poulson, isocreatinine, 346

Pozerski, tryptic digestion, 68; prosecre-
tin, 475; tryptic digestion, 484

Pozzi-Escot, 14*, 15*

Prausnitz, nitrogen excretion in starva-
tion, 379*, 499*, 826*, 837

Pregl, koilin, 106*, 112*, 114*, 115;
hsemochromogen, 124*, 281, 282; de-
hydrocholan, 396; cholic acid, 401;
desoxycholic acid, 403; secretion of
Lieberkuhn's glands, 465; pseudomu-
cin, 595; ovalbumin, 603; polypeptides
in urine, 717; urine, 732

Presch, sulphur compounds in urine, 714;
hyposulphites in urine, 715

Preti, enzymotic processes, 17*, 73

Preusse, pyrocatechinsulphuric acid, 689*,
690; hydroquinone, 691; oxidation of
benzene, 738; mercapturic acids, 743

Prevost, bile absorption, 517, 651*

Preyer, globin, 281, 608*

Preysz, phosphates in urine, 724

Pribram, potassium and sodium in urine,
106*, 727; fate of benzene derivatives
in the body, 737; melanins, 794

Pringle, histone-peptone, 109; prota-
mines, 110; protein absorption, 502;
influence of leucocytes on proteoses,
505; influence of leucocytes in protein
assimilation, 507

Prochownick, amniotic fluid, 609

Profitlich, liver constituents, 367

Proscher, bilirubin, 407; human milk,
626*, 633

Prutz, indican elimination, 692

Prym, spleen, 353; pancreatic juice, 472;
activation of trypsinogen, 473

Przibram, muscle proteins, 545

Pugliese, retarding blood coagulation,
306; gastric juice, 453

Pupkin, 297*

Quevenne, lymph, 329; human milk, 632
Quincke, colloids, 45; haematoidin, 290;

poikilocytosis, 324; milk fat globules,

613 ' .
Quinquand, urea in blood, 317, 319;

muscle metabolism, 563; fate of

volatile fatty acids in body, 733
Quinton, 35*

Raaschow, guanylic acid, 179*, 180
Rachford, amylopsin, 478; trypsin diges-
tion, 483; action of bile on steapsinogen,
489

Radenhausen, milk globules, 613
Radziejewski, fat absorption, 533
Radzikowski, gastric juice, 438
Rahlmann, urea in vitreous humor, 586
Raikow, sulphur in proteins, 79
Raineri, enzymes in placenta, 608
Ramsden, adsorption, 51; proteid modi-
fication, 96; coagulated proteins, 106

Rane, pigments in blood serum, 260

Ranke, quantity of blood, 326; bile
secretion, 392

Ranke, J., 665*

Ranvier, potassium in cells, 22

Ransom, haemolysis, 266; cholesterin,
424

Raper, peptones, 136

Rapp, 9*

Raske, K., d-alanine, 139; cystine, 147

Rauchwerger, cholesterin, 421

Raudnitz, casein, 611*, 616, 618*

Reach, pepsin, 442; muscle work, 569

Reale, oxalic acid, 680; glycosuria, 384;
sulphur compounds in urine, 714

Reemlin, artificial digestion with trypsin,
492

Reese, r-alanine and glycocoll in urine,
717

Regnault, absorption of oxygen by
the skin, 800; determining respiratory
gas exchange, 819, 825*

Reh, A., proteid reactions, 104; lymph
glands, 347; casein, 619

Rehn, fibrinogen, 244, 245*

Reich, estimating ammonia in urine, 728,
729

Reich-Herzberge, gelatin cleavage, 485

Reichel, enzymes, 64, 617*

Reichert-Meissl, volatile fatty acids equiv-
alent, 231

Reid, osmotic pressure, 39; sugar absorp-
tion, 509

Reinbold, sugar test, 209, 762- tryptic
digestion, 284

Reinecke, fatty tissue, 532

Reiset, absorption of oxygen by the sk.n,
800; determining respiratory gas ex-
change, 819, 825*

Reiss, globulins, 252

Reitzenstein, purine bases, 676

Rekowski, elimination of phenols 742

Ren wall, calcium and magnesium in
urine, 729

de Renzi, glycosuria, 384

Retinger, hsemopyrrol, 288

Rettger, activation of trypsinogen, 473

Reuss, transudates and exudates, 336

de Rey-Pailhade, oxidases, 11

Reye, W., fibrinogen, 246

Reynold, acetone test, 776

von Rhorer, urine, 643

Ribaut, 14*

Richards, elastin, elastoses, 115, 116;
elastin, 117; 161*, 430*, 431*

Richet, Ch., gastric juice, 437*, 441; fat
formation, 537; urea, 648; uric acid,
670; thalassin, 797; determining res-
piratory gas exchange, 820; metab-
olism, 865

Richter, osmotic pressure, 36; lysine in
blood serum, 260; liver autolysis, 365,
652*

Ricker, 626*

Riecke, 687*

INDEX OF AUTHORS

917

Rieder, nitrogen excretion, 825

Riegel, gastric juice, 440; triglycerides
in butter, 614

Riegler, haemochromogen, 282

Riehl, reducing property of lungs, 821

Riess, oxyacids in urine, 697; paralactic
acid, 710

Riesser, arginine, 159

Ringer, calcium salts in blood coagulation,
303, 6.43*

Ringstedt, urine, 641

Ritter, glycogen, 377; taurocholic acid
in bile, 379*, 414; pigmentary acholia,
415; cholesterin stones, 420; choles-
terin, 425, 512*

Ritthausen, hydrolytic cleavage, 80; leu-
cmimide, 93*, 142 ; milk carbohydrate,
622; method of determining proteins
in milk, 623

Riva, urochrome, 704; uroerythrin, 709;
haematoporphyrin, 754

Rivalta, transudates, 335, 336*

Roaf, H. E., cell-electrolytes, 21; adsorp-
ates, 34; osmotic pressure, 37, 38, 39;
adsorption, 50; preparing lecithin, 238

Roberts, pancreatic rennin, 487; esti-
mating proteid, 750; estimating sugar,
766

Robertson, T. B., casein, 21*, 615, 616

Roch, proteid test, 746

Rockwood, action of bile on fatty acids,
489; reaction of intestinal contents,
497; protein absorption, 502; fat
absorption, 512; solvent action of bile
on fatty acids, 513; uric acid, 667;
phosphocarnic acid in urine, 718

Rod en, antitryptic action, 68; antiren-
nins, 450

Rodier, fibrin in blood, 320

Roeder, uracil, thymine, 190

Roger, liver, 360; ptyalin, 432; saliva,
433

Rogozinsky, peptone, 136

Rohde, proteid detection, 100

Rohmann, oxidases, 11, 12; leucine, 142;
isomaltose, 219; glycolysis, diastase in
blood, 258; fat in blood, 318; lymph,
328; glycogen formation, 372; glyco-
gen, 377; ptyalin, 432; intestinal
secretion, 466; antiputrid action of
bile, 495; influence of fat on protein
digestion, 496: lactase formation, sugar
absorption, 509, 510; effect of sugar in
intestines, 511 ; fat absorption, 514, 515;
muscle reaction, 536; casein, 560, 616;
sebum, 723*, 795; coccygeal secretion,
pennacerin, 797; carbon dioxide elimi-
nated by the skin, 800; metabolism,
826; influence of phosphates in metab-
olism, 845; metabolism, 847

von Rohrer, proteids, 94

Rohrig, metabolism in muscles, 562

von Rokitansky, volatile fatty acids in
urine, 710

Rona, proteid, 96; proteid test, 101;

histone, 108; gelatin, 119; proteoses,
133; sugar in blood serum, 257; sugar
in blood corpuscles, 314; permeability
of blood corpuscles, 316; secretion of
Brunner's glands, 317*, 465; protein
absorption, 502; protein synthesis,
506; homogentisic acid, 698, 699;
gelatin as a protein sparer, 854

Ronchi, carbon dioxide eliminated by the
skin, 800

Roos, iodothyrin, 356; phenylhydrazine
test, 723*, 762; sugar test, 763

Roosen, uric acid, 663, 664*

Rorive, sugar reaction, 211

Rosa, respiratory gas exchange, 820

Rose, C., tooth degeneration, 532, 617*

Rosemann, gastric juice, 442; pyloric
secretion, 454; alcohol in metabolism,
637*, 852*, 863

Rosenbach, test for biliary pigments, 756;
urine test, 780

Rosenbaum, 387*

Rosenberg, B., 380*

Rosenberg, S., glycosuria, 384; olive oil
as a cholagogue, 393; antiputrid action
of bile, 472*, 495; pancreas destruc-
tion, 496*, 508; starch absorption, 511;
fat absorption, 515

Rosenberger, inosite fermentation, 551;
heptose in urine, 770

Rosenblatt, 438*

Rosenfeld, sugar in blood serum, 257;
haamatin, 283, 286; liver fat, 363;
digestion, 459; fat formation and
absorption, 534; uric acid, 665; indican
elimination, 692; volatile fatty acids
in urine, 710; phenylhydrazine test,
761; acetone elimination, 773; 855*

Rosenheim, proteid detection, 99; cere-
brospinal fluid, 342; muscle metab-
olism, 563; protagon, 577, 578; catab-
olism limits, 858

Rosenstein, A., fat in chyle, 329; chyle,
330, 331; glycosuria, 381; protein
assimilation, 503; sugar absorption,
510; passage of fat into chyle, 513;
fat absorption, 514

Rosenthal, respiratory gas exchange, 819

Rosenthaler, asymmetrical synthesis, 65

Rosenqvist, sugar formation from fat,
391

Rosin, detecting levulose, 211; indican,
694; skatol chromogen, 696; levulose
in urine, 768, 780*

Rosing, E., protein oxidation, 6*, 14

Rossi, 342*

Rost, elimination of gallic and tannic
acids, 742; effect of salts on metab-
olism, 862

Rostoski, proteid, 749

Roth, lymph formation, 333

Rothberger, urea formation, 361*, 650

Rothera, cystine, 146; acetone test, 776

Rothmann, creatine, 547

Rotmann, 337*

918

INDEX OF AUTHORS

Rotschky, hsematoporphyrin, 287

Rouiller, 154*

Roux, starch, 220

Rovida, hyaline substance, 267

Rovighi, putrefaction, 495, 688*

Rowland, spleen, 9*, 351 ; muscle enzymes,
545

Rowntree, guanylic acid, 178

Rubbrecht, blood serum, 262

Rubner, hydrolytic cleavage, 79; sugar
test, 208; food utilization, nitrogen
loss, 507; sugar in intestine, 508*;
511; fat absorption, 514; fat formation,
538; human milk, 628, 630; sugar test,
762; metabolism, 823; nitrogen excre-
tion, 825; energy theory of metab-
olism, calorific value of foods, 829;
protein heat value, 830; physiological
food combustion, 833, 834, physiological
availability, 835; decomposition of fat
during starvation, 838; protein mini-
mum, specific dynamic action, 859;
metabolism, 864, 865; metabolism in
old and young, 866; influence of
sleep on metabolism, 869; influence of
temperature on metabolism, 870; in-
fluence of food ingestion on metabolism,
871; dynamic action of foods, 872;
food requirements, 873

Rubow, 557*

Riidel, uric acid, 672

Rudinger, adrenalin glycosuria, 384

Ruff, carbohydrates, 196; hydrazones, 199

Ruge, marsh-gas in intestine, 493

Rulot, fibrin, fibrinolysis, 247

Humpf, sugar formation from fat, 391;
ethereal sulphuric acids, 689, 728*

Ruppel, protagon, 576; human milk, 627;
vernix caseosa, 796

Russel, milk, 620

Russo, chitin, 791

Rutherford, sulphur in hair, 790

Rywosch, blood corpuscles, 258*, 265

Saarbach, methsBmoglobin, 276

Sabanejew, 137*

Sabbatani, calcium salts in blood coagu-
lation, 303

Saccharjin, 314*

Sachs, Fr., nucleases, 176; pentose tests,
203; gastric secretion, 453; nuclease,
469; tryptic digestion, 484; action of
trypsin on nucleic acids, 485

Sachsse, sugar test, 208

Sackur, casein, 615, 616

Sadikoff, gelatins, 117

Sagelmann, rapidity of digestion, 457

Sahli, haemometer, 292; determining
acidity of gastric juice, 465; 764*

Saiki, magnesium in muscle, 573; para-
lactic acid, 710, 711*

Saillet, hsematoporphyrin, 287; uro-
bilinogen, 703, 708; urobilin, 705, 706,
707, 709; urospectrin, haematopor-
phyrin, 754

Sainsbury, calcium salts in blood coagu-
lation, 303

Saint Martin, 278*

Saint Pierre, quantity of oxygen in blood,
818

Saito, lactic acid in blood, 318; lactic acid
elimination, 554

Salaskin, peptic digestion, 131; anti-
albumid, 134; leucinimide, 142; am-
monia in blood, 297*, 317; erepsin,
469; urea, 505*, 648, 649; urea elimi-
nation, 651 ; urea, 652 ; estimating urea,
656, 727*

Salkowski, oxidases, 11; enzymes, 17;
putrefactive products, 81; proteid
reactions, pseudonuclein, 96*, 100*,
104; pentoses, 129*, 201, 202; test for
pentoses, 203; glucuronic acid, 215;
cerebrospinal fluid, 341, 342; synovial
fluid, 343; ferratin, 362; glycogen, 372;
cholesterin reaction, 422; saliva, 435;
trypsin, 481; putrefaction of proteins,
492; adipocere, 495*, 534; meat con-
stituents, 572; ovomucoid, 603; casein,
618, 619; urea, 648, 649; estimating
urea, 651*, 657; creatinine reaction,
661; uric acid, 670; estimating purine
bases, 678, 679; oxalic acid, 680, 681;
allantoin, 681; hippuric acid, 684;
phenaceturic acid, 687; ethereal sul-
phuric acid, 688; indican, 694; skatol
formation, 695; indolacetic acid, 696;
urobilin, 707*, 709; volatile fatty
acids in urine, 710; carbohydrates in
urine, 711; sulphur combinations in
urine, 714; sulphuretted hydrogen in
urine, 715; determining sulphuric acid
in urine, 726; phenylacetic acid elimi-
nation, 737; fate of oxalates in the
body, 733; fate of ethylsulphates in
the body, 735; elimination of taurine
in urine, 736; uraminobenzoic acid in
the urine, 740; detecting proteoses,
748; blood test, 753; hsematoporphy-
rin, 754; phenylhydrazine test, 761*,
762; pentoses in urine, 769; aceto-
acetic acid test, 778; urine fermenta-
tion, 782; effect of water on metab-
olism, 862

Salkowski-Ludwig, estimating uric acid,
674

Salomon, xanthine bodies, 182; pus cells,
295; carbohydrate formation, 390;
purine bases, 676; heteroxanthine,
methylxanthine, paraxanthine, episar-
kine, 677; epiguanine, 678; acetone
bodies in urine, 552

Salomone, 78*

Salomonsen, urochrome, 703, 704

Salvioli, fate of peptone in intestine, 504

Samec, leucine, 140

Sammis, 877*

Samuely, uric acid, 107*, 670; r-alanine
and glycocoll in urine, 717;- elimination
of cystine in urine, 736; melanins, 794

INDEX OF AUTHORS

919

Sandberg, digestion, 459

Sandemyer, pancreas extirpation, 383,
508; fat absorption, 515, 517; metab-
olism, 826

Sandstrom, thyroid, 355

Sasaki, adialyzable bodes in urine, 717

Satta, acetone elimination, 647*, 773

Sauer, curare-glycosuria, 381

Savare, protein and enzymes in placenta,
608; adialyzable bodies in urine, 717

Sawitsch, pancreatic juice, 473; pancrea-
tic secretion activator, 476; solids in
pancreatic juice, 477

Saw j alow, antialbumid, 134; plastein
135; proteid digestion, 451

Saxl, autolysis, 18; liver autolysis, 365;
muscle stroma, 542

Schardinger, levolactic acid, 553

Schade, 200*

Schafer, thrombin, 303; adrenal pressure
raising action, 357

Schaffer, ammonia in urine, 78*, 729,
780*, 781*

Schaffidi, ferratin, 362; iron in liver, 366

Schalfejeff, hsemin, 285, 286

Scheele, 430*

Scheermesser, gelatin, 120; peptic-gluten
peptone, 135

Scheibe, human milk, 626*, 630

•Scheibler, saccharose, 218

Schemiakine, pyloric secretion, 454; gas-
tric digestion, 455

Schenck, Fr., sugar in blood, 316, 317*,
378; oxalic acid, 680

Schenck, M., protein oxidation products,
82; martamic acid, 178

Schepowalnikoff, enterokinase, 472

Scherer, lymph, 329; inosite, 551; metal-
bumin, paralbumin, 594; metabolism
in nurslings, 866

Scheuer, gastric juice, 440

Scheunert, gastric digestion, 455*, 458;
diastatic enzyme in Brunner's glands,
465; pancreatic calculi, 487

Schierbeck, ptyalin, 432; gases of stom-
ach, 461; tryptic digestion, 483; feces,
498; carbon dioxide eliminated by the
skin, 800

Schiff, spleen, 353; liver, 360; glycogen,
377; bile secretion, 394; cholesterin
reaction, 423; pepsin test, 446; tryp-
sinogen activation, 453*, 473; bile
absorption, 517; urea reaction, 653

Schiff, H., proteid detection, 24*, 77*
100

Schindler, thymus, 349

Schittenhelm, xanthineoxidase, 13; nucle-
ic acids, 176; purine bases, 183; aden-
ine, 188; fluoride plasma, 305; spleen,
310*, 351, 354; urea, 498*, 648; uric
acid, 665, 666, 667, 668, 670; glycocoll
in urine, 717, 718*; ammonia in urine,
728; estimating ammonia in urine,
729; pyridine elimination, 743 ; 780*

Schlatter, 460*

Schlesinger, 432*

Schilep, estimating acetone, 778

Schloessing, colloids, 44

Schlosing, ammonia in urine, 728

Schlossberger, human milk, 632, 637

Schlosse, 852*

Schlossmann, determining casein in milk,
624; milk, 626; human milk, 630

Schmey, mineral bodies of muscles, 55S,
572*

Schmid, purine bases estimation, 679

Schmidt, buccal mucus, 50*, 428; influ-
ence of bile on digestion, 434*, 476*,
496; fat absorption, 514; isocreatinine,
546; purine bases, 676; acetone bodies
in urine, 772

Schmidt, Ad., putrefaction of proteids,
491, 500*

Schmidt, Alex., thrombin, 249; prothrom-
bin, 248, 304; fibrinoplastic substance,
250; fibrinogen in blood corpuscles,
268; leucocytes, 294; cytoglobin and
praeglobulin, 295; blood coagulation,
301; constituents of leucocytes, zymop-
lastic substances, cytoglobin preglobin,
action of cell constituents on blood
coagulation, 302; fibrinogen transfor-
mation, 303; retarding blood coagu-
lation, 306; serum reactivation, 307;
zymoplastic substances, action of tissue
extracts, 307; blood coagulation, 308;
intra vascular coagulation, 310; cytin,
349; spermine, 590; blood gases, 803

Schmidt, C., blood analysis, 314; lymph,
330; protein in tissue fluids, 335; gas-
tric juice, 441; solids hi pancreatic
juice, 477; bone, 530

Schmidt, C. H. L., protein cleavages, 81

Schmidt, C. W., mineral bodies in lungs,
821

Schmidt-Mulheim, blood coagulation, 243 ;
protein assimilation, 503; protein ab-
sorption, 852

Schmidt-Nielson, chymosin, 59; casein,
616*, 617; paracasein, 618

Schmiedeberg, active oxygen, 8; pro-
tamines, 93*, 109*, 110; desaminp-
albuminic acid, 126, 167*; nucleic
acid, 175; thymonucleic acid, 177;
nucleotin, thymonucleic acids, 178;
nucleic acids, 180; thymine, 190;
glucuronic acid, 215; ferratin, 362;
chondroitin-sulphuric acid, chondrosin,
522 ; preparing chondroitin-sulphuric
acid, 523; urea, 649; hippuric acid,
686; benzoic acid, 687; indol, 695,
camphoglucuronic acid, 689*, 743;
sarcomelanin, melanoidic acids, 793

Schmitz, putrefaction, 495, 688*

Schneider, tyrosinases, 12; blood cor-
puscles, 314; saliva, 430; melanins,
902*, 794

Schoffer, leuctne, 140

Scholz, indican elimination, 692

Schonbein, nitrates in urine, 726, 727*

920

INDEX OF AUTHORS

Schondorff, urea in blood, 317; glycogen
formation, 356*, 383; glycogen, 369,
553; urea in muscles, 546; urea, 645;
estimating urea, 657; sugar test, 666*,
759; sugar estimation, 763; protein
destruction, 850*, 851

Schone, pentoses, 202

Schottelius, digestion, 494

Schotten, fellic acid, 404; hippuric acid,
684; volatile fatty acids in urine, 710;
damaluric and damolic acid in urine,
719; fate of volatile fatty acids in body,
733; fate of amino-acids in the body, 737

Schoubenko, hydrolytic cleavage, 79

Schoumowa, action of gastric juice on
toxalbumins, 461

Schoumow-Simanowski, pepsin, 443; gas-
tric juice, 453

Schreiber, 666*

Schreiner, crystals in semen, 590

Schreuer, meat nitrogen, 572; calorific
food values, 835; protein metabolism,
852; protein fattening, 860

Schrodt, bone analysis, 529

von Schroder, urea in blood, 317; uric
acid in blood, 318; urea, 645*, 649;
uric acid, 651*, 668

Schroter, nucleic acids, 176

Schrotter, crystalline proteoses, 129, 130;
proteoses and peptones, 137; oxgyen
tension of blood, 812; carbon dioxide
tension in blood, 815

Schiile, 431*

Schulz, oxyprotein, 82; globulin, 94*,
107; valine, 139; galactosamine, 164*,
215; ceralbumins, 253; globulin, 281;
hsematoporphyrin, 289; protein catab-
olism in starvation, 838

Schulz, Fr., sulphur in proteins, 79, 97*

Schulz, H., connective tissue, 520

Schulz, N., fat in blood, 83*, 321

Schulze, isocholesterin, 22*, 423; fatty
tissue, 532, 617*

Schulze, C., 432*

Schulze, E., hydrolytic cleavage, 80;
leucine, 141; phenylalanine, 150; tyro-
sine, 151; histidine, 157; arginine, 158;
lysine, 160; hemicelluloses, 224; leci-
thins, 237

Schultz, compound protein, 170

Schultze, B., fat formation, 537

Schultze, E., urine nitrogen, 646

Schultze, H., colloids, 36*, 43, 44

Schultzen, diabetes, 382; urea, 648, 649;
oxyacids in urine, 697; paralactic acid,
710; fate of acid amides in the body,
734; fate of amino-acids in the body,
735; oxidation of benzene and toluene,
738; oxidation of xylene into toluic
acid, 739

Schumburg, parachymosin, 450; oxygen
heat value, 832,867*

Schumm, O., fat .in chyle, 324*, 329;
spleen, 351 ; sugar formation from fat,
352*, 391, 753*

Schunck, blood pigment, 269; luteins,
600; uromelanins, 703

Schur, uric acid, 661, 667, 676*

Schurig, 366*

Schuurmanns-Stekhoven, 275*

Schuster, utilization of foods, 508, 880

Schutz, E., rule of enzyme action, 64;
HC1 in gastric juice, pepsin test, 446;
digestion products, 447; gastric diges-
tion, 455; bile salts, 479*, 489, 711*

Schutz, J., pepsin test, 446; digestion,
447

Schiitz-Borissow's rule for formation of
digestion products, 447 ; rule of ferment
action, 479

Schiitze, precipitines in blood, 259

Schiitzenberger, protein decomposition
products, 80; proteins, 85*, 128

Schwalbe, hsemerythrin, 292; blood coag-
ulation, 301; fat formation, 363*, 534
| Schwann, biliary fistula operation, 392;
influence of bile in digestion, 496

Schwarz, preparing dextrose, 209; choline,.
239; iodothyrine, 354*, 356; secretin
476; acetonuria, 774

Schwarz, C., choline, 239

Schwarz, H., elastin, 115, 116, 117

Schwarz, L., HC1 secretion, 453

Schwarz, O., antipepsin, 462

Schwarz, S., 126*

Schwarzchild, trypsin, 480; action of
trypsin on acid-amides and polypep-
tides, 485

Schweitzer, cellulose reagent, 224
| Schwinge, haemoglobin, 320, 321*

Scofield, biliverdin, 409

Scott, nucleoproteins, 172

Sebauer, bone, 530

Sebelien, milk, 129*, 611; casein, 618;
lactoglobulin, 619; lactalbumin, 620;
lactose, 622; method of determining
proteins in milk, 623, 624, 625*

Seegen, liver constituents, 257*, 365:
glycogen, 377; sugar formation in
liver, 378; ptyalin, 383*, 431; muscle
metabolism, 432*, 563; muscle work,
568; respiratory gas exchange, 819;
effect of water on metabolism, 825*,
862

Seelig, 495*

Seeman, protein oxidation products, 82,
83; gelatin oxidation products, 118;
oxidation products of nucleic acids, 178;
erepsin, 469; putrefaction of proteins,
491 ; amino-acids and proteoses in blood,
504; ovomucoid, 604; creatinine, 547,
659

Segale, 22*

Seitz, protein in liver, 360*, 368 ,

Seliwanoff, detecting levulose, 211

Selmi, ptomaines, 24

Seltrenny, gelatin, 118

Semmer, fibrinogen in blood corpuscles,
268

Senkowski, cholic acid, 401

INDEX OF AUTHORS

921

Senator, 693*

Senter, 62*, 63

Seo, uric acid, 673

Sertoli, blood gases, 806

Sestini, L., uric acid, 664

Sestini, F., uric acid, 664

Setschenow, blood gases, 802, 804, 806

Sezelkow, blood gases, 802

Shepherd, hippuric acid, 684

Sherer, inosite test, 552

Shiile, gastric juice, 440

Shur, uric acid, 665

Siau, isomaltose in blood serum, 257;
phlorhizin action on kidney, 379

Sieber, N., hydrolytic cleavage, 78; gly-
colysis, 79*; 258; diabetes, 273*, 283;
haemin, 285, 286; hsematoporphyrin,
287, 288; bile pigments, 418; gastric
juice, 441, 442; pepsin, 443; pepsin
digestion, 452; action of gastric juice
on toxalbumins, 461 ; putrefaction in
intestines, 490; human milk, 630;
urorosein, 697*, 703; urobilinoids ;
705; fate of metanitrobenzaldehyde in
the body, 741; hippomelanin, 793;
reducing property of the lungs, 820,
821*

Siedentopf, colloids, 40

Siegert, human fat, 533

Siegfried, hydrolytic cleavage, 80; kyrin,
glutokyrin, protokyrins, caseinokyrin,
136; glutamic acid, 145; ammo-acids,
160*, 162; protein cleavage, 163; re-
ticulin, 120, 121; peptone cleavage
products, 135; antipeptone, 131; pro-
teoses, 132; pseudohaemoglobin, 276;
reticulin, 364*, 519; nucleon, 550;
sarcolactic acid, 556; phosphocarnic
acid, 550, 564; muscle energy, 565*,
569; milk, 620; human milk, 629, 806

von Siewert, haemin, 286

Sikes, human milk, 630

Silbergleit, glycosuria, 384

Silbermann, isoserine, 144; oxyamino-
succinic acid, 146; bile pigments, 419

Simacek, 555, 386*

Simon, ptyalin, 432; proteoses and pep-
tones in lungs, 625*, 821

Simon, O., pus cells, 18*, 346

de Sinety, lactose in urine, 768

Siven, uric acid, 665, 667; purine bases,
6^6; protein metabolism, 844; nitrog-
enous equilibrium, 858, 875

Siwertzow, lecithin, 235

Sjoqvist, pepsin, 63; proteins, 94; deter-
mining activity of gastric juice, 137*;
465; urine nitrogen, 646, 652*

Skita, serine, 143

Skraup, proteins, 77*, 78; hydrolytic
cleavage, 80; glutinic acid, 118; casein-
okyrin, 136; glutamic acid, 145; oxy-
aminosuccinic acid, 146; dioxydiami-
nosuberic acid, 161

Slator, 200*

Slavu, Z-adrenalin, 358

Slosse, urea elimination, 651

Slowtzoff, pentoses, 201; metals in liver,
367; semen analysis, 589; paracresin,
618, 826*, 868*

van Slyke, D., casein, 615, 616*

van Slyke, L., casein, 615, 616*

Smale, uric acid, 672

Smirnow, fate of .metanitrobenzaldehyde
in the body, 741

Smith, ptyalin, 432; saliva, 433; per-
spiration, 798, 799; oxygen tension in
blood, 812

Smith, Herbert, bone cells, 526

Smith, Lorraine, quantity of blood, 325

Smith, W., sulphur in urine, 714; oxida-
tion of sulphur in the body, 736

Smolenski, sugar reaction, 769

Socin, 375*

Socoloff, 412*

Soetber, phosphates in urine, 724

Soldner, casein, 614*, 016, 617; mineral
bodies in milk, determining proteins
in milk, 623; determining lactose in
milk, 624; mineral bodies of milk, 625;
human milk analysis, 629; human milk,
630, 631, 633

Solera, saliva, 431

Solley, gelatin, 117*, 119

Sollmann, T., muscle plasma, 329*, 415*,
539*, 540; muscle proteins, 544

Solomon, lactic acid in blood, 318, 649*,
680*

Sommer, 363*

Sommerfeld, mucin in bile, 413; gastric
juice, 441

Sonden, determining respiratory gas
exchange, 479*, 820; metabolism in old
and young, 866, 867; metabolism during
sleep, 878

Sorby, oorodein, oocyan, 604

Sorensen, glycocoll, 138; phenylalanine,
150; proline, 152; ornithine, 159;
protein hydrolysis, 162; ammo-acids
in urine, 718

Soulie, adrenalin, 359

Sourdat, human milk, 631

Southgate, trypsin digestion, 483

Soxhlet, preparing dextrose, 209; galac-
tose, 210; maltose, 219; fat formation,
537; milk plasma, 614; casein, 617;
milk fat formation, 636; sugar test,
764

Spampani, milk fat formation, 636

Spangaro, blood coagulation, 242

Speck, determining respiratory gas ex-
change, 820; influence of work on me-
tabolism, 868; influence of light and
external temperature on metabolism.
870

Spiegler, proteid test, 746; melanin, 793,.
794 ; results of lack of water, 842

Spiro, colloids, 46, 51; gelatin solutions,
52; enzymes, 64; proteids, 94; proteid
test, 101; gelatin, 118; glycocoll, 138;
amino-sugars, 213; serglobulins, 251;

922

INDEX OF AUTHORS

retarding blood coagulation, 305*, 306,
307; substances retarding blood coagu-
lation, protozyme as a cause of coagu-
lation, 311; blood coagulation, 475;
lactic acid in muscles, 564; casein, 617;
acetone bodies in urine, 775

Spitzer, oxidases, xanthine-oxidase, 11,
12; nucleoprotein, 13; glycolysis, 258;
oxidases active in glycolysis, 362*, 386;
uric acid, 667

Spriggs, pepsin test, 446

Spring, 36*

Staal, urine, 641; skatol red, 696

Stade, 64*, 452*

Stadeler, bilifuscin, biliprasin, bilihumin,
410

Stadelmann, adrenals, 153*, 290*, 357;
bile secretion, 393; cholagogues, 394;
bilirubin, 413 ; bile acids, 416; bile
pigments, 418, 419; putrefaction in
intestines, 497; bile absorption, 652*,
517; ammonia in urine, 727; blood
gases, 748*, 807

Stadthagen, adenine, 24*, 187; xantho-
creatinine, 663; sulphur compounds in
urine, 714; cadaverine in urine, 719,
780*

Staehelin, transudates, 335

Stahl, 642*

Stanek, choline, 240

Stangassinger, creatine, 547; creatinine,
659, 663

Stangc, 226*

Starke, J., globulins, 96*, 102, 103

Starke, K., seralbumin, 254, 255

Starkenstein, inosite, 551; inosite in urine,
552*, 771

Starling, osmotic pressure, 39; lymph
secretion, 61*, 332; lymph formation,
333; hormones, 385; kinase and secre-
tin in intestine, enterokinase, 468, 472;
erepsin, 469; action of trypsin on
trypsinogen, 474; pancreatic juice ex-
citants, secretin, prosecretin, 475; en-
zyme in pancreas, 481

Stassano, enterokinase, 472; pepsinogen
conversion, 474

Stavenhagen, 9*

St. Bugarsky, blood corpuscles and serum,
313

Steel, estimating ammonia in urine, 729*

Steensma, modified Giinzburg's test for
HC1 in gastric juice, 156*, 463

Steiff, 688*

Steiger, 158*

Steil, fat in muscle, 570

Stein, cholesterin, 420

Steinitz, urine, 723*, 732; lactose in urine,
768; metabolism, 826; nitrogen-carbon
relation, 827; metabolism, 847

Stejskal, 317*

Stenger, oxyhaemoglobin, 274; methaemo-
globin, 277; carbon-monoxide haemo-
globin, 279; haemochromogen, 282;
lutein, 600

Stenson, test, 560

Stepanek, enzyme in egg yolk, 597

Stern, phosphatides, 55*, 233; blood
pigments in bile, 415; bile pigments,
417; yolk phosphatides, 590*, 599,
688*

Steudel, mucin, 165; nucleic acid, nucleo-
tides, 175; thymonucleic acids, 176,
177; nucleotin, thymonucleic acids,
178; cytosine, 179*, 189; uracil, 190;
glucosamine, 214

Stewart, muscle-plasma, 448*, 539*, 540;
muscle proteins, 544

Steyrer, musculin, 545; musculin in
muscle juice, 566; urine, 643

Sticker, buccal saliva, 430 ; saliva, 434

Stiles, 379*, 433*

Stintzing, rigor mortis, 561

St. Kozlowski, 640*

St. Mostowski, haemopyrrol, 288

Stockman, 566*

Stoeltzner, W., 529*

Stoelzner, H., 529*

StofTregren, A., 748*

Stohmann, calorific value of foods, 829

Stokes, haemoglobin, 275; reduced haema-
tin, 281 ; haemochromogen, 282

Stoklasa, sugar fermentation, 200; glycol-
ysis, alcoholic fermentation in animal
tissues, 236*, 386; enzymes in pan-
creatic juice, 387*, 487; lactic acid, 555;
milk enzyme, 620

Stokvis, bilirubin reactions, 408; benzoic
acid, 687; urobilin, 705; detecting
pentoses, 707*, 748; blood test, 753

Stolnikow, estimating proteid, 750

Stolte, amino-sugars, 213; glucosamine
in glycogen formation, 376; urea, 648

Stolz, sphygmogenin, 358

Stone, pentoses, 201

^tookey, gelatin, 120; peptones, 136;
glycogen formation, 373

Stoop, serine, 144; cystine, 147

Storch, V., milk globules, 613; tubercu-
lous milk, 637

Strada, pus cells, pus corpuscles, 345

Stradomsky, fate of oxalates in body,
680*, 733

Strashesko, gastric juice, 438

Strassburg, carbon dioxide tension in
lymph, 328; oxygen tension in blood,
811; carbon dioxide tension in blood,
814, 815, 819

Strassburger, 500*

Straub, glycosuria, 381 ; results of lack
of water, 842; effect of water and salts
on metabolism, 862

Strauss, sponginoses, 114*, 122; levulose,
210; levulose in blood serum, 211*,
257; alkalinity of blood, 296; ascitic
fluid, 337*, 340; molecular concentra-
tion of bile, 412; lactic acid fermenta-
tion, 460; hippuric acid, 461*, 643*,
685

Strauss, H., 297*

INDEX OF AUTHORS

923

Strecker, preparing lecithin, 234*, 238;
cholic acid, 400

Strickler, 625*

Strigel, lung concretions, 821

Stromer, fat formation, 537

Strusiewicz, 855*

Struve, blood test, 753

Strzyzowski, haemin compounds, 287

Stiibel, 838*

Stutz, proteid test, 747

St. Zaleski, liver proteins, 362; iron in
liver, 366, 367*

Subbotin, haemoglobin, 321; alcohol in
metabolism, 863

Succi, starvation experiments, 836

Sugg, milk enzyme, 621

Suida, cholesterin, 420

Suleima, putrefaction of proteins, 491

O'Sullivan, enzymes, 10; invertin, 61*,
63

Sundberg, pepsin, 443, 444

Sunde, lactose, 622

Sundvik, glucuronic acids, 214*, 664*,
713; glucuronic acid conjugation, 736;
chitin, 791 ; psylla-alcohol, 796

Suter, hydrolytic cleavage, 78; kera-
tin, 79*, 113; thiolactic acid, 148, 149*

Suto, estimating fat, 231

Suwa, fish flesh constituents, 550

Suzuki, cystine, 146; phytase, 551

Svedberg, metallic sols, 37; molecular
movement, 41

Swain, skatosine, 156

Symmers, organic combined phosphoric
acid in urine, 718

Syniewski, dextrin, 221*, 223

von Szontagh, milk enzyme, 621; milk,
626; human milk, 628

Szydlowski, 450*

Szymonowicz, adrenal pressure raising-
action, 357

Takaishi, phytase, 551

Takamine, adrenalin, 358

Tallquist, carbohydrates as protein spar-
ers, 857

Tammann, enzymotic action, 66

Tangl, glycerin in blood serum, 256; molec-
ular concentration of sera, 263; blood
corpuscles in serum, 313; sugar for-
mation in liver, 378; egg development,
606; casein, 615; urine, 732; nitrogen-
carbon relation, 827

Tanret, lactose, 621

Tappeiner, enzymes, 59; cellulose fer-
mentation, 488; fermentation of car-
bohydrates, 489*, 493; absorption of
bile acids, 517

Tarchanoff, bile pigments, 417; tatalbu-
min, 601

Tarugi, perspiration, 799

Tarulli, urine, 641

Taveau, 358*

Tawara, 874

Taylor, fat formation, 61*, 62*, 363*, 534;

results of insufficient mineral substances,
843

Tebb, reticulin, 121; glycogen, 370;
ptyalin, 432; intestinal juice, 467; prp-
tagon, 478*, 577, 578; cholesterin in
brain, 582

Tedesko, 642*

Teeple, bilirubin, 405, 406

Tengstrom, bile constituents, 396

Terrat, 719*

von Terray, antiputrid action of bile,
429*, 495; oxalic acid, 496*, 680;
paralactic acid, 710, 711*

Terroine, lecithalbumin, 105; maltose in
pancreatic juice, 478, 479*

Teruuchi, urea, 648

Tezner, 430*

Theissier, choline, 239

Thelen, creatinine, 658

Thesen, J., isocreatinine, 546; potassium
indoxylsulphate, 693

Thevenot, choline, 239

Thiele, uroferric acid, 716

Thiemich, 363*

Thierfelder, galactose, 22, 210; glucuronic
acid, 216; phosphatides, 233; digestion,
494; protagon, 577; brain sugar, 579;
cerebron, 580; yolk phosphatides, 599;
mammary gland, 610; origin of milk
sugar, 611*, 636

Thies, Z-adrenalin, 358

Thiroloix, internal secretion in pancreas,
385

Thiry, secretion of Lieberkuhn's glands,
465; intestinal secretion, 466; pro-
tein in intestinal secretion, 467

Thomas, 464*, 467*, 654*

Thompson, enzymes, 10; invertin, 61*,
63; urea, 648

Thorner, milk, 611

Thudichum, J. L. W., phosphatides, 232,
233; lecithin, 234; preparing lecithin,
cephalin, 238; bilirubin, 407; brain
phosphatides, 575; protagon, 577
cerebrosides sphingomyelin, 578; phre-
nosin, kerasin, brain sugar, 579; luteins,
600; paraxanthine, 677; uromelanins,
urochrome, 703; alcohol in metabolism,
863

Tichomirow, pepsin, 444

Tiemann, globulin, 620, 625*

Tigerstedt, determining respiratory gas
exchange, 820; nitrogen excretion hi
starvation, 837; metabolism in old and
young, 866, 867; effect of food inges-
tion on metabolism, 871; metabolism
during sleep and rest, 878

Tigerstedt, R., phosphates in urine, 723

Tissot, J., 563*

Tobler, digestion, 459; gastric absorption,
460, 724*

Toepfer, pseudoglobulin, 507; determining
chlorine in urine, 721

Tollens, B., pentoses and their deter-
mination, 202; xyloses, 204; prepar-

924

INDEX OF AUTHORS

ing dextroses, 206*, 209; sugar reac-
tion, 211; glucuronic acid, 216; naph-
thoresorcin reaction, 654*, 771

Tolmatsheff, determining casein and
albumin in milk, 623; human milk,
629*, 632

Tomasinelli, perspiration, 799

Tonjan, adrenalin, 359

Toppelius, creatinine, 658

Torup, carbon -monoxide haemoglobins,
279; carbon-dioxide haemoglobin, 280;
blood gases, 804

Tower, 121*

Toyonaga, calcium oxide in liver, 367

Traube, bradoxidizable substance, 4;
auto-oxidation, 6; oxidation ferments,
7; precipitation membrane, 26; cell
permeability, 34

Treupel, sugar test, 711*, 763

Treves, 78*

Trifanowski, 412*

Trillat, oxidase action, 13

Tritschler, fate of oxalates in body, 733

Troller, gastric juice, 440

Trommer, sugar test, 207, 758

Triimpy, perspiration, 798

Tschenloff, 852*

Tscherwinsky, fat formation, 537

Tschirjew, blood corpuscles, 325, 326*

Tshuschija, estimating proteid, 750

Tsuboi, haemaglobin, 321; nitrogen ex-
cretion, 825

Tuczek, 434*

Tiillner, uric acid, 664

Turby, intestinal juice, 467

Turk, glutamic acid, 145

Tiirkell, liver constituents, 365, 366*

Tyndall, optical phenomenon, 40

von Udransky, modified Pettenkofer test,
25*, 397; humin substances, 703; dex-
trose in urine, 711; ptomaines in
urine, 719; bile acids, 755; sugar test,
763; cystine in urine, 780

Uffelmann, reagent for free lactic acid in
gastric juice, 463

Uhlik, haemoglobin, 273*, 275

Ulenhaut, precipitine in blood, 259

Ulpiani, lecithin, 236; uric acid, 664

Ulrich, 219*

Ultzmann, calculi, 785

Umber, proteoses, 132; nucleins, 172;
transudates, 335; serosamucin, 336;
gastric juice, 440, 441; a-proteid, 470;
levulose in urine, 768

Umikoff, milk reaction, 630

Underhill, protozym as a cause of coagu-
lation, 14*, 311; hyperglycaemia, 380;
piperidine-glycosuria, 381 ; paralactic
acid, 429*, 682*, 710, 711*

Unna, vernix caseosa, 796; keratin, 797*,
789

Urano, creatine, 547; muscle salts, 558

Urbain, blood gases, 802

Ure, hippuric acid formation, 740

Ussow, tryptic digestion, 483
Ustjanzew, effect of removing caecum, 518

Vahlen, catalytic substance in pancreas,

387; specific rotation of cholic acid,

402, 403*
Valenciennes, creatine in muscle, 573;

cumydin, ichthin, ichthidin, ichthulin,

605

Valenti, nucleon in human milk, 629, 630*
Vamossy, 361*
Vandegrift, analyses of ligamentum nu-

chie, 520
Van de Velde, milk enzyme, 621; benzoic

acid, 687; ethereal sulphuric acid, 688
Vanlair, bilirubin, 406; stereobilin in

feces, 499
Vasilin, hippuric acid, 684; phenaceturic

acid, 687

Vussule, thyroid, 355
Vaubel, putrefactive products, 81, 98*
Vauquelin, allantoin, 681
Vay, metabolism in muscles, 362*, 563
Velichi, muscle proteins, 572; nucleon in

muscle, 573
Vella, secretion of Lieberkuhn's glands,

465

Veraguth, 852*
Ver Eecke, A., 867*
Verploegh, creatine, 574; creatinine, 574,

658-660, 663

Verneuil, gastric juice, 437
Vernois, human milk, 631
Vernon, enzymes, 63; tryptic digestion,

68; behavior of erepsin, 468; erepsin,

469; action of trypsin on trypsinogen,

474; amylopsin, 478; trypsin, 480;

enzyme in pancreas, 481 ; pancreatic

rennin, 487; tryptic digestion, 483
Verworn, biogens, 4
Viault, blood corpuscles, 322
Vierordt, estimating blood pigments, 291 ;

carbon dioxide in respired air, 813
Vietorow, fermentation sugar test, 760
Vigno, protein assimilation, 502
Vignon, 122*
Vila, stromata, 267; oxyhaemoglobin,

274; haemin, 285; musculamine, 550
Ville, methaemoglobin, 274; fat absorp-
tion, 397*, 515, 516; splitting of fats,

516, 719*, 749*

Villiers, leucomaines in urine, 719
Villiger, animal oxidations, 6
Vincent, nucleon in muscle, 573
Vines, enzymes in plants, 469; enzymes,

480
Virchow, amyloid, 168; hasmatoidin,

290; bile acids, 416
Vitali, blood test, 719*, 720*, 753
Vitek, 386*

Voegtlin, tryptic digestion, 484
Vogel, pentoses, 201; isomaltose, 219;

ptyalin, 432; lactase in intestinal

juice, 467; amylopsin, 478; rigor

mortis, 562 ; pentoses in urine, 769

INDEX OF AUTHORS

925

Vogelius, glycogen, 369

Vogelsohn, enzymes, 15

Voges, urine nitrogen, 646

Vohl, inosite fermentation, 551

Voirin, homogentisic acid, 698

Voit, antiputrid action of bile, 495; in-
fluence of fat on protein digestion, 496;
feces, 498; protein assimilation, 499*,
502 ; lactase formation, 509 ; fat absorp-
tion, 514, 534; muscle work, 562*, 565,
568; phosphates in urine, 571*, 651*,
723; lactose in urine, 768*, 769; metab-
olism, 823; nitrogen equilibrium, 825;
tissue loss in starvation, 839, 840;
mineral bodies in tissue, 842*, 843;
meat metabolism, 848; fat metabolism,
849; protein catabolism, tissue pro-
teins, circulating proteins, 850; pro-
tein destruction, 851 ; gelatin as protein
sparer, 853; nutritive value of anti-
peptone, 854 ; effect of water on metab-
olism, 862; influence of work on
metabolism, 868; influence of tem-
perature on metabolism, 870; effect
of food ingestion on metabolism, 871 ;
daily average adult diet, 874; daily
protein requirement, 875-880

Voit, C., glycogen, 369; glycogen for-
mation, 372, 374; results of absence of
proteins from food, 375*, 846, 867*

Voit, E., glycogen formation, 374; bone
analysis, 375*, 529; bone, 530; fat
formation, 535, 536, 537; oxygen heat
value, 832; heat of combustion of foods,
835; protein metabolism during starva-
tion, 837; decomposition of fat during
starvation, 838; fat as protein sparer,
855*, 856; catabolism limits, 858; flesh
deposition, 859; metabolism, 865

Voit, Fr., glycogen formers, 210*, 375;
acetone elimination, 773

Voitinovici, ichthylepidin, 106*, 114*, 121

Volhard, F., gastric lipase, 452; pepsin
test, 446; estimating purine bases, 679;
determining chlorine in urine, 721

Volkmann, mineral content of tissues, 842

Voltz, milk fat-globules, 613, 855*

van Voornveld, 323*

Vossius, bile pigments, 417

Voswinckel, carminic acid, 795

Vozarik, urine, 641, 643

Vulpian, adrenal chromogen, 357; bile
acids, 416

de Vries, semipermeable membranes, 27;
plasmolysis, 30, 31

Waage, calalysis, 53

Wachsmann, amylopsin, 478; pericardial

fluid, 338

deWaele, milk enzyme, 621
Wagner, muscle extractives, 546; carnine,

548; estimating sugar, 767
Wahlgren, bile mucin, 395; glycocholic

acid, 399; bile, 415
Wait, muscle work, 566

Wakeman, liver in phosphorus poison-
ing, 365

Walbum, enzymes, 59; antibodies, 69;
estimating proteid, 750

Waldvogel, acetonuria, 774

W^alker, adsorption, 48, 49

Wallace, polypeptides, 89

Walter, ichthulin, 170, 598; urea, 649;
blood gases, 802, 807

Walther, passage of fats into chylous
vessels, 513; pancreatic juice, 471

von Walther, 512*

Walton, 55*

Wanach, blood corpuscles, 293

Wang, indican, 694, 695*

Wanklyn, 614*

Wanner, proteoses and peptones in lungs,
821

Warburg, leucine, 141

Warmbold, 614*

Warren, muscle reaction, 563, 564*

Washbutski, putrefaction in intestine, 497

Wassermann, precipitines in blood, 259

Wassiliew, quantity of pancreatic secre-
tion, 476

Waymouth, sugar absorption, 509

Weber, S., muscle work, 565; creatinine,
659, 660; mineral bodies of egg white,
604

Weber, O., bone, 530

Wechselmann, indolacetic acid, 696

Wedenski, carbohydrates in urine, 711

Weidel, xanthine reaction, 185; carnine,
548

Weigert, lysine, 160; lipolysis, 259

Weil, carbon-monoxide methaBmoglobin,
279

Weinland, glycogen, 369; glycogen for-
mation, 375; sugar formation from
protein, 390; antipepsin, 443; auto-
digestion, antipepsin, 462; lactase hi
intestinal juice, 467; antienzymes in
intestinal juice, 468; lactase formation
in intestine, 473; lactase formation,
509; fat formation, 535

Weintraub, determining acidity of gas-
tric juice, 465; 652*, 723*

Weintraub, respiratory quotient in dia-
betes, 382; glycogen, 383

Weisbach, brain composition, 581

Weiser, St., glycerin in blood serum, 256

Weisgerber, carbon dioxide tension in
blood, 816

Weiske, bone, 489*, 529*, 530, 855*

Weiss, protamines, 109; glycogen for-
mation, 297*, 372, 374; trypsin estima-
tion, 482; glycogen in muscles, 563;
hippuric acid, 684, 685

Weissberg, animal oxidations, 6

Wellmann, elimination of phorphorus in
starvation, 839

Wells, liver autolysis, 365

Wendel, leucine, 141, 142

von Wendt, sulphur excretion, 826; in-
fluence of phosphates, 846

926

INDEX OF AUTHORS

Wenz, J., 128*

Wenzel, carnine, 548

Werenskiold, reindeer milk, 626

Werigo, carbon-dioxide elimination, 94*,

816

Werncken, 618*
Wertheimer, blood pigments in bile, 415;

pancreatic juice-excitants, 475; chloral

secretion, 476
Werther, saliva, 435; rigor mortis, 561;

lactic acid in muscle, 564
Westenrijk, 297*
Wetzel, conchiolin, 122, 157*
Weydemann, protein cleavage products,

83
Weyl, muscle activity, 93*, 565; protein

in amniotic fluid, 609; creatinine

reaction, 661; benzoic acid, 687;

nitrates in urine, 726, 727*
Wheeler, iodogorgonic acid, 123; nucleic

acid, 174; plant nucleic acids, 181;

cytosine, 189; uracil, 190
White, uric acid, 670
Whitney, pancreas diabetes, 385
Wichmann, seralbumins, 83*, 253; lactal-

bumin, 620
Widiscombe, 467
Wiechowski, uric acid, 656*, 670, 671;

allantoin, 681, 683; hippuric acid, 685
Wiener, xanthine-oxidase, 13; autolysis,

18; uric acid, 667, 668, 669, 681*, 685*
Wild, 7*
Wilenko, 718*
Wilhelmy, catalysis, 54
Willcock, ovalbumin, 603
Willdenow, C., 160*
Willebrand, carbon-dioxide eliminated by

the skin, 800
Williams, action of bile on diastatic

action of pancreas infusion, 488
Willstadter, proline, 152; lecithin, 235;

glycerophosphoric acid, 240 ; beta-choles-

tanol, 421

Wilson, protagon, 576, 578
Wiman, pseudonuclein, 104
Windaus, histidine, 157; methylimidazol,

197; cholesterin, 420, 424
Windrath, protein synthesis, 506
Winkler, human fat, 533
Winogradow, cholagogues, 394
Winter, human milk, 627
Winterberg, ammonia in blood, 318;

urea, 641*, 650, 652*
Wintemitz, purine bases, 183; haemo-
globin, 290*, 320; spleen, 415*, 351;

putrefaction, 495; fat absorption, 534;

milk fat formation, 635, 667*, 688*
Winterstein, valine, 139; tyrosine, 143*,

151; arginine, 158; lysine, 160; lysat-

ine, 161; lecithins, 237; phytin, 551;

tunicin, 625*, 790; chitin, 791
Wirsung, pancreatic calculi, 487
Wislicenus, muscle work, 567
Witt, caseinokyrin, 136
Wittmaack, human milk, 629

Woerner, creatinine, 658

Wohl, monosaccharides, 196, 200*

Wohler, synthesis, 3; uric acid, 670?
allantoin, 681 ; hippuric acid formation,.
740

Wohlgemuth, diastatic power of saliva,
73; oxyaminosuberic acid, 146; oxy-
aminosebacic acid, 161; sugar fer-
mentation, 199; pentoses, 201; arab-
inose, 204; ferratin, 362; glycogen for-
mation, 372; glycogen, 373; gastric
secretion, 417*, 453; pancreatic secre-
tion, 471; trypsinogen, 474; pancreatic
rennin, 487; enzyme in egg yolk, 597;
human casein, 627*, 628; glucuronic
acids, 713

Wolf, 780*, 781*

Wolff, glucosamine, 40*, 214; glycogen
formation, 376; protein metabolism,
566; melanins, 646*, 793

Wolff, H., transudates, 340

Wolff, J., oxidase action, 13

Wolffberg, glycogen formation, 374; oxy-
gen tension in blood, 811; carbon
dioxide tension in blood, 814, 815

Wolkow, homogentisic acid, 698, 699

Woltering, 362*

Woods, determining lecithin and cephalin,
238

Wooldridge, stromata, 267; tissue fibrino-
gen, 295; blood coagulation, 301, 308;
a-fibrinogen, 302; intra vascular coagu-
lation, 310, 311; negative and positive
phases of coagulation, 311

Worm -Midler, blood corpuscles, 321, 322;
quantity of blood, 325; sugar test, 759;
sugar estimation, 765, 766, 777

Worner, cerebrpn, 580; protagon, 577;
estimating uric acid, 675

Wright, coagulation of blood, 248*, 297*,
300; intravascular coagulation, 310,
379*

Wroblewski, pseudonuclein, 9*, 104;
pepsins, 221*, 442; human milk,
opalisin, 446*, 460*, 628

Wulff, guanine, 186, 189*

Wurm, tetronerythrin, 795

Wurster, ammonia in urine, 728

Wiirtz, urea in chyle, 328, 594*

Yakuwa, 855*

Yoshimura, phytase, 551

Young, enzymes, 60; dextrins, 223; gly-
cogen precipitants, 370; bile constit-
uents, 411

Yvon, sulphur compounds in urine, 714

Zadik, 826*

Zahor, estimating proteid, 750

Zaitschek, casein, 618; milk enzyme, 621;
milk, 626; human milk, 628

Zak, glycosuria, 335*, 384, 748*

Zaleski, haemopyrrol, 269; haemin, 285,
286; hsemin compounds, " haemato-
porphyrin, 287; mesoporphyrin, hsemo-

INDEX OF AUTHORS

927

pyrrol, 288; hydrogenized haemin, 289;
ammonia in blood, 317; reaction of
intestinal contents, 497; ammonia
formation in digestive apparatus, 505;
bone earth, 527; human milk, 633, 634*;
urea, 649, 652; urea elimination, 651;
estimating urea, 656; uric acid, 668;
ammonia in urine, 727, 728; saman-
darin, 797; protein metabolism, 853

Zander, chitin, 791

Zanetti, seromucoid, 252, 256; bile acids,
396; ovomucoid, 604

Zangerle, pseudomucin, 595

Zangermeister, hsematin, 290; amniotic
fluid, 609

Zaudy, 666*

Zdarek, sarcomelanin, 793

Zebrowski, 430*

Zeehuisen, 735*

Zeidlitz, sugar test, 759

Zeitler, muscle activity, 565

Zeller, chlorides in urine, 720, 735*

von Zeynek, R., ethal, 232; methsemo-
globin, 276; cyanmethaemoglobin, 277;
haemochromogen, 281; haematin, 283;
metals hi liver, 367; dermoid cysts,
43*, 596; sarcomelanin, 793; dermo-
cerin, 796

Zickgraf, protein oxidation products, 82;
guanidine, 118; lung concretions, 821

Ziegler, allantoin, 681; oxidation of
cymene into cumic acid, 739

Zillesen, lactic acid in muscles, 564

de Zilwa, mineral constituents of pan-
creatic juice, 477

Zimmermann, spleen, 353

Zink, oleic acids, 230 ; animal fat, 533

Zinoffsky, haemoglobin, 270

Zinsser, A., 452*

Zobel, sugar of muscles, 553

Zoja, R., elastin, 82*, 115, 116; ovalbu-
min, 602; uroerythrin, 706*, 709; hae-
matoporphyrin, 753*, 754

Zsigmondy, colloids, 37, 40, 42, 44; col-
loids, molecular movement, 41

Zuco, Marino, choline, 239

Zuelzer, preparing lecithin, 238; adrena-
lin glycosuria, 384; internal secretion
in pancreas, 385; absorption of oxygen
by the skin, 800

von Zumbusch, bilifuscin, 410

Zuntz, N., metabolism in muscles, 262;
sp.gr. of blood, 296; alkali in blood,
298; haemoglobin, 320; blood corpus-
cles, 323; glycogen, 369, 377; sugar
formation in kidney, 378*, 379; tryptic
digestion, 483 ; protein absorption, 502 ;
effect of removing the caecum, 518;
fatty acids, 557; protein metabolism,
566; muscle work, 567, 568; perspira-
tion, 799; carbon dioxide eliminated by
the skin, 800; blood gases, 802, 803,
804; lymph gases, 807; respiration,
809, 810 ; carbon dioxide in respired air,
813; carbon dioxide elimination, 816;
determining respiratory gas exchange,
819*, 820; metabolism, 823; respira-
tory quotient, protein heat value, 831;
milk protein calorific value, oxygen
heat value, 832; starvation experi-
ments, 836; nutritive value of pro-
teoses, 854; alcohol in metabolism,
863; metabolism, 864; influence of work
on metabolism, 867*, 868; respira-
tory quotient during work, 869; diges-
tion work, 872; effect of food and altitude
on metabolism, digestion work, 871

Zuntz-Geppert, determining basal require-
ment in metabolism, 841

Zunz, E., peptic digestion, 131, proteoses,
132, 133; separating proteoses and
peptones, 137; rapidity of digestion,
457; extent of digestion, 459; gastric
absorption, 460; enterokinase, 472; pan-
creatic juice, 473; trypsinogen activa-
tors, 474; absorption of proteoses and
peptones, 503; meat digestion, 509;
food absorption, 516; hexone bases in
meat, 549

Zweifel, ptyalin, 431; amylopsin, 478;
paralacitc acid, 710, 711*

Zwerger, caseinokyrin, 136

GENERAL INDEX

Abiuret products, 131, 491, 503
Absorption, 460, 495, 497, 501-518
in intestine, 501-518
ratio, of the blood pigments,

291

Acceptor, 6
Acethaemin, 285

Acetone bodies, formation, 771-774, 841
Acetyl equivalent of fats, 231

number, 231

Acetylene haemoglobin, 280
Acetylglucosamine, 741
Acetylpara-amido phenol, 738
Achilles tendon, formation of, 520.
Acholia, pigmentary, 415
Achroodextrin, 222, 434
Acid, abietinic, 423

acetic, occurrence in intestine, 491
in stomach, 441
in urine, 710, 733

aceto-acetic, in urine, 728, 771-778
acetylaminobenzoic, 741
adenylic, 179
albuminates, 91, 124

absorption of, 502
in peptic digestion, 448
alloxyproteic, 714, 716

action of trypsin on, 485
amides, behavior in animal body, 734
aminoacetic, see Glycocoll, 3, 138
aminocaproic. See Leucine, 140
aminobenzoic, behavior in animal

body, 740

aminocerebrinic, chloride, 581
aminocinnamic, 737, 739
aminoethylsulphonic. See Taurine,

149

aminoglucuronic, 523
a-aminoglutaric. See Glutamic

acid, 145

aminohippuric, in urine, 740
a-aminoisobutylacetic. See Leu-
cine, 140
/?-amino-n-oxypropionic. See Iso-

serine, 144
a-amino-/9-oxypropionic. See Se line,

143
aminophenylacetic, behavior in

organism, 738
Q'-aminopropionic. See Alanine, 139

Acid, aminosuccinic. See Aspartic acid,

144
a-amino-5-thiolactic. See Cystine,

146, 148

a-amino valeric. See Valine, 139
antoxyproteic, 714-717, 779
arachidic, 224, 596, 614, 796
aspartic, 144

quantity in proteins, 106,

107, 114, 124
relation to formation of

urea, 648, 734
relation to formation of uric

acid, 668
benzoic, formation from proteins, 82,

118, 683, 738
behavior in the animal body,

3, 683, 713, 728, 738
in perspiration, 799
occurrence in the urine, 683,

687

substituted benzoic acids in
the animal body, 683, 738
benzoicglucuronic, 713
benzoylaminoacetic. See Hippuric

acid, 683
bilianic, 401

bromphenylmercapturic, 743
butyric, fermentation, 200, 207, 493
camphoglucuronic, 215, 712, 743
capric, 225, 614
caproic, 225, 614
caprylic, 225, 614
carbamic, 657

in the blood, 260, 650
in the urine, 650, 658
toxic action, 650, 651
carbamino-acetic, 162, 806-807

salts of, 162, 806-

807

carboglobulinic, 806
carbolic, effect upon peptic digestion,

447. See also phenols,
carminic, 795
carnaubic, 232
carnic, 550
caseanic, 80, 84, 162
caseinic, 80, 84, 162
cephalic, 238, 575
cerebrinic, 581

929

930

GENERAL INDEX

Acid, cerebrinic-phosphoric, 581
cerebronic, 580
cerotic, 232

cheno-taurocholic, 400, 404
chitaminic, 214
chitaric, 214
chlorhodinic, 347
chlorphanylmercapturic, 743
chondroitic. See Chrondoitinsul-

phuric

chondroitinsulphuric, 98, 164, 168,
198, 522, 523
occurrence in
kidneys, 639
occurrence in
urine, 714,
717, 751

cholalic. See Cholic acid, 400
cholanic, 402
cholecampheric, 410
choleic, 399, 400, 402^04
cholesterinic, 401
cholic or cholalic, 395, 400, 401, 404,

498,

choloidic, 404
cholylic, 401
chrysophanic, elimination in urine,

744

cilianic, 401
cinnamic, behavior in animal body,

683

citric, in milk, 614, 625, 630
coccinic, 111, 795
cochenillic, 795
cresolsulphuric, 687, 688
crotonic, 778-779
cumic, 738

behavior in animal body,

739, 740
cuminuric, 740
cyanuric, 653, 664
cysteinic, 147
damaluric in urine, 719
damolic in urine, 719
dehydrocholic, 401, 402
desaminoalbuminic, 126
desoxycholic, 401, 403
diacetic. See Acid, aceto-acetic.
dialuric, relation to formation of uric

acid, 669

diaminoacetic, 84, 159
a-e-diaminocaproic. See Lysine, 159
diaminotrioxydodecanoic, 80, 84,

162

diaminovalerianic. See Ornithine
dimethy laminobenzoglucuronic ,713,

743

p-dimethylaminobenzoic, 743
dioxydiaminosuberic, 161
dioxyphenylacetic, 698. See Homo-

gentisic acid,
dioxyphenyl-lactic, 701
dioxystearic, 229, 614
doeglic, 230
elaic, 229

Acid, ellagic, 501

ethylidenelactic, 553. See various

lactic acids,
equivalent, 230
erucic, 225
euxanthic, 216, 742
euxanthoglucuronic, 713, 742
excretolic, 500
fellic, 404
fermentation, lactic, 553-556

in blood, 318
in coagulation of
milk, 554, 612
in muscle, 573
in the stomach,

463, 464

formic, fate in organism, 710, 733
furfuracrylic, 741
furfuracryluric, 741
gadoleic, 230
• galactonic, 210
gallic, 697

elimination of, 742
gentisic, 698, 700

behavior in animal body,

742
gluconic, 194, 205

in diabetes, 382
glucosaminic, 197, 214
glucothionic 198, 295, 346, 351, 365,

449, 522, 610, 639
glucuronic, 202, 203, 215-217

conjugated glucuronates,

215, 216, 712-714
in the bile, 395, 411
in the blood, 257
in the urine, 215, 712,

742, 770, 771
relation to formation of

glycogen, 372
glutamic, 81, 84, 106, 107, 114, 124,

145

glutinic, 118
glyceric, 144, 390, 734
glycerophosphoric, 233-235, 240, 411
in the urine, 711,

718

p-glycocholic, 399
glycocholic, 395, 397

absorption of, 517
in feces, 494

in various animals, 413
glycollic, behavior in animal organ-
ism, 680

glycophosphothymic, 178
glycosuric, 698

glycuronic, 215. See Glucuronic.
glyoxylic, 680, 683

as reagent, 99
behavior in various ani-
mals, 680

guanidine acetic. See glycosamine.
guanidine-a-amino valeric, 158. See

Arginine.
guano bile, 399

GENERAL INDEX

931

Acid, guanylic, 174, 175, 176, 178, 362, 470
, hsematopyrrolidinic, 284
haemoglobin, 278
hsemopyrrol carboxylic
heminucleic, 177
hippuric, 3, 683-687, 737, 739
homogentisic, 690, 697, 698-702
hydrochloric, secretion in stomach,

441, 453
action upon ptya-

lin, 432

action upon secre-
tion of bile, 394
action upon secre-
tion of pan-
creatic juice, 474
action upon the
pylorus, 456, 458
antifermentive ac-
tion, 461
hydrocinnamic, behavior in animal

body, 684

hydrocyanic, effect upon blood pig-
ments, 278
effect upon pepsin

digestion, 447
effect upon trypsin

digestion, 483
hydroparacoumaric, 151, 697

in intestinal pu-
trefaction, 492

hydroquinone-carboxylic, 700
hydroquinonesulphuric, 687, 691
hyoglycpcholic, 399, 404
hypogaeic, 232

imidazolaminoacetic in urine, 719
indolacetic, 81, 153, 155, 696-697
indolaminopropionic, 81, 153, 155
indol-carboxylic, 692
indol-proprionic, 81, 153, 155
indoxyl- carboxylic, 692
indoxylglucuronic, 215, 691, 694, 712
indoxyl-sulphuric, 687, 691-695
inosinic, 175, 176, 179, 180, 550
iodogorgonic, 123
isobilianic, 401
isocholanic, 402
iso valeric, 734
kynurenic, 697, 702, 719
I- lactic, 553

lactophosphocarnic, 612, 620
lanoceric, 797
lanopalmitic, 797
lauric, 225, 226, 614, 797
lepidotic, 795
leucinic, 141
levulinic, 202, 205, 621
linolenic, series, 225
linolic, 223, 224
lithobilic, 501
lithofellic, 404, 501
lithuric, in urine, 719
lysalbinic, 126
lysuric, 160
malic, fate in organism, 642, 634

Acid, maleic, 283
mandelic, 738
mannonic, 205
margaric, 228
martamic, 178
menthol-glucuronic, 770
mesitylenuric, in urine, 740
metaphosphoric as precipitant of
proteids, 98, 746

in nucleins, 172
in pseudonucleins, 173
methylethyl-a-aminopropionic, 142,
See Isoleucine.
methylguanidine-acetic, 546. See

Great ine.

methylhydantoic, 735
monoxystearic, 226, 229
mucic, 210, 372, 621
myristic, 225, 226, 614, 797
naphtholglucuronic, 744
neurostearic, 580
nitrobenzoic, 741
nitrohippuric, 741

nitrophenylpropiolic, test for dex-
trose, 209,
763

behavior in
animal body,
691, 694

norisosaccharic, 214
nucleotinic, 175
oleic, 225, 229
ornithuric, 159, 160; 740
orotic, 614, 621
orylic, 620
oxalic, in urine, 679

fate in organism, 679, 733
oxaluric, 664, 679
oxonic, 664
oxy-tf-pyrolidinecarboxylic, 153. See

Oxyproline.

oxyaminosuberic, 80, 84, 146
oxyaminosuccinic, 80, 84, 146
oxyanhydroparacoumaric, 697
oxybenzoic, behavior in animal

body, 740
/?-oxybutyric, 778

hi the blood, 807
detection and estima-
tion, 779

occurrence in the
urine, 728, 771-779
oxydiaminosuberic, 80, 84, 161
oxyethylsulphonic, behavior in ani-
mal body, 736
oxymandelic, 697, 702
oxyhydroparacoumaric, 697
oxy iso valeric, 734

oxymethylpyrazine-carboxylic, 213
oxyphenylpyroracemic, 700
p-oxyphenylacetic, 492

in urine, 697, 742
p-oxyphenyl a-aminopropionic. See

Tyrosin, 150
p-o :yphenylpropionic, 492, 697

932

GENERAL INDEX

Acid, p-oxyphenylpropionic, in the urine,

742

oxyproteic, 640-641, 714, 715
oxyprotosulphonic, 82
oxypyrrolidine carboxylic. See oxy-

proline.
oxyquinoline carboxylic, 697, 702.

See Kynurenic acid,
palmitic, 225, 228, 22(J
parabanic, 664
paralactic. See Sarcolactic.
paranucleic, 619

paraoxyphenylpropionic, 492, 697
pepsin-hydrochloric, 449
peroxyproteic, 82
phenaceturic, 687

in urine, 738, 740
phenolglucuronic, 215, 689, 711,

713

phenyl acetic, formation in putrefac-
tion, 81
behavior in animal

body, 687, 738
phenylaminoacetic, 738
phenylaminopropionic, 150. See

Phenylalanin.
phenylbutyric, 739
phenylcaproic, 739
phenylketopropionic, 777, 739
phenyllactic, 699, 737, 739
phenyl propionic, formation in putre-
faction, 81, 492,
684
behavior in animal

body, 684, 739
phenyl oxypropionic, 739
phenyl pyroracemic, 700
phenylsulphuric, 687, 688
phenylvaleric, 739

in perspiration, 799
in urine, 687, 688
phosphocarnic, 260, 546, 550, 576,

612, 620
in urine, 718
relation to muscular

work, 564, 569
phosphoric, in urine, determination

of, 722-725, 729
, formation in muscle, 565
phthalic, fate in body, 737
physetoleic, 232
plasminic, 181
polypeptid phosphoric, 619
protalbinic, 126
protic, 465
protocatechuic, behavior in animal

body, 690

pseudonucleic. See paranucleic acid,
psyllic, 796
pulmotartaric, 820
pyinic, 347

pyridinecarboxylic, 738, 741
pyridinuric, 741
pyrocatechinsulphuric, 687, 690
pyroracemic, 149

Acid, a-pyrolidinecarboxylic, 152. See

Proline.
pyromucic, 741
pyromucinornithuric, 741
pyromucuric, in urine, 741
pyrrolidine carboxylic. Se'e a-

proline.
quinic, 684

racemic, fate in organism, 734
rhodizonic, 552
saccharic, 194,215

behavior in diabetics, 382
relation to glycogen for-
mation, 372

salicylic, behavior in animal body, 740
salmonucleic, 174, 176, 177, 178
sarcolactic, 20, 553-556, 561
in brain, 576

in muscular work and
rigor, 560, 561, 563, 564
in the bones, 530, 531
in vitreous humor, 586
passage into urine, 554.

555, 564, 710
relation to uric acid for-
mation, 668
sarcomelanic, 793
scymnol-sulphuric, 395
sebacic, 229

silicic, occurrence in connective tis-
sue, 518
in blood, 261
in bones, 529
in feathers and hair, 790
hi hen's egg, 600, 604, 606
in urine, 730
skatolacetic, 81, 153
skatolaminoacetic, 153
skatolcarboxylic, 153, 696
skatoxylglucuronic, 215, 695, 712
skatoxylsulphuric, 687, 695
stearic, 228

succinic, in fermentation of milk, 612
from phosphocarnic, 550
in intestine, 491
in perspiration, 799
in spleen, 351
in thyroid, 354
in transudates, 337, 341, 343
passage in urine, 711, 734
sulphosalicylic as a reagent, 746
sulphuric, effect on pepsin digestion,

446.

ethereal sulphuric and
sulphate-sulphuric, 687,
688, 726

in perspiration, 799
tannic, elimination of, 742
tartaric, relation to glycogen forma-
tion, 372
in organism, 733
in perspiration, 799
tartronic, 669
taurocarbamic, 736
taurocholeic, 400

GENERAL INDEX

933

Acid, taurocholic, 395, 399, 414, 494

as precipitant of pro-
teins, 98, 751

tetraoxyaminocaproic, 523
thiolactic, 78, 84, 113, 148
thiophenic, 741
thiophenuric in urine, 742
thymic, 177
toluic, 738, 739, 740
toluric in urine, 740
trichlorethylglucuronic. See acid,

urochloralic.

trioxybenzoic, 742. See Gallic acid,
triticonucleic, 175, 180
turpentine glucuronic, 742, 770
tyrosinesulphuric, 151
uraminoberizoic, 740
urea glucuronic, 712
uric, 182-184, 663-675

decomposition products, 664
elimination, 665
formation of, 666
in blood, 259, 318, 665, 667
in brain, 576
in perspiration, 800
in sediment, 640, 762, 782-783
properties and reactions, 671-

675

quantitative estimation, 674
urocanic in urine, 719
urochloralic, 215, 736
uroferric, 714, 716
uroleucic, 701
uronitrotoluolic, 743
uroproteic, 716
uroxanic, 654
ursalicylic, 740
ursocholeic, 404
valeric, 80, 141
vanillinic, 738
whey, 612

yeast-nucleic, 175, 180
Acids, amino, 76-80, 84, 85, 89-90, 138-163
amounts in proteins, 106-107,

112, 122
breaking down of, 734-735,

737

chlorination of, 87
combining of, 85-90
in the intestine, 492, 503, 504
in the stomach, 459
in urine, 364, 717, 734, 735,

780

occurrence in blood, 260
relation t9 carbohydrate
metabolism, 372, 375, 390
relation to urea, 647-650
relation to uric acid, 668
desaminoproteic, 82
diamino, 84, 157-161
ethereal sulphuric, in the bile, 395,

411, 413

in urine, 492-493,
687-694, 742,
799

Acids, ethereal sulphuric, in perspiration,

799
synthesis in liver,

360
fatty, 225-229, 533

absorption, 512, 515-517
in brain, 575
in perspiration, 799
in perspiration fat, 797
in urine, 710,734, 782
volatile, 225

inorganic. See Mineral acids,
kyroproteic, 82
lactic, 144, 553, 554

detection iu organs and tis-
sues, 557
in bones, 530

in relation' to formation of uric
acid, 668, 669. See also Sar-
colactic acid,
in urine, 668-669, 710
melanoidic, 793
mercaptic, elimination of, 743
mineral, alkali-removing action of,
and action on the elimination of
ammonia, 641, 642, 728, 807, 844
monoamino. See acids, amino.
nucleic, 171, 174-181, 673
in urine, 718, 751

organic, behavior in animal body,
728, 733, 734, 737, 738
oxyamino, 79, 80, 84, 146, 211
oxyfatty in animals fat, 225, 231
oxypropionic, 553
proteic, in blood, 260

, in urine, 714-716
thymonucleic, 174, 176-179, 470
thymus nucleic, preparation of, 180
ureido glucuronic, 712 .

Acethsemin, 285

Acetanilide, elimination in the urine, 739
Acetone, 82, 775

in urine, 771-777
quantitative estimation, 778
Acetophenone, elimination of, 738, 742
Acetyldiglucosamine, 791
Acidosis, 773, 841
Acrite, 205
Acrolein, 227, 230
Acrose-o- and /9, 205
Actiniochrom, 795
Activators, 60
Adaptation of the glands, 429, 430, 438-

439, 471^72

Adamkiewiez reaction for proteids, 99, 154
Addison's disease, 357, 359
Adelomorphic cells, 436
Adenase, 16, 183, 346, 347, 667
Adenine, 182, 183, 187
in liver, 364
in urine, 676

preparation and detection of, 188
Adenosin, 181
Adialyzable bodies, 717
Adipocere, 534

934

GENERAL INDEX

Adrenalin, 240, 358, 359

relation to glycosuria, 359, 380
relation to melanin formation,

359, 795
Adrenal bodies, 235, 357-359

relation to diabetes, 382,

384

Adsorpates, 34

Adsorption, 49, 50-52, 70-71, 94
^Egagrophilae, 501
jErotonometric method, 814
Age, effect upon metabolism, 864, 866
Agglutination and agglutinins, 70, 265, 307
Agglutinins, 70

Air, alveolar, composition of, 811, 814-816
Air bladder, of fishes, gases of, 817

guanin, 185
Alanine, 84, 106, 107, 111, 122, 139, 144,

390, 556, 734
Alanylalanine, 88, 486
Alanylalanineglycin, 86
Alanylglycine, 87, 486
Alanyl leucine, 86, 486
Albamine, 214
Albumin, 90-91, 102, 103
in urine, 747, 749

See also proteins.
Albuminates, 94, 103, 124, 125
in the spleen, 351
Albuminose, 592
Albuminous bodies, in general, 93-97

table for, 90-91, 102-

111

See also various al-
buminous bodies of
juices and tissues.
Albuminoids, 91, 111-124, 524, 587

in lungs 820

Albumins, 90, 91, 92, 106-107
properties of, 102

See also various albumins.
Albuminuria, 731, 744

elmentary, 502

Albumoids. See albuminoids.
Albumoses. See proteoses,
Alcapton, 690
Alcaptonuria, 700-704
Alcohol . See ethyl alcohol .
Alcohols, high molecular, 226
Alcoholase, 58
Aldehydase, 12
Aldehydes, fate in organism, 735, 740,

741

Aldoses, 193, 194, 205
Aleuron grains, 597
Alexines, 259

Alimentary glycosuria, 378-389, 510, 758
Alizarin, elimination in urine, 530, 744
Alkali albuminates, 91, 124, 502
Alkali earths, elimination through intes-
tines, 723

deficient in, 530, 844, 845
in bones, 527
in urine, 722, 723, 729
Alkaline fermentation of urine. 782

Alkali albumose, 126

carbonates, physiological impor-
tance for metabolism
of gases, 803-806,844
effect upon secretion of

gastric juice, 438
effect upon secretion of
pancreatic juice, 474.
See also various tissue
and juices,
phosphates, in urine, 722-725. See

also various tissues and fluids,
urate, 640, 672

in calculi, 787
in sediments, 640
Alkaloids, effect upon muscles, 560

effect upon peptic digestion, 448
passage of into urine, 744
retention by the liver, 360
Alkyl sulphides in skunks, 797
Allantoin, 664,681,682

in transudates; 337, 340, 609,

381
formation from uric acid, 664,

670, 681

Alloisoleucine, 143
Alloxan, 664

Alloxuric bases. See Purines.
Almen's test for sugar, 208, 759
Altitude, effect on metabolism, 871
Alveolar air, composition of, 814
Ambergris, 501
Amboceptors, 71
Ambrain, 501
Amidases, 668
Amidoguanidine, 681
Amicrons, 40
Amidulin, 221, 433
Amino acids. See Acids, amino.
Aminoaldehyde, 212
Aminocerebrinic-acid glucoside, 581
Aminophenol, 739
Amino-oxypurine. See Guanine.
Amino-oxypyrimidine. See Cytosine.
Aminopurine, 186
Aminosuccinic-acid amide. See Aspara-

gine.

Amino sugars, 212. See also glucosamine.
Ammonia, after extirpation or atrophy of

the liver, 651

after partaking of food, 505,852
elimination after administra-
tion of mineral acids, 641,
642, 728

estimation in urine, 727
in blood, 317, 649, 651, 652, 727
in sickness, 646
in urine, 641, 646, 652, 727-729,

841
increased elimination during

starvation, 841
Ammonium magnesium phosphate, in,

intestinal calculi-, 501
in urinary calculi, 786
in urinary sediment, 782, 784

GENERAL INDEX

935

Ammonium salts, relation to glycogen

formation, 372
relation to urea forma-
tion, 649, 727
relation to uric acid for-
mation, 668

sulphate, methods of separa-
ting proteoses, 94,
97, 102, 128-130,
132, 133

methods of separa-
ting carbohy-
drates, 223, 370
urate, in urinary calculi, 786
in urinary sediment,

782, 783

Amniotic fluid, constituents of, 609
Amphicreatine, 550
Amphopeptone, 128
Amygdalin, 198

action of emulsin on, 67
action .of yeast on, 67
Amylase, 58
Amylases, 16
Amylodextrin, 221, 222
Amyloid, 168, 224, 352, 522

preparation and reactions of, 170
Amylolytic enzymes, 16. See also various

tissues and secretions.
Amylopectin, 220, 223
Amylopsin, 478
Amylose, 220, 223
Amylum. See Starch.
Anaerobic metabolism, 386, 556
Aniline, elimination in the urine, 738
Animal oxidation, 3
Anisotropous muscle substance, 538
Antedonin, 795
Anthozoa, axial system, 122
Antialbumate, 448
Antialbumid, 134, 448
Anti-bodies, 69-74

artificial and natural, 69
Antienzyme, 18. Also see various enzymes.

for emulsin, 69
Antigens, 69
Antimony, passage into milk, 637

effect upon elimination of nitro-
gen, 646

Antipathies, 122
Antipepsin, 443

in intestinal juice, 468
Antipeptone, 128, 130, 131, 135

nutritive value of, 854
Antipyrine, elimination in urine, 743, 760
relation to glycogen formation,

372

Antirennin, 69
Antirennins, 450
Antithrombin, 309

occurrence in peptone-
plasma, 312
Antithrombins, 306
Antitoxins, 70
Antitrypsin in intestinal juice, 468

Anuria in cholera, 799

Aorta elast in, 115, 116

Apatite, in bone ash, 527

Aqueous humor and its constituents, 342

Arabinoses, 194, 175, 199, 203, 224

relation to glycogen forma-
tion, 372
in urine, 769
Arabinosimine, 197
Arabite, 194
Arachnoidal fluid, 335
Arbacin, 108
Arbutin, 372, 691
Arginase, 16, 90, 158, 346, 648
Arginine, 77, 78, 108, 110

amounts in proteins, 106-107.

111-114, 161
properties of, 158, 159
relation to urea formation, 648
relation to creatinine, 659
relation to uric acid, 668
Arginine-histone peptone, 137
Argon, in blood, 801
Arnold's creatinine reaction, 662
Arnold-Lipliawsky's test for aceto-acetic

acid, 777

Arnold's urine reaction, 662
Aromatic combinations, behavior in ani-

mal bodies, 736-744
Arsenic, 22, 261, 316, 320, 349, 790

action on elimination of nitrogen,

646

in perspiration, 799
poisoning, 417-419
Arterin, 268

Artificial starch. See starch.
Ascitic fluid and its constituents, 339
Asparagin, 144

in metabolism investigations,

855
relation to glycogen formation,

0*7*)

Asparagus, effect of on odor of urine, 4Hr

Assimilation limit, 510

Atheromatous cysts, 140

Atmidalbumin, 129

Atmidalbumose, 129

Atmidkeratin, 113

Atmidkeratose, 113

Atropine, effect upon uric acid elimination,

666

upon saliva secretion, 435
Autodigestion. See Autolysis.
Autolysis, 17, 335, 336, 555, 589. Also

see various organs.
Autooxidation, 4-6, 11-13
Autotoxines, influence of on putrefaction

in intestines, 498
Availability, physiological, 835
Avpgadro's law, 28
Axis cylinders, 574

Bacillus, common of cholera, 461
Bacteria, influence of on putrefaction in
intestines, 497

f *\ T"

936

GENERAL INDEX

Bacterial action in intestinal canal, 495

Bacteriolysins, 70

Bacterium urese, 782

Balsam copaiba, elimination in urine, 744

Bang's method of estimating sugar, 764

Barium, 22

Basal requirements in metabolism, 841,

864

Basedow's disease, 355
Baumann's test for dextrose, 209
Beer vinegar bacteria, enzymes of, 10
Beeswax, 232

Bela v. Bitto's reaction for acetone, 776
Bence- Jones protein, 749
Benzaldehyde, oxidation to benzoic acid,

6
Benzene, behavior in animal body, 736-

737

Benzidine blood test, 753
Benzoylation of carbohydrates, 208-209,

711, 762

Benzoylchloride test for dextrose, 208
Benzoyl cystine, 148
Betaine, 239, 550
Bezoar stone, oriental, 404, 501
Bial's reagent for pentoses in urine, 203,

770

Bifurcated air, 812
Bile acid test, Pettenkofer's, 396
Bile acids, 395-405

absorption, 394, 517

detection in animal fluids , 405,

755

in blood, 260, 316
in urine, 517, 755
place of formation, 416
preventative of putrefaction in

intestines, 495
Bile, 392-419

action on proteins, 490

as activator of steapsinogen, 489

as preventative of peptic zymolysis,

489
characteristics and constituents of,

394,395,411

chemical formation of, 415
concretions, 405, 419
crystallized Plattner's, 396
decomposition in intestine, 493, 494
diastatic action, 488
effect of exclusion from intestines,

394, 515

effect on protein digestion, 447
Hufner's, 398

in stomach contents, 489, 490
its formation, 392
occurrence in urine, 517, 755, 756,

757

on absorption of fats, 489, 495, 515
Pettenkofer's test for, 396
preventative of putrefaction in in-
testines, 495
properties in disease, 415
quantitative composition of, 412, 413
solvent action on fatty acids, 512.

Bile mucus, 394, 415

Bile pigments, 395, 405-412

reactions of, 407, 408, 756,

757

passage into urine, 756
Gmelin's test for, 407
Bile salts, 395
Biliary fistula, 392, 496
Bilicyanin, 405, 408, 410
Bilifulvin, 405
Bilifuscin, 405, 410
Bilihumin, 405, 410
Biliphaein, 405
Biliprasin, 405, 410
Bilipurpurin, 410
Bilirubin, 405-409

Ehrlich's test for, 407
Hedenin's test for, 411
Huppert's test for, 407
occurrence in serum, 260
putrefaction of, 494
relation to blood-pigments, 288,

290, 405, 417, 418
relation to haematoiind, 290,

406, 416-418
transformation into urobilin,

494

Biliverdin, 405, 407, 409, 604
in excrements, 499
in feces, 499
Biogen molecule, 4
Biological protein reactions, 259, 502
Bismuth, passage into rnilk, 637
Bitter substances, effect upon secretion,

438, 458
Biuret, 99, 653
Biuret base, 85

reaction, 653
test for proteids, 99
Blister-fluid and its constituents, 342
Blood, 242-326

after partaking food, 321, 507
analyses, quantitative, 312-318
arterial and venous, 269, 318, 801-

802

asphyxiation, 269, 803
behavior in starvation, 262, 320,

324, 840

buffy coat of, 299
casts, 752.
clot, 244, 246

composition under various con-
ditions, 318-324
defibrinated, 244
detection of, 290
enzymes and antienzymes, 247,

248, 249, 252, 259
general behavior, 242, 296-299
in gastric contents, 449
in invertebrates, 248, 292, 307
in urine, 752-754
quantity of in body, 324
Blood corpuscles, 264-268, 292-296
changes in volume, 32
composition of, 229,293

GENERAL INDEX

937

Blood corpuscles, and blood fluid relation,
of weight between,
313, 314
number of red, 264, 321-

322
number of white, 294,

324
passage of into urine,

752
permeability, 32, 33,

268
red, 242, 264-268, 292,

293
red, decrease in number

in anaemia, 323
red, increase in number
after blood transfu-
sion, 322

red, quantitative con-
stitution of, 292
relation to coagulation,
301, 302, 306, 310-
311
relation to high altitude,

322

white, 242, 293-295
Blood, analysis of, 261

distribution of in various organs,

326

enzymes in, 259
gases of, 261, 312, 801-807
in bile, 415
in urine, 752, 753
menstrual, 261, 319
of portal vein, 318, 378, 509,

649

pigments of, 260
pigments, 268-289
plasma, 242, 244-256
plates, 242, 293, 295
proteins in, 262
relation to coagulation of blood,

293-294, 303, 306
serum, 243, 256-264, 298
transfusion, 322, 325
Blueberry, pigments of in urine, 744
Blue milk, 637
Blue stentorin, 795
Boar sperms, 591
Boas' test, for lactic acid in gastric juice,

463

for HC1, 462-463
for lactic acid, 463

Body, relation of weight and age of to
absolute consumption of material
864
weight decrease during starvation,

836
Boiling-point, 28-29

elevation of, 28-29
Bombicesterin, 424
Bondi-Schwarz's test for acetoacetic acid,

777

Bone ash, composition of, 527
Bone earths, 526, 527

Bone marrow, 225, 226, 235, 244, 245, 528,

Bonellin, 795

Bones and bone tissue, 526-532, 724

digestion, 448, 449
in starvation, 839,

804
Borneol, behavior in animal organisms, 712

elimination in urine, 743
Bottcher's spermine crystals, 590
Bottger-Almen's test, 208, 759
Bowman's disks, 538
Boyle Marriot's law, 28
Bradoxidizable substances, 4
Brain, 574-584

constituents of, 574-576

phosphatides, 575

proteins in, 574

quantitative composition of, 581-

583

-sugar, 479
British gum, 222
Bromhajmin, 287
Bromides, in saliva, 435

relation to formation of gastric

juice, 453
Bromine, 22, 123

Bromoform, behavior in animal body, 735
Bromthymine, 191

Bromtoluene, behavior in animal body, 740
Brownian molecular motion, 41
Briicke's method of testing pepsin, 445
Brunner's glands, 465, 476
Buffy coat, 299
Bufidin, 797
Bufonin, 797
Bufotalin, 797
Bufotenin, 797
Bufotin, 797
Butalanine, 471
Butter, constituents, 614
Butter-fat, 614

absorption, 515

Butterflies, pigments of, 665, 795
Butylmercaptan, 797
Butyric acid, in intestine, 493
in milk fat, 614
in stomach, 441
in urine, 710
Butyrinase in blood, 259
Burbot, sperms of, 177
Bursse mucosse, 344
Byssus, 91, 121, 122

Cadaverine, 24, 160, 550, 780

in urine, 719
Csecum, 491, 492

effect of extirpation of, 518
Caffeine, 182, 560

behavior in animal body, 676
Calcium, deficiency in the food, 530, 531,

844, 845

carbonate in urine, 783
oxalate, in calculi, 786, 788

in sediments, 782, 783
in urine, 679, 783

938

GENERAL INDEX

Calcium, oxide in liver, 367

phosphate in urinary sediments,

782, 784
in urinary calculi, 786,

788 '

salts, elimination, 723, 729, 839
deficiency in food, 530,

531, 844, 845
See also various fluids and

tissues.
See also various calcium

salts.

importance to enzymotic
processes, 248, 249, 302-
307, 308, 474, 6,16, 617
salts in blood coagulation, 303,

304

sulphate in urine, 784
Calculi, cystine, 786

Heller's scheme for investigating,

788

intestinal, 500
mulberry, 786
pancreatic, 487
salivary, 436
urinary, 784
Caliphora larvae, fat formation, 535

sugar formation, 390
Calomel, effect upon excrement, 499
Calorific value, of carbon dioxide, 832

of diets, 829, 830, 834-836
of foods, 829
of oxygen, 832, 833
various diets, 873-876, 878

879

Calorific values, variations in, 865
Calorific urine quotient, 835
Cammidge's reaction for sugar, 769
Camphor, elimination in urine, 712, 743
Camphors, fate of in the body, 742
Cane-sugar. See Saccharose.
Capsule of crystalline lens, 167, 586
Caramel, 207, 218

Carbazol, behavior in animal body, 738
Carbohaemoglobin, 280
Carbohydrate (phosphatides), 237
Carbohydrates, 192-224

absorption, 509-512
action on intestinal putre-
faction, 495, 496, 688
action on protein-metab-
olism, 856, 857
as sparers of protein, 856
calorific value, 829, 833
combustion in body, 829
fate in stomach and in-
testines, 492

importance in acetone for-
mation, 772

inadequate supply of, 846
influence on protein met-
abolism, 833
in proteins, 83, 84, 164,

253, 254
in urine, 708-709

Carbohydrates, importance in fat forma-
tion, 536, 861
importance in glycogen
formation, 371, 372,374,
375

importance in muscular
activity, 562, 567, 568.
See also various carbo-
hydrates.

Carbolic urines, 691
Carbon dioxide, assimilation, 1, 2

action on secretion of

gastric juice, 440
calorific value, 831
determining tension in

blood, 814, 816
elimination during work,

869

elimination following car-
bohydrate introduc-
tion, 872

elimination in lungs, 817
in blood, 802-806, 814-

816

in intestines, 493
in lymph, 328, 805
in muscles, 561, 562, 567
in secretions, 806, 817
in stomach, 461
in transudates, 805-806
quantity in arterial blood,

802-807 "

elimination, during work
and rest, 562
567,686,869
dependence
upon diet,
537 800,872
dependence
upon tem-
p e r a t u re,
870

ellimination
t hrough
skin, 800

Carbon-dioxide haemoglobin, 280, 804
Carbon excretion, 827
Carbon monoxide blood, test for, 279
Carbon monoxide haemochromogen, 281
Carbon monoxide hemoglobin, 272, 278,

281, 291

Carbon-monoxide methsemoglobin, 279
Carbon monoxide poisoning, 278, 381, 554,

646

Carnaubyl aclohol, 797
Carniferrine, 550
Carnine, 182, 545, 548
Carnitine, 545, 549
Carnomuscarine, 549
Carnosine, 546, 549
Carp eggs, 170

sperms, 110
Cartilage, 117,521-525

digestion of, 449, 485
Cartilage gelatin, 524

GENERAL INDEX

'939

Caseid, 616

Casein, 90, 106,1-61,615

behavior to gastric juice, 104, 446,

459, 619
behavior towards rennin, 10, 248,

451, 616

behavior towards trypsin, 63
calorific value, 829
from cow's milk, 615-620
from woman's milk, 627, 628
hexonebases in, 161
products of digestion of, 619
Caseinokyrin, 136
Caseoses, 129
Castoreum, 797
Castorin, 797
Castor lipase, 227
Catabolism, protein, upper and lower

limits of, 857
Catalases, function of, 7
Catalases, 12. Also see Fluids and tissues.
Catalysis, 53
Catalytes, 11,53-58
Catalytic reactions, laws of, 61

processes in homogeneous

systems, 56
processes in heterogeneous

systems, 57
processes, reversibility of, 64

reactions, 54
Cataphoresis, 42
Cataract of lens, 588
Cat milk, 626
Cell, animal, 19-24

constituents, primary and secondary,

20

fibrinogen, 349
globulin, 267
membrane, 24, 449
Cells, animal and plant, action of gastric

juice on, 449
boundary layer of, 21
delomorphic and adelomorphic, 436
lymphoid, 293
mineral constituents of, 22
permeability of, 34
Cellobiose, 224
Cellose, 224
Cellulose, 220, 224, 790, 821

digestion of, 488, 493, 518
fermentation of, 224, 489, 493
in lungs, 821
Celluloses, 220
Cement, of teeth, 531
Cephalin, 238, 575, 582, 629
Cephalopoda, flesh of, 546, 573
collagen, 117
liver, 367
Cerebrin, 346, 577, 579

in pus, 346

Cerebron, 575, 577, 580
Cerebrosides, 575, 577, 578
Cerebrospinal fluid and its constituents,

335, 341, 583
Cerolein, 232

Cerumen, 797

Cetin, 231

Cetyl alcohol, 232, 596, 796

Chalaza, 601

Charcoal bone, ability to absorb trypsin,

669

Charcot's crystals, 324, 590, 821
" Chemical tonus," 562
Chief (adelomorphic) cells, 436, 452, 454
Chitin, 121, 214, 790, 791

, action of trypsin on, 485
Chitose, 214
Chitosamine, 164, 213
Chitosan, 164, 791
Chloral hydrate, behavior in animal body,

712, 736, 737
effect upon secretions,

394, 466, 476
Chloral secretin, 394, 476
Chloramine, 40
Chlorbenzene, behavior in animal body,

743

Chloresterin fat as a protection, 796
Chlorides, action on protein metabolism,

862
excretion through perspiration,

799
excretion through urine, 719-

722

insufficient supply of, 844
See also various fluids and

tissues.

Volhard's method of determin-
ing chlorides, 721
Chlorine in blood, 316
Chloroform, influence upon elimination

of chlorine, 720
behavior in animal body, 720,

735

influence upon muscles. 560
influence upon protein, 96
Chlorometer, 722
Chlorosis, 323
Chlortoluene, behavior in animal body,

740

Chlorine, in teeth, 532
Chlorochrome, 363
Chlorocruorin, 292
Chlorophan, 586
Chlorophyll, 2, 795

relation to blood pigments,

269, 288
relation to bile pigments,

410

Cholagogues, 394
Cholecyanin, 408
Choleprasin, 405, 410
Cholepyrrhin, 405
Cholera bacilli, behavior toward gastric

juice, 461
Cholera, blood, 317

perspiration, 799
a-Cholestanol, 421
/?-Cholestanol, 421
Cholestenon, 421

940

GENERAL INDEX

Cholesteriline, 420
Cholesterin, 420-425

behavior toward saponin,

424

in bile, 395, 41 1-414
in blood, 256, 267, 292
in brain, 575, 581,582
in cells, 20-21
in feces, 498, 499
in gallstones, 419-420
in skin secretions, 796
in urine, 780
reactions for, 422, 423
. stones, 419, 420
Cholesterin ester, 257, 340, 422, 423, 582,

796

Cholesterone, 420
Chondrigen, 117, 521
Chondroalbuminoid, 523-526
Choletelin, 405, 705

Choline, 235, 239, 342, 354, 475, 549, 583
Cholohsematin, 410
Chondrin, 120
Chondrin-balls, 524
Chondroitin, 522
Chondromucoid, 168, 521-523, 526

preparation of, 523
Chondroproteins, 163, 168, 522, 523
Chondrosin, 522

from sponges, 167
Chorda saliva, 427
Choroid coat, 588

pigment of, 792, 793, 794
Chromhidrosis, 800
Chromogens, urinary, 702
Chromoproteins, 91, 92, 163
Chyle, 327-329, 512, 514

constituents of, 328
quantitative composition of. 328
Chyluria, 780
Chyme, 454-462

in intestines, 456, 465
investigation of, 462-464
Chymosin, 10, 16, 259, 450-453, 463
action of light upon, 59
action of on casein, 10
Circulating proteins, 850
Cleavage, hydrolytic, 16
Cleavage processes, 15
Clupeine, 110, 152

Coagulation of blood, 242, 243, 299-311
bodies retarding,

311

intravascular, 310
Coagulation, method of estimative pro-

teid, in urine, 749"
Coagulation test for proteids, 97
Coagulins, 307
Coaguloses, 134
Coapeptides, 135
Coaproteoses, 135
Cobra-poison, 300, 310
Cochineal, 795
Codfish, eggs, 599

sperms, 107

Co-enzymes, 60

Coffee, action on metabolism, 863

Collagen, 91, 117, 118, 485, 519, 521, 524,

525

Colloid, 165, 354, 593
Colloid cysts, 593
Colloid envelope, 45
Colloid from uterine fibroma, 596
Colloid polysacharides, 193, 229
Colloids, 36-54, 95

filterability, 40
hydrophile and suspension, 37
internal friction, 42
opt. properties, 41
osmotic pressure, 39
precipitation of, 43-52
protective, 44
Conglutin, 829, 830
Colon, effect of extirpation of, 518
Coloring matters, elimination in urine,

744

Coloring matters, medicinal, in urine, de-
tecting, 757. Also see various pig-
ments.

Colostrum, 625, 631

Combustion, physiological heat of, 830
Complements, 70
Conalbumin, 602
Conchiolin, 121, 122
Concrements. See various calculi.
Concretions, lung, 821
Conjugated glucuronates, in urine, 712
Connective tissue, 519-521
Copper, occurrence in blood, 261, 316
in bile, 395, 411, 420
in liver, 367
in pigments, 292
Cornea, 525, 588
Cornea mucoid, 525, 526
Cornein, 9 1,12 1,122
Cornicrystallin, 122
Corpora lutea, 290, 592
Corpse-wax, 534
Corpus callosum, 582, 583
Corpuscles, blood, effect of various solu-
tions on, 32, 33. See also Blood cor-
puscles.

Corpuscles, Gluge's, 593
Corpuscula amylacea, 581
Cover cells, 436, 452, 454
Crab extract, 550
Crangitine, 550
Crangonine, 550
Creatine, 161, 546, 547, 548, 659
anhydride, 658
detection of, 548
in brain, 576
in muscle, 547
relation to muscular activity,

547, 565
relation to urea formation, 547,

648
Creatinine, 161, 547, 548, 658-663

relation to muscular activity,
565, 567, 659

GENERAL INDEX

941

Creatinine, in muscle, 573

preparation and estimation, 662
zinc chloride, 661
Croner-Conheim's test for lactic acid in

gastric juice, 464
Crude fibre, digestion of, 518
Crude silk, 121, 122
Cruor, 244
Cruorin, purple, 275
Crusocreatinin, 550
Crustaceorubin, 795
Crusta inflammatoria, 299
Crusta phlogistica, 299
Crystalbumin, 587
Crystalfibrin, 587
Crystallin, a and ?, 587
Crystalline lens, 586, 588
Crystalloid, 36

spherules, 93
Cuorin, 233, 241, 557
Curare, 331, 381, 562, 871
Cyanha^moglobin, 277
Cyanhydrins, formation of, 196
Cyanmethaemoglobin, 277
Cyanocrystallin, 605, 795
Cyanurin, 703
Cyclopterine, 110

Cymene, oxidation of into cumic acid, 739
Cyprinine, 110, 160, 162
Cyprinine ( i ), hexone bases in, 161
Cysteine, 15, 78, 146-148

behavior in body, 736, 743
disulphide, 146

Cystic fluid, protein bodies in, 595
Cystine, 79, 80, 81, 113-114, 146, 148, 780
amounts in proteins, 106, 107,

113, 114, 122

behavior in animal body, 417, 736
in perspiration, 800
in sediment, 780, 781
in urinary calculi, 780, 786
in urine, 714, 780, 784
Cystinuria, 25, 147, 718, 780
Cysts, 593-596

colloid, 593
dermoid, 596
echinococcus, 343, 792
intraligamentary, 596
myxoid, 593
ovarian, 592
papillary, 596
paraovarial, 596
proliferous, 593
serous, 592
tubo-ovarial, 596
Cytin, 349

Cytoglobin, 292, 302, 349
Cytosine, 174-175, 689
Cytotoxins, 70, 259
Cytozym, 306

Deamidation, 390, 505, 556, 668, 734
Dehydrocholon, 396, 401
Dehydrochloride haemin, 286
Delomorphic cells, 436

Denige's reaction for uric acid, 673

test for tyrosine, 152
Dentin, 528, 531, 532
Dermocerin, 796
Dermoid cysts, 232, 596, 796
Dermolien, 796

Descemet's membrane, 167, 525, 587
Desamidoprotein, 78
Desoxyhsematoporphyrin, 284
Deuterocaseoses, 129
Deuteroelastose, 116
Deuterogelatose, 120
Deuteromyosinose, 130
Deuteroproteose, 129
Deuterosponginose, 122
Deuterovitellose, 130
Development, work of, 609
Dextrin, 222, 432, 433

effect on gastric secretion, 454
in gastric contents, 456
in portal blood, 509
Dextrins, 220, 222

separation of, 223
Dextrose, 192-196, 204-209

absorption, 509-511
calorific power, 829
detection of, 209, 758-763
in blood, 257, 314-316, 328.

737-381
in lymph, 328
in urine, 378, 711, 757-758
in vitreous humor, 586
pure preparation of, 209
quantitative estimation, 762-767
Diamine, 24, 131, 159, 719, 781
Diabetes mellitus, 382-392, 757

elimination of am-
monia by the urine,
in, 640, 728, 731, 757-
758

origin of sugar eliminated in, 388
Diabetic sugar,. 206
Dialysis, 36
Diastase, malt, 9

pancreatic, 478
Diastases, 16, 222. See also various

fluids and tissues.
Diastatic enzyme in urine, 718
Diazobenzenesulphonic-acid test for dex-
trose, 209

Diazo-reaction, Ehrlich, 715
Dibenzoylornithine, 159
Diet, average daily adult, 874
Diets of laborers, 874, 875

for people in poorhouses, 880

for prisons and workhouses, 880

of people in different vocations, 874,

875, 878
of prisoners, 880
Diffusion, 26, 95, 332
Digestibility of foods, 457, 458, 460, 508,

511, 515

Digestion, Chapter 9
Digestion work, 871

influenc.e of, 872

942

GENERAL INDEX

Diglucosamine, 214
Diglycyl-glycine, 85
Dihydrocholesterin, 421, 423
Di-isobutyldiacy'piperazine, 142
Di-iodotyrosin, 123
Di-leucyl-cystine, 85
Di-leucyl-glycyl-glycine, 85
Dimethylaminobenzaldehyde, reaction,

99-100,
708, 779
behavior in
animal or-
ganism,
743

Dimethylfulvene, 7
Dimethyl guanidine in urine, 663, 718
Dimethylindol, 693
p-Dimethyltoluidine, 743
Dimethylxanthine, 677
Dioxyacetone, 205
Dioxybenzene, 738
o-Dioxybenzene, 690
p-Dioxybenzene, 691
Dioxymethylene creatinine, 548
Dioxynaphthaline, 738
Dioxypurine, 184
Dioxypyrimidine, 190
Dipalmitylolein, 226
Dipentosamine, 213
Dipeptides, 85-89

behavior towards enzymes,

88, 486

Diphosphatides, 232
Disaccharides, 193, 217-219

relation to formation of gly-

cogen, 375
Disperse phase, 47
Dispersion means, 47
Dissociation molecular, calculation of de-
gree of, 29, 30

Di-stearyl lecithin, 146, 234, 235
Di-stearyl-palmitin, 226
Di-stearylolein, 226
Di-tetraoxybutylpyrazine, 213
Donne's pus test, 755
Dotterplattchen, 93, 597, 605

drawing blood, 317, 323,

325-326
Dulcite, 194

relation to formation of glycogen,

372

Duodenal diabetes, 384
Dyslysins, 404

in feces, 498

Dysoxidizable substances, 4
Dyspeptone, 448
Dysproteoses, 129

nutritive value of, 855

Ear fluids, 588

Earths, results of lack of, 854

Earthy phosphates, elimination through
urine, 723-725, 729
solubility of, in pro-
tein-rich fluids, 531

Earthy phosphates, absorption, 517, 723
occurrence in bone
ash, 527, 529-531
See also various
earthy phosphates.
Echinococcus cyst, 167, 92

constituents, 343
Echinochrom, 292

Eck's fistula operation, 376, 506, 511, 650
Edestan, 108, 446
Edestin, 83, 102, 105, 158, 161, 446
absorption, 502
hexone bases on, 161
Eel-meat, 570

serum, 243, 310
Egg, 592

absorption in intestine, 507
albumin. See ovalbumin.
globulin, 601
hen's, 596-606
incubation of, 608
shell, 112, 114,604
shell membrane, 604
white, 601

absorption, 502
albumin of the, 601-603
calorific value, 829
mineral bodies of, 604
yolk, 596

Ehrlich's diazo reaction, 715, 779
glucosamine test, 214
side-chain theory, 71
test for bilirubin, 407
urine test, 779
Eicosyl alcohol, 796
Elaidin, 229
Elastin, 78, 91, 115, 116, 123, 161, 519

behavior with gastric juice, 449

behavior with trypsin, 485

composition of, 115

hexone bases in, 161

peptone, 116

properties of, 115

proteoses, 116

putrefaction products of, 116

absorption of, 255, 503
table of decomposition products

of, 124

Elastoses, 116, 129
Electrolytes amphoteric, 90, 93
Elephant, bones of, 527
milk of, 626
teeth of, 532
Emulsin, 198, 713

action of on isomaltose and

maltose, 65
Emulsoids, 37
Emydin, 605
Enamel of teeth, 531, 842
Encephalin, 577, 578, 580
Endoenzymes, 17
Endolymph, 588

Energy exchange in metabolism, calculat-
ing, 835
Energy metabolism, calculating, 830

GENERAL INDEX

943

Energy of various foods, 829
Enterokinase, 60, 472

in intestinal juice, 468
Enzymes, absorption of, 517
fat splitting, 478
glycolytic, 258
in egg yolk, 597
in general, 9-11, 16-18, 58-69
in liver, 365
in lungs, 820
in milk, 620
in urine, 718
laws of action, 60
lipolytic, 16

in lungs, 821
in blood, 259
of placenta, 608
proteolytic action of, on poly-

peptides, 88

proteolytic, action of, on pro-
teins, 80

See also various enzymes in
tissues, organs and fluids,
retardation., 67
reversibility, 64
specificity of, 66
zymogens, 11
Enzymotic cleavages, 64

processes, influence of salts

upon, 73
syntheses, 65

Epidermis, 112, 788, 789, 796
Epiguanine, 182, 676, 678
Episarkine, 182, 677
Epinephrin, 358
Epitoxoid, 71

Equilibrium constant, 53, 55
Erepsin, 442, 467-469

action of, on proteins, 60
action upon polypeptides, 486
in intestinal juice, 467
Ereptases, 480
Erythrite, relation to glycogen formation,

372

Erythrocytes. See Red blood corpuscles.
Erythrodextrin, 222, 434
Ery thr opsin . See Visual purple .
Esbach's method for estimating protein

in urine, 750
Ethal, 232
Ether, action on blood, 266

action on muscles, 560
action on proteins, 96
action on secretions, 439, 466
diazoacetic, decomposition by dif-
ferent acids, 55
Ethyl benzene, behavior in animal body,

738

Ethyl alcohol, action on metabolism, 863
action on muscles, 560
action on peptic digestion,

447

action on proteins, 95-97
action on secretions, 438,
440, 454, 458, 476

Ethyl alcohol, behavior in animal body.

735, 863

calorific value, 839, 863
food value, 863
in intestine, 491
passage of, into milk, 637
production by fermenta-
tion, 9, 199,207, 209,386r
38 , 655

butyrate, formation of, 65
mercaptan, behavior in animal

body, 736
secretin, 476
sulphide, from protein, 79

behavior in animal body,
736
Ethyl-sulphuric acid, behavior in animal

body, 736

Ethylene glycol, glycogen formation, 372
Euglobulin, 251
Euxanthon, 742
Excelsin, 108, 158
Exchange of force, 822, 828, 830. See also

Chapter 18, Metabolism.
Excrements, 498, 518, 824, 825

in biliary fistulas, 496
Excreta, amount for twenty-four hours,

824

regular and constant, 823
Excretin, 500
Exostosis, 530
Expectorations, 820, 821
Extinction coefficients, 291
Extirpation of large intestine, effect of,

517

Exudates, 327, 334-344
Eye, 584-588

Fat action on catabolism of proteins, 855
formation, from protein, 534-536, 849
from carbohydrates, 3, 536,

849

in muscle, 570, 577
in urine, 780
metobolism, in work and rest, 567.

568

in starvation, 836-839
with various diets, 849,

856

metabolism of, 849
milk, determining, 624
passage into blood, 511
passage into chyle, 513
perspiration, 797
Fats, 225-232

absorption, 512-517
action on gastric juice, 439, 458
action on pancreatic juice, 474
action on secretion of bile, 393
and sugar formation, 390-392
calorific value, 829, 830, 833
combustion in body, 829
decomposition of during starvation,

838
. digestion of, 449, 452, 478

944

GENERAL INDEX

Fats, emulsion, 227, 479, 489, 512-514

influence on protein matabolism, 833
iodized, behavior in animal body,

534, 635

neutral, in brain and nerves, 575
nutritive value, of 833, 855-857, 861
relation to formation of acetone, 774,

775
relation to formation of glycogen,

372 373

relation to work, 569, 568
saponification of, 16, 227, 478, 512
sparing action of, on proteins, 856
synthe es, 65, 479, 512, 534
Fattening, 850, 860, 861
Fatty degeneration, 363, 534
Fatty migration, 363, 534-535
Fatty tissue, 532

relation to gastric juice, 532,

449
Feathers, 112, 114

pigments of, 795
Feces, alcoholic, 499

and their constituents, 498
formation, 499

reaction, 499, also see Excrements.
Fehling's solution, 207
Fermentation, 8, 9, 199, 200
alcoholic, 8

alcoholic, procedure of, 59
in gastric contents, 460, 461
See also various kinds of

fermentation,
in intestine, 490, 509
, in urine, 710, 782, 785
Fermentation lactic acid, in the stomach,
9, 200, 207,
460, 554
in intestines, 489

491, 509
in milk, 612, 621

Fermentation processes in intestine, 490
Fermentation saccharometer, 760, 767
Fermentation test in urine, 760, 761, 766,

767, 769

Fermentation test for sugar, 760
Ferments, 8-11, also see various enzymes.
Ferratin, 362
Ferrine, 363

Fertilization, artificial of, eggs, 74, 607, 608
Fever, elimination of ammonia, 728
elimination of creatinine, 660
elimination of potassium salts, 727
elimination of urea, 646
protein metabolism in, 646
Fibrils, composition of , 519
Fibrin, 83, 106, 243, 246, 255
calculi, 501, 787
coagulation, 248-250, 301-311
constituents, 303
digestion, 445,-447, 481
digestion, products of, 129
ferment, 16, 244, 246, 248-250,

301-311
globulin, 246, 250, 251, 255

Fibrin, Henle's, 589

Fibrinogen, 90, 244-248, 255,262, 302-304,

307, 310, 528
quantity of, in blood 319
tissue, 295

Fibrinolysifc, 247, 309
Fibrinoplastic substance. See serglobulin

and zymoplastic substances.
Fibroin, 89,91, 122,123

table c-f decomposition products,

124

Filtration, 331, 332, 334, 335
Fischer- Weidel's reaction, 185
Fish, eggs, 93, 105, 235, 605, 607
. air bladder, 185, 817
bones, 529
scales, 121, 185
sperms, 109, 177, 591, 592
FleischTs haemometer, 292
Flesh, calorific value, 571, 830, 834
composition of, 569-572
deposition of in the body, 860
nitrogen in, 571
"quotient," 571
Florence's sperm reaction, 590
Fluid, animal, osmotic pressure of, 34

of echinococcus cyst, 343
Fluids of eye, 484

of inner ear, 588
Fluorine and fluorides, 22, 243, 261, 305,

527, 532

Fly maggots, fat formation, 535
Folin's method of determining urea, 656
Food, artificial, 847

effect on blood, 321

effect on urine, 505, 645-646, 659,

665,684,688,710
influence of, on composition! of

human milk, 634
influence on metabolism, 8.71
influence on secretion of bile, 393
influence on secretion of gastric

juice, 438
influence on secretion of intestinal

juice, 465

influence on secretion of milk, 634
influence on secretion of pancreatic

juice, 470-472

insufficient supply of, 840-847
.necessity of, under various condi-
tions, 873

rapidity of digestion, 457
required in work and rest, 879
various, 841-846, 848, 855-816
Foods, digestibility of various, 875
energy of various, 829
influence of, on acidity of urine, 641
various, isodynamic value of, 834
Foodstuffs, necessity of, 822

determining heat value of, 834
heat of combustion, 829, 830
Formaldehyde, 1, 2, 205

relation to glycogen for-
mation, 375
titration method, 162

GENERAL INDEX

945

Formaldehyde, formation by silent electric

charge, 1

formation of from car-
bonic acid, 2
transformation into sugar,

1
Freezing-point, calculating the depression

of, 29, 32

Frog egg, 164,215,605
Fructosazine, 213
Fructose. See Levulose.
Fruit-sugar, 210 See Levulose
Fuld-Levison's method of testing pepsin,

446

Fundus glands, 436
Fiirbringer's test for proteid, 747
Furfurol, 741

from pentoses, 202
behavior in animal organism, 741
Fuscin, 586

Gaduhistone, 108
Galactase, 621, 769
Galactosamine, 83, 171, 215
Galactose, 194, 105, 199, 205, 210, 222, 621
absorption, 509, 510
from cerebrins, 578, 579
passage into urine, 768
relation to formation of glyco-

gen, 375

Gallacetophenone, 742
Gall bladder, secretion of, 394, 415
Gallois' inosite test, 552
Ganassini's reaction for uric acid, 673
Gas, exchange between blood and pul-
monary air and the tissues, 808
exchange, determining, 827
exchange during starvation, 838
exchange in the lungs, 809
exchange in the tissue, 809
exchange in various ages, 866
exchange of, through the skin, 800
in basal requirement, 841
in rest and work, 869
Gases, in bile, 414, 808, 818-819

in blood serum, 261, 312, 801-806
in gastric contents, 461
' in hen's eggs, 605, 607
in lymph, 328, 807
in muscles, 560, 567
in saliva, 427-428, 807
in transudates, 336, 807
in urine, 730, 808, 818-819
secretion of, in animals, 819
Gastric juice, 437-455

action of 444-452, 454
action of mechanical irri-
tation on, 466
antifermentative and anti-
toxic action of, 461
composition of, 440-442
detecting free hydrochloric

acid in, 463

1 determining acidity, 463-
464

Gastric juice, secretion of, 437-441, 454-

455, 476

Gastric lipase, 442, 452
Gastric secretion, excitants of, 439
Gay-Lussac's law, 28
Geissler's albumin-test papers, 747
Gelatin, 77, 78, 95, 116-119, 124, 161

action of acids and alkalies upon.

52

conversion into protein, 853
digestion of, 449, 481, 485
hexone bases in, 161
nutritive value, 853, 854
oxidation products of, 82, 118
.peptones, 119

physiological availability of, 853
preparation of, 117
properties of, 119
relation to coagulation of blood,
310
relation to formation of glycogen,

372, 373
sugar, 118, 138
table of decomposition products

of, 124
value of, 854

Gelatin- giving tissues. See Collagen.
Gelatinous tissue, 520, 586
Gelatone-peptone hydrochlorides, 119-

121, 136, 448
Gelatoses, 119,243,448
Gels, 51

Gerhardt's test for acetoacetic acid, 777
" Ghosts," 266
Gliadin, 89, 105, 107

digestion of, 457, 459
Globan, 103

Globin, 107, 157, 256, 281, 290
Globulicidal bodies, 266
Globulins, 90, 91, 92, 102, 103, 106
in brain, 574
in hunger, 261, 262
in urine, 744, 747, 749

See also various globulins.
Globuloses, 129
Glucocyanhydrin, 196
Glucopeptose, 196
Glucoproteins, 92, 163
Glucoproteose, 133

Glucosamine, 164-166, 197, 213, 252, 254,
256, 594, 601, 602, 604,
791
relation to formation of

glycogen, 375-376
Gluconose, 199
Glucosan, 206
Glucose. See Dextrose.
Glucosides, 19, 198, 713
Glucoside-splitting enzymes, 16, 198, 467
Glucosoxime, 196

Glucuronates, conjugated, in urine, 712
Glucurone, 216

Glucuronic acid. See Acids, glucuronic.
Gluge's corpuscles, 592
Gluteins, 118

946

GENERAL INDEX

Glutelins, 91, 92

Gluten proteid, 77, 106, 107, 160, 161

Glutin, 91

properties of , 119
proteids, hexone bases in, 161
^-Glutin, 119
Glutinase, 481

Glutin casein, hexone bases in, 161
Glutokyrin, 136
Glycine. See Glycocoll.
Glycerine, in blood, 256

relation to glycogen formation,

372, 375, 390
Glyceroses, 205
Glycin anhydride, 86
Glycin. See Glycocoll.
Glycocoll, 84, 102, 138

amounts in proteins, 106-107,

114, 124
inbk>od,260
in urine, 718
relation to formation of urea,

648, 649, 734
relation to formation of uric

acid, 669, 670
synthesis with, 3, 395, 685, 686,

738, 739

Glycocyamine, 659
Glycogen, 20, 222, 295, 328, 346, 368-377,

553, 573, 608
carbohydrates in the formation

of, 374
formers, 374
in liver, 360
in lungs, 820
in muscles, 368
in placenta, 688
in starvation, 369, 553, 837
preparation and quantitative

estimation of, 371
proteins in the formation of, 373
relation to muscular work, 563,

567

relation to rigor mortis, 561
Glycolaldehyde, formation by silent elec-
tric charge, 1

Glycolysis, 258, 386-388, 544
Glycolytic enzyme, 258
Glycoproteins, 91, 92, 163-170

relation to formation of

glycogen, 373, 376
Glycosuria, 379-390, 758

alimentary, 380, 510
Glycylalanine, 85, 89, 486
Glycylasparaginyl leucine, 86
Glycylglycin, 62, 86
Glycyl leucine, 89
Glycyl proline anhydride, 89
Glycyl tryosine, 89, 487, 699
Glycyl valanine anhydride, 89
Glyoxyldiureide, 681. See also Allantoin
Gmelin's test, for bile pigments, 407

in urine, 756
Goitre, 354
Gold equivalent, 44, 96

Gorgonin, 122

Gout, 666, 718

Graafian follicles, 592

Grape-mole, 609

Grape sugar. See Glucose.

Guaiactest, 11, 12

for blood, 274, 752

Guanadine, 77, 82, 118, 159, 484, 595, 65S
Guanase, 16, 183, 360, 351, 668
Guanine, 182, 183, 185
in urine, 676

preparation and detection of, 188-
Guano, 184, 185, 665
Guanosin, 179, 181
Guanovulit, 605
Guldberg and Waage's law, 53
Gulonic acid lactone, 215
Gulose, 204

Gums, 204, 205, 220, 222, 224
animal, 165,622, 711
vegetable, 223

Gunning's modified Lieben's test, 775
Gunzburg's test for HC1 in gastric juicer
Steensma's modification of, 46S
Gynesin in urine, 718
Haemagglutination, 268
Hsemaphilia, 312
Hsemase, 62
Hsemataerometer, 812
Hsematin, 269, 273, 282-284
neutral, 208
reduced, 281
Haematingen, 289
Haematinometer, 290
Haematoblasts, 295
Hsematocrit, 313
Haematocrystallin, 271
Hsematogen, 598

HaematogloDulin, See Oxyhaemoglobin*
Hsematoidin, 290

in urine, 784

relation to bilirubin, 290r

405, 406, 416

Haematoporphyrin, 282, 284, 287-289, 795
in lower animals, 795
in urine, 287, 702, 753,

754
relation to bilirubin,

288, 406, 418
relation to chlorophyl,

269, 288
relation to urobilin,

287, 418, 705

Haematoscope, Henocque's, 292
Haematuria, 27G, 7.YJ
Haemerythrin, 292
Hsemin, 283, 285-287, 290, 753
crystals, 285, 286, 753
Haemochrom, 268, 272
Haemochromogen, 269, 281, 282
alkaline, 282
Haemocyanin, 91, 292
Haemoglobin, 3, 91, 92, 267, 269, 270, 275
amounts in blood, 269, 316,
318, 320, 321

GENERAL INDEX

917

Haemoglobin, in muscles, 573

See also Oxy haemoglobin,
-nitric oxide, 280
Hsemoglobinuria, 276, 752
Hsemolysines, 70, 259, 266, 424
Haemolysis, 266
Haemometer, Fleischl's, 292

Sahli's, 292

Haemopyrrol, 269, 284, 288, 406, 794
Haemorrhodin, 280
Haemoverdin, 280
Hair, 111-113, 790
Hair balls, 501

pigments, 792, 793, 794
Halogenized proteins, 81, 121
Hammarsten's test for bile pigments, 408,

757

Haptogen membrane, 613
Haptophore group, 71
Harriot-Richet's method of determining

respiratory gas exchange, 820
Haser's coefficient, 731
Hatching of eggs, 606, 607
Heat regulation, 870, 871
Hedenius' bilirubin test, 411
Helicoproteid, 170
Heller's blood test, 753

scheme for investigating calculi,

788

Heller-Teichmann's test, 753
in urine, 745
test for proteid, 98
Hemicelluloses, 222, 224
Hemicollin, 119
Hemielastin, 116, 256, 503
Hemindigotin, 694
Hemipeptone, 128
Hemolysins, 70
Hemp seed calculi, 786
Henle's fibrin, 589
Henocque's hsematoscope, 292
Hen's egg, 596, 608

incubation, 606, 607

white of, 601-603

white of, digestion of, 447, 459,

460, 484, 491
Heparphosphatide, 364
Hepatopancreas, 469
Hepatothrombin, 308
Heptapeptides, 85, 87
Heptose, 193

in urine, 770

Herring, spermatozoa, of 110, 177, 592°
Heterocasepses, 129
Heterocyclic compounds, passage into

urine, 736
Heterolysis, 18
Heteroproteose, nutritive value of, 108,

129, 130, 132, 133
food value, 855
Heterosponginose, 122
Heterosyntonose, hexone bases in, 161
Heterogeneous systems, 56
Heteroxanthine, 182, 676
Hexapetides, 85

Hexobioses, 217

Hexone bases, 110, 111, 116, 157-162

in proteins, table of, 161
Hexoses, 193,204-211
High altitudes, effects on blood, 322

effects on metabolism,

871
effects on perspiration,

799

effects on urine, 718
Hippokoprosterin, 423
Hippomelanin, 793
Hirudin, 245, 305
Histidine, 84, 157, 158

amounts in proteins, 106, 107,

111, 122, 161
in urine, 719
Histidyl-histidine, 85
Histone-peptone, 109
Histones, 90, 91, 92, 107, 161, 267, 303,

348, 349

hexone bases in, 161
in urine, 751
Histozyme, 16, 687
Hoffmann's tyrosine test, 152
Holosym, 306
Holothuria, mucin of, 167
Homocerebrin, 577, 578
Homocyclic compounds, passage into

urine, 736

Homogeneous systems, 56
Hoppe-Seyler's CO blood test, 279

colorimetric method, 290
test for bile acid, 175
Hordein, 107, 152
Hormones, 385
Horn. See Keratin.
Hiifner's bile, 398
Human fat, 226, 228, 533
Humor, aqueous, 335, 342, 586
Huppert's test for bile pigments, 407,

408, 756

Huppert-Messinger method of estimat-
ing acetone, 778

Huppert-Schiitz's method of testing pep-
sin, 446

Hyaline, 167, 792
Hyaline substance, 267
Hyalogens, 163, 167
Hyalomucoid, in vitreous humor, 586
Hydraemia, 323, 335
Hydramnion, 609
Hydrazines, asymmetric, as test for

ketoses, 212

Hydrazine poisoning, 682
Hydrazones, 198
Hydrobilirubin, 406, 705
Hydrocele fluids and their constituents,

335, 337, 340
Hydrogel, 36
Hydrogenases, 14

Hydrogen, in putrefaction and fermenta-
tion, 5, 81, 493
sulphuretted, in urine, 715
peroxide in urine, 730

948

GENERAL INDEX

Hydrolysis, general, 16. See also various

cleavages.

Hydronephrosis fluid, 639
Hydroquinone, 691
Hydrosol, 36

Hydroxylainine poisoning, 682
Hyperacidity, 462
Hyperglycaemia, 380
Hyperisotonic solutions, 265
Hyperthyreoidismus, 356
Hypertonic solutions, 30
Hypnotics, and glycogen formation, 372
Hypoisotonic, 265 .
Hyposulphites, in urine, 715, 736
Hypotonic solutions, 30
Hypoxanthine, 179, 182, 183, 185, 573

in urine, 676

preparation and detection,
of, 188

Ichthidin, 598, 605

Ichthin, 605

Ichthulin, 91, 170, 598, 605

Ichthylepidin, 121

Icterne, 392, 406, 409, 418, 419

urine, 756
Ignotine, 549

Ileum, effect of extirpation of, 518
Imidazol, derivatives in urine, 719, 779
Imidazole, 181, 213
Immune bodies, 70, 71, 252, 259
Indican elimination, 494, 691, 692

elimination during starvation, 494
excretion of, 692. See also Acids,

indoxylsuphuric.
test, Jaffe-Obermeyer's, 693
Indigo blue, 693

in urine, 784
red, 694-696
Indirubin, 694

Indol, 155, 156, 260, 492, 687, 691
Indophenol, synthetic production of, 12
Indoxyl red. See Indol.
Infraproteins, 92
Inosine, 548
Inosite, 551, 552

in brain, 576

in muscle, 573

in urine, 771

relation to formation of glycogen,

371

test, Gallois', 552
Scherer's, 552
Inositogen, 551

" Integral Factor " in uric acid elimina-
tion, 671

Intestinal concrements, 500
gases, 493

juice, investigations on, 465-469
proteins, 468

Intestine, absorption, 501-519
digestion, 488-499
extirpation, 517, 518
large, 469
putrefaction, 490-497

Intestine, reaction of contents, 497, 513

small, 465-468,491,492
Intraligamentary cysts, 596
Intravascular coagulation of blood, 310
Inulin, 210, 221

relation to formation of glycogen,

371

relation to secretion of pepsin, 454
Inversion 9, 10, 10, 217, 449, 467, 509
Invertase, in intestinal juice, 467
Invert sugar, 217
Iodine, 22

in blood, 261, 316, 320
in glands, 356, 357
in perspiration, 799
in proteins, 81, 123
number of fats, 231
Iodine and gastric juice, 453

combinations, passage into the

milk, 637

passage into the

perspiration, 800

passage into the

saliva,435
haemin, 287
Iodized fat, 534, 635

lodoform, behavior in animal body, 735
lodospongin, 122
lodothyreoglobulin, 354, 356
lodothyrin, 349, 354, 356
Ion-action and enzyme action, correspond-
ence between, 73
Ions, action, 73-75

permeability, 32-33
theories, 29, 30

Iron, and formation of blood, 322, 606
absorption, 321-322
and spleen, 353
elimination of, 411, 418, 730
formation of bile pigments, 418
in blood, 261
in newly born, 366, 632
in urine, 730
presence in oxidases, 13
results in lack of, 846. See also

various tissues and fluids,
starvation, 846
Isobutyl alcohol, behavior in animal body,

736

Isocasein, 616
Isocholesterin, 423, 796, 797
Isocreatinine, 546
Isocysteine, 147
Isocytosine, 190

Isodynamic value of various foods, 834
Isoleucine, 84, 142
Isomaltose, 65, 217, 219, 257, 711
Isosaccharine, 372
Isoserine, 144
Isotonic solution, 30, 265
Isotropous muscle substance, 538

Jacoby's method of testing pepsin, 446
Jaffe-Obermayer's indican test, 693
Jaffe's creatinine reaction, 661

GENERAL INDEX

949

Janthinin, 795
Japanese, diet for, 875
Jaime indien, 216
Jecorin, 257, 351, 364
in brain, 575

Jejunum, effect of extirpation of, 518
Jerusalem's test for lactic acid in gastric

juice, 464
Juice, gastric, 437. Also see Gastric

juice.

Karyogen, 592

Kathsemoglobin, 280

Kephir,495,621,626

Kephir-lactase, action of, on galactose

and dextrose, 65
Kerasin, 577, 578, 582 .
Keratin, action of gastric juice on, 449

cleavage products of , 113
Keratins, 91, 112-115, 789
properties of, 112
table of various, 114

Ketone, behavior in animal body, 736, 737
Ketoses, 193, 194, 205
Kidneys, 638, 639

relation to formation of urea, 652
relation to formation of hippuric

acid, 686

Kinase, in thrombin formation, 306
occurrence in intestine, 468
Kinases, 11. Also see Blood coagulation

and Pancreatic juice.

Kjeldahl's method of determining nitro-
gen, 654
Klupeovin, 605
Knapp's method of estimating sugar, 765

reaction for dextrose. 208
Knop-Hufner's method of determining

urea, 657
Kolin, 115

table of decomposition products of,

124

Koprosterin, 421, 423, 498
Kramm's creatinine reaction, 662
Krinosin, 581
Kumyss, 621, 626
Kyestein, 784
Kyrin, 136

Laccase, 12
Lactacidase, 200
Lactalbumin, 90, 620
Lactase, 467, 473, 509, 621

formation of in intestine, 473

in intestinal juice, 467
Lactocrit, 624
Lactoglobulin, 619
Lactoprotein, 620
Lactose, 217, 219, 621, 622

absorption, 509-510

calorific value, 829

in urine, 721, 767

origin of, 610, 636

reactions, 622
La^tosuria, 768

Laiose in urine, 768

Lake color of blood, 299

Lambs, intestinal juice, 465-467

Langerhans, islands of, 470

Lanocerin, 797

Lanolin, 424

Lanugo hair, 609

Lard, absorption of, 514

Large intestine, effect of extirpation oL

517

Latebra, 596
Lead, in blood, 316

in the liver, 367
passage into milk, 637
Lecithalbumin, 103-105, 437, 638
Lecithin, in bile, importance of, 511
in brain, 575
in the lungs, 820. See also

various tissues and fluids.
Lecithins, 232, 234-238

preparation and estimation oL

238

relation to absorption, 512
relation to haemolysis, 265
relation to protoplasm boun-
dary layer, 21, 22
relation to sj^nthesis of cell

constituents, 845
Lecithin sugar, 237, 257, 364
Lecithoproteins, 91
Leech, infusion of, 243, 300, 305, 332
Legal's test for acetone, 776
Legumin, 107
Lens fibers, 578
Leo's sugar, 767
Lepidoporphyrin, 795
Lethal, 231

Leucaemia, blood, 183, 324, 664
spleen, 353
urine, 676, 724
Leucine, 84, 141-143

amounts in proteins, 106, 107,

114, 124
breaking down of, 14, 468, 649,

668, 734, 735
in brain, 576
in urine, 717, 780
relation to formation of acetone.

772

synthetic, 140
Leucinimide, 142

synthetic, 142
Leucocyte protoplasm, constituents of,

295
Leucocytes, action of peptone or proteoses

on, 312

constituents, 302
increase in number, 324
influence of, on taking up pro-
teoses, 505
polynuclear, 293
relation to absorption, 505,

507

See also White blood cor-
puscles.

950

GENERAL INDEX

Leucomaines, 25

, in urine, 550, 718
Leuconuclein, 303, 348
Leucyl alanine anhydride, 89
alanyl glycine, 85
diglycyl glycine, 88
leucine, 87-88, 88
pentaglycyl gylcine, 87
tyrosine, 85
Levulin, 222
Levulose, 190, 191, 199, 205, 206, 210, 211,

212, 257

in transudates, 210, 337, 609
in urine, 767
Lichenin, 322

Lieben's iodoform test for acetone, 775
Liebermann-Burchard's reaction for cho-

lesterin, 422

Liebig's method of determining urea, 654
Lienase,d-, 18, 351

Lifschiitz's reaction for cholesterin, 423
Ligamentum nuchse, 115, 116, 520
Lignin, 224

Linseed oil, feeding with, 534, 635
Lipanin, absorption of, 514
Lipase, 58, 60

gastric, 451
in blood, 258
in fat-tissue, 537
in intestine, 467, 468
in milk, 620

in pancreatic juice, 477, 479
Lipochrome, 260, 600
Lipoid,21,233,265,424

relation to development of the egg,

608
relation to protoplasmic boundary

layers, 21, 22

Lipoid sulphur, 575, 581, 582
Lipuria, 780
Lithium, 22, 3 16

lactate, 556
urate, 572
Liver, 360-368

action upon urine, 652
atrophy, acute, yellow, 18, 365
atrophy, effect on urine, 365, 554,

652,710,780
blood of, 318,378
cirrhosis of, ascitic fluid, in 340
elimination of lactic acid, 554, 668
elimination of urea, 651
elimination of uric acid, 668
extirpation of, elimination of

ammonia in, 651, 668
proteins of, 361,368
relation to coagulation of blood,

309, 312
relation to formation of fibrinogen,

244, 245
relation to formation of lymph, 333,

334

relation to formation of urea, 651 , 652
relation to formation of uric acid,
668, 670

Liver, sugar formation, 377, 378, 380-384

Livetin, 598

Lotahistone, 108

Lucerne-lacease, 13

Lung catheter, 815

Lung concretions, 821

Lungs, 808-809

and their expectorations, 820
. pigment and other bodies in, 821
Luteins, 260, 290

in blood serum, 260
. in corpora lutea, 290, 592
in yolk of egg, 599
relation to hsematoidin, 290,

592

Lymph, 327-334, 807
Lymphagogues, 331

and its constituents, 329, 330
extent of secretion, 331
formation of, 332
gases of, 807
Lymphatic glands and their constituents,

120, 244, 347
Lymphocytes, 293
Lymphoid cells. Also see white blood

corpuscles, 293
Lysatine, 160°
Lysatinine, 160
Lysine, 80, 85, 159, 160

amounts in proteins, 106, 107,

114, 124

Lysine peptone, 136
Lysylglycylpeptide, 89
Lysyllysine, 85

Mackerel, meat, 570

, spermatozoa, 107, 109
Magnesium salts, in urine, 723, 729, 782,

784, 839

in bones, 527, 531
.in calculi, 501,784

See also various tisues

and fluids.

in muscles, 558, 572, 573
Maintenance value, 841
Maltase, 65, 219, 258, 430, 431, 434, 467
Maltodextrin, 223
Maltose, 212-214, 218, 223, 375, 432, 434,

478, 488

absorption, 509-510
in urine, 768
Malt sugar, 218

Mammary gland, constituents of, 610
Manganese, 13, 261, 316, 420
Mannite, 194, 205, 210, 372
Mannose, 197, 200, 205, 206, 210, 224, 375
Manonose, 199
Marcitine, 24
Margarin, 228
Marrow, bone, 528
Marsh-gas as decomposition product in

intestine, 493

Maschke's reaction for creatinine, 661
Mass action, 53
Mastic emulsion, 96, 101, 133

GENERAL INDEX

951

Meat extract, 546, 548, 549

effect upon gastric secre-
tion, 438

Meat, metabolism of, 848. Also see flesh.
Meconium, 500
Medulla, 576

Medullary fibers, composition of, 583
Melanin, 359, 792-794
in the eye, 586
in urine, 755, 792
Melanogen, 755
Melanoidins, 77, 80, 794
Melanotic cancers, 755, 793, 794
Melissyl alcohol, 232
Membrane, semi-permeable, 26
Membranin, 167, 525

of capsule of lens, 586
Menthol, behavior in animal body, 742

elimination in urine, 743
Mercaptans, from protein substances, 78,

112, 492

Mercuric chloride in perspiration, 799
Mercuric salts poisoning, 23
Mesitylene, oxidation of into mesitylenic

acid, 739
Mesoinosite, 551
Mesoporphyrin, 288
Mesoxalyl urea, 664
Metabolism, 822

calculation of extent of, 826-

828, 832

dependence of external tem-
perature upon, 870
dependence of, on various

conditions, 854
influence of altitude on, 871
influence of hunger on, 836-

841

influence of light on, 870
influence of mental activity,

on, 870

influence of sex on, 867
influence of sleep on, 869
influence of various ages on,

865, 866*
influence of various foods on,

848-862, 872
influence of work and rest on,

562-569, 869
in muscles, 562
Metacasein reaction, 618
Metalbumin, 341, 594
Metanitrobenzaldehyde, fate of in the

body, 741
Metaproteins, 92
Metazym, 307

Metha3moglobin, 273, 276, 277
in urine, 752
Methal, 231
Methose, 205
Methylamine, 237
Methylenitan, 205

Methylethylmaleic acid anhydride, 283
Methylglycocoll. See Sarcosin.
Methylglyoxal, 197, 200

Methylguanidine, 548, 550, 661, 663, 678

in urine, 718
Methylimidazol, 197
Methylmercaptan, 80, 492, 493, 744

as decomposition prod-
uct in intestine, 493
Methylindol, 155. SeeSkatol.
Methylindolin, 693
Methyl pentoses, 192, 201
Methylphenylhydrazine test for levulose,

211
Methyl pyridine, behavior in animal body,

738, 741

chloride in urine, 718

Methylpyridylammonium hydroxide, 743
Methylthiophene, 741
Methyluracil, 190
Methyluramine, 548, 661
Methylurea, 658
Methylxanthine, 677
Z-Methylxanthine, 182
Mett's method of testing pepsin, 445
Micro-organisms in intestines, 488, 491,

494, 497

Microtonometer, 812
Milk, 610-637

behavior in stomach, 456, 459, 627
blue and red, 637

coagulation retarded by horse-
serum, 68
consumption of intestine, 508, 515,

627-628

fat, constituents, 614, 624, 627, 635
gases of, 625
globules, 612

human, characteristics and con-
stituents of, 627-633
lactic acid fermentation of, 612
mineral bodies of, 623, 625
of the ass, 626
of cow, 611-626

analysis, 623-626
coagulation with rennet, 612,

616

composition of, 624
general behavior, 611, 612
of the dog, 626, 632
of horses, 626
of pig, 626
of reindeer, 626
of sheep, 626
of various animals, composition of,

626

of woman. See Human milk,
plasma, constituents of, 614
proteins, determining, 623
secretion, chemistry of, 634-637
solids, determining, 623
sugar. See Lactose.
Millimoll, 43

Millon's reaction for proteids, 98, 100
Mineral bodies, in connective tissue, 521

in serum and plasma, 260,

263
of human milk, 633

952

GENERAL INDEX

Mineral substances, elimination in starva-
tion, 723, 725, 839
excretion of, 828
insufficient supply of,
842-846

See also various tis-
sues and fluids.
Mingin in ur e, 718
Mohr's reagent for HC1 in gastric juice,

463

Mole, 53

Molecular movement, 41
Molisch's test for sugar, 209
Monaminophosphatides, 234
Monobutyrin, 65
Monomethylxanthine, 676
Mononucleotides, 175
Monophosphatides, 232
Monosaccharides, 193, 200
Moore's test for sugar, 207, 217
Morner-Sjoqvist method of estimating

urea, 655
Morner's tyrosine test. See also Denige's

test.
Morphine, elimination in urine, 712, 744

in milk, 637
Moss starch, 222
Mucilages, 220

vegetable, 223. See also

mucin substances.
Mucin substances, 91, 163, 164

in urine, 718, 745, 746,

750

digestion of, 165

Mucin, action of gastric juice on, 449
action of trypsin on, 485
from tendons, 165,519
of holothuria, 167
Mucin-like substances, in bile, 394-395,

413

in kidneys, 639
in urine, 718-719,

746-746

Mucinogen, 165, 427, 605
Mucinoid, 167. See Mucoids.
Mucoids from tendons, 449, 519, 520, 526
Mucoid substances, 91, 164, 165, 167, 519
action of gastric juice

on, 449

in transudates, 339
See also various tis-
sues.

Mucoproteoses, 449
Mucous membrane of stomach, 436
tissues, 520
glands, 436
Mucus, buccal, 428
Mulberry calculi, 786
Multi rotation, 195
Murexide test, 673
Muscarine, 239
Muscle, fat in, 570
Muscle fibers, 538

permeability of, 34, 559
pigments, 545

Muscle-plasma, 539

proteins of, 543
-power, source of, 567, 568
-serum, 539
-snow, 539
-stroma, 542

substance, composition of, 569, 570
sugar, 553

-syntonin, 132, 162, 543
water in, 571

Muscles, amphoteric reaction of, 563
autolysis, 555
blood of, 3 19, 562
calorific value, 830
composition of, 569-572
extractive bodies of, 545-546,

549, 550
gases of, 559
metabolism in, 562
mineral constituents of, 558, 572
non-striated, 572
proteins in, 539, 565
rigor mortis of, 560
striated, 538-572
Musculamine, 550
Muscular work, chemical processes in, 15,

562-569

effect upon metabolism,
565-567, 660, 663, 724,
867

Musculin, 540, 541, -543-560
Mutton tallow, feeding with, 533

absorption, 514, 515
Myeline, 236, 239, 576, 582

" forms," 576
Myoalbumin, 540, 542
Myofibrin, 542
Myogen, 543

fibrin, 540, 544, 560, 561
Myoglobulin, 542
Myohsematin, 545
Myoproteid, 545
Myosin, 294, 540-545

absorption, 502
ferment, 543, 544
fibrin, 542
preparation of, 541
Myosinogen, 543
Myosinoses, 129
Myricin, 232
Myricyl alcohol, 232
Mytolin, 543
Myxcedema, 355, 520
Myxoid cysts, 593

Nails, 112, 113, 790

Naphthalene, conversion into oxy-
naphthalene in the body,
738-743
elimination of in urine, 743,

744
Naphthalene sulfo-derivatives of amino-

acids. See various ammo-acids.
Naphthol reagent for sugar, 209, 763

elimination of in urine, 712, 744

GENERAL INDEX

953

Naphthoresorcin, reaction for conjugated
glucuroric acid, 211,
216, 771
reaction for levulose,

211
Naphthylisocyanate compounds of amino-

acids. See various amino-acids.
Naphtindol, 693
Narcotics, relation to glycogen formation,

372

Navel cord, 164, 520, 521
Neosine, 549
Neossin,167
Neottin, 233, 599
Neozym, 307
Nepenthes enzyme, 442
Nerves, 22, 112, 574, 575, 583
Neuberg-Rauchwerger's reaction for cho-

lesterin, 422

Neuberg's test for levulose, 211
Neuridine, 576,581, 597
Neurine, 239, 549
Neurochitin, 583
Neuroglia, 575
Neuroglobulin a and 9, 574
Neurokeratin, 112, 575, 583
Neutral fats. See Fats.
Nicotine, effect upon gases of stomach, 461
Nitrates in urine, 5, 15, 726
Nitric-oxide haemoglobin, 280
Nitriles, fate in organism, 735
Nitrobenzaldehyde, behavior in animal

body, 739, 740
Nitrobenzene, conversion of into amido-

phenol, 739

Nitrobenzyl alcohol, 743
Nitrogen-carbon relation, 827
Nitrogen deficit, 825

elimination, during work and
rest, 565-567, 867
868
during starvation,

825, 837, 838, 841
relation to digest-
ive activity, 505,
852

through the intes-
tine, 507, 508, 824

through keratin
formations, 824

through perspira-
tion, 799, 825

through the urine,
646, 714, 723, 824

826, 837, 841, 852
transitory progress,

852

with various foods,
848-859
form of occurrence in proteins,

76
Nitrogen- (free), amounts in blood, 802

See also gases of various
fluids,
colloidal in the urine, 711

| Nitrogen-(free), determination of, 654

in flesh, 571

j Nitrogenous constituents, different, in
urine, 646, 665, 714

equilibrium, 825. See also

Chapter XVIII.
Nitrophenol conversion of into amido-

phenol, 739

Nitroso-indol nitrate, 156
Nitrotoluol, behavior in animal body, 743
Nitrotoluolsulfo compounds of amino-
acids. See various amino-acids.
Novaine, 549

in urine, 718
Nubecula, 639, 718, 781
Nuclease, action of trypsin on, 485
identity with erepsin, 469
in intestinal juice, 467
Nucleuses, 176, 350, 481
Nucleic acid. See Acids nucleic,
bases. See Purine bases,
in brain, 575
plates, 296
Nucleins, 172, 173

action of gastric juice on, 173,

449

action of trypsin on, 485
Nucleoalbumins, 20, 90, 103, 173, 348

action of pepsin-acid on>

448

behavior towards peptic
digestion, 104, 173,
448

in bile, 395, 415
in kidneys, 639
in transudates, 335
in urine, 750, 751
Nucleohistone, 107, 295, 348, 349

in blood coagulation, 303
in urine, 751
Nucleon, 550, 573

in semen, 589
in milk, 620, 629
Nucleoprotamines, 171, 592
Nucleoprotein, from spleen, 145, 350

Spitzer's, 13
Nucleoproteins, 20, 91, 92, 104, 163, 171^

173, 244, 250, 542

See also various organs.

action of enzymes and

microorganisms on, 176

action of pepsin-acid on,

448

action of trypsin on, 485
cleavage products of, 174
in brain, 574
Nucleoside, 181
Nucleosin, 190
Nucleotides, 175
Nucleotin, 175, 178
Nutritive requirement, 850, 875-880
Nylander-Alrne"n's test, 208
Nylander's sugar test, 759

Obermayer's indican test, 693

GENERAL 1NDKX

Oberm filler's cholestorin reaction, 423

Oblitino. 540

Oehronose. 525

Octadecyl alcohol. 707

Octodeeapeptide. 85, 87

Oedematous fluid, and its constituents.

343

Ooqdphageal fistula. 437
Oil-turpentine, behavior in animal bodv,

711. 712
effect upon bile secretion.

394
elimination in urine, 743,

760

Olein, 237, 229
Oleyl-loeithin, 234
Oligaemia, 323
Oligocythaemia.
Oliguria, 731
Olive oil, absorption, 514

effect upon bile secretion, 394
Onuphin, 167
Oocyan, 604
Oorodein, 604
Opalisin, 620, 628
Opium, passage of, into milk, 637
Optograms, 585

Orcin-hydrochloric acid test, 202, 216
Orcin test for pentoses in urine, 770
Organs, loss of weight in, during starva-
tion, 839

Oriental bezoar stone, 501
Ornithine, 84, 89, 159, 740, 741
Orthonitrobenzaldehyde, fate of on the

body, 741

Orthonitrophenylpropiolic acid, test for
dextrose, etc. See Acid, nitrophenyl-
propiolic.

Orthonitrotoluine, elimination of, 743
Osamines, 197
Osazones, 198, 199
Osimines, 197
Osmometer, 38
Osmosis, 30

Osmotic experiments, 30, 34
pressure, 26, 27, 38

of colloids, 38-39
See also various animal
fluids

Osones, 199
Ossein, 117, 526, 530
Osseoalbuminoid, 526
Osseomucoid, 164, 526
Osteomalacia, 530, 531
Osteosclerosis, 530
Otoliths, 588
Ovalbumin, 83, 106, 601, 602

conversion into globulin, 103
relation to glycogen forma-
tion, 343, 502
Ovin, 599
Ovoglobulin, 601
Ovomucin, 601
Ovomucoid, 601, 603

bodies, 167

Ovovitellin. S3, 90. 104. 106. 507
Ovum. 500
Oxuhite calculi, 7S(>
Oxanthranol. oxidation of. 7
Oxidase. 5S
Oxidases. 12,183

primarv or direct, and indirect,

11
See also various tissues and

fluids

Oxidation ferments. See Oxidases.
Oxidations, 3-8, 11-14. 733, 734, 738, 808

, in diabetes, 382
Oximes, 196
Oxyacids, aromatic, in urine. 402, 097

in perspiration, 799
Oxybenzol, 738
Oxycarbazol, 738
Oxycholesterin, 797
Oxygen, activation, 4-8

amount, specific, 819
amounts in blood, 802, 809, 810
See also various organs and
fluids.

as decomposition product in in-
testine, 493
calorific value, 831-833
capacity, 835
capacity, specific, 818
carriers, 8, 13
combustion value of, 835
consumption, 831
effect upon lactic acid elimination,

554,555,564,710
effect upon sugar elimination,

381, 554

in alveolar air, 814
scarcity of, effect upon protein
decomposition. 554. 040. 714
tension in blood, 810, 81 1,813, 818
Oxj^genases, 12
Oxyhaematin, 282-284
Oxyhaemocyanin. 202
Oxyhscmoglobin, 269, 270-275

action of gastric juice on,

449

action of trypsin on, 485
amounts in blood, 269,
315, 317, 319, 321-322
crystals, preparation of,

dissociation, 271, 272,

809,810,818-819
in urine, 752
Oxynaphthalene, 738
Oxyphenylethylamine, 80, 131, 484, 794
Oxyproline, 80, 84, 153

amounts in proteins, 106, 107,

124

Oxyprotein, 82
Oxypurine, 186
Oxypyrimidine, 190

Oxyquinolines, elimination in urine, 743
Ozone, occurrence in the organism, 4
Ozone transmitter, 274

GENERAL INDEX

Palmitin, 227, 228
Palmityl-lccithin, 234
Pancreas, 409-47 1

charging, 473
diabetes, 383-386
enzyme, action of on ethyl
alcohol and butyric acid, 65
relation to absorption, 508, 511,

515, /ill)

relation to diabetes, 383-386
relation to glycolysis, 258, 386-

388

results of extirpation, 511, 516
proteid, 172,201,470
Pancreatic calculi, 487
casein, 487
diastase, 478, 488
juice, 469, 471-287

excitants of secretion

471-476
action on foodstuffs, 478-

486
action on petides, 89,

486-487

constituents of, 477
mineral constituents of,

477

rennin, 474, 476, 487
Papain, action of, 134, 484
Paralbumin, 341, 354, 593
Paraminophenol, 738
Paracasein, 617
Parachymosin, 450, 541
Paracresol, formation in putrefaction, 492,

687, 688

Paraglobulin. See Serum globulin.
Parahaemoglobin, 273
Parahistone, 109
Paralytic saliva, 427
Paranuu'in, 594
Paramyosinogen, 541, 544, 545
Paranuclein. See Pseudonuclein.
Paruovarial cysts, 596
Paraoxypropiophenone, 742
Parapeptone, 448
Parathyroids, 355
Paraxanthine, 6, 182, 677

in urine, 676
Parenteral canal, introduction of protein

by way of, 510-502
Parotid, 426

saliva, 428
Peas, percentage taken up in intestine,

511

Pectin bodies, 222, 224
Pemphigus chronicus, 342
Pennacerin, 797
Pennatulin, 122
Pentacrinin, 795

Pentamethylenediamine. See Cadaverin.
Pentapeptides, 85
Pentosamines, 213
Pentosans, 201

digestion of, 518
Pentoses, 175, 193, 210-205

Pent oses, relation to formation of glyco-

gen, 201, 372
in blood, 257
in milk, 622

in nucleic acids, 171, 17 ;
in urine, 203, 769, 770, 771
Pentoside, 548

Penzoldt's test for acetone, 776
Pepsin, 16, 442-449, 451, 465
Pepsin glands, 436

in urine, 718
-related enzvmes, 442
test, 445-447

testing in gastric juice, 462
Pepsinogen, 452
Pepsinogenic action, 454
Peptases, 480
Peptic digestion, 446-449

cleavage products of>

128, 131, 132, 448
Peptic-glut in peptone, 135
Peptic-peptones, 135
Peptides, 68, 85-89, 126-127, 154

behavior toward enzymes, 8.

259, 468, 486, 487
in urine, 717

relation to alcaptonuria, 699
relation to urea formation, 734}
Peptinuria, 748
Peptochondrin, 523
Peptoid, 484
Peptone plasma, 243
Peptones, 79, 80, 88, 91, 92, 126-132, 135-

137,448

absorption, 460, 503-507
in lungs, 821
in urine, 744, 748
nutritive value of, 854
preparation and separation of^

137
Witte's products of fractional

precipitation of, 132
Percaglobulin, 605
Perch eggs, 105, 164, 599, 605
Pericardial fluid and its constituents, 335.

338

Perilymph, 588
Peritoneal fluid and its constituents, 335,,

339-341
Permeability, 31-33

of the blood corpuscles, 32-

33, 268, 316
of the muscles, 34, 559
of the vascular walls, 340
Peroxidases, 11, 12

Peroxides, formation of in living cell, 7
" Perspiratio insensibilis," 824
Perspiration, character and constituents,.

93, 798-801
Pettenkofer's method of determining

respiration gas exchange, 819
Pettenkofer's test for bile, 396

Pit test for bile-acid, 755
Pfaundler's method of precipitating urine,
647

956

GENERAL INDEX

Phacozymase, 587
Phaseomannite, 551
Phenol, in urine, 492, 687-690, 742, 744
as precipitant of proteids, 98
behavior in animal body, 492, 688,

689, 742

in starvation, 494
production in putrefaction, 81, 492,

688
Phenylalanine, 80, 81, 85, 118, 150

amounts in proteins, 106,

107, 114, 121
behavior in animal body,

684, 738, 740

in alcaptonuria, 698, 699
Phenylglucosazone, 198, 208
Phenylglucosehydrazone, 198
Phenylhydrazine acetate test for dex-
trose, 198, 208
test for sugar in urine,

761
Phenylisocyanate compounds of amino-

acids. See various acids, amino.
Phenylisocyanate peptone, 136
Phenylmethylketone, 742
Philothions, 14
Phlebin, 268
Phlorhizin diabetes, 379, 389, 392, 712

poisoning, 363, 379,
692, 712
Phloroglucin as a reagent, 202, 216, 463,

770

Phosphate calculi, 786
Phosphates, in urine, 640, 722-725, 782,

784, 839
relation to synthesis of organic

cell constituents, 845
results of lack of, 845

See also various phosphates.
Phosphatides, 225, 232-241, 363. See
also various tissues and
fluids.

in muscles, 557
of yolk, 599
Phosphaturia, 724
Phosphoglycoproteins, 163, 17.0
Phosphoproteins, 90, 91, 92

properties of, 103

Phosphorus compounds, organic in urine,
t 718

in nucleic acids, 175
metabolism, 826-827, 845
poisoning, action on the elim-
ination of am-
monia, 652
action on bile, 415,

419
action on blood,

245, 247

action on lactic
acid elimination,
554, 710

action on urea
elimination, 646,
652

Phosphorus poisoning, changes of urine,

365, 554, 646,

652, 710, 715,

780

liver autolysis in

18, 363, 365
resulting in fatty
degeneration,
363, 534

Phosphorus-containing urinary constit-
uents, 718, 826
Photomethaemoglobin, 277
Phrenin, 581
Phrenosin, 579, 580, 582
Phyllocyanin, 288
Phylloporphyrin, 269, 288
Phylloverythrin, 410
Phymatorhusin, 755, 793
Physiological availability of food, 835
heat of combustion, 830
Phytase, 551
Phytin, 551
Phytosterines, 421

a-Picolin, behavior of, in animal body, 741
Pig meat, 572

guanine-gout, 15
Pigmentary acholia, 415
Pigments, in lungs, 821

medicinal, in urine, 744, 757
of bile, 405-411,413-414
of blood, 268-291
of blood serum, 260, 600
of corpora lutea, 290, 592
of egg shells, 604
of eye, 584-586
of liver, 362
of lobster, 605, 795
of lower animals, 292, 795
of skin, 792-793
'of urine, 702-710
stones, 419
Pike flesh, 571, 572

stomach, enzyme of, 444, 450
Pilocarpine, effect upon elimination of

CO2 in stomach, 461
effect upon elimination of

uric acid, 666

effect upon secretion of in-
testinal juice, 466
effect upon secretion of saliva,

435

Pine seeds, protein of, 158, 161
Piperdine-glycosuria, 381
Piqure, 380

Piria's tyrosine test, 151
Placenta, 409, 416, 608

sanguinis, 244
Plant gums, 220, 222, 224
Plants, chemical processes, 1, 2
Plasma. See Blood plasma.
Plasmolysis, 30
Plasmoschisis, 301
Plasmozym, 306
Plastein, 134, 452, 487
Plasteinogen, 135J

GENERAL INDEX

957

Plastin, 174

Plattner's crystallized bile, 396

Plethora polycythaemia, 322

Plcural fluid and its constituents, 335, 338

Pneumonic infiltrations, solutions of, 18,

346, 820

Poikilocytosis, 324
Polarization test for sugar, 762, 767
Polycythocmia, 322
Polynucleotides, 175
Polypeptides. See Peptides.
Polyperythrin, 795
Polysaccharides, 193, 220, 224
Polyuria, 731

Potassium salts, excretion, 434, 727, 839.
See also various organs

and fluids.

Potatoes, absorption-in intestine, 511
Precipitation membranes, 26

phenomenon, theories of, 46
Precipitins, 70, 259, 502
Preglobulin, 295, 302, 303, 349
Prepuce of beaver, 797
Preputial secretion, 797
Pressure, osmotic. See Osmotic pressure.
Productive increase, 841
Proenzymes, 11, 60
Prolamins, 91
Proline, 80, 84, 152

amounts in proteins, 106, 107,

114, 124, 152
Propepsin, 452
Propeptone, 126
Propylalanine, 85
Propvl benzene, behavior in animal body,

738
Propylene glycol, relation to glycogen

formation, 372
Prosecretin, 439, 475

in intestinal juice, 468
Prostatic concrements, 168, 580, 592
Prosthetic group, 171
Protagon, 575, 576-578
Protamines, 77, 90, 91, 92, 109, 111, 136,

592

Proteans, 92
Protease, 58

Protective colloids, 37, 44, 131
Proteid, approximate estimation in urine,

750

crystalline, 93
detection of in urine, 744
quantitative determination in

urine, 749
Proteids, 90, 163-173. See also various

protein groups,
crystalline, 93
native, 95

vegetable, table of cleavage prod-
ucts of, 107
Protein absorption, extent of, 507

catabolism, upper and lower limits

of, 857
-cystine, 146
destruction, 851-853

Protein, digestion after extirpating the

pancreas, 508
digestion in intestines, 491
fattening, 850, 851, 860, 861
hydrogel, 95, 96
hydrosols, 95, 96
metabolism, endo- and exogenous,

853
in starvation, 837,

838, 846
in various diets, 846,

847

in various stages, 866
in work and rest, 565-

570

premortal rise, 383
results following de-
crease of, 851
minimum, 859
molecule, 4

of milk, calorific value of, 832
substances containing a carbo-
hydrate group, 83
synthesis, place of, 506
Proteinchrome, 153
Proteins, absorption of digestion products,

503

action of bile on, 490
action of enzymes upon, 80
action of fat on catabolism of,

855

action of gastric juice on, 449
action of microbes on, in intes-
tines, 491

action of pepsin-acid on, 448
action of pepsin on, 444
action of trypsin on, 481
active, 4
adsorption of, 96
aminoacid isolated from, 8
a taking up of, by cells, 861
calorific value of, 831
circulating, 850
color reactions for, 98-101
combustion in the body, 829
different groups, properties of,
102

See Chapter III, and various
protein substances,
formation of, 3

horse-blood, table of composi-
tion of, 255
in brain, 574

influence on metabolism, 833
in lung, 820
in muscles, 565
living and non-living, 4
metabolism with foods rich in,

418

of crystalline lens, 586, 588
of dead muscle, 540
of kidneys, 638
of liver, 361,368
of milk, determining, 623
of muscle plasma; 539, 543

958

GENERAL INDEX

Proteins, precipitation reactions for, 97-98
products of hydrolytic cleavage,

80

products of putrefaction -in in-
testines, 492

putrefaction of in intestines, 491
reactions for in urine, 744-752
results of putrefaction of, 80
salting out, 94
synthetic, 85

undigested, assimilation, 502
Proteoses, 79, 80, 88, 91, 126-132,135,137
absorption, 503-507
absorption of, 503
action of on blood coagulation,

311

action of on leucocytes, 312
conversion into amino-acids, 505
differences from peptones, 139
food value, 854, 855
in blood, 255, 256, 503, 504
in lungs, 821
in stomach, 459, 460
in urine, 744, 746, 748, 749
nutritive value of, 854
precipitation properties of, 133
preparation and separation of,

137
presence and detection in urine,

748
relation to coagulation of blood,

298, 300, 310, 311
Prothrombin, 248, 250, 302, 304-307

in blood, 302
Protocaseoses, 129
Protoelastose, 116
Protogelatose, 119
Protogen, 126
Protokyrins, 136
Protones, 110
Protoplasm, 20-22

leucocyte-constituents of, 295
Protoproteose, 129, 132-133
Protosyntonose, hexone bases in, 161
Proto-toxoids, 71
Pseudocerebrin, 580,
Pseudoglobulin, 251
Pseudoglycogen formers, 375
Pseudoha3moglobin, 276
Pseudomucin, 167, 593, 594, 595

in ascitic fluids, 340
in gallbladder, 415
Pseudonuclein, 104, 173, 598, 618
Pseudopepsin, 442, 465
Pseudoxanthine, 550
Psittacofulvin, 795
Psylla-alcohol, 796
Ptomaines, 24

in urine, 718
Ptyalin, 431-434

action of gastric juice on, 488
velocity of reaction, 433
Purine, 181

Purine bases, 181, 686. See also nuclein
bases.

Purine bodies, composition of, 182
Purple, 795

cruorin, 275
Pus, 344-347
blue, 347,

corpuscles and their constituents, 345
in urine, 765

serum and its constituents, 344.
Putrefaction processes in intestines, 490-

498, 687, 688, 691, 695, 696
Putrefactive processes, 8-10, 25, 80, 81
Putrescine, 24, 159, 780
in urine, 719
Putrine, 24
Pyin, 338, 344, 346
Pyloric glands, 436

secretion, 454
Pylorus reflex, 456
Pyocyanin, 347

in perspiration, 800
Pyogenin, 346, 579
Pyosin, 346, 579
Pyoxanthose, 347
Pyridine, elimination of, 743
Pyrimidine, 181

bases, 171, 174, 175, 189-191
Pyrin, 338
Pyrocatechin, 690

intransudates, 337,342
Pyrodine, action of, 353
Pyrrol derivatives, 152-156, 269, 283, 703

Quercite, 372
Quince mucilage, 224
Quinine, effect upon spleen, 353

effect upon uric acid elimination,

666

elimination in urine, 744
in perspiration, 799
Quotient, C to N, 731

flesh, 57 1,827

nitrogen to homogentisic acid,

nitrogen to sugar, 388-390, 391
respiratory quotient, 391, 537,

567, 830, 838, 869
urea to nitrogen, 731, 828
urine, calorific, 828, 835

Rachitis, 530, 531

Rape-seed oil, 533

Reaction velocity, 53

Receptors, 71

Reductases, 14, 58

Reduction processes, 3, 5, 13-15. See

also various chapters.
Reductonovain in urine, 718
Regnault-Reiset's method of determining

respiratory gas exchange, 819
Reichert-Meissl's equivalent for fats, 231
Rennin, 8, 436, 450 See also Chymosin.
Rennin glands, 436

cells, 436

Resacetophenone, 742
Reserve cellulose, 210

GENERAL INDEX

959

Resin acids in urine, 744, 746
Resorcinol test for levulose, 211, 768
Respiration, external, 801, 809, 813-817
internal, 801,809, 818
See also exchange of gas
under various conditions,
of hen's eggs, 606

See also Chapter XVII.
Respiratory gas exchange, determining,

819

quotient. See " Quotients."
Rest, influence of, on metabolism, 562-567,

841,846,867,869
Rest nitrogen, in serum, 260

in stomach, 459, 460
Retardation of enzyme action, 18, 67
Reticulin,91, 120,519
Retina, composition of, 584-586
Retrogradation, 220
Reversibility of catalytic processes, 53,

56, 64-66, 217
Reversion, 217

Reynolds test for acetone, 776
Rhamnose, 201

relation to glycogen formation,

372

Rhodophan, 586
Rhodopsin, 584
Rhubarb, elimination of coloring matter

of in urine, 744
Ricin, 446, 482

in testing pepsin, 446
Rigor mortis, of muscles, 560
Ringer-Locke's solution, 75
Ritthausen's method of determining pro-
teins in milk, 623
Roch's test for proteid, 946
Rodents, bile acids of, 404
Rosenbach's, bile pigment test, 756

urine test, 780

Rovida's hyaline substance, 267, 295, 345
Rubner's, reaction for dextrose, 208

sugar test in urine, 762
Rye bread, utilization of, 511

Saccharose, 217-218

absorption, 509, 510
calorific value, 829, 832, 833
inversion, 9-10, 449
passage into urine, 769
Sachsse's reaction for dextrose, 288
Sahli's hsemometer, 292
Salicylase, 12
Saliva, 426-435

bahavior in stomach, 435, 456
physiological importance of, 435
quantitative composition of, 434
Salivary concrements, 436
glands, 426

relation to gastric secre-
tion, 435, 439
Salkowski's, creatinine reaction, 662 .

reaction for cholesterin, 422
Salmine, 110, 143, 152
Salmon, flesh of, 546, 570, 845

Salmon, sperms, 109, 177, 591, 592
Salt action, 73-75
glycosuria, 380
plasma, 243
Salts, action upon metabolism, 862, 863.

See also various salts.
Samandarin, 797
Sandmeyer's method of inducing pancreas

diabetes, 383

Santonin, elimination of in urine, 744, 757
Sapokrinin, 476
Saponification, 231, 453, 479, 489, 513, 516

equivalent, 231
Saponin, 266, 424, 425
Sarcolemma, 538
Sarcomelanin, 793
Sarcosine, fate in organism, 735
Sarkine. See Hypoxanthine, 186
Schalfejeff's hsernin, 285
Scherer's inosite test, 552
SchifFs reaction, 653

reaction for cholesterin, 423
Schreiner's bases, 590
Schutz-Borissow's rule of ferment action,

64,447,452,479,482
Schweitzer's reagent, 224
Scleroproteins, 92
Sclerotic tissue of eye, 588
Scombrin, 110, 111, 136, 152
Scombron, 107
Scyllite, 351, 552
Scymnol, 396
Seal, fat of, 230, 232
Sea-urchin, spermatozoa of, 108, 177
Sebelien's method of determining proteins

in milk, 623
Sebum, 795
Secretin, 394, 439, 475

in intestine, 468
Secretion of the prostate, 590
Sediments. See Urinary sediments.
Sedimentum lateritium, 640, 672, 709
Segregation, 52
Self digestion of stomach, 461. See also

Autolysis.

Seliwanoff's reaction for levulose, 211, 768
Semen, constituents and characteristics

of, 589-592

Semicarbazide, poisoning therewith, 682
Semiglutin, 119
Seminose, 210
Senna, elimination of coloring matter of,

in urine, 744, 757, 780
Sensibilizators, 70
Sepia, 359, 792, 794
Sepsine, 24

Seralbumin, 90, 105, 244, 253-255, 603 '
in urine, 744, 747, 749
absorption, 494
Serglobulin, 90, 105, 244, 250-253, 255,

261-262

in urine, 744, 747, 749
Sericin,90, 121,122, 123

table of decomposition products of,
124

960

GENERAL INDEX

Serine, 84, 143

amounts in proteins, 105, 114, 124,

144

Serine anhydride, 143
Serolin, 256
Seromucoid, 252, 256
Serosamucin, 336
Scrum. See Blood serum.
Serum casein, 250. See Serum globulin.
Sex, influence of, on metabolism, 867
Sham feeding, 437, 442, 872
Shark, bile, 395, 411, 645
urea, 317, 41 1,645
Shell membrane, of hen's egg, 112-114,

604

Side-chain theory, 71
Silk gelatin, 123, 124
Sinistrin, animal, 170
Skatol, 81, 155, 156, 492, 695. 696

behavior, in animal oody, 492, 695
-chromogen, pigments of, 695
Skatosine, 165
Skatoxyl, 155, 687, 695
Skeletins, 121

composition of, 122
Skeleton, 529, 840, 842
Skin, 788-794

and its secretions, 789
coloring matters of, 792
exchange of gas through, 800
excretion through, 794-799
Sleep, influence on metabolism, 869
Smegma prseputii, 797
Snake poison, effect upon blood, 248, 300,

311
Soaps, importance of, for secretions, 457,

476
importance of, for emulsification

of fats, 489
importance of, for their absorption,

Sodium alcoholate, as a saponification

agent, 227, 230
chloride, physiological solution,

263, 265

behavior with food poor

in potassium, 452-843

behavior with food rich

in potassium, 843-844

effect upon intestinal

secretions, 466
effect upon peptic diges-
tion, 447
effect upon trypsin diges-

483

elimination in perspira-
tion, 799
elimination in urine, 719,

720

influence upon gastric
juice secretion, 438, 452
quantitative determina-
tions, 720, 722

^compounds, elimination through
urine, 727

Sodium compounds, division among form
elements and fluids
22, 23

See also various

juices and tissues.

salicylate, as a cholagogue, 394,

395

Solanin, 266

Soldiers, diets of, 874, 879
Sorbin, 202
Sorbinose, 199, 205, 212

d-, 212
Sorbite, 194
Specific dynamic action, 859, 871-872

rotation, 195
Specificity, of enzyme action, 66,

, of anti bodies, 69

Speck's method of determining respira-
tory gas exchange, 820
Spectrophotometric, method, 291, 292
Spermaceti, 231

oil, 231, 232
Spermatin, 592
Spermatocele fluid and their constituents,

340

Spermatozoa, 23, 33, 591, 608
of bull, 591
Spermine, 590

crystals, 590

Sperm reaction, Florence's, 590 .
Sphingomyelin, 578
Sphingosin, 580
Sphygmogenin, 357
Spider silk, 123
Spiegler's test for proteid, 746
Spinal marrow, 583
Spirographin, 167
Spleen, 350-354

blood of, 319

relation to digestion, 353, 473

relation to formation of uric acid,

353, 354, 670

relation to metabolism of iron, 353
Spongin, 91, 121, 122
Sponginoses, 122
Spongosterin, 424
Sputum, 821
Stachyose, 220
Starch, 220-222

absorption, 509-511
calorific value, 429
cellulose, 220
granulose, 220
gum, 222

hydrolysis, 221, 222, 432, 478
Starvation, effect on blood, 262, 321, 325
effect on urine, 494, 645, 660,
665, 684, 692, 724, 727, 772
effect on metabolism, 836-841
Steapsin, 478

stomach, 451

Steapsinogen, activation of, by bile, 489
Stearin, 227

absorption, 514
Stearyl-lecithin, 234

GENERAL INDEX

961

Steensma's modification of Giinzburg's

test, 463

Stenson's test, 560
Stentorin blue, 795
Stercobilin, 406, 706

in feces, 499
Stercorin, 423
Stethal, 231

Stokes' reduction fluid, 275
Stomach contents. See Chymus.
digestion in, 454-462
fistulas, 437, 348
glands, 436
importance for digestion, 458-

461

relation to intestinal putrefac-
tion, 461, 497
self-digestion, 459
Stone-cystine, 146, 148
Stroma-fibrin, 268

Stroma, of blood corpuscles, 264-266
of muscles, 542
of ovaries, 592
Stromata, 266, 267
Struma cystia, 354
Strychnine, and glycogen transformation,

369, 372

and glycosuria, 380
in urine, 744

Sturgeon, spermatozoa of , 110, 177
Sturine, 110, 152, 157, 161
Stutz's test for proteid, 747
Sublingual glands, 426

saliva, 428
Submaxillary glands, 424

mucin, 165,^16 i
saliva, 427, 428
Submicrons, 40
Substrate, 58

reformation of, 65
Sugar, absorption of, 509

alcoholic fermentation of, 200
behavior after subcutaneous in-
jection, 375
calorific value, 829, 833. See also

various sugars.

cane, action of gastric juice on, 449
eliminated in diabetes, origin of ,388
entrance into the blood stream, 510
formation from glycogen, 377, 378
formation from fat, 390-392
formation of, from formaldehyde, 2
in blood, 314, 315,316
in perspiration, 799
of muscles, 553
out of proteins, 387-390
passage into urine, 511
quantitative determination in

urine, 763-767

relation to work, 563, 567, 568
simple and complex, 193
tests for, in urine, 757-764
Sulphsemoglobin, 279
Sulphates in urine, and their determina-
tion, 725-726

Sulphocyanides, occurrence in urine, 714,

735

in gastric juice, 442
in saliva, 428, 429,431
Sulphonal intoxication, urine, 287, 754
Sulphur, behavior in animal body, 714
elimination, 566, 825, 826
in proteins, 78, 79. See also vari-
ous proteins.

in urine, 714, 825, 826, 841
methsemoglobin, 279
Sulphuretted hydrogen, as decomposition

product in intestine, 493
Suprarenin, 358. See Adrenalin.
Surface rule, 865, 866
Surface tension, 28, 50, 51
Suspension colloids, 37

electrolyte precipita-
tion of, 43
filterability of, 45
Suspensoid, 37
Swallow's nest, edible, 167
Symphthetic saliva, 427
Synovia, 343
Synovial cavities around joints, fluid in,

343

fluid and its constituents, 343
mucin, 166, 336, 343
Synovin, 343
Synproteose, 133

Syntheses, 1, 2. See also various chem-
ical substances.
Synthetical processes, 1, 2, 3. See also

various chemical substances.
Syntonin (meat), hexone bases in, 125, 161

543

calorific value, 830
Syntoxoids, 71

Table salt. See Sodium chloride.

Tamia, 369

Talose, 204

Tartar, 436

Tatalbumin, 601

Taurine, 147, 149, 395, 417, 456, 573

behavior in animal body, 735

in the lungs, 820
Tea, action on metabolism, 863
Tears, composition of, 93, 588
Teichmann's crystals, 285, 753
Temperature, external, influence on met-
abolism, 870
Tendon, mucin, 165, 168

mucoid, 519, 526
Tension, of CO2in blood, 815-817

in lymph, 328

in tissues, 819

of O in blood, 819-813
Terpenes, cyclic, fate of in the body, 742

elimination in urine, 743
Testes, protein bodies in, 589
Testudo, egg membrane, 114, 152
Tetanolysin, 72

Tetanotoxine and gastric juice, 461
Tetraglycylglycine, 85, 486

962

GENERAL INDEX

Tetramethylenediamine, 24. See Putres-

cine.

Tetramido monophosphatide, 233
Tetrapeptides, 85, 87, 89, 486
Tetronerythrin, 292, 795
Tetroses, 193
Thalassin, 797
Theobromine, 182

behavior in animal body,

676
Theophylline, 182

behavior in animal body,

675
Thioalcohols, behavior in animal body,

736

Thiophene, behavior in animal body, 741
Thiotolene, 741
Thrombin, 16, 248-250, 249, 302, 304-307,

308-310

Thrombogen, 305, 308-309
Thrombokinase, 306, 308-309
Thromboplastic substances, 309
Thrombosin, 303

-lime, 303
Thrombozym, 308
Thymamin, 109*
Thymine, 174, 176, 190
Thymus, 348-350
Thyreoglobulin, 356
Thyreoproteid, 356
Thyroid, gland and its constituents, 354-

357

effect of extirpation, 355
Thyroids, 354-357, 384
Tissue fibrinogen, 295, 310, 349
Tissue-proteins, 850
Tollen's reaction for pentoses, 202
Tollen's-Rorive test for levulose, 211

test for glucuronic acid,

216
Toluene, behavior in animal body, 683,

738

Toluylenediamine, poisoning with, 419
Tonus, chemical, 562
Tooth, degeneration, 532
structure, 528, 531
Tortoise shell, 112
Toxins, 18, 22, 24, 70-74, 461
Toxoid, 71
Toxons, 71
Toxophore group, 71
Transudates, 327, 334-344

pathological, gases of, 808
Trichohyalin, 789
Triglycerides, 225
Triglycylglycin, 85, 486
Triglycylglycinethylester. See Biuret

base.
Trimethylamine, 239

in urine, 718
Trimethylvinylammonium hydroxide.

239. See Neurine.
Triolein, 226, 229
Trioses, 193, 220
Trioxypurine, 663. See Uric acid.

Tripalmitin, 226, 228
Tripeptides, 85, 485, -486
Triple phosphates, in urinary concrements,
m 784,786
in urinary sediments,

782-784

Trisaccharides, 193
Tristearin, 226, 227
Tropics, inhabitants of, metabolism in,

870

Trommer's test, 207, 749, 758
Trout egg, development of, 607
Trypsin, 16, 472-474, 479-486

action of, on casein, 63
action of, on other bodies, 485
action on peptides, 89, 485, 486
action on proteins, 128, 131, 481-

485

digestion, speed of, 282
in urine, 718

quantitative determination of , 482
Trypsinogen, 60, 472-474
Tryptic digestion, 482

products of, 484
retardation of, 69
peptones, 131, 135
Tryptophane, 81, 84, 131, 150-151

amounts in proteins, 106,

107, 153
reaction, 99
Tubo-ovaria, 596
Tunicin, 790
Turacin, 795
Turacoverdin, 795
TyndalTs phenomenon, 40
Tyrosinases, 12, 151, 359, 794
Tyrosine, 81, 84, 150-153, 492, 687

amounts in proteins, 106, 107,

114, 124, 150
behavior in animal body, 492,

687,698, 699, 737, 739
in urine, 780, 784
mellinin formation, 794
transformation into homo-
gentisic acid, 700

Uffelmann's test for free lactic acid in

gastric juice, 463
Ultramicroscope, 40
Umikoff's reaction, 630
Uracil, 174, 190

Uranium salts and glycosuria, 380
Urates, 672

in sediment, 640, 672, 781, 783
Urea, 645-657

decreased elimination during starva-
tion, 841

effect upon metabolism, 862
formation and origin, 82, 647-652
in ascitic fluids, 340
in bile, 411, 415
in blood, 260, 317, 319, 651-652
in brain, 576

in lymph and transudates, 328, 337,
340,341,342,343,609

GENERAL INDEX

963

Urea, in muscle, 546

in perspiration, 799
m-nitrohippurate, 741
oxalate, 654

properties and reactions, 652
quantitative estimation, 654
quantity eliminated per day, 866
Urease, 16, 782
Urein, 657
Urethane, 658
Ureotheobromine, 677
Uric acid stones, 785
Uricolysis, 670

Urinary calculi, 146, 781-787
Urinary pigments and chromogens, 702-

710

purines, 667, 676-679
sand, 785
sediments and calculi, 639-640,

781-784

sugar. See Glucose.
Urine, 638-787

acid fermentation of, 782

acidity, 641-643

alkaline fermentation of, 642, 710,

782, 785

carbohydrates and reducing sub-
stances in, 711
casual, 733-744

constituents, inorganic, 719-730
constituents, organic, pathological,

744-781
detecting acetone and acetoacetic

acid in, 728, 771-778
gases in, 730
indican, 691
influence of foods on acidity of,

641

of children, 640, 646
of lion, 663

osmotic pressure of, 643
physical properties, 639-645, 732
physiological, 645-719
solids, calculation of, 732, 733,, 758
specific gravity of, 644
toxic power of, 719
Urinometer, 645
Urobilin, 405, 406, 702, 705-709

relation to blood pigments, 287,

288, 418, 705
relation to bile pigments, 405,418,

494, 705

Urobilinoids, 287, 705
Urobilinogen, 405, 702, 705, 707
Urochrome, 702, 703
Urocyanin, 703
Uroerythrin, 702, 709
Urofuscohccmatin, 754, 755
Uroglaucin, 703
Urohjcmatin, 703
Urohodin, 703
Urornelanins, 703
Urophain, 703
Uropyrryl, 704
Urorosein, 696, 697, 703

Urorubin, 703

Urorubrohaematin, 754

Urospectrin, Saillet's, 754

Urostealith calculi, 787

Urotheobromine, 677

Urotoxic coefficient, 719

Uroxanthine, 691

Uterine milk, 609

Uterus colloid, 596

Utilization of foods, 507, 508, 835

Valine, 84, 139

amounts in proteins, 106, 107, 114,

124, 139

Vanillin, behavior in animal body, 739
Van'tHoff's rule, 194
Vasodilatin, 475
Vegetable gums, 322

mucilages, 223
Vegetarian, diet of, 858, 876°

excrements of, 498
Velocity coefficient, 55

coefficients in monomolecular re-
actions of various substances,
62

constant, 56

constant of ester, formation, 66
Vernix caseosa, 796
Vesicular calculi, 785-788
Vesiculase, 590
Viridinine, 24
Virtual sugar in blood, 257
Viscosity, of proteins, 95

of blood, 298, 299
Visual purple, 584

red, 584

Vitali's pus-blood test, 753
Vitellin, properties of, 598. See Ovovi-

tellin.

Vitellolutein, 600
Vitellorubin, 600
Vitelloses, 129
Vitianine, 549
Vitiatin, in urine, 718
Vitreous humor, 520, 521, 685
Volhard's method of determining chlor-
ides, 721

Walden's reversion, 87, 139
Walls of vessel, relation to coagulation,
300, 301, 306-307, 309,
311
relation to transudates,

332-335

Walrus, bile of, 400
Water, elimination in urine, 730-732, 824,

861
effect on metabolism, 842, 861,

862

elimination during starvation, 839
elimination, importance of, for

animal body, 22

elimination through skin, 797, 821
lack of, effect upon metabolism,
842, 843, 862

964

GENERAL INDEX

Water, in muscle, 571
Wax, 232

in plants, 796

" Wear and tear " quota, 859
Weiss' sparing theory, 374
Weyl's creatinine reaction, 661
Wheat bread, 511
Whey protein, 617
Witch's milk, 632
Wool-fat, 424, 797
Work, action of the necessity for food,

878, 879
action on metabolism, 562-569,

867, 868

influence on elimination of nitro-
gen, 476, 565, 566, 867, 868
influence on elimination of chlorine,

719-720
influence on elimination of sulphur,

567

Worm-Miiller's sugar test, 759
Wound secretion, 343

Xanthine, 182-184

in urinary calculi, 786, 787

in urinary sediments, 784

in urine,- 676

oxidase, 12, 351, 667

preparation and detection of,

188

Xanthocreatinine, 550, 565, 663
Xanthomelanin, 82

Xanthophan, 586

Xanthophyll group, 600

Xanthoproteic reaction for proteid, 99

Xylene, behavior in animal body, 739

Xyliton, 793

Xylose, 179, 194, 204, 224, 362

relation to formation of glycogen,
372

Yeast cells, 9, 17, 369
Yolk, constituents, 597

membrane, 597

mineral bodies of, 600

of hen's eggs, 596-601

phosphatides of, 599

spherules, 93, 596, 605

Zein, 77, 105, 160
Zinc, in the bile, 411
in the liver, 367
passage into milk, 637
Zooerythrin, 795
Zoofulvin, 795
Zoorubin, 795
Zuntz-Geppert's method of determining

respiratory gas exchange, 820
Zymase, 9, 17, 58, 200
Zymogens, 11, 60. See also respective

enzymes.
Zymoplastic substances, 302, 305-307,

310

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