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WORKS OF
PROFESSOR J. A. MANDEL
PUBLISHED BY
JOHN WILEY & SONS.
Handbook for Biochemical Laboratory.
12mo, cloth, $1,50.
TRANSLATIONS.
A Text=bool4 of Physiologrical Ctiemistry.
By Olof Hammarsten, late Professor of Medical and
Physiological Chemistry in th3 University of Upsala
Authorized Iranslafon, from the author's ei-larged
and revised Gth Ge man ed t on, by John A. Man-
del, Professor of Chemisty iu the New York Univer-
sity and Bc-Uevue Hospital Medical College. 8vo,
viii + 845 pages, cloth, $4 OU.
A Compendium of Chemistry, Including: General, In-
org:anic, and Orsranic Chemistry.
By Dr Carl Ari old Professor of C hem stry in the
Royal Veterinary School of Hannover. Au horized
translation from the eleventh enlirged and revised
German edition, by John A Mandel. Small 8vo,
xii + 6_'7 pages, cloih, $3.50.
A TEXT-BOOK
PHYSIOLOGICAL CHEMISTRY.
OLOF HAMMARSTE:^^,
Late Professor of Medical and Physiological Chemistry in the
University of Upsala .
FMOM THE AUTHOR'S ENLARGED AND REVISED
SIXTH GERMAN EDITION
JOHN A. MANDEL, Sc.D.,
Professor of Chemistry in the Xew York University and
Bellevue Hospital Medical College.
FIFTH EDITION,
FIRST THOUSAND.
NEW YORK:
JOHN WILEY & SONS.
LoxDOx: GHAPMAK^ & HALL, LnriTED.
1908.
Copyright, 1893, 1898, 1900, 1904, 1908,
BY
JOHN A. MANDEL.
a "F ^'1 "^
Sl)e Sri fttllfir ^rpBa
i&abrrt Qrummanb anh Qlomtiang
TRANSLATOR'S PREFACE TO THE FIFTH AMERICAN
EDITION.
Since the appearance of the First American Edition of Hammarsten's
Physiologischen Chemie in 1893 it has become more and more popular as
a text-book in our medical schools, and has acquired much favor among
the biochemical research workers of this country.
A special debt of gratitude is owing to Professor Hammarsten for his
many revisions, which have enabled this work to keep jjace with all the
advances of this department of chemical science. The same sound
critical survey of the subject characterizes this as well as the past
editions.
I am under obligations to Dr. Charles B. Robinson for much assistance
in proof revision.
John A. Ma.ndel.
New York, December, 1907.
V
PREFACE TO THE SIXTH GERMAN EDITION.
Although only a short time has elapsed since the appearance of the
Fifth Edition, the enormous advances in the different domains of physio-
logical chemistry have necessitated a thorough revision of each of the
chapters and a rewriting of most of them. B3cause of these facts it has
been impossible to prevent an increase in the size of the book, and hence
this edition is somewhat larger than the preceding ones. Acceding to the
wishes expressed by many friends, an Authors Index has been added to
this edition, but in other respects the plan of the work has not been
changed.
Olof Hammarsten.
IJpsAiA, September 8, 1906.
CONTENTS.
CHAPTER I.
PAGE
I STPODUCTION 1
CHAPTER II.
The Protein Substances 26
CHAPTER III.
The Carbohydrates 104
CHAPTER IV.
The Animal Fats 131
CHAPTER V.
The Animal Cell 139
CHAPTER VI.
The Blood 170
CHAPTER VII.
Chyle, Lymph, Transudates, and Exudates 250
CHAPTER VIII.
The Liver 280
CHAPTER IX.
Digestion 339
CHAPTER X.
Tissues of the Connective Substance 428
vii
VlU CONTENTS.
CHAPTER XI.
PAGE
Muscles 447
CHAPTER XII.
Brain and Nerves 479
CHAPTER XIII.
Organs of Generation 495
CHAPTER XIV.
Phe Milk 514
CHAPTER XV.
The Urine 541
CHAPTER XVI.
The Skin and its Secretions 685
CHAPTER XVII.
Chemistry of Respiration 696
CHAPTER XVIII.
Metabolism 715
General Index 779
Index to Authors 813
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 already
existing material and to transform it into new forms of energy.
Out of a few relatively simple combinations, especially carbon dioxide
and water, together with ammonium compounds or nitrates, and a few
mineral substances, which serve as its food, the plant builds up the extremely
complicated constituents of its organism, proteins, carbohydrates, fats,
resins, organic acids, etc. The chemical work w^hich is performed in the
plant must therefore, in the majority of cases, consist in syntheses; but
besides these, processes of reduction take place to a great extent. The
radiant energy of the sunlight induces the green parts of the plant to split
off oxygen from the carbon dioxide and water, and this reduction is generally
considered as the starting-point of the following syntheses. According
to a hypothesis suggested by A. Baeyer,i at first formaldehyde is pro-
duced, C02 + H20 = CH20 + 02, which by condensation is transformed into
sugar, and this then serves in the structure of other bodies. The energy
of the sun, which produces this splitting, is not lost; it is only transformed
and is stored as chemical energy in the new compounds produced in the
synthesis. W. Loeb^ has been able to obtain formaldehyde as a direct
reaction product from CO2 and H2O by the aid of the silent electric dis-
charge. Th6 formation of aldehyde takes place in the three following
* Ber. d. d. chem. Gesellsch., 3. ' Zeitschr. f. Elektrochem., 12.
2 INTRODUCTION.
phases: first, 2C02 = 2CO + 02; second, CO+H20 = C02+H2; and third,
C0+H2 = HC0H. The formation of sugar from CO2 and H2O with the
introduction of energy can be expressed by the following:
1. C02+H20 = c6 + H2 + 02.
2. H2+C0 = HC0H.
3. 2(H2 + CO) = CH20H.CHO.
4. 6HCOH = C6Hi206.
5. 3CH20H.CHO = CoHi206.
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 derive the three chief groups of organic nutritive,
matter — proteins, carbohydrates, and fats. These bodies, of which the
protein substances and fats form the chief mass of the animal body, undergo
within the animal organism a cleavage and oxidation, and yield as final
products exactly the above-mentioned chief components in the nutrition of
plants, namely, carbon dioxide, water, and ammonia derivatives, which are
rich in Oxygen and have little energy. The chemical energy, which is
partly represented by the free oxygen and partly stored up in the above-
mentioned more complex chemical compounds, is transformed into other
forms of energy, principally heat and mechanical work. While in the plant
we find chiefly reduction processes and syntheses, which by the introduc-
tion 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 chloro-
phyll, 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 carbon dioxide, while in the
light the oxidation processes going on in the green parts are overshadowed
or hidden beneath the more intense reduction jirocesses. As in the animal,
we also find a heat production in fermentation produced by plant organisms;
and even in a few of the higher plants — as the aroideoe when bearing fruit —
a considerable development of heat has been observed. On the other
hand, in the animal organism, besides oxidation and splitting, reduction
processes and syntheses also takes 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
ANIMAL OXIDATIONS. 3
in the plant chiefly those of reduction and synthesis have thus far Ijeen
studied.
WoHLER 1 in 1824 was the first to observe an example of synthetical
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 glycocoU (aminoacetic acid). Since the discovery of
this synthesis, which may be expressed by the following equation:
CeHs.COOH + NH2.CH2.COOH = NH (C6H5.CO).CH2.COOH +H2O,
Benzoic acid GlycocoU Hippuric acid
and which is ordinarily considered as a type of an entire series of syntheses
occurring in the body where water is eliminated, the number of known
syntheses in the animal kingdom has increased considerably. ]\Iany of
these syntheses have also been artificially produced outside of the organism,
and numerous examples of animal syntheses of which the course is abso-
lutely clear will be found in the following pages. Besides these well-studied
syntheses, there occur in the animal body also similar processes unquestion-
ably of the greatest importance to animal life, but of which we know
nothing with positiveness. We enumerate as examples of this kind of
synthesis the re-formation of the red-blood pigment (the haemoglobin), the
formation of the different proteins from simpler substances, and the produc-
tion 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,^ that it is connected with the
form-elements and the tissues. The question as to how this oxidation in
the form-elements is induced and how it proceeds cannot be answered with
certainty.
When a substance is oxidized by neutral oxygen at the ordinar\' tempera-
ture or at the temperature of the body, the substance is said to be easily oxidized
or autooxidized, and the process is considered as a direct oxidation or auto-
oxidation. 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, fat, 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.
' Berzelius, Lehrb. d. Chemie, ubersetzt von Wohler, 4, p. 356, Abt. 1, Dresden, 1831.
^ Pfluger, Pfliiger's Archiv. 6 and 10; Finkler, ibid., 10 and 14; Oertmann, itid.t
14 and 15; Hoppe-Seyler, ibid., 7.
4 INTRODUCTION.
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 bod}'.
In explanation it is very generally admitted that the oxygen is made
active and this causes a secondary oxidation. It is generally conceded that
in autooxidation a cleavage of neutral oxygen takes place. The autooxidiz-
able 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 sup-
position that the oxygen is made active and thus produces secondary
oxidation.
The cause of the animal oxidation is considered, by Pfltjger and
several other investigators, to be dependent upon the special constitution 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-li\dng proteins," and considers them to be
somewhat different from those occurring in living protoplasm. The latter
are called "living proteins" (PFLiJGER), "active proteins " (Loew), or "bio-
gens" (Verworn). The living protoplasmic molecule differs from the
ordinary non-living protein b}^ being more unstable and therefore having a
greater inclination towards 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,i on the contrary, claims
an intramolecular introduction of oxygen into a large hypothetical proto-
plasmic 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,^ who bases his claim upon special investigations
and numerous toxicological observations, the unstability of the active
proteid molecule is due to the simultaneous presence of aldehyde and
unstable amino 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.
* Pfliigcr, Pfliiger's Archiv, 10; Latham, Brit. Med. Journal, 1886; Verworn,
Die Biogenhypothese, Jena, 1903.
' Loew and Bokomy, Pfliiger's Archiv, 25; 0. Loew, ibid., 30; and specially
0. Loew, The Energy of Living Protoplasm, London, 1896.
ANIMAL OXIDATIONS. 5
LoEW has also shown, in conjunction with Bokorxy, that in many
plants a verj' unstable reserve-protein substance occurs, which to a cer-
tain extent occupies an intermediate position between protein and organizec
living substance.
The explanation as to the oxidation process differs entirely accord-
ing to the conception of the structure of the unstable protoplasmic mole-
cule. If the living protoplasmic protein is not, like protein in the ordinary
sense, indifferent to neutral oxygen, we can admit of a cleavage of the
oxygen molecule by this change. The protein would be itself oxidized,
while on the other hand a secondary oxidation of other difficultly oxidiza-
ble substances could be brought about by the oxygen atoms set free.
Another very wideh' difTused view exists in regard to the origin of the
activity of the oxygen, namely, that by the decomposition processes in the
tissues, reducing substances are formed which split the neutral oxygen
molecule, miiting with one oxygen atom and setting the other free.
The formation of reducing substances during fermentation and putre-
faction is generally known. The butyric fermentation of dextrose in which
hydrogen is set free — C6Hi206 = C4H802-f 2C02-r2H2 — 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 ordi-
narily explained by the statement that reducing, easily oxidizable bodies
are formed which split oxygen molecules, liberating oxygen atoms which
aftenvard 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-Seyler 1), are produced.
In accordance with what has been stated above on the oxidations of
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 substances.
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 and then succumb
to consecutive oxidations, until after repeated cleavages and oxidations
the final products of metabolism are formed.
An activation of the oxygen may be produced according to 0. Nasse^
by a hydroxylization of the constituents of the protoplasm with the split-
ting off of molecules of water. If benzaldehvde is shaken with water and
' Pflliger's Archiv, 12.
- 0. Xasse, Rostocker Zeitung, No. 534, 1S91, and No. 363, lS9o,
6 INTRODUCTION.
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 ^ have also
found that certain varieties of protein have the property of being hydroxy 1-
ized in the presence of water. According to Nasse a whole series of oxida-
tions 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^).
By quantitative methods van't Hoff and his pupils ^ have shown
that molecular oxygen can be divided in two parts by certain autooxida-
tion processes. One of these unites wuth the autooxidizer and the other
with a body simultaneously present but not directly oxidizable, which, ac-
cording to the suggestion of Engler,^ is called the acceptor, a^an'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 two halves has also been shown by other investigators, such
as Maxchot, Engler, and his collaborators.^ These investigators never-
theless consider that autooxidation takes place in another way, namely, by
the formation first of peroxides by the taking up of oxygen molecules.
Traube ® 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 atoms
set free on the decomposition of the water unite with the neutral oxygen,
forming hydrogen peroxide, which may naturally also have an oxidizing
action.
A+2H20 + 02 = A(OH)2 + H202.
' E. Rosing, Untersuchungen liber die Oxydation von Eiweiss in Gegenwart von
Schwefel. Inaug. Dissert. Rostock, 1891.
' Baeyer and Villiger, Ber. d. d. chem. Gesellsch. , 33; Engler and Weissberg, ibid., 33.
^ van't Hoff, Zeitschr. f. physikal. Chem., 16; Jorissen, Ber. d, d. chem. Gesellsch.,
30, and Zeitschr. f. physikal. Chem., 22; Ewan, ibid., 16.
■* Ber. d. d. chem. Gesellsch., 33.
' Manchot, tJber freiwillige Oxydation, Leipzig, 1900; Engler and Weissberg,
Ber. d. d. chem. Gesellsch., 33; Engler and Frankenstein, ibid., 34.
' Ber. d. d. chem. Gesellsch., 15, 18, 19, 22, and 26.
ANIMAL OXIDATIONS. 7
According to the view of Engler and his collaborators, which corre-
sponds in great measure with those of Bach and of Manchot,^ 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):
A + 02 = A02 and A02 + B = AO + BO.
If this is so, still we do not know to what extent such peroxides are
formed in the oxidation in the living cell. The possibility of a production
of peroxides, and also of hydrogen peroxide, in animal oxidation is still
generally admitted, andCnoDAT and Bach ^ 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 decompose the hydrogen
peroxide with the production of molecular oxygen. According to Loew^
the physiological importance of the catalases is to protect the cell from
hydrogen peroxide, which acts as a protoplasmic poison.
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 proteins. The
active movement of the atoms within the active protein molecule is trans-
mitted to the oxygen and to the oxidizable substance, and when the dis-
solution 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,^ who also denies the supposition that the oxygen
becomes active, is 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,
' Engler and Wild, Ber. d. d. chem. Gesellsch., 30; Bach, Le Moniteur scientifique,
1897, and Compt. rend., 124; Manchot, 1. c.
^ Ber. d. d. chem. Gesellsch., 3o u. 36.
' 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, and Kastle and Loeven-
hart, Amer. Chem. Joum., 29.
*0. Loew, The Energy of Living Protoplasm, London, 1896.
*Arch. f. exp. Path. u. Pharm., 1-t.
8 INTRODUCTION.
according to the older views, without being oxidized themselves, act in an
analogous manner to the nitric oxide in the manufacture of sulphuric acid
by alternately taking up and giving off oxygen in the oxidation of dys-
oxidizable bodies. Traube has for a long time explained the oxidations
of the animal body in this way, and he calls these c^uestionable oxygen-
carriers oxidation ferments}
It has also been positively proved by the researches of Jaquet, Sal-
KOWSKi, Spitzer, Rohmann, Abelous and Biarnes, Bertrand, Bour-
QUELOT, De Rey-Pailhade, Medwedew, Pohl, Jacoby, Chodat and Bach.^
and others that in the blood and different tissues of the animal body, as also
in plant-cells, substances occur which have the property of causing certain
oxidations and are therefore called oxidation ferments or oxidases. The
nature and mode of action of these bodies will be discussed elsewhere in
this volume, hence it will be sufficient here to state that in general two
different groups of oxidation ferments are recognized. 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 from these latter by decomposition.
The many different views in regard to the oxidation processes 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 fer-
ments important means of bringing about oxidative decomposition of various
substances, and the occurrence of numerous intermediary metabolic prod-
ucts 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. Most investigators are agreed that
these decompositions are similar to certain oxidations studied by Drechsel ^
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.*
The oxidations in the animal body have long been designated as a
' M. Traube, Theorie der Ferment wirkungen, Berlin, 1858.
^Jaquet, Arch. f. exp. Path. u. Pharm., 29; Salkowski, Centralbl. f. d. med. Wis-
sensch., 1892 and 1894, and Virchow's Arch., 1-47; Spitzer, Pfliiger's Archiv, 60 and
07; 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; Jacoby, Ergebnisse der Physiologie, Jahrg. I, Abt.
1, which contains the literature of the subject; Chodat and Bach, 1. c,
3 Joum. f. prakt. Chem. (N. F.), 22, 29, 38, and Festschrift fiir C. Ludwig, 1887.
■* See M. Nenckl Arch, des sciences biol. de St, P6tersbourg, 1, 483; Abelous and
Aloy, Compt. rend., 136, 137; Kastle and Elvove, Amer, Chem. Joum., 31; Underhill
and Closson, Amer. Joum. of Physiol., 13.
CLEAVAGES IX THE ANIMAL BODY. 9
combustion, and such a conception is easily reconcilable with the above-
mentioned views. In combustion in the ordinary sense, as, for example,
the burning of wood or oil, we must not forget that the substances them-
selves do not com]:)ine with oxygen. 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 essential source of heat and mechanical work developed in the
organism is to be found in the oxidations. Chemical energy is transformed
into the above-mentioned forms of energy in cleavage processes, where
complicated chemical compounds are reduced to simpler ones, and there-
fore the atoms change from an imstable to a stabler equilibrium, 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 living
muscle are an example of this kind. A removed muscle, which gives off
no oxA'gen when in a vacuum, may, as Hermann ^ has shown, work, at least
for a time, in an atmosphere devoid of oxygen, and give off carbon dioxide
at the same time.
Cleavage processes which are accompanied by a decomposition of water
and then a taking up of its constituents are called hydrolytic cleavages.
These cleavages, which play an important role within the animal body,
and which are most frequently met with in the processes of digestion, are
exemplified by the transformation of starch into sugar and the splitting
of neutral fats into the corresponding fatty acids and glycerine:
C3H5(Ci8H350o)3 + 3H20 = C3H5(OH)3 + 3(Ci8H3602).
Tristearin Glycerine Stearic acid
As a rule the hydrolytic cleavage processes as they occur in the animal
body may be performed outside of it by means of higher temperatures
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 glycerine 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 disposal which act similarh', but in such a
manner that they may work without endangering the life or normal con-
stitution of the tissues. Such means have been recognized in the so-called
unorganized ferments or enzymes.
Untersuch. iiber den Stoffwechsel der Menschen, Berlin, 1867.
10 INTRODUCTION.
Alcoholic fermentation and other processes of fermentation and putre^
faction are dependent upon the presence of living organisms, ferment
fmigi, 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 fer-
ments 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 which are products of the
<:• hemic al work within the cell, and which after they are removed from the
t'ell still have their characteristic action. Such bodies — for example, malt
diastase, rennin, and the digestive ferments — are capable in the very small-
est quantity of causing a decomposition or cleavage in very considerable
quantities of other substances, without entering into permanent chemical
combination with the decomposed body or with any of the cleavage or
decomposition products. These formless or unorganized ferments are
generally called enzymes, according to KtJHXE.
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 severed from this ferment, and
still remain active even after the death of the latter. Consequently fer-
ments and enzymes are capable of manifesting a different behavior towards
<'ertain 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 i and his
pupils. He has been able to obtain from beer-yeast, by grinding and
strong pressure, a cell fluid rich in protein which when introduced into
' E. Buchner, Ber. d. devitsch. chem. Gesellsch., 30 and 31; E. Buchner and Rapp,
xbid., 31, 32, 34; H. Buchner, Sitzungsber. d. Gesellsch. f. Morphol. u. Physiol, in
Miinchen, 13, 1897, part 1, which also contains the discussion on this topic. See also
E. and H. Buchner and M. Hahn, Die Zymasegarung, Miinchen, 1903; Stavenhagen,
Ber. d. deutsch. chem. Gesellsch., 30; Albert and Buchner, ibid., 33; Buchner, ibid.,
33; Albert, ihid., 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 Joum. f. prakt. Chem. (N. F.), 64.
FERMENTS AND ENZYMES. 11
a solution of a fermentable sugar caused a violent fermentation. The
objections raised from several sides that the fluid expressed still contained
dissolved living cell substance has been so successfully answered by Buch-
NER and his collaborators that there is at present no question but that
alcoholic fermentation is caused by a special enzyme called zymase which
is formed in the yeast-cell.
As from the yeast-cell so also from other lower organisms, indeed from
the lactic-acid bacilli and beer-vinegar bacteria, we have recently been able
to isolate enzymes (E. Buchner and Meisexheimer, Herzog i) which
produce the specific fermentative action of the mother organism. The
question whether there exist ferment processes which, in Pasteur's sense,
are the result of the biological phenomena connected with the metabolism
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 are organic substances formed in the cells, whose chemical
nature has unfortunately not been established at the present time. Thus
far no enzyme has been prepared in a pure state with positiveness. The
enzymes are considered as protein bodies by many investigators, but this
opinion has not sufficient foundation, and is disputed at least for certain
enzymes. It is indeed true that the enzymes isolated by certain investi-
gators acted like genuine protein bodies; but it is uncertain whether or not
the products isolated in these instances were pure enzymes or were com-
posed of enzymes contaminated with proteins.
The enzymes may be extracted from the cells and tissues by means of
water or glycerine, especially by the latter, which forms very stable solu-
tions and hence is extensively used as a means of extracting them. The
enzymes, generally speaking, do not appear to be diffusible, and Bredig ^
has given several reasons, which will be given later, for considering them
not as true solutions but rather colloidal ones. The enzymes are also
absorbed by other colloids and are carried down by fine precipitates, and
this property is extensively taken advantage of in their preparation.^
' E. Buchner and J. Meisenheimer, Ber. d. d. chem. Gesellsch., 36; Herzog, Zeitschr.
f. physol. Chem., 37.
^ Anorganische Fermente, Leipzig, 1901.
' See Briicke, Wien. Sitzungsber., 43, 1861,
12 INTRODUCTION.
The manner of combination of the enzymes with the colloids has not been
explained and is no doubt not the same in all cases.^ They are precipitated
from their solutions by alcohol. All enzymes lose their specific action on
Ijoiling their aqueous solutions, and this is generally considered as an im-
portant criterion as to the ferment nature of a body. The continued heating
of their solutions above 80° C. generally destroys the enzymes. In" the
dry state, however, certain enzymes may be heated to 100° or indeed
to 150-160° C. without losing their activity. Light can also destroy en-
zymes in watery solution, as shown wuth malt diastase (Emmerling) and
chymosin (Emmerling, Schmidt-Nielsen).-
The action of the enzymes may be markedly influenced by external con-
ditions. The reaction of the liquid is of special importance. Certain
enzymes act only in acid; others, and the majority, on the contrary, act
only in neutral or alkaline liquids. Certain of them act in very faintly
acid as well as in neutral or alkaline solutions, but best at a specific reac-
tion. They are all destroyed by concentrated mineral acids and alkalies.
The temperature exercises also a very important influence. In general
the activity of enzymes increases to a certain limit with the temperature.
This optimum is not always the same, but, as shown by Tammann, depends,
like the destructive action of high temperatures, essentially upon the
quantity of enzyme and other conditions. The products of the enzymotic
processes exercise a retarding influence in proportion as they accumulate,
and indeed the enzymotic process may thereby be entirely stopped. In
such cases of "false equilibrium" (Bredig) we may, as shown by Tam-
mann,^ often start the reaction again by removing the products of the re-
action, by diluting with water, by raising the temperature, by the addition
of more substance, or by the addition of more of the enzyme. The addition
of neutral salts and other substances of various kinds has in some cases
an accelerating, and in other cases a retarding action.^
The velocity of the enz3'me action and the final condition at the conclu-
sion of the enzymotic processes is not only dependent upon the reaction,
the temperature, and the presence of transformation products or of foreign
bodies, but also upon the amount of enzyme present and the concentration
of the solution. The velocity increases regularly with an increase in tlie
amount of enzyme, but not in the same proportion with all enzymes, as it
has been shown for different enz3'mes that they require different times for
' Dauwe, Hofmeister's Beitrage, 6.
^ Emmerling, Ber. d. d. chem. Gesellsch., 34; Schmidt-Nielsen, Hofmeister's Bei-
trage, o.
^ The work of Tammann may l)e found in Zeitschr. f. physiol. Chem., 16, and
Zeitschr. f. physikal. Chem., 3 and IS.
* See Fermi and Pernossi, Zeitschr. f. Hygiene, 18; also in regard to the enzymes
in general see C. Oppenheimer, Die Fermente, 2. Aiid.. IJOo.
ENZYMES. 13
action. This will be discussed later. The concentration of the solution
is also of great importance, and the result of a change of this durino- enzy-
motic action is of special importance in the study of the kinetics of enzyme
reactions.
We have no characteristic reactions for all enzymes 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 combinations in definite pro-
portions by weight with the bodies upon which they act, or their decompo-
sition products; and, secondly, that an insignificantly 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 ^), and 1 part of chymosin can in a short time
decompose more than 400,000 parts of casein (Hammarsten^). This does
not exclude the possibility of a primary, but temporary, combination of the
enzymes with the substances acted upon. Such an assumption is, indeed,
substantiated by the work of Hanriot, Henri, Armstrong,^ and others,
while, according to Oppenheimer,* we can represent ferment action as
consisting of a first phase where combination of the enzyme and the sub-
stance occurs, and a second phase where after this combination a chemical
decomposition of the substance occurs according to the laws of catalysis.
This view coincides best with the specificity of enzyme action.
The specific action of the enzymes is of special importance, as one and
the same enzyme acts only upon one substance or a definite group of sub-
stances. Their action seems to be entirely dependent upon the stereo-
metric construction of the substance acted upon, and we may assume
that the enzyme attacks only specially arranged stereometric atomic
groups, where the enzyme fits the substance in a manner similar to a key
fitting a lock (E. Fischer). E, Fischer ^ has given a positive proof for
the great importance of a different stereometric configuration by his inves-
tigations upon the artificially prepared series of stereoisomeric glucosides
which he calls a- and /9-glucosides. The enzymes of yeast infusions act
only upon the glucosides of the a-series, while emulsin, on the contrarj',
acts only upon those of the /3-series.
Of especially great importance for a deeper insight into the manner
of enzyme action, we must mention the investigations which have been
' O'SuUivan and Thompson, Joum. of Chem, Soc, 57.
' See Maly's Jahresbericht, 7.
' Hanriot, Compt. rend., 132; Henri, Lois generates de Taction des diastases, Paris,
1903, and Arch, di Fisiol., 1 and 2; Armstrong, Proc. Roy. Soc. London, 73.
*Die Fermente, 2. Aufl., 1903, p. 66.
"Zeitschr. f. physioL Chem., 26.
14 INTRODUCTION.
carried on recently on the relationship of inorganic catalyzers to the
enzymes, which have thrown light upon the correspondence between
catalysis and enzyme action. The catalyzers, like the enzymes or their
derivatives, 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 propor-
tionate to the quantity of substance transformed is infinitesimally small
in enzyme action as well as in catalysis. In both, the reaction velocity
also seems to be independent of the quantity of the active substance added,
and this indicates that the enzyme action is not to be considered as the
starting of a reaction which would not of itself take place, but rather as
an acceleration of a slowly proceeding, often not noticeable, chemical
change. According to this conception enzyme action comes in line with
catalysis, for, according to Ostwald,i bodies are called catalyzers which
b}' their presence cause a change in the reaction velocity of chemical proc-
esses, and indeed positive or negative, according as they produce accelera-
tion or retardation. The striking correspondence between enzymes and
inorganic catalyzers has been shown especially by Bredig and his collaljo-
rators, v. Bernek, Ikeda, and Reinders,^ by their very important in-
vestigations.
Bredig has been able to prepare colloidal solutions of platinum, gold,
and silver by allowing the electric arc to play between two poles of the
respective metal beneath water. These solutions of colloidal metals,
metallic sols, show in their activity and the dependence of this activity
u})on external influences, and especially in their destruction by poisons,
such strong resemblance to the enzymes that Bredig has indeed called them
inorganic ferments.
Still it is nevertheless true that the manner of action of catalyzers
has not been explained, and we must be careful not to draw too positive
conclusions from the remarkable correspondence of the manner of action
of metallic sols and certain ferments. In studying the action of enzymes
one is repeatedly struck with the marked deviation from the laws of reac-
tion underlying inorganic catalyzers,^ and this has called forth a series
of hypotheses and attempts at explanation, which on account of space
cannot be entered into, but we must refer the reader to special works on
the subject. On the other hand, we must not forget that the enzymes are
not pure substances, but are habitually mixtures whose action may be
' Gruiidriss d. allgemoin. Chcmie, 3. Aufl., 1899.
* See Bredig, Anorganische Fermente, Leipzig, 1901, and also Die Elemente d..
chemischen Kinetik, etc., Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 1902.
3 See Brown and Glendinning, Proc. Chem. Soc. 18, 1902; Tammann, Zeitsehr. f.
physikal. Chem., 3 .~.i.^ 18, and Zeitsehr. f. physiol. Chem., 16; Henri, Zeitsehr. f.
physikal, Chem., 31), and Lois g6n6rales, etc. See also the work of H, Euler, Zeitsehr.
f. physiol. Chem. , 45.
ENZYME ACTIONS. " 15
modified by an apparently insignificant admixture, and for this reason
the study of the mode of action is made very difficult. Although the
question as to whether enzymes follow the same laws as the inorganic
catalyzers is still an open one, nevertheless we know that in a great
many regards the enzymes correspond with catalyzers. The comparison
of these two has opened up in the study of enzyme action new points
of elucidation and attack which have been very fruitful in result, and
which have no doubt helped very much in the explanation of these difficult
questions.
It is not within the scope of this book to enter more in detail into the
various theories of catalysis. Still it seems important at least to present
in a few words one of these, namely, that of H. v. Euler.i This theory
explains the mode of action of enzymes and the inorganic catalyzers by
assuming an increased concentration of the active molecules producing the
reaction, i.e., by increasing the ions occurring in the solution.
The action of enzymes presupposes the presence of water, and the best-
studied enzymotic processes, the hydrolyses, are comparable with the
+
action of acids and bases, i.e., the action of H and HO ions. In the hydroly-
ses by enzymes an activation of the water takes place, and the assumption
+ -
that the enzymes act by an increased concentration of the H and HO ions,
which bring about the reaction, seems to be attractive. The enzymes
acting analogously to mineral acids have been assumed, according to this
+
view, to be producers of H ions, which strongly accelerate cleavages which
would otherwise take place very slowly or with immeasurable velocit}'.
This explanation may, as developed by Friedenthal,^ be applied to the
oxidation enzymes, the oxidases, which will be treated of later. Water is
also imperative for animal oxidations, and the reaction of the fluid is in this
case also important because oxidations are regularly accelerated by an
alkaline reaction, i.e., by the presence of HO ions. We can, according to
Friedenthal, consider the oxidases as producers of hydroxy 1 ions, just as
we can consider pepsin as a producer of hydrogen ions. It is apparent that
this view, that the oxidases are producers of hydroxyl ions, is in harmony
with the previously mentioned views of Traube and Nasse,^ that the
hydroxyl ions of the water combine with the oxidizable substance.
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
catalyzers, but only upon certain bodies. Some restriction must be put
to this, as the cells do not always produce a complete enzyme, but oftener
only the mother-substance thereof. These mother-substances of the
* Zeitschr. f. physikal. Chem., 36. "^ Salkowski's Festschrift, 1904. ^ See pp. 5 and 6.
16 INTRODUCTION.
enzymes are called proenzyines 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 Chapters VI and IX),
The enzymes are, as above mentioned, not characterized by chemical
reactions in the ordinary sense, but by their action. From this stand-
point most of the enzymes which have been studied can be divided into
two chief groups, namely, those enzymes having a hydrolytic action and
those having an oxidizing action.
Among the hydrolytic enzymes we must mention in the first place the
jyroteolytic or those which dissolve proteid, whose representatives, pepsin and
trypsin, occur in the animal kingdom; the lipolytic or fat-splitting; and
the amylolytic or diastatic enzymes, which act upon the starches. In this
group we must include the invertases, which split the disaccharides into
simpler forms of sugar. In close relationship to these enzymes we may
mention the ghicoside-splitting enzymes, which occur especially in the
higher plants. Among the hydrolytic enzymes of the animal kingdom
we must also include arginase, which splits arginineinto urea and ornithine;
the two desaniinating enzymes adenase and guanase, which convert the two
bodies adenine and guanine, with the splitting off of ammonia, into hypo-
xanthine and xanthine respectively; and the hippuric-acid-splitting/iis^02;i/m
and the urea-splitting urease. The proteid-coagulating enzymes, chymosin
■or casein-coagvilating, and thrombin or blood-coagulating enzyme, belong to
a special though not clearly defined group.
The best-known and most carefully studied enzyme actions, the hydroly-
ses, are exothermal processes, and therefore the sum of the new products
produced has a lower heat of combustion than the original substance.
Now, as syntheses are generally endothermal reactions, i.e., are processes
requiring a taking up of heat where external energy must be supplied before
they take place, and also as the enzymes are not a source of energy, it
used to be generally considered that the enzymes could not bring about
any syntheses. This view is nevertheless untenable, and it has also been
shown that enzymotic hydrolyses may be reversible processes which pro-
duce syntheses. Croft Hill has shown that maltase, which, as is well
known, has a splitting action upon maltose, also has the power of regener-
ating from glucose two isomeric bioses, one a new body called revertose and
another which is probably maltose (see also Emmerling i). E. Flscher
and E. F. Armstrong ^ were able to obtain a dissaccharide, isolactose,
from galactose and glucose by means of kephir lactase. Hanriot,' Kastle
» Hill, Ber. d. d. chem Gesellsch., 34, and Transactions Chem. Society, 1903, 83;
Emmerling, Ber. d. d. chem. Gesellsch., 34.
' Ber. d. d. chem. Gesellsch., 35.
» Compt. rend., 132.
ENZYME ACTIONS. 17
and LoEVENHART 1 have shown that the Upases can bring about syntheses,
and finally Emmerling ^ has been able to synthesize amygdalin from raan-
delic-aeid-nitrile glucoside and glucose by means of the yeast maltase.
According to Abelous and Ribaut^ the pig and horse kidneys contain
an enzyme which produces hippuric acid from benz^d alcohol and glycocolL
These investigators are of the opinion that the benzyl alcohol is first oxi-
dized to benzoic acid and then that the synthesis is brought about by the
aid of the energy set free in this process. There is more and more tendency
to accept the view that the intracellular enzymes, which will be discussed
later, are of importance for the S3'ntheses in the animal body.
The second group of enzymes include the so-called oxidation fer-ments,
which, as above remarked, are recognized as of great importance in bringing
about oxidations in the animal body. These enzymes do not all act in the
same way, and correspondingly we differentiate between direct oxidases
or oxidases proper, and indirect oxidases or peroxidases. Certain inves-
tigators include among the oxidation enzymes still a third group, the
catalases, which split peroxides into hydrogen and oxygen.
Those enzymes which transfer oxj^gen to other bodies and oxidize
them are called oxidases or direct oxidases. Peroxidases or indirect oxi-
dases are, on the contrary, enzymes having an oxidizing action onl}^ in the
presence of hydroperoxides or another peroxide, as they decompose the
peroxide and bring about oxidation by the oxygen set free. Correspondingly
the oxidases turn tincture of giiaiacum blue directly, while the peroxidases
only have this action in the presence of a peroxide. The catalases do not
give any reaction with giiaiacum either directly or indirecth* in the presence
of peroxides.
According to the investigations of Bach and Chodat ■* the conditions 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 takmg up oxygen. These peroxides 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 molecular oxygen is first converted
by the oxygenase into peroxide. This peroxide is activated by the peroxi-
dase and then has strong oxidizing power. The oxidizing power of the
* Amer. Chem. Journ., 24.
2Ber. d. d. chem. Gesellsch., U, 3810.
^ Compt. rend. Soc. biol., 52; Maly's Jahresber., 30.
* Biochem. Centralbl., 1, pp. 417 and 457.
18 INTRODUCTION.
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 supposed to be nucleoproteids (Spitzer), others globulins (Abelous
and BiARNEs), and still others, like the liver aldehydase (Jacoby) and
laccase (Bertrand), are of a non-protein nature. The materials upon
which the oxidation enzymes act may also be very different from each
other. Thus the oxidases studied by Rohmann and Spitzer may Vjy
synthetical oxidation produce indophenol from a-naphthol and p-phenyl-
enediamine in the presence of alkali. The salicylase or aldehydase detected
in the liver and many other organs oxidizes many aldehydes to their cor-
responding 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 p-phenols, such as hydroquinone, but not upon tyrosine.
The bodies called tyrosinases, first found by Bertrand * in certain fungi
and later also found by Biedermann, v. Fijrth, and Schneider in the
animal kingdom, have, on the contrary, an action upon tyrosine, converting
it into homogentisic acid (Gonnermann 2) or other colored compounds.
Another oxidase occurring in the liver and spleen, and called xanthine
oxidase by Burian, has the property, as shown by Spitzer, Wiener,
Schittenhelm, and Burian,^ of transforming xanthine and hypoxanthine
into uric acid by oxidation.
The oxidases and peroxidases as well as the catalases occur very widely
distributed in the animal and plant kingdoms.
Like other enzymes, the oxidation enzymes show also a pronounced
specificity; thus a certain oxidase, for instance laccase, oxidizes only certain
substances and not others. This behavior, which is difficult of explana-
tion according to the common hypotheses as to the action of oxidation
enzymes, indicates, according to Medwedew,^ that in the oxidation the
active substances do not act upon the oxygen, but rather upon the sul-
stance 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 in all cases where oxidation
enzymes have been claimed to have been found we were actually dealing
with enzymes.
' In regard to the work of the various authors cited, see foot-note, p. 8.
' Biedermann, Pfliiger's Archiv, 72; v. Fiirth and Schneider, Hofmeister's Beitrage,
1; Gonnermann, Pfliiger's Archiv, 82.
^Spitzer, Pfliiger's Archiv, 76; Wiener, Arch. f. exp. Path. u. Pharm., 42; Schit-
tenJielm, Zeitschr. f. physiol. Chem., 42 and 43; Burian, ibid., 43.
*Pfliiger'.s Archiv, 81.
OXIDATION ENZYMES. 19
In investigations with hydroperoxides and vegetable peroxidases Bach
and Chodat ^ 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 enzy-
motic nature. Aso ^ has also shown that in certain cases where an apparent
oxidase action was present ver}- probably we w^ere dealing only with nitrites
which were present; and finally, attention must be called to the fact that
manganese or iron, sometimes in considerable amounts, has been found
in many oxidases. As manganous and ferrous salts are active as cataly-
zers in certain other oxidations, so also in certain cases important roles
as' oxygen-carriers have been ascribed to these metals, for instance in
laccase, which contains manganese (Bertrand), and the oxidases contain-
ing iron (Spitzer's nucleoproteid). Manchot^ by his work on the auto-
oxidation of ferrous sulphate has called attention to the apparently great
importance of iron for physiological oxidations, and the work of Trillat,*
who has prepared colloidal solutions of protein-manganese which had
great similarity to oxidase solutions, is also of special interest.
Our knowledge of the reducing enzymes,^ the so-called reductases or
hydrogenases, is even still more meagre. 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 and consider the enzymotic nature of the philothions
as doubtful.^ There is no doubt that reductions occur to a great extent
in the animal body and often hand in hand \nth oxidations; nevertheless
the question as to how far special reduction enzymes take part in these
reductions is still an open one. According to Abelous and Aloy ' 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 Ijy remo^•ing
it from another substance through reduction.
The property of decomposing h3^drogen peroxide has been observed with
many enzymes, but this property does not Iselong to them,^ depending
' Ber. d. d. chem. CJesellsch., 37.
' Beihefte zum botan. Centralbl., 18.
' Zeitschr. f. anorg. Chem., 27.
' Compt. rend., 137, 138.
^ Abelous and Gerard, Compt. rend., 129; Pozzi-Escot, Bull. Soc. chim. (3), 27.
• De Rey-Pailhade, Recherches exper. sur le Philothion, etc., Paris (G. Masson),
1891, and Nouvelles recherches sur le Philothion, Paris (G. Masson), 1892; Pozzi-Escot,
1. c, and Chem. Centralbl., 1904, 1, S. 1645; Chodat and Bach, Ber. d. d. chem.
Gesellsch., 36; Abelous and Ribaut, Compt. rend., 137, and Bull. Soc. chim., Paris (3),
31.
' Compt. rend., 136, 137, and 138.
^ See Al. Schmidt, Zur Blutlehre, Leipzig, 1892; Jacobson, Zeitschr. f. physiol.
Chem., 16.
20 INTRODUCTION.
rather upon another enzyme, a catalase, vhich often adheres to other
enzymes as an impurity. The catalases were first closely studied by O.
LoEW,i and he has investigated two different catalases — the a- and /3-catalase.
The first, which is not soluble in water, is a nucleoproteid, while the other,
^-catalase, is soluble in water and is a proteose.
The catalases, whose action consists in decomposing hydrogen peroxide
into oxygen and hydrogen, occur widely diffused in the animal and plant
kingdoms. According to L. Liebermaxn^ the fatty tissues among the
animal structures seem to be richest in catalases, an observation which has
been substantiated and developed by Euler.^ The liver, kidneys, and
spleen are relatively rich in catalases, while the brain and muscles are
poor therein; still the proportions vary somewhat for different varieties
of animals.'* As has long been known, the blood also contains a catalase,
which has been called hcemase by Sexter.^
The physiological importance of the catalases is still unknown. Ac-
cording to LoEW ^ they have the function of destroying the hydrogen per-
oxide, which occurs perhaps as an intermediary product in oxidations and
which has a destructive action as protoplasmic poison; but this assumption
is disputed by Euler "^ and others. Euler calls attention to the parallelism
which exists between the fat-splitting and the peroxide-splitting action of
plant and animal extracts and claims that the lipolytic extracts have the
property of decomposing hydrogen peroxide.
The glycolytic or svigar-destroying enzyme, which occurs in the blood
and tissues and which takes part in the decomposition of the sugars, stands
in close relationship to the oxidases. We will discuss this enzyme in a
following chapter in speaking of glycosuria and the question of diabetes,
and it is here sufficient to remark that certain investigators, like Spitzer,
consider this enzyme as an oxidase, while others, on the contrary, consider
the decomposition of the sugar in the tissues to be a process analogous to
alcoholic fermentation.
Alcoholic fermentation by means of yeast or zymase is not an oxidation
in the ordinary sense, where the sugar takes up free oxygen. It is rather
an internal oxidation where a part of the molecule is oxidized at the cost
' U. S. Dept. of Agriculture, Rep. 68, Washington, 1901, and Ber. d. d. chem.
Gesellsch., 35.
2 Pfliiger's Arch., 104.
^ Hofmeister's Beitrage, 7, which also gives the references to the literature.
* See Battelli and Stern, Compt. rend., 138; BatteUi and HalifT, Compt. rend. Soc.
biol., o".
^ Senter, Zeitschr. f . physikal. Chem., 44, also A. Jolles and Oppenheim, Virchow's
Arch., ISO; Ville and Moitesnier, Bull. Soc. chim. (3), 29; A. Rosenbaum, Salkow-
ski's Festschrift, 1904.
•See foot-note, 3, p. 7.
' Hofmeister's Beitrage, 7.
GLYCOLYSIS AND ALCOHOL FERMENTATION. 21
of another part, and finally a destruction into alcohol and carbon dioxide
takes place. According to the recent investigations of Buchxer and
Meisexheimer, Stoklasa, and Maze,^ we are dealing here with the united
action of two enzymes, one the lactolase (Stoklasa) or ladacidase (Buch-
xer and ]\Ieisexheimer), which converts the sugar into lactic acid, while
the other, the zymase (Buchxer and ]\Ieisexheimer) or alcoholase (Stok-
lasa), splits the lactic acid into alcohol and carbon dioxide. According to
several investigators, the sugar passes into lactic acid, with methylglyoxal,
CH3.CO.CHO, as an intermediary body.
Stoklasa and his collaborators ^ believe that an alcoholic fermentation
b}' means of a zymase or perhaps a mixture of the two above-mentioned
enzymes, lactolase and alcoholase, also takes place in animal tissues. Ob-
jections to these investigations have been made by several experimenters
who claim essentialh' that in these cases we are dealing only with the action
of micro-organisms.^ Hammarstex considers that the views of Stoklasa
and his collaborators have not been disproved, and one cannot exclude the
possibility that an alcoholic fermentation may also occur in the animal
tissues in anaerobic respiration.
The enzymes, in certain instances, may also act upon one another, and
as an examj^le of this kind of action we may mention Buchxer's zymase,
which can be destroyed by the proteolytic enzyme of the yeast-cells. Pepsin,
which has a destructive action upon diastases and especially upon trj'psin,
is another example. Of special interest is the action of the anti-enzymes
upon the enzymes, wliich consists in retarding or arresting the specific
action of the enzyme by a corresponding anti-body. This subject will be
discussed later.
Unfortunately considerable confusion exists in the nomenclature of the
enzymes. In most cases the enzyme is named after the substance upon which
it acts, thus amylase, lipase, arginase, urease; in other cases according to its
action, thus oxidase, reductase; while in certain cases the products produced
are the basis for the name, thus alcoholase, lactacidase, glucase. In order to
obtain a clear and concise nomenclature of the enz}Tnes v. Lippmann " has suggested
that we construct the name of the enzyme out of two words, one of which rep-
resents the substance acted upon by the enzyme, while the second is the im-
portant or chief product produced by the enzyme. Thus maltoglucase is an
enzyme which produces d-glucose from maltose, amylmaltase one that forms
maltose from starch (amylum), etc.
* Buchiaer and Meisenheimer, Ber. d. d. chem. Gesellsch., 3" and 38; Stoklaha,
Ber. d. d. botan. Gesellsch., 22, pp. 358 and 460; Maze, Compt. rend., 13S.
^ Hofmeister's Beitrage, 3; Centralbl. f. Physiol., 16, 17, 18; Ber. d. d. chem.
Gesellsch., 38; see also Czemy, ibid., 36, with Jelinek, Simacek, and Vitek, Pfliiger's
Arch., 101.
' See the work of O. Cohnheim, Zeitschr. f. physiol. Chem., 39, 42, 43; BatteUi,
Compt. rend., 137; Portier, Compt. rend. Soc. biol., 57.
* Ber. d. d. chem. GeseUsch., 36.
22 INTRODUCTION.
Many enzymes are secreted by the cells as such or as proenzymes. They
act outside of the cells in which they were formed, or they act after having
been transformed into the enzyme, and hence are called secretion enzymes
or e:xtracellular enzymes.
Besides these extracellular enzymes we also have another group which
act within the cells, hence are intracellular and therefore are called intra-
cellular 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 proteo-
lytic enzymes, which were first observed by Salkowski and his pupils, and
which bring about the self-digestion or autodigestion of organs in the
absence of micro-organisms. This autodigestion has been the subject of
numerous investigations, principally by the Hofmeister school and esj^e-
cially by Jacoby.^ 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, oxi-
dations 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
ocour 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,^ the products obtained by the hydrolytic 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 conjectures 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 are
' A complete summary of the literature of intracellular enzymes and autolysis may
be found in Jacoby, Uber die Bedeutung der intrazellularen Fermente, etc., Ergeb-
nisse der Physiologic, Jahrg. I, Abt. 1, 1902.
2 Levene, Amer. Joum. of Physiol., 11 and 12, and Zeitschr. f. physiol. Chem., 41;
"W. Jones, ibid. 42.
AUTOLYSIS. 2a
inactive in neutral or alkaline reaction. The observations of Lane-Claypon
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. It is possible that this is due to the fact that the
enzymes are first formed from the proenzymes after the death of the organ,
or perhaps certain conditions tending to retard the enzymotic action are
removed. Recent investigations of Wiener ^ show that the post-mortem
formation of acid is the important factor in this. 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 pathological conditions.
The changes of the liver and blood in acute phosphorus intoxication
and in acute yellow atrophy of the liver, where we find in the urine the
enzymotic decomposition products of the proteins, 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 pneumonic
infiltrations by the enzymes of the migrated and inclosed leucocytes as
studied by Fr. ]\Iuller ^ and this is at the same time an example of hete-
rolysis, i.e., of a solution or a destruction in an organ by enzymes not
belonging therein but introduced from without. An autol3'sis, although not
very marked, occurs in those organs or parts of organs which have not
been normally nourished because of a disturbance in the circulation, and
they are gradually consumed by this action. The part injured undergoes
solution, while the healthy part remains unattacked. By this solvent
action as well as by the formation of bactericidal bodies, as observed by
CoNRADi,* and of antitoxins (Blum ^) 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 role. Numerous observations show this to be true, and
we tend more and more toward the view that the chemical transforma-
tions in the living cells are brought about by enzymes and that these latter
are to be considered as the chemical tools of the cells (Hofmeister and
others ^).
* Joum. of Physiol., 31.
'Centralbl. f. Physiol., 19, p. 349.
' Verhandl. d. naturforsch. Gesellsch. zu Basel, 1901. See also O. Simon, Deutsch.
Arch. f. klin. Med., 1901.
* Hofmeister's Beitrage, 1.
^Ibid., 5, p. 142.
* F. Hofmeister, Die chemische Organisation der Zelle, Braunschweig, 1901.
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. Pfluger says
that there exists a blood-relationship 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 pro-
cesses 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 biochemical 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
products produced by them must be of infuiite importance in their chemical
investigation.
The products produced by micro-organisms may be of very different
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.^ Certain of these are poisonous, designated as tox-
ines, while the 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
pentamethylenecUamine, C5H14N2, and jmtrescine or tetramethylenediamine,
C4H12N2, which have awakened special interest because they occur in the
contents of the intestine and in the urine in certain pathological condi-
tions, especially in cholera and cj^stinuria.^ Among the bodies produced
by putrefaction, the bacterial poison sepsine, C5H14N2O2, recently isolated
by E. Faust,^ is of especially great interest because to this substance we
ascribe the characteristic toxic action of putrefactive masses. Sepsine was
prepared by Faust as a crystalline sulphate which on repeated evaporation
of its solution was readily converted into cadaverine sulphate.
Those substances of basic nature which are incessantly and regularly
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 Gautii:r
' Selmi, Sulle ptomaine od alcaloidi cadaverici c loro importanza in tossicologia,
Bologna, 1878; Ber. d. deutsch. chem. Gesellsch., 11, Correspond, by H. S hiff;
Brieger, Ueber Ptomaine, Parts 1, 2, and 3, Berlin, 1885-1886; A. Gautier, Traite
de chimie appliqu6e k la physiologic, 2, 1873, and Compt. rend., 94.
^ See Brieger, Berlin, klin. Wochenschr., 1887; Baumann and Udransky, Zeitschr.
f. physiol. Chem., 13 and 15; Brieger and Stadthagen, Berlin, klin. Woohcnschr., 1889.
^ Arch. f. exp. Path. u. Pharm., 51.
PTOMAINES AND LEUCO:\IAL\ES. 25
in contradistinction to the ptomaines and toxines produced by micro-
organisms. These bodies, to which belong several well-known animal
extractives, were isolated by Gautier ^ from animal tissues such as the
muscles. The hitherto known leucomaines, of which a few are poisonous
in small amounts, belong to the choline, the uric acid, and the creatinine
groups.
The leucomaines are considered as being of certain importance in caus-
ing disease. It has been contended that when these bodies accumulate on
account of an incomplete excretion or oxidation in the system, an auto-
intoxication 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 which have an unmistakable relationship
to the enz3-mes. A closer study of these various bodies, Ij-sines, agglutinines,
toxines, etc., as well as of the antitoxines and the theory of immunit}', does
not lie within the scope of this work, and although the subject is of the
greatest importance, it cannot be treated here. We can only call atten-
tion to one similarity between many toxines and enzymes, and this is
important in connection with what we have already stated in regard to
the enzymes. As by the repeated introduction of a toxine into an animal
body we can excite a formation of the corresponding antitoxine, so, as first
shown by ^Iorgexroth,^ it is also possible, by the m creasing introduction
of an enzyme (rennin, for example), to produce an antienzyme (an antiren-
nin) in the body. Similar antienzymes have been produced in several
other cases, but this is not suiprising, as this is onl}' a special case of the
general immunity theor}', according to which the animal bod}' has the
power of making foreign substances non-destructive by reaction products
formed by the body.
' Bull. Soc. chim., 43, and A. Gautier, Sur les alcaloides derives de la destruction
bacterienne ou physiologique des tissus animaux, Paris, 1886.
^ Bouchard, Logons sur les auto-intoxications dans les maladies, Paris, 1887. See
also the various text-books of clinical laedicine.
^ Centralbl. f. Bakteriol. u. Parisitenkunde, 26.
CHAPTER II.
THE PROTEIN SUBSTANCES.
The chief mass of the organic constituents of animal tissues consists of
amorphous, nitrogenized, xery complex bodies of high molecular weight.
These bodies, which are either proteids in a special sense or bodies nearly
related thereto, take first rank among the organic constituents of the ani-
mal 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 npaorevo, I am the first, or take the first place). The bodies
belonging to these several groups are called protein substances, although in
a few cases the protein bodies in a special sense are designated by the
same name.
The several protein substances ^ 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 nitrogenized
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 products.
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 com-
bined 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 belongs) ; (4) monamino-acid nitrogen ;
•See "Eiweisskorper," Ladenburg's Handworterbuch der Chemie, 3, 534-589,
which gives a very complete summary of the Hterature of protein substances up to
1885. The more recent Hterature up to the year 1903 may be found in O. Cohnheim,
Chemie der Eiweisskorper, Braunschweig, 1904. See also Mann, Chemistry of the
Proteids, London, 1906.
26
NITROGEN OF THE PROTEINS. 27
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 moreover can-
not be given ^\ith certainty, because of the above-mentioned melanoidin
formation and the errors in the methods used.^ The follo\Wng gives at least
an approximate idea of this di^ision.2 The loosely combined so-called amide
nitrogen seems to be entireh' absent in the protamines. In the gelatines we
find 1-2 per cent, and 5-10 per cent in other animal protein substances; in
the plant gluten-proteids. 13-20 per cent of the total nitrogen is amide nitro-
gen. 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 gelatines
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, 15-25 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, ^ith
the exception of the protamines, as the monamino-acid groups. The result-s
for the melanoidin nitrogen vary so considerably that they will not be
mentioned.
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 xery small part, 1-2 per
cent, of the nitrogen is evolved,^ which seems to indicate that NII2 groups
exist only to a slight extent in protein substances. This assumption 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. In ^^ew of several observations, it is generally ad-
mitted that the amino-groups occurring in the cleavage products exist in
the original protein substance chiefly as imino-groups.
The sulphur occurs in the different protein bodies in very' different
* See the work of Hausmann, Zeitschr. f. physiol. Chem., 27 and 29; Henderson,
ibid., 2"; Kossel and Kutscher, ibid., 30; Kutscher, ibid., 31, 38; Hart, ibid., 33;
Giimbel, Hofmeister's Beitrage, 0; Rothera, ibid.
^ See the works given in foot-note 1 and Blum, Zeitschr. f. physiol. Chem., 30;
Kossel, Ber. d. d. chem. Gesellsch., 34, 3214; Hofmeister, Ergebnisse der Physiol.,
Jahrg. I, Abt. 1, 759, which also contains the literature; Osborne and Harris, Joum.
Amer. Chem. Soc, 25; and Giimbel, 1. c.
2 See C. Paal, Ber. d. d. chem. GeseUsch., 29; H. SchifT, ibid., 1354; O. Loew,
Chemiker Zeitung, 1896; and O. Xasse, Pfliiger's Arch., 6.
^ Levites, Zeitschr. f. physiol. Chem., 43.
28 THE PROTEIN SUBSTANCES.
amounts. Certain of them, such as the protamines and apparently also
certain bacterial proteids/ are free from sulphur; some, such as gelatine
and elastin, are very poor in sulphur; while others, especially horn sub-
stances, are relatively rich in sulphur. On hydrolytic cleavage with min-
eral acids, the sulphur of the protein substances is regularly, at least in
part, split off as cystine (K. ^Iorner) or, with bodies poorer in sulphur, as
cystein (Embden), but this, according to ^Morn'er and Patten, is a second-
ary^ formation. From certain protein substances a-thiolactic acid (Suter,
Friedmann, Frankel), wliich]\IORXER claims is also produced secondarily,
mercaptans and sulphuretted hydrogen (Sieber and Schoubenko, Rub-
xer), and a body having the odor of ethyl sulphide (Drechsel) have been
obtained .2
A part of the sulphur separates as potassium or sodium sulphide on
boiling with caustic potash or soda, and may be detected by lead acetate
and quantitatively determined (Fleitmann, Daxilewsky, Kruger, Fr.
ScHULZ, Osborne, K. ^Morner^). What remains can be detected only
after fusing with potassium nitrate and sodium carbonate and testing for
sulphates. The ratio between the sulphur spUt off b}' alkali and that not sj^lit
off is different in various proteins. No conclusions can be dra\\ai from
this in regard to the number of forms of combination which the sulphur
has in the protein molecule. As shown by K. ]\Iorxer, onh' 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
I, we obtain the quantity corresponding to the cystine sulphur in the body.
By such calculation ]Morxer found in certain bodies, such as horn sub-
stance, seral])umin and serglobulin, that the quantity of cystine sulphur and
total sulphur were identical, and therefore we have no reason for consider-
ing the sulphur in these bodies as existing in more than one form of com-
bination. 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 ■* keratin-like proteins split off sulphur dioxide on
' See Nencki and Schaffer, Joum. f. prakt. Chem. (N. F.), 20, and M. Nencki,
Ber. d. d. chem. GeselLsch., 17.
2 K. Morner, Zeitschr. f. physiol. Chem., 28, 34, and 42; Patten, ibid., 89; Embden,
ibid., 32; Suter, ibid., 20; Friedmann, Hofmeister's Beitrage, 3; Sieber and Schou-
benko, Archiv d. sciences biol. de St. Petersbourg, 1; Rubner, Arch. f. Hygiene, 19;
Drechsel, Centralbl. f. Physiol., 10, 529; Frankel, Sitzungsber. d. Wien. Akad., 112,
II b, K03
' Fleitmann, Annal. der Chem. und Pharm., 60; Danilcwsky, Zeitschr. f. physiol.
Chem., 7; Kriiger, Pfliiger's Archiv, 43; F. Schulz, Zeitschr. f. physiol. Chem., 25;
Osborne, Connecticut Agric. Expt. Station Report 1000; Morner, 1. c.
^ See Biochem. Centralbl., 4, p. 353.
CLEAVAGE PRODUCTS OF THE PROTEINS. 29
treatment witli phosphoric acid at ordinan* temperatures; hence it follows
that a part of the sulj^hiir in the proteins, esi:)ecially in the keratins, exists
in direct combination ^\'ith 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 yesLTs 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 the proteids in the true sense have
been primarily used, esjiecially those that can be prepared in the cr^'stalline
form, first large atomic complexes — proteo-ses and peptones — are obtained
which still have protein characteristics, and these then suffer further de-
composition until finally we obtain simpler, generally cr\-stalline, or at
least well characterized end products.
On heating protein T\-ith barium hydrate and water m sealed tubes to
Io0-2o0° C. ScHUTZEXBERGER ^ obtauied a mixture of products among
which were ammonia, carbon dioxide, oxalic acid, acetic acid, and, as chief
product, a mixture of amino-acids. The conclusion he drew from this
experiment, that the proteid is a complex ureide or oxamide, cannot be con-
sidered for several reasons .2
On fusing proteins with caustic alkali we obtain ammonia, methyl mer-
captan, and other volatile products; also leucine, from which then volatile
fatty acids, such as acetic acid, valerianic acid, and also butyric acid are
obtamed, and also tvrosine, from which latter phenol, indol, and skatol are
produced. As to the products prepared by hydrolytic cleavage ^dth mm-
eral acids we have a number of investigations by various experimenters,
especialh^ Hlasiwetz and Habermanx, Ritthausex and Kreusler. E.
ScHULZE and his collaborators, Drechsel, Siegfried, R. Cohx, Kossel
and his pupils, K. Morxer, Abderhaldex, Skraup, and recently E. Fis-
cher and his collaborators.^ The chief products thus obtained are mon-
amino-acids, such as glycocoll, alanine, amino valerianic acid, leucine, tyro-
sine, 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; oxymonamino-acids, such as serine,
oxyaminosuccinic acid, and oxyaminosuberic acid; ox^-diamino-acids, such
as oxydiaminosuberic acid, ox^^diaminosebacic acid, diaminotrioxvdodeca-
' -Ajinal. de chim. ct phys. (5), 16, and Bull. Soc. chim., 23 and 24.
^ See Habermann and Ehrenfeld, Zeitschr. f. physiol. Chem., 30.
^ In regard to the literature see O. Cohnheim, Chemie der Eiweisskorper, Braun-
schweig, 1904, and F. Hofmcister, Ergebnisse der Phy.siologie, Jahr^. I, Abt. 1, 7.59,
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.
30 THE PROTEIN SUBSTANCES.
noic acid, caseanic and caseinic acids; a-pyrrolidine and oxypyrrolidine car-
boxylic acids; indolaminopropionic acid; sulphuretted hydrogen, ethyl
sulphide, leucinimide, ammonia, and melanoidins,^ 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 dif-
ferent kmds, hexone bases, tr^^ptophane (proteinochromogen), which is
indolaminopropionic acid, and finally oxyphenylethylamine, diamines, and
a little ammonia and other substances.
A great many substances are produced in the putrefaction of proteins.
First the same bodies as are formed in the decomposition by means of
proteolytic enzymes are produced, and then a further decomposition 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,
valerianic, 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 jDroducts of the aromatic and
heterocyclic series into three groups : (a) the phenol group, to which tyrosine,
the aromatic oxy acids, phenol, and cresol belong; (b) the phenyl group,
including phenylacetic acid and phenylpropionic acid; and lastly (c) the
indol group, which includes indol, skatol, skatolacetic acid, and skatolcar-
boxylic acid. These various products are formed during putrefaction with
access of air. Nencki and Bovet^ obtained only p-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 skatolamino-
acetic acid (indolaminopropionic acid), and according to Nencki these three
last-mentioned amino-acids exist preformed in the protein molecule.
By the moderate action of chlorine, bromine, or iodine upon proteins these
halogens enter into more or less firm combination with the molecule (Loevv,
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 (Hop-
kins and PixKUs). The proteins are so changed that they do not split off
sulphur on treatment with alkali, nor do they respond to Millon's reaction,
' See Samuely, Hofmeister's Beitrage, 2.
' Salkowski, Zeitschr. f. physiol. Chem., 12, 215, and 27, 302; Nencki and Bovet,
Monatshefte f, Chem., 10.
OXIDATION PRODUCTS OF THE PROTEINS. 31
nor do they yield tyrosine as a cleavage product. This is ordinarily ex-
plained by the supposition that a substitution of hydrogen by iodine takes
place in the aromatic t3-rosine nucleus; but since according to Oswald the
heteroproteoses, which yield only vers- little tyrosine, take up about the
same quantity of iodine as the protoproteoses, which yield considerable
tyrosine, it appears that the iodine is united to other groups besides the
tyrosine-yielding atomic complex. By the action of iodine an oxidation alsc^
occurs, and Schmidt ^ has shown that a continuous splitting off of amino-
groups takes place. According to him phenol and p-cresol, cleavage prod
ucts of tj^rosine, besides benzoic acid, are produced by the oxidation of
phenylaminopropionic acid.
By the oxidation of proteid 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 oxida-
tion product in which the group SH is changed into SOo.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 oxidation of proteid 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. FuRTH^ there exist at least three different peroxyproteic acids (from
casein) which differ from each other by a different division of the nitrogen
in the molecule. On treatment with baryta-water we find that they split
off basic complexes and oxalic-acid groups, and new bodies, the desamino-
proteic acids, which give the biuret reaction, are produced. These acids,
which on hydrolysis give benzoic acid but no diamino-acids, ma}^ 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.
• Loew, Joum. f. prakt. Chem. (N. F.), 31; Blum, Miinch. med. Wochenschr.,
1896j Blum and Vaubel, Joum. f. prakt. Chem. (N. F.), 5"; Liebrecht, Ber. d. deutsch.
chem. Gesellsch., 30; Hopkins and Brook, Joum. 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, 38, 37.
2 Maly, Sitzungsber. d. k. Akad. d. Wissensch. Wien, 91 and 97. Also Monatshefte f.
Chem., 6 and 9. See also Bondzjaiski and Zoja, Zeitschr. f. physiol. Chem., 19;
Bemert, ibid., 26; Schulz, ibid., 29; v. Furth, Hofmeister's Beitrage, 6.
32 THE TROTEIN SUBSTANCES.
On the oxidation of gelatine or proteid witli permanganate we obtain
also oxaminic acid, oxamide, oxalic acid, oxaluric-acid amide, succinic acid,
several volatile fatty acids, and giianidine, which was first shown liy Lossen
as an oxidation product (Kutscher, Zickgraf, Seemaxx. Kutscher and
Schexck).^
On the oxidation of gelatine by ferrous sulphate and hydrogen peroxide
Blumenthal and Neuberg have obtained acetone as a product, and Orgler - the
same from ovalbumin. Jolles ' claims to have obtained large ciuantities of urea
in the oxidation of various proteins by potassium permanganate in acid solu-
tion, but this has been disputed by other investigators. On the oxidation of
protein in acid liquids, volatile fatty acids, their aldehydes, nitriles and ketones,
also hydrocyanic acid, benzoic acid, and other bodies, have been obtained.
Nitric acid gives various nitro-products. A melanoidin substance, xaniho-
}nela>u)i, has been obtained by v. FVrth.* Habermanx and Ehhexfeld ^
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-
benzoic acid, and other bodies. With aqua regia, fumaric acid, oxalic acid, chor-
iizol, and other bodies are obtained. The recent investigations of Habermanx
^nd Ehrexfeld and Panzer '^ upon the action of chlorine upon jiroteins 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
^MfLLER "^ and his students. They have shown that it is always an amino-
sugar and generalh' glucosamine. That so-called true proteids also }'ield
a carbohydrate on hydrolytic cleavage was first shown by Pavy, using
ovalbumin. The continued investigations of Fr. Muller, Weydemaxx,
'Lossen, 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., 3" and 38.
^ Bhimenthal and Neuberg, Deutsch. med. Wochenschr., 1901; Orgler, Hofmeister's
Beit rage, 1.
^ Zeitschr. f. physiol. Chem., 32 and 38.
''Sec Maly's Jahresbcr., 30, 24.
^ Zeitschr. f. physiol. Chem., 35.
* Hahermann and Ehrenfeld, ibid., Panzer, ibid., 33 and 34.
' iiiiller, Sitzungsber. d. Ges. d. Naturw. zu Marburg, 1896 and 1898, and Zeitschc
f. Biologie, 42.
CARBOHYDRATE MOIETY OF THE PROTEINS. 33
Seemann, Frankel, Hofmeister, and Laxgsteix ^ have demonstrated
that in these cases the carbohydrate is also glucosamine. A carbohydrate
complex, although sometimes only to a very slight amount, has also been
det€cted in other proteins, ovoglobulin, serglobulin, seralbumin, pea-
globulin, albumin of the graminese, yolk-proteid, and fibrin. In other
proteins, on the contrary, such as edestin (of the hemp-seed) and casein,
myosin, pure fibrinogen, and ovovitellin, carbohydrates have been sought
for with negative results. All proteins hence do not contain a carbohy-
drate 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 observa-
tions ^ show that in working with crystalline proteins a contamination with
other protein substances is unfortunately 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 war-
ranted in considering the carbohydrate groups as belonging to the carbon
nucleus produced on the destruction of the real protein complex.
The previously mentioned methods used in studying the structure
of the protein substances are not of the same value, but they in part sub-
stantiate 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 molecule. 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 monamiiio-acids: Glycocoll (aminoacetic
acid), alanine (aminopropionic acid), aminovalerianic acid, leucine (isobutylamino-
acetic acid), and isoleucine. 3. Bibasic monamino-acids: Aspartic acid (amino-
succinic acid) and glutamic acid (aminoglutaric acid). 4. Oxymonamino-acids:
serine (oxyaminopropionic acid) oxyaminosuccinic acid and oxyaminosuberic
acid. 5. Monobasic diamino-acids: Diaminoacetic acid, ornithine (diaminovaleri-
anic acid) and lysine (diaminocaproic acid). 6. Oxy diamino-acids: Oxydiamino-
suberic acid, oxydiaminosebacic acid, diaminotrioxydodecanoic acid, caseanic and
caseinic acids.
B. Sulphurized: Cysteine (aminothiolactic acid) and its sulphide cystine, thio-
lactic acid, mercaptans, and ethyl sulphide.
II. The Nuclei belonging to the Carbocyclic Series.
Phenylaminopropionic acid and tyrosine.
' In regard to the literature on this subject see the work of Fr. Miiller, Zeitschr.
f. Biologie,42, and Langstein, Ergebnisse der Physiologie, Jahrg. I, Abt. 1,63, Zeitschr.
f. physiol. Chem., 41, and Hofmeister's Beitrage, 6. See also Abderhalden, Bergell,
and Dorpinghaus, Zeitschr. f. physiol. Chem.. 41.
'See Wichmann, Zeitschr. f. physiol. Chem., 23, and N. Schulz, Die Grosse des
Eiweissmolekiils, Jena, 1903, 51.
34 THE PROTEIN SUBSTANCES.
III. The Nuclei belonging to the Heterocyclic Series.
A. Oi the pyrrol group: Pyrrolidine carboxylic acid (n-proline) and oxypyrro-
lidine carboxylic acid.
B. Of the indol group: Tryptophane or indolaminoproijionic acid, from which
iiidol and skatol are produced by putrefaction.
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, leu-
cine, tyrosine, or cystine, is obtained in very variable amounts from differ-
ent protein substances. It is very difhcult to say to what extent all the
above-mentioned carbon nuclei exist in the jirotein 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 Loew,' 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 neigh-
boring groups.
Even if we admit the above, still it is undoubtedly true that the chief
cleavage products of the protein substances are amino-acids. Emil Fischer
has shown that the amino-acids have the property of readily grouping
together when water is split off and the amide group of one amino-acid
unites with the carboxyl group of the other. In accord with this behavior
w'e can, as Hofmeister 2 has explained, consider the proteins as chiefly
formed by the condensation of amino-acids, where the amino-acids are united
to each other by means of imino-groups according to the following scheme:
— NH.CH.CO— NH.CH.CO NH.CH.CO— NH.CH.CO—
I I I I
C4H9 CH2.C6H4(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 Schijtzenberger
and Pickering have been able to prepare substances which in many prop-
erties 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,
' Loew, Die chem. Energie d. lebenden Zellen, Miinchen, 1898, and Hofmeister's
Beitrago, 1.
^"Uber den Bau des Eiweissmolekiils." Gesellsch. deutsch. Naturforscher and
Artze, Verhandl. 1902, and Ergebnisse der Physiologie, Jahrg. I, Abt. 1, 759.
POLYPEPTIDES. 35
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 ^ and his pupils. They hav&
prepared a large number of complex bodies called polypeptides, 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 ^ill mention — dipeptides: glycylalanine. leucyW-tyrosine, propylalanine,
diaminopropionic-acid dipeptide, lysyl-lysine, liistidyl-histidine; tripep-
tides: diglycyl-glycine, leucyl-alanyl-glycine, dileucyl cystine; tetrapeptides:
triglycyl-glycine, dileucy 1-glycyl-glycine ; penta}>eptide : tetraglycyl-glycine.
In connection with these syntheses it is important to note that E.
Fischer and Bergell,^ by the decomposition of a protein substance,
fibroin, by successive action of acid, proteolytic enzyme (tr}-psin), andbaryta-
water, were able to obtain a dipeptide. probably glycylalanine. Levexe
and Beatty have obtained a dipeptide anhydride, prolineglycyl anhydride,
in the tryptic digestion of gelatine, and Fischer and Abderhaldex ^ have
also isolated from silk fibroin a dipeptide composed of glycocoU and l-
tyrosine. In the hydrolysis of elastin with sulphuric acid they obtained a
third dipeptide, which, like the others, was an anhydride, namely, glycyl-Z-
leucine anhydride. Of the synthetically prepared polypeptides several give
the biuret reaction, and in this regard, as well as their behavior towards
other reagents, they are similar to the peptones, which will be discussed later-
Certain polypeptides, like the biuret base 'according to Schwarzschild)^
the glycyl-Z-tyrosine, and alanyl-glycine, are split by trypsin, while others,
like glycyl-glycine and glycylalanine, are not attacked by thii me (see
Chapter IX).
It is admitted that the atomic chaining in the protein consists of a
union of a-amino-acids by means of the imide bonds. It is probable that
also other Unkings occur, and besides the above-mentioned bondage we
certainly have one other, namel}', the urea-forming group (the guanidine
residue) united by the imide Unkings \\'ith the ornithine (diaminovalerianic
acid). This imide linlvage is not ruptured, like that of the a-amino-acids,.
' See Pickering, Kiag's College, London, Physiol. Lab. Collect. Papers, 1897, whichr
also cites Grimaux's work; also Joum. of Physiol., 18, and Proceed. Roy. Soc, 60,,
1897; Schiitzenberger, Compt. rend., 106 and 112; Curtius, Joum. f. prakt. Chem..
(X. F.), 26 and 70, and Ber. d. d. chem. Gesellsch.. 37; Fischer and collaborators,
ibuL, 35, 36, 37, 38, 39, and .\nnal. d. Chem. u. Pharm., 340. AU the work of E. Fischer
and his collaborators on this subject may be found in E. Fischer's Untersuchimgen
iiber Aminosauren, Polj-peptide and Proteine (1899-1906), Berlin, 1906.
2 See Biochem. Centralbl., 1, p. 84.
' Levene and Beatty, Ber. d. d. chem. Gesellsch., 39, p. 20G0; Fischer and
Abderhalden, ibid., 39, p. 2315.
"36 THE PROTEIN SUBSTANCES.
by trypsin, but it is by another enzyme discovered by Kossel and Dakix/
called arginase.
If we consider the proteins as composed chiefly of amino-acids combined
together in imide-like complexes containing also several NH2 groups at the
ends of the chains, it is easy to understand that the proteins, like the amino-
Acids, are amphoteric electrolytes, combining with bases as well as with
acids to form salts which are strongly dissociated hydrolytically. As we
must also admit of the presence in the protein molecule of a large number
of COOH as well as XH2 groups, it follows that the protein bodies may be
polybasic acids, as well as polyacidic bases. In this regard the various
proteins behave somewhat differently, as some of them, like the protamines,
are strongly basic, while others, like casein, behave chiefly like acids, while
others take a certain intermediate position. On this behavior as well as on
their chemical constitution it is unfortunately impossible to base a proper
classification of the protein substances. Their general properties, such as
solubilities and precipitation properties, are too uncertain to aid us in the
construction of a proper classification. On the other hand a classification
is important, and we cannot do without one, so we will give the following
systematic summary of the chief groups of the protein bodies as suggested
by Hoppe-Seyler and Drechsel, which will be of some aid to us.
I. Simple Proteids or Albuminous Bodies.
, I Seralbumin,
( Lactalbumin, and others. )
r Fibrinogen,
-Globulins -< Myosin,
f Serglohvlins, and others. )
Nucleoalbumins -I ^ . ' . , ^,
( Ovovitellin, and others.
.„ . ^ ( Acid albuminate,
Albuminates 1 , i? 7 ■ 71, • ^
I Alkali albuminate.
Proteoses (and Peptones).
C 1 t d P t 'd i Fibrin,
^ I Proteids coagulated by heat, and others.
Histones (Protamines).
II. Compound Proteids.
Haemoglobins.
! Mucins and Mncinoids,
Amyloid,
Ichthidin, and others.
_, , ^ . - ( Nucleohi stone,
Nucleoproteids -^ „ , , , . , , ,
( Cytoglobin, and others.
* Zeitschr. f. physiol. Chem., 41.
SIMPLE PROTEIDS. 37
III. Albumoids or Albuminoids.
Keratins.
Elastin.
Collagen.
Reticulin.
(Fibroin, Sericin, Comein, Spongtn, Conchiolin, Byssus, and others.)
To this summary must be added that we often find in the investigation?
of animal fluids and tissues protein substances which do not fall in with
the above scheme, 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.
I. Simple Proteids or Albuminous Bodies.
The simple proteids are never-failing constituents of the animal and
vegetable organisms. They are especially found in the animal bod}- , where
they form the solid constituents of the muscles and of the blood-serum, and
they are so generally distributed that there are onl}- a few animal secre-
tions and excretions, such as the tears, the perspiration, and perhaps the
urine, in which the}' are entirely absent or occur only in traces.
All proteids 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 proteids the follow-
ing 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
0 21.50—23.50
The animal proteids are odorless, tasteless, and ordinarily amorphous.
The crystalloid spherules {D otter pldttchen) occurring in the eggs of certain,
fishes and amphibians do not consist of pure proteids, but of proteids
containing large amounts of lecithin, which seem to be combined with
' See foot-note l,p.28.
S8 THE PROTEIN SUBSTANCES.
mineral substances. Crystalline proteids ^ have been prepared from the
seeds of various plants, and crystallized animal proteids (see seralbumin
and ovalbumin, Chapters YI and XIII) can be readily prepared. 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 questionable whether there exists any
proteid body which is soluble in water without the aid of mineral sub-
stances. Nevertheless it has not been thus far successfully proved that
a native proteid body can be prepared perfectly free from mineral sub-
stances without changing its constitution or its properties.^
As previously stated, the albuminous bodies are amphoteric electrolytes
and are poly acidic bases as well as poly basic 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 differentiation of the
various proteids (Cohnheim, Erb, and others).
The acid-combining power of the proteids has been studied by means of
physical methods by Sjoquist, Bugarsky, and Liebermann and with the aid
of chemical methods by Spiro and Pemsel, Erb, Cohnheim and Krieger,
V. Rhorer. The methods pursued by Cohnheim and Krieger consisted in
precipitating the proteid from acid solution (HCl) with an alkaloid reagent
(calcium phosphotungstate). The reaction took place as follows: proteid
hydrochloride + calcium phosphotungstate = proteid phosphotungstate + calcium
chloride. The acid remaining in the filtrate was determined, and when this
quantity was subtracted from the known original amount in the proteid solu-
tion, the difference represented the acid combined with the proteid. If sodium
picrate or potassium-mercuric iodide is used instead of the phosphotungstate
we have, according to v. Rhorer,^ a method which is the best of all heretofore
suggested.
The proteids can be salted out from their neutral solutions by neutral
salts (NaCl, Na2S04, MgS04, (NH4)2S04, and many others) in sufficient
■concentrations. While by other precipitants they are often changed or
modified, their properties remain unchanged on salting out, and the process
is reversible, as on diminishing the concentration of the salt the precipitate
redissolves. Spiro •* has shown that we are not dealing here with the
' See Maschke, Journ. f. prakt. Chem., 74; Drechsel, ibid. (N. F.), 19; Griibler,
ibid. (N. F.), 23; Ritthausen, ihul. (N. F.), 25; Schmiedeberg, Zeitschr. f. physiol.
Chem., 1; Weyl, ibid., 1.
^ See E. Hamack, Ber. d. d. chem. Gesellsch., 22, 23, 25, and 31; Werigo, Pfliiger's
Archiv, 48; Billow, ibid., 58; Schulz, Die Grosse des Eiweissmolokiils, Jena, 1903.
^ Pfliiger's Arch., 90. In regard to the literature on this subject see Cohnheim,
Chemie der Eivveisskorper, 2. Aufl., pp. 107-109.
* Hofmeister's Beitrage, 4.
PROPERTIES OF THE PROTEIDS. 39
formation of a proteid combination, but rather that this is an instance of
a division of a body between two solvents. The various proteids act in an
essentially different manner towards the same salt, and also for one and
the same proteid the behavior towards different neutral salts is different,
as some cause a precipitate, while others on the contrary do not
]^recipitate.
According to Pauli ^ this can be explained by the fact that we have to do
■with ion action and that the precipitation action is the algebraic sum ot the antago-
nistic properties. If we ascribe the precipitating action to the cations and a
retarding action upon precipitation to the anions, then, according as a salt has
the positive cations or the negative anions in excess, we obtain a precipitation
action or not, that is, it is accelerated or retarded.
The behavior of various proteids with one and the same salt, su^h as
M,!;S04 or (NH4)2S04, is often made use of in the isolation of the proteid,
and special methods of separation are based upon fractional precipitation.
Haslam^ 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 proteid
solution is precipitated by salts of the heavy metals. Here the precipi-
tates (often called metallic albuminates) are not true combinations in con-
stant proportions, but are rather to be considered as loose absorption
compounds of the proteid with the salt.^ These reactions are irreversible
ir. 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 precipi-
tate, 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 re-
versible one.
The precipitation of proteids by salts stands in close relationship to their
colloidal nature. The protein bodies 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. Certain of them,
especially the peptones and a few proteoses, which will be discussed later,
seem to occupy an intermediate position between colloids and crystalloids,
as their solutions are characterized by a lesser viscosity and greater dif-
fusibility, are not readily precipitable by alcohol, not coagulable by heat,
and only slightly precipitable by salts.
The solutions (or suspensions) of proteids in water, the proteid hydrosols,
are converted by various means into proteid hydrogels. Of these means
' Hofmeister's Beitriige, 3.
'See Cohnheim, Chemie der Eiweisskorper, 2. Aufl., 1904, pp. 144-148; Pinkus,
Journ. of Physiol., 27; Pauli, Hofmeister's Beitrage, 3, p. 225; Haslam, Joum. of
Physiol., 32.
' See Galeotti, Zeitschr. f. physiol. Chem., 40, 42, and 44.
40 THE PROTEIN SUBSTANCES.
we must specially mention the following: flocking out with salts, precipi-
tation with alcohol, and coagulation by means of heat.
The precipitation with alcohol is a reversible reaction, as the precipitate
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.
Those proteids which occur, according to the common views, preformed
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 {" denatu-
rierte ") proteids, to differentiate them from the native proteids. In the
scheme given on page 36 the albumins, globulins, and nucleoalbumins
belong to the native proteids, and the acid or alkali albuminates, the pro-
teoses, the peptones, and the coagulated proteids to the modified proteids.
On heating a solution of a native proteid it is modified at a different
temperature for each different proteid. With proper reaction and other
favorable conditions, for instance in the presence of neutral salts, most
proteids can in this w^ay be precipitated in a solid form as coagulated pro-
teid. 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 containing salt
have in many cases given us good means for detecting and separating
proteids. The views in regard to the use of these means are somewhat
divided.^
A modification can be brought about also by the action of acids, alka-
lies, or salts of the heavy metals, in certain cases by water alone, also by
the action of alcohol, chloroform,^ and ether, by violent shaking, etc.
As colloids 3 the proteids can, like other protein substances, to a more
* See Halliburton, Joum. of Physiol., 5 and 11; Corin and Berard, Bull, de I'Acad.
roy. de Belg., 15; Haycraft and Duggan, Brit. Med. Joum., 1890, and Proc. Roy.
Soc. Edin., 1889; Corin and Ansiaux, Bull, de I'Acad. roy. de Belg., 21; L. Fredericq,
Centralbl. f. Physiol., 3; Haycraft, ibid., 4; Hewlett, Joum. 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; PauH, Pfliiger's Arch., 78.
2 See Salkowski, Zeitschr. f. physiol. Chem., 31; Fr. Kriiger, Zeitschr. f. Biologie,
41; Loew and Aso, Bull. Coll. Agric. Tokio, 4.
'The study of colloids and especially their changes of state is of the greatest
importance for the chemistry of the proteids as well as for biochemistry in general.
As the views on important points in this extensive subject are so very divergent, it is
PRECIPITATION REACTIONS. 41
or less degree, prevent the precipitation of a colloidal metallic solution
(gold solution) by means of an electrolyte (see gold equivalent according
to ZsiGMoxDY and Schulz).^
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:
A. Precipitation Reactions of the Proteid Bodies.
1. Coagulation Test. An alkaline proteid solution does not coagulate
on boiling, a neutral solution only partly and incompletely, and the reaction
must therefore be acid for coagulation. The neutral liquid is first boiled
and then the proper amount of acid added carefull}'. A flocculent precipi-
tate is formed, and if properly done 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 c.c, depending on the
amount of proteid present, and boiled before the addition of each drop. If
dilute nitric acid be used, then to 10-15 c.c. 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 proteid 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. Behavior towards Mineral Acids at Ordinal^ Tem-
peratures. The proteids are precipitated by the three ordinary mineral
acids and by metaphosphoric acid, 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). 3. Precipitation by
Metallic Salts. Copper sulphate, neutral and basic lead acetate (in small
amounts), mercuric chloride, and other salts precipitate proteid. On this
is based the use of proteids as antidotes in poisoning by metallic salts. 4.
Precipitation by Ferro- or Ferricyanide of Potassium in Acetic-acid Solution,
In these tests the relative quantities of reagent, proteid, or acid do not
interfere with the delicacy of the test. 5. Precipitation by Neutral Salts, such
as Na2S04 or NaCl, when added to saturation to the liquid acidified with
acetic acid or hydrochloric acid. 6. Precipitation by Alcohol. The solutioa
must not be alkaline, but must be either neutral or faintly acid. It must^
impossible to give a short review of the subject, hence we can only refer for the litera-
ture to Hamburger, Osmotischer Druck und lonenlehre in den med. Wissenschaften,
Wiesbaden, 1902-1904; H. Aron, Uber organische Kolloide, Biochem. Centralbl., 3»
pp. 461 and .501.
' Hofmeister's Beitrage, 3.
42 THE PROTEIN SUBSTANCES.
at the same time, contain a sufficient quantity of neutral salts. 7. Precipi-
tation 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. 8. Precipitation by
Phosphotungstic or Phosphomolybdic Acids in the presence of free mineral
acids. Potassium-mercuric iodide and potassium-bismuth iodide precipitate
proteid solutions acidified with hydrochloric acid. 9. Precipitation by
Picric Acid in solutions acidified by organic acids. 10. Precipitation by
Trichloracetic Acid in 2-5 per cent solutions. 11. Precipitation by Sali-
cylsulphonic Acid. The proteids are precipitated by nucleic acids, tauro-
cholic and chondroit in-sulphuric acid in acid solutions.
B. Color Reactions for Proteid Bodies.
1. Millon's Reaction.^ A solution of mercury in nitric acid containing
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 0. Nasse 2 it is best to use a solution
of mercuric acetate which is treated with a few drops of a 1 per cent solu-
tion of potassium or sodium nitrite; previous to use 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 ^ 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 b}' adding sodfum amalgam to a concentrated solution
of oxalic acid and filtering after the discharge of the gas. • A dilute aqueous
' The reagent is obtained 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.
' See O. Nasse, Sitzungsb. d. Naturforsch. Gesellsch. zu Halle, 1879, and Pfliiger'a
Arch., S3; see also Vaubel and Blum, Joum. f. prakt. Chem. (N. F.), 57.
* Proceed. Roy. Soc, 68,
COLOR REACTIONS. 43
solution of the acid or some of the soHd acid is added to the proteid s6lu-
tion and sulphuric acid allowed to flow do\\Ti the side of the test-tube, when
the reddish-violet color will appear at the point of contact of the two
liquids. Gelatine does not give this reaction. 4. Biuret test. If a proteid
solution be first treated with caustic potash or soda and then a dilute cop-
per-sulphate solution be added drop by drop, first a reddish, then a red-
dish-violet, and lastly a violet-blue color is obtained. 5. Proteids are
sofuble on heating with concentrated hydrochloric acid, producing a violet
color, and when they are pre\iously boiled with alcohol and then washed
with ether (Liebermaxx \) they give a beautiful blue solution. This blue
color is due, according to Cole, 2 to a contamination of the ether ^^•ith gly-
oxylic acid, which reacts ^^■ith the tryptophane groups split off by the hydro-
chloric acid. 6. With concentrated sulphuric acid and sugar (in small quan-
tities) the alljuminous bodies give a beautiful red coloration. 7. With
;)-dimethylaminobenzaldehyde and concentrated sulphuric acid the pro-
teids give a beautiful reddish-\iolet or deep-violet coloration (0. Neubauer
and E. Rohde^).
Many of these color reactions are obtained, as shown by Salkowski,' 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
reaction by the phenol group and skatol or skatolcarbonic acid. Liebermaxn's
reaction depends, according to Cole, upon the skatol (indol) group, and the reac-
tions with sulphuric acid and sugar (Cole) and with dimethylaminobenzaldehyde
(Rohde) are also caused by this group. Ada.mkiewicz's reaction is given only by
the bodies of the indol group. The biuret reaction is not only given by pro-
tein substances but also by many other bodies. According to H. Schiff ^ this
reaction occurs with those bodies containing amino groups, COXHo, CSNH,,
C(XH)XH2 or also CHoXH,, united either directly by their carbon atoms or by
means of a third carbon or nitrogen atom. As examples of such bodies we can
mention several diamines or aminoamides, such as oxamide, biuret, glycinamide,
a- and ,5-aminobutyramide, aspartic-acid amide, etc., although we are not dear
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 reaction, and
' Centralbl. f. d. med. Wissensch., 1887.
^ Joum. of Physiol., 30.
' Zeitschr. f. physiol. Chem., 44.
* Ibid. ,12.
^ Ber. d. d. chem. Gesellsch., 29 and 30.
44 THE PROTEIN SUBSTANCES.
because it can be performed so easily. Among the precipitation reactions,
that with basic lead acetate (when carefully and exactly executed) and the
reactions 6, 7, 8, 9, and 11 are the most delicate. The color reactions 1 to
4 show great delicacy in the order in which they are given. ^
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 ciuantitative estimation of coagulable proteids the determina-
tion by boiling with acetic acid can be performed with advantage, since, 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 determine the quantity
of acetic acid necessary to completely precipitate the proteids in small
measured portions o the neutralized liquid which have previously been
heated on the water-bath, so that the filtrate does not respond to Hel-
ler's test. Now warm a larger weighed or measured 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 filtrate.
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 glyco-
gen, 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 i^recipitating 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 performed
by boiling with acetic acid. Small amounts of proteid which remain in the
filtrates may be separated by boiling with freshly precipitated lead car-
bonate or with ferric acetate, as described by Hofmeister.^ If the liquid
cannot be boiled, the proteid may be precipitated by the very careful addi-
' In regard to the precipitation and coloration reactions of proteids with aniline
dye.s see Heidenhain, Pfliiger's Arch., 90, 9G.
Zeitschr. f. physiol. Chem., 2 and 4.
ALBUMINS AND GLOBULINS. 45
tion 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 alternate addition of potassium-mercuric
iodide and hydrochloric acid (see Chapter VIII, on Glycogen Estimation),
or also by trichloracetic acid as suggested by Obermayer and Frankel.^
In the precipitation of proteid as well as the quantitative estin lation by means
of heat, it must be borne in mind, as shown by Spiro," that several nitrogenous
substances, such as piperidine, pyridine, urea, etc., disturb the coigulation of the
proteids.
Synopsis of the Most Important Properties of the Different Groups of
Proteids.
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 characteri-
zation. 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 solu-
tion in water coagulates on boiling in the presence of neutral salts, but a
weak saline solution does not. If NaCl or i\IgS04 is added to saturation
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 in
substance to saturation to an albumin solution a complete precipitation
occurs at the ordinary temperature. Of all the native proteids the albumins
are the richest in sulphur, containing from 1.6 per cent to 2.2 per cent.
Globulins. These substances are 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 addition of very little
acid or alkali, and on neutralizing the solvent they precipitate again. The
solution in a minimum amount of alkali is precipitated by carbon dioxide,
Ixit the precipitate may be redissolved by an excess of the precipitant.
The neutral solutions of the globulins containing salts are partly or com-
pletely precipitated on saturation with NaCl or J\IgS04 in substance at
normal temperatures. 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.
' Obermayer, Wien. med. Jahrbiicher, 1888; Frankel, Pfliiger's Arch., 52 and 55.
^ Zeitschr. f. physiol. Chem., 30.
46 THE PROTEIN SUBSTANCES.
According to J. Starke ' the globulins are not soluble in dilute salt solutions,
but form alkali proteid compounds whose solubility in salts is brought about by
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 allmmins and globnlins
follows from the fact that the albumins can be converted into giob-
iilins. The possibility of a conversion of ovalbumin into globulin is based
upon the observations of Starke. That a transformation of seralbumin
into serglobulin by 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 ^Ioll^ by experimenting with blood-serum as well as with crys-
talline seralbumin. According to Moll first pseudoglobulin 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 just as difficult to draw^ a sharp line between the globulins and
albuminates as it is betw'een the globulins and albumins. Several globulins
are very readily changed by the action of vers' little acid, as also by standing
under water when in a precipitated condition, into albuminates, and then
become insoluble in neutral salt solutions. Osborxe,^ who has closely
studied this property in connection with edestin (from hemp-seed), considers
the globulin, "globan, " which has been made insoluble in salt solution,
as an intermediate step in the formation of the albuminate w'hich is pro-
duced by the hydrolytic action of the H ions of water or of the acid.
Nucleoalbumins. This group of phosphorized proteids is found widely
distributed in both the animal and vegetable kingdoms. The nucleoalbumins
behave like weak acids; they are nearly insoluble in water, but dissolve
easih- with the aid of a little alkali. The nucleoalbumins resemble certain
of the globulins and albuminates in solubility and precipitation properties,
but differ from these two groups by containing phosphorus. They stand
close to the nucleoproteids by their content of phosphorus, but differ from
these in not yielding any purine bases on cleavage. It has not j'et been
found possible to obtain from the nucleoalbumins any proteid-free pseudo-
nucleic acids corresponding to the nucleic acids, but only acids rich in
phosphorus, which always give the proteid reactions (Levene and Alsberg,
Salkowski "*). For this reason the nucleoalbumins cannot be classed as
' Zeitschr. f. Biologie, 40 and 42. In regard to the various views on this subject
see Wolff and Smits, ibid., 41; Osborne, 1. c; Hammarsten, Ergebnisse der Physiologie,
Jahrg. I, Abt. 1.
^ Hofmeister's Beitrage, 4 and 7.
' Zeitschr. f. physiol. Chem., 33.
* Levene and Alsberg, ibi/1., 31; Salkowski, ibid., 32; Levene, ibid., 32.
XUCLEOALBUMIXS AND LECITHALBL^uXS. 47
compound proteids. In peptic digestion a proteid rich in phosphorus can
be split off from most nucleoalbumins, and this has been called para- or
pseudonudein. The claim made by LiEBERiL\xx that the pseudonuclein
is a combination of proteid with metaphosphoric acid has been shown to
be incorrect by the investigations of Giertz.^ The nucleoalbumins always
seem to contain some iron.
The separation of pseudonuclein in peptic digestion is no doubt characteristic
of the nucleoalbumin group, but the non-appearance of the pseudonuclein precij^i-
tate does not entirely exclude the presence of a nucleoalbumin. The extent of
such a cleavage is dependent upon the intensity of the pepsin digestion, the
degree of acidity, and the relationship 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 i^seudonuclein at all in the digestion of the casein from human milk.
WiMAX - has also .shown in the digestion of vegetable nucleoalbumin that the
obtainment of considerable pseudonuclein or none is de})endent 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 xanthine bases are
absent in their cleavage products.
The nucleoalbimiins are often confoimded with nucleoproteids and
also witli phosphorized glucoproteids. From the first class they differ by
not yielding any xanthine bodies when boiled with acids, and from the
second group by not yielding any reducing substance on the same treat-
ment.
Lecithalbumins. In the preparation of certain protein substances
products are often obtained containing lecithin, and this lecithin can only
be removed with difficulty or incompletely by a mixture of alcohol and
ether. Ovovitellin (Chapter XIII) is such a protein body containing
considerable lecithin, and Hoppe-Seyler considers it a combination of
proteid and lecithin. Similar substances occur in fish-eggs. These last
lecithalbumins often have the solubilities of the globulins and are readily
soluble in dilute salt solutions. The behavior of the nucleoalbumin of
the eggs of the perch shows how easily this solubility may be changed. This
nucleoalbumin, which contains considerable amounts of lecithin, is readily
soluble in dilute NaCl solution, but at ordinary temperatures it is changed
by 0.1 per cent HCl nearly instantaneou.sly and without splitting off
lecithin, so that it becomes insoluble in dilute salt solutions (Hamm.\rsten).
LiEBERMAXx^ has obtained proteids containing lecithin as an insoluble
' Liebermann, Ber. d. deutsch. chtm. Gesellsch.. 21; Giertz, Zeitschr. f. physiol.
Chem.. 28.
- Salkowski, Pfliiger's Arch., 63; Wioljlewski, Beit rage zur Kenntnis des Frauen-
ka?eins, Inaug.-Diss. Bern, 1894; Wiman, Upsala Lakaref. Forh. (X. F.), 2.
' Hoppe-Seyler, Med. chem. Untersuch., 1868; also Zeitschr. f. physiol. Chem., 13,
479; Hammarsten, Skand. Arch. f. Physiol., 1"; Liebermann, Pfliiger's Archiv, 50
and 54.
48 THE PROTEIN SUBSTANCES.
residue on the peptic digestion of the mucous membrane of the stomach,
liver, kidneys, lungs, and spleen. He considers them as combinations of
proteid and lecithin and calls them lecithalbumins. Further investigation
of these bodies is desirable.
Alkali and Acid Albuminates. The native proteids are modified by the
action of sufficiently strong acids or alkalies. By the action of alkalies
all native albuminous bodies are converted, with the elimination of nitro-
gen, 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 substance or in strong
solution be allowed to act on a concentrated proteid solution, such as
l)lood-serum or egg-albumin, the alkali albuminate may be obtained as a
solid jelly which dissolves in water on heating, and which is called " Lieber-
kuhn's solid alkali albuminate." By the action of dilute caustic alkali
solutions on dilute proteid solutions we have alkali albuminates formed
slowly at the ordinary temperature, but more rapidly on heating. These
solutions may vary with the nature of the proteid acted upon, and also
wnth 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-hydro-
■chloric 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 gelatinous 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 albuminate, which is obtained by extracting muscles with hydrochloric
acid of 1 p. m.
The alkali and acid albuminates have the following reactions in com-
mon: They are nearly insoluble in water and dilute common-salt solu-
tion (see page 46), but they dissolve readily in water on the addition 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 tem-
perature on neutralizing the solvent by an alkali or an acid. A solution of
an alkali or acid albuminate in acid is easily precipitated 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. IMineral acids in excess
})recipitate solutions of acid as well as alkali albuminates. The nearly neu-
tral solutions of these bodies are also precipitated by many metallic salts.
Notwithstanding this agreement in the reactions, the acid and alkali
albuminates are essentially different, for by dissolving an alkali albuminate
in some acid no acid albuminate solution is obtained, nor is an alkali al-
ALKALI AND ACID ALBUMINATES. 49
buminate 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 solul)le 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 ])roducts are of a different
kind. The alkali albuminates are relatively strong acids. They may be
dissolved in water with the aid of CaCOg, with the elimination of COo,
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 nitrogen, and often also the
sulphur, is split off, and from this property we may obtain an alkali albu-
minate by the action of an alkali upon an acid albuminate; but we cannot
obtain an acid albuminate by the obverse reaction (K. MorxerI). For
this reason the designation of the modified proteid obtained by the action
of alkali or acid as proteiti, the combination of this protein with alkali
as alkali albuminate, and the combination with acid as acid albuminate,
leads to a misunderstanding or to a wrong conception.
The preparation of the albuminates has been given above. The cor-
responding albuminate obtained by the action of alkalies or acids upon a
proteid solution may be precipitated by neutralizing with acid or alkali.
The washed precipitate is dissolved in water by the aid of a little alkali or
acid, and again precipitated by neutralizing the solvent. If this 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 ^ belongs to this class. The lysalbinic acid and protalbinic acid obtained
by Paal ^ from ovalbumin are likewise alkali albuminates. Desaminoalhuminic
acid is an alkali albuminate which Schmiedeberg ^ 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 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.^
'Pfliiger's Arch., 17.
- Zeitschr. f. physiol. Chem., 30.
' Ber. d. d. chem. Gescllsch., 35.
' Arch. f. exp. Path. u. Pharm., 39.
^ 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. Schwartz, Zeitschr. f. physiol. Chem., 30;
Bliss and No%y, Joum. of Exper. Med., 4.
50 THE PROTEIN SUBSTANCES.
Proteoses and Peptones. Peptones were formerly designated as the
final products of the decomposition of protein bodies by means of pro-
teolytic enzymes, in so far as these final products are still true proteins,
while tlie intermediate products produced m 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
l>e 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 pre-
formed under physiological conditions requires very careful investiga-
tion.
Between the peptone, which represents the final cleavage product , and
the proteose, which stands closest to the original protein, we have undoubt-
edly 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 conception of
peptones and proteoses in an exact and satisfactory manner.
The proteoses (or albumoses) used to be considered as those protein bodies
whose neutral or faintly acid solutions do not coagulate on boiling, 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 disappearing 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 com-
pletely 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
readil}' soluble in water and which do not coagulate by heat, whose solutions
are precipitated neither by nitric acid, nor by acetic acid and potassium
ferrocyanide, nor by neutral salts and acid.
The reactions and properties which the proteoses and peptones have in
common were formerly considered as the following: They give all the color
reactions of the proteins, but with the biuret test they give a more beautiful
red color than the ordinary proteids. They are precipitated by ammoniacal
lead acetate, by mercuric chloride, tannic, phosphotungstic, and phospho-
molybdic 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 solul^le 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
PROTEOSES AXD PEPTONES. 51
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
Heyxsius' 1 observation that ammonium sulphate was a general precipi-
tant for proteins, and for peptones in the old sense, Kuhxe and his pupils ^
proposed this salt as a means of separating proteoses and peptones. Those
products of digestion which separate on saturating their solution \\ith
ammonium sulphate, or can indeed be salted out at all, are considered
by Kuhxe and also by most of the modern investigators as proteoses, while
those which remain in solution are called peptones or true peptones. These
true peptones are formed in relativeh' large amounts in pancreatic diges-
tion, while in pepsin digestion they are formed only in small quantities
or after prolonged digestion.
According to Schutzexberger and Kuhxe ^ the proteins yield 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, nameh%
the hemi grouj). These two groups are, according to Kuhxe, 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 ampho peptone.
In tryptic digestion a cleavage of the amphopeptone takes place into anti-
peptone 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.
KtJHXE and his pupils, who have conducted extensive investigations
on the proteoses and peptones, classify the various proteoses according
to their different solubilities and precipitation properties. In the pepsin
digestion of fibrin * they obtained the following proteoses: (a) Hetero-
proteose, insoluble in water but soluble in dilute salt solution; (6) Proto-
proteose, soluble in salt solution and water. These two proteoses are
precipitated by NaCl in neutral solutions, but not completely. Hetero-
proteose may, by being in contact with water for a long time or by drj-ing^
be converted into a modification, called (c) Di/sproteose, which is insoluble
' Pfliiger's Archiv, Si.
^ See Kiihne, Verhandl. d. naturhistor Vereins zu Heidelberg (N. F. ), 3; J. Wenz,
Zeitschr. f. Biologie, 22; Kiihne and Chittenden, Zeitschr. f. Biologie, 22; R. Xeu-
meister, ibid., 23; Kiihne, ibid., 29.
' Schiitzenberger, Bull, de la Soc. chimique de Paris, 23; Kiihne, Verhandl. d.
naturhist. Vereins zu Heidelberg (N. F.), 1, and Kiihne and Chittenden, Zeitschr, f.
Biologie, 19. See also Paal, Ber. d. deutsch. chem. Gesellsch., 27.
* See Kiihne and Chittenden, Zeitschr. f. Biologie, 20.
52 THE PROTEIN SUBSTANCES.
in dilute salt solutions, (d) Deuteroproteose is a proteose 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 (Herth^). The deuteroproteose 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, myosinoses, etc. These
various proteoses are further distinguished, as proto-, hetero-, and deutero-
caseoses, for example. Chittenden ^ has suggested the common name
proteoses for the products formed intermediary between the proteins and
peptones in the digestion of animal and vegetable proteins. We have
made use of it in this sense in preference to the word albumose (which is
used in the German and by some other waiters), but which will be used
in this book as indicating the intermediary products in the hydrolysis
of albumins and not as a general term. Certain proteoses have also been
obtained in a crystalline state (Schrotter).
Neumeister ^ designates as atmidalbumose that body which is obtained by
the action of superheated steam on fibrin. At the same time he also obtained a
substance called atmidalbumin, which stands between the albuminates and the
proteoses.
Of the soluble proteoses Neumeister designates the protoproteose and
heteroproteose as primary proteoses, while the deuteroproteoses, which are
closely allied to the peptones, he calls secondary proteoses. As essential
differences between the primary and secondary proteoses he suggests the
following:'* The primary proteoses are precipitated by nitric acid in salt-
free solutions, while the secondary proteoses are precipitated only in salt
solutions, and certain deuteroproteoses, such as deuterovitellose and deu-
teromyosinose, are precipitated by nitric acid only in solutions saturated
with NaCl. The primary proteoses are precipitated 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 pre-
' Monatshefte f. Chem., 5.
^ Kiihne and Chittenden, Zeitschr. f. Biologie, 22 and 25; Neumeister, ibid., 23;
Chittenden and Hartwell, Joum. of Physiol., 11 and 12; Chittenden and Painter,
Studies from the Lalx)ratory, etc., Yale University, 2, New Haven, 1887; Chittenden.
ibid., 3; Sebelien, Chem. Centralblatt, 1890; Chittenden and Goodwin, Joum. of
Physiol. 12.
^ Zeitschr. f. Biologie, 26. See also Chittenden and Meara, Joum. of Physiol.,
15, and Salkowski, Zeitschr. f. Biologie, 34 and 37.
* Neumeister, Zeitschr. f. Biologie, 21 and 26.
PROTEOSES AND PEPTONES. 53
cipitated 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.i 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 ammo-
nium 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 ferro-
cyanide and acetic acid, picric acid, trichloracetic acid, potassium-mercuric
iodide, or hydrochloric acid. They are precipitated by phosphotungstic acid,
phosphomolybdic acid, corrosive sublimate (in the absence of neutral salts),
absolute alcohol, and tannic acid, but the precipitate may redissolve on the
addition of an excess of the precipitant. As an important difference between
amphopeptone and antipeptone we must also mention that the former gives
Millon's reaction, while the antipeptone does not.
In regard to the precii^itation 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. Schr5tter - 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. As this question of peptones
is at the present time undergoing active development, and as it is also
very complicated and still not clear in many points, it is at present not
possible to give a clear, short, and still comprehensive discussion of the
subject. We can give here only the most important results.
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, Law^row', Langstein,^ and
others have shown that simpler products can be produced, some whose
■ Zeitschr. f. physiol. Chem., 2i.
' Frankel, Zur Kenntnis der Zerfallsprodukte des Eiweisses bei peptischer und
tryptischer Verdauung, Wien, 1896; Schrotter, Monatshefte f. Chem., 14 and 10.
' 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.
54 THE PROTEIN SUBSTANCES.
nature is still unknown, while others are known, such as alanine, leucine,
leucinimide, aminovalerianic acid, aspartic and glutamic acids, phenyl-
alanine, tyrosine, pyrrolidine-carboxylic acid, and lysine and on further
cleavage indeed also oxyphenylethylamine, tetra- and pentamethylene-
diamine. It has not been possible to cause a disappearance of the biuret
reaction, and the occurrence of tryptophane is somewhat disputed. Mal-
FATTi obtained tryptophane in peptic digestion only when he used a certain
apparently impure preparation of jiepsin, and on using pepsin ])urified
according to Pekelharing it was absent. According to Pekelharixg.^
purified pepsin also yields tryjjtophane when the solution is rich in pepsin,
and also when the acidit\' 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 H2SO4) was continued for a very long time, indeed for an
entire year, without controlling the influence of the acid alone upon the proteoses.
KtJHNE's view that in tryptic digestion always a peptone, so-called
antipeptone, remains which cannot be further split is not strictly true.
By sufficiently long autodigestion of the pancreas Kutscher^ was able
to obtain as final products a mixture of digestion products which failed to
respond to the biuret test. In this connection we must remark that the
pure antipeptone (see below), isolated by Siegfried, could be split b}'
trypsin only with great difficulty, and also that the complete disappearance
of the biuret reaction in tryptic digestion does not show that a complete
decomposition into amino-acids has taken place. According to E. Fischer
and Abderhalden,^ polypeptide-like bodies are produced, especially in
tryptic digestion, and these bodies resist the prolonged action of the enzyme^
but yield several different amino-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 i^iuret reaction remains and no forma-
tion of tryptophane takes place.
B}^ the use of the methods specially worked out by the Hofmeister
s^chool, of fractionally salting out with ammonium sulphate or zinc sul-
' Malfatti, Zeitschr. f. physiol. Chem., 31; Pekelharing, Archives d. scienc. biolog.
de St. P6tersbourg, 11; Pawlow Festband.
"^ Zeitschr. f. physiol. Chem., 25, 26, 28, and Die Endprodukte der Trypsin-
verdauung, Habilitationsschrift Strassburg, 1899.
^Zeitschr. f. physiol. Chem., 39.
PROTEOSES AND PEPTONES. 55
phate, numerous attempts to separate the various proteoses and peptones
have recently been made by Umber, Alexander, Pfaundler, and espe-
cially by Pick and Zunz.^ Not onh^ 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 ]:)eptic digestion, a splitting of the protein molecule
into several complexes takes place. In opposition to the view of Huppert,^
that the proteoses, in pepsin digestion, are always derived from the pri-
marily formed acid albuminate. Pick and Zuxz have shown that several
proteoses, as well as acid albuminate, appear as primary products at the
commencement of the digestion. According to Goldschmidt ^ a splitting
off of proteoses and the formation of acid albuminate takes place simul-
taneously b}^ the action of dilute acids alone. Besides the proteoses we
have, according to Zuxz and Pfauxdler, even at the beginning, also
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 pep-
tones and the amino-acids, and they correspond probably to the poly-
peptide bodies obtained by Fischer and Abderhaldex 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 cent satu-
ration with ammonium sulphate solution, i.e., the presence of 2-4-42 c.c. of the
saturated ammonium sulphate solution in 100 c.c. of the liquid. Then follows
a fraction A at 54-62 per cent saturation, then a third fraction B, with 70-9.5
per cent saturation, and finally fraction C, which precipitates from the saturated
solution on acidification with sulphuric acid saturated with the salt.
The hetero- and protoproteoses are not, according to our present views,
the only primary proteoses. In the proteose fraction B, obtained on saturat-
ing with ammonium sulphate in neutral liquids, primary proteoses are
also found. As examples we may mention the glucoproteose (Pick) which
contains a carbohydrate group, and Hofmeister's ■* synproteose. Unequal
responsiveness to the salting-out process is no longer sufficient to differen-
tiate between the primary and secondary- proteoses, especially as Haslam ^
has shown that the products obtained by fractionation are not units l)ut
' Umber, Zeitschr. f. phj'siol. Chem., 2o; Alexander, ibuL, 25; Pfaiindler, ibid.,
30; Zunz, ibid. 28, and Hofmeister's Beitrage, 2; Pick, ibid., 2, and Zeitschr. f. physiol.
Chem., 24 and 28.
^ Schiitz and Huppert. Pfliiger's Arch., 80.
' F. Goldschmidt, Ueber die Einwirkiing von Sauren auf Eiweissstoffe, Inaug,-
Diss. Strassburg, 1898.
^ Ueber Bau imd Gnippirung der Eiweisskorper, Ergebnisse der Physiol., Jahrg. I
Abt. 1, 78.3. " '
* Journ. of Physiol., 32.
56 THE PROTEIN SUBSTANCES.
mixtures. According to Haslam, it is possible to separate the various pro-
teoses by salting out only by following the directions he suggests.
Under these circumstances we cannot enter into a discussion of the
properties of various proteoses thus far prepared. The differences which
exist between the hetero- and protoproteoses obtained from fibrin (Pick^)
are of great interest. The heteroproteose is insoluble in 32 per cent alcohol^
yields only very little tyrosine or indol, but gives abundant leucine and
glycocoU, and contains about 39 per cent of the total nitrogen in a basic
form. The protoproteose, on the contrary, is soluble in 80 per cent alcohol,
yields considerable tyrosine and indol, only little leucine but no glycocoU,.
and contains only about 25 per cent basic nitrogen. Friedmanx, Hart^
and Levene have obtained very similar results in regard to the quantity
of basic nitrogen in the two proteoses, although Levene did not find the
same results as Pick in regard to the amounts of leucine, tyrosine, and
glycocoll in the two proteoses. However, Haslam has shown that Pick's
heteroproteose was contaminated by a proteose which was readily pre-
cipitated by alcohol and called a-protoproteose and that there also exists
Ijesides this a second protoproteose, /?-protoprot€ose, which is probably
identical with Pick's proteose; still this does not change the fundamental
fact that we have a hetero- and a protoproteose which differ essentially
in chemical constitution. Hart^ also showed that the heteroproteose
(from muscle syntonin) is considerably richer in arginine and poorer in
histidine than the protoproteose.
According to Pick, the heteroproteose is also more resistant towards
trypsin digestion than the protoproteose, a behavior which coincides with
KtJHNE's view of a resistant atomic complex, an antigroup, in the protein
bodies. Kuhne and Chittenden ^ obtained regularly on the trjqDtic
digestion of heteroproteose a separation of so-called antialbumid, a body
which is attacked with great difficulty in tryptic digestion, but which sepa-
rates as a jelly-like mass and which is richer in carlDon (57.5-58.09 per cent),
but poorer in nitrogen (12.61-13.94 per cent), than the original protein.
This antialbumid has recently attracted further attention, because, as
first found by Danilewsky and as other investigators, Okunew, Saw-
JALOW, Lawrow, and Salaskin and Kurajeff, have further shown,
solutions of rennin, gastric juice, pancreatic juice, and papain cause a
coagulum in not too dilute proteose solutions. These coagula, called
plasteines (coagulum 1)}^ rennin) by Sawjat.ow, and coaguloses (coagu-
lum by papain) by Kurajeff,^ are similar in many respects to antialbumid,
' Zeitschr. f. pliysiol. Chem., 28.
^ P'riedmann, ibul., 21); Hart, ibid., 33; Haslam, 1. c; Levene, Joum. of BioL
Chem., 1.
^ Kiihne and Chittenden, Zeitschr. f. Biologie, 19, 20.
* The works of Danilewsky and Okunew are cited and reviewed in the following:
PLASTEINES. 57
having a higher content of carbon (57-60 per cent) and nitrogen (13-14.6
per cent). They are produced only from proteoses and not from peptones,
and form only a small fraction of the related proteose. We cannot state
anything with positiveness at present in regard to their importance. It
is evident from their composition that they do not represent the reforma-
tion of protein from the proteoses, as claimed by some investigators, and
their protein natm'e has indeed been disputed.
By fractional treatment of Witte's peptone with alcohol and acetone
H. Bayer ^ has shown that the substance plasteinogen, from which plastein
is produced, is not a true protein. This substance was soluble in alcohol-
acetone and gave on further purification neither the Millon reaction nor
the biuret test. Its composition also differs from the proteins, contain-
ing C 38.43, H 7.01, N 8.05 per cent, and the relation of C:N was 4.755:1.
According to these investigations plasteinogen is not a proteose but may
rather be considered as a peptoid.
It is also generally admitted that the peptones are mostly mixtures
of various bodies.^ Only those peptones isolated by Siegfried and his
pupils MiJHLE, Fr. JMiJLLER, BoRKEL, Krijger, and ScHEERMEssER ^ must
be considered as chemical individuals. All these peptones have a pro-
nounced acid character and form salts with carbonates with the evolution
of carbon dioxide; they are levorotatory and show a constant degree of
rotation. The pepsin-fibrin peptones, a and /?, isolated and studied by
Siegfried, ^Iijhle, and Borkel, have the formulae C21H34N6O9 and
CoiHseNfiOio, respectively. The /J-peptone seems to be converted into
a-peptone on the splitting off of water. These pepsin peptones give the
biuret test as well as ]Millon's reaction. Their solutions are not pre-
cipitated by tannic acid, picric acid, corrosive sublimate, phosphotungstic
acid, or alcohol, but are precipitated by basic lead acetate, metaphosphoric
acid, and acetic acid and potassium ferrocyanide. The pepsin peptone
may be considered as an amphopeptone in Kuhne's sense, for in trypsin
digestion amino-acids are formed, and all the tyrosine and arginine are
split off and antipeptone is formed. The a-pepsin-fibrin peptone is, like
the pepsin-glutin peptone, a tribasic acid as well as a biacidic base
(Neumann) .4
The trypsin-fibrin antipeptones studied by Siegfried and Muller have
Sawjalow, Pfliiger's Arch., 85, and Centralbl. f. Physiol., Ifi; Lavvrow and Salaskin,
Zeitschr. f. physiol. Chem., 36; Kurajeff, Hofmeister's Beitrage, 1 and 2; see also
Sacharow, Biochem. Centralbl., 1, 233.
' Hofmeister's Beitrage, 4.
^ See Kutscher, 1. c; Friiiikel and Langstein, Wien. Sitzungsber. Math.-Naturw.
Klasse, 110, 1901; Pick, Hofmeister's Beitrage, 2.
^Siegfried, Arch. f. (Aaat. u.) Physiol., 1894;' also Zeitschr. f. physiol. Chem., 21,
43, and 4n; Siegfried and his pupils, ibid., 38; Scheermesser, ibid.. 41.
* Zeitschr. f. physiol. Chem., 45.
58 THE PROTEIN SUBSTANCES.
the formulae a, C10H17N3O5, and /?, C11H19N3O5. They have a different
specific rotation and are at the same time, according to Neumann, bibasic
acids and monoacidic bases. The fact that two different antipeptones are
formed from the pepsin-fibrin peptone shows that this latter contains at
least two antigroups, and not, as Kijhne claimed, only one. The antipep-
tones do not give the biuret test, but respond to Millon's reaction, and
contain no tyrosine groups. They are precipitated by alcohol, but are pre-
cipitated less readily or less completely by the reagents which precipitate
the pepsin peptones. They have a persistent resistance towards further
cleavage by trypsin. On hydrolysis with mineral acids they yield arginine,
lysine, glutamic acid, and it seems also aspartic acid. The quantity of
basic nitrogen is less than 2.5 per cent, and the nitrogen split off as ammonia
in antipeptone is /? 16.1 and a 21.9 per cent of the total nitrogen.
The glutin peptones isolated by Siegfried and Kruger have the formulae
C21H39N7O10 for the pepsin-glutin peptone and C19H30N6O9 for the /?-tryp-
gin-glutin peptone. The composition of the apparently very pure pepsin-
glutin peptone as prepared by Scheermesser was C21H39N7O10. It gave
the biuret test, but did not give the other protein color reactions. Its
solution became cloudy with picric acid but was not precipitated. Tannic
acid gave a precipitate which was soluble in acetic acid, while phosphotung-
stic acid produced a precipitate only in concentrated solution. Of the total
nitrogen 25 per cent existed as basic and 69.85 per cent as amino-acid
nitrogen. It yielded arginine, lysine, glutamic acid, and glycocoU as hydro-
xy tic cleavage products; this peptone contained no histidine.
From glutin peptone, Siegfried, on warming with hydrochloric acid,
obtained a base, C21H39N9O8, which can also be directly obtained from
gelatine. 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 com-
plete 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. Similar basic nuclei, protokyrins, have recently
been obtained by Siegfried 1 from fibrin and casein, using the same method.
Casein okyrin gives a non-crystalline sulphate but a crystalline phospho-
tungstate. The free caseinokyrin has an alkaline reaction, gives the biuret
test, and its composition corresponds to the formula C23H47N9O8. It
yields arginine, lysine, and glutamic acid on cleavage. The basic nitro.aen
amounts to about 85 per cent of the total nitrogen, and hence caseinokyrin
behaves in this respect like a protamine.
Skraup and Zwerger have presented certain doubts, based upon their
* Kgl. Sachs. Ges. d. Wiss., Math.-Phys. Klasse, 1903, and Zeitschr. f. physiol.
Chem., 43.
BASIC NUCLEUS OF THE PROTEINS. 59
own investigations, as to the individuality of the products designated
kvrins by Siegfried. Siegfried ^ repudiates the claims made by thase
investigators, criticises their methods and presents new claims for the
indi\aduality of the kyrins, describing exactly the properties of their phos-
photungstates and picrates. The constant composition of the sulphate
is of importance. It was obtained thus only after repeated recr\-stalliza-
tion (reprecipitation nine times of a caseinokyrin sulphate), but it remained
unchanged on recrystallization up to fifteen times.
Among the known clea\'age 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 wliich contain, tesides chained
monamino-acids, also arginine, or, more simply, show the above two kinds
of imide bindings. Hence caseinokyrin. which yields only arginine, lysine,
and glutamic acid, and scombrin (see below), which yields only arginine,
a-pyrrolidine-carboxyUc acid, and alanine are the simplest known proteins.
Scombrin belongs to the group of substances called protamines, which
will be treated of later, and these substances are strongly basic, simple of
constitution, give the biuret reaction, and are similar to the proteids.
According to Kossel^ we can conceive of the formation of the protamines
by a successive cleavage of the typical proteins, and the occurrence of
basic protokyrins in the hydrolytic cleavage of genuine proteins like gelatine
has given valuable support to Kossel's theor}' as to a basic nucleus in
the protein bodies. We must not infer from tliis that each protein con-
tains only one nucleus. It is, on the contrary', possible and not improbable
that each protein is composed of several larger complexes and that each
of these contains a special nucleus. The proteoses may be considered as
large complexes of this kind which, at least in part, do not separate but seem
to stand together. The cleavage of proteins, according to ScHiJrzENBERGER
and Kt'HXE, into a hemi- and an antigroup, of which the first contains,
among other complexes, the readily split tyrosine and tiyptophane, while
the antigroup contains a-proUne (a-pyrrolidine-carboxyUc acid), glycocoU,
and phenylalanine; the different beha\'iors of proto- and heteroproteoses ;
and the occurrence of non-biuret-gi\-ing polypeptides in digestion, coincide
well ^^ith such a view.
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. The molecular weight of the different proteins has
not been determined with certainty .^ but it is generally considered as about
• Skraup and Zwerger, Monatshefte f. Cheniie, 26; Siegfried, Zeitschr. f. Physiol
48.
- Zeitschr. f. physiol. Chem., 44.
'See especially F. N. Schulz, Die Grosse des EiweissmolekiiLs, Jena, 1903.
60 THE PROTEIN SUBSTANCES.
5000-10 000 for the albumins and for casein. The molecular weight for
protoproteoses was found by Sabanejew to be 2467-2643, and 3200 for
the deuteroproteoses. The peptones have a still lower molecular weight,
being between 400 and 250 for the different peptones (Sabanejew, Paal,
Sjoqvist 1).
The elementary analj^ses ^ have not given us much information as to
the characteristic differences between the various proteoses and most
so-called peptones. Certain proteoses, especially those that can be salted
out with difficulty, and the peptones differ very materially in composition
from the mother-substances and often have a lower carbon content.
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 ^ 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 proteose
(in KiJHNE'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 KtJHNE'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 protopro-
teoses 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. As
most of the older methods do not give pure substances but rather mixtures,
it is perhaps sufficient simply to call attention here to other methods,
such as those suggested by K. Baumann and Bomer, P. ^Muller, Frankel,
Schrotter, and Paal. The only method which seems thus far to have
led to a pure preparation of peptone is the iron method used by Siegfried.^
For the detection of proteoses and peptones in animal fluids we proceed
as follows, according to Devoto: The coagulable proteins are removed by
prolonged heating, and the solution is then saturated with ammonium sul-
phate. True peptones (besides deuteroproteose not precipitated) may be
detected in the cold filtrate by means of the biuret test. The proteoses
' Sabanejew, Ber. d. d. chem. Gesellsch., 20, 385; Paal, ibid., 27, 1827; Sjoqvist,
Skand. Arch. f. Physiol., 5.
^ Elementary analyses of proteoses and peptones will be found in the works of
Kiihne and Chittenden and their pupils, cited in foot-note 2, p. 52; also by Herth,
Zeitschr. f. physiol. Chem., 1, and Monatshefte f. Chem., 5; Maly, Pfliiger's Arch.,
9, 20; Hcnninger, Compt. rend., 80; Schrotter, 1. c; Paal, 1. e.
^ Schrotter, Monatshefte f. Chem., 14 and 10; Frankel, Zur Kenntnis der Zerfalls-
produkte des Eiweiss bei peptischer und tryptischer Verdauung, Wien, 1896.
* Kiihne, Zeitschr. f. Biologic. 28; Pick, I. c; Haslam, 1. c.
^ Baumann and Bomer, Chem. Centralbl., 1898, 1, 640; Miiller, Zeitschr. f. physiol.
Chem., 20; Frankel, 1. c., Zur Kenntnis, etc.; Schrotter, Monatshefte f. Chem., 14
and 10; Paal, 1. c; Siegfried, 1. c.
HISTONES. 61
are contained in the mixture of precipitate and salt crystals collected on
the filter. The proteoses are dissolved from this mixture by washing with
water, and may be detected in the wash-water by means of the biuret
test. According to Halliburton and Colls ^ traces of proteoses may
be formed from other proteins in this method by prolonged heating. As
the best method they suggest either the precipitation of the native proteids
b}' the addition of a 10 per cent trichloracetic-acid solution, or the con-
version of the native proteins to the insoluble form by the continued action
of alcohol. The last method is hardly applicable to blood-serum, as the
so-called fibrin-ferment, which also gives the biuret test, is not made insol-
uble by this procedure.
If a solution saturated with ammonium sulphate is to be tested for the
biuret reaction, it must first be treated with a slight excess of concentrated
caustic-soda solution, the solution being kept cold, and after the sodium
sulphate has settled, the liquid is treated with a 2 per cent solution of copper
sulphate, drop by droj).
The estimation of nitrogen, the biuret test rcolorimetric), and the polari-
scopic method have been used in the quantitative estimation of proteoses
and peptones. These two last-mentioned methods do not yield exact results.
Coagulated Proteins. Proteins ma}^ be converted into the coagulated
condition by different means: by heating, b}" 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 (Ramsdex^)^ and in cer-
tain 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 in dilute acids or alkalies, at normal
temperature. They are dissolved and converted into albuminates by
the action of less dilute acids or alkalies, especially on heating.
Coagulated proteins also seem to occur in animal tissues. We find, at
least in many organs such as the liver and other glands, proteins which are
not soluble in water, dilute salt solutions, or verv' dilute alkalies, and only
dissolve after being modified by strong alkalies.
Histones are basic proteins which stand to a certain extent between
the strongly l^asic protamines (see below) and the tme 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 proteins.
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
* Devoto, Zeitschr. f. physiol. Chem., 15; Halliburton and Colls, Joum. of Path,
and Bact., 1895.
2 Arch. f. (Anat. u.) Physiol., 1894.
62 THE PROTEIN SUBSTANCES.
it histone. At the present time a number of very different bodies are
described as histones, such as those obtained from nucleohistone (Ltliex-
feld), from hannoglobin (globin according to Schulz). from mackerel si)er-
matozoa (scombron according to Bang), from the codfish (gadushistor.e
according to Kossel and Kutscher), from the burbot, (lotahistone. Ehr-
STROm), and from the sea-urchin (arbacin, jMathews).^
Sulphur has been found in those histones in which it has been tested for.
They give the biuret test, but as a rule only a faint ]\Iillon's reaction. The
goose-blood histone first studied by Kossel gives the three following reac-
tions: The neutral salt-free solution 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 disap-
pears on heating and reappears 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. Time proteins,
as Osborne's 2 edestan, also give these two" reactions; therefore it is im-
possible by qualitative tests alone to identify a substance as a histone
with posit iveness. The large content of basic nitrogen and of argininc 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 p:r
cent arginine (gadushistone), and the lotahistone only 12 per cent. The
plant -globulin edestin ^ yields a much larger amount of arginine, namely,
14.0? per cent. On hydrolytic cleavage the histones, like other proteins,
but unlike the protamines, yield a large number of monamino-acids.
Abderhalden and Rona^ obtained from thymus histone the following:
leucine 11.8, alanine 3.46, glycocoll 0.50, a-proline 1.46, phenylalanine 2.20,
tvrosine 5.20, and glutamic acid 0.53 per cent. According to Kossel the his-
tones are probably intermediate bodies between the protamines and pro-
tein bodies on the demolition of the latter, and if this is true, then it is not
1 Kossel, Zf'itschr. f. physiol. Chem., 8, and Sitzungsber. der Gosellsch. zur Beford.
d. ges. Naturwiss. zu Marburg, 1S97; Kossel and Kutscher, ibid., 1900, and Zeitschr.
f. physiol. Chem., 31; Lawrow, i6m/., 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., 28.
2 Zeitschr. f. physiol. Chem., 33.
3 See Kossel, Ber. d. d. chem. Gesellsch., 34, 3236.
" Zeitschr. f. physiol. Chem., 41,
PROTA^IINES. 63
to be expected that histones should have perfectly specific reactions, and
for this reason it is hardly possible for the present to give a precise defi-
nition 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 ^).
Protamines. In close relationship to the proteins stands a group of
substances, the protamines, discovered by Miescher, which are desig-
nated by Kossel as the simplest proteins or as the nucleus of the jDro-
tein bodies. Thus far they have been found only in comljination with
nucleic acids in fish spermatozoa. They differ essentially from the proteins
by the fact that they yield chiefly diamino-acids (always abundant arginine)
as cleavage products, and oidy a small amount of monamino-acids. They
are strongly basic substances rich in nitrogen (about 30 per cent or more)
and have high molecular weight.
Protamine was discovered by Miescher 2 in salmon spermatozoa. 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, scomhritie,
sturine, cyprinine, cyclopterine, etc.
The percentage composition of these bodies has not been satisfactorily
determined. As probable formulae we have for salmine C32H56N18O4
(Miescher-Schmiedeberg) or C30H57N17O6 (Kossel and Goto), for clu-
peine C30H62N14O9, and for sturine C36H69N19O7 (Kossel) or C34H71N17O9
(Goto). On boiling with dilute mineral acids, as also by tryptic digestion,
the protamines first yield peptone-like substances called protones, from
which simpler products are derived on further cleavage. All protamines
yield arginine, the four protamines salmine, clupeine, cyclopterine, and
sturine yielding 87.4, 82.2, 62.5, and 58.2 per cent respectively. 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 protamine, cyprinine, occurs in two different modifications, namel}',
a- and ^9-cyprinine. The a-cyprinine yields only little arginine, 4.9 per
' Fleroff, Zeitschr. f. physiol. Chem., 2S; Kossel and Kutscher, 1. c.
^ In regard to protamines, see Miescher, Histochemische und Physiologische Ar-
beiten, Leipzig, 1897; Piccard, Ber. d. deutsch. chem. Gesellsch., 7; Schmiedeberg,
Arch. f. exp. Path. u. Pharm., 3"; 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; (^-nto, ibid., S7 ; Kurajeff, i6id., 32; Morkowin, *«/., 28; Kossel and Dakin,
iow/., 40, 41, and 44.
64
THE PROTEIN SUBSTANCES.
€ent, 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, aminovalerianic 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. aminovalerianic acid, and 2 mol. a-proline.
Scombrine contains only arginine, alanine, and a-proline. The following
summary according to Kossel ^ gives a view of the cleavage products of
the protamines thus far investigated.
Alanine
iiJerine
Aminovalerianic acid
Leucine
Arginine
Lysine
Histidine
«- Proline
Tyrosine •
Tryptophane
Scom-
Salmine.
Clupeine.
Sturine.
Cyclop-
a-Cyp-
brine.
terine.
rinine.
+
0
+
+
?
?
0
+
+
0
?
?
0
+
+
0
?
+
0
0
0
+
?
•?
+
+
+
+
+
+
0
0
0
+
0
+
0
0
0
+
0
0
+
+
+
c
?
9
0
0
0
0
+
0
0
0
0
0
+
0
/J-Cyp-
rinine.
Solutions of these bases in water are alkaline and have the pro]3erty
of giving precipitates with ammoniacal solutions of proteins or primary pro-
teoses. These precipitates are considered as hist ones by Kossel. 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, levog3'rate. They
give the biuret test beautifully, but with the exception of cyclopterine and
_,i9-c3'prinine do not give Millon's reaction. The protamine salts are pre-
cipitated in neutral or even faintly alkaline solutions by phosphotungstic
acid, picric acid, chromic acid, and alkali ferrocyanides.
The protamines are prepared, according to Kossel, by extracting the
heads of the spermatozoa, which have previously been extracted with
alcohol and ether, with dilute sulphuric acid (1-2 per cent), filtering, and
precipitating with 4 vols, of alcohol. The sulphate may be purified by
repeated solution in water and precipitation with alcohol, and if necessary,
conversion into the ]:)icrate. For more details see the works of Kossel.
The double-platinum salt is best suited for analysis and can be obtained,
according to Goto, by j^recipitating the methyl-alcohol solution of the
protamine hydrochloride with platinum chloride. Miescher also precipi-
tates the base as a douLle-platinum salt.
' Zeitschr. f. } hy.sio). Chcni., 44.
COMPOUND PROTEIDS. 65
II. Compound Proteids.
With this name we designate a class of bodies which are more complex
than the proteids, and which yield as primary splitting products proteids
on the one side and non-proteid bodies, such as pigments, carbohydrates,
nucleic acids, etc., on the other.^
The compound proteids known at the present time are di\-ided into
three chief groups. These are the hcemoglobins, the glucoproteids, and the
nudeoproteids. The haemoglobins will be discussed in a following chapter
(Chapter YI, on the blood) .
Glucoproteids are those compound proteids which on decomposition
yield a proteid on the one side, and a carbohydrate or derivatives of the
same on the other, but no purine bodies. Some glucoproteids are free from
phosphorus (mucin substances, chondroproteids, and hyalogens), and
some contain phosphorus (phosphoglucoproteids).
The glucoproteids 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 chondroproteids. The first yield on hydrolytic
cleavage an amino-sugar, which has been shown to be glucosamine in all
cases except one.- In the chondroproteids, on the contrary, the proteid
is united to chondroit in-sulphuric acid.
Mucin Substances. These bodies contain carbon, hydrogen, nitrogen,
sulphur, and oxygen. Compared with proteids they are poorer in nitrogen
and as a rule have also considerably less carbon. The carbohydrate complex,
M^hose nature has been showm by the investigations of Fr. MiJLLER ^ and
his pupils, occurs, as it seems, in the mucin substances as a pol3'saccharide
related to chitosan, which on hydrolytic cleavage yields glucosamine
(chitosamine), and, at least in most cases, also acetic acid. The mucin
substances differ ver\^ markedly among one another, hence we divide them
into two groups, the mucins and the mucoids.
The true yyiucins are characterized by the fact that their natural solu-
tions, or solutions prepared by the aid of a trace of alkali, are mucilaginous,
ropy, and give a precipitate with acetic acid which is insoluble in excess of
acid or soluble only with great difficult3^ The mucoids do not show these
' Hoppe-S.eyler has given the name prote'ide to these compound proteids, but as
this term is misleading in EngHsh we do not use it in English classifications in this
sense.
^ See Schulz and Ditthorn, Zeitschr. f. physiol. Chem., 29. When both carbo-
hydrate groups are simultaneously combined with one body, then probably we are not
dealing with a chemical individual, but rather with a mixture.
^ See Fr. Miiller, Zeitschr. f. Biologie, 42, which contains all the pertinent litera-
ture, and also L. Langstein, Die Bildung von Kohlenhydraten aus Eiweiss, Ergebnisse
der Physiologie, Jahrg. I, Abt, 1.
66 THE PROTEIN SUBSTANCES.
physical properties and have other soIubiUties and precipitation properties.
As we have intermediate steps between different protein 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-
teids and the mucins or mucoids, since we have been able to split off carbo-
hydrate complexes from several proteids, and the proteids of the white
of egg are undoubtedly glucoproteids. It is immaterial whether we con-
sider these glucoproteids as belonging to the mucoids or to a special group.
From a comparative chemical standpoint, they undoubtedl}' belong to the
mucoid group, representatives of which occur in eggs to a considerable
extent.
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 i), a mother-substance of mucin, a mucinogen,
has been found which may be converted into mucin by alkalies. I\Iucoid
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 recent investigations of Levexe and of Cutter, and
GiES,2 contains chondroitin-sulphuric acid or a related substance, cannot
be classified as a mucin, but must, like the chondromucoid and the osseo-
mucoid, be classified as chondroproteid. As the mucin question has not
been sufficiently studied, it is at the present time impossible to give any
positive statements in regard to the occurrence of mucins and mucoids,
especially as without doubt in many cases non-mucinous sul^stances have
been described as mucins.
I. 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 10.7 1.4 (Fr. Muli.er)
Mucin from submaxillary 48.84 6.80 12.32 0.84 (H.\mm.\rsten*)
Mucin from snail 50.32 6.84 13.65 1 .75 (Hammarstex^)
Synovial mucin 51 .05 6.53 13.01 1 .34 (v. Holst ")
MtJLLER obtained 35 per cent glucosamine from mucous-membrane
mucin and 23.5 per cent from the submaxillary mucin.
'Giacosa, Zeitschr f physiol. Chem. 7; Hammarsten, Pflliger's Archiv, 30, and
Skand. Arch. f. Physiol., 17.
2 Levene, Zeitschr. f. physiol. Chem. 31; Cutter and Gies, Amer. Journ. of Physiol., 6.
3 Fr. Miiller, Zeitschr. f. Biologic, 42; Hammarsten, Zeitschr. f. physiol. Chem., 12,
and Pfluger's Arch., 3G.
* Zeitschr. f. phy,?iol Chem., 43.
MUCINS. 67
By the action of superheated steam on mucin a carbohydrate, animal
gum (Landwehp", is split off. This has not been substantiated by other
investigators, such as Folin and Fr. iMtJLLER.^ 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 (Steudel^). By the action of strong acids upon
mucins or mucoids Otori ^ 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 submaxil-
lary mucin, are easily changed by very dilute alkalies, as lime-water, while
others, such as tendon-mucin, are not affected. If a strong caustic-alkali
solution, such as 5 per cent KOH solution, is allowed to act on submaxillary
mucin, we obtain alkali albuminate, bodies similar to proteoses and peptones
and one or more substances 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 tryptophane
(PosNER and Gies*). 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
MlLCHNER^).
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 drj^ 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 solu-
tion with water with the aid of small amounts of alkali. Such a solution
' 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. Naturwiss. zu Marburg, 1896.
' Zeitschr. f. physiol. Chem., 34.
Ubul., 42 and 43.
*Amer. Journ. of Physiol., 11.
* Berl. klin. Wochenschr., 1904.
68 THE PROTEIN SUBSTANCES.
does not coagulate on boiling, but acetic acid gives at the normal tempera-
ture 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 care-
fully acidified with acetic acid without giving a precipitate. Such acidified
solutions are copiously precipitated by tannic acid; with potassium ferro-
cyanide they give no precipitate, but on sufficient concentration 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 hydro-
chloric 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 submaxillary
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. HCl. On the addition of the acid the mucin is immediately pre-
cipitated, but dissolves on stirring. If this acid liquid is immediately
diluted with 2-3 vols, of water, the mucin separates and may be purified
by redissolving in 1-5 p. m. acid, and diluting with water and washing
therewith. The mucin of the navel-cord may be prepared in the same way.
As a rule the mucins can be prepared by precipitation with acetic acid and
repeated solution in dilute lime-water or alkali and reprecipitation with
acetic acid. Finally they are treated with alcohol and ether. In the
preparation of sputum mucin a very complicated method is necessary (Fr.
MtJLLER).
The precipitation by acetic acid, as shown by Hammarsten,^ is not applicable
in the preparation of submaxillary mucin, because another proteid substance is
precipitated with the mucin, but remains in solution on using the hydrochloric-
acid method above described. Posner and Gies ^ have by special experiments
shown the power of mucins of precipitating proteids, and this makes the ordi-
nary method of precipitating with acetic acid questionable.
2. Mucoids or Mucinoids. In this group we must include those non-
phosphorized glucoproteids which are neither true mucins nor chondro-
proteids, even though they show amongst themselves such differences in
behavior that they can be divided into several subgroups of mucoids.
To the mucoids belong pseiidomucin, 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 ^ has designated a number of
differing 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-
' Zeitschr. f. physiol. Chem., 12.
* Amer. Journ. of Physiol., 11.
3 Verh. d. physik.-med. Gesellsch. zu Wiirzburg, 1883; also Zeitschr. f. Biologie, 22.
AMYLOID. 69
bohydrate by further decomposition. We find that very heterogeneous substances
are included in this group. Certain of these hyalogens seem undoubtedly to
be glucoproteids. Neossin ^ of the Chinese edible swallow 's-nest, mernbranin ^
of Descemet's membrane and of the capsule of the crystalline lens, and spiro-
'jraphin ^ of the skeletal tissue of the worm Spirograplais seem to act as such.
Others on the contrary, such as hyalin * of the walls of hydatid cysts, and onu-
phin^ from the tubes of Onuphis tubicola, do not seem to be compound proteids.
The so-called mvcin of the holothurcs ^ and chondrosin ^ 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.
3. Chondroproteids are those glucoproteids which as primary cleavage
products yield proteid and an ethereal sulphuric acid containing a carbo-
hydrate, chondroitin-suliphuric 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 chon-
droitin-sulphuric acid of precipitating proteid, it is also possible that under
certain circumstances combinations of this acid with proteid may be pre-
cipitated from the urine and be considered as chondroproteids.
The chondromucoid, the so-called tendon-mucin, and the osseomucoid
have greatest interest as constituents of cartilage, of the connective tissues,
and of the bones, and on this account these bodies and their cleavage prod-
uct, chondroit in-sulphuric acid, will be treated in a following chapter (X).
On the contrary, amyloid, which has alwaj^s 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 con-
centric la^^ers. It probably also occurs as a constituent of certain prostate
calculi. The chondroproteid occurring under physiological conditions in
the Avails of the arteries is perhaps, according to Krawkow, very nearly
related to the amyloid substance, but not identical with it, as shown by
Neuberg.^
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.S per cent.
The aorta amyloid of man and of the horse contained respectively C 49.6
' Krukenberg, Zeitschr, f, Biologie, 22.
' C. Th. Morner, Zeitschr. f. physiol. Chem., IS.
'Krukenberg, Wiirzburg, Verhandl. 1883; also Zeitschr. f. Biologie, 22.
*A. Liicke, Virchow's Arch., 19; also Krukenberg, Vergleichende physiol. Stud.,
Series 1 and 2, 1881.
^ Schmiedeberg, Mitth. aus d. zool. Stat, zu Neapel, 3, 1882.
^ Hilger, Pffiiger's Archiv, 3.
' Krukenberg, Zeitschr. f. Biologie, 22.
" Krawkow, Arch. f. exp. Path. u. Pharm., 40, which contains the literature; Neu-
berg, Verhandl. d. d. Pathol. Gesellsch. 1904.
70 THE PROTEIN SUBSTANCES.
and 5U.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 follo\Aing:
Monamino-X Diamino-N Amide-N
Liver amyloid 43 .2 ol . 2 4.9
Spleen amyloid 30. 6 57. U 11.2
Aorta amyloid 54.9 36.0 8.8
From liver amyloid Neuberg obtained glycocoll 0.8; leucine 22.2; glu-
tamic acid 3.8; tyrosine 4.0; a-proline 3.1; arginine 13.0, 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,
ether, 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 and in agree-
ment with most of the older statements. 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 trj^psin, although more slowly than fibrin,
and that it is also destroyed in autolysis, so that in life an absorption
is possible. Amyloid gives the xanthoproteic reaction and the reactions
of MiLLOx 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.
The preparation of amyloid may be performed as follows according
to Modrzejewski and Krawkow.^ The finely divided organ is exhausted
first with water and then with dilute ammonia, which leaves the insoluble
' Modrzejewski, Arch. f. exp. Path. u. Pharm., 1; Krawkow, I. c.
NUCLEOPROTEIDS. 71
amyloid and removes the free or the combined chondroit in-sulphuric acid,
besides other substances. The product, after being washed with water, ia
digested ^^ith pepsin for several days at 38° C. The residue, after washins
"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 bar\'ta-water, which leaves the
nucleins undissolved, and the barium filtrate precipitated "^nth hydrochloric
acid, and then washed with water, alcohol, and ether.
Phosphoglucoproteids. This group includes the phosphorized glucoproteids.
They yield no xanthine substances (nuclein bases) as cleavage products. They
are not nucleoproteids and therefore they must not be considered together
with the gluconucleoproteids (nucleoglucoproteids; or mistaken for them. On
pepsin digestion they ma}', like certain nucleoalbumins, yield pseudonuclein,
but they differ from the nucleoalbumins in that they yield a reducing substance
on boiling with dilute acid. They differ from the gluconucleoproteids in that
they do not, as above mentioned, yield any xanthine bodies.
Only two phosphorized glucoproteids are known at the present time, namely,
ichthidin, occurring in carp eggs and studied by Walter ^ and which was con-
sidered as a vitellin for a time. Ichthulin has the following composition: C .53.52;
H 7.71; X 15.64; S 0.41; P 0.4.3; Fe 0.10 per cent. In regard to solubilities it
is similar to a globulin. Walter has prepared a reducing substance from the
paranuclein of ichthulin which gave a highly crystalline compound with phenyl-
hydrazine.
Another phosphoglucoproteid is helicoproteid, obtained by Hammarsten -
from the glands of the snail Helix pomatia. It has the following composition:
C 46.99 ; H 6.78 ; N 6.08 ; S 0.62 ; P 0.47 per cent. It is converted into a gumn.y,
levorotatory carbohydrate, called atnmal sinistrin, by the action of alkalies.
On boiling with an acid it yields a dextrorotatory reducing substance.
The compound proteid found by Schulz and Ditthorx ^ in the spawn
of the frog probably belongs to this group, but instead of glucosamine it
gives galactosamine on cleavage.
Nucleoproteids. With this name we designate those compound pro-
teids which yield true nucleins (see Chapter V) on pepsin digestion, and
on cleavage ^^•ith dilute caustic alkali yield proteid and nucleic acid.
The nucleoproteids seem to be widely diffused in the animal body. They
occur chiefly in the cell-nuclei, but they also often occur in the proto-
plasm. They may pass into the animal fluids on the destruction of the
cells, hence nucleoproteids have also been found in blood-serum and other
fluids.
They may be considered as combinations of a proteid nucleus with a
side chain, which Kossel calls the prcsthetic group. This side chain,
which contains the phosphorus, may be split off as nucleic acid (see Chapter
Y) on treatment with alkali. As we have several nucleic acids, it follows
that we must have different nucleoproteids, depending upon the nucleic acid
' Zeitschr. f. physiol. Chem., l.>.
* Hammarsten, Pfliiger's Arch., 3B.
' Zeitschr. f . physiol. Chem., 23.
72 THE PROTEIN SUBSTANCES.
united with the proteid. Certain nucleic acids contain a readily split-off
sugar (pentose or hexose); others, on the contrar}^ do not. In the first
case we obtain from the corresponding nucleoproteid a reducing sugar
on boiling with dilute mineral acid, while in the other case this is not pos-
sible. Corresponding to this different behavior we may divide off a special
group of nucleoproteids, the gluconucleoproteids or nucleoglucoproteids.
Such gluconucleoproteids, yielding pentoses, occur in yeast-cells, and, as it
appears, are widely distributed in the animal organism (Blumenthal,
Gruxd 1).
The native nucleoproteids contain a variable but not a high percen-
tage of phosphorus, which Halliburton 2 found to vary between 0.5 per
cent and 1.6 per cent. On heating their solutions, as well as by the action
of dilute acids, a modification of the compound proteid takes place, and
nucleoproteids of strong acid character, poorer in proteid but richer in
phosphoi-us, are formed. The native nucleoproteids have faint acid proper-
ties and are insoluble in water, but their alkali compounds, which are
soluble in water, split on heating their solutions into coagulated proteid and
a nucleoproteid rich in phosphorus, which remains in solution. In peptic
digestion they yield so-called true nuclein, which is also a nucleoprotjeid
poor in proteid. The proteid can be precipitated by acetic acid from its
alkali compound, and the precipitate dissolves with more or less readiness
in an excess of the acid. A confusion may occur here with nucleoalbumins
and also with mucin substances. This confusion may be avoided by
warming the ])ody for some time on the water-bath with dilute sulphuric
acid, nearly neutralizing the boiling-hot fluid with barium hydrate, filtering
as quickly as possible while boiling hot, and testing the filtrate for purine
bodies with copper sulphate and bisulphite according to the method given
on page 163. Any precipitate formed is examined more closely by the
method there given. The nucleoproteids give the color reactions of the
proteins, but those which have been investigated are dextrorotatory^ and
not levorotatory (Gamgee and Jones 3).
The properties of the various nucleoproteids are given in detail in the
various chapters which follow.
III. Albuminoids or Proteinoids.
Under this name we collect into a special group all those protein bodies
which cannot be placed in either of the other two groups, although they
differ essentially among themselves, and from a chemical standpoint do
' Blumenthal, Berlin, klin. Wochenschr., 1897, and Zeitschr. f, klin, Med., 34;
Grund, Zeitschr. f. physiol. Chem., 35. See also Bendix and Ebstein, Zeitschr. f.
allgem. Phys., 2; Levene and Mandel, Zeitschr. f. physiol. Chem., 47.
^ Journ. of Physiol., IS.
^ Hofmeister's Beitrage, 4.
KERATINS. 73
not show any radical difference from the true protein bodies. The most
important and abundant of the bodies belonging to this group are important
constituents of the animal skeleton or the cutaneous structure. They occur
as a rule in an insoluble state in the organism, and they are distinguished
in most cases by a pronounced resistance to reagents which dissolve proteins
or to chemical reagents in general.
The Keratin Group. Keratin is the chief constituent of the homy
structure, of the epidermis, of hair, wool, of the nails, hoofs, horns, feathers,,
of tortoise-shell, etc., etc. Keratin is also found as neurokeratin (Kuhxe)
in the brain and nerves. The shell-membrane of the hen's egg seems
also to consist of keratin, and according to NeumeisterI the organic
matrix of the egg-shells 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 ^\ithout a partial decomposi-
tion, is sufficient explanation for the variation in the elementary composi-
tion given below. As examples the analyses of a few tissues rich in keratin
and of keratins are given.^
c H N s 0
Human hair. .. . 50.65 6.36 17.14 5.00 20 . 85 (v. L.var)
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)
Horn (average).. 50.86 6.94 3.20 (Horbaczewski)
Tortoise-i^hell. . . 54.89 6.56 16.77 2.22 19.56 (Mulder)
Shell-membrane. 49.78 6.94 16.43 4.25 22.90 (Lindvall)
!MoHR 2 has determined the quantity of sulphur in various keratin sub-
stances. 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 dissolves, with the
elimination of sulphuretted hydrogen or mercaptan (Bauer), and the
solution contains proteose-like substances (Krukenberg) called atmidkera-
tin and atmidkeratose by Bauer.^ Keratin is dissolved by alkalies, especially
on warming, producing besides alkali sulphides also proteose substances.
> Kiihne and Ewald, Verh. d. naturhistor.-med. Vereins zu Heidelberg (N. F.), 1;
also Kiihne and Chittenden, Zeitschr. f. Biologic, 2(>; Neumeister, ibid., 31.
^ V. Laar, Annal. d. Chem. u. Pharm., 4o; Mulder, Versuch einer allgem. physioL
Chem., Braunschweig, 1844-51; Kiihne, Zeitschr. f. Biologic, 20; Horbaczewski, see
Drechsel in Ladenburg's Handworterbuch d.. Chem., 3; Lindvall, Maly's Jahres-
bericht, 1881.
^Zeitschr. f. physiol. Chem.. 20.
* Krukenberg, Untersuch. iiber d. chem. Bau d. Eiweisskorper, Sitzungsber. d.
74 THE PROTEIN SUBSTANCES.
Besides the well-known cleavage products such as leucine, tyrosine,
aspartic acid, glutamic acid, arginine, and lysine, Fischer and Dorping-
HAUS ^ have recently found glycocoll, alanine, a-aminovalerianic 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. Morner2 was the first to positively prove the abundant occur-
rence 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. From the
amount of sulphur split off by alkali, he concludes that, at least in ox-horn
and human hair, all the sulphur exists as cystine. Galimard ^ was able
to get only a qualitative test for cystine in the keratin of the adder eggs.
SuTER, Morner, and Friedmann* have obtained a-thiolactic acid as a
hydrolytic cleavage product of the keratin substances. The last-men-
tioned investigator was also able to detect thioglycolic acid in the cleavage
products of wool.
Bodies occur in the animal kingdom which form to a certain extent
intermediate substances between coagulated protein and keratin. C. Th.
Morner ^ has detected such a body {alhumoid) in the tracheal cartilage,
which forms a net-like trabecular tissue. This substance appears to be
related to the keratins on account of its solubilities and the quantity of
the sulphur (lead-blackening) it contains, while according to its solubility
in gastric juice it must stand close to the proteins. Another substance,
more similar to 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. It contains only 1 per cent sulphur and yields
on decomposition only a very little tyrosine but considerable leucine.
Keratin is amorphous or takes the form of the tissues from which it was
prepared. On heating it decomposes and generates an odor of burnt horn.
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, espe-
cially on heating. It is not dissolved by artificial gastric juice or by tryp-
.sin solutions. Keratin gives the xanthoproteic reaction, as well as the
reaction with Millon's reagent, although the latter is not always typical.
Jenaischen Gesellsch. f. Med. u. Naturwissensch., 1886; Bauer, Zeitschr. f. physiol.
Chem., .3.}.
' Zeitschr. f. physiol. Chem., 3G, which contains also the older literature.
2 Morner, ibid., 34 and 42; EmmerHng, Ref. in Chemiker Zeitung, 1894.
3 Chem. Centralbl. II., 1905.
♦Suter, Zeitschr. f. physiol. Chem., 20; Morner, ibid., 42; Friedmann, Hofmeister's
Beitrage, 2.
^ See Maly's Jahresber. , 18.
*Skand. Arch. f. Physiol., S.
ELASTIN. 75
In the preparation of keratin a finely divided horny structure is treated
first with boihng 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 abun-
dantly in the cervical ligament (ligamentum nuchse).
Elastin used to be generally considered as a sulphur-free substance.
According to the investigations of Chittenden and Hart, it is a question
whether or not elastin does not contain sulphur, which is 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 changing
the properties of the elastin ; and recently Zoja, Hedin, Bergh, and Richards
and GiEs ^ have found that elastin contains sulphur. The most trust-
worthy analyses of elastin from the cer\'ical ligament (Nos. 1 and 2) and
from the aorta (No. 3) have given the following results, which compare
well with each other:
s o
.... 21 .94 (HORBACZEWSKI ^)
.... 21 . 79 (Chittenden and Hart)
0.38 (H. Schwarz)
Zoja found 0.276 per cent sulphur and 16.96 per cent nitrogen in elastin.
Hedin and Bergh found different quantities of nitrogen in aorta-elastin,
depending upon whether Horbaczewski's or Schwarz's method was
used in its preparation. In the first case they found 15.44 per cent nitro-
gen 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. Abundant leucine, but very little
tyrosine, some glycocoll, and perhaps aminovalerianic acid, but no aspartic
acid or glutamic acid, used to be considered amongst the hydrolytic cleavage
products of elastin. Abderhalden and Schittenhelm ^ have obtained
glycocoll 25.75; leucine 21.38; alanine 6. 58; phenylalanine 3.89; a-pro-
line 1.74; glutamic acid 0.76, and aminovalerianic acid 1.0 per cent. The
three 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.
' Chittenden and Hart, Zeitschr. f. Biologic, 25; Schwarz, Zeitschr, f, physiol.
Chem., 18; Zoja, ibuL, 23; Bergh, ibid., 25; Hedin, ibid.; Richards and Gies, Amer.
Journ. of Physiol., ".
^Zeitschr. f. physiol. Chem., (5.
^ Ibid., 41.
C
h
Is
r
1.
54,
32
6.99
16,
75
2.
54
,24
7.27
16.
70
3.
53
.96
7,03
16,
,67
76 THE PROTEIN SUBSTANCES.
On putrefaction by anaerobic micro-organisms, Zoja found carbon
dioxide, hydrogen, methane, mercaptan, butyric acid, valerianic acid,
ammonia, and possibly also phenylpropionic acid and aromatic oxyacids.
Indol and skatol have not been found in putrefaction,^ 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 products, called by Horbac-
ZEWSKi hemielastin and elastinpeptone. According 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 precipitated by mineral acids 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 peptone 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 insoluble 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 concentrated hydrochloric acid.
It responds to the xanthoproteic reaction and to that with Millon's reagent.
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 gelatine-forming substance, occurs very extensively in
vertebrates. The flesh of cephalopods is also said to contain collagen.^
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
'See Walchli, Journ. f. prakt Chem. (N. F.), 1".
* Hoppe-Seyler. Physiol. Chem., p. 97.
6.47
17.86
24
.92
(Hofmeister)
6.80
17.97
0.
t
25.13
(Chittenden)
6.56
17.81
0.
26
25.26
(van N.\me)
6.71
17.90
0,
57
24.33
(Richards and GiEs)
6.76
17.68
(Faust)
COLL AG EX. 77
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 prob-
ably there are several varieties of collagen.
By continued boiling with water (more easily in the presence of a
little acid) collagen is converted into gelatine. Hofaieister ^ found that
gelatine on being heated to 130° C. is again transformed into collagen; and
this last may be considered as the anhydride of gelatine. Collagen and
gelatine have about the same composition.^
c H N s o
Collagen 50 . 75
Gelatine (commercial). ... 49.38
Cielatine from tendons.. . . 50.11
Gelatine from ligaments. . 50.49
Fish glue 48 . 69
Gelatines of different origin show a somewhat variable composition,
which seems to indicate the occurrence of different coUagens. 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 com-
bined sulphur during the purification. C. JMorner ^ has prepared a typical
gelatine containing only 0.2 per cent of sulphur by a method which elim-
inated any possible changes due to reagents.
Sadikoff^ has prepared gelatines by various methods from tendons
and from cartilage. Those from tendons, some of which were prepared
after previous tryptic digestion, some after treatment with 0.25 per cent
caustic potash, 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 0.34-0.526 per
cent sulphur. Sadikoff seems to think that the gelatines 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 gelatines or glutins. They
were poorer in carbon and nitrogen, 17.7 to 17.87 per cent, but somewhat
richer in sulphur, 0.53-0.712 per cent, than the tendon glutin. The glu-
teins 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 reac-
tion with phloroglucin-hydrochloric acid. The glutins differ from the
gluteins by a different behavior with certain salts.
^ Zeitschr. f. physiol. Chem., 2.
2 Hofmeister, 1. c; Chittenden and SoIIey, Journ. of Physiol., 12; van Name,
Journ. of Exper. Med., 2; Richards and Gies, Amer. Journ. of Physiol., S; Faust,
Arch. f. exp. Path. u. Pharm., 41.
5 Zeitschr. f. physiol. Chem., 28.
*Ibul , 39 and 41.
78 THE PROTEIN SUBSTANCES.
The decomposition products of the collagens are the same as those of
the gelatines. Besides the leucine, glycocoll, aspartic acid, and glutamic
acid found by the earlier investigators as hydrolytic cleavage products,
E. Fischer and collaborators ^ have obtained alanine, phenylalanine, and
a-proline. Gelatme does not give any tyrosine, but does ^neld considerable
glycocoll (16.5 per cent according to E. Fischer), which because of its
.sweetish taste has received the name gelatine-sugar. Skraup ^ has obtained
on the h^'drolytic cleavage of gelatine a crystalline acid having the for-
mula C12H25N5O10, which he calls glviinic acid. Gelatine yields consider-
able basic nitrogen, according to Hausmaxx ^ 3o.S3 per cent of the total nitro-
gen. DRECHSELand Fischer found lysine; Hedix, Kossel and Kutscher *
found also arginine, which amounted to 9.3 per cent (Kossel and Kutscher).
On putrefaction gelatine gives neither tyrosine, indol, nor skatol. According
to Seltrexxy ^ it yields phenylpropionic acid and phenylacetic acid.
The aromatic group in gelatine is therefore, as directly shown by Fischer
(see above) and also by Spiro,^ represented by phenylalanine.
On the oxidation of gelatine ^\'ith potassium permanganate, Seemaxx
obtained, besides volatile fatty acids (formic, acetic, butyric acids), benzoic
acid, oxalic acid, .succinic acid, oxaluramide and probably also oxaluric
acid. Zickcraf '' produced guanidine from the arginine.
Collagen is insoluble in water, salt solutions, and dilute acids and alka-
lies, but it swells up in dilute acids. By continued boiling with water it is
converted into gelatine. It is dissolved by the gastric juice and also by the
pancreatic juice (trypsin solution) when it has previously been treated with
acid or heated with water above 70° C.^ By the action of ferrous .sul-
phate, corrosive sublimate, or tannic acid, collagen shrinks greatly. Col-
lagen treated by these bodies does not putrefy, and tannic acid is there-
fore of great importance in the preparation of leather.
Gelatine or glutin is colorless, amorphous, and transparent in thin layers.
It swells in cold water without dissolving. It dissolves iii warm water,
forming a sticky liquid, which solidifies on cooling when sufficiently con-
centrated. As Pauli and Roxa ^ have .shown, various bodies may have
a different influence upon the gelatinization-point of a gelatine solution;
' Fischer, Levene and Aders, Zeitschr. f. physiol Chem., 35. In regard to the
older researches, see O. Cohnheim, Chemie der Eiweisskorper, 2. Aufl., 1904.
^ Monatshefte f. Chem., 26.
' Zeitschr. f. physiol. Chem., 2".
* Drechsel, Arch. f. Anat u. Physiol., 1891; Hedin, Zeitschr. f. physiol. Chem., 21;
Kossel and Kutscher, ibid., 31,
5 Monatshefte f. Chem., 10.
* Hofmeister's Beitrage, 1.
' Seemann, Zeitschr. f. physiol. Chem., 44; Zickgraf, ibid., 41.
* Kiihne and Ewald, Verh. d. Naturhist Med. Vereins in Heidelberg, 1877, 1.
* Hofmeister's Beitraeo. 2.
GELATINE. 79
thus certain substances such as sulphates, citrates, acetates, and glycerine
may accelerate, while the chlorides, chlorates, bromides, alcohol, and urea
retard this power.
Gelatine solutions are not precipitated on boiling, nor by mineral acids,
acetic acid, alum, basic lead acetate, nor metallic salts in general. A gela-
tine solution acidified with acetic acid ma}' be precipitated by potassium
farrocj-anide on carefully adding the reagent. Gelatine solutions are precipi-
tated by tannic acid in the presence of salt; by acetic acid and common
salt in substance; mercuric chloride in the presence of HCl and NaCl;
metaphosphoric acid and phosphomolybdic acid in the presence of acid; and
lastly also by alcohol, especially when neutral salts are present. Gelatine
solutions do not diffuse. Gelatine gives the biuret reaction, but not Adam-
KiEwicz's. It gives ^IiLLOx's reaction and the xanthoproteic reaction
so faintly that they probably occur from impurities consisting of pro-
teids. According to C. ^Iorxer, pure gelatine gives a beautiful ^Iillox'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 ^^•ith water gelatine is converted into a non-gelat-
inizing modification called ,5-glutin by Nasse. According to Xasse and
Kruger the specific rotator}^ power is hereby reduced from — 167.5° to
about — 136°.^ On prolonged boiling with water, especially in the presence
of dilute acids, also in the gastric or trj-ptic digestion, the gelatine is trans-
formed into gelatine proteoses, so-called gelatoses and gelatine peptones,
which diffuse more or less readily.
According to Hofmeister two new substances, semighitin 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^ have obtained in the peptic and tryptic digestion a proto- and
a deiiterogelatose, besides a true peptone. The elementary- composition of
these gelatoses does not essentially differ from that of the gelatine.
According to Levene the proto- as well as the deuterogelatoses yield
a larger amount of glycocoll, as much as 20.3 per cent, than the gelatine
itself. On prolonged tryptic digestion a further demolition takes place, so
that the peptone 3-ields only about the same amount of glycocoll as the
gelatine. 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 and Stookey).^ Paal ^has prepared gelatine-peptone
' Xasse and Kriiger . Maly's Jahresber., 19, p. 29. In regard to the rotation of ;9-glutin,
see Framni, Pfliiger's Arch., 6S.
-Hofmeister, 1. c; Chittenden and SoUey, 1. c.
^ Levene, Zeitsehr. f. physioL Chcm., 37; Levene and Stookey, ibid., 41.
'' Ber. d. devitsch. chem. Ge.sellsch., 2o.
80 THE PROTEIN SUBSTANCES.
"hydrochlorides from gelatine by the action of dilute hydrochloric acid.
These salts a"8 partly soluble in ethyl and methyl alcohol, and partly insolu-
ble therein. The peptones obtained from these salts contain less carbon and
more hydrogen than the gelatine from which they originated, showing that
hydration has taken place. The molecular weight of the gelatine peptone as
determined by Paal, by Raoult's cryoscopic method, was 200 to 352, while
that for gelatine was 878 to 960. The gelatine peptones isolated by Sieg-
fried and his pupils Scheermesser ^ and Kruger, and which have already
been mentioned, are of the greatest interest.
Collagen (contaminated with mucoid) may be obtained from bones by
extracting them with hydrochloric acid (which dissolves the earthy phos-
phates) 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
\/ater. Gelatine is obtained by boiling collagen with water. The finest
<;ommercial gelatine always contains a little proteid, which may be removed
by allowing the finely divided gelatine 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 gelatine
for many days with 1-5 p. m. caustic potash, as suggested by C. Morner.
The typical properties of gelatine are not changed by this.
Chondrin or cartilage gelatine is only a mixture of gelatine with the specific
•constituents of the cartilage and their transformation products.
Reticulin. The reticular tissues of the lymphatic glands contain a
variety of fibres which have also been found by Mall in the spleen, intestinal
mucosa, liver, kidne3's, and lungs. These fibres consist of a special sub-
stance, reticulin, investigated by Siegfried.^
Reticulin has the following composition: C 52.88; H 6.97; N 15.63;
S 1.88; P 0.34; ash 2.27 per cent. The phosphorus occurs in organic com-
bination. It yields no tyrosine on cleavage with hydrochloric acid. It yields,
■on the contrary, sulphuretted hydrogen, ammonia, lysine, arginine, and
aminovalerianic acid. 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-hydro-
chloric acid or trypsin does not dissolve it. Reticulin responds to the biuret,
xanthoproteic, and Adamkiewicz's reactions, but not to ^Iillon's reagent.
* Zeitschr. f. physiol. Chem., 37 and •41; Kruger, 1. c. See foot-note 3, p. 57.
^ Mall, Abhandl. d. math.-phys. Klasse d. Kgl. sachs. Gesellsrh. d. Wiss., 1891;
Siegfried, Ueber die chem. Eigensch. der retikulirten Gewebe, Habil.-Schrift, Leipzig,
1892.
SKELETINS. 81
According to Tebb reticulin is only a somewhat changed, impure collagen,
but this is disputed by Siegfried.'
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. Morner,^ which occurs
■with collagen in fish-scales and forms about i 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 Millox's
reagent, the xanthoproteic reaction, and the biuret test. At least a part of
the sulphur is split off by the action of alkali.
As skeletins, Krukenberg^ has designated a number of nitrogenized
substances which form the skeletal tissue of various classes of invertebrates.
These substances are chitin, spongin, conchiolin, cornein, and fibroin (silk).
Of these chitin does not belong to the protein substances, and fibroin
(silk) is hardly to be classed as a skeletin. Only those so-called skeletins
will he discussed that actually belong to the protein group.
Spongin forms the chief mass of the ordinary sponge. It gives no gelatine. On
boiling with acids, according to the older statements, it yields leucine and glyco-
coll, but not tyrosine. Zalocostas claims to have found tyrosine and also amino-
isovalerianic acid and glucalanine (C5Hi2N204). After Hundeshagen had shown
the occurrence of iodine and bromine in organic combination in different sponges
and designated the albumoid containing iodine, iodospongin, Harnack^ later iso-
lated from the ordinary sponge, by cleavage with mineral acids, an iodospongin
which contained about 9 per cent iodine and 4.5 per cent sulphur. On the hydrol-
ysis of spongin Abderhalden and Strauss ^ obtained abundance of glutamic acid,
18.1, and glycocoll, 13.9 per cent, also leucine, 7.5, a-proline,6.3,and aspartic acid,
4.1 per cent. Very remarkable was the fact that neither tyrosine nor phenylalanine
could be detected. Strauss * has obtained sponginoses of various kinds from
spongin by dilute acids. The heterosponginose contained the greater part of
the iodine and sulphur, while the deuterosponginose contained the carbohydrate
groups. Iodospongin is considered as a derivative of the heterosponginose.
Conchiolin is found in the shells of mussels and snails and also in the egg-shells 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
»Tebb, Journ. of Physiol., 27; Siegfried, ibid., 28.
' Zeitschr. f. physiol. Chem., 24 and 37. See also Green and Tower, ibid., 35.
' Grundziige einer vergl. Physiol, d. thier. Geriistsubst., Heidelberg, 1885.
* Zalocostas, Compt. rend., 107; Hundeshagen, Maly's Jahresber., 25; Harnack,
Zeitschr. f. physiol. Chem., 24.
' Zeitschr. f. physiol. Chem., 48.
' Biochem. Centralbl., 3.
'Zeitschr, f. physiol. Chem., 29, and Centralbl. f. Physiol,, 13, 113.
82
THE PROTEIN SUBSTANCES.
substance, closely relat3d to conchiolin, which is soluble with difficulty. Comein
forms the axial system of the Antipathes and Gorgonia. It gives leucine and a
crystallizable substance, cornicrystalUne. According to Drechsel the axial sys-
tem of the Gorgonia cavolini contains nearly 8 per cent of the dry substance as
iodine. The iodine occurs in organic combination with an iodized albumoid, gor-
gonin, which is a comein. Drechsel obtained leucine, tyrosine, lysine, ammonia,
and an iodized amino-acid, iodogorgonic acid, as cleavage products of gorgonin.
According to Wheeler and Jamieson ' iodogorgonic acid is diiodotyrosine, prob-
ably 3,5-diiodotyrosine, C6H2(CH,CH(NH2)COOH)(OH)I., and was prepared by
them by the action of iodine upon tyrosine and alkali. Henze ' could obtain this
acid only in very small quantities, and by acid cleavage of gorgonin he obtained
the three hexone bases, abundance of tyrosine, and very little 1 sucine. On cleavage
with barium hydrate he obtained only lysine besides tyro.iine and glycocoU in
larger amounts.
Fibroin and sericin are the two chief constituents of raw silk. By the action
of boiling water the sericin (silk gelatine) dissolves and can be obtained by a
method suggested by Bondi,^ while the more difficultly soluble fibroin remains
undissolved in the shape of the original fibre. 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. As cleavage
products E. Fischer and Skita obtained alanine, serine, very little glycocoll,
tyrosine, arginine, and probably also lysine. Leucine had been found previously.
From fibroin they obtained, besides the previously known cleavage products,
glycocoll, tyrosine, and alanine (Weyl *), also leucine, phenylalanine, serine,
a-proline (Fischer), and a small amount of arginine. The chief products were
glycocoll, 36 per cent, alanine, 21 per cent, and tyrosine, 10 per cent. The com-
position of the above-mentioned albuminoids is as follows : ^
Gjnchiolin (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
6.54
16.60
0
85
(Wetzel)
6.88
17.86
0
31
(Krukenberg)
6.30
16.20
0
50
(Croockewitt)
6.35
16.40
(Posselt)
5.90
16.81
(Krukenberg)
6.27
18.31
(Cramer)
6.50
19.20
(Vignon)
6.18
18.30
(Cramer)
6.32
17.14
(Bondi)
' Amer. Chem. Journ., 33.
^ Drechsel, Zeitschr. f. Biologie, 33; Henze, Zeitschr. f. physiol. Chem., 38.
^ Zeitschr. f. physiol. Chem., 34.
* Fischer and Skita, ibid., 33; Fischer, ibid., 39; Weyl, Ber. d. d. chem. Gesellsch., 21.
'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., llo; Wetzel, 1. c, and Bondi, 1. c.
GLYCOCOLL. 83
Appendix to Chapter II.
HYDROLYTIC CLEAVAGE PRODUCTS OF THE PROTEIN SUBSTANCES.
I. Monamino-acids.
GlycocoU (aminoacetic acid), C'2H5N02= ?^L ^ , also called glvcine
or gelatine sugar, is found in the muscles of the invertebrates, but has
chief interest as a hydrolytic decomposition product of protein bodies,
especially gelatine, fibroin, and spongin, as well as of hippuric acid a<nd
glycocholic acid. It is also formed in the decomposition of uric acid,
xanthine, guanine, and adenine.
GlycocoU has been most abundantly obtained thus far from the protein
substances fibroin ^ (36 per cent), elastin^ (25.75 per cent), gelatine and
gelatoses^ (16.5 and 20.3 per cent respectively).
GlycocoU 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). It is insoluble in alcohol and ether and dissolves
with difficulty in warm alcohol. GlycocoU combines with acids and alkalies.
With the latter compounds we must mention those with copper and
silver. GlycocoU dissolves cupric hydrate in alkaline liquids but does
not reduce at boiling heat. A boiling-hot solution of glycocoll dissolves
freshly precipitated cupric hydrate, forming a blue solution, which, in
proper concentration, deposits blue needles of copper glycocoll on cooling.
The compound with hydrochloric acid is readily soluble in water but less
soluble in alcohol.
SoRENSEN^ finds that phosphotungstic acid does not precipitate glyco-
coll from dilute solutions but only from concentrated ones. By the action of
gaseous HCl 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 ^ 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 (Ch. Fischer, Gonnermann, Spiro ^). The melting-
' Fischer and Skita, Zeitschr. f. physiol. Chem., 33.
^ Abderhalden and Schittenhelm, ibid., 41.
^ Fischer, Levene and Aders, ibid., 35; Levene, ibid., 37 and 41.
* Meddelelser, fraa Carlsberg-laboratoriet, 6, 190.5.
^ Ber. d. d. chem. Gesellsch., 34.
' Ch. Fischer, Zeitschr. f. physiol. Chem., 19; Spiro, ibid., 2S; Gonnermann,
Pfliiger's Arch., 59.
84 THE PROTEIN SUBSTANCES.
point of glycocoll-/3-naphthalenesulphonate is 156° (corr. 159°), of glycocoU
4-nitrotoluene-2-sulphonate 177.5° (corr. 178°), of the phenylisocyanate
compound 195°, and of the a-naphthylisocyanate compound 190.5-191.5°.
GlycocoU can be best prepared from hippuric acid by boiUng it with
4 parts dihite 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 precijHtated by
BaCOa, and the filtrate evaporated to the point of crystallization. (In regard
to its preparation from protein substances see below.)
CH3
Alanine (a-aminopropionic acid), C3H7N02 = CH(NH2), was first obtained
COOH
by Weyl as a cleavage product of fibroin. This d-alanine has been
isolated by E. Fischer and his collaborators^ still more abundantly from
fibroin (21 per cent) and also from sericin (5 per cent), horn substance
(1.20 per cent), gelatine (0.8 per cent), haemoglobin (2.87 per cent), and
elastin ^ (6.58 per cent).
Alanine has a sweet taste, is readily soluble in water, and dissolves cupric
hydrate on boiling, producing copper alanine, which has a deep blue color.
The specific rotation of the hydrochloride (9-10 per cent solution) is (a)D =
+ 10.3°. In regard to the synthetical preparation of {-alanine, its separa-
tion as the benzoyl compound, and the preparation of r-alanine ethyl
ester we must refer to E. Fischer.^
The d-alanine-/?-naphthalenesulphonate melts at 78-80° (79-81° corr.),
the racemic alanine-4-nitrotoluene-2-sulphonate at 96° (uncorr.), the
phenylisocyanate compound at 168°, and the a-naphthylisocyanate com-
pound at 198° C.
CH3CH3
Aminovalerianic acid, C5HiiN02= • has been detected several times
CH(NH2),
COOH
among the cleavage products of protein substances. Kossel and Dakin ^ ob-
tained 4.3 per cent from salmine. The acid isolated by E. Fischer from horn
substance (5.70 per cent) and casein, as well as that obtained by Schulze and
WiNTERSTEiN ^ from lupin sprouts, seems to be dextrorotatory a-aminovalerianic
acid. The copper salt of aminovalerianic acid is, according to Schulze and
WiNTERSTEiN," readily soluble in methyl alcohol.
^Weyl, Ber. d. d. chem. Gesellsch., 21; Fischer and Skita, Zeitschr. f. physiol.
Chem., 33; Fischer and Dorpinghaus, ibid., 36; Fischer, Levene and Aders, ibid.,
35; Fischer and Abderhalden, ibid., 36.
^ Abderhalden and Schittenhelm, Zeitschr. f. physiol. Chem., 41.
3 Ber. d. d. chem. Gesellsch., 32 and 34.
^Zeitschr, f. physiol. Chem., 41.
' Fischer, iind., 36 and 33; Schulze and Winterstein, ibid., 35.
""Ibid.A^.
LEUCINE. 85
Leucine (aminocaproic acid, or, more correctly, a-aminoisobutylacetic
CH3CH3
CH
acid),C6Hi3N02= 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 hydrates, and by putrefaction.
Because of the ease with which leucine (and tyrosine) are formed in the
decomposition of protein substances, it is difficult to decide positively
whether these bodies when found in the tissues are constituents of the living
body or are to be considered only as decomposition products formed after
death. Leucine, it seems, has been found as a normal constituent of the
pancreas and its secretion, in the spleen, thymus, and lymph glands, in the
thyroid gland, in the salivary glands, in the kidneys and in the liver. It also
occurs in the wool of sheep, in dirt from the skin (inactive epidermis), and
between the toes, and its decomposition products have the disagreeable odor
of the perspiration of the feet. It is found pathologically in atheromatous
cysts, ichthyosis scales, pus, blood, liver, and urine (in diseases of the
liver and in phosphorus poisoning). Leucine occurs often in invertebrates
and also in the plant kingdom. On hydrolytic cleavage various protein
substances yield different amounts of leucine. Erlenmeyer and Schoffer
obtained 36-45 per cent of leucine from the cervical ligament, Abderiialden
and ScHiTTENHELM 2L3S per cent from elastin, Cohn 32 per cent from
casein, and Nencki L5-2 per cent from gelatine. E. Fischer and Abder-
HALDEN obtained 20 per cent of leucine from haemoglobin, Fischer and
DoRPiNGHAUS 18.3 per cent from horn substance, Nencki 1..5-2 per cent
from gelatine, and Fischer and Skita 1.5 per cent from fibroin.^
Leucine occurs, like other monamino-acids, in the /-, d-, and ^-modifica-
tions. The leucine obtained by cleavage of protein substances is generally
levorotatory in watery solution and dextrorotatory Z-Ieucine in acid solution.
The leucine prepared synthetically by HtJFNER^ from isovaleraldeh3'de,
ammonia, and hydrocyanic acid is optically inactive. Inactive leucine may
also be prepared, as shown by E. Schulze and Bosshard,^ by the cleavage
of proteins with baryta at 160-180° C, or by heating ordinary leucine with
baryta-water to the same temperature. The levorotatory modification
'Erlenmeyer and Schoffer, cited from Maly, Chem. d, Verdauungssafte, in Her-
mann's Handb. d. Physiol., 5, Theil 2, p. 209; Abderhalden and Schittenhehn,
Zeitschr. f, physiol. Chem., 41; Cohn, ibid., 22; Nencki, Journ. f. prakt. Chem. (N.
F.), 15; Fischer and his collaborators, see p. 84, foot-note 1.
^ Journ. f. prakt. Chem. (N. F.), 1.
^ See Zeitschr. f . physiol. Chem., 9 and 10.
86 THE PROTEIN SUBSTANCES.
may be formed from the inactive leucine by the action oi Penicillum glauciim.
On benzoylating i-leucine we obtain i-benzoyl leu cine, from whose cinchonine
and quinidine salts first d- and then Z-benzoy lieu cine are prepared, and
then by hydrolytic cleavage d- and Z-leucine may be obtained (E. Fischer).
On oxidation the leucines yield the corresponding oxyacids (leucinic acids).
Leucine is decomposed on heating, evohang carbon dioxide, ammonia, and
amylamine. On heating with alkalies, as also in putrefaction, it 3'ields
valerianic 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 alter-
nating light and dark concentric layers which consist of radial groups of
crystals. By slow heating, leucine melts and sublimes in 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 verv^ 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 t-leucine is much less solu-
ble. According to Habermann and Ehrenfeld ^ 100 parts of boiling
glacial acetic acid dissolve 29.23 parts of leucine. The specific rotation of
the ordinary leucine, dissolved in hydrochloric acid, is about (a)D= +17.5°.
The solution of leucine in water is not, as a rule, precipitated by metallic
salts. The boiling-hot solution may, however, be precipitated by a boiling-
hot solution of copper acetate, and this fact is made use of in separating
leucine from other substances. If the solution of leucine 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
hydrate, but does not reduce on boiling.
Leucine is readily soluble in alkalies and acids. It gives crystalline com-
pounds with mineral acids. If leucine hydrochloride is boiled with alcohol
containing 3-4 per cent HCl, long narrow crystalline prisms of leucine ethyl
ester hydrochloride, melting at 134° C, are formed (Rohmann). The
same is produced by the action of gaseous HCl upon leucine in alcohol,
and the free ethyl ester can be obtained from this by the method suggested
by E. F1SCHER.2 This ester can be separated from the other amino-acid
esters by distillation. The pure leucine can be prepared from the ester by
boiling with water for a long time. The picrate of the leucine ester melts at
128° C. The phenylisocyanate compound of i-leucine melts at 165° C.
and its anhydride at 125° C. The a-naphthylisocyanate compound melts at
163.5° and Z-leucine /3-naphthalenesulphonate melts at 67° (corr. 68°).
' Zeitschr. f. physiol. Cheni., 37.
^Rohmann, Ber. d. d. chem. Gesellsch., 30; E. Fischer, ibid., 34.
ISOLEUCINE AND ASPARTIC ACID. 87
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 by heatin;;;
with baryta-water, the a-naphthylisocyanate compound and the leucine
^-naphthalenesulphonate. Leucine must first be isolated before it can be
detected, and this is best done by preparing the ethyl ester and then dis-
tilling it.
Leucinimide, CoHzzNaOj = ^ "Viz-i attlt V^u n tt > was first obtained by Ritt-
LU . J\ rl .Lrl . U4ri9
HAtJSEN' in the hydrolytic cleavage products on boiling proteins with acids, and
subsequently by R. Corn. Salaskin ' obtained it in the peptic and tryptic
digestion of haemoglobin. As an anhydride of leucine (2.5-diacipiperaznie) 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 ^ from leucine ethyl
ester melted at 271° C.
Isoleucine, an isomer of leucine, has recently been discovered by F. Ehr-
LiCH, but its constitution is still unknown. Ehrlich first isolated it from
the mother-liquor after removing the sugar from molasses, and found it also
on the hydrolysis of several proteins, and considers it as regularly asso-
ciated with ordinary leucine. Winterstein and Pantanelli obtained
it on the hydrolysis of the protein of lupin seeds, and it has also been found
by Schulze and Winterstein ^ in sprouts.
Isoleucine is more soluble in water than Z-leucine (1 : 25.8). It is dextro-
rotatory in aqueous as well as in acid solutions and in the presence of hydro-
chloric acid it acts more than twice as strongly as ordinary leucine. In
aqueous solution the specific rotation is (a:)D= +9.74°, in hydrochloric-acid
solution = +36.8°. Isoleucine melts at 280°, and the benzoyl compound
has a melting-point of 116-117°. Its copper salt is rather soluble in water
and, like the copper salt of aminovalerianic acid, is readily soluble in methyl
alcohol.
COOH
Aspartic Acid (aminosuccinic acid), C4H7N04=^^^ , has been
COOH
obtained on the cleavage of protein substances by proteolytic enzymes
as well as by boiling them with dilute mineral acids. Hlasiwetz and
Habermann obtained 23.8 per cent from ovalbumin and 9.3 per cent
from casein, although the product was not quite pure. E. Fischer and
' Ritthausen, Die Eiweisskorper der Getreidearten, etc., Bonn, 1872; R. Cohn,
Zeitschr. f. physiol. Chem., 22 and 29; Salaskin, ibid., 32.
^ Ber, d d. chem. Gesellsch., 34.
^ Felix Ehrlich il>id,, 37; Winterstein and Pantanelli, Zeitschr. f. physiol. Chem.,
45; Schulze and Winterstein ibid., 45
88 THE PROTEIN SUBSTANCES.
his co-workers ^ obtained 3.29 per cent aspartic acid from haemoglobin,
2.50 per cent from horn substance, and 0.56 per cent from gelatine. This
acid also occurs in secretions of sea-snails (Hexze ~) and is verj- widely
diffused in the vegetable kingdom as the amide Asparagixe (aminosuccinic-
acid amide), which seems to be of the greatest importance in the develop-
ment and formation of the proteins in the plants.
Aspartic acid dissolves in 256 parts water at 10° C. and in IS. 6 parts
boiling water, and crystallizes on cooling as rhombic prisms. The acid'
prepared from protein substances is optically active, and its 4 per cent solu-
tion acidified with HCl has the rotation (q:)d= +25.7°; but it is either
dextrogyrate or levogyrate in a watery solution, depending upon the tem-
perature. It forms with copper oxide a crA'stalline 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.
In regard to the benzoylaspartic acids and the diethylester we must
refer to the work of E. Fischer and his collaborators. For identification
we make use of the analysis of the free acid and the copper salts, as well
as the specific rotation.
COOH
CH(XH2)
Glutamic acid fa-aminoglutaric acid), C5H9N04 = CH2 , is obtained
CH2
COOH
from the protein sul)stances under the same conditions as the other mon-
amino-acids and from the peptones (Siegfried). Hlasiwetz and Haber-
MANX obtained 29 i:)er cent from casein by cleavage with hydrochloric acid,
while KuTSCHER could oljtain only 1.8 per cent glutamic acid by cleavage
with sulphuric acid. Horbaczewski has obtained 15-18 per cent glu-
tamic acid from gelatine and about the same amount from horn, while
Fischer and Dorpixghaus obtained only 3 per cent from horn. Fischer
and Abderhaldex obtained 1.06 per cent from haemoglobin, Kutscher
3.66 per cent from thymus hist one, and Abderhaldex and Pregl ^ ob-
tained 8 per cent from ovalbumin.
Glutamic acid crystallizes in rhomljic tetrahedra or octahedra or in
small leaves. It melts at 202-203° C. with partial decomposition. It
dissolves in 100 parts water at 16° C. and in 1500 parts 80 per cent alcohol.
' Hlasiwetz and Habermann, Annal. d. Chem. u. Pharm., 159 and 169; E. Fischer
and collaloorators, see foot-note 1, p. 84.
^ Ber. d. d. chem. Gesellsch., 34.
^ Hlashvetz and Habermann, 1. c, 159; Kutscher, Zeitschr. f. physiol. Chem., 28
and 38; Horbaczewski, Maly's Jahre.'-ber., 10; Fischer and collaborators, L c;
Abderhalden and Prcgl, Zeitschr. f. physiol. Chem., 46.
TYROSINE. 89
It is insoluble in alcohol and ether. The rf-glutamic acid obtained from
proteins by boiling with an acid or from the mother-liquor from molasses
is dextrorotatory, and in water has a rotation of (a)D= +12.04° according
to AxDRLiK.i Strong acids increase the rotation, and a 5 per cent solution
of glutamic acid containing 9 per cent HCl has a rotation (a)D= +31.7°,
while that obtained by heating with barium hydrate is optically inactive.
The (/-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 hydrate a beautiful crystalline copper salt, which is soluble with
difficulty, is obtained. Like the monamino-acids in general, glutamic acid
is not precipitated by phosphotungstic acid. In regard to the benzoylglu-
tamic acids and the diethylester we must refer to the works of Fischer.^
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.
C6H4(OH)
... -^ CH.,
Tyrosine (7>oxypheny.-Q:-ammopropionic acid). C9H1 1X03 = ptt-.^tt n, is
COOH
produced from most protein substances (not from gelatine and reticulin)
under the same conditions as leucine, which it habitually accompanies. The
largest quantity of tyrosine obtained from animal proteins was obtained
by Fischer and Skita from fibroin, namely, 10 per cent. The maxi-
mum obtained from thymus histone (Kutscher) was 6.3 per cent, from
horn substance (R. Cohx) 4.6 per cent, from casein (Reach) 4.55 per cent,
from fibrin (Kuhxe) 3.86 per cent, from ovalbumin, seralbumin, and ser-
globulin (K. ^Iorxer) 2.4. 2.0, and 3.0 per cent respectively, from syntonin
(Reach) 1.37 per cent, from haemoglobin (Fischer and Abderhaldex)
1.5 per cent, and from elastin (Schwarz^) 0.34 per cent. It is especially
found with leucine in large quantities in old cheese (Tvpoi). form which
it derives its name. Tyrosine has not with certainty been fomid in per-
fectly fresh organs. It occurs in the intestine in the digestion of protein
substances, and it has about the same physiological and pathological im-
portance as leucine.
Tyrosine was prepared by Erlexmeyer and Lipp from p-aminophenyl-
alanine bv the action of nitrous acid, and according to another method bv
' See Biochem. Centralbl., 3, p. 469.
M. c.
^ Fischer and Skita, 1. c; Kutscher, Zeitschr. f. physiol. Chem., 3S; R. Cohn, ibid.,
26; Reach, Virchow's Arch., loN; Kiihne, ibid., 39; K. Morner, Zeitschr. f. physiol.
Chem., 34; Fischer and Alxlerhalden, ibid., 1. c.; Schwarz, ibid., 18.
90 THE PROTEIN SUBSTANCES.
Erlenmeyer and Halsey.^ On fusing with caustic alkali it fields p-oxy-
benzoic acid, acetic acid, and ammonia. On putrefaction it may yield
jD-hydrocoumaric acid, oxyphenylacetic acid, and p-cresol.
Naturally occurring tyrosine and that obtained by the cleavage of pro-
tein substances is generally Z-tyrosine, Avhile that obtained by decomposition
"vvith baryta-water or prepared synthetically is inactive, v. Lippmann^
has obtained d-tyrosine from beet-sprouts. The statements as to specific
rotation of tyrosine are somewhat variable. For tyrosine from jjroteins E.
Fischer has found a rotation of (a)D = — 12.56 to 13.2° for the hydrochloric-
acid solution, while Schulze and Winterstein^ 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 may be 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 am-
monia. One hundred parts glacial acetic acid dissolve on boiling only 0.18
parts 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 Ehrenfeld). The /-tyrosine ethyl ester crys-
tallizes in colorless prisms which melt at 108-109° C. The naphthyliso-
cyanate-Z-tyrosine melts at 205-206°. Tyrosine can be oxidized with the
formation of dark-colored products by various plant as well as animal
oxidases, so-called tyrosinases (see Chapter I). By the enzyme occurring
in beet-juice tyrosine can be converted into homogentisic acid (Gonner-
MANN ■*). 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 BaCOs, and filtered. On the addi-
tion of a solution of ferric chloride the filtrate gives a beautiful violet color;
' Erlenmeyer and Li])p, Ber. d. d. chem. Gesellsch., 15; Erlenmeyer and Halsey,
ibid., 30.
' Ibid., 17.
^ See Hoppe-Seyler-Thierfelder, Handb. d. physiol. u. pathol. chem. Analyse, 7.
Auflage, 190.3. Also E. Fischer, Ber. d. d. chem. Gesellsch., 32; Schulze and Winter-
stein, Zeitschr. f. physiol. Chem., 45.
^Pfliiger's Arch., 82.
PHENYLALANINE. 91
This reaction is disturbed by the presence of free mineral acids and by the
addition of too much ferric chloride.
Hofmann's Test. If some water is poured on a small quantity of
tyrosine in a test-tube and a few drops of JMillon'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,i is performed as follows: To
a few cubic centimetres of a solution consisting of 1 vol. formaline, 45 vols,
water, and 55 vols, concentrated sulphuric acid add a little tyrosine in sub-
stance or in solution and heat to boiling. A beautiful permanent green
coloration is obtained.
CH2.C6H5
Phenylalanine (phenyl-a-aminopropionic acid), C9HnN02= CH(NH2),
COOH
was first found by E. Schulze and Barbieri 2 in etiolated lupin sprouts.
It is produced in the acid cleavage of protein substances. E. Fischer
and his collaborators ^ obtained 3.38 per cent phenylalanine from haemo-
globin, 3.0 per cent from horn substance, 2.5 per cent from ovalbumin and
casein, 1.5 per cent from fibroin, and 0.4 per cent from gelatine. Abder-
HALDEN and ScHiTTENHELM obtained 3.89 per cent from elastin.
The /-phenylalanine crystallizes in small, shining leaves or fine needles
which are rather difficultly soluble in cold water but readily soluble in
hot water. A 5 per cent solution acidified with hydrochloric acid or sul-
phuric acid is precipitated by phosphotungstic acid, while a more dilute
solution is not precipitated. On putrefaction, phenylalanine yields phenyl-
acetie acid. On heating with potassium dichromate and sulphuric acid
(25 per cent) an odor of phenylacetaldehyde is produced and benzoic acid
is formed.
The separation and preparation of the three amino-acids, aspartic
acid, glutamic acid, and tyrosine, from a mixture of hydrolytic decomposi-
tion products of protein substances is performed essentially according
to the method suggested by Hlasiwetz and Habermann with the modi-
fications and changes proposed by other investigators. The isolation and
purification of the amino-acids can be best accomplished according to E.
Fischer's method, which consists essentially in esterifying the acids first
with hydrochloric acid and alcohol, separating the esters from their hydro-
chloride by means of alkali, and then fractionally distilling the esters mider
very low pressure, and finally saponifying the different fractions by
boiling with water or by heating with baryta-water. It is not ^^•ithin the
scope of this book to give a detailed description of these methods, there-
^ Deniges, Compt. rend., 130; C. Morner, Zeitschr, f. physiol. Chem., 37.
^ Ber. d. d. chem. Gesellsch., 14, and Zeitschr. f. physiol. Chem., 12.
^ See foot-note 1, p. 84.
92 THE PROTEIN SUBSTANCES.
fore we must refer for further information to Hoppe-Seyler-Thierfel-
der's " Handbuch der physiologisch- unci pathologisch-chemischen Analyse,'^
7. Auflage. and to Fischer's ^ collected works on this subject.
We must here add that the preparation of the /?-naphthalenesulpho-
derivatives according to Fischer and Bergell, of the 4-nitrotoluenc-2-
sulpho-compounds according to Siegfried, and of the a-naphthyliso-
cyanate compounds according to Neuberg and Manasse^ are also of
importance in the detection and isolation of many of the amino-acids.
Cystine, C6H12N2S2O4 (the disulphide of a'-amino-,5-thiolactic acid),
CH2— S— S— CHo
CH(NH2) CH(NH2), was first obtained with positiveness as a cleavage
COOH COOH
product of protein substances by K. Morxer, and then also by Embden.
KtJLz 3 obtained it also once as a product of tryptic digestion of fibrin.
Morner obtained 6.8 per cent cystine from ox-horn, 13.92 per cent from
human hair, 7.62 per cent from the membrane of the hen egg, 2.53 per
cent from seralbumin, 1.51 per cent from serglobulin, 1.17 per cent from
fibrinogen, and 0.29 per cent from ovalbumin.
According to Neuberg and Mayer ^ two kinds of cystine occur in nature,
namelv, stone-cystine and protein-cystine. Stone-cystine is the disulphide of
CH2NH2 CH2NH2
/S-amino-a-thiolactic acid, CH — S — S — CH
COOH COOH
It is difficult to say which cystine we have had in the various cases
where it has been found. The protein-cystine has been chiefly obtained
from the protein substance, but also from calculi, while the stone-cystine has
only been obtained from urinary calculi. Rothera could not find any
difference between the stone-cystine and the cystine prepared from hair, and
Fischer and Suzuki ° arrived at similar results, which seems to place the
existence of stone-cystine in doubt. The occurrence of two stereoisomeric
cystines is not improbable, and important observations of ^Iorxer show that
the cystine-yielding group of the protein substances contains two cystines.
Cystine occurs in rare cases in the urine or as a calculus, and has also
been found in ox-kidneys, in the liver of the horse and dolphin, and in
traces in the liver of a drunkard. Abderhaldex^ has found cvstine in
' Ber. d. d. chem. Gesellsch., 39, p. 530. His collected Avorks on this subject
may be found in Fischer's " Untersuchungen iiber Amino. Lauren, PoI\'peptide und Pro-
teine 1899-1900," Berlin, 1906.
^ Fischer and Bergell, Ber. d. d. chem. Gesellsch., 35; Neuberg and Manasse, ibid.,
38; Siegfried, Zeitschr. f. physiol. Chem., 43.
K. Morner, ilrul., 28, 34, and 42; Embden, ibid., 32; Kiilz, Zeitschr. f. Biologic, 27.
* Zeitschr. f. physiol. Chem., 44.
* Rothera, Journ. of Physiol., 32; Fischer and Suzuki, Zeitschr. f. physiol. Chem., 45.
* Zeitschr. f. physiol. Chem., 38.
CYSTINE. 93
the urine and also alnindantly in the organs (spleen) in a case of parental
cystine diathesis.
The constitution of cystine has Ijeen explained by Friedmaxx.^ and he
has also established the relationship between cystine and taurine. Cystine
is the disulphide of cysteine, which is a-amino-.9-thiolactic acid. From
cysteine Friedmaxx obtained cysteinic acid (aminosulphopropionic acid),
CHoSOoOH
C3H7XS05 = CH(XH2). from which taurine is produced by splitting off CO2.
COOH
Cystine has also been prepared s}-nthetically. Starting from ethyl
formyl hippurate. Erlexmeyer. Jr.. and Stoop first prepared the benzoyl-
serine ester, and then with phosphorus pentasulphide they obtained the
benzoylcystine ester. On splitting the latter with HCl they obtained
cysteine, and then inactiye cy.stine on oxidation. Gabriel 2 has also pre-
pared an isocysteine by the cleayage of rhodandihydrouracil with hydro-
chloric acid, and then inactiye cystine by the oxidation of this isocysteine.
Cystine crystallizes in thin, colorless, hexagonal plates. It is not soluble
in water, alcohol, ether, or acetic acid, but dissolyes in mineral acids and
oxalic acid. It is also soluble in alkalies and ammoni^, but not in ammo-
nium carbonate. Cystine is optically actiye. being leyorotator}-. Morxer
found it to be (a)^ = —224.3°. On heating with hydrochloric acid it
can, according to Morxer. be changed into a modification crj'staUizing in
needles and with a weaker leyorotator\- power, and indeed it can be
changed into a dextrorotatory modification. On heating with HCl to
165° for 12-15 hours Xeuberg and Mayer obtained inactiye cystine. It is
questionable whether this is identical with the inactiye cystine prepared
by Erlexmeyer s^-nthetically. By fungus fermentation with Aspergillus
niger they obtained dextrorotatory- cystine. Cystine has no melting-point
but slowly decomposes at 258-261°. On boiling cystine with caustic alkali
it decomposes and yields alkali sulphide, which can be detected by lead
acetate or sodium nitroprusside. According to Morxer 75 per cent of
the total sulphur is separated. On treatment of cystine with tin and hydro-
chloric acid it deyelops only a little sulphuretted hydrogen, and is con-
yerted into cysteine. On shaking a solution of cystine in an excess of sodmm
hydrate with benzoyl chloride, a yoluminous precipitate of benzoyl cystine
is obtained (Baumaxx and Goldmaxx^). The benzoyl compoimd melts
at 182-184°. The phenylcyanate compoimd melts at 160° and on boiling
with 25 per cent HCl is transformed into its anhydride, a hydantoin melting
at 119°. Cystine forms crystalline salts with mineral acids and with bases.
For isolating and separating cystine the precipitation with mercuric acetate
' Hofmeister "s Beitrage, 3. p. 1.
- Erienmeyer and Stoop, Ber. d d. chem. Gesellsch., 36; Gabriel, ibiJ., 38.
' Morner, Zeitschr. f. physiol. Chem., 34; Baumann and Goldmann, ibid., 12.
94 THE PROTEIN SUBSTANCES.
is especially suited. 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.
Stone-cystine, according to Neuberg and ^Mayer, differs in many regards
from the ordinary cystine, among which the following will be mentioned:
The optically active stone-cystine crj^stallizes in needles, the specific rota-
tion is (q:)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 crystal-
line form, the behavior on heating on platinum-foil, and the sulphur reac-
tion after boiling with alkali. As to its preparation from protein substances
see K. MoRNER.i In regard to the detection of cystine in the urine see
Chapter XV.
CH2.SH
Cysteine (a'-amino-,/?-thiolacticacid),C3H7NS02=CH(NH2), is formed from cys-
COOH "
tine 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 ^ as a second-
ary formation, while Embden considers it as primary from proteins poor in sul-
phur. Besides a-amino-,5-thiocysteine occurring in the proteins we may prob-
ably also have a /?-amino-«-thiocysteine. According to Morner the thiolactic acid
which he obtained on the decomposition of cystine probably originates from the
latter, while the a-amino-;?-thio cysteine is probably the mother-substance of the
alanine obtained at the same time. Cysteine can be readily converted into cj'stine.
Towards 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.
CH3
Thiolactic acid (a-thiolactic acid), C3HtiS02= CH.SH, has been found once as a
COOH
cleavage product of ox-horn by Baumann and Suter. It has been shown by
Friedmann that this acid is a regular cleavage product of keratin substances, and
that it can also be obtained from the proteins. Frankel ^ 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.
CTT NTT-T
Taurine* (aminoethylsulphonic acid), C2H7NS03 = ,v„^' ,^ ^r^xj- ^^^ ^lot
Crl2.k)U2.Uxl
boen obtained as a cleavage product of protein substances; still its origin
' Zeitschr. f. physiol. Chem., 34.
2 See foot-note 2, p. 28.
'Suter, Zeitschr. f. physiol. Chem., 20; Friedmann, Hofmeister's Beitrage, 3;
Frankel, Sitzungsber. d. Wicn. Akad., 112, II, b, 1903.
* Taurine does not belong to the cleavage products of the proteins, but for practical
reasons it will be described in connection with cvstine.
TAURINE. 95
from proteins has been shown by Friedmann by the close relationship
that taurine bears to cysteine. 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 spirits of wine it dissolves slightly, but better when warm. Taurine
yields acetic and sulphurous acids, but no alkali sulphides, on boiling with
strong caustic alkali. The content of sulphur can be determined as sul-
phuric acid after fusing with saltpetre and soda. Taurine combines with
metallic oxides. The combination with mercuric oxide is white, insoluble,
and is formed when a solution of taurine is boiled with freshly precipitated
mercuric oxide (J. Lang ^). This compound may be used in detecting the
presence of taurine. Taurine is not precipitated 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 dissolved in 5 per
cent hydrochloric acid, and precipitated with 10 vols. 95 per cent alcohol.
The crystals are readily purified by recrystallization 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 de-
colorized by animal charcoal and the solution then evaporated to crystalli-
zation.
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.
Oxymonamino-acids.
CH2(0H)
Serine (a-amino-^^-oxypropionic acid),C3H7N03 = CH(NH2), was obtained
COOH
by E. Fischer and his collaborators 2 as a cleavage product from fibroin (1.6
per cent), horn substance (0.68 per cent), sericine, gelatine (0.4 percent),
and casein. Kossel and Dakin ^ obtained 7.8 per cent from salmine.
Synthetically it was prepared by E. Fischer and Leuchs * from ammonia.
' See Maly's Jahresber., 6.
^ See foot-note 1, p. 84.
' Zeitsehr. f. physiol. Chem., 41.
'' Ber. d. d. chem. Gesellsch., 35, and Sitzungsber. d. Akad. d. Wiss., Berlin, 1902-
96 THE PROTEIN SUBSTANCES.
hydrocyanic acid, and glycolyl aldehyde. Serine has also been prepared by
Erlenmeyer, Jr., and Stoop ^ by starting with ethyl formyl hippurate
and converting it by reduction into benzoylserine ester, from which
benzoylserine was obtained by saponification with alcoholic potash, and
then from this, serine was obtained by boiling with sulphuric acid.
Isoserine (/9-amino-a-oxypropionic acid) has been prepared by Ellinger from
<liaminopropionic hydrobromide and silver nitrite, and by Neuberg and Silber-
MANN ^ from diaminopropionic hydrochloride.
Serine does not dissolve readih' in cold water (23 parts water at 20° C),
but more easily in hot water. The solution is inactive and has a sweet
taste. Serine crystallizes from water in thin plates, which melt at 240°
with the generation of a gas.
According to Skraup, oxyaminosuccinic acid, CnHyNOs, is very jorobably a
hydrolytic cleavage product of the proteins. This acid has been prepared syn-
thetically by Neuberg and Silbermann from diaminosuccinic acid and barium
nitrite in sulphuric-acid solution. Oxyaminosuberic acid, CgHisNOj, has been
found by Wohlgemuth as a cleavage product of a liver nucleoproteid, and the
acid CeHisNOe, isolated by Orgler and Neuberg ^ from chondroitin-sulphuric
acid, but not from protein, and considered by them as tetraoxyaminocaiaroic acid,
seems also to belong to the oxyamino-acid group.
2. Diamino-acids (Hexone Bases).
Arginine (guanidine-a-amino valerianic acid),
C6Hi4N402= (CH2)2 ,
CH(NH2)
COOH
first discovered by Schulze and Steiger in etiolated lu])in- and pumpkin-
sprouts, has later been found in other germinating plants, in tubers and
roots. GuLEWiTSCH has found arginine in the ox-spleen. It was first
found by Hedin as a cleavage product of horn substance, gelatine, 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 proteins
(edestin and the protein from pine seeds) also yield abundant arginine.
Arginine also occurs among the products of tryptic digestion (Kossel and
Kutscher).
On boiling with baryta-water as well as by the action of an enzyme,
' Ber. d. d. chem. Gesellsch., 35.
^Ellinger, ibid., 87; Neuberg and Silbermann, ibid., 37.
'Skraup, Zeitschr. f. physiol. Chem., 42; Neuberg and Silbermann, ibid., 44;
"Wohlgemuth, ibid., 44; Orgler and Neuberg, ibid., 37.
ARGIXIXE. 97
arginase, discovered by Kossel and Dakix/ arginine yields urea and
ornithine. Arginine has been prepared s}Tithetically from ornithine (a-o-di-
aminovalerianic acid) and cyanamide by Schulze and Wintersteix.^
Arginine cr}'stallizes in rosette-like tufts, plates, or thin prisms, is readily
soluble in water and nearly insoluble in alcohol. With several acids and
metallic salts it forms cr\-stalline salts and double salts respectively. Its
acidified waters' solution is precipitated by phosphotungstic acid. The most
important salts are the copper-nitrate (C6Hi4X402)2.Cu(X03)2+3H20 and
the silver salts C6H14X4O2.HXO3 + AgXOs (the more readily soluble) and
C6Hi4X402.AgX03 + ^H20 (the more difficultly soluble) and its compound
with picrolonic acid (Steudel^).
Arginine is dextrorotatory-,. but the arginine obtained Ijy Kutscher in
the tr}'ptic digestion of fibrin was inactive. On oxidation with perman-
ganate it splits off guanidine, which can be precipitated with sodium picrate.
Orglmeister ^ bases his method for the quantitative estimation of arginine
in mixtures obtained by hydrolysis upon this fact.
(:h2.(xh,)
Ornithine (a-5-diaminovalerianic acid),C5Hi,X202= (^jj('>^-jj \, is not a primary
COOH "
cleavage product of proteins, but is formed from arginine on boiling with barvta-
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 Sorexsex ^ have prepared s}"!!-
thetically yields, as shown by Ellixger, putrescine (tetramethylenediamine),
C4Hg(XH2)2, on putrefaction. A. Loewy and X'euberg ' have shown that orni-
thine is split into putrescine and CO, in the organism of cystinuria patients.
OrnitMne 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 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 Sorexsex has .sho\\Ti that
the naturally occurring ornithuric acid is identical with the dextrorotatory a-3-
dibenzoyldiamino valerianic acid.
Diaminoacetic acid, CoHeX'oO, =CH(NH2)2COOH, was obtained by Drechsel*
as a cleavage product of casein by boiling with tin and hydrochloric acid. It
^ 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;
Kossel and Dakin, ibid., 11.
2 Ber. d. d. chem. Gesellsch., 32, and Zeitschr. f. physiol. Chem., 34.
'Zeitschr. f. physiol. Chem., 3" and 44.
* Hofmeister's Beitrage, 7.
^ Ber. d. d. chem. Gesell.sch., 10 and 11.
'Fischer, ibid., 34; Sorensen, Zeitschr. f-. physiol. Chem., 44.
' EUinger, Zeitschr. f. physiol. Chem., 29; Loewy and Xeuberg, ibid., 43.
*Ber. d, sachs. Ges. d. Wissensch., 44.
98 THE PROTEIN SUBSTANCES.
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)
Lysine (a-£-diaminooaproic acid), C6Hi4N202= r;TJVTy-^TT x- was first
Lrl(jNrl2)
COOH
obtained by Drechsel as a cleavage product of casein. Later he and his
pupils, as well as Kossel and others, found it among the cleavage products
r)f various proteins. It has not been detected in some vegetable pro-
teins such as zein and gluten-protein. E. Schulze found lysine in ger-
minating plants of the Lupinus luteus, and Winterstein found it in ripe
cheese. It has been obtained in largest amounts (28.8 per cent) by Kossel
and Dakin ^ from the protamine a-cyprinine.
Lysine has been synthetically prepared by E. Fischer and Weigert.^
This lysine was inactive, w^hile that prepared from protein is always optic-
ally active and dextrorotatory. On heating with barium hydrate it is
converted into the inactive modification. According to Ellinger^ lysine
yields cadaverine (pentamethylenediamine), C5HiofNH2)2. f^n putrefaction,
and this base is formed from the lysine in the organism of those with cysti-
nuria and at the same time CO2 is split off (A. Loewy and Neuberg).
Lysine is readily soluble in water but is not crystalline. The aqueous
solution is precipitated by phosphotungstic acid Init not by silver nitrate
and baryta-water (differing from arginine and histidine). It gives two
hydrochlorides \vith hydrochloric acid, and with platinum chloride a
chloroplatinate w^hich is precipitable by alcohol and has the compo-
sition C6Hi4N202.H2PtCl6 +C2H5OH. It gives two silver salts with
AgNOs; one has the formula AgN03+C6Hi4N202 and the other AgN03 +
CeHi4N202.HN03. With benzoyl chloride and alkali, lysine forms an acid,
lysuric acid, C6Hi2(C7H50)2N202 (Drechsel), which is homologous with
ornithuric acid and whose difficultly soluble acid l)arium salt may be used
in the sejjaration of lysine.^ The rather insoluble picrate, which is pre-
cipitated from a not too dilute solution of the hydrochloride by sodium
picrate, may also be used in the detection of lysine.
' Drechsel, Arch. f. (Anat. u.) Physiol., 1891, and Ber. d. d. chem. Gesellsch., 25;
Siegfried, Arch. f. (Anat. u.) Physiol., 1891, and Bor. 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; Winterstein,
cited in Schulze and Winterstein, Ergebnisse der Physiologie, I, Abt. 1, 1902; Kossel
and Dakin, Zeitschr. f. physiol. Chem., 40.
^ Ber. d. d. chem. Gesellsch., 35.
' Zeitschr. f. physiol. Chem., 29.
•* Drechsel, Ber. d. d. chem. Gesellsch., 2-S; .see also C. Willdenow, Zeitschr. f.
physiol. Chem., 25.
«
HISTIDINE. 99
KuTSCHER and Loiimann ' 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 CeH gNjO^ or
CgHiiNsO +Hv;0. In the first case this base would appear to be a homologue of
creatine, C4H6N3O2, and in the other case a homologue of creatinine, 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.^
Histidine, C6H9N3O2, is perhaps not a diamino-acid, as Fraxkel^
first showed, but, according to the investigations of H. Pauly, Kxoop
CH— NH\
C N/^^
and Windaus,^ is an a(?)-amino-/?-imidazolpropionic acid, CH2
CHNH2
COOH
Fraxkel ^ has made several objections to Pauly, Knoop and Windaus's
view that histidine is an imidazol derivate, which seem to be well founded,
therefore the question as to the constitution of histidine remains still an
open one.
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 diiTerent protein sub-
stances. It does not occur in the protamines, with the exception of sturine.
Of the protein bodies globin (from horse-haemoglobin) seems to be richest in
histidine, as Abderhalden found 10.96 per cent. It also occurs in germi-
nating plants (E. ScHULZE^).
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 precipitated ])v mr;-
' Zeitschr. f. physiol. Chem., 41.
^ Hedin, ihiil., 21; Siegfried, ibid., 35.
3 Sitzunosber. d. Wien. Akad., 112, II, b, 190.3.
'' Pauly, Zeitschr. f. physiol. Chem., 42; Knoop and Windaus, Hofmeister's Bci-
trilge, 7.
^ Hofmeister's Beitrage, 8.
' As histidine is always ol)tained with the diamino-acids it is called a hexone base,
hence it will be treated here with the diamino-acids.
' Kossel, Zeitschr. f. physiol. Chem., 22; Hedin, ibid., Kutscher, ibid., 25; Wetzel,
ibid., 20; 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.
lOO TEIE PRCVTEIS: SITR^TAATES.
cumic fWksmfe^ or^ sItiM belrter, by tlte swIfp'&Latie andtfi!e<i witii sulphuric acid,
amd eaaa ia tlafe tray be sep^iateti frojaat th© tltammo^cids as well as from
Ijne inoiiiaiimiDi«>-a(ekfe (Kosjel and Pattex). The Ii>-drocMoride cn-stal-
Ifiaes; III Ibeawritiffiaill plate? (,R\rEE). dissolves ratker readily in water, but is
iBBseJhfflfete BBi afedlMji aini«J et!i:er> "Wifelt !mii:oeMoirt<j ajftd. aad metliyl akohol
• :. - :1ft «MiiyT!fcro«W®rtde of kistldinje metbLyl ester. wMch. melts at 1%"^.
v. - -i' is tevenrodatiiMnr,, while its solutitm in hydrocMortc acid is dextro-
KjiftaltocT. Qtt ■wramniMinig it ^ve? the bitiret test (HEsaoG ^). and it also gi\'es
"' '^ ••■■ "■ " ed as su^:^:ested by Feschee (,st^e Xanthine.
i giv\es a "very beautiful diazonreaction with
;<ailpjho£twe a«fM m soiutkuois made atkaEne with sodium carbon-
vjig t© Paitly is deep chern.'-red in dilutions of 1:20 000
;. . V red im 1:100 000' (tyrosine gtv-es a similar reaction).
■••.^tt«i-«L of the abo've basses -we can first precipitate all the
Ijsjus^tj, t - ".i^tic acid, w-hen the monamino-acids remain in solu-
tk«iu T e is^ dewimpose^-i in boiling water by barium hyi.irate
amd tht" - - :amed as sih"er compounds from this tiltrate. In regard
tO' furth^; ......~^ we must refer to the woifcj of D-rechsejl and Hkdlx
aUre^ady cited. KiotssasiL and KrcscHBua and recently Wixteksthiix - have
<•-.;-• •^,,-,1 a method of sepaorating histidiiae and arginine sus sil\-er compoimct?
-:-ae'. and KosS'EL. and Pattbix have proposed a method of separating
hijCiUiae fftofln argintne by means of mercuric sulphate.
We give betow a tabulation of the antjounts of the three hexone basses
iodaiBttiii in cectain piiro>tein substamj^es (in weight per cent) :
Arpxiiae Lysine Histidine
SfttEmH?* 5!v.:i 12.0 12. d
CNcpctniae^ ^-JB'^ 4.^ 3J<.{^ 0.0'
4L^b«- p.w«3imin«es * 62-5 — ST. 4 0'. © 00
Hkfiyasfs*. 14.315— 15.K 1.7— S. 3 1.21—2.34
C^»titt*. 4.Il)--4.S4 l.ftJ— 5.JJ0 2.53-^2.59
SSfTBtewiiEa! ('ftens! BBoeailt)!*. ....... . 5. 0<§ 52!$ 2.66
Hirt.«etgni<K.Okii« *. . S.5S 3.06— T.0S 0.3^-1.12
P^wftuxs^^niitHijiBWtie' *..,............ . 455 3 OS 3. 35
E&sftBBi *. ...-.■. 11.0^—14.07 1.3 1.17
FwteM ftwm WNSsSfesat seed* '. ms-— 11.3; 0.25— 0.7^ 0.(52— 0.7S
CStelwa. «taB«£ii ' 44 2.15 1.16
IG&alWffi Mw*««ns *. 2-75 — 3;..13: 00 0.43 — 1.5S
Grih*£Bif»aaiii*.... T.«Ki— 9i.S 2.4!J— «.0 0 40
Ofcstecnt*.. 0 3 -t- 0 027
"^ Ktfcsswii aoui Fitfititicu Ziiit^br.. L p&jjnsajL ChKCU.., 3J>; Bauer. iJbid.. 22; Hensug,
- KotssfJ ami KoittsvJiBir, iAa£.. Si.;. Wtatersfeeoi. iM£^^ -IS,"; Koisel and Pafetea, L G-.
* Koisail: ami Ktrtscfter. Z&ifiistf&ir. ff. p&ijrsajL C&mul.. SL
*H!art. ii6«i£.. SL
* 5s«f&db8 ami Wtoiwcstieai, e6»i.^ 3S; see aLsci Kocsssel, Ber. d. d. chem. CteseBiseh.,
ai,?2a5^
* Bv^-iiisell ami Ktxb^&er. 2^s«i&r. fi. p&jjrsbL Chem.. 2S», ami Richards ami Gies,
AnjBtr. Jbairtii. <<?£ PfecyswL.. T.
^ Koesfll ami PtiMa. 2ufi.t:<«t&ir. f. phystpL Cbem.. -Ml
« -PROLINE. 101
Oxydiamino-acids.
Oxydiaminosebacic acid, C,„H.,,N.,05, has been isolated as a copper salt by
WoHi-ciEMi'TH ' from a iiueleoproteid oi the liver. The free acid was obtained
as small white plates. It is soluble with diftieulty in hot water, insoluble in cold
water and in aleohul. It was optically inactive in hydrochloric acid. The
beautifully crystalline phenylcyanate compound had a meltitifj-puint of 200'"\
lyiox yd Laminate id ni-ir acid, C\Hi,,N,;0,„ has been obtained by Skkaup - on the
hydrolysis of casein with hydrt)chloric acid. The copper salt crystallizes in beauti-
ful deep bluish-violet rosettes which are composed of long, irregular, right-
angled plates. It is cjuite soluble in cold water. The free acid crystallizes iu
fern-like formations. Besides this acid Skkaip obtained two other acids which
he calls cascanic acid, C„H,uN^,C)7, and caseirdc acid, Civll.'-tN-Os. The caseanic acid
crystallizes, melts at 190-191", is tribasic, and is probably an oxydiamino-acid.
The caseinic acid is tlibasic 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-ileflned crystals. Caseinic acid seems also to be an oxydiamino-acid.
Diaminotrioxydudccarioic acid, Vy.li._^^.Oi, is an acid obtained by Fischkk and
.\BnKKnALOEN ^ Oil the hydrolysis of cast>iu and seems to stand close to Skr^vup's
caseinic acid, but ditTers from it in its optical properties. This acid is fauitly
levorotatory : (ci:)p =- 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.
3. Pyrrol and Indol Derivatives.
«-Pyrrolidine-carboxylic acid, abbreviated to tt-ProUne, C5II9NO2,
I I
CH3 CH.COOH,
Ml
was prepared by E. Fischkk as a cleavage product from casein (3.2 per
cent) and ovulbutnin (1.55 per cent), and by him and his collaborators in
the tryptic digestion of casein, and as a cleavage product of haemoglobin
(1.46 per cent), gelatine (5.2 per cent), horn substance (3.60 per cent), and
from silk fibroin.^ The acid thus obtained was generally the levorotatory
moditicatioii. Kosskl and Dakin ^ obtained 11 i)er cent a-proline from
salmine, while Abdkrhalden and his co-workers*^ obtained 2.25 per cent
from ovalbumin, 1.46 from thymus hist one, 1.74 from elasthi, and 3.4-3.5
per cent from keratin substances. a-ProUne also occurs in scombrine
' Ber. d. d. chem. Cesellsch., 37, and Zeitschr. f. physiol. Chem., 44.
• Zeitschr. f. physiol. Choni., 42.
' Ibid.
* Fischer, ibv.1., 33 and 3o. >See also foot-note I, p. 84.
' Zeitschr. f. physiol. Chem., 41.
' Abderhalden and I'regl, ibid., 46; with Rona, ibid., 41; with Schittenhelni, ibid.,
41; with Wells and Le Count, ibid., 40.
102 THE PROTEIN SUBSTANCES.
and clupeine, but not in sturine, which according to Kossel seems to
contradict the view as to the common origin of ornithine and a-proline.
SORENSEN 1 by means of a general method of preparing a-amino-acids
synthetically has prepared a-amino-o-oxyvalerianic acid from phthalimide-
malonic ester and has obtained a-proline from this by evaporating with
hydrochloric acid, at the same time splitting off water.
This acid is readily soluble in water and alcohol and crystallizes in flat
needles which melt at 203-206° C. with an odor of pyrrolidine. The solu-
tion acidified with sulphuric acid is precipitated by phosphotungstic acid.
In the detection of this acid we make use of the copper salt, the anhy-
dride of the phenylisocyanate compound (melting-point 144°), and the
picrate (Alexandroff^). The inactive acid and its compounds show some-
what varying properties. In regard to the detection of this acid we refer
to p. 91.
In the hydrolysis of gelatine and casein E. Fischer ' obtained an amino-
acid having the formula CjHgNOg, which on reduction yielded a-pyrrolidine-carbo-
xylic acid, and which according to Fischer is an oxypyrrolidine-a-carboxylic
acid. Leuchs ^ has synthetically prepared two similar, inactive acids.
Indolaminopropionic acid (tryptophane, proteinochromogen),CiiHi2N202,
C.CH2.CH(NH2)COOH C.CH(NH2).CH2.Co6h
C6H4/VH or C6H4<^CH
NH NH
is one of the cleavage products of the proteins formed in tryptic digestion
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 ^ considered
tryptophane, which name is generally given to this acid, as the mother-
substance of various animal pigments.
Trvptophane was first prepared in a pure form by Hopkins and Cole,®
and they considered it as skatolaminoacetic acid. After Ellinger^ showed
that skatolcarbonic acid (Salkowski) and skatolacetic acid (Nencki)
were indolacetic acid and indolpropionic acid respectively, we have con-
sidered tryptophane as indolaminopropionic acid.
■ Zeitschr. f. physiol. Chern., 44.
^ In regard to the preparation of the phenylisocyanate compounds of the amino-
acids, see Paal, Ber. d. d. chem. Gesellsch., 27; Mouneyrat, ibid., 33, and Hoppe-
Seyler-Thierfelder's Handbuch, 7. Aufl.; Alexandroff, Zeitschr. f. physiol. Chem., 46.
' Ber. d. d. chem. Gesellsch., 35 and 86.
5 In regard to trytophano, sec Stadelmann, Zeitschr, f. Biologie, 26; Neumeister,
ibid., 26; Nencki, Ber. d. d. chem. Gesellsch., 2S; Beitler, ibid., SI; Kurajeff, Zeitschr.
f. physiol. Chem., 26; Klug, Pfliiger's Arch., 86.
'Journ. of Physiol., 27.
'Ber. d. d. chem. Gesellsch., 37 and 38.
INDOLAMINOPROPIONIC ACID. 103
Tryptophane crystallizes in shining plates which are readily soluble in hot
Avater, less soluble in cold water and in alcohol. On heating sufficiently,
it yields indol and skatol. It gives the Adamkiewicz-Hopkins reaction
and a rose-red coloration on the addition of bromine-water (tryptophane
reaction). If a pine stick moistened with hydrochloric acid and then washed
off be introduced into a concentrated tryptophane solution, it becomes
purple-colored on drying (pyrrol reaction). Tryptophane, as Hopkins and
Cole ^ shoAved later, yields skatolacetic acid (indolpropionic acid) on anae-
robic putrefaction, and skatolcarbonic acid (indolacetic acid), skatol, and
indol on aerobic putrefaction.
In regard to the somewhat complicated method of preparation we must
refer to the original work of Hopkins and Cole.
Skatosine, CiiiHieNjOo, is a base first obtained by Baum in the pancreas auto-
digestion and later studied by Swain. It develops an indol- or skatol-like odor
on fusing with potassium hydrate. Langstein ^ obtained a substance, which is
perhaps identical with skatosine, in the very lengthy peptic digestion of blood
proteid.
The putrefactive products of the proteins will be in part treated in
Chapter IX (intestinal putrefaction) and in part in Chapter XV (putre-
factive products in the urine).
' Journ. of Physiol., 29; see also EUinger and Gentzen, Hofnaeister's Beitrage, 4.
^ Baum, Hofmeister's Beitrage, 3; Swain, ibid.; Langstein, see Hofmeister, t'ber
Bau und Gruppierung der Eiweisskorper, in Ergebnisse der Physiologie, I, Abt, 1, 1902,
CHAPTER III.
THE CARBOHYDRATES
We designate by this name bodies which are especially abundant
in the plant kingdom. As the protein bodies form the chief portion of
the solids in animal tissues, so the carbohydrates form the chief portion
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 proteids.
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 w^ater, but we also have a sugar (rham-
nose, C6H12O5) which has these two elements in another proportion. At
one time it was thought possible to characterize as carbohydrates those
bodies which contained 6 atoms of carbon, or a multiple, in the molecule,
but this is not considered tenable at the present time. We have true car-
bohydrates containing less than 6, and also those containing 7, 8, and 9
carbon atoms in the molecule. The carbohydrates have no properties or
characteristics in general which differentiate them from other bodies;
on the contrary, the various carbohydrates are in many cases very different
in their external properties. 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 aldehyde
or ketone derivatives of such alcohols, and the more complex carbohydrates
seem to be derived from these by the formation of anhydrides. It is a
fact that the more complex carbohydrates yield two or even more molecules
of the simple sugars when made to undergo hydrolytic splitting.
104
MONOSACCHARIDES. 105
The carbohydrates are generally divided into three chief groups, namely,
monosaccharides, disacchandes , and polysaccharides.
Our knowledge of the carbohydrates and their structural relationships
has been verj^ much extended by the pioneering investigations of Kiliani ^
and especially those of E. Fischer.^
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 re\'iew of those carbohydrates which
occur in the animal kingdom or are of special importance as food for man
and animals.
Monosaccharides.
All varieties of sugars, the monosaccharides as well as disaccharides,
are characterized by the termination "ose," to which a root is added signi-
fving their origin or other relations. According 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 kefoses. Ordinary
dextrose is an aldose, while ordinary fruit sugar (levulose) is a ketose. The
difference may be shown by the structural formulse of these two varieties
of sugar:
Dext rose = 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 ha\dng less carbon. On mild oxidation the aldoses yield mono-
basic oxyacids and dibasic acids on more energetic oxidation. Thus ordi-
nary dextrose yields gluconic acid in the first case and saccharic acid in
the second.
Gluconic acid =CH2(OH).[CH(OH)]4.COOH;
Saccharic acid = Co6H.[CH(OH)]4.COOH.
The monobasic oxyacids are of the greatest importance in the artificial forma-
tion of the monosaccharides. These acids, as lactones, can be converted into
' Ber. d. deutsch, chem. Gesellsch. 18, 19, and 20.
^ 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 Chemie der Zucker-
arten, Braunschweig, 1904.
106 THE CARBOHYDRATES.
their respective aldehydes (corresponding to the sugars) by the action of nascent
hydrogen. On the other hand, they may be transformed into stereoisomeric
acids on heating with quinoline, pyridine, etc., and the stereoisomeric sugars may
be obtained from these by reduction.
Xumerous 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 asymmetric
carbon atoms.
The monosaccharides are converted into the corresponding polyhydric
alcohols by nascent hydrogen. Thus arabixose, which is a pentose,
C5H10O5, is transformed into the pentatomic alcohol, arabite, C5H12O5.
The three hexoses, dextrose, levulose, and galactose, C6H12O6, are
transformed into the corresponding three hexites, sorbite, mannite, and
dulcite, C6Hi406- In these reductions a second isomeric alcohol is also
obtained; in the reduction of levulose we obtain besides mannite also
sorbite. Inversely, the corresponding sugars may be prepared from
polyhydric alcohols by careful oxidation.
Like the ordinary aldehydes and ketones, the sugars may be made to
take up hydrocyanic acid. Cyanhydrins are thus formed. These addition
products are of special interest in that they make possible the artificial prepara-
tion 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)],.CH(OH).CX.
On the saponification of glucocyanhydrin the corresponding oxyacid is formed:
CH2(OH).[CH(OH)],.CH(OH).CN+2H,0
= CH2(OH).[CH(OH)],.CH(OH).COOH + NH3.
By the action of nascent hydrogen on the lactone of this acid we obtain gluco-
heptose, C7H14O7.
The monosaccharides give the corresponding oximes with hvdroxylamine ;
thus glucose yields glucosoxime, CH,(OH).[CH(OH)],.CH:N.OH.*^ These com-
pounds are of importance on account of the fact, as found by Wohl,' 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.
The monosaccharides are strong reducing bodies, similar to the alde-
hydes. They reduce metallic silver from ammoniacal silver solutions, and
also several metallic oxides, such as copper, bismuth, and mercury oxides,
on warming their alkaline solutions. This property is of the greatest
importance in their detection and quantitative estimation.
With phenylhydrazine or substituted phenylhydrazines, 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.
» Ber. d. d. chem. Gesellsch., 2G. p. 730.
MONOSACCHARIDES. 107
The reaction takes place as follows:
(a) CH,(0H).[CH(0H)]3.CH(0H).CH0 + HoN.XH.CH^
= CH,(0H).[CH(bH)]3.CH(0H)CH:N.NH.C,H5 + H,0.
Phenylglucosehydrazone.
(b) CH,(0H)[CH(0H)]3.CH(0H).CH:N.NH.C,H,+H,N.XH.C,H5
= CH,{0H).[CH(0H)]3.C.CH:N.NH.C6H,
N.NH.C,H5 + H,0 + H,.
Phenylglucosazone.
The hydrogen is not evolved, but acts on a second molecule of phenylhy-
drazine and splits it into aniline and ammonia:
H,N.NHAH5 + H, = H,N.C6H, + NH,.
The osazones 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 carbohydrates for other reasons. Thus they are a ver}^ good
means of precipitating sugars from solution in which they occur mixed
with other bodies, and they are of the greatest importance in the artificial
preparation of sugars. On cleavage, by the brief action of gentle heat
and fuming hydrochloric acid (for disaccharides still better with benzalde-
hyde) ^ 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
(ketoses) in other ways, namely, by direct reduction of the osazones \Aith
acetic acid and zinc dust. The corresponding osamine is first formed
(from phenylglucosazone we obtain isoglucosamine), which on treatment
with nitrous acid yields the sugar (in this case le\ailose).
The sugars can be prepared from the hydrazones by decomposition
with benzaldehyde (Herzfeld) or with formaldehyde (Ruff and Ollen-
dorff 2). This latter method is especially applicable if substituted hydra-
zines, especially benzylphenylhydrazine, are used.
With ammonia the glucoses may form compounds which have been
considered as osamines by Lobry de Bruyn, but to differentiate them from
the true osamines have been called osimines by E. Fischer.^ The corre-
sponding 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 c?-glucosa-
mine, which occurs in the animal kingdom and is an isomer of the above-
' E. Fischer and Arinstronsr, Ber.. d. d. chem. Gesellsch., 35.
2 Herzfeld, ibid., 28; Ruff and OllendorlT, ibid., 32.
'Lobry de Bruyn, ibid., 2S; E. Fischer, ibid., 35.
108 THE CARBOHYDRATES.
mentioned isoglucosamine, by starting from d-aral)inose, then obtaining
rf-aral^inosimine, then d-gkicosaminic acid, and finally the glucosamine
from the lactone of this acid. They ^ also have prepared Z-glucosamine
from Z-arabinose in a similar manner.
Knoop and Windaus - have obtained large amounts of methylimidazol,
CH3
C — NHv , from glucose by the action of ammonium-zinc hydroxide
I! >H
CH— N^
at ordinary temperatures. A genetic relationship of the carbohydrates to
histidine and the purine bodies is thus made probable by the imidazol for-
mation.
By the action of hydrochloric acid upon alcoholic sugar solutions E.
Fischer and his pupils have obtained ether-like compounds which have
been called gincosides. Compounds with aromatic groups similar to the
glucosides occur widely distributed in the vegetable kingdom. The more
complex carbohydrates may be considered, according to Fischer, as
glucosides of the sugars. Thus maltose, for example, is the glucoside
and lactose the galactoside of dextrose.
By the action of alkalies, even in small amounts, as also of alkaline
earths and lead hydroxide, a reciprocal transformation of the sugars, such
as dextrose, levulose, and mannose, may take place (Lobry de Bruyn and
Alberda van Ekenstein^).
Four other sugars, among them two ketoses, are produced by the action of
potash or soda on each of the three sugars, dextrose, levulose, and galactose.
For example, from dextrose two ketoses, levulose and pseudolevulose, are pro-
duced, also mannose and a non-fermentable sugar, glutose. From galactose
are formed talose and galtose, besides two ketoses, tagatose and pseudotagatose.
The transformation of the different varieties of sugar into each other
also occurs in the animal body. Neuberg and Mayer'* have shown by
experiments on rabbits the partial transformation of various mannoses into
the corresponding glucoses.
The monosaccharides are colorless and odorless bodies, neutral in re-
action, with a sweet taste, readily soluble in water, generally soluble with
difficulty in absolute alcohol, and insoluble in ether. Some of them crys-
tallize well in the pure state. They are optically active, some levorotatory
and others dextrorotatory; but there are also optically inactive modi-
' Ber. d. d. chem. Gesellsch., 35, p. 3787, and 36, p. 24.
^ Ibid., 38, and Hofmeister's Boitrage, 6.
'Ber. d. d. chem. Gesellsch., 2S, 3078; Bull. see. chim. de Paris (3), 15; Chem.
Centralbl., 1896, 2, and 1897, 2.
•" Zoitschr. f. physiol. Chem., 37.
MONOSACCHARIDES. 109
fications (racemic), which are formed from two opti'^-ally opposed compo-
nents.
We designate the optical activity of the carbohydrates with the letter
I- for levogyrate, d- for dextrogyrate, and i- for inactive. These are only
partly indicative. Thus dextrorotatory glucose is designated cZ-glucose,
levorotatory /-glucose, and the inactive ^'-glucose. 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 Z-le^allose,
but rf-le^1llose, showing its close relation to dextrorotatory d-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 gn. substance
dissolved in 1 c.c. 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 (a)D, and is expressed by the following formula:
(a)D= it — -, in which a represents the reading of degrees, 1 the length of the
tube in decimetres, and p the weight of substance in 1 c.c. 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
s.l
rotation.
A freshly prepared sugar solution often shows a different rotation from one
Avhich has been allowed to stand for some time. If the rotation gradually
diminishes, this is called birotation, while a gradual increase in the rotation is
called half -rotation.
]\Ian3' monosaccharides, but not all, ferment with 3'east, and it has been
shown that only (hose varieties of sugar containing 3, 6, or 9 atoms of
carbon in the molecule are fermentable with yeast. We must state, how-
ever, 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. The
restriction of fermentation to only certain monosaccharides is, accord-
ing to E. Fischer, like the action of the inverting enzymes upon disac-
charides and glucosides, dependent upon the stereometric configuration of
the sugars (see Chapter I). This difference in configuration is important
not only for the action of lower living organisms upon the sugars, but
also upon the behavior of the sugars within more highly developed organ-
isms. Thus the investigations of Neuberg and Wohlgemuth 1 upon ara-
binose and of Neuberg and Mayer 2 on mannoscs have shown that in
rabbits the /-arabinose and the tZ-mannose are much better utilized than d-
and /-arabinose or /- and /-mannose, and they have also shown that the
lower organisms have the tendency toward decomposing inactive sub-
* Zeitschr. f. physiol. Chem., 35. 'Ibid., 37.
110 THE CARBOHYDRATES
stances into their optically active components to a much higher degree
than the higher organisms.
By the action of lower organisms of various kinds the sugars may be
made to undergo fermentations of different kinds, such as lactic-acid and
butyric-acid fermentation and mucilaginous fermentation.
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, dextrose
and levulose. They also occur in great abundance in nature as more
complex carbohydrates (di- and polysaccharides); also as ester-like com-
binations 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 imjjortance in biochemistry, although the}^ are of high scien-
tific interest. Of the other tw^o groups the hexoses are of the greatest
importance, hence in the past only those carbohydrates with six carbon
atoms w^ere considered as tnie carbohydrates. As the pentoses have been
the subject of numerous biochemical investigations of late, they will also
be discussed in brief.
Pentoses (C5H10O5).
As a rule the pentoses do not occur as such in nature, but are formed in
the hydrolytic splitting of several more complex carbohydrates, the so-
called pentosanes, especially on boiling gums with dilute mineral acids.
The pentosanes exist very widely distributed in the plant kingdom and aro
especially of great importance in the building up of certain plant con-
stituents. The pentoses were first found b}' Salkowski and Jastrowitz
in the animal kingdom in the urine of a person addicted to the morphine
habit, and later by Salkowski and others in normal human urine. Small
quantities of pentoses have been detected by KiJLZ and Vogel ^ in the
urine of diabetics, as also in dogs with pancreas diabetes or phlorhizin
diabetes. Pentose has also been found by Hammarstex amongst the
cleavage products of a nucleoproteid obtained from the pancreas, and
saems also, according to the observations of Blumenthal, to be a constit-
uent of nucleoproteids 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 Gruxd and of Bexdix
and Ebsteix.2
'Salkowski and Jastrowitz Centralbl i, d. med. Wissensch. 1892. 337 and 593;
Salkowski, Berl. klin. Wochenschr., 1895, Bial Zeitschr. f. klin. Med.. 39; Bial and
Blumenthal, Deutsch. med. Wochenschr., 1901, No. 2; Kiilz and Vogel, Zeitschr. f.
Biologie, 32.
^ Hammarsten Zeitschr f. physiol. Chem., 19; also Salkowski, Berl. klin. Wochen-
schr., 1895; Blumenthal Zeitschr. f. klin. Med., 34; Grund, Zeitschr. f. physiol. Chem..
3.'; Bendix and Ebstein Zeitschr. f. allgemein. Physiol., 2.
PENTOSES. 11]
The pentosanes (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 Wohlgemuth ^ upon rabbits and hens show that these animals car-
utilize the pentoses. The question whether the pentoses are active as
glycogen-formers is still an open one (see Chapter VIII). The pentoses
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 .^
The natural pentoses are reducing aldoses, and as a rule do not belong
to the sugars fermentable with yeast. Still, the observations of Salkowski.
Bendix, Schoxe and Tollens seem to indicate that the pentoses are
fermentable.^ 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 le\ailinic acid. The furfurol passing 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 quan-
titative estimation we can use the method suggested by Tollex.s, which
consists of converting the furfurol in the distillate into phloroglucide by
means of phloroglucin and weighing (see Tollens and Krober, Grund,
Bendix and Ebsteix).^ These methods are still not quite accurate, to
say nothing of the fact that glucuronic-acid compounds also yield furfurol
under the same conditions. The two following pentose reactions, as sug-
gested by Tollexs, are especially applicable.
The orcin-hydrochloric acid test. Mix with the solution or the sub-
stance introduced into water an equal volume of concentrated hydrochloric
acid, add some orcin in substance, and heat. In the presence of pentoses
the solution becomes reddish blue, then bluish green, and on spectroscopic
examination an absorption-band is observed between C and D. If i:
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
' S'one, Amer. Chem. Journ., 14; Slowtzoff, Zeitschr. f. physiol. t^Jhem., 34; Sal-
kowski, 1. c, Centralbl.; Cremer, Zeitschr. f. Biologie, 29 and 42; Neuberg and Wohl-
gemuth, Zeitschr. f. physiol. Cliem., 3.5.
^ See Ebstein, Virchow's Arch., 129; Tollens, Ber. d. deutsch. chem. Gesellsch.. 29,
1208; Cremer, 1. c; Lindemann and May, Deutsch. Arch. f. khn. Med., 56; Sal-
kowski, Zeitschr. f. physiol. Chem.. 30.
'Salkowski. Zeitschr. f. physiol. Chem., 30;. Bendix, see Chem. Centralbl., 1900, 1;
Schone and Tollens. ibid., 1901, 1.
^Bendix and Ebstein, 1. c, which contains the literature.
112 THE CARBOHYDRATES.
solution becomes cherry-red on heating and then becomes cloudy and hence
a shaking out with amyl alcohol is very necessary. The red amyl-alcohol
solution shows an absorption-band between D and E. The orcin test is
better for several reasons than the phloroglucin test (Salkowski and
Neuberg ^). In regard to the use of these tests in urine examination
see Chapter XV.
Many modifications of these tests have been suggested. Brat ^ performs
the orcin reaction by the addition of NaCl and heating to only 90-95°. Bial ^
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 may
occur (Bial, van Leersum).* According to R. Adler and O. Adler the phlo-
roglucin and orcin tests can be made with glacial acetic acid and a few drops
hydrochloric acid instead of with the hydrochloric acid alone. These investigators
also use a mixture of equal volumes of aniline and glacial a etic acid as a reagent
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 ^ 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.
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 2-arabinose. It can be isolated from the urine as the diphenylhydrazone,
from w^hich the arabinose can be separated by splitting with formaldehyde.
The 2-arabinose is crystalline, has a sweetish taste, is optically inactive,
and melts at 163-164° C. Its di])henylhydrazone, which, according to
Neuberg and Wohlgemuth,'^ 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 arable or
cherry gum with dilute sulphuric acid. The c?-arabinose is prepared syn-
thetically. The diphenylhydrazone of Z-arabinose has according to Neuberg
' Salkowski, Zeitschr. f. physiol. Cbem., 27; Neuberg, ihid., 31.
^Zeitschr. f. klin. Med., 47.
^Deutsch. med. Wochenschr., 1902 and 1903, and Zeitschr. f. klin. Med., 50.
* Bial, Zeitschr. f. klin. Med., 50; van Leersum, Hofmeister's Beitrage, 5.
* R. and O. Adler, Pfliiger's Arch., 106; A. Neumann, Berl. klin. Wochenschr.,
1904.
" Ber. d. d. chem. Gesellsch., 33.
' Zeitschr. f. physiol. Chem., 35. ,
HEXOSES. 113
a melting-point of 216-218° C, while according to Tollens and ^Iauren-
brecherI i^g melting-point is 204-205°.
Xyloses. The only pentose thus far isolated from the animal tissues
is /-xylose, obtained by Neuberg from the pancreas proteins, and is
identical with the xylose found widely distributed in the plant kingdom
and obtained from wood-gum by boiling with dilute acids. Xylose is
crystalline, melts at 153-154° C, dissolves very readily in water but ^^•ith
difficulty in alcohol, is faintly dextrorotatory, (o)d= +18.1°, and gives a
phenylosazohe which melts at 159-160° C, and according to Tollexs and
^MiJTHER^ a diphenylhydrazone which melts at 107-108°. Xylose can
be transformed into xylonic acid, CH20H(CHOH)3COOH, by bromine-
water, and the brucine salt of this acid is, according to Neuberg, well suited
for the detection and isolation of xylose.
Hexoses (C6Hi206).
The most important and best-known simple sugars belong to this group,
and most of the other bodies which have been considered as carbohydrates
in the past are anhydrides of this group. Certain hexoses, 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.
All hexoses, as also their anhydrides, yield levulinic acid, C5H8O3.
besides formic acid and humus substances on boiling with dilute mineral
acids. Some of the hexoses are fermentable \Wth yeast, while the artificially
prepared hexoses are not, or at least only incompletely and with great
difficulty.
Some hexoses are aldoses, while others are ketoses. Belonging to the
first group we have mannose, dextrose, gulose, galactose, and talose,
and to the other levulose, and possibly also sorbinose. We differen-
tiate also between the d, I, and i modifications; for instance, d-, 1-, and
t-dextrose; hence the number of isomers is very great.
The most important syntheses of the carbohydrates have been made by
E. Fischer and his pupils chiefiy within the members of the hexose group.
A short summar}^ 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 ob-
' Neuberg, Zeitschr. f. physiol. Chem., 35^ and Ber. d. d. chem, Gesellsch., 33;
ToUens and Maurenbrecher, ibid., 38.
'Neuberg, Ber. d. d. chem. Gesellsch., 35; Tollens and Muther, ibid., 37.
114 THE CARBOHYDRATES.
tained a faintly sweetish syrup called methylenitan. Loew ' later obtained a
jnixture 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.^
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 [i-acrose on the oxidation of glycerine with
bromine in the presence of sodium carbonate and treating the resulting mixture
with alkali. On the oxidation with bromine a mixture of glvcerine aldehvde,
CH^OH.CHCOHj.CHO, and dioxyacetone, CH^COHj.CO.CH^OH, is obtained.
These two bodies may be considered as true sugars, nameh', 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 its osazone and then retransforming this into the sugar.
«-acrose is identical with ?'-levulose. With yeast one half, the levogyrate d-levu-
lose, ferments, while the dextrogyrate Z-levulose remains. The i- and /-levulose
may be prepared in this waj'.
On the reduction of a-acrose we obtain a-acrite, which is identical with {-man-
nite. On oxidation of ?'-mannite we obtain i'-mannose, from which only /-man-
nose remains on fermentation. On further oxidation of {-mannose it j^elds
1-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 107, using
the osazone as an intermediate step. The d- and /-mannonic acids are |3artly
converted into d- and /-gluconic acids on heating with quinoline, and d- or /-glucose
is obtained on the reduction of these acids; /-glucose is best prepared from
/-arabinose by means of the cyanhydrin reaction, using /-gluconic acid as the
intermediate step. The combination of /- and d-glueonic aeids, forming {-glu-
conic acid, yields ?-glucose on reduction.
The artificial preparation of sugars by means of the condensation of formalde-
hyde has received special interest because, according to Baeyer's assimilation
hypothesis, in plants formaldehyde is first formed by the reduction of carbon
dioxide, and the sugars are produced by the condensation of this formaldehyde.
BoKORNY ■* has shown, by special experiments on algse Spirogyra, that formalde-
hyde .sodium sulphite was split by the living algse cells. The formaldehyde set
free is immediately condensed to carbohydrate and precipitated as starch.
Among the hexoses kno^^Tl at the present time only dextrose, levulose
and galactose are really of physiological-chemical interest; therefore the
other hexoses will be only incidentally mentioned.
Dextrose (d-glucose) — glucose, grape-sugar, and diabetic sugar —
occurs abundantly in the grape, and also, often accompanied with le^1dose
(J-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.
' Butlerow, Ann. d. Chem. u. Pharm., 120; Compt. rend., 53; O. Loew Jouin. f.
prakt. Chem. (N. P.), 33, and Ber. d. deutsch. chem. Gesellsch. 20, 21, 22.
^ Ber. d. d. chem. Gesellsch., 21, and 1. c, p. 105.
*Biolog. Centralbl., J2, pp. 321 and 481.
DEXTROSE. 115
It occurs only as traces in urine under normal conditions, while in diabetes
the quantity is ^•er^' large. It is formed in the hydrolytic cleavage of
starch, dextrin, and other compound carbohydrates, as also m the splitting
of glucosides. The question whether dextrose can be formed in the bodj^
from proteins or from fats is disputed and will be discussed in a following
chapter (VIU).
Properties of Dextrose. Dextrose ciystallizes sometimes with 1 molecule
of water of crj'stallization in warty masses or small leaves or plates, and
sometimes when free from water in needles or prisms. The sugar contain-
ing water of cr\-stallization melts even below 100° C. and loses its water
of crystallization at 110° C. The anhydrous sugar melts at 146° C, and
is converted into glucosan, CeHioOs, at 170° C. -uith the elimination of
water. On strongly heating it is converted into caramel and then de-
composes.
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
52.74° at 20° C.^ Dextrose dissolves sparingly in cold, but more freely
in boiling alcohol. 100 parts alcohol of sp. gr. 0.837 dissolves 1.95 parts
anhydrous dextrose at 17.5° C. and 27.7 parts at the boilmg temperature
(AxTHOx^). Dextrose is insoluble in ether.
If an alcoholic caustic-potash solution is added to an alcoholic solution
of dextrose, an amorphous precipitate of insoluble sugar-potash compound
is formed. On warming this compound it decomposes easih' with the
formation of a yellow or bro'WTiish color, which is the basis of !\Joore's
test. Dextrose forms also compounds with lime and bars'ta.
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 brouTi. It has
at the same time a faint odor of caramel, and this odor is more pronounced
on acidification.-^
Dextrose forms several cr\-stallizable combinations vdth. NaCl, of which
the easiest to obtain is (C6Hi206)2-NaCH-H20, which forms large colorless
six-sided double pyramids or rhomboids with 13.52 per cent NaCl.
Dextrose in neutral or verj^ faintly acid (organic acid) solution under-
goes alcoholic fermentation ^ith beer-yeast: C6Hi206 = 2C2H5.0H + 2C0o.
Besides the alcohol and carljon dioxide there are formed, especiallv at
* For further information see Tollens, Handbuch der Kohlehydrate, 2. Aufi. 44.
^ Cited from ToUens' Handbuch.
^ In regard to the products formed in this reaction, see Framm, Pfliiger's Arch., 64-
and especially Gaud, Compt. rend., 119.
116 THE CARBOHYDRATES.
higher temperatures, small quantities of homologous alcohols (amyl
alcohol), glycerine, and succinic acid. In the presence of acid milk or
cheese the dextrose undergoes lactic-acid fermentation, especially in the
presence of a base such as ZnO or CaCOs. The lactic acid may then
further undergo butyric-acid fermentation: 2C3H603 = C4H802 + 2C02 + 4H.
Dextrose reduces several metallic oxides, such as copper oxide, bismuth
oxide and mercuric oxide, in alkaline solutions, and the most important
reactions for sugar are based on this fact.
Trommer's test is based on the property that dextrose possesses of re-
ducing cupric hj^drate in alkaline solution into cuprous oxide. Treat the
dextrose solution with about -|-J vol. caustic soda and then carefully add
a dilute copper-sulphate solution. The cupric hydrate is thereby dissolved,
forming a beautiful blue solution, and the addition of copper sulphate
is continued until a ver}^ small amount of hydrate 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 yello\\ish brown in color, as in Moore's
test; but if an excess of copper salt has been added, the excess of hydrate
is converted on boiling into a dark-brown hydrate which interferes with
the test. To prevent the.se difficulties the so-called Fehlixg'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 (see Quantitative Estimation of Sugar in the Urine in regard to
concentration). This solution is not reduced or noticeably changed by
boiling. The tartrate holds the excess of cupric hydrate in solution, iand an
excess of the reagent does not interfere in the performance of the test. In
the presence of sugar this solution is reduced.
BoTTGER-ALMiix's tcst is based on the property dextrose possesses of
redvicing bismuth oxide in alkaline solution. The reagent best adapted for
this purpose is obtained, according to Nylander's ^ modification of Alm^ix's
original test, by dissolving 4 grams of Rochelle salt m 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 ^^ "^'ol-, 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 j-ellow, then yel-
lowish brown, and finally nearly black, and after a time a black deposit
of bismuth (?) settles.
The property of dextrose 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.
* Zeitschr. f. physiol. Chem., 8.
DEXTROSE. 117
On heating with phenylhydrazixe 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 crj^stalline precipitate
consists of phenylglucosazone (see page 107). This compound melts when
pure at 204-205° C.,. dissolves readily in pyridine (0.25 gram in 1 gram),
and precipitates again from this solution as crystals on the addition of
benzene, ligroin, or ether. According to Neuberg ^ this behavior can be
used in the purification of the osazone.
Dextrose is not precipitated by a lead-acetate solution, but is almost
completely precipitated by a solution of ammoniacal basic lead acetate.
On warming, the precipitate becomes flesh-color or rose-red (Rubner's
reaction^).
If a watery solution of dextrose is treated with hcnzoylchloride and
an excess of caustic soda, and shaken until the odor of benzoylchloride
has disappeared, a precipitate of benzoic-acid ester of dextrose will be pro-
duced which is insoluble in water or alkali (Baumaxx^).
If i-l c.c. of a dilute water}^ 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 c.c. of con-
centrated sulphuric acid (Molisch). According to Reixbold^ 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 raspberr}^-
red to ruby-red coloration.
Diazoeenzexesulphonic 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 towards certain pentose reactions has already been given on page 112.
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 following
simple method of Soxhlet and Tollexs, being a modification of Schwarz's ^
method :
'Ber. d. d. chem. Gesellsch., 32, 3384.
2 Zeitschr. f. Biologic, 20.
^ Ber. d. deutsch. chem. Gesellsch., 19; also Kueny, Zeitschr. f. physiol. Chem., 14,
and Skraup, Wien. Sitzungsber., 98 (1888).
'' Molisch, Monatshefte f. Chem., 7, and Centralbl. f. d. med. Wissensch., 1887,
pp. 34 and 49; Reinbold, Pfiiiger's Arch., 103.
^Tollens, Handbuch der Kohlehydrate, 2. Aufl. I, 39.
118 THE CARBOHYDRATES.
Treat 12 litres 90 per cent alcohol with 480 c.c. 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 teen inverted. To incite crystallization, some crystals of anhydrous
dextrose are added, and after several days the crystals are sucked dr}^ 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 al)Ove-mentioned reduction tests, the optical determination,
fermentation, and phenylhydrazine tests. For the quantitative estima-
tion the reader is referred to the chapter on the urine. Those liquids contain-
ing 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
l^lood and serous fluids we refer the student to the works of Schenck,
RoHMAXN, Abeli^s, and Seegen.^
The guloses are stereoisomers of dextrose and may be prepared artificially.
.rf-Gulose is obtained on the reduction of rf-gulonic acid, which is obtained on the
reduction of glucuronic acid.
Mannoses. — d-Mannose, also called seminose, is obtained with d-levulose
on the careful oxidation of rf-mannite. It is also obtained on the hydrolysis
of natural carbohydrates, such as salep sli meand reserve cellulose (especially
from the shavings from the ivory-nut). It is dextrorotatory, readily fermente
with beer-yeast, gives a hydrazone not readily soluble in water, and an osazone
■"which is identical with that from d-glucose.
Levulose, also called ^-fructose and fruit-sugar, occurs, as above
stated, mixed with dextrose extensively distributed in the vegetable king-
»dom and also in honey. It is formed in the hydrolytic cleavage of cane-
.sugar and several other carbohydrates, but it is especially readily obtained
'.by the hydrolytic splitting of inulin. In extraordinary cases of diabetes
mellitus we find levulose in the urine. Neuberg and Strauss ^ have
'detected le\ailose with positiveness in human blood-serum and exudates in
'Certain cases.
Le\ailose crystallizes with difficulty in needles partly anhydrous and
partly containing water. It is readily soluble in water, but nearly insoluble
in cold absolute alcohol, though rather readily in boiling alcohol. Its
: aqueous solution is levogyrate. Le\mlose 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 dex-
trose compound. Levulose is not precipitated by sugar of lead or basic
lead acetate.
' Schenck, Pfluger's Arch., 40 and 47; Rohmann, Centralbl. f. Physiol., 4; Abeles,
2eitschr. f. physiol. Chem., 15; Seegen, Centralbl. f. Physiol,, 4.
^ Zeitschr. f. physiol. Chem., 36, which also contains the older literature.
LEVULOSE. 119
L€\'iilose does not reduce copper to the same extent as dextrose.
Under similar conditions the reduction relationship of dextrose to le\'ulose
is 100:92.08.
In detecting le\'ulose and those varieties of sugar which yield le\'ulose
on cleavage we make use of the following reaction suggested by Seli-
WANOFF. To a few cubic centimetres of fummg hydrochloric acid, add
an equal volume of water and a small quantity of the sugar solution or
of the solid substance and a few cr\-stals of resorcin 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 Ofxer ^ the
nyxture must not contain more than 12 per cent HCl, and the boiling must
not be continued longer than twenty seconds, otherwise glucose, mannose,
and indeed maltose, may give a similar reaction. R. and 0. Adler^ per-
form the test with glacial acetic acid and a drop of hydrochloric acid and
some resorcin, in which case a reaction with aldoses is not obtained. Seli-
waxoff's reaction, which according to Rosix may be made more delicate
by a combination with the spectroscopic examination, is, as Neuberg ^ has
shown, a general reaction for ketoses.
According to Neuberg,'* methylphenylhydrazine is an excellent sub-
stance to vise for the separation and detection of le\idose, as it gives a
characteristic levulose-methylphenylosazone. This osazone when recr^-s-
tallized from alcohol melts at 153°. It shows a dextrorotation of 1° 40'
when 0.2 gram of the osazone is dissolved in 4 c.c. pyridine and 6 c.c.
absolute alcohol.
Ofxer has made objections to the use of methylphenylhydrazine in the
detection of levulose. He has obtained the osazone from dextrose and
methjdphenylhydrazine, although the osazone is formed much more quickly
with le\n.ilose than with dextrose. Only when the separation of the osazone
cr\'stals with methylphenylhydrazine after the addition of acetic acid
takes place within five hours at ordinar}- temperatures is the presence of
le\T.ilose positively proven (Ofxer ^).
The use of secondary asjTnmetric hydrazines as a general reagent for ketoses
and as a means of separation from aldoses is objected to by Ofxer.
Levulose, as above stated, is best obtained by the hydrolytic cleavage
of inulin, by warming with faintly acidulated water.
Sorbinose (sorbin) is a ketose obtained from the juice of the berry of the
mountain ash under certain conditions. It is cr}'stalline and levogjTate, and
is converted into sorbite by reduction.
' Monatshefte f. Chem., 25.
^ See foot-note 5, p. 112. .
'Zeitschr. f. physiol. Chem., 31; Rosin, ibid., 3S.
* Ber. d. d. chem. Gesellsch., 35; also Neuberg and Strauss, ibid., 36.
^ Ibid., 3", and Zeitschr. f. physiol. Chem., 45.
120 THE CARBOHYDRATES.
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 than dextrose in water. It is dextrogyrate and shows
multirotation. With ordinary yeast the galactose is slowly, but neverthe-
less completely, fermented. It is fermented by a great variety of yeasts
(E. Fischer and Thierfelder), but not by Saccharomyces apiculatus,-
which is of importance in physiological-chemical investigations. Galactose
reduces Fehling's solution to a less extent than dextrose, and 10 c.c.
of this solution are reduced, according to Soxhlet, by 0.0511 gram galac-
tose in 1 per cent solution. Its phenylosazone melts at 193° C, and is
soluble with difficulty in water, but wdth 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 the
pentoses, but the solution does not give the absorption spectrum. On
oxidation it first yields galactonic acid and then mucic acid. Both I- and
?!-galactose have been artificially prepared.
Talose is a sugar which is artificially prepared by the reduction of talonic
acid. Talonic acid is obtained from d-galactonic acid by heating it with quinoline
or pyridme to 140-150° C.
Appendix to the Hexoses.
CH2OH
a -Glucosamine 2 (chitosamine), C6Hi3N05= Att tsttt ^, whose synthet-
COH
ical preparation has already been given on page 108, was first prepared by
Ledderhose 3 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 33 and 65). Glucosamine is, as E.
Fischer and Leuchs"^ have shown, a derivative of glucose or rf-mannose
(probably dextrose), and as an intermediary member between the hexoses
and the oxyamino-acids obtainable from the proteins, it forms in certain
regards a bridge between the proteins and the carbohydrates.
The free base is readily soluble in water with an alkaline reaction and
■See F. Voit, Zeitschr. f. Biologie, 28 and 29.
^ According to E. Fischer's suggestion we shall use the term glucosamine instead
of the term chitosamine which has lately been generally used.
^ Zeitschr. f. physiol. Chem., 2 and 4.
^ Ber. d. d. chem. Gesellsch., 36.
GLUCOSAMINE. GLUCURONIC ACID. 121
quickly decomposes. The characteristic hydrochloride forms colorless
Ctystals which are stable in the air and readily soluble in water, difficultly
soluble in alcohol, and insoluble in ether. The solution is dextrorotaton-,
(a)D=+"0.15° to 74.64°, at various concentrations. ^ Glucosamine has a
reducing action similar to glucose, gives the same osazone, but is not fer-
mentable. With benzoyl chloride and caustic soda it gives a cr\'stalline
ester. In alkaline solution it gives with phenylisocyanate a compound
which can be converted into its anhydride by acetic acid, and is used in
the separation and detection of glucosamine (Steudel^). On oxidation
with nitric acid it yields norisosaccharic acid, whose lead salt can be
separated and whose salts ^N^th cinchonine or quinine are difficultly
soluble in water and can also be used very- successfully in the detection
of glucosamine (Neuberg and Wolff 3). On oxidation with bromine
chitaminic acid (c?-glucosaminic acid) is produced, and this is converted
into chitaric acid, CeHioOe, b}' nitrous acid. On treatment with nitrous
acid glucosamine yields a non-fermentable sugar called chitose.
Ehrlich ■* 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 treating
with hot concentrated hydrochloric acid.^ In regard to its preparation
from protein substances we must refer to the works cited on page 33, foot-
note 1.
Galactosamine has been prepared by Schulz and Ditthorx ^ from a
glucoproteid of the spa\\"n of the frog.
CHO
Glucuronic acid (glycuronic acid), C6Hio07= (CH.0H)4, is a derivative
COOH
of dextrose and has been sjmt helically prepared by E. Fischer and Piloty '
by the reduction of the lactone of saccharic acid. On oxidation with
bromine it forms saccharic acid, and on reduction it yields gidonic-acid
lactone. Salkowski and Neuberg ^ have obtained /-xvdose from glu-
curonic acid by splitting off CO2 by means of putrefaction bacteria.
• See Hoppe-Seyler-Thierfelder's Handbuch, 7. Aufl.; Sundwik, Zeitschr. f. physiol.
Chem., 34.
^ Zeitschr. f. physiol. Chem., 34.
^ Ber. d. d. chem. Gesellsch.. 34.
^ Mediz. Woche, 1901, Xo. 15; see Langstein, Ergebm'sse der Physiol., I, Abt. 1, SS.
* See Hoppe-Seyler-Thierfelder's Handbuch, 7. Aufl.
"Zeitschr. f. physiol. Chem., 29.
' Ber. d. d. chem. Gesellsch., 24.
^Zeitschi. f. phj'siol. Chem., 36.
122 THE CARBOHYDRATES.
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 conjugated
acid after the ingestion of certain aromatic and also aliphatic substances,
especially camphor and chloral hydrate. It was obtained first by
ScHMiEDEBERG and Meyer from camphoglucuronic acid, and then by
V. Merino ^ from urochloralic acid by cleavage with dilute acids. Accord-
ing to P. Mayer,2 on the oxidation of dextrose a partial formation of glu-
curonic 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 glucuronic acids
may also occur in the blood (P. Mayer, Lepine and Boulud^), in
the faeces and in the bile.* Neuberg and Neimann ^ have prepared
certain conjugated glucuronic acids (see Chapter XV) synthetically, among
them being euxanthic acid. The most abundant source of glucuronic 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 solu-
tion is boiled for an hour the acid is partly (20 per cent) converted into
the crystalline lactone, glucurone, CeHgOe, which is soluble in water and
insoluble in alcohol. The alkali salts of the acid are cr>'stalline. If a
concentrated solution of the acid is saturated with barium hydrate the
basic barium salt is obtained as a precipitate. The neutral lead salt is
soluble in water, while the basic salt is insoluble. The readily crystallizable
cinchonine salt can be used in isolating glucuronic acid (Neuberg ^).
Glucuronic acid is dextrorotatory, while the conjugated acids are levo-
rotatory; 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 yield abundant furfurol on distillation
with hydrochloric acid. With the phenylhydrazine test they give crystal-
line compounds which are not sufficiently characteristic (Thierfelder,
P. ]\Iayer'^). By the action of 3 mol. phenylhydrazine and the necessary'
amount of acetic acid upon 1 mol. glucuronic acid at 40° for a few days
' Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29; Schmiedeberg u. Meyer, ibid.,
3; V. Mering, ibid., 6-
2 Zeitschr. f. klin. Med., 47. See Chapter XV.
3 Zeitschr. f. physiol. Chom., 32; Lepine and Boulud, Compt. rend., 133, 134, 138.
* See Bial, Hofrnoister's Beitrilge, 2, and v. Leersum ibid., 3.
'Zeitschr. f. physiol. Chem., 44.
« Ber. d. d. chem. Gesellsch., 33.
'Thierfelder, Zeitschr. f. physiol. Chem., 11, 13, 15; P. Mayer, ibid., 29.
DISACCHARIDES. 123
Neuberg and Neumann obtained the glucuronic-acid osazone, which was
very similar to glucosazone and melted at 200-205°. With 2>bromphenyl-
hydrazine hydrochloride and sodium acetate, glucuronic acid gives 7>brom-
phenylhydrazine glucuronate, which is characterized by insolubility in
absolute alcohol and by a very prominent levorotatory action. This com-
pound is very well suited for the detection of glucuronic acid.^ Dissolved
in a mixture of alcohol and pyridine (0.2 gram substance in 4 c.c. pyri-
dine and 6 c.c. alcohol) the rotation is 7° 25', which corresponds to
(«)f=-369°.
Glucuronic acid is best prepared from euxanthic acid, which decomposes
by 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 Neimann.^
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 complicated carbohydrates. Isomaltose is besides
this also obtained from dextrose by reversion (see page 125).
The disaccharides or hexobioses are to be considered as anhydrides,
derived from two monosaccharides with the exit of 1 molecule of water.
Corresponding to this, their general formula is Ci2H220n. On hydrolytic
cleavage and the addition of water they yield 2 molecules of hexoses,
either 2 molecules of the same hexose or one each of two different hexoses.
Thus
Saccharose + H2O = dextrose + le\ailose ;
Maltose 1 -1- H2O = dextrose -f- dextrose ;
Lactose + H2O = dextrose -|- galactose.
The levulose turns the polarized ray more to the left than the dextro.se
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
accovmt 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
'See Neuberg, Ber. d. d. chem. Gesellsch., 32, and Mayer and Neuberg, Zeitschr.
f. physiol. Chem., 29.
^ Tollens, Zeitschr. f. physiol. Chem., 44, which cites also the older work; Neu-
berg and Neimann, ibid., 44; Neuberg, ibid., 45.
124 THE CARBOHYDRATES.
compound sugars into monosaccharides. The reverse reaction, whereby
monosaccharides are condensed into complex carbohydrates, is called
reversion.
We subdivide the disaccharides into two groups: first, the group to
which saccharose belongs, where the members do not have the property of
reducing certain metallic oxides; and the second group, to which tlie 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.
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 extraordinarily 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 Scheibler ^ 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 rotation is (a)D= -1-66.5°.
Saccharose acts indifferently towards Moore's test and to the ordinary
reduction tests. 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. Con-
centrated 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 material 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 glycogen
under the same conditions (see Chapter VIII). Maltose is also produced
transitorily in the action of sulphuric acid on starch. ]\laltose forms the
fermentable sugar of the potato or grain mash, and also of the beerwort.
^laltose cr^^stallizes with 1 molecule water of crystallization in fine
white needles. It is readily soluble in water, rather easily in alcohol, but
'See Tollens' Handbuch der Kohlehydrate, 2, Atifl. 1, 124.
ISOMALTOSE. LACTOSE. 125
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.^ 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
1-^- hours. This phenylmaltosazone melts at 206° C. and is more soluble
than the glucosazone. Maltose differs from dextrose chiefly in the follow-
ing: 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
c.c. Fehling's solution is, according to Soxhlet,^ 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,^ is
produced, besides 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 Emmerlinc*). 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 maltose.^ It is also produced,
with maltose, by the action of saliva or pancreatic juice (KtJLZ and Vogel)
or blood-serum (Rohmann ^) 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 mal-
tose. 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.
It is rather easily soluble in hot water and dissolves 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).
* See Hoppe-Seyler-Thierfelder's Handbuch, 7. Aufl.
^ Cited from Tollens' Handbuch der Kohlehydrate, 2. Aufl. 1, 154.
^ Ber, d. deutsch. chem. Gesellsch., 23 and 28.
■"Emmerling, ibid., 34; Hill, ibid., 34, and 1. c., foot-note 1, p. 16.
^ 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.
° Kiilz and Vogel, Zeitschr. f. Biologic, 31; Rohmann, Centralbl. f. d. med. Wis-
sensch., 1893, 849.
126 THE CARBOHYDRATES.
Polysaccharides.
If we exclude the hexotrioses and the few remaining sugar-Uke poly-
saccharides, 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 ordmary 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 are neither dissolved
nor visibly changed. Polysaccharides are ultimately converted into mono-
saccharides by hydrolytic cleavage.
The polysaccharides (not sugar-like) are ordinarily divided into the
following chief groups: starch group, gum and vegetable-mucilage group,
and cellulose group.
Starch Group, (C6H]o05)x.
Starch, amylum, (C6Hio05)x. This substance occurs in the plant king-
dom very extensively distributed in the different parts of the plant, espe-
cially as reserve food in the seeds, 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. According to the ordinar}^ opinion the starch granules
consist of two different substances, starch granulose and starch cellu-
lose (v. Nageli), corresponding to ]\Iaquexne and Roux's ^ amylose and
amylopectin, of which the first alone is converted into sugar on treatment
with diastatic enzymes.
Starch is considered insoluble in cold water. The grains swell up in
warm water and burst, yielding a paste. Starch is insoluble in alcohol and
ether. On heating starch with water alone, or heating with glycerine to
190° C, or on treating the starch grains with 6 parts dilute hydrochloric
acid of sp. gr. 1.07 at ordinary temperature for six to eight weeks,^ it is
converted 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.^
Starch granules swell up and form a pasty mass in caustic potash or
soda. This mass gives neither ^Ioore's nor Trommer's test. Starch
paste does not ferment with yeast. The most characteristic test for starch
* V. Nageli, Botan. Mitteil,, 1863; Maquenne and Roux, Compt. rend., 140, and
Bull. Soc, chim. de Paris (3), 33.
^ See ToUens' Handb., 191. In regard to other methods, see Wroblewsky, Ber. d.
deutsch. chem. Gesellsch., 30; Syniewski, ibid.
^ In regard to the compounds of soluble starch and dextrins with barium hydrate,
see Biilow, Ffliiger's Arch., 62.
STARCH. INULIN. 127
is the blue coloration produced by iodine in the presence of hydriodic
acid or alkali iodides.^ 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
met hod, 2 by converting it into dextrose by hydrochloric acid and then
determining the dextrose by the ordinary methods.
Inulin, (C6Hio05)x + H20, occurs in the underground parts of many
compositiE, especially in the roots of the Inula helenium, the tubers of the
dahlia, the varieties of helianthus, etc. It is ordinarily obtained from the
tubers of the dahlia.
Inulin forms a white powder similar to starch, consisting of spheroid
crystals which are readily soluble in warm water, without forming a paste.
It separates slowly on cooling, but more rapidly on freezing. Its solutions
are levogyrate and are precipitated by alcohol, and are colored only yellow
with iodine. Inulin is converted into the levogyrate monosaccharide levu-
lose on boiling with dilute sulphuric acid. Diastatic enzymes have no or
only very slight action on inulin .^
According to Dean ^ inulin occurs together with other substances, levulins,
which are more soluble and have less rotation. He suggests that we limit the
name inulin to that of carbohydrate (or mixture of carbohydrates), which is
readily precipitable by 60 per cent alcohol and shows a specific rotation 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 ^).
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).
' See Mylius, Ber. d. deutsch. chem. Gesellsch., 20, and Zeitschr. f. physiol. Chem., 11.
2Tollens' Handb., 2. Aufl. 1, 187.
Ubid., 208.
^ Amer. Chem. Journ., 32.
'Upsala Lakaref. Forh., 28.
128 THE CARBOHYDRATES.
The Gums and Vegetable Mucilages, (C6Hio05)x.
These bodies may be di^'ided into two chief groups, accordmg to their
origin and occurrence, namely, the dextrin group and the vegetable gums or
mucilages. The dextrins stand in close relationsliip to the starches and
are formed therefrom as intermediate products in the action of acids and
diastatic enzymes. The various kinds of vegetable gums and vegetable
mucilages occur, on the contrar}^, as natural products in the vegetable
kingdom, and some may be separated from certain plants as amorphous,
transparent masses, and others may be extracted from certain parts of the
plant, such as the wood and seeds, by proper solvents.
The dextrins yield as final products only hexoses, indeed only dex-
trose, on complete hydrolysis. The vegetable gums and the mucilages
yield, on the contrarj% not only hexoses, but also an abundance of pentoses
(gum arabic and wood-gum). d-Galactose occurs often among the hexoses,
and accordingly as a differentiation from the dextrins, they yield mucic
acid on oxidation with nitric acid. The dextrins, as svell as the ordinary
varieties of gums and mucilages, are precipitated by alcohol. Basic lead
acetate precipitates the gums and mucilages, but not the dextrins.
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.
We are not quite clear in regard to the steps taking place in the above
processes, but the ordinary views are as follows: The first product, which
gives a blue with iodine, is soluble starch or amylodextrin, which on further
hydrolytic cleavage yields sugar and erythrodextnn, which is colored red
by iodine. On further cleavage of this erj-throdextrin more sugar and
a dextrin, achroodextrin, which is not colored by iodine, are formed. From
this achroodextrin after successive splittings we have sugar and dextrins
of lower molecular weights formed, until finally we have sugar and a dextrin,
maltodextrin, which refuses to split further, as final products. The views
are rather contradictory in regard to the number of dextrins which occur
as intermediate steps. The sugar formed is isomaltose, from which mal-
tose and only verv' little dextrose are produced. Another \'iew is that
first several dextrins are formed consecutively in the successive splittings,
with hydration, and then finally the sugar is formed by the splitting of
the last dextrin. According to Moreau, in the first stages of saccharifica-
tion amylodextrin, ery^throdextrin, achroodextrin and sugar are formed
simultaneously. Other investigators, especially Syniewski, have recently
suggested other views on this subject.^
' In regard to the various views on the theories of the saccharification of starch,
CELLULOSE. 129
The various dextrins have not as yet been separated from each other,
nor isohited as chemical individuals. Recently Youxo ^ has tried their
separation by means of neutral salts, especially ammonium sulphate, and
MoREAU by the aid of a bar\-ta-alcohol method. We cannot enter into
the differences as to the dextrins so separated, and only the characteristic
properties and reactions will be given for the dextrins in general.
The dextrms appear as amorphous, white or yellowish-white powders
which are readily soluble in water. Their concentrated solutions are \'iscid
and stick}', similar to gum solutions. The dextrins are dextrog}'rate.
They are insoluble or nearly so in alcohol, and insoluble in ether. Waters-
solutions of dextrins are not precipitated by basic-lead acetate. Dextrins
dissolve cupric hydrate in alkaline liquids, forming a beautiful blue solu-
tion, which, as is generally admitted, is reduced by pure dextrins. Accord-
ing to !\IoREAU 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
those varieties of gum which do not or onh' 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 arable, wood-gum, cherry-gum, salep, and Cjuince 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, 'C6Hio05)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 lea.st the walls of the young cells, while in the
walls of the older cells the cellulose is extensively incmsted with a sub-
stance called LiGxix.
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 ammoniacal
solution of copper oxide called Schweitzer's reagent. The cellulose may
be precipitated from this solvent by the addition of acids, and obtained as
an amorphous powder after washing with water.
Cellulose is converted into a substance, so-called amyloid, which gives
a blue coloration with iodine by the action of concentrated sulphuric acid.
see Musculus and Gruber, Zeitschr. f. physiol. Chem., 2; Lintner and Didl, Ber. d. d.
chem. Gesellsch., 26 and 2S; Biilow, 1. c; Brown and Heron, Journ. of Chem. Soc, 1879;
Brown and Morris, ibid., 1885 and 1889; Moreau, Biochem. Centralbl., 3, 648; Sy-
niewski, Annal. d. Chem. u. Pharm., 309, and Chem. Centralbl., 1902, 2.
* Journ. of Physiol., 22, which contains the older researches of Nasse, Kriiger,
Neumeister, Pohl, and Halliburton. Moreau, 1. c.
130 THE CARBOHYDRATES.
B}^ the action of strong nitric acid or a mixture of nitric acid and concen-
trated sulphuric acid celluloses are converted into nitric-acid esters or nitro-
celluloses, 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. We also have celluloses which behave
differently, namely, those which yield mannose on the above treatment.
Hemicelluloses are, according to E. Schui-ze, those constituents of the
cell-wall related to cellulose which differ from the ordinary cellulose by dissolv-
ing on heating with strongly diluted mineral acids, such as 1.25 per cent sulphuric
acid, and of yielding arabinose, xylose, galactose, and mannose instead of dextrose.
The hemicelluloses (from lupin seeds) are hydrolized even by 0.1 per cent hydro-
chloric acid and are dissolved, although only slowly, by diastatic enzymes
(ScHULZE and Castoro ')•
The cellulose, at least in part, undergoes decomposition in the infest iral
tract of man and animals. A closer discussion of the nutritive value of
cellulose will be given in a future chaj^ter (on digestion). The great im-
portance of the carbohydrates in the animal economy and to animal metab-
olism will also be given in the following chapters.
' E. Schulze, Zeitschr. f. physiol. Chem., 16 and 19, with Castoro, ibid., 36.
CHAPTER IV.
THE ANIMAL FATS.
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 contains 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 con-
nective tissues. In plants, the seeds and fruit and in certain instances
also the roots, are rich in fat.
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, glycerine, with monobasic fatty acids. These esters are triglycerides,
that is, the hydrogen atoms of the three hydroxyl groups of the glycerine
are replaced by the fatty-acid radicals, and their general formula is there-
fore C3H5O3.R3. The animal fats consist chiefly of esters of the three fatty
acids, stearic, palmitic, and oleic acids. In certain fats, especially in milk-
fat, glycerides of fatty acids such as butyric, caproic, caprylic, and capric
acids also occur in considerable amounts. Besides the above-mentioned
ordinaiy fatty acids, stearic, palmitic, and oleic acids, we also find in human
and animal fat, exclusive of certain fatty acids only little studied, the fol-
lowing non-volatile fatty acids, as glycerides, namely, lauric acid, C12H24O2,
myristic acid, C14H28O2, and arachidic acid, C20H40O2. In the plant king-
dom 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 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,
' Erben, Zeitschr. f. physiol. Chem., 30; Bernert, Arch. f. exp. Path. u. Pharm., 49.
131
132 THE ANIMAL FATS.
althoii,2;h ordinarily only in small amounts, has on the contrary been posi-
tively 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 cent.
It must be remarked that in animal fat (mutton and beef tallow) as well
as in plant fat (olive-oil) mixed triglycerides, such as dipalmityl-olein,
distearyl-palmitin and distearyl-olein, occur and that these mixed glycerides
may also be prepared synthetically.^
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 tripalmitin and 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 .2 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.
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, and chloroform. The fluid neutral fats give an emulsion
when shaken \\ith a solution of gxim or albumin. With water alone they
give an emulsion only after vigorous and prolonged shaking, but the
emulsion is not persistent. 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 decomposition, and bums 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 glycerine, C3H5(0H)3 — 2H20 = C3H40, when heated alone, or more
*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.
^ See Knopfelmacher, "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.
STEARIN. 133
easily when heated with potassium bisulphate or with other dehydrating
substances.
The neutral fats may be spUt by the addition of the constituents of
water according to the following equation : C3H5(OR)3 + 3HoO = C3H5(OH)3
+ 3H0R. This splitting may be produced by the pancreatic enzyme and
other enzymes occurring in the animal and vegetable kingdoms, or by
superheated steam. We most frequently decompose the neutral fats by
boiling them with not too concentrated caustic alkali, or, still better (in
biochemical researches), with an alcoholic potash solution or -uith 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 pro-
duced. By saponification is to be understood not only the cleavage of
neutral fats by alkalies, but also the splitting of neutral fats into fatty
acids and glycerine 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 mto fatty acids and gl3'cerine, 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 kmgdom are stearin,
palmitin. and olein.
CH2O.C18H35O
Stearin or tristearm, CoyHnoOe^CHO.CisHsoO, occurs especially in
CH2O.C18H30O
the solid varieties of tallows, but also in the vegetable fats. Stearic acid,
C18H36O2, is foimd 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 excrements 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 "U'ith great difficult}'
in cold ether (225 parts). It separates from warm alcohol on coolmg as
rectangular, less frequenth^ as rhombic plates. The statements in regard
to the melting-point are somewhat varied. Rire stearin, accordmg to
Heixtz.i melts transitority at 55° and permanently at 71.5°. The stearin
from the fatty tissues (not pure) melts at 63° C.
CH3
Stearic acid, (CH2)i6. cr\'stallizes (on cooling from boiling alcohol) m
COOH
* Annal. d. Chem. u. Pharm., 92
134 THE ANIMAL FATS.
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.
CHaO.CieHsiO
Palmitin, or tripalmitin, C5iH9806 = CHO.Ci6H3iO. Of the two solid
CH2O.C16H31O
varieties of fats, palmitin is the one which occurs in predominant quan-
tities in human fat (Langer i). 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, C16H32O2,
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 expectorations 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 palmitin and
stearin, called margarin, crystallizes, on cooling from a solution, as balls or
round masses which consist of short or long, thin plates or needles which
often appear like blades of grass. Palmitin, like stearin, has a variable
melting and solidifying point, depending upon the way it has been pre-
viously treated. The melting-point is often given as 62° C. According
to other statements,^ it melts at 50.5° C, solidifies on further heating,
and melts again at 66.5° C.
CH3
Palmitic acid, (CH2)i4, crystallizes from an alcoholic solution in tufts
COOH
of fine needles. It melts at 62° C; still the admixture mth .stearic acid,
as Heixtz has shown, essentially changes the melting- and solidify ing-points
according to the relative amounts of the two acids. Palmitic acid is .some-
what more soluble in cold alcohol than stearic acid; but they have about
the same solubility in boiling alcohol, ether, chloroform, and benzene. Its
barium salt contains 21.17 per cent barium, and the silver salt contains
29.72 per cent silver.
CH2O.C18H33O
Olein, or triolein, C57Hio406 = CHO.Ci8H330, is present in all animal
CH2O.C18H33O
fats, and in greater quantities in vegetable fats. It is a solvent for stearin
and palmitin. The oleic acid (elaic acid), C18H34O2, has as soaps probably
about the same occurrence as the other fatty acids.
Olein is, at ordinary temperatures, a nearly colorless oil of a specific
' Monat.shefte f. Chcm., 2; see also Jaeckle, Zeitschr. f. phy.siol. Chem., 36.
' R. Benedikt, Analyse der Fette, 3. Aufl., 1897, p. 44.
OLEIC ACID. 135
gravity of 0.914, without odor or marked taste, and solidifies in cr}'staliine
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, bv nitrous acid.
CHs
(CHo)7
/iTT
Oleic acid, -„ , forms on heating, besides volatile acids, sebacic acid,
m-2)7
COOH
CioHi804, which crv'stallizes in shining leaves and melts at 127° C. With
nitrous acid oleic acid is transformed into the isomeric solid elaidic acid,
which melts at 45° C. Oleic acid forms at ordmary- temperature a colorless,
tasteless, and odorless oily liciuid which solidifies in crystals at about 4° C,
which then melt again at 14° C. Oleic acid is insoluble in water, but dis-
solves in alcohol, ether, and chloroform. 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. Oleic acid is an unsaturated fatty acid which can take up halogens.
Chi heating with hydriodic acid and amorphous phosphorus it takes up
hydrogen and is converted into stearic acid. Oleic acid readily oxidizes
in the air, yieldmg acid products. The monoxystearic acid found in
certain animal fats may be formed from oleic acid by oxidation. 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 towards
ether and benzene is made use of in separating oleic acid from the other
fatty acids.
An acid related to oleic acid, doeglic acid, which is solid at 0° C, liquid at
16° C, and soluble in alcohol, is found in the blubber of the Baloena rostrata.
KrRBATOFF ' has demonstrated the presence of I'noleic acid in the fat of the silurus,
sturgeon, seal, and certain other animals. Drying fats have also been found by
Amthor and Zink ^ 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 the acrolein test must not be neg-
lected. If this test gives positive results, then neutral fats are present;
if the results are negative, then only fatty acids are present. If the above
» Maly's Jahresber., 22. ' Zeitschr. f. anal>^. Chem., 36.
136 THE ANIMAL FATS.
residue after evaporation gives the acrolein test, then a small portion is
dissolved in alcohol-ether free from acid and which has been colored bluish
violet by tincture of alkanet. if the color becomes red, a mixture of
neutral fat and fatty acids is present. In this case the fat is treated while
warm with a soda solution and evaporated on the water-bath, with constant
stirring until all the water is removed. The fatty acids hereby combine
with the alkali, forming soaps, while the neutral fats are not saponihed
under these conditions. If this mixture of soaps and neutral fats is
treated with water and then shaken with pure ether, the neutral fats are
dissolved, while the soaps remain in the watery solution. The fatty acids
may be separated from this solution by the addition of a mineral acid
which sets the acid free.
The neutral fats separated from the soaps by means of ether are often
contaminated with cholesterin, which must be separated in quantitative
determinations by saponification with alcoholic caustic potash. The
cholesterin is not attacked by the caustic alkali, while the neutral fats
are saponified. After the evaporation of the alcohol the residue is dissolved
in water and shaken with ether, which dissolves the cholesterin. The fatty
acids are separated from the watery solution of the soaps by the addition
of a mineral acid. If a mixture of soaps, neutral fats, and fatty acids is
originally present, it is treated first with water, then agitated with ether
free from alcohol, which dissolves the fat and fatty acids, while the soaps
remain in the solution, with the exception of a very small amount which is
dissolved by the ether.
To detect and to separate the different varieties of neutral fats from
each other it is best first to saponify them with alcoholic potash, or still
better with sodium alcoholate, according to Kossel, Obermuller, and
Kruger.i After the evaporation of the alcohol the salts of the fatty acids
are dissolved in water and precipitated with sugar of lead. The lead oleate
is then separated from the other two lead salts by repeated extraction with
ether, but it must be remarked that the lead salts of the other fatty acids
are not quite insolu])le in ether. The residue insoluble in ether is decom-
posed on the water-bath with an excess of soda solution, evaporated to
dryness, finely pulverized, and extracted with boiling alcohol. The alco-
holic solution is then fractionally precipitated by barium acetate or bariimi
chloride. In one fraction the amount of barium is determined, and in the
other the melting-point of the fatty acid set free by a mineral acid. The
fatty acids occurring originally in the animal tissues or fluids as free acids
or as soaps are converted into barium salts and investigated as above.
According to Jaeckle,^ it is better to isolate the fatty acids as silver salts.
This same experimenter also considers it more advisable to dissolve the lead
salts in warm benzene, as suggested by Farnsteiner, and to obtain the
crystalline lead salts of the solid fatty acids by cooling.
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 and is determined by
titrating the fat dissolved in alcohol-ether with N/10 alcoholic caustic potash,
usmg phenolphthalein as indicator. 2. The sajwnification equivalent, which gives
^ Zeitschr. f. physiol. Chem., 14, 15, and IQ.
2 Ibid . 36.
INVESTIGATIOXS OF FATS. 137
the milligrams of caustic potash united -with the fatty acids in the saponification
of 1 gram fat with X/2 alcoholic caustic potash. 3. Reichert-Meissl's equiva-
lent, which gives the quantity of volatile fatty acids contained in a given amount
of neutral fat (5 grams). The fat is saponified, then acidified with mineral acid
and distilled, whereby the volatile fatty acids pass over and the distillate is
titrated with alkali, -i. ludine equivalent is the cjuantity of iodine absorbed by a
certain amount of the fat by addition. It is chieflj' a measure of the quantity
of unsatm-ated 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. Oxyacids, alcohols such as cetyl alcohol or cholesterin,
and those constituents of fats containing the OH group are transformed into the
corresponding acetyl ester on boiling v>'ith acetic anhydride, while the fatty acids
remain unchanged, and in this way the estimation of these bodies is possible. The
fat is saponified, the soaps decomposed by an excess of acid, and the mixture
of fatty acids, oxyfatty acids, cholesterin, etc., boiled with acetic anhydride.
The acid equivalent is determined in a weighed part of the carefully washed
acetic-acid-free mixture by titration with alcoholic caustic potash. This acid
equivalent represents all the acids (fatty acids as well as the acetylated oxyacids),
and it is designated the acetyl-acid equivalent. The neutral fluid is now titrated
with an exactly measured, sufficient quantity of the same alkali and the acetyl
compounds saponified by boiling. On retitrating we find the quantity of alkali
used in saponification, and this number, calculated to 100 parts of the fat, repre-
sents the acetyl equivalent. In regard to the performance of the above-mentioned
different estimations we must refer the reader to more complete works, such as
"Analysis of Fats and Waxes," R. Bexedikt, 1897.
In the quantitative estimation of fats the finely divided dried tissues
or the finely divided residue from an evaporated fluid is extracted Anth
ether, alcohol-ether, benzene, or any other proper extraction medium. The
investigations of Dormeyer ^ and others, carried on in PpLiJGER's labora-
toiy . have shouii that even ^\-ith veiy prolonged extraction with ether all the
fat is not extracted. First extract the greater part of the fat b}^ ether.
Then digest with pepsin-hydrochloric acid, collect the insoluble residue on
a filter, dr\', and extract with ether. The fat is extracted from the filtrate
by shaking uith ether, evaporatmg the extract and the fat separated from
other bodies by extracting the residue with petroleum ether. Lecitliui
and other bodies are dissolved by the various solvents, hence the results
for the fats may be too high. This is especially the case on using the saponi-
fication method - suggested by Liebermaxx and Szekely, whereby the leci-
thins as well as the fats are saponified. Glikix 3 recommends as the best
procedure the extraction with boiling petroleum ether and the removal of
the lecithin by acetone, in which it is insoluble.
The fats are poor in oxygen, but rich in carbon and hydrogen. They
therefore represent a large amount of chemical potential energy, and yield
correspondingly large quantities of heat on combustion. The}^ take first
^ On fat extraction for quantitative estimation see Dormeyer, Pfliiger's Arch., 61
and 65; Bogdanow, ibid., 65, 6S, and Arch. f. (Anat. u.) physiol., 1897, 149; N. Schulz,
Pfliiger's Arch., 66; Voit and Krummacher, Zeitschr. f. Biologie, 35; O. Frank, ibid.,
35, Polimanti, Pfliiger's Arch., 70; J. Nerking, ibid., 71.
^ Pfliiger s Arch., 72, and Liebermann, ibid., lOS.
'' Ibid. , 95.
138 THE AXIMAL FATS.
rank among the foods in this regard, and are therefore of \evy great
importance in animal life. We will speak more in detail of this signifi-
cance, also of fat formation and of the beha\'ior of the fats m the body, in
the following chapters.
The LECITHINS, which stand in close relationship to the fats, will be
treated in a subsequent chapter (Y). The following bodies are related
to the ordinary- animal 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, CjHas.OH, methal, C,4H29.0H, and stethal, CisH3;.0K.
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 w^ater, but dissolves
easily in cold ether or volatile and fatty oils. It dissolves in boiling alcohol,
but crystallizes on cooling. It is saponified with diSiculty by a solution of caustic
potash in water, but with an alcoholic solution it saponifies readily and the above-
mentioned alcohols are set free.
CH3
Ethal or cetyl alcohol, CeHg^O = (CH2)i4, which occurs in smaller quar titles
CH2.OH
in beeswax, and was found by Ludwig and v. Zeynek ' in the fat from dermoid
cysts, 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 valerianic acid, small amounts of
solid fatty acids, and physetoleic acid. This acid, which has, like hypogaeic
acid, the composition CibH»)02, occurs also, as found by Ljubarsky,^ 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, CjeHsjOj,^ which occurs as cetyl
ether in Chinese wax and as free acid in ordinary w^ax. It dissolves in boiling
alcohol and separates as crystals on cooling. The cooled alcoholic extract of
wax contains (2) cerolein, which is probably a mixture of several bodies, and
(3) MYRiciN, which forms the chief constituent of that part of w-ax which is in-
soluble in warm or cold alcohol. Myricin consists chiefly of palmitic-acid ester
of melissji (myricyl) alcohol, C^ Hgi.OH. This alcohol is a silky, shining, crys-
talline body melting at 85° C.
' Zeitschr. f. physiol. Chem., 23.
2 Journ. f. prakt. Chem. (N. F.), 57.
'See Henriques, Ber. d. deutsch. chem. Gesellsch., 30, 1415.
CHAPTER V.
THE ANIMAL CELL.
The cell is the unit of the manifold variable forms of the organism; it
forms the simplest physiological apparatus, and as such is the seat of
chemical processes. It is generally admitted that all chemical processes
of importance do not take place in the animal fluids, but transpire in the
cells, hence the cell may be considered as the chemical laboratory of the
organism. It is also principally the cells wliich, through their greater or
less activity, regulate or govern the range of the chemical processes, and
also the extensiveness of the total exchange of material.
It is natural that the chemical investigation of the animal cell should
in most cases be in reality a study of those tissues of which it forms
the chief constituent. Only in a few cases can the cells, by relatively
simple manipulations, be directly isolated in a rather pure state from the
tissues, as, for example, in the investigation of pus or of tissues very rich
in cells. But even in these cases the chemical investigation may not lead
to any positive results in regard to the constituents of the uninjured living
cells. By the process of chemical transformation new substances may be
formed on the death of the cell, and at the same time physiological con-
stituents of the cell may be destroyed or transported into the surrounding
medium and therefore escape investigation. For this and other reasons
we possess only a veiy limited knowledge of the constituents and the com-
position of the cell, especially of the living one.
While young cells of different origin in the early period of their exist-
ence may show a certain similarity in regard to form and chemical com-
position, they may, on further development, not only take the most varied
forms, but may also offer from a chemical standpoint the greatest diversity.
As a description of the constituents and composition of the different cells
occurring in the animal organism is nearly equivalent to a demonstratioQ
of the chemical properties of most animal tissues, and as this exposition
will be found in the corresponding chapters, we ^^dll here discuss only the
chemical constituents of the young cells or cells in general.
In the study of these const Huerts we are confronted with another
difficulty, namely, we must different iate by chemical research between
139
140 THE ANIMAL CELL.
those constituents which are essentially necessar}- for the life of the cells
and those which are casual, i.e., stored up as reserve material or as meta-
bolic products. In tliis connection we have only been able, thus far, to
learn of certain substances wliich seem to occur in every developing cell.
Such bodies, called pri^l\ry by Kossel,i are, besides water and certain
mineral constituents, proteins, nucleoproteids or nucleins, lecitliins, gly-
cogen (?), and cholesterin. Those bodies which do not occur in every
developing cell are called secondary. Among these we have fat, gly-
cogen (?), pigments, etc. It must not be forgotten that it is still possible
that other primar}- cell constituents may exist, as yet unkno\\'n to us, and
we also do not know whether all the primary constituents of the cell are
necessary' or essential for the life and functions of the same.
,\nother important question is the division of the various cell constit-
uents 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; ne^■ertheless it is appropriate to differentiate between
the protoplasm and the nucleus.
The Protoplasm of the developing cell consists during life of a semi-
solid mass, contractile under certain conditions and readily changeable,
which is rich in water and whose cliief portion consists of protein sub-
stances, i.e., of colloids. If the cell be deprived of the physiological con-
ditions of life, or if exposed to destmctive exterior influences, such as the
action of high temperatures or of chemical agents, the protoplasm dies. The
protein bodies which it contains coagulate at least partially, and other
chemical changes are found to take place. The alkaline reaction (litmus)
of the living cell may become acid by the appearance of paralactic acid,
and the carbohydrate, glycogen, which habitually occurs in manj^ cells,
may after their death be quickly changed and consumed.
The question as to the internal structure of the protoplasm is still in
controversy. It is of little importance in the study of the chemical com-
position of the cells, as it is impossible to study, especially by chemical
means, the morphologically different constituents of the protoplasm. With
the exception of a few microchemical reactions the chemical analysis has
been restricted to the protoplasm as such, and the investigations have
been directed in the first place to the protein substances which form the
chief mass of the protoplasm.
The proteins of the protoplasm consist, according to the older general
view, chiefly of globulins. Albumins have also been found besides the
globulins. There is no doubt at present that the albumins occur in the
Cells only as traces, or at least only in trifling quantities. The presence of
globulins can hardly be disputed, although certain cell constituents de-
* Verhandl. d. physiol. Geselisch. zu Berlin, 1S90-91. Nos. 5 and 0.
PROTEINS OF THE CELL. 141
scribed as globulins have been sho^\^l on closer investigation to be
nucleoalbumins or nucleoproteids. According to Halliburton ^ the
protein occurring in all cells and coagulating at 47-50° C. is a true
globulin.
In opposition to the ^dew that the chief mass of the animal cell consists
of true proteicls, Hammarstex^ expressed the opinion several years ago
that the chief mass of the protein substances of the cells does not consist
of proteids in the ordmary sense, but consists of more complex phosphor-
ized bodies, and that the globulins and albumms are to be considered as
nutritive material for the cells or as destructive products in the chemical
transformation of the protoplasm. This view has received substantial
support by investigations within the last few years. Alex. Schmidt^ has
come to the \'iew, by investigations on various kinds of cells, that they
contain only ver\' little proteid, and that the chief mass consists of very
complex protein substances.
The protein substances of the cells consist chiefly of compound proteids,
and these are divided between the glucoproteid and the nucleoproteid
groups. It is impossible at present to state to what extent nucleoalbumuis
exist in the cells, because thus far in most cases no exact difference has been
made between them and the nucleoproteids. Hoppe-Seyler "* calls vitelUn
a regular constituent of all protoplasm. This body used to be considered
as a globulin, but later researches have sho\Mi that the so-called \'itellin
bodies may be of various kinds. Certain \'itellins seem to be nucleo-
albimiins, and it is therefore \ery probable that cells habitually contain
nucleoalbumins.
The nucleoproteids take a verj- prominent place among the compound
proteids of the cell. The various substances isolated by different investiga-
tors from animal cells, such as tissue-fibrinogen (Wooldridge), cytoglobin
and prdglobulin (Alex. Schmidt), or nucleohistone (Kossel and Liliex-
FELD^), belong to this group. The cell constituent which swells up to a
sticky mass ^\ith common salt solution and is called Ro^^DA's hyaline sub-
stance also belongs to this group.
The above-mentioned different protein substances have simply been
designated as constituents of the cells. The next question is which of
these belong to the protoplasm and which to the nucleus. At present
we can give no positive answer to this question. According to Kossel
' See Halliburton, On the Chemical Physiology of the Animal Cell, 1893, No. 1,
King's College Physiol. Laboratory.
2 Pfliiger's Arch., 36, 449.
3 Alex. Schmidt, Zur Blutlehre, Leipzig, 1892.
^Physiol. Chem., 1877-1881, 76.
* See L. C. Wooldridge, Die Gerinnung des Blutes, Leipzig, 1891; A. Schmidt,
Zur Blutlehre; Lilienfeld, Zeitschr. f. physiol. Chem., 18.
142 THE ANIMAL CELL.
and LiLiENFELD,^ the cell-nucleus of the leucocytes of the thymus gland
contains a nucleoproteid as chief constituent, besides nucleins, and some-
times perhaps also nucleic acid (see below), while the body of the cells
contains chiefly pure proteids, besides other substances, and a nucleo-
proteid, containing only a very small quantity of phosphorus. As the
lymphocytes of the thymus gland of the calf contain only one nucleus, in
which the mass of the nucleus surpasses that of the cytoplasm, it is natural
that the relative proportion of the various protein substances in these cells
cannot be taken as a standard for the composition of other cells richer in
cytoplasm.
Complete investigations in regard to the distribution of protein sub-
stances in the protoplasm and nucleus of other cells have not been made.
If we consider for the present that the cells rich in protoplasm contain, as
a rule, only ver}' little true proteid, we are hardly wrong in considering it
probable that the protoplasm contains chiefly nucleoalbumins and com-
pound proteids besides traces of albumin and a little globulin. These com-
pound proteids are in certain cases glucoproteids, but otherwise nucleo-
proteids, which differ from the nucleoproteids of the nucleus in being
poorer in phosphorus, besides containing a great deal of proteid and only
a little of the prosthetic group, and hence have no specially pronounced
acid character.
The nucleoproteids of the nucleus are on the contrar}^, as shown by
LiLiENFELD and KossEL, rich in phosphorus and of a strongly acid charac-
ter. These nucleoproteids will be treated in speaking of the nucleic acids
of the nucleus.
In cases in which the protoplasm is surrounded by an outer, condensed
layer or a cell membrane, this envelope seems to consist of albuminoid
substances. In a few cases these substances seem to be closely related to
elastin; in other cases, on the contrary, they seem rather to belong to the
keratin group. Even in cells w-hich do not seem to have any visible special
layers forming boundaries, we still admit of such layers on account of the
behavior of the cells as regards permeability.
Nernst^ has shown by a special experiment that the permeability of a
membrane for a certain substance is essentially dependent upon the sol-
vent 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.^ The behavior of the
living cells towards dyestuffs, also the ready introduction into animal
' Ueber die Wahlverwandtschaft der Zellelemente zu gewissen Farbstoffen, Ver-
handl. d. physiol. Gesellsch. zu Berlin, No. 11, 1893.
^ Zeitschr. f. physikal. Chem., 6.
' Vierteljahrsschr. d. Naturf. Ges. in Zurich, 44 (1899), and Overton, Studien iiber
die Narkose, Jena, 1901.
LECITHLXS. 143
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., with lecithins, cholesterin, and bodies similar
to protagon, and among which lecithin, which also takes up water, must
be of the greatest importance.
The cholesterins and the protagons will be best treated in another
connection (see Chapters VIII and XII). We will discuss here only the
lecithins, which are present in every cell.
Lecithins. These bodies are ester compounds ^ of glycerophosphoric
acid substituted b}' 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 stear}'l-, palmityl-, and oleyl-
lecithins. According to Thudichum ^ two different fatty acids may exist
simultaneously in one lecithin, and according to him ever}- true lecithin
always contains at least one oleic-acid radical. All lecithins are mono-
nitrogenous monophosphatides, which contain 1 atom of nitrogen for
every atom of phosphorus. As an example of a lecithin we give the ore
closely studied by Hoppe-Seyler and Diaconow,^ called distearyl-lecithin,
CH2 — O — C18H35O
I
CH- O-C18H35O
C44H9oNP09= CH,— 0\
HO^PO.
/C,H4— 0/
Nf(CH3)3
\0H
According to Henriques and Hansen ^ the iodine equivalent of the
fluid fatty acids obtained from egg as well as brain lecithin is higher than
that of oleic acid, hence it follows that the lecithins contain other fatty
acids besides stearic, palmitic, and oleic acids.
Erlandsen ^ in a specially thorough ard careful investigation has studied
the phosphatides of the ox heart and ox muscles. The lecithin had the
same composition as that from the egg-yolk. The iodine equivalent as
* Streoker, Annal. d. Chem. u. Fharm., 148; Hundeshagen, Journ. f. prakt. Chem.
(N. F.), 28; Gilson, Zeitschr. f. physiol. Chem., 12.
^ J. L. W. Thudichum, Die chemische Konstitution des Gehirns des Menschen, etc.,
Tubingen, 1901.
^Hoppe-Seyler, Med. chem. I'ntersuch., Heft 2 and 3.
* Skand. Arch. f. Physiol., 14.
^A. W. E. Erlandsen, Undersogelser over Hjertets Phosphatider, Copenhagen, 1906-
144 THE ANIMAL CELL.
well as the analysis show that the fatty acids occurring in the lecithin mole-
cule are very poor in hydrogen and belong in part to the linolic or linolenic
acid series. Diaminomonophosphatides, i.e., compounds in which the
relationship N:P is not, as in lecithin, 1:1, but 2:1, occur in the muscles,
but chiefly in the heart muscle. These phosphatides are isolated as metal-
lie salts, and the cadmium compound of the diaminomonophosphatide
obtained from the heart had the composition C4oH75N2POi2-2CdCl2.
Erlandsen has isolated a new phosphatide from the heart, which he
calls cuorin and which belongs to the group of monaminodiphosphatides,
in which the relation of N:P is 1:2. This cuorin, which occurs only in
traces in other muscles, contains two phosphoric-acid radicals which in
part are united with glyceryl. Besides these it contains two residues of
strongly unsaturated fatty acids and a basic radical, which is not identical
with choline. The empirical formula is C7iHi25NP202i. Cuorin is soluble
in ether but insoluble in alcohol, and is characterized by a very great auto-
oxidizability. It is obtained in the amorphous state. The monaminophos-
phatides (lecithin and cuorin) can be directly extracted from the air-dried
and finely dinded organs, and to all appearances occur in the free state.
The diaminophosphatides are also soluble in ether, but cannot be directly
extracted by ether, but only after a previous treatment with alcohol, and
therefore probably exist in combination with proteins.
WiNTERSTEiN and HiESTAND,! and previous to them Schulze and WiN-
TERSTEiN, have isolated from different parts of plants, lecithin preparations
which are poorer in phosphorus than the ordinary lecithin, containing as a
maximum 2.74 per cent phosphoms, and which on cleavage with dilute
mineral acids yielded, besides fatty acids, glycerophosphoric acid, and
cholme, also considerable quantities of hexoses, indeed 16 per cent. The
hexoses were cZ-glucose and c?-galactose, and besides these small quantities
of pentoses were found. These phosphatides seem to be widely distributed
in the plant kingdom.
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.
CH^.OH
Glycerophosphoric acid, CgHgPOg^CH.OH , is a bibasic acid which prob-
I
CH-0\
oh4po
OH/
ably occurs in the animal fluids and tissues only as a cleavage product of lecithins.
According to Willstatter and Ludecke ^ the glycerophosphoric acid split off
* Zeitschr. f. physiol. Chem., 47.
^Willstatter and Ludecke, Ber. d. d. chem. Gcsellsch., 37.
LECITHINS. 145
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.
/CH,.CH,(OH)
Choline (trimethyloxyethylammoniumhydroxide),C5H,5N02 = N— (0113)3 ,
\0H
which occurs extensively in the plant kingdom, is not identical with the base,
NEURiNE, prepared by Liebreich as a decomposition product from the brain,
which is considered as trimethylvinylammonium hydroxide, C5H13NO. Choline
is a syrupy fluid readily miscible with absolute alcohol. Hydrochloric acid gives a
compound which is very soluble in water and alcohol, but insoluble in ether,
chloroform, and benzene. This compound forms a double combination with plati-
num chloride which is soluble in water, insoluble in absolute alcohol and ether,
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 ciuantitative estimation of this base (Stanek ^) .
On heating the free base it decomposes into trimethylamine, ethylene oxide, and
water.
Lecithin occurs, as Hoppe-Seyler 2 has especially shown, widely diffused
in the vegetable and animal kingdoms. According to this investigator it
occurs also in many cases in loose combination with other bodies, such as
proteins, haemoglobin, and others. Lecithin, according to Hoppe-Seyler,
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 differ-
ent pathological tissues or liquids.
SiWERTZow^ has determined the amount of lecithin in the human
foetus and in children of various ages, and he finds that the quantity of
lecitliin is much greater in the organs (brain, liver, heart, and muscles)
of the ripe foetus as compared with the same organs of children up to ten
years of age. The child according to him has a certain store of lecithin
when it comes into the world and this is consumed during the first months
of its extra-uterine life.
This wide distribution of the lecithins, as also the fact that they are
primary cell constituents, gives great physiological importance to these
substances. We have in lecithin, no doubt, a verj- important material
for the building up of the complicated phosphorized nuclein substances of
the cell and cell nucleus. That the lecithins are of great importance in
the development and growth of li^dng organisms, in fact for the bioplast ic
* In regard to choline and its compounds see Gulewitsch, Zeitschr. f. physiol,
Chem., 24; Stanek, ibicL, -46.
=> Physiol. Chemie, Berlin, 1877-1881, 57.'
^ See Biochem. Centralbl., 2, 310.
146 THE ANIMAL CELL.
processes in general, follows also from several investigations.^ The fact
must not be overlooked that in the animal body we find besides the leci-
thins also other related phosphatides which have been little studied and
which can be readily mistaken for lecithins.
Lecithin may be obtained in grains or warty masses composed of small
crystalline plates by strongly 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 (Ulpiani^). The solution of lecithin in alcohol-
ether or chloroform is precipitated by acetone. 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.
With considerable water, lecithins give an emulsion or indeed a filter-
able colloidal solution, which is precipitated by salts with, divalent cations,
such as Ca, ^Ig, and others (W. Koch). This precipitate dissolves again
in water after the removal from the solution of the electrolytes, and the
formation of this precipitate can be prevented by the presence of salts of
monovalent cations. We are here not dealing with a chemical but rather
with a physical precipitation reaction (Koch 3). In putrefaction lecithins
yield glycerophosphoric acid and choline; the latter further decomposes
with the formation of methylamine, ammonia, carbon dioxide, and marsh-
gas (Hasebroek^). 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 are easily carried down dur-
ing the precipitation of other compounds such as the protein bodies, and
may therefore very greatly change the solubilities of the latter.
Lecithins combine with acids and bases. The compound with hydro-
chloric acid give 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
'See Stoklasa, Ber. d. deutsch. chem. Gesellsch., 29; Wiener Sitzungsber. , 104;
Zeitschr. f, physiol. Chem., 2.); W. Danilewsky, Comp. rend., 121 and 123, and W.
Koch, Zeitschr. f. physiol. Chem., 3"; P. Kyes, ibid., 41, and Berl. klin. Wochenschr.,
1904.
2 Chem. Centralbl., 1901, 2, 30 and 193.
^Zeitschr. f. physiol. Chem., 37.
'Ibid., 12.
LECITHINS. 147
3 molecules of lecithin and 4 molecules of cadmium chloride (Ulpiam i)
is difficultly soluble in alcohol, but dissolves in a mixture of carbon disul-
phido and ether or alcohol. A solution of lecithins in alcohol is not pre-
cipitated by lead acetate and ammonia.
Lecithin may be prepared tolerably pure from the 3'olk of the hen's egg
by the following methods, as suggested by Hoppe-Seyler and Diacoxow.
The yolk, deprived of protein, is extracted with cold ether until all the
yellow color is removed. Then the residue is extracted with alcohol at
50-60° C. After the evaporation of the alcoholic extract at 50-60° C, the
syrupy matter is treated with ether and the insoluble residue dissolved in
as little alcohol as possible. On cooling this filtered alcoholic solution to
— 5° to —10° C. the lecithin gradually separates in small granules. The
ether, however, contains considerable of the lecithin. The ether is dis-
tilled off and the residue dissolved in chloroform and the lecithin precipi-
tated from this solution by means of acetone (Altmann).
According to Gilson ^ a new portion of lecithin may be obtained from
the ether used in extracting the yolk by dissolving the residue after the
evaporation of the ether in petroleum-ether and then shaking this solution
with alcohol. The petroleum-ether takes the fat, while the lecithin re-
mains dissolved in the alcohol and may be obtained therefrom rather
easily by using the proper precautions, as described in the original puljli-
cation.
Zuelzer's method is based upon the precipitability of the lecithin by
acetone, and Bergell's ^ method upon the preparation of the double
salt of cadmium and its decomposition by ammonium carbonate. The
preparations oljtained by the different methods consist generally of a
mixture of lecithins.
The detection and the quantitative estimation of lecithins in animal
fluids or tissues is based on the solubility of the lecithins (at 50-60° C.) in
alcohol-ether, by which the phosphoric-acid or glycerophosphoric-acid
salts which may be present at the -same time are not dissolved. The
alcohol-ether extract is evaporated, the residue dried and fused with soda
and saltpetre. Phosphoric acid is formed from the lecithin, and it can be
used in the detection and quantitative estimation. The distearyl-lecithin
yields 8.798 per cent P2O5. This method is, however, not exactly correct,
for it is possible that other phosphorized organic combinations, such as
jecorin (see Chapter VIII) and protagon (Chapter XII), may have passed
into the alcohol-ether extract. In detecting lecithin the double compovmd
of choline and platinum chloride must also be prepared. The residue of the
evaporated alcohol-ether extract may be boiled for an hour with baryta-
water, filtered, the excess of barium precipitated with CO2, and filtered
while hot. The filtrate is concentrated to a syrupy consistency, extracted
with absolute alcohol, and the filtrate precipitated with an alcoholic solu-
tion of platinum chloride. The precipitate after filtration may be dissolved
in water and allowed to crystallize over sulphuric acid. For the detection
'Chem. Centralbl., 1901, 2, 30 and 193.
^Altmann, cited from Hoppe-Seyler-Thierf elder's Handbuch, 7. Auflage; Gilson,
ibid.
■■' Zuelzer, Zeitschr. f. physiol. CIiem.,27, and Bergell, Ber. d. d.chem. Gesellsch., 33.
148 THE ANIMAL CELL.
and estimation of lecithin we can make use of the method of heating with
hydriodic acid as suggested bv Koch.^ One methyl iodide group is split
ofif at 240° and the two others at about 300° C.
Protagons, which are found in the leucocytes and pus-cells, are also to
be considered as constituents of protoplasm. These phosphorized Ijodies
occur principally in the brain and nerves, and hence will be described in a
following chapter (XII).
Glycogen, first discovered by Cl. Bernard, is found in developing
animal cells and especially in developing embryonic tissues. According
to Hoppe-Seyler it seems to be a never-failing constituent of the cells
which show ama?boid movement, and he found this carbohydrate in the
leucocytes, but not m the developed motionless pus-corpuscles. Salomon
and afterwards others have, however, found glycogen in pus.^ From the
relationship which seems to exist between glycogen and muscular work (see
Chapter XI), it is presumable that a consumption of glycogen takes place
in the movement of animal protoplasm. On the other hand, the extensive
occurrence of glycogen in embryonic tissues, as also its occurrence in patho-
logical tumors and in abmidant cell formation, speaks for the importance
of this body in the formation and development of the cell.
In adult animals glycogen occurs as stored foodstuff in the muscles and
certain other organs, but principally m the liver; therefore it will be com-
pletely described in connection with this organ (Chapter VIII).
Another body or perhaps more correctly a group of bodies which occur
wideh' distributed in the animal and vegetable kingdoms, and which are
present regularly in the cells, are the cholesterins. The best-known repre-
.sentative of this group is ordinan.- choksterin (see Chapter VIII), which is
the chief constituent of certain biliary calculi and exists in abimdant quan-
tities in the brain and nerv-es. It is hardly probable that this body is of
direct importance for the life and development of the cell. It must be
considered that the cholesterin, as accepted by Hoppe-Seyler,^ is a cleavage
product appearing in the cell during the processes of life, but this does not
exclude the possibility that the cholesterin, as a constituent of the lipoids
of the protoplasm-boundary layers (Overton), may be of indirect im-
portance in cell life. According to Hoppe-Seyler. the same is true for the
fats, which do not occur constantly in the cells and have nothing to do in
the ordinary processes of life. There is no doubt that cholesterin exists
as a constituent of the protoplasm, but its existence in the nucleus is ques-
tionable. The intracellular enzymes are undoubtedly constituents of the
protoplasm as well as of the nucleus and must be of the greatest import-
ance for the life and fmictions of the cells.
' Zeitschr. f. physiol. Chem., 36, and Amer. Jour. Physiol.. 11.
^ In regard to the literature on glycogen see Chapter VIII.
3 Physiol. Chem., p. 81.
XUCLEOPROTEIDS. 149
The cell nucleus has a rather complex structure. It consists in part
of fibrils which form a network and another part which is less solid and
homogeneous. The first differs from the second in possessing a stronger
affinity for many dyes. On accoimt of this behavior the first is called the
chromatic substance or chromatin, and the other the achromatic substance
or achromatin.
The homogeneous substance of the nucleus is considered as a mixture
of protein. The network seems to contain the more specific constituent
of the nucleus, namely, the nuclein substances. Besides this it is alleged
to contain another substance also, plastin. This last is less soluble than
the nuclein substances and does not have the property, like them, of fixing
dyes.
The chief constituents of the cell nucleus are the nucleo'proteids, and
in certain cases the nucleic acids.
Nucleoproteids. The most important of these bodies have already
been discussed in a previous chapter (II, page 71). These bodies are either
strong or loose combinations of nucleic acids with proteid. To the latter
class belongs histone, in certain cases, and the compounds between nucleic
acids and protamines should also perhaps be called nucleoproteids. There
is a difference among the nucleoproteids. dependent on the various proteid
complexes as well as upon the nucleic acids. They contain generally con-
siderable proteid in the molecule, hence they give the ordinary proteid
reactions, and therefore are closely related to the protein bodies. The
nucleoproteids occurring in the cell nucleus seem to be characterized by
containing a relatively large amount of phosphorus and a pronoimced acid
character.
In the preceding discussion of the nucleoproteids, attention was called
to the fact that, on their modification b}' heat, b}' weak acid action, and
by peptic digestion, proteid is split off and a nucleoproteid richer in phos-
phorus is formed. These compoimd proteids, rich in nucleic acid, obtained
by peptic digestion from cells, cell-rich organs, or nucleoproteids. have been
called Jiuclcins (Miescher, Hoppe-Seyler i) or true nucleins. But as the
true nuclein seems to be nothing but a modified nucleoproteid poor in
proteid, it seems unnecessaiy to give the name nuclein thereto. On the
other hand, the nucleins have other properties than the nucleoproteids,
and as the nucleins bear the same relationship to the nucleoproteids that
the pseudonuclein does to the nucleoalbumins, 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-
proteids in their peptic digestion or by treatment ^nth dilute acids. It
must be remarked that the nucleins are not entirely resistant towards
' Hoppe-Sej'ler , Med. f^hem. Untersuch., 4.52.
150 THE ANIMAL CELL.
gastric juice, and also that at least one nucleoproteid, namely, the one
obtained from the pancreas, completely dissolves, lea\ing no nuclein residue
on treatment with gastric juice (Umber, Milroy'). The nucleins are
rich in phosphorus, containing in the neighborhood of 5 per cent. Accord-
ing to LiEBERMAXN,- mctapliosphoric acid can be split off from true nucleins
(yeast nuclein). The nucleins are decomposed into proteid and nucleic
acid by caustic alkali, and as different nucleic acids exist, so also there
exist different nucleins. As previously stated, proteids may be precipitated
in acid solutions by nucleic acids, and in this way, as shown by Milroy,
combinations of nucleic acid and proteids may be prepared which behave
quite like true nucleins. All nucleins yield so-called nuclein bases on boil-
ing with dilute acids. The nucleins contain iron to a considerable extent.
They act like rather strong acids.
The nucleins are colorless, amorphous, 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
MiLLOx's reaction. They show a great affinity for many dyes, especially
the basic ones, and take these up with a\ddity from watery or alcoholic
solutions. On burning they yield an acid residue which is ver}^ difficult
to incinerate and which contains metaphosphoric acid. On fusion with
saltpetre and soda the nucleins yield alkali phosphates.
To prepare nucleins from cells or tissues, first remove the chief mass of
proteids by artificial digestion with pepsin-hydrochloric acid, lixiviate the
residue with very dilute ammonia, filter, and precipitate \^^th hydrochloric
acid. The precipitate is further digested with gastric juice, washed and
purified by alternately dissolving in very- faintly alkaline water and re-
precipitating with an acid, washing ^^ith water, and treating ^^ith alcohol-
ether. A nuclein may be prepared more simply by the digestion of a
nucleoproteid. In the detection of nucleins we make use of the above-
described method, testing for phosphorus in the product after fusing with
saltpetre and soda. Naturally the phosphates, lecithins (and jecorin)
must first be removed by treatment with acid, alcohol, and ether, re-
spectively. We must specially call attention to the fact, as shown by
LiEBERMANN,^ that it is very' difficult to remove lecithin by means of
alcohol-ether. No exact methods are known for the quantitative estima-
tion of nucleins in organs or tissues.
Pseudonucleins or Paraxucleixs. These bodies are obtained as an
insoluble residue on the digestion of certain nucleoalbumins or pliospho-
glucoproteids 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 relationship between the
> Umber, Zeitschr. f. klin. Med., 43; Milroy, Zeitschr. f. physiol. Chem., 22.
^ Pfliiger'.s Arch., 4".
' Ibid.
NUCLEIC ACIDS. 151
degree of acidity and the quantity of substance is not properly selected,
the formation of pseudonucleins may be entirely overlooked in the digestion
of certain nucleoalbumins. Pseudonucleins contain phosphorus, which,
as shown by Liebermaxx,^ is split off as metaphosphoric acid by mineral
acids.
The pseudonucleins are amorphous bodies insolul^le in water, alcohol,
and ether, but readily soluble in dilute alkalies. They are not soluble in
\eTy dilute acids, and may be precipitated from their solution in dilute
alkalies by adding acid. They give the protein reactions very strongly,
but do not yield nuclein bases.
In preparing a pseudonuclein, dissolve the mother-substance in hydro-
chloric acid of 1-2 p. m., filter if necessary-, add pepsin solution, and allow
the mixture to stand at the temperature of the body for about twenty-
four hours. The precipitate is filtered off, washed with water, and purified
by alternately dissohdng in very faintly alkaline water and reprecipitating
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 resiaue 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.
Nucleic Acids. All nucleic acids are rich in phosphorus and yield phos-
phoric acid and nuclein bases as cleavage products. The various nucleic
acids are nevertheless very different in regard to the products they yield.
The statements in this regard are somewhat contradictory and it seems
as if in certain cases we were dealing with impure or partly decomposed
nucleic acids. For example, according to Kossel, the nucleic acid from
ox-sperm yields chiefly xanthine, while Levexe obtained only guanine and
adenine. The guanylic acid isolated by Baxg from the pancreas contained
only guanine, while the pancreas nucleic acid investigated by Levexe
contained adenine as well as guanine. The nucleic acids of the thymus
yield, according to most statements, only adenine and guanine, similar to
the acids obtained from the spleen, brain, mammar}' gland, and fish-sperm.
According to Steudel, the thymusnucleic acids yield xanthine, hypoxan-
thine, adenine, and guanine,while according to Baxg the thymus gland con-
tains two different nucleic acids, one containing adenine and guanine, while
the other contains only adenine, hence is an adenylic acid. The nucleic acid
of the intestine yields, according to Ixouye and Kotake, all four nuclein
bases, although it has about the same composition as the salmonucleic
acid, W'hich yields only adenine and guanine.
All nucleic acids thus far investigated, with the exception of guanylic
' Ber. d. d. chem. Gesellsch., 21, and Centralbl. f. d. med. Wissensch., 1SS9.
152 THE ANIMAL CELL.
acid, contain also representatives of the pyrimidine group; there seems to
exist a difference in this regard between animal and plant nucleic acids.
As far as known, in the plant nucleic acids the pyrimidine group is repre-
sented only by cytosine and uracil (Kossel, Ascoli, Kossel and Steudel,
Osborne and Harris), and in the ar.imal (the thymusnucleic acids), on
the contrary, by cytosine, thymine, and uracil (Kossel, Neumann, Levene).
Mandel and Levene ^ have nevertheless isolated a nucleic acid from
haddock eggs which yielded uracil but no thymine, and this acid behaved
in other respects like a nucleic acid from plant-cells. The guanylic acid
contains, as above remarked, neither uracil, thymine, nor cytosine.
The nucleic acids show a different composition also in other regards. A
reducing pentose group can be split off from guanylic acid and the vege-
table nucleic acids (the tritico- and yeast nucleic acid), while from the yeast
nucleic acid also a hexose is claimed to be obtained. No reducing carbo-
hydrate has, on the contrary, been split off from most animal nucleic acids.
Certain observations which were based upon qualitative pentose reactions
seem to show that the various organs contain nucleoproteids containing
pentoses and that we have several nucleic acids which yield pentose (see
Chapter III, p. 110). The preparation of these acids in a pure form has
been attempted only in a few cases, and the qualitative pentose reactions
are not to be relied upon to any great extent. Bang ^ has indeed shown
that a nucleic acid occurs in the thymus gland which gives the phloroglucin
reaction but does not contain any pentose. Those nucleic acids which do
not split off any reducing carbohydrate contain nevertheless a carbohydrate
group which, as Kossel and Neumann first showed, on deep cleavage with
a mineral acid 3nelds le\adinic acid.
We generally admit of 4 atoms of phosphorus in the empirical formula?
of the various nucleic acids. In salmonucleic acid the relationship of phos-
phorus to nitrogen is as 4 to 14, in triticonucleic acid 4 to 16, and in guanylic
acid 4 to 20. The form of combination of the phosphorus is not known
with positiveness, but it seems at least that guanylic and triticonucleic
acids are derivatives of a pentahydroxylphosphorie acid, P(0H)5.
1 Journ. of Biol. Chem., 1, 425, and Zeitschr. f. physiol. Chem., 49, 262.
^ The works of Kossel and his pupils on nucleic acids are found in Arch. f. (Anat. u.)
Physiol., 1892, 1893, and 1894; Sitzungsber. d. Berl. Akad. d. Wissensch., 18, 1894,-
Centralbl. f. d. med. Wissensch., 1893; Ber. d. deutsch. chem. Gesellsch., 26 and 27;
Zeitschr. f. physiol. Chem., 22 and 38. See also Neumann, Arch. f. (Anat. u.) Physiol.,
1S98 and 1899, Suppl.; Miescher, Hoppe-Seyler's Med. chem. Untersuch., 441, and
Arch. f. exp. Path. u. Pharm., 3"; Schmiedeberg, ibuL, 3" and 43; Osborne and
Harris, Zeitschr. f. physiol. Chem., 36; Bang, ibid., 26 and 31; Hofmeister's Beitrage, o,
and Biochem. Centralbl., 1, 29.5; Altmann, Arch. f. (Anat. u.) Physiol., 1899; Ascoli,
Zeitschr. f. physiol. Chem., 28 and 31; Levene, ibid., 32, 3", 38, 39, 43, and 45; Man-
del and Levene, ibid., 46, 47, 49, iiO; Inouye and Kotake, ibid., 46; Steudel, ibid., 42,
43, 46, and 49.
NUCLEIC ACIDS. 153
All nucleic acids are amorphous, white, and have an acid reaction.
They are readily soluble in aramoniacal or alkaline water and form insoluble
salts with the heavy metals, and as a rule also insoluble basic salts with the
alkaline earths. 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 compomids by an excess of acetic acid, but by a slight excess
of hydrochloric acid, especially in the presence of alcohol. In acid solu-
tions these latter nucleic acids give precipitates with proteids, 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 this
acid in dilute acids. All nucleic acids are insoluble in alcohol and ether.
They do not give either the biuret test or Millox's reaction. The nu-
cleic acids are optically active and indeed dextrorotatory (Gamgee and
Jones 1).
The proteolytic enzymes, such as pepsin and trypsin, decompose the
nucleoproteids 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 erepsm (Nakayama) or by other closely
allied enzymes which have been called nucleases (Iwaxoff, Fr. Sachs).
Micro-organisms can also bring about a more or less deep cleavage of the
nucleic acids (Schittexhelm and Schroter^).
Guanylic acid differs essentially from the other animal nucleic acids.
These latter are closely related to each other, and as they all yield thymine
on cleavage and in this regard differ markedly from the guanylic acid and
the plant nucleic acids,^ they can for the present be treated of as one group
which has received the common name of thymonucleic acids.
Thymonucleic Acids. A. Neumaxx has isolated a- and ^-thymusnu-
cleic acids from the thymus gland. The a-acid is soluble with difficulty
and can, according to Kostytschew, be transformed (two thirds), with
the splitting off of purine bases, into the .5-acid. The a-acid gives in
proper concentration a sodium salt which gelatinizes and a barium salt
which is precipitated by barium acetate in substance (Kostytschew).
The barium salt of the ,.5-acid is not precipitated by barium acetate. Ac-
cording to Baxg. the thymus contains both an adenylic acid and a nucleic
acid which contains adenine as well as guanine. This last acid is prob-
' Proceed. Roy. Soc, 72.
- Nakayama, Zeitschr. f. physiol. Chem... 41; Iwanoff, ibid., 39; Fr. Sachs, "1st die
Nuklease mit dem Trj^jsin identisch?" Inaug.-Dissert. Heidelberg, 1905; Schitten-
helm and Schroter, Zeitschr. f. physiol. Chem.,' 41.
^See Mandel and Levene, Jour, of Biol. Chem.. 1. 425, and Zeitschr. f. physiol.
Chem.. 49.
154 THE ANIMAL CELL.
ably the thymusnucleic acid which is identical with the nucleic acid from
the salmon milt (or salmon iicleic acid) (Schmiedeberg and Herlant^).
The salmonucleic acid and the thymusnucleic acid as obtained by
Schmiedeberg's method have the same composition, C40H56N14O16.2P2O6.
Other nucleic acids, such as those prepared by Alsberg. from the sperm of
the burbot (Lota vulgaris), and by Levene from ox sperm, brain, and spleen,
are identical with the th3'musnucleic acid or are at least closely related
acids. To this group belong also the nucleic acids from the kidneys and
the mammary glands (Mandel and Levene), from the intestinal mucosa
(Inouye and Kotake), from the sperm of the sturgeon (Noll), herring
(Mathews, Gulewitsch), and sea-urchin (Mathews 2).
On the decomposition of thymusnucleic acids (or salmonucleic acids) in-
termediate products of various kinds are produced by a more or less com-
plete cleavage of the nuclein bases. One of these is thymic acid, which is
obtained on heating the free acid with water at the water-bath tempera-
ture, when adenine and guanine are simultaneously split off. Thymic acid
is readily soluble in water and yields a barium salt which is also soluble in
water and has the formula Ci6H23N3P20i2Ba (Kossel and Neumann).
On cleavage with acids first a part of the nuclein bases is split off. The
remaining part is more difficult to set free, and in this operation an abundant
formation of melanin and a decomposition of the original substance take
place at the same time. When one half of the purine bases have been split
off we obtain the substance called heminucleic acid by Alsberg, which con-
tains onl}^ 1 molecule of purine bases to 2 P2O5. According to Schmiede-
berg, tlwmusnucleic acid (or salmonucleic acid) is a combination of
purine bases with another substance, the 7iucleot in phosphoric acid,
C30H46N4O15.2P2O5. The non-phosphorized component of this substance,
the nucJcoHn, C30H42N4O13, which is the ground substance of thymus-
nucleic acid, has been isolated by Alsberg. On the decomposition of
nucleic acids with 5 per cent sulphuric acid, Levene was able to split off
the purine bases completely and the pyrimidine bases in part. The car-
bohydrate groups went completely into solution.
KuTSCHER and Seemann obtained guanidine and urea, but no uric acid, as
products on the oxidation of nucleic acid with potassium permanganate.
KuTSCHER and Schenck ' 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 CsHgNgOj or
^ Neumann, 1. c; Kostytschcw, Zeitschr. f. I'hysiol. Chem., 39; Bang, Hofmeister's
Beit rage, 5; Schmiedeberg and Herlant, Arch. f. exp. Path. u. Pharm., 44.
2 Alsberg, Arch. f. exp. Path. u. Pharm., 51; Noll, Zeitschr. f. physiol. Chem., 25;
Mathews, ibid., 23; Gulewitsch, ibid., 27; see also for the other references foot-note 2,
p. 152.
^ Kutscher and Schenck, Zeitschr. f. physiol. Chem., 44; Kutscher and Seemann,
Ber. d. d. chem. Gesellsch., 36, and Centralbl. f. Physiol., 17.
NUCLEIC ACIDS. 155
CsHioXeO;, 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.
Guanylic Acid. This acid, which thus far has been obtained only from
the pancreas, has, according to B.a.xg, the composition C44H66X20P4O34. It
is readily soluble in warm water, but partialh- separates out on cooling. It
is considered as an ester of a glycerophosphoric acid and decomposes on
hydrolytic cleavage with acids, according to Bang, into 4 molecules of
guanine, 3 molecules of pento.se (/-xylose according to Neuberg), 3 mole-
cules of glycerine, and 4 molecules of phosphoric acid.
According to the more recent investigations of Baxg and Raaschou ^
the guanylic acid, which Baxg now designates as ,^-acid is formed, in the
preparation from another acid called a-guanylic acid, by the action of tlie
alkali. The a-guanylic acid, which is readily soluble in water, even in cold
water, contains less phosphorus and nitrogen (6.65 and 15.38 per cent re-'
spectively) as compared with the ,5-acid, which contains 7.64 per cent phos-
phorus and 18.21 per cent nitrogen. By the action of alkalies the a-guanylic
acid splits off a pentose group and is converted into the ,3-acid.
The following acid is also generally included among the nucleic acids :
Inosinic acid, C10H13N4PO8, was first isolated by Liebig from the flesh of
certain animals and then closely studied by Haiser.^ It contains phosphorus,
is amorphous, and gives crystalline salts with barium and calcium. Haiser
obtained hypoxanthine as a cleavage product and probably also trioxyvalerianic
acid, though this has not been positively proven.
The thvmusnucleic acid may be prepared as the copper salt, according
to ScHMiEDEBERG. from the heads of the salmon spermatozoa or from the
residue after the peptic digestion of the thymus glands (Herlaxt). The
protamines are removed by the action of copper chloride and the last traces
of proteid removed by dissolving the residue in dilute caustic potash and
precipitating this solution with alcohol, and this is repeated until it fails to
give the biuret test. The copper salt can be precipitated by copper chloride
from the water}' solution of the potassium nucleate, after acidification with
acetic acid. According to Neumanx, the two thymusnucleic acids, a and ,5,
can be obtained from the gland, after previously boiling the same 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 concentration, the
product is precipitated with alcohol. The nucleic acids can be obtained
from the precipitated sodium nucleates bj' precipitating with alcohol con-
taining hydrochloric acid. In the separation of the two acids, Kostytschew
makes use of tlie difTerent behavior of the barium salts on saturating their
sohition Avith barium acetate (see aboveV Levexe's^ method consists, on
' Hofmeister's Beitrage, 4.
^Liebig, Annal. d. Chem. u. Pharm., 62; F. Haiser, Monatshefte f. Chem., 16.
^ Schmiedeberg, Arch. f. exp. Path. u. Pharm., 43; Herlant, ibid., 44; Neumann,
156 THE ANIMAL CELL.
the contrary, in treating the organs first with 5 per cent sodium hydrate or
Avith S per cent ammonia in the cold, then nearly neutralizing -with acetic
acid, precipitating the proteids with picric acid, and treating the strongly
acidified liquid (acetic acid) Avith alcohol. In the presence of sufficient
acetate the nucleic acids are precipitated. ^lore recently Levene has sug-
gested that the nucleic acid be dissolved in strong c^cetic acid and then
precipitated A\'ith copper chloride or hydrochloric acid.
Guanylic acid may be best prepared, according to Bang and Raaschou,
by the following method: After treating the pancreas with 1 per cent
sodium-hydrate 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 concentrated, and precipi-
tated \Adth alcohol while hot. The proteoses remain in solution, and the
precipitated guanylic acid (a-acid) is purified by repeated solutions in
water and precipitations by alcohol.
Plant Nucleic Acids. Those best known are the yeast nucleic acid and the tri-
ticonucleic acid, C41H61X16P4O3,, isolated by Osborne and Harris from the wheat
embryo, and which according to these investigators is identical with the yeast
nucleic acid. The plant nucleic acids are nearly 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
hydrolysis with acid 1 molecule of guanine, 1 molecule each of adenine and
cytosine (Wheeler and Johnson ')> 2 molecules of uracil, and 3 molecules of pen-
tose for every 4 atoms of phosphorus. Levene has been able to prepare from
the tubercle bacilli nucleic acids whose nature has not been closely studied.
Plasminic acid is an acid which was prepared [by Ascoli and Kossel ^ 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.^
Among the cleavage products of the nucleic acids the purine deriva-
tives and the pyrimidine derivatives are of special interest.
Purine Bases (nuclein bases, alloxuric bases, xanthine bodies). With
these names we designate a group of bodies consisting of carbon, hydrogen,
nitrogen, and in most cases also of oxygen, which, by their composition,
show a relationship not only among themselves, but also with uric acid.
All these bodies, uric acid included, are considered as consisting of an
alloxuric and a urea nucleus, and for this reason Kossel and KRiJGER have
called them alloxuric bases, or the entire group, including uric acid, alloxuric
bodies. According to E. Fischer,^ who has not only shown, in several
ways, the close relationship of uric acid to this group, but has also pre-
Arch. f. (Anat. u.) Physiol., 1S99, Supplb.; Levene, Zeitschr. f. physiol. Chem., 32
and 4o; Kostytschew, ibid., 39,
■ Amer. Chem. Journ., 25).
2 Ascoli, Zeitschr. f. physiol. Chem., 28
^ See foot-note 2, p. 152.
*iiee Fischer, Ber. d. deutsch. chem. Gesellsch., 30 and 32.
PURINE BASES. 157
pared a number of the members of this group synthetically, they are all
N=CH
I I
derived from a compound, €5114X4 = HC C — NH , called 'purine.
II II >CH
N— C— X
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:
IX— C6
2C oC— X7
1 I > C8.
3X— C— X9
4
HX— CO
For example, uric acid, OC C — XH , is 2, 6, 8-trioxypurine, adenine,
I II >C0
HX— C— XH
X=C.XH2 HX— CO
II II
HC C — XH , is 6-aminopurine, and heteroxanthine, OC C — X.CH3 , is
II II >CH I li >CH
X— C— X HX— C— X
7-methyl-2, 6-dioxjTDurine, etc.
The starting-point used by Fischer for the synthetical preparation of the
purine bases was 2, 6, S-trichlorpurine, which is obtained, with 8-oxy-2, 6-dichlor-
purine as an intermediary product, from potassium urate and phosphorus oxy chlo-
ride. The close relation between uric acid and the nuclein bases follows from
the fact, as showm by Suxdvik,' that two bodies may be obtained on the reduction
of uric acid in alkaline solution, which, although not quite identical with xanthine
and hypoxanthine, are at least very similar thereto. Gal'tier claims to have
prepared xanthine sjmthetically by heating hydrocyanic acid with water and
acetic acid. Further syntheses of purine bases have been made by Traube.^
The purine bodies or alloxuric bodies found in the animal body or its
excreta are as follows: Uric acid, xanthine, heteroxanthine, \-methylxanthine,
paraxanthine, guanine, epiguanine, hypoxanthine, episarkine, adenine, and
carnine. The bodies theobromine, theophylline, and caffeine, occurring in the
vegetable kingdom, stand in close relationship to this group.
The composition of the purine bodies most important from a physio-
logical standpoint is as follows:
Uric acid, C3H4X4O3 2, 6, 8-trioxypurine
Xanthine, C5H4N4O2 2, 6-dioxypurme
1-methylxanthine, C6H6N4O0 1-methyl "
Heteroxanthine, C6H6N4O2 7- "
TheophyUine, C7HgN402 1 , 3-dimethyl ' '
Paraxanthine, C7HSN4O0 1,7-
Theobromine, C,HsN402 3,7-
Caffeine, C8H,oN402 1, 3, 7-trimethyl
' Zeitschr. f. physiol. Chem., 23.
^ Gautier, Compt. rend., 98, 1523, and Ber. d. deutsch. chem. Gesellsch., 31; W.
Traube, ibid., 33, and Annal. d. Chem. u. Pharm., 331.
158 THE ANIMAL CELL.
Hypoxanthine, CgH^N^O 6-oxypurine
Guanine, C5H5N5O 2-amino " "
Epiguanine, CgH.Np 7-methyl " " " "
Adenine, C5H5N5. . 6-aminop urine
Episarkine, C4HsN403(?)
Carnine, C7HSN4O3
After Salomon ^ had shown the occurrence of xanthine bodies in young
cells, the importance of the xanthine bodies as decomposition products of
cell nuclei and of nucleins was sho'^Ti by the pioneering researches of
KossEL, who discovered adenine and theophylline. Kossel gave them
the name nuclein bases. In those tissues in which, as in the glands, the
cells have kept their original state, the nuclein bases are not found free,
but in combination with other atomic groups (nucleins). In such tissues,
on the contrary, as in muscles, which are poor in cell nuclei, the nuclein
bases are found in the free state. Since the nuclein bases, as suggested by
Kossel, stand in close relationship to the cell nucleus, it is easy to under-
.stand why the quantity of these bodies is so greatly increased when large
quantities of nucleated cells appear in such places as were before relatively
poorly endowed. As an example of this, the blood, in leucaemia, is ex-
tremely rich in leucocytes. In such blood Kossel ^ found 1.04 p. m. nuclein
l:)ases, against only traces in the normal blood. That the nuclein bases
are also intermediate steps in the formation of urea or uric acid in the
animal organism is probable, and vdW be showTi later (see Chapter XV).
Only a few of the nuclein bases have been found in the urine or in the
muscles. Only four bases — xanthine, guanine, hypoxanthine, and adenine
— have been obtained, thus far, as cleavage products of nucleins. 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. B3' the action of nitrous acid guanine
is converted into xanthine and adenine into hypoxanthine.
C5H4N40.NH+HN02 = C5H4N402 + N2+H20;
Guanine Xanthine
C5H4N4.NH +HNO2 = C5H4N4O -' N2 + H2O.
Adenine Hypoxanthine
Similar transformations may be brought about by putrefaction as well
as by the action of special enzymes. The researches of Schittenhelm,
Levene, Jones, Partridge, Winternitz, and Burian ^ have shown that
in various orjrans desamination enzymes, such as giianase and adcnase,
' Sitzungsber. d. Bot. Verein der Provinz Brandenburg, 1880.
- Zeitschr. f. physiol. Chem., 7.
^ See Chapter XV (uric acid formation).
XANTHINE. 159
occur, which conveH guanine and adenine into xanthine and hypoxanthine
respectively, and also oxidases which oxidize hypoxanthine into xanthine
and this then into uric acid.
On cleavage with hydrochloric acid all four of the bodies are converted into
ammonia, glycocoll, carbon dioxide, and formic acid. On oxidation with hydro-
chloric acid and potassium chlorate, xanthine, bi"omadenine, and bromhypo-
xanthine yield alloxan and urea; guanine yields guanidine, parabanic acid (an
oxidation product of alloxan), and carbon dioxide. According to Burian ' the
nuclein bases give beautiful ued products with diazo-compounds as long as the
imide hydrogen in the 7th position (see structural formula above) is not substi-
tuted. As the nucleic acids do not react with the diazo compounds, Burian
concludes that probably the nucleic-acid residue is combined with the imide hy-
drogen at position 7.
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 somewhat
different. They are all precipitated from acid solution by phosphotungstic
acid; they also separate as silver compounds on the addition of ammonia
and ammoniacal silver-nitrate solution. These precipitates are soluble in
boiling nitric acid of 1.1 specific gravit3^ All xanthine bodies are also
precipitated by Fehling's solution (see Chapter XV) in the presence of a
reducing substance such as hydroxy lamine (Drechsel and Balke). Copper
sulphate and sodium bisulphite may also be used to advantage in their
precipitation (KRtJGER).^ This behavior of the xanthine bases serves just
as well as the behavior with the silver solution for their precipitation and
preparation.
HN— CO
II'
Xanthine, C5H4N407= OC C — NHv (2, 6-dioxypurine), is found in
I II >CH
HN— C— N ^
the muscles, liver, spleen, pancreas, kidneys, testicles, carp-sperm, thymus,
and brain. It occurs in small quantities as a physiological constituent
of urine, and it occasionally has been found as a urinarj^ sediment, or
calculus. It was first observed in such a stone by ^Iarcet. Xantliine is
found in larger amounts in a few varieties of guano (Jar-vds guano).
Xanthine is amorphous, or forms granular masses of crystals, or may
also, according to Horbaczewski,^ separate as masses of shining, thin,
large rhombic plates with 1 mol water of cr}'stallization. It is veiy slightly
soluble in water, in 14 151-14 600 parts at 16° C, and in 1300-1500 parts
at 100° C. (Almen*). It is insoluble in alcohol or ether, but is readily
* Ber. d. d. chem. Gesellsch., 3".
^ Balke, Zur Kenntnis der Xanthinkorper, Inaug.-Diss. Leipzig, 1893; Kriiger,
Zeitschr. f. physiol. Chem., IS.
^ Zeitschr. f. physiol. Chem., 23.
*Journ. f. prakt. Chem., 9G.
160 THE ANIMAL CELL.
dissolved by alkalies and with difficulty by dilute acids. With hydro-
chloric 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 xan-
thine. This precipitate is dissolved by hot nitric acid, and by this means
an easily soluble crystalline double compound is formed. Xanthine in
aqueous solution is precipitated on boiling with copper acetate. At
ordinary temperatures xanthine is precipitated by mercuric chloride and
by ammoniacal basic lead acetate. It is not precipitated by basic lead
acetate alone.
When evaporated to dryness in a porcelain dish with nitric acid, xan-
thine gives a yellow residue, which turns, on the addition of caustic soda,
first red, and, after heating, purple-red. If we place some chloride of 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 mider a bell-jar to the vapors of ammonia, a red or purple-
violet color is produced (Weidel's reaction). E. Fischer ^ has modified
Weidel's reaction in the following way: He boils the xanthine 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. 05115X50 = H9N.C C — NH (2-amino-6-oxvpurine). Gua-
II I! >CH
N— C— N^
nine is found in organs rich in cells, such as the liver, spleen, pancreas,
testicles, and in salmon-sperm. It is further found in the muscles (in very
small amounts), in the scales and in the air-])ladder 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 pathological conditions it has
been found in leucsemic blood, and in the muscles, ligaments, and articula-
tions of pigs with guanine-gout.
Guanine is a colorless, ordinarily amorphous powder which may he
obtained as small crystals by allowing its solution in concentrated am-
monia to spontaneously evaporate. According to Horbaczewski it may
under certain conditions appear in crystals similar to creatinine zinc
' Ber. d. deutsch. chcni. Gesellsch., 30, 2236.
GUANINE. HYPOXANTHINE. 161
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 ^ 100 c.c. of cold am-
monia solution containing 1, 3, or 5 per cent NH3 dissolve 9, 15, or 10
milligrams of guanine respectively. The solubility is relatively increased
in hot ammonia solution. The hydrochloride readily crystallizes, and has
been recommended by Kossel^ for the microscopical detection of gua-
nine, on account of its behavior to polarized light. The sulphate 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, differentiates it from
6-amino-2-oxypurin€, 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 crystalli-
zation, which is not expelled at 120° C. Very dilute guanine solutions are pre-
cipitated by both picric acid and metaphosphoric acid. These precipitates
may be used in the quantitative estimation of guanine. The silver com-
pound 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 satu-
rated 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, crystal-
line precipitate (Capranica). The composition of these and other guanine
compounds has been studied by Kossel and Wulff.^ Guanine does not
give Weidel's reaction.
HN— CO
I I
Hypoxanthine, Sarkine, C5H4N40 = HC C — NH =(6-oxypurine).
II II >CH
N— C— N^
This body is found in the same tissues as xanthine. It is especially abun-
dant in the sperm of the salmon and carp. Hypoxanthine 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 tlie blood
and urine in leucaemia.
Hypoxanthine forms very small, colorless, crystalline needles. It dis-
' Zeitschr. f. physiol. Chem., 17.
^ Ueber die chem. Zusammensetz. der Zelle,.Verh. d. physiol. Gesellsch. zu Berlin,
1890-91, Nos. 5 and 6.
^ Zeitschr. f. physiol. Chem., 17; Capranica, ibid., 4.
162 THE ANIMAL CELL.
solves with difficulty in cold water, but the statements in regard to the
solubility therein are very contradictory. ^ 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 h}-drochloric acid
is crystalline, and is more soluble than the corresponduig 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 possessing an in-
constant composition separates out. On treating this mixture with am-
monia and an excess of silver-nitrate and heating, a silver hypoxanthine
is formed, which when dried at 120° C. has a constant composition,
2(C5H2Ag2N40)H20, and is used in the quantitative estimation of hypo-
xanthine. Hypoxanthine picrate is soluble with difficulty, but if a
boiling-hot solution of the same is treated with a neutral or only faintly
acid solution of silver nitrate the hypoxanthine is nearly quantitatively
precipitated as the compound C5H3AgX40.C6H2(N02)30H. Hypoxan-
thine does not yield an insoluble compound with metaphosphoric acid.
WTien treated, like xanthine, with nitric acid it yields a nearly colorless
residue which, on warming with alkali, does not turn red. Hypoxanthine
does not give Weidel's reaction. After the action of hydrochloric acid
and zinc a hypoxanthine solution becomes first ruby-red and then brownish
red m 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
I !
Adenine, C5H5N5 = HC C — NHv (6-aminopurine), was first found
1! II >H
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 leucsemic urine (Stadthagex "*). It ma}' be obtained
in large quantities from tea-leaves.
Adenine crystallizes with 3 molecules of water of crystallization in long
needles which become opaque gradually in the air, but much more rapidly
when warmed. If the crystals are warmed slowly with a quantity of
water insufficient for solution, they become suddenly cloudy at 53° C, a
characteristic reaction for adenine. It dissolves in 1086 parts cold water,
but is easily soluble in warm. It is insolul)le 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
'See E. Fischer, Ber. d. deutsch. chem. Gesellsch., 30.
^ Kossel, Zeitschr. f. physiol. Chem., 12, 252; E. Fischer, 1. c.
'See Zeitschr. f. physiol. Chem., 10 and 12.
*Vircho'.v's Ann., 100.
ADEMXE. 163
hypoxanthine. The silver compound of adenine is difficultly soluble in
warm nitric acid, and deposits on cooling as a ciystalline mixture of
adenine silver-nitrates. ^Yith picric acid adenine forms a compound,
C5H5N5.C6H2(N02)30H, which is very insoluble and which separates more
readily than the hypoxanthine picrate and which can be used in the quanti-
tative estimation of adenine. We also have an adenine mercurj'-picrate.
Metaphosphoric acid with adenine gives a precipitate which dissolves in
an excess of the acid if the solution is not too dilute. Adenine hydro-
chloride 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 micro-
scopic detection of adenine. With the nitric-acid test and with Weidel's
reaction adenine acts in the same way as hypoxanthine. 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 xanthine bodies in organs and tissues 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 proteid 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 nitrification) is dissolved in not too large a quantity of boiling
nitric acid of sp. gr. 1.1, and this solution filtered boiling hot. On cool-
ing, the silver xanthine remains in the solution, while the double com-
pounds of guanine, hypoxanthine, and adenine crj'stallize out. The
silver xanthine may 1)6 precipitated from the filtrate by the addition of
ammonia and the xanthine set free by means of sulphuretted hydrogen.
The three above-mentioned sih'er-nitrate compounds are decomposed in
water with ammonium sulphide and heat; the silver sulphide is filtered off,
the filtrate concentrated, saturated with ammonia, and digested on the
water-bath. The guanine remains undissolved, while the other two bases
pass into solution. A part of the guanine is still retained by the silver
sulphide, and may be li1)erated by boiling it with dilute hydrochloric acid
and then saturating the filtrate with ammonia. A^ hen the above filtrate
containing the adenine and hypoxanthine, which has been, if necessary,
freed from ammonia by evaporation, is allowed to cool, the adenine sepa-
rates, while the. hypoxanthine remains in solution. According to Balke ^
we can advantageously precipitate the xanthine bases with a copper salt and
hydroxylamine as abo\e mentioned and then further separate the Ijodies.
In cases where the proteids have not been completely separated it is advan-
tageous to precipitate the bases as copper compounds with copper sulphate
and bisulphite. IvRtiGER and Schittexhklm's^ method for the separation
and quantitative estimation of purine bodies in faeces can be followed and
the bases then transformed into silver compounds.
The method of Buriax and Hall ^ is serviceable in the estimation of
'Zeitschr. f. physiol. Chom. 45. 'Ibid., 3S.
164 THE ANIMAL CELL.
the total quantity of purine bodies in animal organs; the quantitative estima-
tion of the various bases is performed in the main according to the method
above described. The xanthine is weighed as silver xanthine. The three
silver-nitrate compoimds are converted into the corresponding silver
derivatives by ammonia and the addition of silver nitrate, and then ammo-
nium sulphide is allowed to act upon the carefully washed silver compounds.
The guanine is weighed as such. The ammoniacal filtrate containing the
adenine and hypoxanthine, which must not be mixed with the hydrochloric-
acid extract of the silver sulphide, is neutralized and a cold concentrated
solution of sodium picrate added until the entire liquid has a pronouncedly
yellow color. The adenine picrate is immediately filtered off, washed on
the filter-paper with water, dried at above 100° C, and weighed. The fil-
trate containing the h^'poxanthine is gradually treated while boiling hot
with silver nitrate, and after cooling more silver nitrate is added to see if
the precipitation is complete. The silver-hypoxanthine picrate is washed,
dried at 100° C, and weighed. In regard to the composition of these com-
pounds see pages 162 and 163. This method of separating adenine and
hypoxanthine presupposes the absence of hydrochloric acid in the liquid.
The above method of separation with ammonia does not give exact
results on account of the not inconsiderable solubility of guanine in warm
ammonia. According to Kossel and Wulff.^ the guanine may therefore
be precipitated fj'om sufficiently dilute solutions by an excess of metaphos-
phoric acid and the nitrogen determined in the washed precipitate by
Kjeld.\hl's method. The adenine and hypoxanthine may be precipitated
from the filtrate by ammoniacal silver nitrate. The silver compound is
decomposed with very dilute hydrochloric acid and the adenine separated
from the hypoxanthine according to the suggestion of Bruhxs.^ In regard
to the complications in the detection and exact estimation of purine bodies
in extracts of organs, we refer to the works of His and Hagen and of
BuRiAX and Hall.^
NH— CO
I I
Uracil, C4H4N202=OC CH (2,6-dioxypyrimidine), was first obtained
I II
NH— CH
by AscoLi and Kossel from yeast nucleic acid and later prepared by Kos-
sel and Steudel from thymusnucleic acid and herring testicles, b}' Levexe
from the spleen and pancreas nucleic acids, and by Levexe and Maxdel from
the nucleic acid of the haddock roe. The synthetical preparation was first
performed by E. Fischer and Roeder.^
Uracil cr}'stallizes in needles which cluster in rosettes. On careful heat-
ing it sublimes in part undecomposed, but develops red vapors and decom-
poses in part. It is readily soluljle in hot water but less so in cold Abater,
' Zeitschr. f. physiol. Chem., 1".
2 76m/., 14, 5.59.
^ His and Hagen, ibid., 30, and Burian and Hall, ibid., 38.
*Ascoli, ibid., 31; Kossel and Steudel, ibid., 3"; Levene, ibid., 38, 39; Levene
and Mandel, ibid., 49; E. Fischer and Roeder, Ber. d. d. chem. Gesellsch., 34.
THYMINE. CYTOSINE.. 165
and is nearly insoluble in alcohol and ether. It is readily soluble in am-
monia. It is precipitated by silver-nitrate solution only after the careful
addition of ammonia or bar>'ta-water, as the precipitate is readily soluble in
an excess of ammonia. Uracil responds to Weidel's test (p. 160). In
regard to the preparation of uracil see Kossel and Stel'del.i
XH— CO
I I
Thymine, CoHeXoO-^^OC C.CH3 (5-methvluracil;. This bodv. which
" ' I
XH— CH
is identical with nuclensin obtained by ScHiHEDEBERG from salmonucleic acid,.
is obtained from the thymusnucleic acids and was first prepared by Kossel
and X'eu^laxx from thymusnucleic acid, and then by other investigators,
especially Levexe and Maxdel. from other animal nucleic acids. Fischer
and RoEDER and recently Gerxgross - have prepared it s}Tithetically.
Thymine crv'stallizes in small leaves grouped in stellar or dendriform
clusters or. rarely, in short needles (Gulewitsch ^). On heating it sublimes.
It is difficultly soluble in cold water, more soluble in hot water, and insolu-
ble in alcohol. It behaves like uracil towards ammonia or bar}-ta-water
and silver nitrate. Thymine is precipitated by phosphotungstic acid, which
does not precipitate uracil. Bromine-water is decolorized by thymine, pro-
ducing bromthymine. For its detection we make use of the sublimation,
the beha\ior towards silver nitrate, and its elementarx* analysis.
In regard to the methods of preparation see Kossel and X'eumaxx
and W. JoxES.-*
HX— C.XH,
I II
Cytosine, C4H5X'30=OC CH (6-aniino-2-oxvpvrimidine) , was first
II
N=CH
prepared by Kossel and X'EUiL\xx from thymusnucleic acid, and then by
Kossel and Steudel, also by Levexe and ^Iaxdel, from the spleen and
many other animal nucleic acids, by Ixouye and Kotake ^ from the nucleic
acid of the intestine, and finalh^ also by Wheeler and Johxsox from tritico-
nucleic acid. "Wheeler and Johxsox ^ have also prepared it s^Tithetically.
The free base is difficultly soluble in water and cr}-stalhzes in thin
leaves with a mother-of-pearl luster. The double compound ^rith pla:i::um
»Zeitschr. f. physiol. Chem., 37, 24.5.
^ Schmiedeberg, 1. c; Arch. f. exp. Path. u. Pharm., 3"; Kossel and Neumann,
Ber. d. d. chem. Gesellsch. , 26 and 2"; Mandel and Levene. Zeitschr. f. physiol. Chem.,
46. 47, 49. oO; E. Fischer and Roeder, ibid.. 34; Gemgross, iW<f., 3S.
^ Zeitschr. f. physiol. Chem., 27.
* Kossel and Neimaann, 1. c, and W. Jones, Zeitschr. f. physiol. Chem., 29, 461.
*In regard to the cited works see foot-note 2, p. 152.
'Amer. Chem. Journ., 29: see also foot-note 2, p. 1"2.
166 THE ANIMAL CELL.
chloride, the crystalline picrate, the nitrate, and the two sulphates are of
importance in the detection of cytosine. This base is precipitated by phos-
photungstic 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). Cytosine gives, like uracil, the murexid reaction with chlorine-
water and ammonia. In regard to the preparation of this base, see
KossEL and Steudel and Kutscher.^
The purine bases and the pyrimidine bodies are closely related to each
other not only from a chemical but also from a physiological point of view,
and for this reason the question has been repeatedly asked whether or not
the pyrimidine bodies might not in part be products produced from the
purine bodies by the action of acid. All researches thus far carried on to
elucidate this question contradict such a possibility.
Mineral Bodies. The mineral substances found habitually in the cells
of higher plants and of animals are potassium, sodium, calcium, magnesium,
iron, phosphoric acid, chlorine, and perhaps also iodine (Justus). In cer-
tain cells we also find manganese, silicic acid, arsenic, barium, and lithium.^
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 so-
dium compounds occur chiefly in the fluids, while the potassium compounds
occur especially in the form-elements. Corresponding to this, the cells con-
tain chiefly potassium as phosphate, while they are less rich in sodium and
chlorine compounds. Still we have some exceptions to this rule, and it
must be remarked that Beebe ^ has found considerably more sodium than
potassium in malignant tumors.
The importance of potassium for the life and the development of the cell
has been shown by several observations. A very instructive and interesting
example of this action has been shown by Loeb * in his investigations on
the pathogenesis of the egg of the sea-annelide Chsetoptenis. The un-
' Kossel and Steudel, Zeitschr. f. physiol. Chem., 37 and 38; Kutscher, ibid.. 38.
^Justus, Virchow's Arch., 170 and 176. In regard to arsenic see the works of
Gautier, Compt. rend., 129, 180, 131, 139; Bertrand, ibid., 134; Segale, Zeitschr. f.
physiol. Chem,, 42; Kunkel, ibid., 44. In regard to the barium see Schulze and Thier-
felder, Sitzungsber. d. Gesellsch. naturforsch. Freunde, 1905, No. 1, and in regard to
lithium see Hermann, Pfliiger's Arch., 109.
*Amer. Journ. of Physiol., 11 and 12.
*Ibid., 3, 4, and Pfliiger's Arch., 87.
MINERAL BODIES OF THE CELLS. 167
fertilized eggs could, in sea-water alone, develop only to the eighth or six-
teenth cell stage; after a short stay in sea-water to which KCl was added
they developed to the trichophora larva. The fact that the KCl could not
be replaced by other chlorides, but could be replaced by other potassium
salts also shows that we are here dealing with a specific action of the
potassium ions.
The division of the potassium in cells and various tissues seems, accord-
ing to Macallum,^ to be peculiar and essentially different. According to
]\1acallum the potassium is absent in the cell nuclei and in the head of
spermatozoa as well as in nerve-cells and their axis-cylinders, while it occurs,
on the contrary, in the medullary sheath and especially in the region of the
nodes of Ranvier. A peculiar division of the potassium also occurs in the
muscle fibers and secreting glandular cells.
The importance of phosphoric acid is not clear; it is possible that
this acid is important for the formation of the lecithins and nucleins, and
thereby indirectly makes possible the processes of growth and division,
which are dependent upon the cell nucleus. Loew ^ has shown, by means
of cultivation experiments on algse Spirogyra, that only by supplying
phosphate (in his experiments potassium phosphate) was the nutrition
of the cell nucleus made possible, and thereby the growth and division of
the cells. The cells of the Spirogyra can be kept alive and indeed produce
starch and proteins for some time without a supply of phosphates, but
their growth and propagation suffer.
As both phosphoric acid and iron are obtained from the nuclein sub-
stances it is likely that these mineral bodies are, at least relatively, richest
in the nucleus. As to the division of the mineral bodies between the
protoplasm and the nucleus we know nothing with positiveness, and the
same is true as to the form of combination of the mineral bodies in the nu-
cleus. On incineration we obtain not only a mixture of the mineral 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 boches. The ordinary chemical analysis is not sufficient for
the study of the mineral constituents of the fluids or tissue, their forms
of combination and action; hence we must resort to physical-chemical
methods.
According to the investigations carried on by these methods the con-
clusion has been reached, irrespective of the importance of the mineral
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
' Journ. of Physiol., 32. ^ Biolog. Centralbl., 11, 269.
168 THE ANIMAL CELL.
of the ions. For example, the permeability of the blood-corpuscles as
well as other cells for neutral alkali salts, which will be treated in the
following chapter, shows an exchange of ions. The investigations of Mail-
LARU on the toxic action of copper salts and of Paul and Kornig^ on
that of mercur}^ salts, acids, and alkalies offer other examples. From these
investigations 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.
Beautiful and instructive examples of the importance of the ions for
cell life have been shown by Loeb 2 and his collaborators. It is not within
the scope of this book to give a detailed account of this important work,
but perhaps it will be sufficient to give at least one example. The develop-
ment of the eggs of the Fundulus can be retarded for a long time by a f
normal NaCl solution. On the addition of CaS04 this retardation is prevented
and the development proceeds. Other calcium salts act like the sulphate,
but alkali sulphates like Na2S04 or other neutral alkali salts do not have
this action, hence it must be a calcium ion action. Small quantities of
other divalent cations, also trivalent ions, act in a similar way to calcium,
while the salts of monovalent cations do not have this action. The fact
that the fresh fertilized Fundulus eggs develop in distilled water as well as in
sea-water shows that we are not dealing simply with a taking up of the salts
necessary for development, but rather with an antagonistic salt action. They
quickly die in a pure NaCl solution (having a concentration equal to that
of sea-water); but if to the NaCl solution a small amount of zinc sul-
phate is added, the eggs are in condition to form an embryo. The common
salt can also retard the toxic action of the zinc salt. According to Loeb
every solution which contains only one electrolyte is poisonous, and this
toxicity can be prevented by another electrolyte, and in certain cases by
two other electrolytes. We are still undecided how the salts act in this
regard; Loeb^ believes that the antagonistic action of two salts may pos-
sibly be brought about by the fact that the diffusion in the egg is slower
when the two are simultaneously in the solution than when each is alone
in the solution. It is a difficult question to decide how the valence of the
ions influences the power of certain ions to act as poisons or as anti-poisons.
The chief mass of the cells consists of colloids, and as the normal func-
tions of the cells are connected with a certain physical condition of the proto-
'Maillard, Journ. de Physiol, et Path., 1; Paul and Kronig, Zeitschr. f. physikal.
Chem., 12, and Zeitschr. f. Hygiene, 25.
^ Loeb. Amer. Journ. Physiol., 3, 4, and 6; Pfliiger's Arch., 80, 87, 88, and 93 (with
Gies) 97, 101, and 107, and University of California Publications, Physiol., 1 and 2.
See also W. Oswald, Pfliiger's Arch., 106.
'" Pfliiger's Arch., 107.
COLLOIDAL CELL SUBSTANCES. 169
plasm it is natural to consider the action of the ions in relationship to the
change in the state of the colloids. The colloids can be precipitated by
electroh'tes, and the investigations of H.irdy and Pauli ^ show that we
are here probabl}' also dealing with an ion action. Negatively charged
colloids are, according to Hardy, precipitated by cations and positively
charged by anions. 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 containmg several salts (Loeb and Gies).
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 ver\^ difficult question, and its solu-
tion is still not answered.2
' See foot-note 1 and 2, p. 168, and Mathews, Amer. Joum. of Physiol., 10 and 12.
^ Hardy, Journ. of Physiol., 24, and Zeitschr. f. physikal. Chem., 33. See in regard
to colloids Hober, Physikal. Chemie der Zelle und der Gewebe, Leipzig, 1906. Ham-
burger, Osmotischer Druck und lonenlehre in den mediz. Wissenschaften, Bd. 3, 1904:
and H. Aron, Biochem. Centralbl,, 3, 505, and 4, 557.
CHAPTER YI.
THE BLOOD.
The blood is to be considered from a certain standpoint as a fluid tissue,
and it consists of a transparent liquid, the blood-plasma, in which a vast
number of solid particles, the red and ivhite 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-ele-
ments.^
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 lea^ing 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) Delezexxe has sho'^n that the blood coagulates very slowly if
it is collected under precautions so that it does not come in contact with
the tissues. On contact with the tissues or with tissue extracts it coagu-
lates in a few minutes. The blood "\nth non-nucleated blood-corpuscles
(mammals) coagulates, on the contrar}-, ver}' 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 (Spaxgaro, Arthus^).
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 cooling; and if we allow equine blood to flow directly
from the vein into a glass cylinder which is not too vdde 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 ^nth 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
'See Latschenberger, Wioo. Sitzungsber. , lOo.
' Delezenne, Compl rend. Soc. de biol., 49, Spangaio, Aich. ital. do Biol. 32;
Arthur, Jouro. de Physiol et Pathol., 4.
170
BLOOD-PLASMA. 171
(towards litmus) liquid which remains fluid for some time when kept at
0° C, but soon coagulates at the ordinary temperature.
The coagulation of the blood may be prevented in other ways. After
the injection of peptone, or, more correctly, proteose solutions into the
blood (in the living dog), the blood does not coagulate on leaving the veins
(Fano, Schmidt-Mulheim 1). The plasma obtained from such blood by
means of centrifugal force is called peptone-plasma. According to Arthus
and HuBER ^ the caseoses and gelatoses act similarly to fibrin proteose in
dogs. Eel serum and certain lymph-forming extracts of organs (see Chapter
VII) also have an analogous action. The coagulation of the blood of w-arm-
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,
herudin (Franz), into the blood current (Haycraft^), 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 adhesive-
ness and elasticity, would otherwise pass easily through the pores of the
filter-paper, are made solid and stiff by the salt, so that they may be easily
filtered. The plasma thus obtained, which does not coagulate spontaneously,
is called salt-plasma.
An especially good method of preventing coagulation of blood consists
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 ^ 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 ^ a non-coagulable blood-plasma may be obtained by drawing the
blood into a sodium-fluoride solution until it contains 0.3 per cent NaFI.
On coagulation there separates in the previously fluid blood an insoluble
or a very difficultly soluble protein substance, fibrin. When this separation
takes place without stirring, the blood coagulates in a solid mass which,
when carefully severed from the sides of the vessel, contracts, and a clear,
generally yellow-colored liquid, the blood-serum, exudes. The solid coagulum
which encloses the blood-corpuscles is called the blood-clot (placenta san-
' Fano, Arch, f (Anat. u ) Physiol, 1881; Schmidt-Mulheim, ihid., ISSO.
^ Arch, de Physiol. (5), 8.
^Haycraft, Proc. Physiol. Soc , 1884, 13, and Arch, f exp. Path. u. Pharm., 18;
Franz, Arch. f. exp. Path. u. Pharm., 49.
* Archives de Physiol. (5), 2, and Compt. rend., 112.
'Jcurn. of Physiol., 19.
' Journ. de Physiol, et Pharm., 3 and 4.
172 THE BLOOD.
guinis). If the blood is beaten during coagulation, the fibrin separates in
elastic threads or fibrous masses, and the defibrinated blood which separates
is sometimes called cruor} and consists of blood-corpuscles and blood-
serum, while uncoagulated blood consists of blood-coipuscles 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, and
the serum is proportionally richer in another body, the fibrin ferment (see
page 175).
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, nucleoproteid, serglobulins, and
sfrnihumins.
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
Mathew^s, the leucocytes, especially of the intestine, according to ]\1uller,
the bone-marrow and probably other lymphoid organs such as the
spleen and lymph glands, and according to Doyon and Nolf, the liver.
The statement that the intestinal wall is a seat of formation of fibrinogen, a
view that had already been held by Dastre, is substantiated not only by
the direct researches of Mathews, but also by the older and confirmed
statement that the blood from the mesentery vein is richer in fibrinogen
than the arterial blood. The occurrence of fibrinogen in the bone-marrow,
as sho^Ti by oMuller, and an increase of fibrinogen in the blood as well as in
the bone-marrow of animals immunized wdth certain bacteria, especially
pus-staphylococci, indicates the formation of fibrinogen in this tissue. That
the liver takes part in the formation of fibrinogen is made probable by the
fact that the quantity of fibrinogen in the blood strongly diminishes after
the extirpation of the liver (Nolf), and that fibrinogen may indeed be
entirely absent in the blood in phosphorus poisoning (Corin and Ansiaux,
Jacoby, Doyon, Morel, and Kareff^).
^ 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.
^ P. Mijller, Hofmeister's Beitrage, 6; Mathews, Amer. Journ. of Physiol., 3; Nolf,
Bull. Acad. Roy. Belg., 1905, and Arch, intern, de Physiol., 3, 1905; Corin and Ansiaux,
FIBRINOGEN. 173
FilDrinogen 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 conglom-
erate into tough, elastic masses or lumps. The solution in 5-10 per cent
NaCI coagulates on heating at 52-55° C, and the faintly alkaline or
nearly neutral weak salt solution coagulates at 56° C, or at exactly the
same temperature at which the blood-plasma coagulates. Fibrinogen
solutions are precipitated by an equal volume of a saturated common
salt solution, and are completely precipitated by adding an excess of NaCl
in substance (thus differing from serglobulin). A salt-free solution of
fibrinogen in as little alkali as possible gives with CaCl2 a precipitate which
contains calcium and soon becomes msoluble. In the presence of NaCl
or by the addition of an excess of CaCl2 the precipitate does not appear.^
A neutral solution of fibrinogen is precipitated b}' a concentrated solution
of sodium fluoride when added in sufficient quantity. Fibrinogens from
different kinds of blood behave somewhat differently in this regard. Ac-
cording to HuiSKAMP 2 fibrinogen from horse-blood hardh' dissolves in NaCl
of 3-5 per cent at ordinar}' 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 t3'pical 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 Mitti^lbach.^
Fibrinogen may be easily separated from the salt-plasma or oxalate-
plasma by precipitation with an equal volume of a saturated NaCl solution.
For further purification the precipitate is pressed, redissolved in an S 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 amouiit of NaCl con-
tained 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
Maly's Jahresber., 24; Jacoby, Zeitschr. f. physiol. Chem., 30; Doyon, Morel, and Ivareff,
Compt, rend., 140; Doyon, Morel, and Peju, Compt. rend. soc. biolog., 58.
'See Hammarsten, Zeitschr. f. physiol. Chem., 22; Cramer, ibid., 23.
^Huiskamp, ibid., 44 and 46. In regard to fibrinogen the reader is referred to
the author's investigations. Pfliiger's Archiv, 19 and 22, and Zeitschr. f. physiol.
Chem., 28.
^Zeitschr. f, physiol. Chem., 19.
174 THE BLOOD.
with 0.05 per cent ammonia, and then neutraUzing this solution treated
with NaCl, and repeating this several times. Fibrinogen may also, accord-
ing to Reye/ be prepared by fractionall}- 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 relationship 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 ma}' be beaten to small, less
elastic, and not particularly fibrous lumps. The typical fibrous and
elastic white fibrin, after washing, stands, in regard' to its solubility, close
to the coagulated proteins. It is insoluble in water, alcohol, or ether. It
expands in hydrochloric acid of 1 p. m., as also in caustic potash or soda
of 1 p. m., to a gelatinous mass, which dissolves at the ordmar}- tempera-
ture only after several days; but at the temperature of the ])ody it dis-
solves more readily although still slowly. Fibrin may be dissolved by
dilute salt solutions after a long time at the ordinary- temperature or much
more readily at 40° C, and this solution takes place, according to Arthur
and HuBER 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 (Rulot^). According to Greex and Dastre-* two
globulins are formed in the solution of fibrm 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 de-
stroyed 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 mammals or man by ■^^■hipping
1 W. Reye, Uber Nachweis und Bestimmung des Fibrinogens, Inaug.-Diss. Strass-
burg, 1898.
^ Arthus and Huber, Arch, de Physiol. (5), 5; Dastre, ibid. (5), 7.
^ Arch, intern, de Physiol., 1.
^ Green, .Jour.i. of Fhysiol., 8; Dastre, 1. c.
FIBRIN. 175
and washing first with water and with common salt solution and then
with water again. The blood of various kinds of animals yields fibrm with
somewhat different properties, and according to FeR.\u ^ pig-fibrin dissolves
much more readily than ox-fibrin in hydrochloric acid of 5 p. m.. Fibrins
of varying purity or originatmg 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 transudates. For the pure preparation, as well
as for the quantitative estimation of fibrui, the spontaneously coagu-
lating liquid is at once, or the non-spontaneously coagulatmg liquid orly
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 ifibrinoh/sis — Dastre^). This fibrinolysis must be
prevented in the exact quantitative estimation of fibrin (Dastre). The
blood constituents that are active in fibrinolysis are still not known, 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 (Nolf), and also when
the coagulability of the blood has been reduced by the injection of pro-
teoses (Nolf, Rulot^).
A pure fibrinogen solution may be kept at the ordinary' temperature
until putrefaction begms without showmg a trace of fibrin coagulation.
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 fibrm 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
BucHAXAX.^ was later rediscovered by Alexaxder Schmidt^ and desig-
nated as fibrin ferment or thrombin. The nature of this enzymotic body has
not been ascertained with certainty. Although many investigators,
especially English, consider fibrin ferment as a globulin, still more recent
experiments of Pekelh.\eixg and others show that it is a nucleoproteid
which according to Huiska.mp ^ occurs in the thymus gland partly as
' Zeitschr. f. Biologic, 28.
- Archives cle Physiol. (.5), 5 and 6.
^Jacoby, Zeitschr. f. physiol. Chem., 30; Xolf, Arch, intern, de Physiol.. 3, 1905;
Rulot, 1. c.
^London Med. Gazette, 1S4.5, 617. Cit. by Gamgee, Journal of Physiol., 1879.
^Pfliiger's Arch., G; see also Zur Blutlehre, 1892, and Weitere Beitriige zur Blut-
lehre, 1895.
' Pekelharing, Verhandl. d. kon. Akad. d. Wetensch. te Amsterdam, 1892, Deel 1;
176 THE BLOOD.
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. ScH^nDT
admits of 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 requires, according to more recent investigations,
the presence of a second, zymoplastic-acting substance (see Coagulation
of the Blood). Thrombin is like other enzymes in that the ver>' 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 Fuld has found that at least within certain limits an
increase of double the quantity of enzyme causes an increase of the coagu-
lation velocity to one and one half. This is true only for experiments with
plasma and solutions containing kinases (see Coagulation of the Blood), and
Martin ^ 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 velocity of coagulation is
inversely proportional to the quantity of ferment. The optimum of the
thrombin action lies at about 40° C; at 70-75° C. the enzyme is destroyed.
The question as to whether the thromljin found in different animals is
the same substance or whether we have several thrombins has not been
decided. The latter is not improbable; nevertheless a definite specificity
of different thrombins has not been observed with certainty.
The isolation of thrombin has been tried in several ways. Ordinarily
it may be prepared by the following method, proposed by Alex. Schmidt.^
Precipitate the serum or defibrinated blood with 15-20 vols, of alcohol and
allow it to stand a few months. The precipitate is then filtered 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.^
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 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 c.c. water, is filtered, and in this way a solu-
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; Uber Leukocyten und
Blutgerinnung, ibid.; Halliburton and Brodie, Journal of Physiol., 17 and 18; Huiskamp,
Zeitschr. f. physiol. Chem., 32; Pekelharing and Huiskamp, ibid., 39.
'Martin, Journ. of Physiol., 32; Fuld, Hofmeister's Beitrage, 2.
^ Pfliiger's Arch., 6.
' Hammarsten, ibid. , 18; Pekelharing, 1. c.
COAGULATION OF FIBRINOGEN. 177
tion is obtained which contains only about 0.3-0.4 p. m. solids and about
0.0007 p. m. CaO (Hamaluisten).
If a fibrinogen solution containing salt, as above prepared, is treated
with a solution of fibrin ferment, it coagulates at the ordinary tempera-
ture more or less quickly and yields a typical fibrin. Besides the fibrin
ferment the presence of neutral salts is necessary, for Alex. Schmidt has
shown that fil^rin coagulation does not take place without them. The
presence of soluble calcium salts is not, as is generally assumed, a positive
condition for the formation of fibrin, because, as shown by Alex. Sch\udt,
Pekelharixg, and Hammarstex,^ 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 (Hammarstex) used to pre-
pare it 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. 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 Dexis, 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 pro-
duced only in small amounts. We find a globulin-like substance which
coagulates at about 64° C. in blood-serum as well as in the serum from
coagulated fibrinogen solutions. This substance is called fibrin-globulin by
Hammarstex. The recent investigations of Huiskamp have sho^\ii 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 beside
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.^
There exist also other views in regard to the processes of coagulation in
the formation of fibrin which are even less positively founded. The fact
that the soluble lime salts are not necessary for the transformation of fibrin-
ogen into fibrin is not in contradiction to the other fact that they must be
present in the coagulation of blood or plasma. This apparent contradiction
may be explained, as shown later, by the special condition of the blood-
plasma, and we must not overlook the fact that the coagulation of the blood
is a much more complicated process than the coagulation of a fibrinogen
'See Hammarsten, Zeitschr. f. physioI.Chem., 22, which also cites the works of
Schmidt and Pekelharing, and ibid., 28.
^ See Hammarsten, Zeitschr. f. phj'siol. Chem., 2S; Heubner, Arch, f, exp. Path,
u. Pharm., 49, and Zeitschr. f. physiol. Chem., 4.5; Huiskamp, ibid., 44 and 46,
178 THE BLOOD.
solution, inasmuch as the first involves other important questions, as, for
instance, the reason for the blood remaming fluid in the body, the origin of
the fibrin ferment, the importance of the form-elements in the coag-ulation
etc. A fuller discussion of the various hypotheses and theories concerning
the coagulation of the blood must therefore be given later.
Nucleoproteid. 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. The difficulty of solution in acetic acid is used by Pekelharing
as an important means of separating the compound proteids from the globulins.
Serglobulins, also called paraglohulin (KiJHNE), fibrinoplastic substance
(Alex. Schmidt), serum-casein (Panum^), 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 quan-
tities. 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 nucleoproteid, fibrin-globulin, and the true serglobulin or mixture of
globulins.
The nucleoproteid has already been discussed. The fibrm-globulin,
which occurs in the serum only in small amounts, can be completel}' pre-
cipitated by NaCl. It has the general properties of the globulins, but
differs from the serglobulins by a lower coagidation temperature, 64-66° C,
and also in that it is precipitated by (NH4)2S04 even at 28 per cent satura-
tion.
Serglohvlins. 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 iMarcus 2 also differentiates between a water-soluble
globulin and one insoluble in water. According to the recent investigations
of HoFMEiSTER and PiCK^ the part insoluble in water corresponds chiefly
to a globulin fraction readily precipitated by (NH4)2S04 (by 28-36 vols.
^ Kiihne, Lehrbuch d. physiol. Chem., Leipzig, 1866-68; Alex. Schmidt, Arch, f
(Anat. u.) Physiol, 1861-62; Panuni, Virchow's Arch., 3 and 4.
' Zeitschr. f. physiol. Chem., 2S.
^ Hofmeister's Beitrage, 1.
SERGLOBULIXS. 179
per cent saturated solution), and the part soluble in water corresponds to a
more difficultly precipitable fraction (by 36-44 vols, per cent saturated solu-
tion). The first fraction is called euglobulin and the second pseudoglobulin.
According to Porges and Spiro ^ the serglobulins can be separated by
(NH4)2S04 into three fractions whose precipitation limits are 28-36, 33-42,
and 40-46 vols, per cent saturated solution. All three fractions contain
globulin insoluble in water. Freund and Joachim ^ have recently 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 numlDer of different globulins in the ssrum 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.^
It must not be forgotten that the globulin fractions are always contaminated
with other serum constituents and that these may influence the solubilities 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
ike casein can also, according to Hammarsten," have the solubilities of a globulin
due to contamination with constituents of the serum, and K. Mqrner ^ has also
shown that a contamination of the serum-globulins with soap can essentiall}' modify
the precipitation of these globulins. Under these circumstances the above state-
ments in regard to the different globulin fractions must be accepted with great
caution.
The mvestigations made thus far upon the so-called serglobulm have
not led to any positive results. That this globulin, with the exception of
the enzymes, immune bodies, and other unknown substances which are
carried douTi 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 senmi by the methods to be described has the following properties.
In a moist condition it forms snow-wliite flaky masses, neither tough
nor elastic, which always contain thrombin and hence can bring about
coagulation in a fibrinogen solution. The neutral solution is only incom-
pletely precipitated by NaCl added to saturation and is not precipitated by
an equal volume of a saturated salt solution. It is only partly precipitated
by dialysis or by the addition of acid. On saturation with magi:esium
sulphate or one-half saturation vdth ammonium sulphate a complete pre-
cijiitation is obtained. The coagidation temperature is, with. 5-10 per cent
NaCl in solution, 69-76°, but more often 75° C. The specific rotation of the
* Hofmeister's Beitrage, 3.
' Zeitschr. f. physiol. Chern., 36.
^ Journ. of Physiol., 32.
* See Hammarsten, Ergebnisse d. Physiol., 1, Abt. 1.
* Zeitschr. f. physiol. Chem., 34.
180 THE BLOOD.
solution containing salt is (a)D= —47.8° for the serglobulin from ox-blood
(FredericqI). The various globulin fractions do not differ essentially
from each other in their coagulation temperatures, specific rotation, refrac-
tion coefficient (Reiss^), and their elementary composition. The average
composition is, according to Hammarsten, C 52.71, H 7.01, N 15.85, S 1.11
per cent. K. Morner^ found 1.02 per cent sulphur and 0.67 per cent lead-
blackening sulphur. All the sulphur seems to exist as cystine.
Serglobulin contains, as K. .Morner first showed, a carbohydrate group
which can be split off. Langstein^ has obtained several carbohydrates
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 the blood-serum contains a glucoproteid,
and the investigations of Eichholz ^ seem to show that the globulins are
contammated 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.
Serglobulm (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 by 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 (NH4)2S04 has been used chiefly. The
serglobulin from blood-serum is always contaminated by lecithin and
thrombin. A serglobulin free from thrombin may l^e 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 ^). In the quantitative estimation the
'Bull. Acad. Roy. de Belg. (2), 50. In regard to paraglobulin, see Hammarsten,
Pfliiger's Arch., 17«and 18, and Ergebnisse d. Physiol., 1, Abt. 1,
^ Hofmeister's Beitrage, 4.
* Zeitschr. f. physiol. Chem., 34.
* Morner, Centralbl. f. Physiol., 7; Langstein, Miinch. med. Wochenschr., 1902,
1876, and Wien. Sitzungsber., 112, Abt. llh, 1903; Monatsheft f. Chem., 25; Hofmeister's
Beitrage, 6; see also foot-note 1, p. 33.
" Zanetti, Chem. Centralbl., 1898, I, p. 624; Eichholz, Journ. of Physiol., 23.
° Hammarsten, 1. c; Hofmeister, Kauder and Pohl. Arch. f. exp. Path. u. Pharm.,
20.
SERALBUMINS. 181
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 Haslajvi.
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 proteids 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 proteid 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 ammonium sulphate
with difficulty, represents two seralbumins (Maximowitsch). According
to the statements of Gl-rber and Michel it would seem that the cr>'s-
talline seralbumin is also a mixture, but this is disproved by the obser-
vations of ScHULZ, WiCHMAxx, and Krieger.^ We know nothing as to
the behavior of the amorphous fraction of the seralbumin in this regard.
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 older investigations of Kauder, as well as the more
recent work of Oppexheimer,^ seem to indicate a non-unit nature of the
seralbumins, but this question is still an open one.
The crystalline seralbumin may perhaps ])e a combination with sulphuric
acid (K. MoRXER, Ixagaki). The coagulated albumin obtained from the
aqueous solution of the crystals by the aid of alcohol has nearly the same
elementary composition (Michel) as the amorphous mixture of albumin
prepared from horse-serum (Hammarstex and K. Starke ^). The average
composition was C 53.06, H 6.98, N 15.99, S 1.84 per cent. K. Morxer,
after the removal of the sulphuric acid from crystalline albumin, found
1.73 per cent total sulphur, which probably exists onh' as cystine. Laxg-
STEix "* has been able to split off a nitrogenous carl)ohydrate (glucosamine)
from crystalline serall)umin. The quantity was so small that the question
' In regard to the literature on the crystalline seralbumins, see Schulz, Die Kristal-
lisation von Eiweissstoffen, Jena, 1901; Maximowitsch, Maly's Jahresber., 31, 35.
^ Halliburton, Journ. of Physiol., 5 and 7; Hougardy, Centrabl. f. Physiol., 15,
665; Oppenheimer, Verhandl. d. physidl. Gesellsch., Berlin, 1902.
' Michel, Verhandl. d. phys.-med. Gesellsch. zu Wiirzburg, 29, No. 3; K. Starke,
Maly's Jahresber , 11; K. Morner, 1. c; Inagaki, Biochem. Cenlralbl., 4, p. 515.
* K. Morner, 1. c; Langstein, Hofmeister's Beitrage, 1.
lo2 THE BLOOD.
is still undecided whether or not the carbohydrate was not a contamination.
The fact that Abderhalden, Bergell, and Dorpinghaus ^ were able to
prepare a seralbumin 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 (a) 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 quantity 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 alco-
hol. On the addition of a little common salt it coagulates in both cases.2
Seralbumin differs from the albumin of the white of the hen's egg in
the following particulars: It is more levogyrate; the precipitate formed by
hydrochloric acid easily dissolves in an excess of the acid; it is rendered
less insoluble by alcohol.
In preparing the seralbumin mixture, first remove the globulins, accord-
ing to JoHANSSOxN, 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, 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 solu-
tion at a gentle temperature or by precipitating with alcohol, which must
be quickly removed. Starke ^ has suggested another method, which is
also to be recommended. The crystalline seralbumin may be prepared
from serum freed from globulin ])y half saturating with ammonium sul-
phate, by the addition of more salt until a cloudiness occurs, and then
proceeding according to the suggestion of Gurber and Michel. By
acidification with acetic acid or sulphuric acid the crystallization may
be considerably enhanced.* In the detection and quantitative estimation
of seralbumin the filtrate from the globulin precijDitated with magnesium
sulphate can be heated to boiling, after acidification with a little acetic acid
if necessary. The quantity of seralbumin is best calculated as the difference
bet\\een the total proteins and the globulin.
' Zeitschr. f. physiol. Chem., 41.
^ In regard to the relationship of neutral saUs to heat coagulation, see J. Starke,
;Sitzungsber. d. Gesellsch. f, Morph. u. Physiol, in Miinchen, 1897.
^ Johansson, Zeitschr. f. physiol. Chem., 9; K. Starke, Maly's Jahresber., 11.
^ See Hopkins and Pinkus, Journ. of Physiol., 23; Krieger, tjber die Darstellung
Icrj'stallinscher tierischer Eiweissstoffe, Inaug.-Dissert. Strassburg, 1899.
BLOOD SERUM. 183
Summary of the elementary composition of the above-mentioned and described
proteins (from horse-blood) :
Fibrinogen 52 . 93 6 . 90 16. 66 1 . 25 22 . 26 (Hammarstex)
Fibrin 52.68 6. S3 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)
Embden and Knoop as well as Langstein have detected in blood-serum
proteose-like substances which, according to them, occur preformed in the blood.
KoLF has also found a small ciuantity of proteoses in the blood after an abundant
absorption of proteoses by the intestine. According to Abderhaldex and Oppen-
HEiMER ' the proteoses cannot be considered as normal blood constituents; even
if we admit of their presence, the cjuantity is too small to be of any physiological
importance.
v. Bergmaxn and Langsteix ^ have determined in dogs the residual nitrogen
in the blood-serum, i.e., the nitrogen of the non-coagulable constituents. They
found after feeding proteids that of the residual nitrogen 25 per cent existed
as proteoses and 5.5 per cent as other products precipitable with phosphotung-
stic acid. In starving animals they found a maximum of 9 per cent of the
residual nitrogen as proteoses.
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 constitu-
ents as the blood-plasma.
Blood-senim is a sticky liquid which is more alkaline towards litmus
than the plasma. The specific gra\'ity in man is 1.027 to 1.032, average
1.028. The color is often strongly or faintly yellow; in human blood-
serum it is pale yellow ^^•ith a shade towards green, and in horses it is often
amber-3^ellow. The serum is ordinarily clear; after a meal it may be
opalescent, 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 amoimt is increased to a great extent. Soaps, cholesterin, and lecithin
are also found. Cholesterin occurs, according to HtJRTHLE,^ at least in
part, as fatty-acid esters (seroh'yi according to Boudet).
' Embden and Ivnoop, Hofmeister's Beitrage, 3; Langstein, ibid., 3; Nolf, Bull.
Acad. Roy. Belg., 1903 and 1904; Abderhalden and Oppenheimer, Zeitschr. f. physiol.
Chem.,42.
^ V. Bergmann and Lang.stein, Hofmeister's Beitrage, 6.
^ 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.
184 THE BLOOD.
Sugar seems to be a physiological constituent of the plasma and serum.
According to the investigations of Abeles, Ewald, Kijlz, v. jMering,
Pavy, Seegen, and Miura^ the sugar found is dextrose. Strauss ^ 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 iso-
maltose (Pavy and Siau) and pentose (L:6pine and Boulud^), in blood-
serum is still undecided. Asher and Rosenfeld* 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 observa-
tions do not exclude the possibility of the existence of a part of the sugar
in a combmed form, as above stated (p. 180). Besides sugar the blood-
serum contains, as first shown by J. Otto, also another reducing non-
fermentable substance. The statements of Jacobsen, Henriques, and
BiNG,^ that this substance is jecorin or lecithin sugar, do not have sufficient
foundation. 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,^ is also undetermined. The virtual
sugar is more abundant in the blood of the right ventricle than m 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 P. ^Iayer,
Lepine and Boulud.^ The last two investigators find two definite glucu-
ronic 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 glucuronic acid coun-
teracts that of the glucose. The second acid also occurs in larger quantities
as compared with the sugar.
Bernard ^ has shown that the quantity of sugar in the blood diminishes
' See V. Mering, Arch. f. (Anat. u.) Physiol., 1877 (this article contains numer-
ous references); Se^gen, Pfliiger's Arch., 40; Miura, Zeitschr. f. Biologie, 32.
2 Fortschritte d. Mediz., 1902.
^ Pavy and Siau, Journ. of Physiol., 2G; Lepine et Boulud, Compt. rend., 133, 135,
and 13G.
■* Centralbl. f. Physiol., 19, p. 449.
^Otto, Pfliiger's Arch., 35 (a good review of the older literature on sugar in the
blood); Jacobsen, Centralbl. f. Physiol., 6 368; Henriques, Zeitschr. f. physiol. Chem.,
23; Bing, Skand. Arch. f. Physiol., 9.
"Compt. rend., 137.
' 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).
* Lemons sur le diabete, Paris, 1877.
BLOOD SERUM. 185
more or less rapidly on lea\'ing the veins. L:fipiNE, associated ^ith Baeral^
has specially studied this decrease in the quantity of sugar and calls it
glycolysis. Lepine and Barral, as well as Arthus, have sho-wn that this
glycolysis takes place in the complete absence of micro-organisms. It
seems to be due to a soluble glycolytic enzyme whose acti\-ity is destroyed
by heating to 54° C. This enzyme is derived, according to the above
investigators, from the leucocytes and, according to Lepine,^ has some
connection ^\-ith the pancreas. The glycolysis is, according to Nasse,
RoHMANN and Spitzer and Sieber,^ an oxidation wliich is produced,,
according to the two last -mentioned investigators, by an oxidation ferment.
It is certainly not connected with the sur\'ival of the cells, but whether it
is a \-ital or a post-mortem process is not decided.'^
The blood-plasma and the serum, as well as the lymph, also contain
enzymes of various kinds. According to Roh^l\xx, Bl\l, Hamburger,*
and others, diastases, which convert starch and glycogen into maltose or
isomaltose, as well as a maltoglucase are found in the blood. Hanriot
has detected a lipase in the senmi which decomposes butyrin, and which,
according to him, decomposes neutral fats and other esters. The occur-
rence of a hutyrinase is generally admitted, while the property of this lipase
of splitting olein and other neutral fats is not generally acknowledged
(Arthus, Doyox and ]\1orel^). Thi« 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 phenom-
enon first observed by Cohxsteix and ^Iichaelis and further studied by
Weigert.^ This property seems to be connected with the form-elements
of the blood.
Besides the above-mentioned enzymes and thrombin, several other
enzymes have been found in the iDlood-serum, namely, oxidases, catalases,
' I'l regard to the numerous memoirs of Lepine and Lepine et Barral, see Lyon
medical., 62 and 63; Compt. rendus, 110, 112, 113, 120, and 139; Lepine, Le ferment
glycolytique et la pathogenie du diabete (Paris, 1891), and Revue analytique et
critique des travaux, etc., in Arch, de med. exper. (Paris, 1892); Revue de medecine,
1895; Arthus, Arch, de Physiol. (5), 3, 4; Xasse and Framm, Pfiuger's Arch., 63,
Paderi, Maly's Jahresber., 26; see also Cremer, Physiologic des Glykogens in Ergebnisse
d. Physiol., 1, Abt. 1.
^ See Chapter I and N. Sieber, Zeitschr. f. physiol Chem., 39 and 44.
^ See Arthus, 1. c; Colenbrander, Maly's Jaliresber., 22; RjTvosch, Centralbl. f.
Physiol., 11, 495.
^ Rohmann; Rohmann and Hamburger, Ber. d. deutsch. chem. Gesellsch., 25 and
27; Pfli'iger's Arch., 52 and 60; Bial, Leber das diast. Perm., etc., Inaug.-Diss. Breslau,
1892 (older hterature). See also Pfliiger's Arch., 52, 54. and 55.
^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. toe. biol., 54;
Achard and Clerr (Lipase in Eisease), Compt. rend., 129, and Arch. d. med. exper., 14.
* Cohnstein and Michaelis, Pfliiger's Arch., 65 and 69; Weigert, ibid., 82.
186 THE BLOOD.
proteolytic enzymes, rennin, and trypsin, and also the corresponding anti-
enzymes. 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 jwecipitines which can be
used as a biological reagent on account of their action upon various pro-
teins. It may be sufficient to state that the works of Bordet, Ehrlich,
Wassermann, Schutze, Uhlenhaut/ 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 towards 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 impor-
tance, due to the work of Uhlenhaut. The various enzymes and anti-
enzymes, toxines 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 pseudo-
globulin fraction.
Among the bodies which are found in the blood, and without doubt are
met with in smaller or greater amounts in the plasma, are to be mentioned
urea, uric acid (found in human blood by Abeles), jihosphocarnic acid (Pa-
NELLA^)^ creatine, carhamic acid, paralactic acid, hippuric acid, and traces of
indol (Hervieux^). Under pathological conditions the following bodies
have been found: xanthine bodies, leucine, tyrosine, lysine (Neuberg and
RiCHTER ^), and biliary constituents.
The coloring-matters of the blood-serum are very little known. In
equine blood-serum the biliary coloring-matter, bilirubin, besides other color-
ing-matters, often occurs. 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 Krukenberg ^ 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
dialysis, the presence of sodium chloride, which forms the chief mass or
' The literature on this subject may be found in bacteriological journals and works.
See also L. Miciiaelis, Biochem. Centralbl., 3, p. 693.
' Abeles, Wien. med. Jahrb., 1887; Panella cited from Virchow's Jahresber. f. 1902,
150.
' Compt. rend. .'oc. biolog., 56.
* Deutsch. n;ed. Wochenschr., 1904.
* Sitzungsber. d. Jen, Gesellsch. f. Med., 1885.
MINERAL CONSTITUENTS OF THE SERUM. 1S7
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.^ 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 coin-
cides also with the fact that the great part of the alkalies does not exist
in the senmi as diffusible alkali compounds, carbonate and phosphate, but
as non-diffusible compounds, protein combinations. According to Ham-
burger ^ 37 per cent of the alkali of the serum from horse-blood was dif-
fusible 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 Bourcet), while
arsenic, which is not found in all blood occurs only in human blood
(Gautier, Bourcet 3). Iodine occurs to a greater extent in menstnial
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.
Because of the difficulty of obtaining plasma only a few analyses have
been made. 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 ]n^Tq_ 2 is the average of the results of three analyses made by
Hammarstex. The figures are given for 1000 parts of the plasma.
No. 1. No. 2.
Water 908.4 917.6
Solids 91.6 82.4
Total proteins 77.6 69.5
Fibrin. 10.1 6.5
Globulin. 38.4
Seralbumin 24 . 6
Fat 1.2
Extractive substances 4.0
Soluble salts 6.4 1 ^^'^
Insoluble salts 1 . 7 J
Lewinsky ^ has determined the total proteins and the individual pro-
teins in the blood-plasma of man and animals with the following results.
' See Giirber, Verhandl. d. phys.-med. Gesellsch. zu Wiirzburg, 23.
^ In regard to method, see Arch. f. (Anat. u.) Physiol., 1898.
^ Gley et Bourcet, Compt. rend., 130; Bourcet, ibid., 131; Gautier, ibid., 131.
* Cit. from v. Gorup-Besanez's Lehrbuch der physiol. Chem., 4. Aufl., 346.
^Pfliiger's Arch., 100.
188 THE BLOOD.
Total Protein. Albumin. Globolin. 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 SO. 5 44.2 29.8 6.5
Abderhaldex has made complete analyses of the blood-.semm of several
domestic animals. From the.=e analyses as well as from those made by
Hammarstex of the serum from htmian, horse, and ox blood it follows that
the amount of solids ordinarily varies betw-een 70-97 p. m. The chief mass
of the solids consists of proteins, about 55-S4 p. m. In hens Hamm.ui-
STEN found much lower values, namely. 54 p. m. solids, with only 39.5 p. m.
protein, and Halliburtox found only 25.4 p. m. protein in frog's bl<x>d.
The relationship between globulin and seralbumin is. as shown by the
analyses of H.odjarstex, H.u.libuhtox, and Rubbrecht.i very different
for various animals, but may also vsiry considerably in the same species of
animal. In human blood-serum HAinL^JBXEx found more seralbumin
than globulin, and the relationship of sei^lobulin to seralbumin was as 1 :1.'5
Lewixskt foimd the relationship in man greater than 1. indeed 1 : 1.39-2.13.
In regard to the quantity of the remaining organic constituents of the serum
we refer the reader to Abderhaldex's complete analyses.
In starvation it .seems, as first found by Brr.CKH.^RDT and recently sub-
stantiated by GiTHExs.2 that the quantity of globulin relative to that of
albumin is mcreased. A change in the relationship with a decrease in the
albumin and increase in the globulin may also occur in animals which have
been made sick or in part immune by inoculation with pathogenic micro-
organisms (Laxgsteix and ^L\yee3). The total protein content is raised
in nearly all cases. The amount of fibrinogen in the plasma is espe-
cially increased by pneumococci, streptococci, and pas^taphylococci
(P. Muller^).
The quantity of nuneral bodies in the .serum has been determined by
manv investigators. The conclusion drawn from the analy.ses is that there
exists a rather close correspondence between htmian and animal blood-
serum, and it Ls therefore sufficient to give here the analysis of C. Schmidt *
of (1) htmian blood, and Buxge and Abderh-axdex's anah-.ses (2) of senun
of ox, bull, sheep, goat, pig, rabbit, dq?, and cat. The results correspond
to 1000 parts by weight of the senun.
' Abderhalden. Zeitschr. f. physiol. Chem.. 25; Hammarsten. Pfluger's Arch., 17;
Halliburton. Jo\im. of Physiol., 7; Rubbrecht, Travaux du laboratoire de I'institut
de physiologie de Liege, 5, 1S96.
2 Burckhardt. Arch. f. exp. Path. u. Pharm., 16; Githens, Hofmeister's Beitrage, 5.
' Hofmeister's Beitrage, 5.
*Ibid., 6.
scit. from Hoppe-Seyler. Physiol. Chem., T SI, p. 439.
MINERAL CONSTITUENTS OF THE SERUM. ISO
1 2
KoO 0.387-0.401 0.226-0.270
Na,0 4.290-4.290 4.251-4.442
CI 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 . 0S5
Even if we bear in mind that certain bodies, such as carbon dioxide,
are driven off durmg uicmeration and that other bodies, such as sulphuric
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 m the serum or in other fluids, a question
which is of the greatest physiological importance. .\n answer to these
questions is obtainable only by physico-chemical investigations, which have
thus far been used cliiefly m determming the molecular concentration, the
amount of electrolytes and non-electrolytes, and the degree of dissociation.
The molecular, or, as Hamburger calls it, the osmotic concentration which gives
the total number of molecules and ions in the litre, is measured bv the osmotic
J
pressure, and it may be expressed bj' 7-^ if we make use of the depression of the
freezing-point (J) instead of the osmotic pressure, as a gram-molecule of a non-
electrolyte, or an equivalent number of ions, when in 1 litre of solution, causes a
depression of the freezing-point of 1 .85°.
The average depression of the freezing-point of human blood-serum is
ordinarily given as J = — 0.526°. According to Th. CohxI the actual
depression of the freezing-pomt of normal human blood is J =—0.537°.
This freezing-point depression, it seems, is a little lower than that of the
sera of other mammals that have been investigated: —0.560° (horse) to
0.619° (sheep). The molecular concentration of the blood-serum of various
mammals also differs only slightly in each case, according to Bugarsky and
Taxgl.- and amounts on an average to about 0.320 mol per litre. The
average freezing-point depression corresponds closely to that of a common
salt solution of 9 p. m. (J=— 0.551° to —0.561°). and at present such a
solution is considered as a physiological salt solution for man and other
mammals.
The conditions are otherwise with sea-animals which live in a medium
rich in salts. According to Bottazzi the blood (or the fluid of the cavities)
of invertebrate sea-animals has an osmotic pressure which corresponds to
an average freezing-point depression of J = — 2.29°, i.e.. exactly the same
^ Mitteil. aus d. Grenzgeb. d. Mediz. u. Chir.. 1.').
■ In regard to the literature on this subject we refer to Hamburger, Osmotischer
Druck und lonenlehre. from which the author obtained most of the facts given. See
also Hober, Physikalische Chemie der Zelle und der Gewebe, 2. Aufl., 1906.
190 THE BLOOD.
as the sea-water in which they Uve. In tlie cartilaginous fishes nearly the
same conditions exist, while in the Teleostei the osmotic pressure is much
lower than that of the sea-water, but is about one half greater than the
blood of land-vertebrates. The Teleostei are the first in the scale of de-
velopment of animals to show an independence of the osmotic pressure of
the inner fluids from the surrounding media.
The researches of Fredericq ^ have led to the same results. In the
sea-invertebrates examined the blood (the hsemolymph) had the same
molecular concentration and same salt content as the exterior medium.
In plagiostoma the blood had, with equal molecular concentration, a con-
siderably lower salt content than the sea-water. The equality of the
molecular concentration was maintained in these cases by a high urea
content. In all bony fishes of salt and fresh waters and in fresh-water
crabs the blood differs markedly in regard to molecular concentration, as
well as in salt content, from the exterior medium.
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.
As seen from the above, blood-serum contains electrolytes as well as
non-electrolytes. Of the latter the proteins and also sugar, fat, lecithin,
urea, and the so-called extracti\e bodies are of the greatest importance.
The electrolytes comprise the various ions and the undissociated molecules
of the salts of the serum. The electrolytes are the only constituents of
the serum which conduct the electric current, while the non-electrolytes
retard the conductivity. The degree of dissociation can also be calculated
from the determination of the conductivity of the blood-serum.
The coefficient of dissociation is, according to Arrhenius, the relationship
between the number of ions in a solution and the number of ions which would
be present if the electrolytes were completely dissociated. As the conductivity
of a solution of electrolytes is determined by the number of ions (admitting that
the migration velocity of the ions is the same for different dilutions), the above
coefficient a can be calculated by the formula n = - — . In this formula h repre-
sents the conductivity of the original dilution (i.e., of the undiluted serum) and
^-jc the conductivity of the completely dissociated molecules (ions) after suffi-
ciently strong dilution of the serum with water.
According to the above principle the degree of dissociation of serum has
been determined by several investigators, especially Bugarsky and Tangl,
Oker-Blom, and Viola. This last .investigator found that the degree of
dissociation of the blood-senim of healthy human beings was equal to
0.68-0.73. According to Hamburger the results thus obtained experi-
' Aich. de Biol., 20. Cited from Centialbl. f. Physiol., 19. p. 21.
PHYSICO-CHEMICAL PROPERTIES. 191
mentally must be a little too low for certain reasons, and we therefore can
consider the dissociation coefficient to be between 0.65 and 0.82.
As above stated, the non-electrolytes have a retarding action upon the con-
ductivity, and according to Bugarsky and Tangl each gram of protein in 100
c.c. of serum diminishes the electrical conductivity of the serum about 2.5 per
cent. By making use of this fact, the corrected conductivity of the electrolytes
present can be determined from the conductivity. The corrected conductivity
is partly dependent upon the chlorides and partly upon the other salts (which are
nearly identical with the quantity of NaaCOg). If the amount of NaCl of the
serum is determined by analysis we can calculate the conductivity of the other salts
by subtracting the calculated conductivity of a solution of NaCl of similar con-
centration (which can be done according to Kohlrausch's method) from the total
corrected conductivity. From these results we can calculate the molecular
concentration of the chlorides and of the non-chlorides. The sum of these two
is subtracted from the molecular concentration of the serum, when the molecular
concentration of the non-electrolytes is obtained.
Bugarsky and Tangl have made physico-chemical analyses of blood-
serum of certain mammals according to the principle given above. They
found that the molecular concentration was, on an average, about 0.320
mol per litre, that about three fourths of the total dissolved molecules
of blood-serum were electrolytes, although the serum contained about
70-80 p. m. proteid and 10 p. m. inorganic bodies, and also that three
fourths of the quantity of electrolytes consisted of NaCl. Viola and Bous-
QUET have recorded less complete osmotic chemical analyses of blood-serum
of diseased and healthy human beings, making use of methods somewhat
different in principle.
In the determination of the alkalinity of blood and blood-serum, up to
the present time we have estimated the amount of alkali by titration 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 Na2C03
is in aqueous solution more or less dissociated into 2Na+ and COs"", depend-
ing upon the dilution. The C03= ions combine partly with the H+ ions
of the dissociated water, forming HCOs", and the corresponding H0~ ions
produce the alkaline reaction. If now, by the addition of a little acid, a
few of the H0~ ions are removed, then the equilibrium is disturbed, a new
quantity of Na2C03 is dissociated, and this process is repeated ever}' 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 physico-chemical
method of determining alkalinity, based upon Nernst's theoiy of liquid
chains. This method was 'used later by Farkas, Franckel, and Hober
after a few changes. The investigations of these last -mentioned experi-
192 THE BLOOD.
menters show that the concentration of the hydroxy! ions in blood-serum
and blood is nearly the same as in distilled water, and that these fluids are
nearly neutral in behavior, which fact ic caused by the presence of carbonic
acid. Friedenthal,! by testing serum with phenolphthalein, arrived at
similar results.
n. THE FORM-ELEMENTS OF THE BLOOD.
The Red Blood-Corpuscles.
The blood-corpuscles are round, biconcave disks without membrane and
nucleus in man and mammalia (with the exception of the llama, the camel,
and their congeners). In the latter animals, as also in birds, amphibia, and
fishes (with the exception of the Cyclostoma), the corpuscles have in general
a nucleus, are biconvex and more or less elliptical. The size varies in
different animals. In man they have an average diameter of 7 to 8 ^
(/i= 0.001 mm.) and a maximum thickness of 1.9 //. They are hea\'ier
than the blood-plasma or sennn, and therefore sink in these liquids. In
the discharged blood they may lie sometimes with their flat surfaces to-
gether, forming a cylinder like a roll of coin (rouleaux). The reason for
this phenomenon, which is considered as an agglutination, has not been
suffic-'^'ntly studied, but as it may be observed in defibrinated blood it seems
proba. le that the formation of fibrin has nothing to do with it.
The number of red blood-corpuscles is different in the blood of various
animals. In the blood of man there are generally 5 million red 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 haemoglobin. We cannot state anything posi-
tive for the present in regard to a more detailed arrangement, 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 hsemoglo]:)in solution,
while the other view considers the stroma as a protoplasmic structure
soaked with haemoglobin. This latter view is in accord with the assump-
tion 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 haemoglobin. This mass represents the water-attract-
ing force of the blood-corpuscles, and besides this it is also considered that
the outer protoplasmic boundary is semi-permeable, i.e., permeable to
water but not permeable to certain crystalloids. The researches of Koppe,
' Hober, PfliJger's Arch., 81 and 99; Farkas, see Biochem. Centralbl., 1, 626;
Franckel, Pfliiger's Arch., 9fi; Friedenthal, Zeitschr. f. allg. Physiol., 1 and 4.
THE RED CORPUSCLES. 193
Albrecht, Pascucci, Rywosch,^ and others indicate the presence of a
special envelope or boundary-layer, and there is no doubt that the outer
layer contains so-called lipoids, such as cholesterin, lecithin, and similar
bodies.
The red blood-corpuscles retain their volume in a salt solution which
has the same osmotic pressure as the serum of the same blood, although
they may change their form in such solutions, becoming more spherical,
and may also midergo a chemical change (Hamburger, Hedin, and others).
Such a salt solution is isotonic^ with the blood-serum, and its concentra-
tion for a NaCl solution is approximately 9 p. m. for human and mam-
malian blood. A solution of greater concentration, a hyperisotonic solu-
tion, abstracts water from the blood-corpuscles imtil osmotic equilibrium
is established, hence the corpuscles shrink and their volume becomes
smaller. In solutions of less concentration, hypisotonic solutions, the cor-
puscles swell up, due to the taking up of water, and this swelling may be
so great, as on diluting the blood with water, that the haemoglobin is sepa-
rated from the stroma and passes into the watery solution. This process
is called Jicemolysis.
A hsBmolysis may also be brought about by alternately freezing and
thawing the blood, as well as by the action of various chemical subst-'nces,
which act as protoplasmic poisons. These bodies are ether, chlo. .orm,
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 haemolysines, which act like toxines. These hsemolysines 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, occurrmg 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
(KoPPE, Ransom and Kobert, Peskind, Pascucci). The action of other
haemolysines, such as snake- venom and tetanotoxine, seems to be an action con-
nected with the lecithin (Kyes, Pascucci ^).
' See Hamburger, Osmotischer Druck und lonenlehre, 1902; Koppe, Pfluger's
Arch., 99 and 107; Albrecht, Centralbl. f. Physiol., 19; Pascucci, Hofmeister's Beitrage,
6; Rywosch, Centralbl. f. Physiol., 19.
^ The work of Hamburger, Hedin, Eykman, Koppe, and others on isotonism, and
the literature on this subject, may be found .in Hamburger, Osmotischer Druck und
lonenlehre, 1902.
^ Koppe, 1. c; Peskind, Amer. Journ. of Physiol., 12; Ransom and Kobert, cited
by Pascucci, Hofmeister's Beitrage, 0; Kyes, Zeitschr. f. physiol. Chem., 11, and Berl.
klin. Wochenschr,, 1904.
194 THE BLOOD.
When the haemoglobin is separated from the so-called stroma by a suffi-
ciently strong dilution with water the stroma is found in the solution in a
swollen condition. By the action of carbon dioxide, by the careful addi-
tion 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 re-
mains after the removal of haemoglobin and other bodies soluble in water.
To fsolate the stromata from the blood-corpuscles, they are washed first
by diluting the blood with 10-20 vols, of a 1-2 per cent common salt
solution and then separating the mixture by centrifugal force or by
allowing it to stand at a low temperature. This is repeated a few times*
until the blood-corpuscles are freed from serum. These purified blood-
corpuscles are, according to Wooldridge, mixed with 5-6 vols, of water,
and then a little ether is added until complete solution is obtained. The
leucocytes gradually settle to the bottom, a movement which may be
accelerated by centrifugal force, and the liquid which separates 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 collected
on a filter and quickly washed. Pascucci,^ on the contrary, treats the
mass of corpuscles with 15-20 vols, of a -g- 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, cholesterin,
nucleoalbumin, and a globulin which, aceordiag to Halliburton, is prob-
ably a nucleoproteid which he calls cell-glohulin. No nuclein substances
or seralbumiri or proteoses could be detected by Halliburton and
Friend. According to Pascucci, the stromata (from horse-blood) consists
of ^ cholesterin and lecithm (besides a little cerebroside), and § protein
substances and mineral bodies. The nucleated red blood-corpuscles of
the bird contain, according to Plosz and Hoppe-Seyler,^ nvjdein and a
protein 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 {hya-
line substance of Rovida, see page 141) occurring in the lymph-cells. In
the mass extracted by alcohol from the blood-corpuscles of the hen,
' Hofmeister's Beitrage, 6.
MVooldridge, Arch. f. (Anat. u.) Physiol., 1881, 387; Halliburton and Friend,
Journal of Physiol., 10; Halliburton, ibid., 18; Plosz, Hoppe-Seyler's Med. chem,
Untensuch., 510.
THE RED CORPUSCLES. l^cp
AcKERMAXx 1 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
hist one. The non-nucleated red blood-corpuscles are, as a rule, ver}^ poor
in protein, but are rich in haemoglobin; the nucleated corpuscles are richer
in protein and poorer in haemoglobin than the non-nucleated.
A gelatinous, fibrin-like protein body may be obtained from the red
blood-corpuscles under certain circumstances. This fibrin-like mass has^
been observed on freezing and then thawing the sediment of the blood-
corpuscles, or on discharging the spark from a large Leyden jar through
the blood, or on dissohing the blood-corpuscles of one kind of animal in
the serum of another (Landois, strorna-fibrin) ; i.e., in the so-called hcem-
agglutination, a clumping of the red blood-corpuscles into clusters takes
place. This agglutination can be brought about by bodies similar to the
hiemolysines and also by serum constituents produced 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 cor-
puscles of frogs' blood (Alex. Schmidt and Semmer^).
Closely related to. the anatomical and chemical structure of the erythro-
cytes is the question which is important, for the metabolism m the blood,,
as to the permeability of the er^^throcytes, that is, their power of taking
up substances of different kinds. On this subject we have the researches
of Gruns, Eykmax, Overton, Koppe, and especially those of Ham-
burger and his collaborators, and of Hedix.^ As a result of these
researches, it has been shown that the blood-corpuscles are completely
impermeable for the ordinary' varieties of sugar, for arabite and mannite.
and, as it appears, also for the cations Ca"*"^, Sr++, Ba"*"*", Mg+"'". On the
other hand, they are permeable for NH4+ ions, as also for acids and alkalies.^
They are also permeable for alcohols (more readily the fewer Iwdrox}!
groups the molecule contains), aldehydes (with the exception of paralde-
hyde), ketones, ethers, esters, urea, bile salts, and other compounds. They
are only slightly permeable for amino-acids. Towards the neutral potas-
sium and sodium salts, according to Koppe and Hamburger, the blood-
corpuscles are impermeable for the cations K+ and Na+, and permeable,
on the contrar}', for the anions when an exchange of an anion, for example
C03=, in the blood-corpuscles is possible -uith an anion in the outer fluid..
for example with Cl~, Br~, NOa", etc. Hober ^ has further shown that
the blood-corpuscles are permeable for anions under the influence df
' Zeitschr. f. physiol. Chem., 43.
'Landois, Centralbl. f. d. med. Wissensch., 1874, 421; Schmidt, Pfliiger's Arch...
11, 550-559.
^ In regard to the hterature, see Hamburger, Osmotischer Druck- und Ionenlehre_
* See Hober, Pfliiger's Arch., 101 and 102.
^Pfliiger's Arch., 102
196 THE BLOOD.
carbon dioxide. Such an exchange of ions can be especially observed,
according to Hamburger, in the erythrocytes suspended in NaCl solution
and treated with CO2, when the outer fluid becomes alkaline, due to the
formation of Na2C03 by the migration of Cl~ ions into the corpuscles and
an outward migration of the COs^ ions. For every one bivalent COa^
ion there must migrate inward two imivalent Cl~ ions; but as every ion
irrespective of whether it is uni- or bivalent has the same osmotic pressure,
therefore the osmotic pressure of the blood-corpuscles must be raised, and
hence a swelling up takes place, due to their taking up water. The question
as to how far these observations can be applied to the blood-corpuscles
in their serum, i.e., to the blood, requires further proofs.^
The mineral bodies of the red corpuscles will be treated in connection
with their quantitative constitution.
The constituent of the blood-corpuscles existmg in greatest quantity is
the red pigment hsemoglobin.
Blood-pigments.
According to Hoppe-Seyler the coloring-matter of the red blood-
corpuscles is not in a free state, but comljined with some other substance.
The crystalline coloring-matter, the hsemoglobin 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 un-
like the questionable compound itself. This compound is insoluble in
water and uncrj^stallizable. 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 hsemoglobin and the oxy haemoglobin, Hoppe-Seyler calls
the compound of the blood-coloring matter of the venous blood-corpuscles
jjhlehin, and that of the arterial arterin. Other investigators, such as
H. U. KoBERT and Bohr,2 the latter calling the pigment of the blood-
corpuscles hcemochrom, are of a similar opinion. Since the above-mentioned
combinations of the blood-coloring matters with other bodies, for example
(if they really do exist) with lecithin, have not been closely studied, the
following statements will apply only to the free pigment, the haemoglobin.
The color of the blood depends in part on hcemoglohin and in part on a
molecular combination of this substance with oxygen, the oxyhcemoglohin.
' See Petry, Hofmeister s Beitrage, 3.
2 Hoppe-Seyler, Zeitschr. f. physiol. Chem., 13, 479; H. U. Robert, Das Wirbeltier-
blut in mikro-kristallogr. Hinsicht, Stuttgart, 1901; Bohr, Centrall)!. {. Physiol., 17,
p. 688.
BLOOD PIGMENTS. 197
We find in blood after asphyxiation almost exclusively haemoglobin, in
arterial blood disproportionately large amounts of oxyhemoglobin, and m
venous blood a mixture of both. Blood-coloring matters are found also in
striated as well as in cert am 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.
Hemoglobin belongs to the group of compound proteids and yields as
cleavage products, besides very small amounts of volatile fatty acids and
other bodies, chiefly a protein globin and a coloring-matter, hcemochromogen
(about 4 per cent), containing iron, which in the presence of oxygen is
easily oxidized into hcematin.
As first shown by Schunck and ^Iarchlewski, and especially by the
work of the latter, a close relationsliip exists between chlorophyll and the
blood-pigment, because a derivative of the first, phylloporphyrin, stands
veiy close in certain regards to a derivative of the blood-pigment hsema-
toporphyrin. By the mvestigations of Nexcki in conjunction ^\ith March-
LEWSKi and Zaleski,^ it was shown that hsemopyrol 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 wdth one another, which
probably depends upon the somewhat varying methods of preparation.
The follo^\ing analyses are given as examples of the constitution of different
hnemoglobms :
Hffimoglobin from the C H N S Fe O P2O5
Dog 53.85 7.32 16.17 0.390 0.430 21. S4 (Hoppe-Seyler)
" 54.57 7.22 16.38 0.568 0.336 20.93 (Jaquet)
Horse 54.87 6.97 17.310.650 0.470 19.73 (Kossel)
" 51.15 6.76 17.94 0.390 0.335 23.43 (Zinoffsky)
Ox 54.66 7.25 17.70 0.447 0.400 19.543 .... (Hufxer)
Pig 54.17 7.38 16.23 0.660 0.430 21.360 (Otto)
" 54.717.38 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 54.09 7.39 16.09 0.400 0.590 21.440 "
Goose 54.26 7.10 16.210.540 0.430 20.690 0.770 "
Hen 52.47 7.19 16.45 0.857 0.335 22.500 0.197 (Jaquet)
^ Schunck and Marchlewski, Annal. d. Chem. u. Pharm., 278, 284, 288, 290; Nencki,
Ber. d. deutsch. chem. Gesellsch., 29; Marchlewski and Nencki, Ber. d. d. chem.
Gesellsch.. 34; Nencki and Zaleski, ibid.; Marchlewski, Chem. Centralbl., 1902, I,
1016; Zaleski, Zeitschr. f. physiol. Chem., 37.
198 THE BLOOD.
The question whether the amount of phosphorus in the haemoglobin
from birds exists as a contamination or as a constituent has not been
-decided. According to Ixoko the hseraoglobin from goose-blood consists
of a combination between nucleic acid and haemoglobin. In the hserao-
globin from the horse (Zinoffsky), the pig, and the ox (Hufxer) 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 loosel}- combined oxygen, HtiFNER*
lias calculated the molecular w^eight of dog-haemoglobin as 14 129 and the
formula CeseH 1025^^1 64FeS30i8i. According to the more recent determina-
tions of HiJFXER and Jaquet,^ ox-haemoglobin contains an average of
0.336 per cent iron, from which a molecular weight of 16 669 may be cal-
culated. The haemoglobin from various kinds of blood not only shows a
diverse constitution, but also a different solubility and crj-stalline form,
and a varj-ing quantity of water of crj'stallization; hence we infer that
there are several kinds of haemoglobin. Bohr is a ver}- zealous advocate
of this supposition. He has been able to obtain haemoglobins from dog-
and horse-blood, by fractional crj'stallization, which had different powers
of combining with oxygen and contained different quantities of iron.
Hoppe-Seyler had already prepared two different forms of haemoglobin
-crj'stals from horse-blood, and Bohr concludes from all these observations
that the ordinary' haemoglobin consists of a mixture of different haemo-
globins. In opposition to this statement, Hufxer ^ has shown that only
one haemoglobin exists in ox-blood, and that this is probably true for
the blood of many other animals.
Oxyhaemoglobin, which has also been called h.ejlatoglgbulix or
H-EiLATOCRYSTALLix. is a molecular combination of haemoglobin and oxy»
gen. For each molecule of haemoglobin 1 molecule of ox\^gen is present;
^nd the amount of loosely combined oxygen which is united to 1 gram of
haemoglobin (of the ox) has been determined by Hufxer* as 1.34 c.c.
(calculated at 0° C. and 760 mm. mercur}-).
According to Bohr, the facts are different. He differentiates between four
oxyhsemoglobins, according to the quantity of oxygen which they absorb, namely,
a-. ;?-, r-. and o-oxyhfemoglobin, all having the same absorption-spectrum and 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, Joum. f. prakt. Chem. (N. F.), 22; Otto, Zeitschr. f. physiol.
Chem., 7; Inoko, ibid., 18.
' Arch. f. (.\nat. u.) Physiol, 1894.
^ Bohr, ''Sur les combinaisons de I'hemoglobine avec I'oxygene," Ext rait du
Bulletin de TAcademie Royale Danoise des sciences, 1890; also Centralbl. f. Physiol.,
1890, 249. Hoppe-Seyler, Zeitschr. f physiol. Chem., 2; H fner. Arch. f. (Anat. u.)
Physiol., 1894.
* Arch, f. (.\nat u.) Physiol.. 1901, Suppl.
OXYHiEMOGLOBIX. 199
gram combining with respectively 0.4, O.S, 1.7, and 2.7 c.c. oxygen at the tem-
perature of the room and with an oxj'gen pressure of 150 mm. mercury. The
/-oxyhsemoglobin is the ordinary one obtained by the customary method of
preparation. Bohr designates as a-oxyhsemoglobin the crystalline powder
obtained by drying /--oxyhsemoglobin in the air. On dissolving a-oxyhaemo-
gJobin in water it is converted into ,5-oxyhaemoglobin witliout decomposition, and
the quantity of iron is increased. On keeping a solution of r-oxyhsemoglobin
in a sealed tube it is transformed into o-oxyhsemoglobin, although the exact
conditions under which this change takes place are not known. According to
HuFNER ' these are nothing but mixtures of genuine and partly decomposed
haemoglobins.
The ability of hsemoglobin 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 hsemoglobin 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 hsemoglobin in solution takes
up more oxygen, until it is completely saturated, when 1 molecule of
ha?moglobin is combined v\ith 1 molecule of oxygen. Still this reaction
is reversible according to the type 1 (Hb) + 1 (Oo) ?^ 1 (OHb) , and v\ith
diminished oxygen pressure a dissociation must take place with the gi^'ing
up of oxygen and a re-formation of hsemoglobin. The equilibrium between
oxyhsemoglobin, hsemogloljin, and oxygen is determined according to the
law of mass-action, and according to the investigations of Hufxer it is
possible to calculate the relationship between ox^-hsemoglobin (OHb) and
hsemoglobin (Hb), at ever}- desired partial pressure of the ox^-gen, by a
formula suggested by him. According to Bohe^ this formula does not
have suflBcient basis and does not correspond to the facts. Bohr found,
m opposition to Hufxer's statements, that with the same oxygen tension
the absorption of oxygen by a hsemoglobin solution changes with the con-
centration, and that a dilute solution combines with more oxygen, calculated
per 1 gram hsemoglobin. than a concentrated solution. Bohr suggested
another formula expressing the relationship between the ox^-gen absorp-
tion and the oxygen tension, based upon the assumption that, besides the
dissociation of the oxygen-hsemoglobin compound, a dissociation of the
hsemoglobin into a part containing iron and a part not containing iron also
takes place. This formula, which in fact accords well with Bohr's findings,
is nevertheless only true for a hsemoglobin solution and not for blood, as,
according to Bohr, the blood-pigment in the blood-corpuscles (the haemo-
chrom) is changed on being converted into hsemoglobin. Hexri also
finds that Hufxer's formula for the dissociation of oxyhsemoglobin is
not useful, basing his claim upon theoretical considerations and upon
unfinished investigations.
'Arch. f. (Anat. u. ) physiol., 1894.
^ Bohr, Centralbl. f. Physiol., 17 pp. 682 and 688; Henri, Compt. rend. toe. biolog.,
o6.
200 THE BLOOD.
The native pigment, the haemochrom, combines, according to Bohr, in
maximo with the same quantity of oxygen as the corresponding haemo-
globin, when the latter is prepared without the use of means ha\'ing a strong
action; still from this it does not follow that the oxygen combination in
haemochrom 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
haemoglobin 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
haemoglobin 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 oxy haemoglobin makes it also possible to completely
expel the oxygen from a haemoglobin solution or from blood by means of
a vacuum or by passing an indifferent gas through the blood.
Oxy haemoglobin, which is generally considered as a weak acid, is dextro-
rotatory, according to Gamgee.^ The specific rotation for light of medium
wave-lengths of C is (a)C= about -1-10°, which corresponds also for carbon-
monoxide haemoglobin. The haemoglobin is also, like carbon-monoxide
haemoglobin (COHb) and met haemoglobin (MHb), diamagnetic, while the
haematin, which is richer in iron, is strongly magnetic (Gamgee^). On
passing an electric current through an oxyhaemoglobin solution, the pig-
ment first separates unchanged at the anode in a colloidal but still soluble
form, and is then gradually transferred to the cathode in the colloidal
state (Gamgee^). This transportation of the colloidal haemoglobin may
also be made to take place through an animal membrane or through parch-
ment paper. According to Gamgee, the haemoglobin probably exists in
such a colloidal condition in the blood-corpuscles.
Oxyhaemoglobm has been obtained in ciystals from several varieties
of blood. These crj'stals are blood-red, transparent, silky, and may be
2-3 mm. long. The oxyhaemoglobin from squirrel's blood crystallizes
in six-sided plates of the hexagonal system; the other varieties of blood
yield needles, prisms, tetrahedra, or plates which belong to the rhombic
' Hofmeister's Beitrage, 4. ' Proceedings of Roy. Society, 68. ^ Ibid., 70.
OXYH.EMOGLOBIN. 201
system.i The quantity of water of ctystallization varies between 3-10
per cent for the different oxyhaemoglobins. When completely dried at a
low temperature over sulphuric acid the crystals may be heated to 110-115°
C. without decomposition. At higher temperatures, somewhat above
160° C, they decompose, giving an odor of burnt horn, and leave, after
complete combustion, an ash consisting of oxide of iron. The oxyhsemo-
globm cr}'stals from difficultly crj'stallizable kinds of blood, for example
from such as ox's, human, and pig's blood, are easily soluble m water^
The oxyhaemoglobins from easily cr}'stallizable blood, as from that of the
horse, dog, squirrel, and guinea-pig, are soluble with difficulty in the order
above given. The oxyhsemoglobin dissolves more easily in a very dilute
solution of alkali carbonate than in pure water, and this solution may be
kept. The presence of a little too much alkali causes the oxy haemoglobin
to quickly decompose. The cry^stals are insoluble without decolorization
in absolute alcohol. According to Nencki,^ it is hereby converted into
an isomeric or polymeric modification, called by him parahcBmoglohin.
Oxy haemoglobin is msoluble in ether, chloroform, benzene, and carbon
disulphide.
A solution of oxyhaemoglobin 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. It is also readily decomposed by acids, alkalies,
and many metallic salts. It gives the ordinary reactions for proteins
with those protein reagents which first decompose the oxyhaemoglobin
with the splitting off of protein. Oxyhaemoglobin, 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 containing peroxide, such as old turpentine.
A sufficiently dilute solution of oxyhaemoglobin or arterial blood shows
a spectrum with two absorption-bands between the Frauxhofer lines D
and E. 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 >^ = 578.1
and the second /^ = 541.7. These bands can be detected in a layer 1 cm.
thick of a 0.1 p. m. solution of oxyhaemoglobin. In a still weaker dilution
the band B first disappears. By increased concentration of the solution
'The observation of Uhlik (Pfliiger's Arch., 104) that the haemoglobin from
horse-blood can also crystalMze in hexagonal six-sided plates seems to be due to the
fact that he had haemoglobin and not oxyhs&moglobin.
' 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.
202 THE BLOOD.
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. The oxy haemoglobin may be differentiated from other color-
ing-matters having a similar absorption-spectrum by its behavior towards
reducing substances.^ (See p. 203.)
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
ill all probability, as also claimed by Ville and Derrien,^ upon a partial forma-
tion of methsemoglobin.
A great many methods have been proposed for the preparation of
oxy haemoglobin crystals, but in their chief features they all agree with
the following one suggested by Hoppe-Seyler: The washed blood-cor-
puscles (best those from the dog or the horse) are stirred with 2 vols.
water and then shaken with ether. After decanting the ether and allowing
the ether which is retained by the blood solution to evaporate in an open
dish in the air, cool the filtered blood solution to 0° C, add while stirring
J vol. of alcohol also cooled, and allow to stand a few days at —5° to — 10°
C. The crj^stals which separate may be repeatedly recrystallized by
dissolving in water of about 35° C, cooling, and adding cooled alcohol as
above. Lastly, they are washed with cooled water containing alcohol
(I vol. alcohol) and dried in vacuum at 0° C. or a lower temperature.-^
For the preparation of oxyhsemoglobin crystals in small quantities
from easily crystallizable blood, it is often sufficient to stir a drop of blood
with a little water on a microscope slide and allow the mixture to evaporate
so that the drop is surrounded by a dried ring. After covering with a
cover-glass, the crystals gradually appear radiating from the ring. These
crj'stals 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
(Stores'*), 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 oxy haemoglobin, and it can
therefore be obtained as crystals only with difficulty. These crystals are
as a rule isomorphous with the corresponding oxyhaemoglobin crystals,
but are darker, having a shade towards blue or purple, and are decidedly
' Zeitschr. f. Biologie, 34, contains the investigations of Gamgee on the absorp-
tion of the ultra-violet rays by the blood pigment. It also contains some of the earlier
investigations.
^ Piettre and Vila, Compt. rend., 140; Ville and Derrien, ibid., 140.
' In regard to the preparation of oxyhsemoglobin, see also Hoppe-Seyler-Thier-
felder's Handbuch, 7. Aufl.; also the works cited in foot-note 1, p. 198; also Schuur-
manns-Stekhoven, Zeitschr. f. physiol. Chem., 33, 296; see also Bohr, Skand. Arch,
f. Physiol., 3.
* Philosophical Magazine, 28, No. 190, Nov., 1864.
HEMOGLOBIN. 203
more pleochromatic. The hsemoglobin from horse-blood has also been
obtained by Uhlik ^ in hexagonal six-sided plates. Its solutions in water
are darker and more \dolet or purplish than solutions of oxyhsemoglobin
of the same concentration. They absorb the blue and the violet rays of
the spectrum in a less marked degree, but strongly absorb the rays lying
between C and D. In proper dilution the solution shows a spectrum with
one broad, not sharply defined band between D and E, whose darkest part
corresponds to the wave-length ^ = 555. Tliis band does not lie in the
middle between D and E, but is towards the red end of the spectrum, a
little over the line D. A haemoglobin solution actively absorbs oxygen
from the air -^ni is converted into an oxyhsemoglobin solution.
A solution of oxyhsemoglobin may be easily converted into a solution
having the spectrum of hsemoglobin by means of a vacuum, by passing an
indifferent gas through it, or by the addition of a reducing substance, as,
for example, an ammoniacal ferrous-tartrate solution (Stokes' reduction
liquid). If an oxyha?moglobin solution or arterial blood is kept in a sealed
tube, we observe a gradual consumption of oxygen and a reduction of the
oxyhsemoglobin into hsemoglobin. If the solution has a proper concen-
tration, a crj'stallization of hsemoglobin may occur in the tube at lower
temperatures (Hufner^).
Pseudohaemoglobin. Ludwig and Siegfried ' have observed that blood
which has been reduced by hyposulphites so completely that the oxyhsemoglobin
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 oxyhsemoglobin
spectrum disappears acts in the same manner. Hence a loose combination of
hsemoglobin and oxygen exists which gives the hsemoglobin spectrum, and this
combination is called pseudohsemoglobin 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 hsemoglobin and
oxyhsemoglobin on the reduction of the latter. The occurrence of pseudohsemo-
globin does not seem to have been positively proved.*
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 haematuria or hsemoglobinuria, also in urine
and blood on poisoning with potassium chlorate, amyl nitrite or alkali
nitrite, and many other bodies.
Methaemoglobin does not contain any oxygen in molecular or dissociable
combination, but still the oxygen seems to be of importance in the forma-
tion of methsemoglobin, because it is formed from 0x3- hsemoglobin and
not from hsemoglobin in the absence of oxygen or oxidizing agents. If
» Pfliiger's Arch., 104.
^ Zeitschr. f. physiol. Chem., 4; see also Uhlik, 1. c.
^ Arch. f. (Anat. u.) Physiol., 1890; see also Ivo Novi, Pfliiger's Archiv, 56.
* See Hufner, Arch, f . (Anat. u.) Physiol., 1894, 140.
204 THE BLOOD.
arterial blood be sealed up in a tube, it gradually consumes its oxj^gen and
becomes venous, and by tliis absorption of oxygen a little methsemogiobin
is formed. The same occurs on the addition of a small quantity of acid to
the blood. By the spontaneous decomposition of blood some methsemo-
giobin is formed, and by the action of ozone, potassium permanganate,
potassium ferricyanide, chlorates, nitrites, nitrobenzene, pyrogallol, pyro-
cateehin, acetanilide, and certain other bodies on the blood an abundant
formation of methamoglobin takes place.
According to the investigations of Hufner, Kulz, and Otto ^ methsemo-
giobin contains just as much oxygen as oxyhsemoglobm, but it is more
strongly coml3ined. By the action of potassium ferricyanide or potassium
permanganate upon oxy haemoglobin first 1 molecule oxygen (i.e., the
entire quantity of loosely combined oxygen) is split off and in the subse-
quent methsemogiobin formation either two oxygen atoms (Haldane) or
two hydroxyl groups are combined (Hufner, v. Zeyxek^). ^ilethsemo-
globin solutions are reduced to haemoglobin by reducing agents. Jader-
HOLM and Saarbach claim that methsemogiobin is first converted into
oxyhsemoglobin and then into hsemoglobin by reducing substances, while
others (Hoppe-Seyler and Araki^) dispute this.
According to HiJFNER and Reinbold* 1 gram methsemogiobin can
take up 2.685 c.c. nitric oxide.
^lethsemoglobin cr}^stallizes, as first showTi by HiJFXER and Otto, in
bro\\Tiish-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 methsemogiobin is,
according to Jaderholm and Bertin-Saxs, ver\' similar to that of hsematin
in acid solution, but is easily distmgiiished from the latter since, on the
addition of a little alkali and a reducing substance, the former jDasses
over to the spectnun of reduced hsemoglobin, while a hsematin solution
under the same conditions gives the spectrum of an alkaline hsemochromogen
solution (see below). [Methsemogiobin in alkaline solution shows two
absorption-bands which are like the two oxyhsemoglobin 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. According to other investigators. Araki and Dit-
* See Otto, Zeitschr. f. physiol. Chem., 7.
2 Haldane, Journ. of Physiol., 22; v. Zeynek, Arch. f. (Anat. u.) Physiol., 1899;
Hiifner, ibid.
^ Jaderholm, Zeitschr. f. Biologie, 16; Saarbach, Pfliiger'.'^ Arch., 28; Araki, Zeit-
schr. f. phy.siol. Chem., 14.
* Arch. f. (Anat. u.) Physiol., 1904, Suppl.
CARBOX-xAIOXOXIDE HiE:\IOGLOBIN. 205
TRICH, a neutral or faintly acid methsemoglobin solution shows only one
characteristic band, a, between C and D, whose middle corresponds to
about i^ = 634. The two bands between D and E are only due to con-
tamination with oxyhsemoglobin (Menzies^).
The statements as to the action of sodium fluoride \\y>o\\ haemoglobin and
naethaemoglobin are somewhat contradictory. -
Crv'stallized methoemoglobin may be easily obtained by treating a con-
centrated solution of oxyha^moglobin with a sufficient c^uantity of concen-
trated potassium-ferrieyanide solution to give the mixture a porter-brown
color. After cooling to 0° C. add \ vol. cooled alcohol and allow the mix-
ture to stand a few days in the cold. The ciystals may be easily purified
by recrv'stallizing from water by the addition of alcohol.
Cyanmethaemoglobin (cyanhsemoglobin) is, according to Haldaxe, identical
with photomethsemoglobin (Bock), which is produced by the influence of sunlight
upon a methsemoglobin solution containing potassium ferricyanide. It was
first carefully described by R. Kobert and obtained in a crystalline form by
V. Zeyxek.^ It is immediately formed in the cold by the action of a hydrocyanic-
acid solution upon methsemoglobin, but is formed by its action upon oxyhsemo-
globin only at the body temperature. The neutral or faintly alkaline solutions
show a spectrum which is very similar to the hsemoglobin spectrum.
Acid haemoglobin is a coloring-matter produced by the action of very weak
acids upon oxyhsemoglobin, which according to Harxack ■* is not, as used to be
admitted, identical with methsemoglobin.
Carbon-monoxide Haemoglobin ^ is the molecular combination between
1 molecule of hsemoglobm and 1 molecule of CO, according to Hufner,®
which contains 1.34 c.c. of carbon monoxide (at 0° and 760 mm. Hg)
for 1 gram hsemoglobin. Tliis combmation is stronger than the oxygen
combination of hsemoglobin. The oxygen is for this reason easily driven
out of oxyhsemoglobin by carbon monoxide, and tliis explains the jDoison-
ous action of tliis gas, which kills by the expulsion of the oxygen of the
blood. In regard to the division of the blood-pigments between the carbon
monoxide and oxygen under different partial pressures of both gases in
' Jaderholm, 1. c; Bertin-Sans, Comp. rend., 106; Dittrich, Arch. f. exp. Path. u.
Pharm., 29; Menzies, Journ. of Physiol., 17. Important references on methsemo-
globin are given by Otto, Pfliiger's Arch., 31.
- Piettre and Vila, Conipt. rend., 140; Ville and Derrien, ibid.. 140.
^ Haldane, Journ. of Physiol., 25; Bock, Skand. Arch. f. Physiol., 6; Kobert,
Pfliiger's Arch., 82; v. Zeynek, Zeitschr. f. physiol. Cheni., 33.
■* Zeitschr. f. physiol. Chem., 26.
'In reference to carbon-monoxide haemoglobin, see especially Hoppe-Seyler, Med.-
chem. Untersuch., 201; Centralbl. f. d. med. Wissensch., 1S64 and 1865; Zeitschr.
f. physiol. Chem., 1 and 13.
'Arch. f. (Anat. u.) Physiol., 1894. On the dissociation constant of carbon-
monoxide hsemoglobin, see ibid., 1895. In regard to the contradictory statements of
Saint-Martin and others and their disproval, see Hi fner, Arch. f. (Anat. u.) Physiol.,
1903.
206 THE BLOOD.
the air, we must refer to the mvestigations of HiJFNER,i 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, oxyhemoglobin, or nitric-
oxide hsemoglol^in are formed. The carbon monoxide is also expelled by
potassium ferricyanide and methsemoglobin is formed (Haldane^).
Carbon-monoxide haemoglobin is formed by saturating blood or a
haemoglobin solution with carbon monoxide, and may be obtained as crystals
by the same means as oxyhaemoglobin. These crystals are isomorphous
with the oxyhaemoglobin cr}'stals, 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 spectnmi shows two bands which are verj' similar to those of ox3^haBmo-
globin, but they occur more towards the violet part of the spectrum. The
middle of the first band corresponds to X = ^12 and the second to ^ = 536.
These bands do not change noticeably on the addition of reducing sub-
stances; this constitutes an important difference between carbon-monoxide
haemoglobin and oxyhaemoglobin. If the blood contains oxyhaemoglobin.
and carbon-monoxide haemoglobin at the same time, we obtain on the
addition of a reducing substance (ammoniacal ferro-tartrate solution)
a mixed spectrum originating from the haemoglobin and carbon-monoxide
haemoglobin.
A great many reactions have been suggested for the detection of carbon-
monoxide haemoglobin in medico-legal cases. A simple and at the same
time a good one is Hoppe-Seyler's alkali test. The blood is treated with
double its volume of caustic-soda solution of 1.3 sp. gr., by which ordinary
blood is converted into a dingy brownish mass, which when spread out
on porcelain iS brown with a shade of green. Carbon -monoxide blood
gives under the same conditions a red mass, which if spread out on porce
lain 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.^
As according to Bohr there are several oxj'^hsemoglobins, so also, according to
Bohr and Bock,'' there are several carbon-monoxide haemoglobins, with different
' Arch. f. exp. Path. u. Pharm., 48.
' Journ. of Physiol., 22.
' In regard to this test (as suggested by Kunkel) and others we refer to Kostin,
Pfii!ger's Arch., S-i, which contains a very excellent summary of the literature on the
subject.
* Centralbl. f. Physiol., 8, and Maly's Jahresber., 25.
CARBON-DIOXIDE HAEMOGLOBIN. 207
amounts of carbon monoxide. As heemoglobin can unite with oxygen and carbon
dioxide simultaneously, as shown by Bohr and Torup, 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 Moitessier.' Sulphur methaemo-
globin is the name given by Hoppe-Seyler to that colormg-matter which is
formed by the action of sulphuretted hydrogen upon oxyhaemoglobin. 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 HjS is passed through an oxygen-free solution of haemoglobin (or carbon-
monoxide haemoglobin). The sulphhaemoglobin thus formed shows one band ia
the red between C and D.
Carbon-dioxide Haemoglobin, Carbohceinoglohin. Hsemoglobin, accord-
ing to Bohr and Torup,^ also forms a molecular combination with carbon
dioxide whose spectrum is similar to that of hemoglobin. According ta
Bohr there are three different carbohsemoglobins, namely, a-, /9-, and
;--carboha3moglobin, in which 1 gram combines with respectively 1.5, 3, and
6 c.c. CO2 (measured at 0° C. and 760 mm.) at 18° C. and a pressure of 60
mm. mercury. If a hsemoglobin solution is shaken with a mixture of
oxygen and carbon dioxide, the hsemoglobin 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 hsemoglobin, that is, the oxygen
with the pigment nucleus and the carbon dioxide with the protein com-
ponent. Bohr has given an equilibrium formula for the carbon-dioxide
absorption of hsemoglobin at different carbon-dioxide tensions, and the
results obtained on calculation, using this formula, correspond veiy well
with the results obtained directly. Attention must be called to the fact
that, as observed by Torup, hsemoglobin is in part readily decomposed
by the carbon dioxide with the splitting off of some protein.
Nitric-oxide Haemoglobin is also a crv'stalline molecular combination
which is even stronger than the carbon-monoxide hsemoglobin. Its solu-
tion shows two absorption-bands which are paler and less sharp than the
carbon-monoxide hsemoglobin bands, and they do not disappear on the
addition of reducing bodies. Hsemoglobin also forms a molecular com-
biration wHh acetylene.
Hasmorrhodin is the name given by Lehmann to a beautiful red pigment
soluble in alcohol and ether, which is extracted from meat and meat products
^ V. Anrep, Arch. f. (Anat. u.) physiol., 1880; Sans and Moitessier, Compt. rend., 113.
^ Med.-chem. Untersuch., 151. See Araki, Zeitschr. f. physiol. Chem., 11; Har-
nack, 1. c.
5 Bohr, Extrait du Bull, de I'Acad. Danoise, 1890; Centralbl. f. Physiol., 4 ami
17; Torup, Maly's Jahresber., 17.
208 THE BLOOD.
by boiling alcohol and which seems to be produced by the action of small amounts
of nitrites. Another pigment isolated by Lewin ' from the blood of animals
poisoned by phenylhydrazine has been called hcemoverdin. By heating a solu-
tion of blood-pigment treated with caustic potash and mixed with alcohol to
60° C. we obtain, according to v. Klaveren, a pigment which he calls kathcemo-
globin, but called by Arnold,^ who first obtained it, neutral hcematin, which is
produced by the splitting off of a ferruginous complex. This pigment still con-
tains protein, but is poorer in iron than the hsemoglobin or methsemoglobin and
probably forms an intermediary product in the conversion of the above into
haematin.
Decomposition j/rodiicts of the blood-pigtnents. By its decomposition
haemoglobin yields, as pre^^ollsly stated, a protein, which has been called
globin (Preyer, Schulz), and a ferruginous pigment as chief products.
According to Lawrow 94.09 per cent protein, 4.47 per cent haematin, and
1.44 per cent other bodies are produced in this decomposition. The globin,
which was isolated and studied by Schulz,^ differs from most other pro-
teins by containing a high amount of carbon, 54.97 per cent, with 1698.
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 coagidated by heat, but the coagulum is readily soluble
in acids. Because of these reactions it is considered as a histone by Schulz.
On hydrolytic cleavage globin (from horse-blood) yields, according to
Abderhalden,^ 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 h(Bmochromogen, by other investigators (Stokes) reduced
hcematin. In the presence of oxygen, haemochromogen is quickly oxidized
to haematin, and there is therefore obtained in this case hcematin as a colored
decomposition product. As haemochromogen is easily converted by
oxygen into haematin, so this latter may be reconverted into haemochromogen
by reducing substances.
Haemochromogen was discovered by Hoppe-Seyler.^ It is, accord-
ing to Hoppe-Seyler, the colored atomic group of haemoglobin and of its
combinations with gases, and this atomic group is combined with proteins
^ K. B. Lehmann, Sitzungsber. d. phys.-med. Gesellsch. Wiirzburg, 1899; Lewin,
Compt. rend., 133.
^ V. Klaveren, Zeitschr. f. physiol. Chem., 33; Arnold, ibid., 29.
^Lawrow, ibid., 26; Schulz, ibid., 24; Preyer, Die Blutkristalle, Jena, 1871.
^ Zeitschr. f. physiol. Chem., 37.
' Ibid., 13.
H.EMOCHROMOGEX. 209
in the pigment. The characteristic absorption of light depends on tlie
haemochromogen, and it is also tliis atomic group which binds in the oxy-
hsemoglobin 1 molecule of oxygen and in the carbon-monoxide hsemoglobm
1 molecule of carbon monoxide ^^■ith 1 atom of iron. Htemochromogen
is produced in an alkaline solution of hipmatin by the action of reducing
bodies. By the reduction of hsematin in alcoholic ammoniacal solution by
means of hydrazine v. Zeyxek ^ was able to obtain the solid bro\\Tiish-red
ammonia combination.
Haemochromogen combines, as Hoppe-Seyler first showed, also with
carbon monoxide. This compound, which hi aqueous solution gives
a spectrum similar to oxy haemoglobin, has been obtained by Pregl- in
the solid condition as a deep-^dolet powder which is insoluble in absolute
alcohol. In opposition to haemoglobin the haemochromogen combines
with oxygen more firmly than with carbon monoxide. The assumption
of Hoppe-Seyler that this compound is a combination of 1 molecule
haemochromogen and therefore contains 1 molecule carbon monoxide
for 1 molecule of iron has been experimentally substantiated by Hufner
and KuSTER and by Pregl.3
An alkaline hsmochromogen solution has a beautiful cherr\'-red color.
It shows two absorption-bands, first described by Stokes, one of which
is dark and whose center corresponds to X = oo6A between D and E,
and a second broader band, less dark, which covers the Frauxhofer
lines E and b. The middle of this band corresponds to / = 520.4. In acid
solution haemochromogen shows four bands, which, according to Jader-
HOLM,* depend on a mixture of haemochromogen and haematoporphyrin
(see below), this last formed by a partial decomposition resulting from
the action of the acid.
MiLROY ° from an alcoholic solution of hsematm containing oxalic acid,
after drivmg out the air by means of hydrogen gas, gradualh' obtained
an acid solution of reduced haematin (haemochromogen) bj' means of zinc
dust. This solution showed one absorption-band between D and E.
Haemochromogen may be obtamed as cr\-stals by the action of caustic
soda on hsemoglobm at 100° C. m the absence of oxygen (Hoppe-Seyler).
By the decomposition of haemoglobin by acids (of coui-se m the absence of
air) we obtain haemochromogen contaminated with a little haematopor-
phyrin. An alkaline haemochromogen solution is easily obtained by the
action of a reducing substance (Stokes' reduction liquid) on an alkaline
haematin solution. An ammoniacal solution of haematin on reduction with
hydrazine yields haemochromogen veiy easih'. An alcoholic, alkaline
' Zeitschr. f. physiol., Chem., 25.
'Ibid.,U.
• H fner and K' ster. Arch. f. (Anat. u.) Physiol., 1904, Suppl.; Pregl, 1. c.
*Nord. Med. Arkiv., 16.
' Journ. of Physiol., 32.
210 THE BLOOD.
hydrazine solution is also recommended by Riegler ^ as a reagent for
blood-pigments, converting them into hsemochromogen.
Haematin, also called Oxyh^m-A-Tin, is sometimes found in old transu-
dates. It is formed by the action of the gastric or pancreatic juices on
oxy haemoglobin, and is therefore also found in the faeces after hemorrhage
in the intestinal canal, and also after a meat diet and food rich in blood.
It is stated that haematin may occur in urine after poisoning uith arseniu-
retted hydrogen As sho\^Ti above, the haematin is formed by the decom-
position of oxy haemoglobin, or at least of haemoglobin, in the presence of
oxygen.
The statements in regard to the composition of haematin are rather
contradictory, which seems to be due to the fact that the substance,
haemin (see below), from which the formula of haematin is derived, has
a somewhat different composition, dependent upon various conditions.
According to Hoppe-Seyler haematin has the formula C34H34N4Fe05.
and from the recent investigations upon haemin, 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 RoSENFELDr haematm has the formula
C3oH34N3Fe03, with 1 atom of iron for ever}' 3 atoms of nitrogen.
Haematin is very resistant towards boiling concentrated caustic potash
as well as towards boiling hydrochloric acid. It dissolves in concentrated
sulphuric acid and is converted into haematoporphyrin with the splitting
off of iron. On heating dr}^ haematin it yields abundant pyrrol. On
reduction with tin and hydrochloric acid a body similar to urobilin is
formed. As an oxidation product of haematin in glacial acetic acid with
potassium bichromate or chromium trioxide, Kuster ^ obtained the
imide of the tribasic haematinic acid, C8HqN04, which is also produced on
theoxidation of haematoporphyrin and bilirubin.
The imide of the tribasic hsematinic acid, which is a derivative of maleic acid
CO
and probably has the formula C5H7(COOH) <r^r\> NH, is readily transformed into
the anhydride of the tribasic hsematinic acid, CsHgOj, having the probable formula
CH3.C.CO
1 1 > O. On heating the imide with alcoholic ammonia to
C00H.CH2.CH,.C.C0
130° C. it spHts off carbon dioxide, and the imide of the bibasic hsematinic acid,
C7H9XO2, is obtained. From this imide on saponification with baryta-water we
' Zeitschr. f. analyt Cfiem., 43.
' Hoppe-Seyler, Med.-chem. Untersuch., p. 525; Cloetta, Arch. f. exp. Path. u.
Pharm., 3(5; Rosenfeld, ibid., 40.
^ Beitriige zur Kenntnis des Hiimatins, Tubingen, 1896; Ber. d. d. chem.
Gesellsch., 2", 30, 32, and 35; Annal. d. ' hem. u. Pharm., 315, and Zeitschr. f. physiol.
Chem., 28, 40, and 44.
HiEMATIN AND H^MIN. 211
obtain the barium salt of an acid whose anhydride is methyl-ethyl male'ic-acid
C^Hj.C.CO
anhydride, || >0.
CH3. C.CO
The yield of haematinic acids is so great that Kuster considers that at least
three if not four molecules CgHgNO^ are formed from one haematin molecule. On
heating haematinic acid ester with alcoholic ammonia in a tube to 130° Kvster
obtained a colored product whose bluish-violet aqueous solution gave a spectrum
with two bands which in position were similar to the oxyhaemoglobin spectrum.
Haematin is amorphous, dark broun 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. Haematin
dissolves in acidified alcohol or ether. It easily dissolves in alkalies, even
when ver\' dilute. The alkaline solutions are dichroitic; 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
alwa5's browii.
An acid haematin solution absorbs the red part of the spectnmi only
slightly and the violet parts strongly. The solution shows a rather sharply
defined band between C and D, whose position may change with the variety
of acid used as a solvent. Between D and F a second, much broader, less
shar^Dly defined band occurs, which by proper dilution of the liquid is con-
verted into two bands. The one between h and F, 13'ing near F, is darker
ai d broader; the other, between D and E, lying near E, is lighter and nar-
rower. Also by proper dilution a fourth ven,- faint band is observed be-
tween T> and E, lying near D. Haematin may thus in acid solution show-
four absorption-bands; ordinarily one sees distinctly onty the bands be-
tween C and D and the broad, dark band — or the two bands — between D
and F. In alkaline solution haematin shows a broad absorption-band,
which lies in greatest part between C and D, but reaches a little over the
line D towards the right in the space between D and E. As the position
of the haematin bands in the spectrum is quite variable, the exact wave-
lengths corresponding thereto cannot be given exactly.
Haemin, H.emin Crystals, or Teichmaxn's Crystals. Hsemin is
the hydrochloric-acid ester of haematin and is the startmg-point m the
preparation of the latter.
The statements as to the composition of ha^min are just as variable
as those for haematin, which is partly due to the fact, as shown by Nexcki
and Zaleski, that the haematin, 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 prepared
according to Xexcki and Sieber's method contains amyl alcohol.
Schalfejeff's haemin, having the formula C34H,33X4Fe04Cl, is supposed
212 THE BLOOD.
to contain an acetyl group and hence is called acethferain. Morner's
heemin, C35H35N4Fe04Cl, is considered as a monoethyl ether of acethsemin.
The investigations of Zaleski, Hetper and INIarchlewski, K. Morner,
and especially those of Kl'Ster have given explanations of these conditions.
The so-called acetha?min does not contain any acetic-acid radical, hence
its name is incorrect. Kuster, by a new method of purification and recrj-s-
tahization, has shown that the older various kinds of hsemins were not
chemical individuals and that we have only one haemin. This view is now
accepted by Morner and most of the other investigators, and the formula
C34H3304N4FeCl is now given to hsemin. Piettre and Vila ^ dispute this
formula, and they claim to have prepared chlorine-free hsemin from pure
cr}'stalline oxy haemoglobin.
Hsemin crj^stals form in large masses a bluish-black powder, but are so
small that they can only be seen by aid of the microscope. They consist
of dark-brown or nearly brownish-black long, rhombic, or spool-like
crj'stals, 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 chloro-
form. 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.
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 HCl and was converted into hsemin. By the action of boiling aniline,
hydrogen is driven out and a combination with aniline, without loss of
iron, takes place.
The principle of the preparation of haemin crystals in large quantities
is as follows: The washed sediment from the blood-corpuscles is coagu-
lated vnth. 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 T^-1 per cent concentrated sulphuric acid, and this is allowed to stand
several hours at the temperature of the room. The filtrate is warmed to
about 70° C, treated with hydrochloric add (for each litre of filtrate add
10 c.c. 25 per cent hydrochloric acid diluted with alcohol — IMorner),
and allowed to stand in the cold. The ciystals, which separate in one
^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, o;
K. Morner, Nord. Med. Arkiv, Festband, 1897, Nos. 1 and 26, and Zeitschr. f. physiol.
Chem., 41; Zaleski, ifeid., 37; Hetper and Marchlewski, i?jxi/., 41 and 42; Kuster , ibid.,
40; Piettre and Vila, Compt. rend., 141, p. 734.
I^MATOPORPHYRIN. 213
or two days, are first washed with alcohol and then with water. For pav-
ticulars as to the various methods of preparation and purification we refer
the reader to the above-cited works of Nencki and Sieber, Cloetta,
MoRXER, Rosexfeld, Nexcki and Zaleski (Schalfejeff) , and especially
to KuSTER.l
Hffimatin is obtained on dissolving the ha?min crystals in ver\- dilute
caustic alkali and precipitating with an acid.
In preparmg ha^min crystals in small quantities proceed m the follo-uing
manner: The blood is dried after the addition of a small quantity of com-
mon salt, or the dried blood may be rubbed with a trace of the same.
The drj^ 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 ver\' small flame, with the precaution that the acetic
acid does not boil and pass with the powder from under the cover-glass.
If no crj'stals 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-browii or nearly black
hajmin crystals of varying forms will be seen.
In regard to the preparation of iodohaematin and the use of the same
for the detection of blood we must refer to Strzyzowski's communication .^
By the action of acids upon hsemochromogen, hsematin, or haemin a
new iron-free pigment, which was first closely studied by Hoppe-Seyler
and called hcBmatoporphi/rin, is produced. According to the method of
preparation hsematoporphyrins having different solubilities and whose
relationship to each other is not perfectly clear are produced, but all show
the same characteristic absorption-spectmm. The best-studied haemato-
porphyrin is the one obtamed according to Nexcki and Sieber's method,
by the action of glacial acetic acid saturated with hydrobromic acid upon
hsemm cr\'stals, best at the temperature of the body (Nexcki and
Zaleski ^) .
Haematoporphyrin, C16H18N2O3, or C34H38N4O6 according to Zaleski.*
Tliis pigment, according to Mac^Iuxx,^ occurs as a physiological pigment
in certain animals. It occurs, as show7i by Garrod and Saillet, as a
normal constituent, although only as traces, of human urine. It occurs
in greater quantities in human urme after the use of sulphonal (see
Chapter XV).
The formation of hseraatoporphyrin from hsematin can be expressed
' Zeitsclir. f. physiol. Chem., 40.
2 Therapeut. Monatshefte, 1901 and 1902.
' 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.
^Zeitschr. f. physiol, Chem., 37, 54.
* Joum. of Physiol., 7.
214 THE BLOOD.
by the following equation if we start with the above formula for hsemin
and Zaleski's formula for hsematoporphyrin :
C34H33N404FeCl + 2HBr + 2H2O = C34H38N4O6 + FeBro + HCl.
On heating ha3matoporphyrin it generates an odor of pyrrol. On oxidation
with bichromate and glacial acetic acid it yields hsematinic acid (see page
210). A pigment closely allied to the urinary pigment urobilin has been
obtained by the action of reducing substances on hsematoporphyrin
(Hoppe-Seyler, Nencki and Sieber, Le Nobel, MacMunn). On the
administration of haematoporpyhrin to rabbits, Nencki and Rotschy ^
observed that a part was reduced to a substance similar to urobilin.
Of especial interest are the recent investigations of Nencki, ^Iarch-
LEWSKi, and Zaleski^ upon the reduction products of hsematoporphyrin
and their relationship to the chlorophyll derivatives. By the action of
glacial acetic acid containing HI and of iodophosphonium upon hsemin or
baemochromogen Nencki and Zaleski obtained a markedly characteristic
pigment, meso porphyrin, having the formula C16H18N2O2, or, according
to Zaleski,^ C34H38N4O4, and which stands in a certain measure between
hsematoporphyrin, C16H18N2O3, and the chlorophyll derivative phyllo-
porphyrin, C16H18N2O, which is very similar to hsematoporphyrin. By
the action of the same reducing agent upon hsemin or baemochromogen,
but under other conditions, we obtain hcBmopyrrol, CsHisN, a colorless
oil, which in the air gradually changes into urobilin. Haemopyrrol 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 relationship between the blood-pigment and
chlorophyll.
According to Nencki and Zaleski haemopyrrol is probably 3-methyI-4-n-
CH3 — C — C — C3H7
propylpyrrol, HC CH. Kuster obtained an imide from hsemopyrrol on
NH
oxidation which was probably a derivative of methylpropylmaleic acid. As
hsematinic acid 's undoubtedly a maleic-acid derivative, it was of interest to
prove the correctness of the above formula of hsemopyrrol, and with this purpose
in view Kuster and Haas ^ have compared the synthetically prepared imide
of methylpropylmaleic acid with the imide obtained from haemopyrrol. The two
bodies were not identical, therefore the above constitutional formula is ques-
^ Hoppe-Seyler, 1. c, 523; Le Nobel, Pfl ger's Arch., 40; MacMunn, Proc. Roy.
Soc, 30, and Journ. of Physiol., 10; Nencki and Rotschy, Monatshefte f. Chem., 10.
2 See foot-note 1, p. 197.
' Zeitschr. f. physiol. Chem., 37.
* Ber. d. d. chem. Gesellsch., 3".
ILEMATOPORPHYRIX. 215
tioned. The attempt of Buraczewski and Marchlewski ^ to prepare hsemo-
pyrrol artificially from methylpropylmaleic-acid imide yielded a product similar
to hsemopyrrol, which on oxidation in the air did not yield a typical uro-
bilin but at least a substance closely related thereto. The assumption that
hsemopyrrol is a pyrrol derivative is best borne out by the property which hsemo-
pyrrol has of reacting with diazonium compounds with the formation of azo
pigments (Goldmaxn, Marchlewski, Hetper ■).
HEBmatoporphyrin 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 crj'stallizes in long bro^^■nish-red needles. If the solution
in hydrochloric acid is nearly neutralized with caustic soda and then treated
with sodium acetate, the pigment separates ovit as amorphous, brown
fiakes not readily soluble in amyl alcohol, ether, and chloroform, but readily
soluble in ethyl alcohol, alkalies, and dilute mineral acids. The com-
pound with sodium crj^stallizes as small tufts of brown cr}-stals. The acid
alcoholic solutions have a beautiful purple color, which becomes \iolet-
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 hsematoporphy rin , acidulated with hydrochloric
or sulphuric acid, shows two absorption-bands, one of which is fainter and
narrow'er and lies betw^een C and D, near D. The other is much darker,
sharper, and broader, and lies midway betw'een D and E. An absorption
extends from these bands towards 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 surrovmding 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 h 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 tW'O bands, one of which
surrounds D and the other lies between D and E. If an acid haematopor-
phyrin solution is shaken wdth chloroform, a part of the pigment is taken
up by the chloroform, and this solution often shows a five-banded spectrum
with two bands between C and D. The position of the hiematoporphyrin
bands in the spectrum differs with the various methods of preparation
and other conditions, so that they do not correspond to the same w^ave-
length. These facts coincide well with the recent investigations of A.
ScHULz;'* according to which the appearance of the spectiaim is not only
^ Zeitschr. f. physiol. Chem., -13.
2 Ibid., 43 and 45.
^ See Hammarsten, Skand. Arch. f. Physiol., 3, and Garrod. Journ. of Physiol., 13.
'Arch. f. (Anat. u.) Physiol., 1904, Suppl.
216 THE BLOOD.
dependent upon the reaction but also upon the character of the solvent
and the method of preparation.
In regard to the preparation of hcematoporphyrin, see Hoppe-Seyler-
Thierfelder's Handbuch, 7. Aufl.. and the works cited on page 213.
Haematinogen is a ferruginous pigment so named by Freund,' which he
obtained by carefully extracting blood with alcohol containing hydrochloric acid.
It is closely related to hoematin, 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 regard 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 haematin, although not identical with it.
Zaleski considers this pigment as a hydrogenized hsemin. A regeneration
of hsemin from hoematoporphyrin 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 haemo-
chromogen is produced, which is changed into haematin by shaking with
air. According to Ham and Balean,^ it is possible to produce haemoglo-
bin from haemochromogen and globin, and it is indeed possible that othor
proteins can replace globin in this formation.
Haematoidin, thus called by Virchow, is a pigment which crystallizes
in orange-colored rhomlDic plates, and which occurs in old blood extrav^a-
tions, and whose origin from the blood-coloring matters seems to be estab-
lished (Langhans, Cordua, Quincke, and others 2). A solution of haema-
toidin shows no absorption-bands, but only a strong absorption from the
\'iolet to the green (E wald ^) . According to most observers, haematoidin
is identical with the bile-pigment bilimbin. It is not identical with the
cr}\stallizable lutein from the corpora lutea of the ovaries of the cow
(Piccolo and Lieben,^ Kijhne 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 ,
* Wien. kiln. Wochenschr., 1903.
^ Zaleski, Zeitschr. f. physiol. Chem., 43; Laidlaw, Journ. of Physiol., 31; Ham.
and Balean, ibid., 32.
' A comprehensive review of the literature pertaining to haematoidin may be found
in Stadelmann, Der Icterus, etc., Stuttgart, 1891, pp. 3 and 45.
* Zeitschr. f. Biologic, 22, 475.
" Cit. from Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl,, 1878.
UANTITATIVE ESTIMATION OF BLOOD PIGMENTS. 217
or hgematin, then the ii reparation of hsemin crv'stals is an absokitely positive
test. The reader is referred to more extended text-books for more exact
methods for the detection of Ijlood in chemico-legal cases, and it is perhaps
sufficient to give here the chief points of the investigation.
If spots on clothes, Hnen, wood, etc., are to be tested for the presence
of blood, it is best, when possible, to scratch or shave off as much as
possible, rub with common salt, and from this prepare the hsemin crystals.
On obtaining positive results the presence of blood is not to be doubted.
When sufficient material is not obtained by the above meaiis, soak the
spot with a few drops of water in a watch-crystal. If a colored solution
is thus obtained, then remove the fibres, wood-shavings, and the like as
far as possible, and allow the solution to dry in the watch-glass. The
dried residue may be partly used for the spectroscopic test directly, and
part may be employed in the preparation of the hsemin crystals. It may
also be used to detect ha^mochromogen in alkaline solution after previous
treatment with alkali and the addition of reducing substances.
If a colorless solution is obtained after soaking with water, or if the spots
are on rusty iron, then digest with a little dilute alkali (5 p. m.). In the
presence of blood the solution gives, after neutralization with hydro-
chloric acid and drjing, a residue which may give the hsemin crystals with
glacial acetic acid. Another part of the alkaline solution shows, after
thQ addition of Stokes' reduction fluid, the absorption-bands of hsemo-
chromogen in alkaline solution. ^
Th3 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 ^ has recently sug-
gested a clinical method based on this procedure.
The physical methods consist either of coloriraetric 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 oxyhsemo-
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 haematinom-
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.^ Instead of an oxyhsemoglobin solution we
row generally use a carbon-monoxide hsemoglobin solution as a standard
'On the use of color reactions for the detection of blood, see O. and R. Adler,
Zeitschr. f. physiol. Chem., 41, and Schumm and Westphal, ibid., 46.
^ Pfliiger's Arch., 65; Monatshefte f. Chem., 17. See also Oerum, Zeitschr. f.
anal. Chem., 48, and the works cited in Maly's Jahresber,, 33.
' F. Hoppe-Seyler, Zeitschr. f. physiol. Chem., 16; G. Hoppe-Seyler, ibid., 21;
Winternitz, ibid., Giacosa, Maly's Jahresber., 26; Zangermeister, Zeitsclir. f. Bio'.o-
gie. 33.
218 THE BLOOD.
liquid because it may be kept for a long time. The blood solution in this
case is saturated with carbon monoxide.^
The quantitative estimation of the blood -coloring matter? by means of
the spectroscope may be done in different ways, but at the present time
the spedrophotometric method is chiefly used, and this seems to be the
most reliable. This method is based on the fact that the extinction coeflB-
cient of a colored hquid for a certain region of the spectrum is directly
proportional to the concentration, so that C:E=C\:Ei, when C and C'l
represent the different concentrations and E and Ei the corresponding
C C
coefl&cients of extinction. From the equation TT = Tr> it follows that for
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 amoimt
of coloring-matter in grams in 1 c.c.) b}' C, then C=A . E.
Different forms of apparatus have been constructed (Yierordt and
HuFXER 2) for the determination of the extinction coefficient, which is equal
to the negative logarithm of those rays of light which remain after the pas-
sage of the licht 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/fif-
ferent regions of the spectrum. Hufner has selected (a) the region between the
two absorption-bands of oxyhaemoglobin, especially between the wave-lengths
5.54 .«,« and .565 ,«,«, and (h) the region between the two bands, especially the inter-
val between the wave-lengths .5.31. .5 ,«,« and .542..5 ,«,«. The constants or the
absorption ratio for these two regions of the spectrum are designated by Hufxer
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 fon-
centratiou or the amount of coloring-matter in 100 parts of the undiluted blood is
C = 100.r .,4 .£:and
C = 100.r ..4'.£'.
The absorption ratio or the constants in the two above-mentioned regions
of the spectrum have been determined for oxyhaemoglobin, haemoglobin, carbon
monoxide haemoglobin, and methaemoglobin, as follows:
Oxvhaemoglobin Ao =0.002070 and A'o =0.001.312
Haemoglobin Ar =0.001.3.54 and A'r =0.001778
Carbon-monoxide haemoglobin. . ..4c =0.001.383 and A'c =0.001263
Methaemoglobin .4 „^^ =0.002077 and A 'm =0.0017.54
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 oxyhaemoglobin and haemoglobin present
in Vjlood at the same time.
In order to facilitate these determinations, Hufxer ' has worked out tables
which give the relation between the two pigments existing in a solution contain-
' See Haldane, Joum. of Physiol., 26.
^ See Vierordt, Die Anwendung des Spektralapparates zu Photometrie, etc. (T bin-
gen, 1873J, and H fner, Arch. f. (.\nat. u.) Fhysiol., 1894, and Zeitschr. f. physiol.
Chem.. 3; v Noorden. jbtV/., 4: Otto. Pfl ger's Arch.. 31 and 36.
-Arch. f. (.\nat. u.) Physiol., 1900.
RED BLOOD CORPUSCLES. 219
ing oxyhi3emoglobin and another pigment (haemoglobin, methsemoglobin, or carbon-
monoxide haemoglobin), and thus allowing of the calculation of the absolute quan-
tity of each pigment.
Among the many apparatuses constructed for clmical purposes for the
quantitative estimation of hix-moglobin, Fleischl's hcemometer, which has
undergone numerous modifications, HExocQUf:'s hcematoscope, and Sahli's
hcemometer are to be specially mentioned. In regard to these apparatuses,
see V. Jaksch, Klinische Diagnostik innerer Krankheiten, 4. Auflage, 1897,
and Jaquet, Korresp.-Blatt f. Schweiz. Aerzte, 1897; Gartner, Miinchener
med. Wochenschr., 1901, and H. Sahli, Diagnostic Methods, Philadelphia,
1905.
Many other pigments are found besides the often-occurring haemoglobin
in the blood of invertebrates. In a few Arachnidae, Crustacea, Gasteropodse,
and Cephalopodse a body analogous to haemoglobin, containing copper, hcerno-
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
l)ecomes colorless again. According to Henze 1 gram haemocyanin combines
-with about 0.4 c.c. oxygen. It is crvstalline and has the following composition:
€5.3.66; H7..3.3; X 16.09; S0.86; Cu0..38; 0 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 spHt 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 color-
ing-matter called chlorocruorin by Lankester is found in certain Chsetopodse.
Hcemerythrin, so called by Krukenberg but first observed by Schw^\lbe, 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,' is a brown coloring-matter occurring in the perivisceral fluid of
a variety of echinoderms.
The quantitative constitution of the red hlood-corpusdes. 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 iV"¥o> of the dried substance consists of hsemoglobin (in human
xmd 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
Dog's " 865 126 5.9 3.6
Goose's " 627 364 4.6 4.8
Snake's " 467 525
Abderhalden found the following composition for the blood-corpuscles
from the domestic animals investigated by him: Water, 591.9-644.3
^ Fredericq, Extrait des Bulletins de l.'Acad. Roy. de Belgique (2), 46, 1878; Lan-
kester, Joum. 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.
^ Med.-chem. Untersuch., 390 and 393.
220 THE BLOOD.
p. m.; solids 408.1-335.7 p. m.; haemoglobin, 303.3-331.9 p. m. ; protein,
5.32 (dog) -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 varjdng proportion of the haemoglobin to the
protein in the nucleated and in the non-nucleated blood-corpuscles. These
last are much richer in haemoglobin and poorer in protein than the others.
The amount of mineral bodies in various species of animals is different.
According to Buxge and Abderhaldex 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 Waxach, about five times as much potash
as soda, on an average 3.99 p. m. potash and 0.75 p. m. soda. The nucleated
er\^throcytes of the frog, toad, and turtle contain, according to Bottazzi
and Cappelli,^ also 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)-0.150 p. m. (pig). The blood-corpuscles
of all animals investigated contain chlorine, 0.460-1.949 p. m. (both in
horse), generalh' 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. -
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 l^lood than the lymphocytes.
' Bunge, Zeitschr. f. Biologie, 12, and Abderhalden, Zeitschr. f. physiol. Chem., 23
and 25; Wanach, Maly's Jahresber., 18, 88; Bottazzi and Cappelli, Arch. Ital. de Biolo-
gie, 32.
2 Hamburger, Arch. f. (Anat. u.) Physiol., 1898; Koeppe, ibid., 1899 and 1900.
LEUCOCYTES. 221
In human and mammalian blood, most of the white blood-corpuscles are
larger than the red blood-corpuscles. They have also a lower specific
gra\ity than the red corpuscles, move in the circulating blood nearer to
the walls of the blood-vessels, and have also a slower motion.
The number of white blood-corpuscles varies not only in the different
b^ood -vessels, but also under different physiological conditions. On an
average there is only 1 white corpuscle for 3.50-500 red corpuscles. Accord-
ing to the investigations of Alex. Schmidt ^ and his pupils, the leucocytes
are destroyed in great part on the discharge of the blood before and during
coagulation, so that discharged blood is much poorer in leucocytes than
the circulating blood. The correctness of this statement has been denied
by other investigators.
From a liistological standpoint we generally, as above indicated, dis-
criminate between the different kinds of colorless blood-corpuscles.
Chemically considered, however, there is no knowTi 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 slim}' mass; the first do not show such behavior.
The protoplasm of the leucocytes has during life amoeboid movements
which S3rve 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. Alex. Schmidt claims to have found serglobulin in
equine-blood leucocytes which have been washed with ice-cold water.
There are also certain leucocytes, as above stated, which yield a slimy mass
when treated with alkalies or NaCl solutions, which seems to be identical
with the so-called hyaline substance of Ro^^DA found in the pus-cells. 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 coagu-
lation of the blood, has been described under different names (see Chapter
V, page 141), and consists, cliiefly, at least, of nucleoproteid. The ordinary'
view^ that this is nucleohistone does not seem to be correct, according to the
recent investigations of Baxg,^ and further proof is necessar}-.
Glycogen, as prexiousl}' stated, is found in the leucocytes. The glyco-
gen found by Huppert, Czerxy, Dastre.^ and others in blood and lymph
■ Pfliiger's Arch., 11. Also Fr. Kriiger, Arch- f. exp. Path. u. Phann., 51.
^ I. Bang, Studier over Nukleoproteider, Kristiania, 1902.
" Huppert. Centralbl. f Physiol., 6, 394; Czerny, Arch. f. exp. Path. u. Pharm.. 31;
222 THE BLOOD.
probably originated from the leucocytes. A glucothionic acid has been
prepared from white cells by Mandel and Levene.^ The constituents of
the leucocytes are the same as the constituents of the cell as described in
Chapter V.
The blood-plates (Bizzozero), hsematoblasts (Hayem), whose nature,
preformed occurrence, and physiological importance have been much ques-
tioned, are pale, colorless, gummy disks, round or somewhat oval in shape
and generally with a diameter two or three times smaller than the red
blood-corpuscles. By the action of different reagents the blood-plates
separate into two substances, one of which is homogeneous and non-refrac-
tive, while the other is highly refractive and granular. Blood-plates
readily stick together and attach themselves to foreign bodies.
According to the researches of Kossel and of Lilienfeld ^ the blood-
plates consist of a chemical combination between protein and nuclein,
and hence they are also called nuclein-plates by Lilienfeld, and are con-
sidered 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.
m. 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 thm layers, having a salty taste and a faint odor differing m differ-
ent 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 growii
men and a little less for women. Lloyd Jones found that the specific
gra\aty is highest at birth and lowest in children when about two years old
and in pregnant women. The determinations of Lloyd Jones, Hammer-
SCHLAG,^ and others show that the variation of the specific gravity, depend-
eai 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
Dastre, Compt. rend., 120, and Arch, de Physiol. (5), 7- See also Hirschberg, Zeitschr.
f. klin. Med., 54.
i Biochem. Zeitschr., 4.
* In regard to the literature of the blood-plates, see Lilienfeld, Arch. f. (Anat. u.)
Physiol., 1892, and "Leukocyten und Blutgerinnung," Verhandl. d. physiol. Gesellsch.
zu Berhn, 1892; and also Mosen, Arch, f (Anat u ) Physiol., 1893, and Maly's Jahres-
ber.. 30 and 31.
"Lloyd Jones, Journ. of Pliysiol., S; Hammerschlag, Wien. klin. Wochenschrift,
1890, and Zeitsclir f. klin. Med., 20.
ALKALINITY OF THE BLOOD. 225
are available, it is best to proceed with tiie method as suggested by Ham-
MERSCHLAG. Prepare a mixture of chloroform and benzene of about 1.050
sp. gr. and add a drop of the blood to this mixture If the drop rises to
the surface then add benzene, and if it sinks add chloroform. Continue
this until the drop of blood suspends itself midway and then determine
the specific gravity of the mixture by means of an areometer. This method
is not strictly accurate and must be performed quickly. In regard to the
necessary details refer to Zuntz and A. Levy.^
The reaction of the blood is alkaline towards litmus. The quantity of
alkali, calculated as NaoCOs, 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, Na2C03. Quantities below 3.3 p. m..
and above 5.3 p. m. are, according to him, to be considered as pathologicaL
V. Jaksch found the quantity of alkali in man to vary between 3.38 and
3.90 p. m., and Brandenburg found 3 p. m. NaOH(=3.98 p. m. Na2C03).
The alkaline reaction diminishes outside of the body, and indeed the more
quickly the greater the original alkalinity of the blood. This depends on
the formation of acid in the blood, in which the red blood-corpuscles seem
to take part in some way or another. After excessive muscular activity
the alkalinity is diminished (Peiper, Cohnstein), and it is also decreased
after the continuous ingestion of acids (Lassar, Freudberg^). 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 statements in regard to-
the physiological alkalinity, require further substantiation .^ Attention
must also be called to what was stated (page 191) in regard to the determina-
tion of the alkalinity of blood-serum — that determinations are made only
of the titratable alkali and not of the true alkalinity caused by hydroxy]
ions.
The alkali of the blood exists in part as alkaline salts, carbonate and
* Zuntz, Pfliiger's Arch., G6; Levy-; Proceed. Roy. Soc, 81.
^ Loewy, Pfliiger's Arch., 58, which also contains the references to the literature;
H. Strauss, Zeitschr. f. khn. Med., 30; v. Jaksch, ibid., 13; Peiper, Virchow's Arch..
116; Cohnstein, ibid., 130, which also cites the works of Minkowski, Zuntz, and Gej)-
pert; Freudberg, ibid., 125. See also Weiss, Zeitschr. f. physiol. Chem., 38, Branden-
burg, Zeitschr f. klin. Med., 45.
' 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, Bifernacki, Beitriige 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 31v
Salaskin and Pupkin, Zeitschr. f. physiol. Chem., 42, and O. Folin, ibid., 43.
224 THE BLOOD.
phosphate^ and partly in combination with protein or haemoglobin. The
first are often spoken of as readily diffusible alkalies, while the others are
not, or are only diffusible with difficulty (see page 187). The quantity
of the first in human blood is about one fifth of the total alkali (Branden-
burg). The readily as well as the difficultly diffusible alkali is divided
between the blood-corpuscles and plasma and the blood-corpuscles seem
to be richer in difficultly diffusible alkali than the plasma or serum. This
division may be changed by the influence of even very small amounts of
acid, even of carbonic acid, and also, as shown by Zuntz. Loew^y and
ZuNTZ, Hamburger, Limbeck, and GiJRBER.^ by the influence of the respir-
atory 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 senun becomes more alkaline. The equilibrium of the osmotic tension
in the blood-corpuscles and in the serum is hereby destroyed; 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 corpuscles 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,^
and they have investigated the relationship between the changes of the
volume of the blood-corpuscles and the electrical conductivity, the refrac-
tivity of the serum and the viscosity of the blood. The refraction coefficient
of the serum is highest with an increase in the amount of carbon dioxide,
while it is lowest when the blood is rich in oxygen and poor in carbon
dioxide. They consider this as an action of acid, as a similar rise is observed
after the addition of acid, while after the addition of alkali a fall in the
refraction coefficient of the serum takes place, and these same changes can
be brought about by 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 0 the viscosity diminishes to a mini-
mum, and on leading in more oxygen it rises again. The changes in vis
cosity of the blood nm parallel with the volume changes of the blood-cor-
puscles, 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).
'Zuntz in Hermann's Handbuch der Physiol., 4, Abt. 2; Loewy and Zuntz,
Pfliiger'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; Gurber,
Sitzungsber. d phys med. Gesellsch zu Wiirzburg, 1895
'Pfliiger's Arch. 110.
COAGULATION OF THE BLOOD. 225
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 dichroitic, dark red
by reflected light and green by transmitted light. The blood-coloring
matters occur in the blood-corpuscles. For this reason blood is opaque in
thin layers. If the hsemoglobin is removed from the stroma and dissolved
by the blood liquid by an}- of the above-mentioned means (see page 193).
the blood becomes transparent and has then a "lake color." "^ 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
abimdance 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 var}-ing quantities of gas contained in these two
varieties of blood, or, better, on the different amounts of oxyhsemoglobin
and hsemoglobin they contain.
The most striking property of blood consists in its coagulating T\ithm a
shorter or longer time, but as a rule ver}- shortly after leading the veins.
Different kinds of blood coagulate viith var}-ing 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 cor-
puscles have time to settle more or less before the coagulation, and the blood-
clot then shows an upper yello\\-ish-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 obser\-ed
in inflammatory- processes and is considered one of the characteristics of
them. This crusta, or "huffy coat," is not characteristic of any special
disease and it occurs chiefly when the blood coagulates slowly or when
the blood-cor]Duscles settle more quickly than usual. A buffy coat is often
observ-ed 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 gimi, glycerine, or much water; also by recei%-ing the blood in
* R Du Bois-Reymond presents objections to the general use of the above terms
in Centralbl. i Physiol., 19, p. 65.
226 THE BLOOD.
oil. Coagulation may be prevented by the injection of a proteose solution
or of an infusion of the leech into the circulating blood, but this infusion
also acts in the same way on l^lood just drawn. Coagulation is also hindered
bv snake poison and toxines (see page 171). The coagulation may be
facilitated by raising the temperature; by contact with foreign bodies, to
which the blood adheres; by stirring or beating it; by admission of air,
by diluting with very small amounts of water, by the addition of platinum-
black or finely powdered carbon; by the addition of laky blood, which
does not act by the presence of dissolved blood-coloring matters, but by
the stromata of the blood-corpuscles; 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 (Dele-
ZENNE, 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 \'iews are derived from the observations
of many investigators. From the observations of Hewson, Lister, and
Fredericq it is known that when a vein full of blood is ligatured at the
two ends and removed from the body, the blood may remain fluid for a
long time. Brucke ^ allowed the heart removed from a tortoise to beat
at 0° C, and found that the blood remained uncoagiilated for some cLays.
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 patho-
logical processes.
What then is the influence which the walls of the vessels exert on the
liquidity of the circulating blood? Freuxd has found that the blood
remains fluid when collected by means of a greased canula under oil or in a
vessel smeared with vaseline. If the blood collected in a greased vessel be
beaten with a glass rod previously oiled, it does not coagulate, but it
quickly coagulates on beating it with an unoiled glass rod or when it is
poured into a vessel not greased. The non-coagulability of blood collected
under oil was confirmed later by Haycraft and Carlier. Freuxd found
on further investigation that the evaporation of the upper layers of
blood or their contamination with small quantities of dust causes a coami-
' Delezenne, .\rch. de Physiol. (5), 8; Wright, Journ. of Physiol., 28; Arthus, Journ.
de Physiol, et Pathol., 4.
■* Hewson's works, edited by Gulliver, London, 1876, cited from Gamgee, Text-
book of Physiol. Chem , 1, 1880; Lister, cited from Gamgee, ibid.; Fredericq,
Recherclies .sur la con.stitution du plasma .sanguin, Gand, 1878, Briicke, Virchov\'s
Arch., 12.
COAGULATION OF THE BLOOD. 227
lation even in a vessel treated with vaseline. According to Freuxd ^ it
is tliis adhesion between the blood or, as the blood shows an adhesion to
the normal vessel walls (Benno Lewy), between its form-elements and a
foreign substance — and the diseased walls of the vessel also act as such —
that gives the impulse towards coagulation, while the lack of adhesion
prevents the blood from coagvilating. Bordet and Gengou ~ have also
shown that the plasma obtamed by centrifuging blood collected in a paraf-
fined vessel, and perfectly free from form-elements, can be kept without
coagulating m a paraffined vessel, and that it does coagulate on being trans-
ferred 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
relationship to the coagulation of the blood.
The views in regard to these changes are, unfortunately, very contra-
dictor}\ Accordmg to Alex. SchxMidt ^ and the Dorpat school an
abundant destruction of the leucocytes, especially polynuclear leucocytes,
takes place in coagulation and important constituents for the coagulation
of the fibrin pass into the plasma. A direct relationship between the de-
struction of leucocytes and coagulation is denied by many investigators,,
while according to other experimenters the essential is not a destruction
of the leucocytes, but an elimination of constituents from the cells into
the plasma. This process is called plasmoschisis by LowaT.'* The passage
of cell constituents into the plasma before coagulation must not neces-
sarily be considered as a phenomenon of death, as it may just as well be
a secretory process (Arthus, [NIorawitz, Dastre^). Great importance
has also been ascribed to the blood-platelets in coagulation, as certain
investigators (Bizzozero, Lilienfeld, Schwalbe, ^Morawitz, Burker)
have found that they cause or accelerate coagulation, while others (Petroxe)
on the contrar}^ fird a retarding action.^
' Freund, Wien. med. Jahrb., 1886; Haycraft and Carlier, Journ. of Anat. and
Physiol., 22; Benno Lewy, Arch. f. (Anat. u.) Physiol., 1899, Suppl.
^ Annal. de I'lnstitute Pasteur, 1".
^ Pflrger's Arch., 11. Tlip works of Alex. Schmidt are found in Arch. f. Anat..
und Physiol., 1861, 1862; Pfl.iger's Arch., 6, 9, 11, 13. See especially Ale.x. Schmidt,
Zur Blutlehre (Leipzig, 1892), wliich also gives the work of his pupils, and Weitere.
Beitrage zur Blutlehre, 1895.
^ Wien. Sitzungsber., 89 and 90, and Prager med. Wochenschr., 1889. referred,
to m Centralbl. f. d. med. Wissenscli., 28, 265.
^ Morawitz, Hofmeister's Beitrage, 5; Arthus, Compt. rend. .'oc. biolog., 55; Dastre,.
ibid., 55.
*• See foot-note 2, p. 222. Also Schwalbe, Unters. z. Blutgerinnvmg, etc., Braun-
schweig, 1900; Morawitz, Deutsch. Arch. f. klin. Med., "9, and Hofmeister's Beitrage^
4 and 5, B .rker, Pfl'ger's Arch., 102, Petrone, Maly's Jahresber., 31, p. 170.
228 THE BLOOD.
WooLDRiDGE ' takes a very peculiar position in regard to this question:
he considers the form-elements as only of secondary importance in coagulation.
As he has found, a peptone-plasma which has been freed from all form-con-
stituents by means of centrifugal force yields abundant fibrin when it is not
separated from a substance which precipitates on cooling. This substance,
which WooLDRiDGE has called A-fibrinogen, seems to all appearances to be a
nucleoproteid, which, according to the unanimous view of several investigators,
originates from the form-elements of the blood, either the blood-plates or the
leucocytes, and the generally accepted view as to the great importance of the
form-elements in the coagulation of the blood is not really contrary to Wool-
dridge's experiments.
The views are greatly di^'ided in regard to those bodies w^hich are elim-
inated 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 contain its antecedent,
prothrombin. The bodies which accelerate coagulation 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 sub-
stances by Alex. Schmidt. The nature of these bodies is unknown, and
Schmidt has given no notice of their beha\dor with 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 proteids and have been called
cytoglobin and preglohulin by Schmidt. The retarding action of these
bodies may be suppressed by the addition of zymoplastic substances, and
the yield of fibrin on coagulation in this case is much greater than in the
absence of the compound proteid 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 twofold. The thrombin first
splits the fibrinogen 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 outside
of the body or by coming in contact with foreign bodies. The parenchy-
mous 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.
^ Die Gerinnung des Blutes (published by M. v. Frey, Leipzig, 1891).
COAGULATION OF THE BLOOD. 229
LiLiENFELD has given further proof as to the occurrence in the form-
elements of the blood of bodies which accelerate or retard the coagulation.
According to this author the nature of these bodies is ver}- markedly differ-
ent from Schmidt's idea. While, according to ScH^noT, the coagulation
accelerators are bodies soluble in alcohol, and the compound proteids
exhausted with alcohol act only retardingly on coagulation, Liliexfeld
states that the substance which acts acceleratingly and retardingly on
coagulation consists of a nucleoproteid, namely, nucleohistone. Nucleo-
histone readily splits into leuconuclein and histone, the first of which acts
as a coagulation-excitant, while the other, mtroduced into the blood-vascular
system, either intravascular or extravascular, robs the blood of its property
of coagulating. Introduced into the circulator}' 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
positive facts.
Beucke 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 Hammarstex, Greex, Rixger and Saixsbury.
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 Sabbataxi ^ have also shown the impor-
tance 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, Pekelharixg, and Hammarstex.' Thrombin acts as well in
the absence as in the presence of precipitable lime salts.
Liliexfeld's theory that the leuconuclein splits off a protein substance,
thrombosinjrom 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 Hammarstex, Sch'afer, and Cramer.^ Liliexfeld's
thrombosin is nothing but fibrinogen which is precipitated by a lime salt from a
salt-poor or salt-free solution.
' Hammarsten, Nova Acta reg. Sec. Scient. Upsal. (3). 10, 1879; Green, Joum. of
Physiol., 8; Ringer and Sainsbury, ibid., 11 and 12, Arthus et Pages and Arthus,
see foot-note 4, p. 171; Hammarsten, Zeitschr. f. physiol. Chem., 22; Sabbatani,
cited, Centralbl. f. Physiol., 16, 665.
^ Hammarsten, Zeitschr. f. physiol. Chem., 22, where the other investigators are
cited.
'Hammarsten, 1. c; Schafer, Journ. of Physiol., 17; Cramer, Zeitschr. f. physiol.
Chem., 23.
230 ' THE BLOOD.
According to Pekelharing ^ thrombin is the lime compound of pro-
thrombin, and the process of coagulation consists, according to him, in
the thrombin transferring the lime to the fibrinogen, which is hereby con-
verted into an insoluble lime compound, fibrin. Of the objections to this
theory can be mentioned, among others, the fact that fibrin has been
obtained not absolutely free from lime, but still so poor in lime (Hammar-
STEN 2) that if the lime belongs to the fibrin, its molecule must be more
than ten times greater than the haemoglobin molecule, which is not prob-
able. 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 the coagulation of blood and plasma. It is very
probable that the lime salts, as admitted by Pekelharing, are a necessary
requisite for the transformation of prothrombin into thrombin.
If we attempt to summarize the more or less contradictory investiga-
tions and views as given m the preceding pages, we can consider the follow-
ing facts as conclusive: In the first place, two bodies, the fibrinogen and
the thrombin, are necessary for the coagulation. Tlie fibrinogen exists
preformed in the plasma. The thrombin, on the contrar}^, 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 necessan,^ 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 thrombm 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 relationship 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 contradictory views
on this question, namely, those of Alex. Schmidt and of Pekelharing.
According to Schmidt prothrombin occurs preformed in the circulating
plasma, and it is transformed into thrombin by the zymoplastic substances
which pass out from the form-elements. Pekelharing, on the contrary,
holds the view that the plasma does not contain appreciable amounts
of prothrombin. This body, according to him, passes before coagulation
' See foot-note 6, p. 175, and especially Virchow's Festschrift, 1, 1891.
* Zeitschr. f. physiol. Chem., 2S.
COAUULATIOX OF THE BLOOD. 231
from the form-elements into the plasma, and is there converted into thrombin
b}' 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 Anew; still
it is not quite conclusive. Leech-extract contains a body, hirudin, which,
according to ^Morawitz, is an antibody towards thrombin and quanti-
tatively 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 m the circulating plasma. Such plasma, according
to Arthus, contains no prothrombin, a statement which has been partly
substantiated by ^Iorawitz, 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 Schit-
TENHELM and BoDONG contradict the statement that fluoride-plasma
contains prothrombin. As Bordet and Gen'gou ^ have sho\\7i that pro-
thrombin 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 it is probable that all plasma contams pro-
thrombin. The absence of prothrombin, as observed by Arthus, in
peritoneal transudates in the horse, can hardly be considered as sufficient
evidence as to the occurrence of this body in blood-plasma.
The unsettled condition of the question of the zymoplastic substances
has recently been somewhat enlightened, 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. The active
constituent of these tissue extracts is called thromhokinase by ^Iorawitz,
and, according to him, this thromhokinase is necessary-, besides Ume-salts,for
the transformation of prothrombin (thrombogen according to Morawitz).
The production of thromhokinase is, according to ^Iorawitz, a general
property of the protoplasm and occurs also in the leucocytes. The throm-
hokinase of drawn blood originates in birds and m part in mammals from
the leucocytes. In mammalian blood the blood-plates are the essential
source. For the formation of thrombin three different substances are
' Arthus, Journ. de Physiol, et Pathol., 3 and 4, and Compt. rend. soc. biol., 56.
The works of Morawitz may be found in ' Hofmeister's Beitrage, 4 and 5, Deutsch.
Arch. f. klin. Med.,"9and 80, and Centralbl. f. Physiol., 17, p. 529; with Spiro, Hof-
meister's Beitrage, 5; Schittenhelm and Bodong, Arch. f. exp. Path. u. Pharm., 51;
Bordet and Gengou, Annal. Institut Pasteur, IS.
232 THE BLOOD.
necessan- according to Morawitz, namely, thrombogen, thrombokinase,
and lime-salts. Thrombogen is, according to Morawitz, not quite
identical with the prothrombin (other investigators), which he calls a-pro-
thrombin, but is a mother-substance of the same. The process of thrombin
formation can be given as follows: the kinase first transforms the throm-
bogen into a-prothrombin, which latter then is converted into thrombin
(a) by the lime-salts.
Thrombokinase is precipitated by alcohol and is not resistant towards
heat. It therefore cannot be identical with Schmidt's zymoplastic sub-
stances, and this point requires further elucidation. The thrombokinase
also does not occur to any appreciable extent in the circulating blood.
The accelerating action upon coagulation of tissues or parts of tissues de-
pends, as above stated, upon their content of kinase; but it also in part
depends upon the fact that the tissue fluids excite the secretory acti\'ity
of the form-elements.
FuLD^ has arrived at about the same results independently of Mora-
wiTz, but he has selected other names. The three substances thrombogen,
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 cytozj-m is only slowly formed therein and the ferment
(holozym) produced thereby is in an inactive form. Another reason is that
the blood contains an antibody for the fibrin ferment. The assumption
of the presence of an antibody, generally antithrombin, in the circulating
blood, which retards coagulation, does not only seem to be probable, but
recently Pugliese - has isolated an antithrombin from blood and tissues.
A serum poor in ferment and ha^Tng a weak action can be reactivated by the
addition of acid or alkali (Alex. Schmidt, Morawitz), and in this action, accord-
ing to MoRA^v^TZ, a thrombin (,3) is produced which is somewhat different from
a-thrombin. The f3-thrombin is produced from a special ^-prothrombin which never
occurs in the plasma, but only in the serum. Fuld explains this by the state-
ment that the a-thrombin is changed in the serum into metazym (/3-prothrombin),
which is then transformed by the alkali or acid into neozym ( = ^-thrombin).
L. LoEB,3 who has also conducted extensive investigations on the coagu-
lation of the blood, ascribes, like other investigators, a great importance
to the bodies existing in the tissue, which accelerate coagulation, and to
which he gives the name tissue coagulins. These coagulins are indeed not
identical with the coagulins of the blood-clot or the blood-serum, but, like
these, act directly upon fibrinogen. Under favorable conditions the com-
bined action of blood and tissue coa2:ulins mav be greater than the sum
' Centralbl. f. Physiol., 17. See also Fuld and Spire, Hofmeister's Beitrage, 5.
' Journ. de Physiol., 7.
^ The work of Loeb may be foimd in Medical News, New York, 1903. Virchow's
Arch., 176, and Hofmeister's Beitrage, 5.
INTRAVASCUL-YR COAGULATION. 233
of the indi\'idual actions. He explains this by stating that an activation
takes place by means of a kinase; still, though this is possible, he has not
proved it.
The coagulins of the blood are, according to Loeb, different from the tissue
coaguiins. The first show no specific action, i.e., not between invertebrates 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.
Based upon recent investigations, a short summary of the coagulation
of the blood would be as follows: In the circulating blood-plasma there
occur fibrinogen, lime salts, and probably also prothrombin. On aecoimt
of the continued destruction of small amoimts of form-elements, probably
small quantities of thrombin are formed, which is destroyed or made
inactive by the simultaneous presence of antithrombin. The reason why
the blood remains fluid in life lies in the lack of thrombin. Under the
influence of foreign bodies or of chemical irritants within or outside of
the body the form-elements of the blood are incited to an increased secretory
acti%-ity. and from them (perhaps also from the leucocytes in o\'ipara or
from the leucocytes but chiefly from the blood-plates in mammalia) an
abundance of kinase passes into the plasma. By this (as well as by the
action of tissue fluids outside of the body) the thrombogen is transformed
into a-prothrombin, which is changed by the lime salts into thrombin
(a-thrombin). The latter transforms the fibrinogen into fibrin.
The bodies accelerating coagulation, like the tissue extracts and the
lime salts, act upon the formation of thrombin. The mode of action of
gelatine, if it has any accelerating action at all, is not knoA^-n. The bodies
retarding coagulation may in certain cases act directly upon the blood,,
either, like the neutral salts, retarding the development of the thrombin,
or. like the oxalate or fluoride, preventing the same; or like the hirudin,^
which, as an antithrombm, makes the thrombin inactive; or like the cobra-
poison, which acts like an antikinase. In other cases they may have an
mdirect action, for they may, like the proteoses and others, cause the
body to produce special bodies which stand in close relation to intra-
vascular coagulation.
Intravascular Coagulation. It has been sho^-n by Alex. Schmidt and
his students, as also by Wooldridge, Wright,- and others, that an intra-
vascular 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 nucleopro-
* The action of hirudin is somewhat doubtful. See Schittenhelm and Bodong, I.e.
'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 Inunimity, etc., Brit. Med. Joiu-nal, Sept., 1S9L
234 THE BLOOD.
teid). Intravascular coagulation may be brought about also under other
conditions, such as after the injection of snake-poison (iNIartin ^ and others)
or certain of the proteid-like colloid substances, sjTithetically prepared
according to Grimaux's method (Halliburton and Pickering 2). If too
little of the above-mentioned bodies be injected, then we observe only a
marked retarding tendency in the coagulation of the blood. According to
WooLDRiDGE it can generally be maintained that after a short stage of
accelerated coagulability, which may lead to a total or partial intravascular
coagulation, a second stage of a diminished or even arrested coagulability
of the blood follows. The first stage is designated (Wooldridge) as the
positive and the other as the negative phase of coagulation. These state-
ments have been confirmed by several investigators.
There is no doubt that the positive phase is brought about by an abun-
dant introduction of thrombin, or by a rapid and abundant formation of
the same. The explanation of the production of the negative phase, which
<;an easily be brought about by pepsin proteoses, by various bodies such as
extracts of crabs' muscles and other organs, eel-serum, enzymes, bacterial
toxines, snake-poisons, etc., has been attempted in different ways. The
best studied is the action of proteoses, but no conclusive results have
been obtained thus far. The assertion of Pick and Spiro that the action
of the proteoses does not depend upon the proteoses themselves, but upon
a contaminating substance, the protozym, has been shown to be incorrect
by Underbill. The bodies retarding coagulation obtained by Conradi^
m autolysis, which are probably antithrombins, seem to act in a different
way from the proteoses, and cannot for the present be made use of in ex-
plaining this question.
There are a large number of researches on the action of proteoses and
oi other retarding substances by different investigators, such as Grosjean,
Ledoux, Contejean, Dastre, Floresco, Athanasiu, Carwallo, Gley,
Pachon, Spiro and Ellinger, Fuld and Spiro, Morawitz and Nolf, but
those of Delezenne * are of the greatest importance. We can say with
certainty that the action is indirect and that the liver, and perhaps also the
leucocytes and vessel walls (Nolf), are important for the process. The
' Journ. of Physiol., 15.
' Ibid., 18.
' Pick and Spiro, Zeitschr. f. physiol. Chem.,31; Conradi, Hofmeister's Beitrage, 1.
"See also Underbill, Amer. Journ. of Physiol., 9.
^ Grosjean, Travaux du laboratoire de L, Fredericq, 4, Liege, 1892; Ledoux, ibid.,
5, 1896; Nolt, Bull. I'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.
Tend. soc. biol., 46, 47, 48, 50, and 51, and Arch. d. Physiol. (6), 7, 8, 9, and 10; see
also especially Delezenne, Arch. d. Physiol. (6), 10; Compt. rend, soc biol., 51, and
Compt. rend., 130.
QUANTITATIVE COMPOSITION OF THE BLOOD. 23o
reasons for the non-coagulability of "peptone blood" are of two kinds: first,
this blood contains an antithrombin, and, secondly, the thrombin for inex-
plicable reasons is absent, although such blood seems to contain thrombogen
as well as kinase. The reason for the insufficient formation of thrombin
is unknown, and only a few observations have been collected on the forma-
tion of antithrombin. According to Xolf. 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 Delezexxe, the proteoses bring
about a destruction of leucocytes, and thereby a substance accelerating
coagulation and another ha^-ing a retarding action are set free. The first
is destroyed by the liver, and hence the action of the retarding substance
(the antithrombin) is obtained. The only thing that is positivel}' proven
is the part taken by the liver in this retardation of coagulation, as sho^-n
by Gley and Pachon; the non-appearance of the thrombin formation is
not explained by the above theories.
The coagulation of the blood of lower animals may be of two kinds, according
to L. LoEB.^ A partial agglutination of the blood-cells may take place, and this
kind of coagulation is the only kind in certain animals; but a true coagulation
of fibrinogen may also take place. This latter coagulation is essentially the same
as occurs in vertebrates, and here also an action of kinase (coagulin) upon throm-
bogen takes place.
The non-coagulability of cadaver blood depends usually, according to Mora-
WITZ,^ upon the fact that it contains no fibrinogen, due to a fibrinolysis.
The gases of the blood will be treated of m 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 relationship of the plasma and blood-corpuscles to
each other, and on the other side 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 li^■ing, non-coagidated blood, have not been
removed. Since the constitution of the blood may differ not only in differ-
ent vascular regions, but also in the same region under different circum-
stances, which renders also a number of blood analyses necessar}', 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 senmi in defibrinated blood
may be determined . according to L. and M. Bleibtreu.^ by various
' Hofmeister's Beitrage, 5 and 6, and Virchow's Arch., 176. See also Ducceschi,
Hofmeister's Beitrage, 3.
' Hofmeister's Beitrage, S.
' Pfliiger's Arch., 51. 55, and 60.
236 THE BLOOD.
methods if the defibrinated blood is mixed with different proportions of
isotonic NaCl solution (1 vol. of the blood to at least 1 vol. salt solution),
the blood-corpuscles allowed to settle to the bottom, which may be facili-
tated by centrifugal force, and the clear supernatant mixture of serum
and salt solution siphoned off. The methods are as follows:
1. Determine the quantity of nitrogen in at least two different portions of
the mixture of serum and salt solution by means of Kjeldahl's method and
calculate the quantity of protein corresponding thereto by multiplying with
6.25: and the relative volume of blood x, and also the volume of the structural
elements (1 —x), are found by the following equation:
/ -, £2 _ fi
In this equation (for mixtures 1 and 2) 6, or h^ represents the volume of blood
in the mixture, s, or 8^ the volume of salt solution, and e-^ or e^ the quantity of
protein in a certain volume of each mixture.
2. Determine the specific gravity of the blood-serum, of the salt solutions, and
of at least one of the mixtures of serum and salt solution by means of a pycnom-
eter. The relative volume of serum x is found in this by the following equation:
a: = —
b 'S,-K'
In this equation s and b represent the volumes of salt solution and blood mixed.
*S' represents the specific gravity of the serum and salt solution obtained on
allowing the blood-corpuscles to settle, So the specific gravity of the serum, and
K that of the salt solution.
For horse's blood two other shorter methods may be made use of (see the
original article).
Important objections have been presented by several investigators, such
as Eykivl^n, Biernacki, and Hedin,i against the above methods, whose
value, therefore, is questionable. The same is also true for another method,
suggested by St. Bugarsky and Tangl and partly corrected, in regard to
the calculations, by Stewaet.2 This method is based upon a difference
in the electrical conductivities of the blood and the plasma. According to
the investigations of P. Franckel,^ the results obtained by determii:ing
the conductivities give the same figures as those by Bleibtreu's method,
at least for human, horse, ox, and dog bloods. Stewart has also worked
out a colorimetric method for the estimation of the volume of the blood-
corpuscles and the plasma, which seems to be worth applying.
For clinical purposes the relative volume of corpuscles in the blood may
be determined by the use of a small centrifuge called a hcematocrit, constructed
bv Blix and described and tested by Hedin. A measured quantity of
1 Biernacki, Zeitschr. f. physiol. Chem., 19; Eykman, Pfl ger's Arch., 60; Hedin,
ibid., and Skand. Arch. f. Physiol., o.
2 Bugarsky and Tangl, Centralbl. f. Physiol., 11; Stewart, Journ. of Physiol., 24.
2 Franckel, Zeitschr. f. klin. Med., 52.
ANALYTICAL METHODS. 237
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, Hedin and Daland have
found that an approximately constant relation exists between the volume
of the layer of blood-corpuscles and the number of red corpuscles under
physiological conditions, so that the number of corpuscles may be calculated
from the volume. Daland ^ 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 ansemia, this method gives such inaccurate results that it cannot
be used.
KoppE^ has recently 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 relationship 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 one
side, and in 100 parts of the blood on the other. If we represent the amount
of this substance in the plasma by f and that in the blood by h, then the
amount of x in the plasma from 100 parts of blood is x= '—.
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.-^ The experimenters just named have tried
to determine the amount of the plasma and blood-corpuscles, respectively,
in different kinds of blood, starting from the above-mentioned substances.
Another method suggested by Hoppe-Seyler is to determine the total
amount of haemoglobin and proteins in a portion of blood, and on the other
hand the amount of 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
' Hedin, Skand. Arch. f. Physiol., 2, 134 and 361, and 5; Pfliiger's Arch., 60;
Daland, Fortschritte d. Med., 9.
2 Pfliiger's Arch., 107.
'Hoppe-Seyler, Handb. d. physiol. u. path. chem. Analyse, 7. Aufl.; Bunge, Zeit-
fichr. f. Biologic, 12; Otto, Pfliiger's Arch., 35.
238 THE BLOOD.
difference between these two determinations corresponds 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 senmi in the blood is easily determined. The
usefulness of this method has been confirmed by Bunge by the control
experiments with the 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 distribution 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 Abderhalden ^ according to Hoppe-Sey-
ler's and Bunge's methods. The analyses of human blood by C. Schmidt ^
are older and were made according to another method, hence perhaps the
results for the weights of the corpuscles are a little too high. All the results
are in parts per 1000 parts of blood.
The relation between blood-corpuscles and plasma may vary considerably
under different circumstances even in the same species of animal. In
animals, in most cases considerably more plasma is found, sometimes twa
thirds of the weight of the blood.^ For human blood Arronet has found
478.8 p. m. blood-corpuscles and 521.2 p. m. seiaun (in defibrinated blood)
as an average of nine determinations. Schneider'* found 349.6 and 650.4
p. m. respectively in women.
The sugar occurs, it seems, only in the serum and not with the blood-
corpuscles. The same is true, according to Abderhalden, for the lime,
fat, and perhaps also the fatty acids. The small traces of bile-acids found
in normal blood are, according to Croftan,^ 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 0X-, 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
senmi, while iron occurs chiefly in the form-elements, especially in the
erythrocytes. As the iiucleoproteids contain iron, some iron always occurs
in the leucocytes, and traces of iron are also found in the serum. This
amount under normal conditions is very small, while in disease the relation
Ijetween hsemoglobin-iron and other blood-iron does not seem to change
' Zeitschr. f. physiol. Chem., 23 and 25.
^ Cited and in part recalculated from v. Gorup-Besanez, Lehrb. d. physiol. Chem.,
4. Aufl., 345.
'See Sacharjin in Hoppe-Seyler's Physiol. Chem., 447; Otto, Pfluger's Arch., 35;
Bun<re, 1. c; L. and M. Bleibtreu, Pfliiger's Arch., 51.
■• Arronet, Maly's Jahresber., 17; Schneider, Centrulbl. f. Physiol., 5, 362.
*Pfliiger'sArch.,90.
COMPOSITION OF THE BLOOD.
239
Water
Solids
Harnoglobin
Proteid
Sugar
Cholesterin
Lecithin
Fat
Fatty acids
Phosphoric acid (
as nuclein '
Soda.
Potash
Iron oxide
Lime.
Magnesia
Chlorine
Phosphoric acid. .
Inorganic P2O5. . .
Pig-blood.
_S - T
ffl
272.20
162.S9
142.20
8.35
0.213
1.504
0.027
0.0455
2.1.57
0.696
0.06.56
0.642
0.89.56
0.7194
518.36
46.. 54
38.26
0.684
0.231
0.805
1.104
0.448
0.0123
2.401
0.152
0.0689
0.0233
2.048
0.1114
0.0296
0.\-blood.
5
192.65
132.85
103.10
20.89
1.100
1.220
0.0178
0.7266
0.2351
0.544
0.0056
0.5901
0.2392
0.1140
616.25
58.249
48.901
0.708
0.835
1.129
0.625
0.0089
2.9084
0.1719
0.0805
0.0300
2.4889
0.1646
0.0571
Horse-blood.
243.87
153.84
125.8
20.05
0.26
1.93
0.02
0.05
1.32
0.59
0.04
0.18
0.98
0.76
551.14
51.15
42.65
0.90
0.31
1.05
0.50
0.36
0.01
2.62
0.15
0.07
0.03
2.20
0.15
0.05
Dog-blood.
a
277.71
165.10
145.6
2.36
0.56
1.02
0.05
1.27'
O.lll
0.71
0.03'
0.60
0.67
0.54
514.30
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
Bull-blood.
206.81
127.50
106.40
15.38
0.610
0.953
608.03
57.66
46.41
0.679
0.599
1.244
2.3.57
0.494
0.0194 0.0089
0.839
0 233
0.562
0.009
0.628
0.236
0.133
2.873
0.174
0.073
0.027
2.453
0.156
0.041
Sheep-blood.
s
200.39
118.82
102.80
12.80
1.147
1.329
0.760
0.236
0.545
0.006
0.575
0.228
0.088
Ed
300
624 1ft
56.63
.46.5&
0.708
0.891
1.088
0.859
0.490S
0.0235 O.OIO*
2.917
0.172
:0.089
0.027
,2.516
0.163
0.057
Goat-blood.
Cat-blood.
Rabbit-blood.
Human Blood,
Man.
Human Blood,
Woman.
o;
lU
<u
V
(U
0
c
M(M
0
, =M
.00
1 S®
.0
3 —
.a>
. 2°
-OJ
, 3n
-t-
£<N
~ "'t
Ed
Tl&ci
£t^
TJ&C^
Ed
~ Ceo
£co
C Of
3 ^'^
C 0^5
3--0
0 0I>
3'=a
2 o-i
300
2 ^=^
C 0 ?0
-0
0 -.i^
-0
0 ccc
-co
0 oiO
>--r
c on
Co
a
■Jl
H
rjl
«
m
«
m
n
XJl
Water
211.35
135.86
592.54
60.25
270.90
163.11
524.17
41.35
235.74
136 37
518.18
46.71
349.69
163.33
439.02
47.96
272 56
123.68
551. 9»
Solids
51.77
Haemoglobin
11 2.. 50
—
143.2
—
123.50
—
Proteid
18.76
0.601
50.96
0.822
0.698
11.62
0.5.56
33.16
0.860
0.3.39
4.55
0.268
33 63
1.036
0.343
Organic
bodies
Cholesterin
Lecithin .
1.339
1.127
1.3.54
0.971
1.722
1.105
159.59
43.82
120.13
46.70
Fat
—
0.0407
0.398
—
0.446
0.282
z
0.749
0.507
Fatty acids
Inorg.
Phosphoric acid |
0.028
0.0117
0.063
0.009
0.040
0.015
3.74
4.14
3.55
5.07
Soda
0.7.55
2.824
1.174
2 512
—
2.789
0.24
1.66
0.65
1.92
Potash.
0.236
0.160
0.112
0.148
1 946
0.162
1.59
0.15
1.41
0 20
Iron oxide ......
0 547
—
0.694
—
0.615
. —
. —
- —
—
—
Lime
—
0.078
—
0.062
—
0.072
—
—
—
—
Magnesia
0 014
0.026
0 035
0.024
0 029
0.028
—
—
—
—
Chlorine
0.514
2 409
0.455
2 360
0.460
2.438
0.90
1.72
0.36
0-14
Phosphoric acid. .
0.243
0.1.54
0.697
0.133
0.835
0.151
—
—
—
—
Inorganic PoOs. . ■
0.097
0.045
0.515
0.040
0.645
0.040
—
—
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 contams 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 extrac-
tive bodies, of proteins and haemoglobin. The amount of this last-men-
tioned 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 Ijlood from the ox, bull, sheep, goat^ and rabbit (Abderhalden).
240 THE BLOOD.
The amount of sugar in the blood is on an average 1-1.5 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. When the quantity of
sugar amounts to more than 3 p. m., then, according to Cl. Bernard,^
sugar occurs in the urine, and a glycosuria appears. An increase in the
quantity of sugar takes place, as first observed by Bernard and lately
substantiated by Fr. Schenck, after removal of blood. According to
Henriques^ this increase of the reducing power, at least in dogs, is not
due to sugar, but chiefly to jecorin, which substance is the cause of more of
the reduction in normal blood than the sugar. It is difficult to judge of the
value of many statements as to the amount of sugar and the reducing
power of the blood, because the experimenters generally have not con-
sidered the presence of a certain quantity of jecorin or conjugated glu-
curonic acids, or they were unable to detect them.
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 (Gr^hant and Quinquaud, Schondorff) and varies between
0.2 and 1.5 p. m. In dogs Schondorff found in starvation a minimum
of 0.348 p. m. and a maximum of 1.529 p. m. at the point of highest
urea formation. Gottlieb obtained much lower results by another direct
method, namely, in starvation 0.1-0.2, and after meat feeding 0.28-0.56
p. m. In man v. Jaksch ^ 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 and the increased urea formation depending thereon.
A more important mcrease in the quantity of urea in the blood occurs in a
retarded elimination of urea, as in cholera, also in cholera infantum and
in infections of the kidneys and the 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* has re-
cently 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.
' Bleile, Arch. f. (Anat. u.) Physiol., 1879; Bernard, Lemons sur le diab^te, Paris,
1877.
' Schenck, Pfliiger's Arch., 57; Henriques, Zeitschr. f. physiol. Chem., 23, See also
Kolisch and Stejskal, Wien. klin. Wochenschr., 1898.
' Gr^hant et Quinquaud, Joum. de I'anatomie et de la physiol., 20, and Compt.
rend., 98; Schondorff, Pfliiger's Arch., 54 and 63j Gottlieb, Arch. f. exp. Path. u.
Pharm., 42; v, Jaksch, Leyden-Festschr., I, 1901.
* V. Schroder, Zeitschr. f. physiol. Chem., 14; Baglioni, Centralbl. f. Physiol., 19.
COMPOSITION OF THE BLOOD. 241
The blood also contains traces of ammonia. According to Horodynski,
Salaskin, and Zaleski.^ who worked with the improved Nencki and
Zaleski method, the quantity in arterial dog-blood was 0.41 milligram 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 is
disputed by Biedl and Wixterberg,^ however. The blood from healthy
persons contains on an average 0.90 milligram per 100 c.c, according to
WiNTERBERG.3 The quantity of uric acid may be 0.1 p. m. in bird's blood
(v. Schroder'*). 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 Salomon 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 K^tsuyama ^ 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 containing
different amounts of gas and different amounts of oxyhsemoglobin and
haemoglobin. The arterial blood is light red; the venous blood is dark red,
dichroitic, greenish by transmitted light through thin laj-ers. The arterial
coagulates more quickly than the venous blood. The latter, on account of
the transudation which takes place in the capillaries, was formerly said to
be somewhat poorer in water but richer in blood-corpuscles and haemo-
globin than the arterial blood ; but this is denied by modern investigators.
According to Kruger ® and his pupils the quantity of dr\^ residue and
ha?moglobin in blood from the carotid artery and from the jugular vein (in
cats) is the same. Rohmann and Muhsam ^ could not detect any differ-
ence in the quantity of fat in arterial and venous blood.
Blood jrom the Portal Vein and the Hepatic Vein. In consequence of
the small quantities of bile and lymph found relatively to the large quantity
^ Zeitschr. f. physiol. Chem., 35, which also gives the older literature.
»Pfliiger's Arch., 88.
' Wien. klin. Wochenschr., 1897, and Zeitschr. f. klin. Med., 35.
* Ludwig's Festschrift, 1887.
* Irisawa, Zeitschr. f. physiol. Chem., 17, which also gives the older literature; Saito
and Katsuyama, ihid., 32.
° Zeitschr. f . Biologie, 26. This also gives the literature on the composition of the
blood in different vascular regions.
' Pfliiger's Archiv, 46.
242 THE BLOOD,
of blood circulating through the liver in a given time^ we can hardly expect
to detect by chemical analysis a positive difference in the composition
between the blood of the portal and hepatic veins. The statements in
regard to such a difference are in fact contradictory. For example, Dros-
DOFF has found more haemoglobin in the hepatic than in the portal vein^
while Otto found less. Kruger finds that the quantities of haemoglobin^
as well as of the solids, in the blood from the vessels passing to and from
the liver are different, but a constant relationship cannot be determined.
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 carbo-
hydrates, the blood of the portal vein not only becomes richer in dextrose^
but may contain also dextrin and other carbohydrates (v. Mering, Otto *),
The amount of urea in the blood from the hepatic vein is greater than
in other blood (Greihant and Quinquaud^). In regard to the quantity
of ammonia, see page 241.
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 haemoglobin than arterial
blood. KriIger ^ and his pupils have found that the blood from the vena
lienalis is generally richer in haemoglobin and solids than arterial blood;
still the contrary is often found. The blood from the splenic vein coagu-
lates slowly.
The Blood from the Veins of the Glands. The blood circulates with
greater rapidity through a gland during acti\aty (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 secretion the
venous blood also becomes somewhat poorer in water and richer in solids.
The blood from the Muscidar 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 statement, has not the power of
coagulating. This statement is nevertheless false, and the apparent un-
coagulability depends in part on the retention of the blood-clot by the
' Drosdoff, Zeitschr. f. physiol. Chem., 1; Otto, Maly's Jahresber ,17; v. Mering,
Arch, f. (Anat. u.) Physiol , 1877, 214.
M c.
'v. Middendorff, Centralbl. 1. Physiol., 2, 753; Kriiger, 1. c.
COMPOSITION OF THE BLOOD. 243
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 coagu-
lation. Menstrual blood, according to Gautier and Bourcet, contains
arsenic and is also richer in iodine than other blood (see blood-senmi,
page 187).
The Blood of the Two Sexes. Woman's blood coagulates somewhat more
quickly, has a lower specific gravity, a greater amount of water, and a
smaller quantity of solids than the blood of man. The amount of blood-
corpuscles and ha3moglobin is somewhat smaller in woman's blood. The
amount of haemoglobm 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 deliverj^ 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 ^ found in most cases an increase in the amount
of haemoglobin m pregnancy in the last months.
The Blood at Different Periods of Life. Foetal 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 l^y
several investigators, such as Cohnstein and Zuntz, Otto, Winternitz,
Abderhalden, Schwinge, and others, immediately or very soon after
birth or at least ^vithin 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-bom infants, as observed by several inves-
tigators. The quantity of haemoglobin and blood-corpuscles sinks gradu-
ally 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, Otto 2). According
' Nasse, Maly's Jahresber., 7; Becquerel and Rodier, Traite de chim. pathol.,
Paris. 1854; Cohnstein, Pfli.ger's Arch., 34,' 233; Mollenberg, Maly's Jahresber., 31,
1S5. See also Payer, Arch. f. Gynak.,. 71.
■ Cohnstein and Zuntz, Pfliiger's Arch., 34; Winternitz. Zeitschn-f. physiol. Chem.,
22; Leichtenstern, Untersuch. iiber den Hamoglobingehalt des Blutes, etc., Leipzig,
244 THE BLOOD.
to older statements, the blood at old age is poorer in blood-corpuscles and
protein bodies, but richer in water and salts.
The Influence of Food on the Blood. In complete starvation, no decrease
in the amount of solid blood-constituents is found to take place (Panum
and others). The amount of hiemoglobin is increased a little, at least in
the early period (Subbotix, Otto, Hermann and Groll, Luciani and
BuFALiNi), and also the number of red blood-corpuscles increases (Worm
MuLLER, BuNTZEN^), 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, Popel found that complete abstinence 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^).
After a rich meal the relative number of blood-corpuscles, after secretion
of digestive juices or absorption of nutritive liquids, may be increased or
diminished (Buntzen, Leichtenstern). The number of white blood-
corpuscles may be considerablv 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 haemoglobin than that of camivora,
and SuBBOTiN has observed in dogs after a partial feeding with food rich
in carbohydrates that the amount of hsemoglobin sank from the physio-
logical average of 137.5 p. m. to 103.2-93.7 p. m. Tsuboi^ has also shown
in experiments on rabbits and dogs that with an insufficient diet of bread
and potatoes, where the body gave up protein and contained relatively
much carl)ohydrate, the amount of htemoglobin decreased and the blood
became richer in water. According to Leichtenstern, a gradual increase
in the amount of hsemoglobin is found to take place in the blood of
himian beings on enriching the food, and according to the same inves-
tigator the blood of lean persons is generally somewhat richer in h<Tmo-
1878; Otto, Maly's Jahresber., 15 and 17; Abderhalden, Zeitschr. f. physiol. Chem.,
34; Schwinge, Pfliiger's Arch., 73 (literature). See also Fehrsen, Journ. of Physiol.,
30.'
^ Panum, Virchow's Arch., 29; Subbotin, Zeitschr. f. Biologie, 7; Otto, 1. c; Worm
Muller. Transfusion und Plethora, Christiania, 1875; Buntzen. see Maly's Jahresber.,
9; Hermann and Groll, Pfliiger's Arch., 43; Luciani and Bufalini, Maly's Jahresber.,
12.
' Popel, Arch, des scienc. biol. de St. Petersbourg, 4, 354; Schulz, Pfliiger's Arch.,
65; Daddi, Maly's Jahresber.,. 30.
^Subbotin, 1. c; Tsuboi, Zeitschr. i. Biologie, 44.
INCREASE IX THE RED CORPUSCLES. 245
globin 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 amoimt of haemoglobin they contain. The action of the iron
salts is obscure.i There does not seem to be any doubt that not only is
the iron contained in the food m the form of organic compoimds active,
but also iron salts and therapeutic iron. Accordmg to Buxge and his
pupils the iron preparations only act indirectly. They may combine with
the sulphuretted hydrogen of the intestinal canal and thereby prevent the
iron associated in the food as assimilable protem compounds from being
eliminated as iron sulphide (Bunge), or they may perhaps act as excitants
upon the blood-forming organs (Abderhaldex).
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 Paxum and Worm Muller.^
the blood-liquid is quickly eliminated and transformed in this case — the
water being eliminated principally by the kidneys and the protein burned
into urea, etc. — while the blood-corpuscles are preserved longer and cause
a " polycijthcvmia." A relative mcrease in the number of red corpuscles
is found after abimdant transudation from the blood, as in cholera and
heart-failure •wdth considerable congestion. An mcrease in the number
of red blood-corpuscles has also been observed under the influence of
diminished pressure or m high altitudes. Viault first called attention to
the fact that the number of red corpuscles was ver\- 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 localities was a very considerable increase in the
number of red corpuscles, in his ovm. case 5-^ millions. A similar increase
of the red blood-corpuscles, as also an increase m the quantity of hemo-
globin under the influence of diminished pressure, has been observed by
many other investigators in human beings as well as in animals. Investi-
gators are not united as to how this increase is brought about. The mcrease
in the blood-corpuscles is not absolute but is only relative, and it is con-
sidered by several observers that there is neither a new formation nor a
dimmished destruction of the blood-corpuscles. A relative increase may
be brought about in different ways. For example, another di\'ision of the
blood-corpuscles in the vascular system has been supposed, whereby the
blood-corpuscles accumulate in the capillaries, from which region the blood
' See Bunge, Zeitsclir. f. physiol. Chem., 9; Hausermann, ibid., 23, where the works
of Woltering, Gaule, Hall, Hoehhaus, and Quincke are cited (the same work con-
tains a table of the quantity of iron in various foods); Kunkel, Pfhiger's Arch., 61;
Macallum, Journal of Physiol., 16; Abderhalden, Zeitsclir. f. Biologic, 39.
' Panum, Virchow's Arch., 29; Worm Midler, 1. c.
246 THE BLOOD.
has been examined most often (Zuntz). It is also claimed that a con-
centration of the blood takes place by increased evaporation (Grawitz),
and finally an increase in the blood-corpuscles has also been explained by
assuming a contraction of the vascular system with the pressing out of
plasma (Bunge, Abderhalden i). 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-raarrow is increased.
A decrease in the number of red corpuscles occurs in anaemia from differ-
ent causes. Every excessive hemorrhage causes an acute anaemia, or, more
correctly, oligaemia. Even during the hemorrhage, the remaining blood
becomes by diminished secretion and excretion, as also by an abundant
absorption of parenchymous fluid, richer in water, somewhat poorer in pro-
teins, and strikingly poorer in red blood-corpuscles. The oligaemia passes
soon into a hydrsemia. The amount of protein then gradually increases
again ; but the re-formation of the red blood-corpuscles is slower, and after
the hydrsemia follows also an oligocythaemia. After a little time the
number of blood-corpuscles rises to normal; but the re-formation of haemo-
globin does not keep pace with the re-formation of the corpuscles, and a
chlorotic condition may appear. A considerable decrease in the number
of red corpuscles occurs also in chronic anaemia and chlorosis; still in such
cases an essential decrease in the amount of haemoglobin occurs without an
essential decrease in the number of blood-corpuscles. The decrease in the
amount of haemoglobin is more characteristic of chlorosis than a decrease
in the number of red corpuscles. The statements on the changes in the
blood in anaemia and chlorosis differ very considerably, and in this con-
nection attention must be called to the findings of Lorrain Smith (based on
his estimation of the oxygen capacity and of the blood-volume) that in
chlorosis an absolute diminution of the amount of haemoglobin does not
occur, but, that on the contrary, the total quantity of haemoglobin may be
normal, with only a relative diminution occurring, due to a pronounced
increase of the blood-plasma and of the total quantity of 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 (J- ^ )
occurs in pernicious anaemia (Hayem, Laache, and others). On the
contrary, the individual red corpuscles are larger and richer in haemoglobin
* The literature on this subject may be found in Abderhalden, Zeitschr. f. Biologie,
43; van Voornveld, Pfliiger's Arch., 92.
^ Hohenklima und Bergwanderungen, by N. Zuntz, A. Loewy, Franz M ller and W.
Caspari, Berlin, 190G.
^ Trans. Path. Soc. London, 51, 1900. Complete analyses of chlorotic blood may
be found in Erben, Zeitschr. f. klin. Med., 47.
NUMBER OF LEUCOCYTES. 247
than they ordmarily are, and the number stands in an inverse relationship
to the amount of hsemoglobin (Hayem). Besides this the red corpuscles
often, but not always, show in pernicious anaemia remarkable and ex-
traordinary irregularities of form and size, which Quincke ^ 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. Leucsemic 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 fre-
quently 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 xanthine bodies, and so-called Charcot's
crystals (see semen. Chap. XIII) have also been found in leucsemic blood.
The peptone (proteose) which is found in the leucsemic blood after death,
and which does not exist in the fresh blood, is, according to Erben, a
digestive product which is produced by a tryptic enzyme which originates
from the leucocytes as well as by traces of a peptic enzyme. These
enzymes, according to Erben, do not occur in normal blood, or are so
firmly combined therein that on the death of the cells they are not set
free, or at least their action does not become evident.^
A great number of investigations have been made on the chemical com-
position of blood in disease. But as we have only a few analyses of the
blood of healthy individuals, and as the possible variations under physio-
logical conditions are little known, it is difficult to draw any positive conclu-
sions from the analyses of pathological blood. Unfortunately, on account
of the large number of contradictory statements of the composition of the
blood of diseased human beings, it is impossible to give a brief summary of
the results, still the changes in the blood in disease must be of the greatest
importance.
The quantity of blood is indeed somewhat variable in different species
of animals and in different conditions of the body; in general we consider
the entire quantity of blood in adults as about ^ j\ of the weight of the
' 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. khn. Med., 40.
^ Erben, Zeitschr. f. Heilkunde, 24, and Hofmeister's Beitrage, S. See also Schumm,
ibid., 4 and 5.
248 THE BLOOD.
body, and in new-bom infants about -^. Haldaxe and Lorraix Smith ,^
who have determined the quantity of blood by a new method, find in
fourteen persons that it varies between ^^ and gV 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^), and it may therefore be also proportionally greater
in star\dng 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 durable sinking of the
blood-pressure in the arteries, because the smaller arteries accommodate
themselves to the small quantities of blood by contracting (Worm ^lijh-
LER 3). 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-bom infants are verj- 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 (Paxum, Laxdois, Worm ]Muller,
Poxfick). According to Worm ^Miiller the normal quantity of blood may
indeed be increased as much as 83 per cent without producing any abnor-
mal 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 ^Ml'ller). If the quantity
of blood of an animal is increased by transfusion 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, Forster, Paxum, Worm ]\1uller4), a polycy-
thsemia is gradually produced.
The quantity of blood in the different organs depends essentially on
their activity. During work the exchange of material in an organ is
more pronounced than during rest, and the increased metabolism is con-
nected ■viith a more abundant flow of blood. Although the total quantity
' Joum. of Physiol., 25.
^ Virchow's Arch., 29.
^ Transfusion und Plethora, Christiania, 1875.
* Panum, Nord. med. Ark., 7; Virchow's Arch., 63; Landois, Centralbl. f. d. med.
Wissensch., 1875, and Die Transfusion des Blutes, Leipzig, 1875; Worm Miiller
Transfusion und Plethora; Ponfick, Virchow's Arch., G2; Tschirjew, Arbeiten aus
der physiol. Anstalt zu Leipzig, 1874, 292; Forster, Zeitschr. f. Biologic, 11; Panum,
Virchow's Arch., 29.
COMPOSITION OF THE BLOOD. 249
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 the same, and from this point of view the
distribution of the blood m the different organs and groups of organs is of
interest. According to Ranke,i to whom we are especially indebted for
our knowledge of the relationship of the acti\dty of the organs to the
quantity of blood contained therem, 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.
Die Blutvertheilung und der Thatigkeitswechsel der Organe, Leipzig, 1871.
CHAPTER VIl.
CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
I. Chyle and Lymph.
The lymph is the mediator in the exchange of constituents 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 theo-
retical 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
Barbera,! 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 nojioubt of a quantitative nature. This differ-
ence consists chiefly in that the lymph is poorer in proteins. No essen-
tial 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 give it a milky appear-
a,nce; hence the old name "lacteal fluid."
Chyle and lymph, like the plasma, contain seralbimiin, serglohulins,
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 the
' Zeitschr. f. Biologic, 3(5.
250
CHYLE AND LYMPH. 251
blood-serum, but in larger quantities than in the blood; this depends on
the fact that the blood-corpuscles contain no sugar. The glycogen detected
by Dastre ^ m the lymph occurs only in the leucocytes. According to
RoHMANN and Bial/ lymph contains a diastatic enzyme similar to that in
blood-plasma, and Lepixe ^ has found that the chyle of a dog during
digestion has great glycolytic activity. The amount of urea has been
determined by Wurtz ^ 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, Munk* 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 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 PFLtJGER and Strassburg,^ smaller than m
venous, but greater than in arterial blood.
The quantitative composition of the chyle must e\idently be very variable.^
The analyses thus far made refer only to that mixtureof 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 composition of human chyle
two analyses will be given. The first is by Owen-Rees, of the chyle
of an executed person, and the second by Hoppe-Seyler,'^ of the chyle in
' Compt. rend, de soc. biol., 47, and Compt. rend., 120; Arch, de Physiol. (5), 7.
^ Rohmann and Bial, Pfliiger's .Arch., 52, 53, and 55; L6pine, Compt. rend., 110.
^ Compt. rend., 49.
" Virchow's Arch., SO and 123. In regard to the analysis of the fat of chyle, see
Erben, Zeitschr. f. physiol. Chem., 30.
* Hanamarsten, Die Gase der Hundelymphe, Arbeiten aus d. physiol. Anstalt zu
Leipzig, 1871; Strasburg, Pfliiger's Archiv, 6.
• See also Panzer, Zeitschr. f. physiol. Chem., 30.
' Owen-Rees, cited from Hoppe-Seyler's Physiol. Chem., 595; Hoppe-Seyler, ibid.,
.597. See also Carlier, Brit. Med. Journ., 1902, 175.
252 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
a case of rupture of the thoracic duct. In the latter case the fibrin had
previously separated. The results are in 1000 parts.
No. 1. No. 2.
Water 904.8 940.72 water
Solids 95 . 2 59 . 28 solids
Fibrin Traces
Albumin 70.8 36.67 albumin
Fat 9.2 7.23 fat
f 2 . 35 soaps
i 0 . 83 lecithin
Remaining organic bodies ... 10.8 -j 1.32 cholesterin
3 . 63 alcohol extractives
[ 0 . 58 water extractives
Q„, . _ ^ . / 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. Muxk and A. Rosenstein ^ have inves-
tigated 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 par-
taking 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 par-
taking of 41 grams of fat the quantity of soaps was only about ^o of the
neutral fats.
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 ob-
tained 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 by C. Schmidt,^ the data being obtained from lymph from the neck
of a colt. The results are expressed in parts per 1000.
12 3 4
Water 939.9 934.8 957.6 955.4
Solids 60.1 65.2 42.4 44.6
Fibrin 0.5 0.6 0.4 2.2
Albumin 42.7 42.8 34.7] ....
Fat, chole.sterin, lecitliin 3.8 9.2 I 35 . 0
Extractive bodies 5.7 4.4 ■••] ....
Salts 7.3 8.2 7.2 7.5
' Virchow's Arch., 123.
* Gubler and Quevenne, cited from Hoppe-Seyler's Physiol. Chem., 591; v. Scherer^
ibid., 591; C. Schmidt, ibid., 592.
LYMPH. 253
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 ^Iuxk 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 relationship between globulin and albumin was as 1:2.4 to 4. The
mineral bodies in 1000 parts lymph (chylous) were: NaCl 5.83; Na2C03 2.17;
K2HPO4 0.28; Ca3(P04)2 0.28; :\Ig3(P04)2 0.09; and Fe(P04) 0.025.
Under special conditions the lymph may be so rich in finely divided fat
that it appears like chyle. Such lymph has been investigated by Hexsen
in a case of lymph fistula in a ten -year-old boy, and by Laxg ^ in a case of
lymph fistula in the upper part of the left thigh of a girl of seventeen.
The lymph investigated by Hexsex varied in the quantity of fat, as an
average of nineteen anah'ses, between 2.8 and 36.9 p. m., while that inves-
tigated by Laxg contained 24.85 p. m. of fat.
The quantity of lymph secreted must naturally change considerably
under various conditions, and there are no means of measuring it. The
size of the flow of lymph is, as Heidenhain suggests, no measure of the
abundance of supply of nutritive material to the organs, and the lymph-
tubes act according to him as "drain-tubes," remo\ang 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
Heidexhaix the quantity averages 640 c.c. for a dog weighing 10 kilos.
Determinations of the quantity of lymph in man have also been
attempted. Noel-Patox^ obtained 1 c.c. 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 ]Muxk and
Rosexsteix, 1134-1372 grams chyle was collected -uithin 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, espe-
cially in the first few hours after powerful muscular exercise.
Several circumstances have a marked influence on the extent of lymph
' Hensen, Pfliiger's Arch., 10; Lang, see Maly's Jahresber., 4.
^ Journ. of Physiol., 11.
254 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
secretion. During starvation less lymph is secreted than after partaking
of food. Nasse 1 has observed in dogs that the formation of lymph 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' deprivation of
food. In this connection mention must be made of the important observa-
tions of AsHER and Barbera ~ that with pure protem diet the lymph
current is increased in the thoracic ca\'ity, 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 movements of the
limbs (Lesser). Under the influence of curare, an increase of the lymph
secretion is observed (Paschutin, Lesser^), and the quantity of solids in
the lymph is also increased.
The bodies inciting lymph-flow, the so-called himphagogues, are of espe-
cially great interest, and they may, accordmg to Heidenhain,^ be divided
into two different chief groups. The lymphagogues of the first series —
extracts of crab-muscles, blood-leech, anodons, liver and intestme of dogs,
as well as peptone and egg albumin, strawberry extracts, metabolic products
of bacteria and others — cause a greatly increased secretion of lymph with-
out raising the blood-pressure, and in tliis way the blood-plasma becomes
poorer in proteins and the lymph richer than before. For the formation
of this lymph, which Heidenhain designates blood-lymph, we must admit
with him that a special secretory activity of the capillary-wall endothelium
exists. The lymphagogues of the second series, such as sugar, urea, sodium
chloride, and other salts, also cause an abundant lymph formation. The
blood, as well as the lymph, thereby becomes richer in w'ater. This
increased amount of water depends, according to Heidenhain, upon an in-
creased delivery of water by the tissue-elements, and this lymph is chiefly
tissue-lymph, according to him. Diffusion is no doubt of great impor-
tance in the formation of this lymph, but the secretory acti\ity of the
endothelium is also of importance, at least for certain bodies, such as sugar.
' Cited from Hoppe-Seyler, Physiol. Chem., 593.
' The works of Asher and collaborators, Barbera, Gies, and Busch, upon lymph
formation may be found in Zeitschr. f. Biologie, 3(5, 37, 40.
^ Lesser, Arbeiten aus der physiol. Anstalt zu Leipzig, Jahrgang 6; Paschutin,
ibid., 7.
^ Heidenhain, Pfiiiger's Arch., 49; Hamburger, Zeitschr. f. Biologie, 27 and 30.
See especially Ziegler's Beitr. zur Path. u. zur allg. Pathol., 14, 443; also Arch. f.
(Anat. u.) Physiol., 189.5 and 1896.
LYMPH FORMATION. 255
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 Hamburger
ascribed a special activity to the capillar}^ endothelium, assuming that
they take part m the formation of lymph in a secretory' manner.
Another view which also besides the physical processes is 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; it&
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 Heidenhain's \dew^
and he explains the increased permeability of the vessel wall by the fact that
these bodies have a poisonous irritating action. On the contran,-, Ashek
explains this increased lymph-flow by the statement that the substance in
question — as well as those influences which incite the acti\'ity of the liver
— produces an increased formation of lymph in these organs. This view is
supported by experiments upon the action of lymphagogues on blood coag-
ulation 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 morpho-
logical changes. The connection between organ activity and lymph for-
mation has also been sho\\ai upon muscles and glands by others besides the
above-mentioned investigators (Hamburger, Bainbridge i).
The extent of organ work certainly essentially influences the quantity
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 ^ upon the lengthy post-mortem lymph-flow, have
' In regard to the works cited, as well as the literature upon lymph formation, see
ElHnger, "Die Bildung der Lymphe," Ergebnisse der Physiol., I, Abt. 1, 1902, and
Asher, Biochem. Centralbl., 4.
^ Anier. Journ. of Physiol., 7.
256 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
shoT\Ti 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 (Koranyi, Starling, Roth, Asher, and others)
have shown clearly 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 dissimilation 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 syn-
thesis build up highly complex constituents from simple molecules, and as
the chief products of catabolism are carbon dioxide and water, it is diffi-
cult to explain these intricate conditions. Still, irrespective of whatever
\aew, a change in one or the other direction in the osmotic pressure upon
both sides of the capillary wall must be produced hereby. Whether this
and other physico-chemical processes are alone sufficient to explain the
lymph formation (Cohnstein, Ellinger) remains an open and disputed
question.*
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 subcutaneoJis tissues,
or under the epidermis; and in this way pathological transudates are
formed. Such tme transudates, which are similar to lymph, are gener-
ally poor in form-elements and leucocytes, and yield only very little or
almost no fibrin, while the inflammatory transudates, the so-called exu-
dates, 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 \aew that all these fluids contain the salts and extractive bodies
' On this question see Ellinger, "Die Bildung der Lymphe," Ergebnisse der Phy-
eiologie, I, Abt. 1, 355, and Asher, Biochem. Centralbl., 4, pp. 1 and 45.
TRANSUDATES AND EXUDATES. 257
occurring in the blood-plasma in about the same quantity as the blood-
plasma, while the amount of proteins is habitually smaller. T\Tiile the
different fluids belonging to this group have about the same quantities of
salts and extractive bodies, they differ from one another chiefly in con-
taining differing quantities of protein and form-elements, as well as vary-
ing quantities of transformation and decomposition products of these
latter — changed blood-coloring matters, cholesterin, etc. The correspond-
ence 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 transu-
date. To what extent filtration is active in the perfectly normal vascular
wall cannot be answered.
The altered permeability of the capillary- walls in disease is a second
important factor in the formation of transudates. The circumstance that
the greatest quantity of protein occurs in transudates in inflammatory
processes, to which is also due the abundant quantity of form-elements in
such transudates, has been explained by this hypothesis. The greater
quantity of protein in the transudates in formative irritation is in great
part explained by the large amount of destroyed form-elements. The
interesting observation made by Paijkull,^ that in such cases in which an
inflammator}- irritation has taken place the fluid contains nucleoalbumin
(or nucleoproteid?), while this substance does not occur in transudates
in the absence of inflammatory' processes, can be explained by the pres-
ence of form-elements. Still, such a phosphorized protein substance does
not occur in all inflammatory^ exudates.
As the secretor}^ importance of the capillar}^ endothelium has been made
probable by the investigations of Heidexhaix, it is a priori to be expected
that an abnormallv increased secretory- acti\'ity of the endothelium 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 capillar}- walls.
The var}-ing quantities of protein observed by C. Schacdt^ in the
tissue-fluids in different vascular regions can perhaps be explained by the
different condition of the capillarv endothelium. For example, the amount
of protein in the pericardial, pleural, and peritoneal fluids is con-
siderably greater than in those fluids which are found in the subarach-
noidal space, in the subcutaxeous tissues, or in the aqueous humor,
which are poor in protein. The condition of the blood also greatly affects
the transudates, for in hydrsemia the. amount of protein in the transudate
• See Maly's Jahresber., 22.
' Cited from Hoppe-Seyler, Physiol. Chem., 607
258 CHYLE, LY^IPH, TRANSUDATES AND EXUDATES.
is very small. With the increase in the age of a transudate, of a hydrocele
fluid for instance, the quantity of protein is increased, probably by resorp-
tion of water, and indeed exceptional cases may occur in which the amount
of protein, without any previous hemorrhage, is even greater than in the
blood-serum.
The proteins of transudates are chiefly seralbumin, serglobulin, and a
little fibrinogen. Proteoses and peptones do not occur, excepting perhaps
in the cerebrospinal fluid, and in those cases where an autolysis has taken
place in the liquid.^ The non-inflammatory transudates as a rule undergo
spontaneous coagulation not at all, or only very slowly. On the addition of
blood or blood-serum they coagulate. Inflammatory exudates coagulate
spontaneously, and Paijkull has shown that these often contain nucleo-
proteid (or nucleoalbumin). In inflammatory' exudates a protein sub-
stance has been habitually observed which is precipitated by acetic acid,
but which does not occur in transudates, or only in very small quantities.
This substance, which has been observed and studied by ]\Ioritz, Staehelin,
Umber, and Rivalta, is claimed by the first three observers to be free
from phosphorus, while Rivalta considers it to be a phosphorized pseudo-
globulin. Umber calls it serosamucin, although it yields only very little
reducing carbohydrate. According to Joachim ^ it is only a part of the
globulin, a view which cannot be correct for all cases, v. Holst^ 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 peri-
toneum, 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 circumstances, 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
complications with ovarial tumors, and which are cleavage products of a
more complicated substance, seem according to Paijkull "* to be regular
constituents of transudates and are closely related to the above-mentioned
serosamucin.
There are numerous investigations on the relationship between globu-
lin and seralbumin, and Joachim has recently determined the relationship
' Umber, Miinch. 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, 3."); and Zak, Wien. klin. Wochenschr., 1905.
'Paijkull, 1. c; Moritz, Miinch. med. Wochenschr., 1903; Staehelin, ibid., 1902;
Umber, Zeitschr. f. klin. Med., 48; Rivalta, Biochem. Centralbl., 2 and 5; Joachim,
Pfliiger's Arch., 93.
^ Zeitschr. f. physiol. Chem., 43.
* Hammarsten, ibid., 15; Paijkull, 1. c.
TRANSUDATES AND EXUDATES. 259
between euglobulin and the total globulin. No conclusive results can be
drawn from these determinations. The relationship between globulin and
seralbumin varies very much in different cases, but, as Hoffmann and
PiGEAND ^ have shown, the variation is in each case the same as in the
blood-serum of the indi\ddual.
The specific gravity runs nearly parallel with the quantity of protein.
The varying specific gravity has been suggested as a means of differentiation
between transudates and exudates by Reuss,^ as the first oftdn show a
specific gravity below 1015-1010, while the others have a specific gra\'ity
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;
but sometimes extractive bodies occur, such as allantoin in dropsical fluids
(MoscATELLi^), which "lave not been detected in the blood. Urea seems
-to occur in vexy variable amounts. Sugar also occurs in transudates, but
it is not known to what extent the reducing power is due, as in blood-
serum, to other bodies. A reducing, non-fermentable substance has been
found by Picil^rdt in transudates. The sugar is generally dextrose, but
le\'ulose seems to have been found* in several cases. Sarcolactic acid has
been found by C. Kulz ^ in the pericardial fluid from oxen. Succinic acid
has been found in a few cases in hydrocele fluids, while in other cases it
is entirely absent. Leucine and tyrosine have been found in transudates
from diseased livers and in pus-like transudates which have undergone
decomposition, and after autolysis. Among other extractives found in
transudates must be mentioned uric acid, xanthine, creatine, inosite, and
jn/rocatechin (?).
The di\isio"n of the nitrogenous substances in human transudates and
exudates has so far been little studied. Otori ^ has 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.
' Joachim, I. c; Hoffmann, Arch. f. exp. Path. u. Pharra., 10; Pigeand, see Maly's
Jahresber., 16.
Reuss, Deutsch. Arch. f. klin. Med., 28. See also Otto, Zeitschr. f. Heilkunde, 17.
' Zeitschr. f. physiol. Chem., 13.
* Pickardt, Berl. klin. Wochenschr., 1897. See also Rotmann, Miinch. med. Woch-
enschr., 1898; Neuberg and Strauss, Zeitschr. f. physiol. Chem., 36.
» Zeitschr. f. Biologic, 32.
' Zeitschr. f. Heilkunde, 25.
260 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
The aniino-acid nitrogen and the urea nitrogen in pus are greater as the
specific gravity rises. In serous exudates and transudates, on the contrary,
the amino-acid nitrogen and the urea nitrogen are not proportional to the
specific gravity, but are dependent upon the general circulator}' condition
of the body.
The investigations upon the molecular concentration have shown that
no essential and constant difference exists between exudates and transu-
dates. The osmotic concentration and the concentration of the electrolytes
are as a rule the same as in blood-serum, although sometimes rather di-
vergent results have been found. The concentration of the electrolytes
shows according to Bodon,^ 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 concentration has shown that the
transudates and exudates in this regard are about as neutral as the blood-
serum (Bodon).
As above stated, irrespective of the varying number of form-elements
contained in the different transudates, the quantity of protein is the most
characteristic chemical distinction in the composition of the various trans-
udates; therefore a quantitative analysis is of importance only in so far
as it considers the quantity of protein. On this account, in the following,
relative to the quantitative composition, 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 anal3'ses per-
formed by v. Gorup-Besanez, Wachsmuth, and Hoppe-Seyler,^ is
37.5-44.9 p. m., and the amount of protein is 22.8-24.7 p. m.' The anal3^sis
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
I Albumin ... 22 . 34
Solublesalts 8.60 NaCl 7.28
Insoluble salts 0.15
Extractive bodies 2 . 00
' Pfliiger's Arch., 104, where the literature on this subject may be found.
^ V. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl.,401; Wachsmuth, Vir-
chow's Arch., 7; Hoppe-Seyler, Physiol. Chem., 605.
PLEUR.IL FLUID. 261
Friend ^ has found nearly the same composition for a pericardial fluid
from a horse, -^ith 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 stop-
page, Hasebroek^ 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
lecitliin, 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 verj^ variable properties. In cer-
tain cases it is nearly serous, in others again sero-fibrinous, and in others
similar to pus. There is a corresponding variation in the specific gra\'ity
and the properties in general. If a pus-like exudate is kept enclosed for a
long time in the pleural ca\dty, a more or less complete maceration and
solution of the pus-corpuscles is found to take place. The ejected 3-elloT\ish-
brown or greenish fluid may then be as rich in solids as the blood-serum;
and an abundant fiocculent precipitate of a nucleoalbumin or nucleopro-
teid (the pyin of early -^Titers) may be obtained on the addition of acetic
acid. This precipitate is soluble with difficulty in an excess of acetic acid.
Numerous analyses, b}' many investigators .^ of the quantitative com-
position of pleural fluids under pathological conditions have teen published.
From these analyses we learn that in hydrot borax the specific gra\dty is
lower and the quantity of protein less than in pleuritis. In the first case
the specific gra\-ity is generally less than 1.015, and the quantity of protein
10-30 p. m. In acute pleuritis the specific g^a^'ity is generally higher than
1020, and the c[uantity 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 wth an abundant accumulation of pus, the specific
gra\'ity may rise even to 1.030, according to the observations of H.^enl^r-
STEN. The quantity of solids is often 60-70 p. m.. and may be even more
than 90-100 p. m. (HA^LMARSTEx). ^lucoid substances have also been
detected in pleural fluids by Paijkull. Cases of chylous pleurisy are also
known; in such a case ^Iehu* found 17.93 p. m. fat and cholesterin in
the fluid.
The quantity of peritoneal fluid is very small under physiological condi-
* Halliburton, Text-book of Chem. Physiol, etc., London, 1904.
- Zeitschr. f. physiol. Chem., 12.
' See the works of Mehu, Runeberg, F. HofTmann, Reuss, all of which are cited in
Bemheim's paper in Virchow's Arch., 131, 274. See also Paijkull 1 c, and Halli-
burton's Text-book, .346; Joachim, 1. c.
*Arch. gen. de med., 1886, 2, cited from Maly's Jahresber.. 16.
262 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
tions. The investigations refer only to the fluid under diseased conditions
(dropsical or ascitic fluid). The color, transparency, and consistency of
these may vary greatly.
In cachectic conditions or a hydrgemic 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-L015. 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 protein
may rise to 35 p. m. In carcinomatous peritonitis it may have a cloudy,
dirty-gray appearance, due to its richness in form-elements of various kinds.
The specific gra\'ity is then higher, the quantity of solids greater, and it
often coagulates spontaneously. In inflammatory processes 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 dropsical fluid may be rich
in very finely emulsified fat (chylous ascites). In such cases 3.86-10.30
p. m. fat has been found in the dropsical fluid (Guinochet, Hay i), and even
17-43 p. m. has been found by Minkowski.
As first shown by Gross, an ascitic fluid may have a chylous appearance
without the presence of fat, i.e., pseudochylous. The cause of the chylous
properties of a transudate is not known, although numerous investigators,
such as Gross, Bernert, Mosse, and Strauss, have studied the subject;
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 molecu-
larly with the euglobulin.
By admixture of ascitic fluid with the fluid from an ovarian cyst the
former may sometimes contain pseudoraucin (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 peri-
tonitis and in cirrhosis hepatis syphilitica in men. According to the
investigations of Paijkull, these substances seem to occur often and perhaps
habitually in the ascitic fluids.
* Guinochet, see Strauss, Arch, de Physiol., 18. See Maly's Jahresber., 16, 475.
^ Gross, Arch. f. exp. Path. u. Pharm., 44; Bernert, ibul., 49; Mosse, Leyden's
Festschrift, 1901; Strauss, cited in Biochem. Centralbl., 1, 4.37; Wolff, Hofmeister's
Beitrjise, 5.
HYDROCELE AND SPERMATOCELE FLUIDS. 263
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 tlie
following example of the composition, taken from Berxheim's ^ 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 (Mosc-vtelli), xanthine, creatine, cholesterin, sugar, diastatic
and proteolytic enzymes, and according to Hamburger ' also a lipase.
Hydrocele and Spermatocele Fluids. These fluids differ essentially
from each other in various ways. The hydrocele fluids are generally colored
light or dark yellow, sometimes brownish with a shade of green. They
have a relatively higher specific gTa\ity, 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 fcwo kinds of fluids we will give the
average results (calculated in parts per 1000 parts of the fluid) of seven-
teen analyses of hydrocele fluids and four of spermatocele fluids made bjy
Hammarstex.3
Hydrocele. Spermatocele.
Water 938 . 85 986 . 83
Sohds 61.15 12.17
Fibrin 0.59
Globulin 13.25 0.59
Seralbumin 35.94 1.82
Ether extractive bodies 4 . 02 "I
Sohible salts 8.60}. 10.76
Insohible salts 0 . 66 J
' 1. c. As it was impossible to derive mean figures from those given by Bernheim,
the author has given the maximum and minimum of the averages given by him.
2 Arch. f. (Anat. u.) Physiol., 1900, 433.
' Upsala Lakaref. Forh., 14, and Maly's Jahresber., 8, 347.
264 CHYLE, LY^IPH, TRANSUDATES AND EXUDATES.
In the hj'drocele fluid traces of urea and a reducing substance have beau
found, and in a few cases also succinic acid and inosite. A hydrocele fluid may,
according to Devillard,' sometimes contain paralbumin or metalbumin (?).
Cases of chylous hydrocele are also knowTi.
Cerebrospinal Fluid. The cerebrospinal fluid is thin, water-clear, of
low specific gravity, 1. 007-1. OOS. 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.
According to Halliburton 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 statements of
Halliburton on the occurrence of ^proteose do not coincide with the ob-
servations of other investigators (Panzer, Salkowski^). In general
paralysis Halliburton and Mott have obtained a nucleoproteid in the
cerebrospinal fluid. Choline occurs in several diseases, as in general paral-
ysis, brain-tumors, tabes dorsalis, and epilepsy (Halliburton and Mott,
DoNATH^). Dextrose, or at least a fermentable sugar, occurs habitually
in the cerebrospinal fluid, while the statements of Halliburton as to the
occurrence of a substance similar to pyrocatechin could not be substantiated
by Nawratzki,^ and hence this substance does not exist in all cerebro-
spinal fluids. Urea occurs in cerebrospinal fluids, but not alwaj's. The
variable relationship between potassium and sodium ^ is probably due,
according to Salkowski, 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 Cavazzani,^ who has es-
pecially 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 towards 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 con-
sists of seralbumin and globulin and very little fibrinogen. According to
Gruenhagen it contains paralactic acid, another dextrogyrate substance,
1 Bull. Soc. chim., 49, 617.
- Halliburton's Text-book; Panzer, Wien. klin. Wochenschr., 1899; Salkowski,
Jaffe Festschrift, 26.5.
^ Halliburton and Mott, Phil. Transact. Roy. Soc. London, Series B, 191; Donath,
Zeitschr. f. physiol. Cbem.. 39 and 42; see also Mansfield, ibid., 42.
* Zeitschr. f. physiol. Chem., 23. See also Rossi, ibid.. 39 (literature).
* See Salkowski, 1. c. New quantitative analyses of cerebrospinal and hydro-
cephalus fluids may be found in the cited works of Xawratzki, Panzer, and Salkowski.
'See Maly's Jahresber., 22, 346, and Centralbl. f. Physiol., 15, 216.
SYNOVIAL FLUID. 265
and a reditcing body which is not similar to dextrose or dextrin. Pautz ^
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
contents of vesicators^ blisters. In a burn-blister K. ^Iorxer^ 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 pyro-
catechin. The fluid of the pemphigus is alkaline in reaction. A wound
secretion collected by Liebleix ^ under aseptic conditions was alkaline in
reaction and contained less protein than the blood-serum. It formed
a slight fibrin clot and contained proteoses only at first or at the beginning
of the abscess formation. As the wound liealed, the relationship 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, xery poor in
solids, purely serous, does not contain fibrinogen, and has a specific gra^■ity
of 1.005-1.013. The quantity of proteins is in most cases lower than 10
p. m., — according to Hoffil^xx 1-8 p. m., — and in serious affections of
the kidneys, generally with amyloid degeneration, less than 1 p. m. has been
shown (HoFFiL\NN *) . The oedematous fluid also habitually contains urea,
1-2 p. m., and sugar.
The FLUID OF THE ECHixococcus cvst 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, creatin-e, succinic acid, and salts (8.3-9.7 p.m.).
Proteins are found only in traces, and then only after an inflammatory irritation.
In the last-mentioned case 7 p. m. proteins have been found in the fluid.
The Synovial Fluid and Fluid in Synovial Cavities around Joints, etc.
The synovia is hardly a transudate, but it is often discussed in an appendix
to the transudates.
The syno\-ia 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. It contains also, besides proteins and salts, a mucin
substance, synoviamucin (v. Holst^). In pathological synovia Haa^l^r-
STEN has found a mucin-like substance which is not mucin. It behaves
like a nucleoalbumin or a nucleoproteid and gives no reducing substance
on boiling with acids. Salkowski ^ also found a mucin-like substance in a
' Gruenhagen, Pfliiger's Arch., 43; Pautz, Zeitschr. f. Biologie, 31.
^ Skand. Arch. f. Pliysiol., 5.
^ Habilitationsschrift Prag, 1902, printed by H. Laupp, Tiibingen.
* Deutsch. Arch. f. klin. Med., 44.
' Zeitschr. f. phj-siol. Chem., 43.
• Hammarsten, Maly's Jahresber., 12; Salkowski. Virchow's Arch., 131.
266 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
pathological synovial fluid, which was neither mucin nor nucleoalbumin.
He called the substance synovin.
The composition of synovia is not constant, but is different in rest and
in motion. In the last-mentioned case the quantity of fluid is less, but the
amount of the mucin-like body, of proteins, and of the extractive bodies is
greater, while the quantity of salts is diminished. This may be seen from
the following analyses by Frerichs.^ The figures represent parts per 1000.
I. Synovia from II. Synovia from
a ytall-fed ox. a Field-fed ox.
Water 969.9 948.;')
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 mucosas, as also the fluid in the synovial cavities
around joints, etc., is similar to synovia from a qualitative standpoint.
m. Pu.s.
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 L020 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 decomposition 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-corpus-
cles must be studied separately.
Pus-serum. Pus does not coagulate spontaneously nor after the addi-
tion 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 towards litmus. It contains, for the
most part, the same constituents as the blood-serum; but sometimes be-
sides these — when, for instance, the pus has remained in the body for a
long time — it contains a nucleoalbumin or a nucleoproteid which is precipi-
tated by acetic acid and is soluble with great difficulty in an excess of the
acid (pi/in of the older authors). This nucleoalbumin seems to be formed
' Wagner's Handworterbuch, 3, Aht., 41 ri3.
PUS. 267
from the hyaline substance of the pus-cells by maceration. The pus-serum
contains, moreover, at least in many cases, no fibrin ferment. According
to the analyses of Hoppe-Seyler ^ the pus-serum contains in 1000 parts :
I. II.
Water 913 . 70 905 . 65
Solids 80 . 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 1 1 . 53 6 . 92
Inorganic salts 7 . 73 7 . 77
The ash of pus-serum has the following conaposition, calculated to 100&
parts of the serum :
I. II.
NaCl 5.22 5.39
Na„S04 0.40 0.31
Na,HPO, 0.98 0.46
NalCOg 0.49 1.13
CagCPOJa 0.49 0.31
MgjCPO,)., 0.19 0.12
PC), (in eicess) 0.05
The pus-corpuscles are generally thought to consist in great part of
emigrated white blood-corpuscles, and their chemical properties have
therefore been given in discussing these. The molecular granules, fat-glob-
ules, and red blood-corpuscles are considered rather as casual form-elements.
The pus-cells may be separated from the serum by centrifugal force, ot
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-cor-
puscles.
The chief constituents of the pus-corpuscles are proteins of wUch
the largest portion seems to be a nucleoproteid 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 quickl}^ 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
the pus-cells contain also a globulin which coagulates at 48-49° C, as well
as serglobulin (?), seralbumin, a substance similar to coagulated protein
(Miescher), and lastly peptone or proteose (Hofmeister^). It is very
remarkable that no nucleohistone or histone has been detected in the pus-
cells.
' Med.-cliem. Untersuch., 490.
2 Miescher in Hoppe-Seyler's Med.-chem. Untersuch., 441; Hofmeister, Zeitschr. f.
physiol. Chem., 4.
268 CHYLE, LY:\IPH, TRANSUDATES AND EXUDATES.
There are also found in the protoplasm of the pus-cells, besides the pro-
teins, lecithin, cholesterin, xanthine bodies, fat, and soaps. Hoppe-Seyler
has found cerehrin, a decomposition product of a protagon-like substance,
in pus (see Chapter XII). Kossel and Freytag ^ have isolated from pus
two substances, pyosin and pyogenin, which belong to the cerebrin group
(see Chapter XII). Hoppe-Seyler ^ claims that glycogen appears only in
the li\'ing, 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 nucleoproteids. ^Iandel and Levene ^
have shuwn the occurrence of glucothionic acid in the pus-cells.
In regard to the occurrence of enzymes in the pus-cells it must be re-
marked that neither thrombin nor prothrombin is found therein, although
these bodies are generally considered as being derived from the leucocytes
and can also be obtained 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 *).
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 anal5'ses of
Hoppe-Seyler is as follows, in parts per 1000 of the dried substance:
I. II.
Proteins 137 .62 1
Nuclein 342.57 [685.85 673.69
Insoluble bodies 205 . 66 J
Lecithin 1 i < o oo 75 . 64
Fat ; ^-^'^-^^ 75.00
Cholesterin 74.00 72.83
Cerebrin 51 .99 \ mo 84.
Extractive bodies 44.33/ lu^.a*
MINERAL SUBSTANCES IN 1000 PARTS OF THE DRIED SUB.STANCE.
NaCl 4.34
CagCPOJ, 2.05
Mg3(P0j; 1.13
FePO^ 1 .06
PO, 9.16
Na 0.68
K Traces (?)
' Zeitschr. f. physiol. Chem., 17, 452.
^ Hoppe-Seyler, Physiol. Chem., 790.
' Biochem. Zeitschrift. 4.
■• Fr. Miiller, Verhandl. Nat. Gesellsch. zu Basel, 1901; O. Simon, Deutsch. Arch,
f. klin. Med., 70.
LY:\rPHATIC GLAXDS. 269
MiEscHER has 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 have stagnated for some time
occur peptone (proteose), leucine and tyrosine, free fatty acids and volatile
fatty acids, such as formic acid, butyric acid and valerianic acid. There are
also found chondrin (?) and glutin (?), wea, dextrose (in diabetes), hile-
pigments and bile-acids ((in catarrhal icterus).
As more specific but not constant constituents of the pus must be men-
tioned the following: pyin, which seems to be a nucleoproteid precipitable
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 pvis. This depends on the presence of micro-organisms {Bacillus pyo-
cyaneiis). From such pus Fordos and Lucre ^ have isolated a crystalline
blue- pigment, pyocyanin, and a yellow pigment, pyoxanthose, which is pro-
duced 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 occurring generally in cells (Chapter V,
pages 141 and 142). According to Baxg^ they also contain histone nucleates
(jiucleohi stone) . but in smaller amounts and of a different variety from ihe
better-studied nucleohistone from the thymus gland. Proteoses occur as
products of autolysis. By a lengthy autotysis of lymph-glands Reh ^ 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, sarcolactic acid, xanthine bodies, and
leucine. In the inguinal glands of an old woman Oidt^l\xn found 714.32
p. m. water, 2S4.5 p. m. organic and 1.16 p. m. inorganic substances. In
the cells of the mesenteral lymphatic glands of oxen Bang^ 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. nucleoproteid, 47.6 p. m. bodies soluble in alcohol, and 10.5 p. m.
mineral constituents.
' Fordo.«;, Corapt. rend., ol and 56; Litcke, Arch. f. kiln. Chirurg., 3; Boland, Cen-
tralbl. f. Bakt. u. Parasit., I, 25.
' Studier over Nucleoproteider, Kristiania, 1902, and Hofmeister's Beitrage, i.
^ Hofmeister's Beitrage, 3.
M. c.
270 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
The Thymus. The cells of this gland are veiy rich in nuclein bodies
and relatively poor in the ordinan,' proteins, but their nature has not been
closely studied. The chief interest is attached to the nuclein substances.
KossEL and Liliexfeld first prepared from the waterj- extract of the
gland, by precipitating ^dth acetic acid and then further purifying, a protein
substance which has been generally called niideohistone. By the action
of dilute hydrochloric acid upon nucleoliistone 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, ^La.lexgreau. and Huisk.\mp ^ upon nucleohistone all show
that tliis nucleoproteid is not a unit substance but a mixture of at least
two bodies. The views of the investigators mentioned differ quite esseu'
tially 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 change-
ability of the substances in question.
Besides the real nucleohistone, B-nucleoalbuminof Malengreau, Liliex-
feld's histone contains a second nucleoproteid which Baxg and Huiskamp
call simple nucleoproteid, while Malexgreau designates it A-nucleoalbumin.
This protein, which contains only about 1 per cent phosphorus and which
is possibly identical with the nucleoproteid found by Liliexfeld in the
thymus, yields a nuclein, but no free nucleic acid, on cleavage. As second
cleavage product it yields, according to Malengreau, the A-histone, which
can be readily precipitated by magnesium and ammonium sulphates from
the ordinary B-liistone of the thymus gland. The occurrence of A-histone
in the gland has been verified by Bang, and according to Bang and Huis-
K-\MP the A-liistone is not derived from the nucleoproteid, as these inves-
tigators claim that it yields no histone. According to Baxg the nucleo-
proteid yields only an albuminate, besides the nuclein, as cleavage products.
The true nucleoliistone, which is much richer in phosphorus (the calcium
salt containing, according to Baxg, on an average 5.23 per cent P), yields
ordinary' histone as one cleavage product and free nucleic acid as the other,
according to the unanimous opinion of the above-mentioned investigators.
According to Baxg, who.se statements on this point have been substantiated
by ^La.lengreau, it splits on saturating with XaCl into nucleic acid and
histone without yielding any other protein. On this account B.an'G does not
consider this body as nucleohistone in the ordinary- sense, i.e., not as a nucleo-
proteid, but as a histone nucleate. The nucleohistone behaves like an acid,
who.se salts, especially the calcium salt, have been closely studied by Huis-
' Lilienfeld, Zeitschr. f. physiol. Chem., IN; Ko.ssel, ibid., 30 and 31; Bang, ibid.,
30 and 31. See also Arch. f. Math, og Naturvidenskab, 25, Kristiania, 1902, and
Hofmeister's Reitrage, 1 and 4; Malengreau, La Cellule, 17 and 19; Huiskamp, Zeit-
schr. f. phj'siol. Chem., 32, 34, and 39.
THYMUS. 271
KAMP. On the electrolysis of a solution of alkali nucleohistone in water Huis-
K.\MP found also that the nucleohistone collected in traces at the anode, and
that the sodium compound is therefore ionized in the solution. The
nucleic acid-calcium-histone compound has been prepared,, it seems, in a
pure state by Bang, and he foimd the follo^^■ing average composition:
C 43.69; Ho.60; X 16.87; S0.47; P5.23; Ca 1.71 per cent. The question
as to what compound contains the A-histone remains to be investigated.
The nucleohistone prepared by Huiskamp's method by precipitating withCaCli
is, according to him, a mbcture of two nucleohistones, of which one, the a-nucleo-
histone, contains 4.5 per cent phosphorus, and the other, .-J-nucleohistone, contains,
on the contrary, only in round numbers 3 per cent phosphorus.^ 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 Malexgreau, 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 isola-
tion of the bodies in question we must refer to the oridnal publications.
In connection with the so-called nucleohistone, attention must be called to
tissue fibrinogen and cdl fibrinogen, which are compound proteins, and are claimed
by certain mvestigators to stand in close relation to the coagulation of the blood.
These may be in part nucleoproteids and in part also nucleohistones. To this
same group belong also the important cell constituents described by Alex.
Schmidt- 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 jat, leucine, succinic acid, lactic
add, sugar, and traces of iodothyrin are present. According to Gautier^
arsenic also occtirs in ver\- small amounts, and no doubt here as well as in
other organs it is related to the nuclein substances. The richness in nuclein
bodies explaias the occurrence of large quantities of purine bases, chiefly
adenine, whose quantity, according to Kossel and Schixdler,-* is 1.79 p. m.
in the fresh organ and 19.19 p. m. in the dr\' substance. The bodies thymine
and uracil (?) obtained, besides lysine and ammonia, by Kutscher, as prod-
ucts of autodigestion of the gland, probably have a similar origin. Liliex-
FELD ^ has 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 Joxes. wliich acts like a nuclease, splitting
off phosphoric acid and ])urine bases from the nucleoproteids. This enzyme,
» Zeitschr. f. physiol. Chem., 39.
^ See foot-note 5, p. 141.
3 Compt. rend., 129.
* Zeitschr. f. physiol. Chem., 13.
-Kutscher, ibid., 34; Lilienfeld, ibid., 18.
272 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
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 th3^mus 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 S6 . 7
Lecithin 75.1
Fat 40 . 2
Cholesteiin 44 . 0
Glycogen S . 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 fouud KH0PO4 ainongst the bodies soluble in alcohol.
Attention must be called to the analyses of Bang 2 which show that the
thymus contains about the same quantity of nucleoproteid, 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
Iw pressure and which ordinarily serves as material for chemical investiga-
tions is therefore a mixture of blood and spleen constituents. For this
reason the proteins of the spleen are little known. The nucleoproteid
isolated by Levene and Mandel is to be considered as a true spleen con-
stituent. The ferruginous albuminate has been considered as a spleen
constituent for a long time, and especially also a protein substance which
does not coagulate on boiling, and which is precipitated by acetic acid
and yields an ash containing much phosphoric acid and iron oxide.*
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 glycei'ophosphoric acid. Besides these two acids there have
been 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, xanthine bodies, and
jecorin. Levene has 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
* Zeitschr. f. physiol. Chem., 41.
'1. c, Arch. f. Math., etc.
' Cited from v. Gorup-Besanez, Lehrb. d. physiol. Chem., 4. Aufl., p. 732.
* See V. Gorup-Besanez, Lehrbuch, 4. Aufl., p. 717.
SPLEEN. 273
acid. The question whether this glucothionic acid originates from the
above-mentioned nucleoproteid or from the mucoid substance has not been
decided (Levene and Mandel^).
]\Iany 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
(Burian), which occurs in the spleen of oxen and horses, but not in man,
dogs, and pigs (Schittexhelm), 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 the adenine into hypo-
xanthine by the latter. The guanase occurs also in the spleen of the ox
and horse, but not (Jones), or only in small amounts (Schittenhelm), in
the pig-spleen.2 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, >5-lienase, is active only in acid reaction.
These enz3^mes not only act autolytically upon the proteins of the spleen,
but they also dissolve fibrin and coagulated blood-serum. In the autolysis
of the spleen Leathes found proteoses, lysine, arginine, histidine, leucine,
arainovalerianic acid, aspartic acid, and tr}^ptophane among the cleavage
products. ScHUMM ^ found, in the 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 leucsemic 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 especialh^ abun-
dant in the spleen of the horse. Nasse ^ on analyzing the grains (from the
si)leen of a horse) obtained 840-630 p. m. organic and 160-370 p. m. inor-
ganic substances. These last consisted of 566-726 p. m. Fe203, 205-388
p. m. P2O5, and 57 p. m. earths. The organic substances consisted chiefly
of proteins (660-800 p. m.), nuclein (52 p. m. maximum), a yellow color-
ing-matter, extractive bodies, fat, cholesterin, and lecithin.
In regard to the mineral constituents, it is to be observed that in compari-
son with sodium and phosphoric acid the amount of potassium and chlorine
' Levene, Zeitschr. f. physiol. Chem., 3"; Levene and Mandel, ibid., 45 and -t".
^ See Chapter XV for the literature.
' Hedin and Rowland, Zeitschr. f. physiol. Chem., 32, and Hedin, Journ. of Physiol. .
30; Leathes, Journ. of Physiol., 28; Schumm, Hofmeister's Beitrage, 3 and 7.
* Maly's Jahresber., 19, p. 315.
274 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
is ^mall. The amount of iron in new-born and 3'oimg animals is small
(Lapicque, Kruger, and Pernou), in adults more appreciable, and in old
animals sometimes very coasiderable. Nasse found nearly 50 p. m. iron
in the dried pulp of the spleen of an old horse. Guillemonat and La-
picque 1 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 ^ 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 leucaemia and
the appearance of amyloid substance (see page 69).
The physiological functions of the spleen are little known, with the
exception of its importance in the formation of leucocytes. Some consider
the spleen as an organ for the dissolution of the red blood-corpuscles, and
the occurrence of the above-mentioned deposit rich in iron seems to con-
firm this view. 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 investi-
gations of Herzen seem to confirm this relation, but the recent work of
Prym^ 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 relationship between the spleen and
the formation of uric acid. This relationship has been studied by Horbac-
ZEWSKi. He has shown that when the spleen-pulp and blood of calves
are allowed to act on each other, under certain conditions and temperature,
in the presence of air, large quantities of uric acid are formed. Under
other conditions he obtained from the spleen-pulp only xanthine bodies
with very little or no uric acid. Horbaczewski ^ has also shown that the
* Lapicque, ibid., 20; Lapicque and Guillemonat, Compt. rend, de soc. biol., iS,
and Arch., de Physiol. (.5), 8; Kriiger and Pernou, Zeitsclir. f. Biologie, 27; Nasse,
cited from Hoppe-Seyler, Physiol. Chem., 720.
^ Cited from v. Gorup-Besanez, Lehrbuch, 4. Aufl., p. 719.
3 Schiff, cited by Herzen, Pfliiger's Arch., 30, 295, 308, and 84, and Maly's Jahr-
esber., IS; Prym, Pfliiger's Arch., lO-l and 107; see also Chapter IX.
* Monatshefte f. Chem., 10, and Wien. Sitzungsber. Math. Nat. Klasse, 100, Abt, 3.
THYROID GLAXD. 275
uric acid originates from the nucleins of the spleen, which yield uric acid
and xanthine bodies according to the experimental conditioas. This
behavior is explained by the above-mentioned investigations of Buriax,
ScHiTTEXHELM, JoxES, and others on the enzj-motic uric-acid formation
and the deamidization of the purine l^odies, and a relationship between the
spleen and uric-acid formation is indisputable. Still we cannot say that
the spleen shows a special relationship to the uric-acid formation as com-
pared with other organs (see Chapter XX).
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 thj-roid 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, iodothyreoglobulin, behaves like a globulin, while the other is a
nucleoproteid (see also Gourlay^). 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 Bertraxd,^ seems to be re-
lated to the nuclein substances.
According to 0sw^\ld 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-lDorn contain thyreoglobulin free
from iodine. The thyreoglobuhn first becomes iodized into iodothyreo-
globulin on passing from the follicle-cells. Besides these mentioned bodies
leucine, xanthine, hypoxanthine, iodothyrine, lactic and succinic acids occur
in the thyreoidea. Oidtivl^xx ^ 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 "struma cystica" Hoppe-Seyler found hardly any protein in the smaller
glandular vessels, but an excess of mucin, while n the larger he found a great
deal of 'protein, 70-80 p. m.* Cholesterin is regularly found in such cj'sts, 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, viethcemoglohin (and hsematin?). Bile-coloring matters have also been
found in such cysts. (In regard to the paralbumins and colloids which have been
foimd in struma cysts and colloid degeneration, see Chapter XIII.)
^ Gourlay, Journ. of Physiol., 16; Oswald, Zeitsclir. f. i^hysiol. Chem.. 32, and
Biochem. Centralbl., 1, 429.
'Gautier, Compt. rend., 129. See also ibid., 130, 131, 134, 135; Bertrand, ibid.,
134, 135.
3 1. c, 732.
* Physiol. Chem., p. 721.
276 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
Those substances which bear a close relationship to the functions of
the gland seem to be of special interest.
The complete extirpation, as also the pathological destruction, of the
thyroid gland causes great disturbances, ending finally in death. In dogs,
after the total extripation, a disturbance of the nervous and muscular sys-
tems occurs, such as trembhng and con\adsions, and death generally super-
venes shortly after, most often during such an attack.^ In human beings
different disturbances appear, such as nervous symptoms, diminished in-
telligence, diyness of the skin, falhng out of the liair, and, on the whole,
those symptoms which are included under the name cachexia thyreopriva,
and death follows gradually. Among these symptoms must be mentioned
the peculiar slimy infiltration and exuberance of the connective tissue
called myxcedema. It has been proved that the destructive action of the
removal of the thyroid can be counteracted by the artificial introduction
of extracts of the thyroid gland into the body, and even by feeding with
the substance of the gland. On the other hand, it has been observed on
administering too large quantities of gland substance that threatening
symptoms and disturbances occur in man as well as in animals. From a
physiologico-chemical standpoint the abnormally increased destruction of
body protein, occurring on continuous feeding witli thyroid preparations,
is of the greatest importance.
From this it follows that the glands contain specifically active sub-
stances. It is impossible for the present to state anything about the im-
portance of the bases found by certain investigators, such as S. Frankel,
Drechsel, and Kocher;^ these bodies have not been characterized suf-
ficiently. It seems positively proven that the specifically active sub-
stance is, in greater part, if not entirely, as first show^n b}' Notkix,^ a protein
substance: 'Sotkis^s thyreoproteid, Oswald^s thyreoglobulin. This does not
contradict the views of BAU^L\xx and Roos that the active substance is
iodothyrin, as this is produced as a cleavage product from the iodothyreo-
globulin.
Iodothyrin is considered by Baumaxx, who first showed that the thjToid con-
tained iodine and who with Roos * showed the importance of this substance for the
physiological activity of the gland, as the onh* active substance. Iodoth\Tin was
obtained by Baumaxn by boiling the finely di\'ided gland with dilute sulphuric
' The divergent statements as to the necessity of the thyroid gland can be found in
H. Munk, Virchow's Arch., 150.
^ Frankel, Wien. med. Blatter, 1895 and 1896; Drechsel and Kocher, Centralbl.
f. Physiol., 9, 705.
^ Wien. med. Wochenschr., 1895, and Virchow's Arch., 144, SuppL, 224.
* In regard to this subject, see Baumann and Roos, Zeitschr. f. physiol. Chem., 21
and 22; also Baumann, Mimch. med. Wochenschr., 1896; Baumann and Goldmann,
ibid.; Roos, ibid. An extensive review of the literature on the action of iodothyrin
SUPRAREXAi. CAPSULE. 277
acid as an amorphous, brown mass nearly insoluble in water but readily soluble
in alkali and precipitated again by the addition of acid. The iodothjTin, which
is not a unit body, has a variable content of iodine and is not a protein substance.
Thyreoglobulin was obtained by Oswald from the water}- extract of
the gland by half saturating with ammonium sulphate. It has the proper-
ties of the globulins and with the exception of the iodine content it has
about the same composition as the proteins. The amount of iodine varies:
0.46 per cent in pigs, 0.86 per cent in oxen, and 0.34 per cent in man. In
young animals, whose glands contain no iodine, the thyreoglobulin is
iodine-free. Thyreoglobulin on taking up iodine is converted into iodo-
thyreoglobulin. By introducing iodine salts the iodine content of the
iodothyreoglobulin can be raised in Uving animals and thereby also the
physiological acti^-ity increased (Oswald). The amount of iodine in the
gland is markedly dependent upon the food.
According to Oswald iodothyreoglobulin, as a physiological excitant upon the
nervous system, has a regulating action upon metabolism. The exclusion of this
action, after destruction or extu-pation of the gland, explains, according to Os-
wald, the injurious results produced by these changes upon the gland. Accord-
ing to Blum the th\Toid gland removes from the blood a poisonous body, the
ihyreotoxalhumin, and makes it non-injurious by taking up iodine. Kishi ^ also
believes that the thjToid gland has the power of removing poisons from the blood.
We cannot enter further into this and other related questions.
The Suprarenal Capsule. Besides proteins, substances of the connective
tissue, and salts, there occur in the suprarenal capsule inosite, purine bases,
especially xanthine (Oker-Blom), a protagon-like substance (Orgler),
relatively considerable lecithin and neurine, and glycerophosphoric acid,
which are probably decomposition products of the lecithin. The older
statements on the occurrence of benzoic acid, hippuric acid, and bile-acids
are, on the contrary-, doubtful and are not substantiated by recent inves-
tigations (STADELiL\xx). Older investigators, like Vltlpiax and Arxold,^
have found in the medulla a chromogen which was considered to be con-
nected with the abnormal pigmentation of the skin in Addison's disease.
This chromogen, which is transformed by air, light, alkahes, iodine, and
other bodies into a red pigment, seems, on the contrary-, to be related to
the substance of the gland producing an increase in the blood-pressure.
and the thyroid preparations can be found in Rocs, Zeitschr. f. physiol. Chem., 22, IS.
In regard to their action in protein destruction and metabolism, see F. Voit, Zeitschr.
f. Biologie, 35; Schondorfif, Pfliiger's Arch., 67, and Andersson and Bergman, SkanJ.
Arch. f. Physiol., S; Magnus-Levy, Zeitschr. f. klin. Med., 52.
* Kishi, Virchow's Arch., 176. A summary of the thyroid hterature for the last
years is found in Maly's Jahresber., 24 and 25. See also the works of Blum and Oswald,
cited by Oswald in Biochem. Centralbl., 1, 249.
■ Oker-BIom, Zeitschr. f. physiol. Chem., 2S; Stadelmann, ibid., IS, wliich also
contains the literature on this subject; Orgler, Salkowski's Festschrift, 1904,
278 CHYLE, LYMPH, TRANSUDATES AND EXUDATES.
Adrenalin (siiprarenin, epinephrin). That the watery extract of the
suprarenal capsule has a blood-pressure-raising action was shown by
Oliver and Schafer, Cybulski and Szymonowicz.^ The substance which
is here active was formerly called sphygmogenin and has also other actions
besides bringing about a marked increase in blood-pressure by the strong
contraction of the muscles of the periphery vessels; for instance, it can bring
about glycosuria. This body has been chemically investigated by several
experimenters and has received different names, v. FtJRTH calls it supra-
renin, Abel epinephrin, and Takamine adrenalin. This last name seems
to be the most generally accepted one. The chemical constitution of
adrenalin has not been positively decided upon. Aldrich gives the
formula C9H13NO3 for adrenalin, and this formula has been accepted as
correct by a large number of investigators, such as v. Furth, Jowett,
Pauly, Abderhalden and Bergell, Bertrand, Friedmann, and Stolz,
basing their opinion upon their own researches.^ Abel disputes the correct-
ness of this formula and considers adrenalin as a hydrate of a substance,
C10H13NO3, called epinephrin by him, hence it is epinephrin hydrate,
CioHi3N03 + §H20. The general opinion in regard to the constitution of
adrenalin is that it contains a pyrocatechin complex, three OH groups, of
Avhich one is found in the side-chain, and one CH3NH group. The formula
(HO)2.C6H3.CH(OH).CH2.NH.CH3, given by Pauly, according to the in-
vestigations of Friedmann, can be accepted as correct. Based upon these
facts it has been possible to prepare compounds synthetically whose phy-
siological action was more or less similar to adrenalin, namely, by starting
from pyrocatechin, especially by treating chloracetopyrocatechin with
ammonia, alkylamines, and other basic bodies (Stolz, Meyer, Friedmann,
Dakin).3
Adrenalin is soluble in water, precipitated by ammonia, and thereby
separates as crystals. It gives an emerald-green color with ferric chloride
in acid solution and a carmine-red coloration in alkaline solution. It re-
duces Fehling's solution and an ammoniacal silver solution. Epinephrin
(Abel) is precipitated by several alkaloid reagents and gives color reac-
tions with JNIandelin's alkaloid reagent and with permanganate and sul-
' Oliver and Schafer, Proceed, of Physiol. Soc, London, 1895. Further literature
on the function of the suprarenal capsule may be found in Szymonowicz, Pfliiger's
Arch., 64.
^ 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., 2S; .A.mer. Journ. of Physiol., 1899, and Tlie Johns Hopkins Hospi-
tal Bull., No. 76 (1897), 90 and 91 (1898), 120 and 12S (1901), 131 and 132 (1902);
Rer. d. d. chem. Gesellsch., 36; Abel and Taveau, Journ. of Biol. Chem., 1, and Fried-
mann, Hofmeister's Beitrage. 6 and 8.
* Stolz, Ber. d. d. cliem. Gesellsch., 37; Friedmann, Hofmeister's Beitrage, 6 and
8; Dakin, Proc. Roy. Soc, 1905, Ser. B, Vol. 76.
ADRENALIN. 279
phuric acid. On this point the conditions are not quite clear. According
to Abel the crj'stalline substance (his epineplirin hydrate) precipitated by
ammonia, which corresponds to the adrenaUn of the other investigators,
does not have the alkaloid properties of epinephrin, but is converted by
the action of mineral acids into epinephrin. The epinephrin is probably a
transformation product of adrenalin. Further investigation is necessar}'-
before this can be explained.
The glycosuria first observed by Blum after the injection of the extract
of the suprarenal capsule is due to an action of the adrenalin, and it is
hardly possible that the diastatic enzyme found in the suprarenaJ capsule
by Croftan ^ takes any part in this change.
' Blum, Pfliiger's Arch., 90; Croftan, ibid., 90.
CHAPTER VIII.
THE LIVER.
The liver, which is the largest gland of the body, stands in close rela-
tionship to the blood-forming glands. The importance of this organ for
the physiological 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. It has been proved, at least for the carbo-
hydrates, that an assimilation of the absorbed nutritive substances which
are brought to the liver by the blood of the portal vein takes place in this
organ, and there is no doubt that synthetical processes also occur. The
occurrence of synthetical processes in the liver has been positively proved
by special observations. It is possible that 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
(Pflijger and Kochs, Embden and Glaessner), probably also converted
into conjugated glucuronic acids (Embden i). The hver has also the prop-
erty of removing and retaining heterogeneous bodies from the blood, and
this is not only true of metallic salts, which are often removed by this
organ, but also, as Schiff, Heger, and others, but especially Roger, have
shown, the alkaloids are retained, and are probably also partially decom-
posed in the liver. Toxines are also withheld by the liver, and hence this
organ has a protective action against poisons.^
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
^ Pfliiger and Kochs, Pfliiger's Arch., 20 and 23; Embden and Glaessner, Hof-
meister's Beitrilge, 1; Embden, ibid., 2.
^ Roger, Action du foie sur les poisons (Paris, 1887), whicli 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
Vamossy, Centralbl. f. Physiol., IS, and Rothberger, Wien. klin. Wochenschr., 1905,
Rothborjrer and WintprKoro-, Biochem. Centralbl., 4.
280
PROTEINS OF THE LIVER. 281
which the red blood-corpuscles are destroyed, or at least one of their con-
stituents, the haemoglobin. It is generally admitted that the liver acts
contrari^\ise during foetal life, at that time forming the red blood-cor-
puscles.
There is no doubt that the chemical operations going on in tliis organ
are manifold and must be of the greatest importance for the organism.
Our knowledge on this subject has been essentially advanced by the recent
investigations on the enzymes of the liver, as well as on the autolytic pro-
cesses in tliis organ,! i^^^ nevertheless it must be admitted that our knowl-
edge of the character and extent of these changes is still small. 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 Uver is necessary-.
The reaction of the Uver-cells is alkaUne towards litmus during hfe, but
becomes acid after death, due to a formation of lactic acid, chiefl}- fer-
mentation lactic acid and other organic acids (^IoRiSHnL\, ^Iagxus-Le\'y2).
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 Plosz. He
found in the water}- extract of the liver an albuminous substance w^hich
coagulates at 45° C, also a globulin which coagulates at 75° C, a nu.cleo-
albumin which coagulates at 70° C, and lastly a protein body which is
nearly related to the coagulated albumins and which is insoluble in dilute
acids or alkalies at the ordinarv^ temperature, but dissolves on the applica-
tion of heat, being converted into an albuminate. Halliburton ^ has
found two globuUns in the hver-cells, one of which coagulates at 68-70°
C, and the other at 45-50° C. He also found, besides traces of albumin,
a nucleoproteid which possessed 1.45 per cent phosphorus and a coagula-
tion-point of 60° C. PoHL has obtained an "organ plasma" by extracting
the finely di^ided liver which had pre\"iously been entirely freed from
blood by washing with 8 p. m. XaCl solution, in which he was able to
detect a globulin having a low coagulation temperature. The ver}- varia-
ble phosphorus content (0.28-1.3 per cent) of this globuUn as well as the
insolubility of the precipitates produced by Httle acid in an excess of acid
and in neutral salts seem to indicate that we have here a mixture which
consists chiefly of nucleoproteids and not of globulins. The nearly com-
• See especially the works of Jacoby, Zeitschr. f. physiol. Chem., 30; Conradi, Hof-
meister's Reitrage, 1; Magnus-Le\'y, ihid., 2.
2 Morishima, Arch. f. exp. Path. u. Pharm., 43; Magnus-Le%'y, 1. c.
^ Plosz. Pfl'iger's Arch., 7; Halliburton, Joarn. of Physiol., 13, Suppl. 1892.
282 THE LIVER.
plete digestibility with pepsin-liydrochloric acid does not contradict this
assumption, because, as is known, nucleoproteids may on digestion yield
no residue (see Chapter V). It is also impossible to state anything
positive about the nature of the liver-globulin found by Dastre,i hav-
ing a coagulation temperature of 56°. The proteins extractable from the
liver without modification must be thoroughly investigated.
Besides the above-mentioned proteins which are very soluble, the liver-
cells contain large quantities of difficultly soluble protein bodies (see
Plosz). The liver also contains, as first shown by St. Zaleski and then
substantiated by several other investigators, ferruginous proteins of dif-
ferent kinds. 2 The chief portion of the protein substances in the liver
seems to consist in fact of ferruginous nucleoproteids. On boiling the
liver with water, such a nucleoproteid or perhaps several are spht, and a
solution is obtained containing a nucleic-acid-rich nucleoproteid or a mix-
ture of these which are precipitable by acids. This protein or protein
mixture, which has been called ferratin by Schmiedeberg,^ has been care-
fully studied by Wohlgemuth.^ The quantity of phosphorus 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, oxy-
aminosuberic acid, and oxydiaminosebacic acid (see Chapter II).
The yellow or brown pigment of the liver has been little studied. Dastre
and Floresco ^ differentiate in vertebrates and certain invertebrates between a
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 sucking animals, as also after food rich
in fat) as rather large fat-drops. The occurrence of a fatty infiltration, 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 posioning with phosphorus,
phlorhizin, and certain other bodies (Leo, Lebedeff, Rosenfeld, and
* Pohl, Hofmeister's Beitrage, 7; Dastre, Compt. rend. see. biolog., 58.
2 St. Zaleski, Zeitschr. f. physiol. Chem., 10, 486; Weltering, ibid., 21; Spitzer,
Pfliiger's Arch., 67.
^ Arch. f. exp. Path. u. Pharm., 33; see also Vay, Zeitschr. f. physiol. Chem., 20.
* Wohlgemuth, Zeitschr. f. physiol. Chem., 37, 42, and 44, and Ber. d. d. chem.
Gesellsch., 37. See on liver nucleoproteids also Salkowski, Berl. klin. Wochenschr.,
1895;' Hammarsten, Zeitschr. f. physiol. Chem., 19; Blumenthal, Zeitschr. f. klin. Med.,
84.
•Arch, de Physiol. (5), 10.
JECORIX. 283
others ^). The fatty infiltration occurring in poisoning and which is accom-
panied ^\ith 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 corre-
sponcUngly, while the quantity of the other solids remains little changed.
Changes of such a kind may occur, so that, because of the opposition
(Rosexfeld) existing between glycogen and fat, a Uver rich in fat is habit-
ually poor in glycogen. The reverse occurs after feeding -^ith carbohy-
drr.te-rich food, namely, the liver is rich in glycogen and poor in fat.
The composition of the liver-fat not onl}^ seems to var}- in different
animals, but is variable ^^ith changing conditions. Thus Xoel-Paton
found that the hver-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, wliile Rosexfeld ~ has observed the
opposite condition on feeding dogs with mutton-fat.
Lecithin is a normal constituent of the hver, and amounts to about 23.5
p. m. according to Xoel-Patox.^ In starvation the lecithin, according to
Xoel-Patox, forms the greatest part of the ethereal extract, while with
food rich in fat, on the contrary-, it forms the smallest part. Cholesterin
occurs only in small quantities. The ethereal extract also contains a
protagon-hke body, jecorin.
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 it solidifies after boiling with alkalies to a gelatinous mass.
^Iaxasse has detected dextrose as osazone in the carbohydrate complex of jecorin.
It may lead to errors in the investigations of organs or tissues, for it can easily
be mistaken for lecithin on account of its solubilities and because it contains
phosphorus.
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
sulphur, even as much as 2.75 per cent, and also the relation of P:X in lecithin is
1:1, while in jecorin it is quite different, 1:2 to 1:6.
The variable composition and divergent properties of the jecorin isolated and
analyzed by various investigators * make it very possible that jecorin is a mixture
' .Voel-Paton, Journ. of Physiol., 19; Leo, Zeitschr. f. physiol. Chem., 9; Lebedeff,
Pfliiger's Arch., 31; Athanasiu, Pfluger's Arch., 74; Taylor, Journ. of Exp. Med., 4;
Kraus u. Sommer, Hofmeister's Beitrage, 2; Rosenfeld, Zeitschr. f. klin. Med., 36.
See also Rosenfeld, Ergebnisse der Physiologic, 1, Abt. 1, and Berl. klin. Wochenschr.,
1904; Schwalbe, Centralbl. f. Phy.siol., 18, p. 319.
^ Cited by Lummert, Pfhiger's Arch., 71. In regard to the liver-fat of children, see
Thiemich, Zeitschr. f. physiol. Chem., 26.
^ 1. c. See also Hefter, Arch. f. exp. Path. u. Pharm., 28.
^ Drechsel, Ber. d. sachs. Gesellsch. d. Wissensch., 18S6, p. 44, and Zeitschr. f. Biol-
ogie, 33; Baldi, Arch. f. (Anat. u.) Ph>siol., 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
284 THE LIVER.
of several suHstances, among which perhaps occurs a sulphurized and phosphor-
ized substance (Sifgfried and Mark).
Among the extractive substances besides glycogen, which will be treated
later, rather large quantities of the xanthine bodies occur. Kossel ^ found
in 1000 parts of the dried substance 1.97 p. m. gimnine, 1.34 p. m. hypo-
xanthine, and 1.21 p. m. xanthine. Adenine is also contained in the liver.
In addition there have been 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 tyrosine have been detected. The
occurrence of bile-coloring matters in the liver-cell under normal conditions
is doubtful; but in retention of the bile the cells may absorb the coloring-
matter and become colored thereby.
A large number of enzymes are found in the liver, among which (besides
the cataloJies, the oxidases, the glycolytic enzyme, 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) we must mention the so-called Zijmse and the proteo-
lytic enzyme. The liver has the power of splitting various esters, an action
which has been recently studied by Dakin,2 and this action is due to an
enzyme which is considered as a lipase. The nature of this lipase, whose
cleavage action upon the amyl ester of salicylic acid was first observed by
Chanoz and Doyen, has been closely studied by Magnus,^ and it has been
shown that this action is the result of the combined action of two sub-
stances. The lipase solution becomes inactive by dialysis, a thermostable
substance soluble in absolute alcohol passing into the diffusate, and this
body acts as a co-enzyme, making the solution which had been made inac-
tive by dialysis active again.
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 intra vitally increased autolysis. In these cases a softening
of the organ takes place, and proteoses, mon- and diamino-acids, and other
bodies are produced, which may also in part be found in the urine, and
although they may not all be derived from the liver (Neuberg and Rich-
ter), are at least in part derived from this organ.
Wakeman* has found in phosphorus poisoning that not only is the
quantity of nitrogen markedly diminished in the liver (of dogs), but also
* Zeitschr. f. physiol. Chem., 8.
2 Journ. of Physiol., 30 and 32.
' Chanoz and Doyen, Journ. de physiol. et de path, g^n^ral, 2; Magnus, Zeitschr. f.
physiol. Chem.. 42.
* Neuberg and Richter, Deutsch. Med. Wochenschr., 1904; Wakeman, Zeitschr. f.
physiol. Chem., 44.
MINERAL CONSTITUENTS OF THE LIVER. 285
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 elimi-
nated under these conditions. The increased consumption of glycogen under
the above-mentioned pathological conditions may also be considered as an
increased autolysis.
Besides the above-mentioned organic constituents in the liver we must
mention the glucothionic acid found by Mandel and Levene, whose rela-
tionship to the carbohydrate metabohsm in this organ, as well as to the
nitrogenous carbohydrate found by Seegen and Neimann ^ in the liver^
requires further investigation.
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 has found 0.01-0.355 p. m. iron in the blood-
free Uver of young cats and dogs. This was calculated on the liver sub-
stance 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. 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,2 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 Novi^ 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.*
A destruction of blood-pigments, with a splitting off of compounds rich
in iron, seems to take place in the hver in the formation of the bile-pig-
ments. Even in invertebrates, which have no haemoglobin, the so-called
' Mandel and Levene, Zeitsphr. f. physiol. Chem., 45; Seegen, Ceutralbl. f. Physiol.,
12 and 13, with Neimann, Wiener Sitzungsber. Math. Klasse, 112.
^ 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.
* See Centralbl. d. Physiol., 16, 393.
* See Lapicque, Compt. rend., 124, and Schurig, Arch. f. exp. Path. u. Pharm., 41.
286 THE LIVER.
liver is rich in iron, from which Dastre and Floresco ^ concKicle that the
quantity of iron in the liver of invertebrates is entirel}' independent of the
decomposition of the blood-pigment, and in vertebrates it is in part so.
According to these authors the Uver has, on account of the quantity of
iron, a specially important oxidizing function, which they call the " fond ion
martiale" of the liver.
The richness in iron of the Uver of new-born animals is of special inter-
est— a condition which was shown by the analyses of St. Zaleski, but was
especially studied by Kruger and ]\Ieyer. In oxen and cows they found
0.246-0.276 p. m. iron (calculated on the drj^ substance), and in the cow-
fcetus 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 sinks in
the first four weeks of life, when it reaches about the same amount as in the
adult. Lapicque - has 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 abundance of iron into the world to be used up, -v\dthin a
certain time, for a purjDOse not well known." A part of the iron exists as
phosphate, but the greater part is in combination in the ferruginous pro-
tein bodies (St. Zaleski).
The quantity of calcium oxide in the fresh, moist liver of the horse, ox,
and pig, according to Toyonaga, amounts to 0.148-0.193 p. m., or 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 Hvers of the
horse, ox, and pig respectively. Kjiuger^ has 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 (Henze).* Foreign
metals, such as lead, zinc, and others (also iron), are easily taken up and
combined by the Hver (Slowtzoff, v. Zeynek, and others S).
v. Bibra 6 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.
' Arcfi. de Physiol. (.')), 10.
-St. Zaleski, 1. c; Kruger and collaborators, Zeitschr. f. Biologic, 2"; Lapicque,
Maly's Jahresber., 20.
^ Zeitschi. f. Biologic, 31; Toyonaga, Bull, of the College of Agriculture, Tokio, 6.
* Zeitschr. f. physiol. Chem., 33.
* Slowtzoff, Hofmeister's Beitriigc, 1; v. Zeynek, see Ccntralbl. f. Physiol., 1,5.
' See V. Oorup-Besanez, Lelirljucli d. physiol. Chem., 1. Aufl., p. 711.
GLYCOGEN FORMATIOX. 287
protein, gelatine-forming and insoluble substances, and 61 p. m. extractive
substances.
The quantitative composition of the liver may show great variation,
depending upon the kind and amount of the food supplied. The amount
of carbohydrate (glycogen) and fat may var}' considerably, which is due to
the fact that the liver is a storage-organ for these bodies, especiall)'- for the
glycogen.
Based upon special experiments, Seitz ^ claims that the hver is a store-
house also for protein. In experiments on hens and ducks which had pre-
viously been starved, he fotmd that the Hver took up abimdant protein on
feeding meat and that its weight as compared with the weight after star-
vation was doubled or quadrupled. As it is characteristic of storage or
reserve bodies that their amoimt iu the storage-organs on feeding \^ith such
bodies strongly increases in percentage, it is remarkable in Seitz's feeding
experiments that the percentage of protein in the Uver did not increase but
rather diminished sHghtly. In this case we did not have a higher percen-
tage of protein, but an increase in the weight of the total cell mass of the
organ, probably brought about by increased work of the U^-er due to the
protein feeding. It is also difficult to decide as to how far in these experi-
ments we were dealing '^\ith an increase in the number or the size of the
hver-celLs or with a deposition of reserve protein in the same sense as of
glycogen or excessive fat.
There is an unanimous beUef that the hver is an especially important
storage-organ for glycogen.
Glycogen and its Formation.
Glycogen was first discovered by Beexaed. It is a carbohydrate closely
related to the starches or dextrins, with the general formula (CeHioOslj..
Its molecular weight is unknown, but seems to be verc' large (Gatix-Gru-
ZEWSK.^ and v. Ivxaffl-Lexz^). The largest quantities are found in the
liver, and smaller quantities in the muscles (Berxard. Xasse). It is found
in ver\- small quantities in nearly all tissues of the animal body. Its occur-
rence in lymphoid cells, blood, and pus has been mentioned in a pre\"ious
chapter, and it seems to be a regiilar constituent of all cells capable of
development. Glycogen was first shown to exist in embrj-onic tissues by
Berx.ard and Kuhxe. and it seems on the whole to l^e a constituent of
tissues in which a rapid cell formation and cell development is taking
place. It is also present in rapidly forming pathological swelUngs (Hoppe-
Seyler). Certain animals, as certain mussels (Bizio'). tienia and ascarides
iPfluger's Arch.. 111.
^ Gatia-Gruzewska, Pfliiger's Arch., 103; v. Ivnaffi-Lenz, Zeitschr. f. physiol. Chein.,
46.
2S8 THE LIVER.
(Weinland ^), 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 nearly completely
after a short time, but more rapidly in small than in large animals, and it
disappears earlier from the liver than from the muscles. After partaking
of food, especially such as is rich in carbohydrates, the liver becomes rich
again in glycogen, the greatest increment occurring 14 to 16 hours after
eating (Kxjlz). 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 on glycogen Schondorff and Gatin-Gruzewska
found still higher results, even more than 180 p. m. Ordinarily it is con-
siderably less, namely, 12-30 to 40 p. m. According to Cremer the quan-
tity of glycogen in plants (yeast-cells) is, as in animals, dependent upon the
food. According to him 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 de-
pendent upon rest and activity, because during rest, as in hibernation, it
increases, and during work it diminishes. KtJLZ has shown that by hard
work the quantity of glycogen in the liver (of dogs) is reduced to a minimum
in a few hours. The muscle-glycogen does not diminish to the same extent
as the liver-glycogen. Kulz, Zuntz and Vogelius, Frentzel, and others
have been able to render rabbits and frogs glycogen-free by suitable strych-
nine poisoning. The same result is produced by starvation followed by
hard work.
Glycogen forms an amorphous, vvhite, 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 pelUcle over the surface that disappears again on
cooling. It is undecided whether we have here 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. A solution of glycogen, es-
pecially on the addition of NaCl, is colored wine-red by iodine. It may
' Zeitschr. f. Biologic, 41. The extensive literature on glycogen may be found in
E. Pfliiger, Glykogen, 2. Aufl., Bonn, 1905; and in Cremer, " Physiol, des Glykogens," in
Ergebnisse der Physiologic, 1, Abt. 1. In the following pages we shall refer to these
works.
PREPARATION OF GLYCOGEN. 289
hold cupric hydrate 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 necessarjO 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. Tan-
nic acid also precipitates glycogen. It gives a white granular precipitate of
benzoyl glycogen with benzoyl chloride and caustic soda. Glycogen is
completely precipitated by saturating its solution at ordinary tempera-
tures with magnesium or ammonium sulphate. It is not precipitated by
sodium chloride or by half saturation with ammonium sulphate (Nasse, Neu-
MEiSTER, Halliburton, Young i). 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 of BrDcke's reagent
(see below) (PFLtJCER). On boiling with stronger caustic potash (even of
36 per cent) it is not injured (Pflijger). 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. Ac-
cording to Tebb^ 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 glycogen from
various animals and different organs is the same in this regard has not been
sufficiently investigated. Nor has it been decided whether all the glycogen
in the liver occurs as such or whether it is in part combined with protein
(Pflijger-Nerking) . The recent investigations of Loeschcke ^ have
shown that we have no positive reasons for this assumption.
The preparation of pure glycogen (most easily from the liver) is generalh^
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 wash-
ing first ^vith 60 per cent and then ^dth 95 per cent alcohol, then treating
vnih ether and drvdng 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
' Young, Journ. of Physiol., 22, citing the other investigators.
^ Journ. of Physiol., 22.
3 Pfliiger's Arch., 102.
290 THE LIVER.
for two hours with strong caustic potash (30 per cent) on the water-bath.
As the glycogen changes in this purification, according to Brucke, it is
better, for quantitative determinations of glycogen, to precipitate it directly
from the alkahne solution by alcohol (Pfluger ^).
The quantitative estimation is best performed according to PflIjger's
method, which is based upon the following: 100 grams of the finely divided
organ and 100 c.c. of 60 per cent caustic-potash solution are heated on the
water-bath for two hours. After evaporating the water to 400 c.c. it is
filtered through glass wool and the glycogen precipitated from 100 c.c. of
the filtrate by 100 c.c. of 96 per cent alcohol. The glycogen is washed on
the filter first with dilute alkali and alcohol and then with alcohol alone.
It is then dissolved in water, exactly neutralized, treated with 25 c.c.
hydrochloric acid (1.19 sp. gr.) and water added to 500 c.c, when the
amount of HCl will be 2.2 per cent. On heating for three hours the glycogen
will have been converted into dextrose, whose quantity can be determined
according to Allihn-Pfluger's method by reduction of an alkaline copper
solution and weighing the cuprous oxide. As a control the weighed cuprous
oxide is dissolved in nitric acid and the copper titrated according to Vol-
hard's method. In regard to the detailed steps, which must be exactly
observed, compare Pfluger's original work. Other methods of esti-
mating glycogen, such as those of BRiJCKE-KiJLZ, Pavy, and Austin, are
descrilDed in PFLtJGER's Archiv, 96. See also the new method as suggested
by Salkowski and the short quantitative analysis of glycogen by Pflijger.^
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 ^ that the varieties of sugars and their
anhydrides, dextrins and starches, have the property of increasing the quan-
tity of glycogen in the body. The action of inulin seems to be somewhat
uncertain."* The statements are questioned in regard to the action of the
pentoses. Cremer found in rabbits and hens that various pentoses, such
as rhamnose, xylose, and arabinose, have a positive influence on the gly-
cogen formation, and Salkowski obtained the same result on feeding
Z-arabinose. Frentzel fdund, on the contrar}^, no glycogen formation on
feeding xylose to a rabbit which had previously been made glycogen-free
by strychnine poisoning, and Neuberg and Wohlgemuth ^ obtained simi-
lar negative results on feeding rabbits with d- and r-aral)inose.
The hexoses, and the carbohydrates derived therefrom, do not all possess
the ability of forming or accumulating glycogen to the same exteijt. Thus
' See also the method suggested by Gautier, Compt. rend., 129.
2 Salkowski, Zeitschr. f. physiol. Chem., 36; Pfluger, Pfliiger's Arch., 103.
^ In reference to the literature on this subject, see E. Kiilz, Pfliiger's Arch., 24, an 1
Ludwig-Festschrift, 1891; also the cited works of Pfliiger and Cremer, foot-note 1, .
288.
* See Miura, Zeitschr. f. Biologic, 32, and Nakaseko, Amer. Journ. of Physiol., 4.
* Salkowski, Zeitschr. f. physiol. Chem., 32; Neuberg and Wohlgemuth, ibid., 35.
See also Pfliiger, 1. c, and Cremer, 1. c.
GLYCOGEN FORMATION. 231
C VoiT 1 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, le\'ulose, cane-sugar, or maltose. The following substances
when introduced into the body also increase the quantity of glycogen ia
the liver: glycerine, gelatine, arbutin, and like^\ise, according to the investi-
gations of KtJLZ, erythrite, quercite, dulcite, mannite, inosite, ethylene and
projyylene glycol, glucuronic anhydride, saccharic acid, mucic acid, sodium
tartrate, saccharine, isosaccharine, and urea. Ammonium carbonate, glycocoll^
and asparagine may similarly, according to Rohmann, cause an increase in
the amount of glycogen in the liver. According to Xebelthau other
ammonium salts and some of the amides, as well as certain narcotics, hyp-
notics, and antipyretics, produce an increase in the glycogen of the Uver,
This action of the antipyretics (especially antipyrine) had been shown by
Lepine and Porteret.^
Pfluger has conclusively shown that we have no positive proofs as to
the action of these various bodies as glycogen-formers. That glycerine 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 \<\\\ be mentioned in connection with the experiments on
the relationship of glycerine to the sugar formation.
The fats, according to Bouchard and Desgrez, increase the glycogen
content of the muscles but not of the liver, and, according to Coua'reur,^
the glycogen is increased at the expense of the fat in the silkworm larva as
it changes into a chrs'saUs. In general it is believed that fat does not in-
crease the amount of glycogen in the liver or in the animal bod}-, although
a carbohydrate formation from glycerine, but not a glycogen formation, is
probable. Pfluger explains this by the fact that the extent of fat metab-
olism is not dependent upon the quantity of fat supplied, but upon the
amount of fat required conditioned by work. If more fat is supplied, then
it is not destroN^ed, but is stored up. Even when 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.
The views in regard to the influence of the proteins are somewhat con-
tradictor}-. From several investigations the conclusion has been drawn
that the proteins cause an increase in the glycogen of the liver. Amongst
these investigations must be included certain feeding experiments with
boiled beef (Xauxyx) or blood-fibrin (v. ^Ierixg), and especially the very
careful experiments made by E. Kulz on hens, with pure protems. .'^uch as
' Zeitschr. f. Biologie, 2S.
- Rohmann, Pfliiger's Arch., 39; Nebelthau, Zeitschr. f. Biologie, 28; Lepine ami Por-
teretjCompt. rend., 107.
^ Bouchard et Desgrez, Compt. rend., 130; Couvreur, Compt. rend, de soc. biol., 47.
292 THE LIVER.
casein, seralbumin, and ovalbumin. The value of these experiments is
disputed by Pflijger, and as a direct proof against the formation of gly-
cogen from protein he refers to Schondorff's investigations when feeding
carbohydrate-free protein (casein) to frogs without finding the least in-
crease in the total glycogen. Later Blumenthal and Wohlgemuth
arrived at similar results. They found no glycogen accumulation in frogs
after feeding with casein or gelatine, but did find it after feeding with oval-
bumin, which contains a carbohydrate group. On the contrary, Bendix
was able to show an increase in the glycogen in dogs by feeding casein and
gelatine, as well as ovalbumin, and in fact a greater increase by casein than
by ovalbumin. Stookey ^ arrived at similar results in hens as he found
a glycogen formation after feeding casein, while he obtained no posi-
tive results after feeding glucoproteids. It seems as if the conditions in
cold-blooded animals were different from those in warm-blooded ones.
According to Pflijger, the experiments of Bendix are not conclusive, and
he doubts the formation of glycogen from protein. He claims it is only
formed from carbohydrates or from the carbohydrate complex of the
glucoproteids.
]Many investigators are still of the opinion that an increase in the gly-
cogen of the liver as well as of other organs can be brought about by feeding
animals with carbohydrate-free proteins.
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 formation
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 transfor-
mation of glycogen in the liver, while others perhaps are more combustible
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-formcrs 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 itself 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
lias given rise to the opinion that the glycogen in the liver is produced from
' Schondorff, Pfliiger's Arch., 82 and 88; Blumenthal and Wohlgemuth, Berl. klin,
Wochenschr., 1901 ; Bendix, Zeitschr. f. physiol. Chem., 32 and 34; Stookey, Amer.
Journ. of Physiol., 9.
GLYCOGEN FORMATION. 293
sugar by a synthesis in which water separates with the formation of an
anhydride (Luchsixger and others). This theor}^ {anhydride theory) has
found opponents because it neither explains the formation of glj^cogen
from such bodies as proteins, carbohydrates, gelatine, 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. It used to be the opinion of many investigators that all gly-
cogen is formed from protein, and that this splits into two parts, one con-
taining 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 i).
In opposition to this theory C. and E. Voit and their pupils have shown
that the carbohydrates are "true" glycogen-formers. After partaking of
large quantities of carbohydrates the amount of glycogen stored up in the
body is sometimes so great that it cannot be covered by the proteids de-
composed during the same time, and in these cases a glycogen formation
from the carbohydrates must be admitted. According to Cremer only the
fermentable sugars of the six carbon series or their di- and polysaccharides
are true glycogen-formers. For the present, only dextrose, levulose, galac-
tose (Weinland^)^ and perhaps also rf-mannose (Cremer) are designated
as true glycogen-formers. Other monosaccharides may indeed, according
to Cremer, influence the formation of glycogen, but they are not converted
into glycogen and hence are called only pseudoglycogen-formers.
The poly- and disaccharides may, after a cleavage into the correspond-
ing 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
le\'ulose, serve as glycogen-formers after subcutaneous injection, but re-
appear almost entirely in the urine (Dastre, Fr. Voit). ^laltose, 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 subcutaneous injection, be used in the forma-
tion of glycogen (Fr. Voit 3).
After Pavy ^ showed the glucoproteid nature of ovalbumin and, as dis-
' In regard to these two theoiies, see especially Wolffberg, Zeitschr. f. Biologie, KJ.
2 E. Voit, Zeitschr. f. Biologie, 25, 543, and C. Voit, ibid., 28. See also Kausch
and Socin, Arch. f. exp. Path. u. Pharm., 31; Weinland, Zeitschr. f. Biolog'e, -10 and
38; Cremer, ibid., 42, and Ergebnisse der Physiol., 1.
' Dastre, Arch, de Physiol. (5), 3, 1891; Dastre and Bourquelot, Compt. rend., 98;
Fritz Voit, Verhandl. d. Gesellsch. f. Morph. u. Physiol, in Miinchen, 1896, and Deutsch.
Arch, f klin. Med., 58.
*The Physiology of the Carbohydrates, London, 1891.
294 THE LIVER.
cusssd later, that glucosamine could be split off from ovalbumin as well as
from certain other protein substances (see Chapter II), the question arose
whether the amino-sugar could serve in the formation of glycogen. The
investigations carried out in this direction by Fabian, Fraxkel and Offer,
Cathcart and Bial,^ have shown that the glucosamine introduced into the
organism is in part eliminated unchanged in the urine and has no glycogen-
forming action. No definite conclusions can be drawn from this on the
beha\-ior of the carbohydrate groups wliich exist not as free groups but
combined wth the protein molecules.
Whether or not, or to what extent, the glucoproteids 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 ver}' meagre.
From the weight of the various organs and the relationship of the weight
of the organs to the total weight of the body, as well as from the qualitative
and quantitative composition of the various organs as far as known for the
present, we can calculate the carbohydrates of the body (excluding the gly-
cogen), although the results ma}- not be exact, but no doubt are too high
or at least are not too low. These calculations of Hammarsten for man
arid dogs have shown that in the nucleoproteids, glucoproteids, and other
substances which are not sugar nor glycogen, but for the sake of brevity
are called glucosides, the maximum carbohydrate supply of the body is 5
grams per 1 kilo of body-weight.
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 pseudoglycogen-formers or are they direct glycogen-
formers which can serve as material for the formation of glycogen or sugar?
This question stands in close relationsliip to the sugar formation and sugar
elimination in the various forms of glycosuria and will be discussed best
below in connection udth the question of diabetes.
Like the carbohydrates in general, glycogen has without any doubt a
great importance in the formation of heat and development of energ}' in
the animal body. The possibility of the formation of fat from glycogen
cannot be denied.^ Glycogen is generally considered as reserve food
accumulated in the liver and formed in the liver-cells. 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 yet be answered with posi-
' Fabian, Zeitschr. f. physiol. Chem., 2"; Frankel and Offer, Centralbl. f. Physiol.,
13; Cathcart, Zeitschr. f. physiol. Chem., 39; Bial, Berl. klin. Woclienschr., 1905.
^ See especially Noel-Paton, Journ. of Physiol., 19.
VITAL SUGAR FORMATION. 295
tiveness, and the investigations on this subject l)y different experimenters
have given contradictor}^ results. The experiments of KiJLz/ in which he
studied the glycogen formation by passing blood containing cane-sugar
through the muscle, have led to no conclusive results. Still the formation
of glycogen from sugar in the muscles is probable. There is no doubt that
glycogen is formed in the muscles during embryonic life.
If it is 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 carriers, it is formed
on the spot from the sugar.^ The glycogen formation seems to be a general
function Cf the cells. In adults, the liver, which is very rich in cells, has
the property, on account of its anatomical position, of transforming large
quantities of sugar into glycogen.
The question now arises whether there is any foundation for the state-
ment that the liver-glycogen is transformed into sugar.
As first shown by Bernard and redemonstrated by many investigators,
the glycogen in a dead hver is gradually changed into sugar, and this sugar
formation is caused, as Bernard supposed and Artiius and Huber, Pavy,
and recently also Pick and Bial,^ proved, by a diastiatic enzyme which,
according to Rohmann and Borchardt,^ is identical with a diastatic
enzyme of the blood.
This post-mortem sugar formation led Bernard to the assumption of
the formation of sugar from glycogen in the liver during life. Bernard
suggested the following arguments for this theory : The liver always con-
tains 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. The correctness of either or both of these statements has
been disputed by many investigators. Pavy, Ritter, Schiff, Eulen-
BERG, Lussana, Abeles, and others deny the occurrence of sugar in the
liver during life, and the greater amount of dextrose in the blood from the
hepatic vein is likewise disputed by them and certain other investigators.^
' See Minkowski and Laves, Arch. f. exp. Patli. ii. Pliarm., 23; Kiilz, Zeitsclir. f.
Biologic, 27.
^ See Dastre, Compt. rend, de Soc. bid., 47, 280, and Kaufmann, ibid., 316.
^ Arthus and Huber, Arch, de Physiol. (5), 4, 659; Pavy, Journal of Physiol., 22;
Pick, Hofmeister's Beitr., 3; Bial, Arch. f. (Anat. u.) Phy.siol., 1901.
^ Rohmann, Verh. d. Ges. deutsch. Naturf. u. Arzte, Breslau, 1903; Borchardt,
Pflfiger's Arch., 100.
^ In regard to the literature on sugar formation in the liver see Bernard, Legons sur
le diab^te, Paris, 1877; Seegen, Die Zuckerbildtmg ira Tierkorper, 2. Aufl., Berlin,
1900; M. Bial, Pfliiger's Arch., 55, 434.
296 THE LIVER.
It can be said that at present there are two contradictory views on the
destruction of the glycogen in the Hving organism: Pavy's view, that the
glycogen is directly used without being previously transformed into sugar,
and Bernard's view, which is accepted by most investigators, that the
glycogen is first transformed into sugar by the aid of diastatic enzymes.
According to certain experimenters (Dastre, Noel-Paton, E. Cavazzani ^),
who also admit a destruction of the glycogen with the formation of sugar,
the change is not brought about by an enzyme, but by a special protoplasmic
activity.
The doctrine as to the physiological formation of sugar in the liver has
obtained an energetic advocate in Seegen. He maintains, after numerous
experiments, that the liver regularly contains considerable amounts of
sugar. He has observed an increase of 3 per cent in the quantity of dex-
trose in the liver of a dog kept alive by passing arterial blood through the
organ, and lastly he has also found in a very great number of experiments
on dogs that the blood from the hepatic vein always contains more — even
double as much— sugar than the blood from the portal vein. Mosse and
ZuNTZ^ have recently made objections as to the correctness of this last
statement, and it follows from the various researches on this question that
when disturbing influences are prevented, the blood from the hepatic vein
is only very little richer in sugar than the blood from the portal vein.
Although Seegen energetically espouses the doctrine of Bernard as to
the vital sugar formation in the liver, still he deviates essentially from
Bernard in that he claims the sugar is not derived from the glycogen.
According to Seegen, the sugar is formed from protein and fat. His older
idea, that this protein was peptone, he has discarded. Of importance for
the study of the sugar formation in the liver is, on the contrary, the fact
that Seegen has found a substance in the liver, besides glycogen, which
yields dextrose on heating with dilute acids. He, in connection with
Neimann, has isolated this substance in the form of a nitrogenous carbo-
hydrate. O. Simon ^ has also recently isolated from the liver a proteose-
like substance which reduces directly and yields a fermentable sugar on
boiling with acids, and this sugar gives an osazone melting at 190°.
Seegen claims to have shown a formation of sugar from fat by a direct
experiment with surviving liver tissue. Certain investigations of Weiss
seem to substantiate this view, while other experiments of Montuori, Abder-
halden, and Rona and Hesse contradict this assumption. Hildesheim and
* In regard to the literature see Pick, Hofmeister's Beitrage, 3.
* Seegen, Die Zuckerbildung, etc., and Centralbl. f. Physiol., 10, 497 and 822;
Zuntz, ibid., 561; Mosse, Pfluger's Arch., 03; Bing, Skand. Arch. f. Physiol., 9.
^ Seegen, Arch. f. (Anat. u.) Physiol., 1903; Seegen and Neimann, Wien. Sitzungs-
ber., 112 (1903); Simon, Arch. f. exp. Path. u. Pharm., 49. (See glucothionic acid,
page 285.)
GLYCOSURIAS. 297
Leathes^ have made experiments with hver pulp which indicate tiie
reverse, i.e., the formation of fat from gl^'cogen.
The circumstance that the blood-sugar rapidly sinks to |-^ of its orig-
inal quantity, or even disappears when the Uver is cut out of the circulation,
speaks for a \dtal formation of sugar in the hver (Seegex, Bock and
HoFFMAXx; K\UF.\L\xx; Taxgl and Harley; Pavy). In geese whose
hvers were removed from the circulation, AIixkowski 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 on drawing is not increased (Schexck^). We shall also learn shortly
of certain poisons and operative changes which ma}' cause an abundant
ehmination of sugar, but only when the liver contains gl3^cogen. If we re-
call the fact shown by Rohmann and Bial ^ that the lymph as well as the
blood contains a diastatic enz^^me, then several reasons speak for' the view
of Berxard that the post-mortem formation of sugar from the glycogen
in the liver is a continuation of the vital process.
The relationship of the sugar eliminated in the urine under certain
conditions, such as in diabetes melUtus, certain intoxications, lesions of
the nervous system, etc., to the glycogen of the liver is also an important
question.
It does not enter into the plan and scope of this book to discuss in
detail the various x-iews in regard to glycosuria and diabetes. The appear-
ance of dextrose in the urine is a S3'mptom which may have essentially
different causes, depending upon different circumstances. Only a few of
the most important points will be mentioned.
The blood contains always about an average of 1.5 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. The
kidneys have the property to a certain extent of preventing the passage of
blood-sugar into the urine; and it follows from this that an elimination
of sugar in the urine may be caused partly b}- 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. AIerixg and Minkow^ski, to be the
case in phlorhizin diabetes, v. ]Merixg has found that a strong glycosuria
appears in man and animals on the administration of the glucoside phlor-
* Weiss, Zeitschr. f. physiol. Chem., 24; Montuori, Maly's Jahresb., 26; Abder-
halden and Rona, Zeitschr. f. physiol. Chem., 41; Hesse, Zeitschr. f. exp. Path. u.
Therap., 1; Hildesheim and Leathes, Journ. of Physiol., 31.
^ Seegen, Bock, and Hoffmann, see Seegen, 1. c; Kaufmann, Arch, de Physiol. (5),
8; Tangl and Harley, Pfliiger's Arch., 61; Pavy, Journ. of Physiol., 29; Minkowski,
Arch. f. exp. Path. u. Pharm., 21; Schenck, Pfliiger's Arch., 57.
' See foot-note 4, p. 295.
298 THE LIVER,
hizin. The sugar eliminated is not derived from the gkicoside alone. It
is formed in the animal body, and in fact, at least on prolonged starvation,
from the protein substances of the body. The quantity of sugar in the
blood is not increased, but rather diminished, in phlorhizin diabetes (Min-
kowski), but this is disputed by Pavy. Tliis last investigator found,
although only to a slight degree, that the sugar in the blood was increased,
but he holds the same \new that v. Mering does, that phlorhizin diabetes
is a kidney diabetes. That after extirpation of the kidney in phlorhizin
diabetics no rise in the blood-sugar is observed, and that after the injection
of phlorhizin in the renal arteiy of one side the urine secreted by this kidney
contains sugar sooner and more abundantly than the urine from the other
kidney (Zuntz), speaks in favor of this view. The experiments especially
performed by Pavy, Brodie, and Siau ^ upon blood containing phlorhizin
and surviving kidneys also indicate the same, namely, that the phlorhizin acts
upon the kidneys. While v. jMering believes in an increased permeabihty
of the kidneys for sugar, produced by the phlorhizin, Pavy is, on the con-
trary', 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
proteid with loosely combined carbohydrate groups.
With the exception of phlorhizin diabetes, which is dependent, accord-
ing to the ordinaiy views, upon a change or special processes in the kidneys,
and in which no essential rise in the blood-sugar occurs, all other forms of
glycosuria or diabetes, as far as known at present, depend on a hypergluccemia.
A hyperglucsemia 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 hmit. If too much sugar is introduced into the
intestinal tract at one time, so that the so-called assimilation Hmit (see
Chapter IX, on absorption) is overreached, then the excess of absorbed
sugar passes into the urine. This form of glycosuria is called alimentary
glycosuria,^ and it is caused by the passage of more sugar into the blood
than the liver and other organs can destroy.
* 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. Biologie, 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. Biologie,
36; 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.) Physiok, 1895; Stiles and Lusk, Amer. Journ. of Physiol.,
10; Cremer, Ergebnisse der Physiol., 1, Abt. 1, and the monographs upon diabetes.
^ In regard to alimentary glycosuria see Moritz, Arch. f. klin. Med., 46, which also
contains the older literature; B. Rosenberg, Ueber das Vorkommen der alimentaren
Glykosurie, etc. (Inaug.-Dissert. Berlin, 1897); van Oondt, Miinch. med. Wochen-
schr., 1898; v. Noorden, Die Zuckerkrankheit, 3. Aufl., 1901.
GLYCOSURIAS. 299
As the liver cannot transform into glycogen all the sugar which comes
to it in ahmentan,- glycosuria, it is possible that a glycosuria may be pro-
duced also under pathological conditions, even by a moderate amount of
carbohydrate (100 grams dextrose), which a healthy person could overcome.
Tliis is the case, among others, in various affections of the cerebral system
and in certain chronic poisonings. Certain observers include the lighter
forms of diabetes in this class of glycosiiria.
We differentiate between Ught and severe forms of diabetes. In the
first the urine contains sugar only when carbohydrates are taken as food,
wliile in the other case the urine contains sugar even -uith food entirely
free from carbohydrates. According to the views of several in^•estigators,
in light forms of diabetes the hver is incapable of transforming into
gh cogen all the carbohydrates introduced, or to utilize this glycogen in a
normal way, and the acti\ity of the Hver-cells is also reduced or changed in
these cases.
A hyperglucsemia which passes into a glycosuria may also be brought
about by an excessive formation of sugar from the glycogen and other
substances within the animal body.
The so-called piqilre, and also probably those glycosurias which occur
after other lesions of the ner^'ous system, belong to the above group of
glycosurias. The glycosuria produced on poisoning Anth carbon monox-
ide, adrenahn, curare, str}-chnine, morphine, etc., also belongs to this group.
That the glycosuria produced in certain cases, as after piqilre. 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 pre\'ioush- made free from glycogen by star^'ation or
other means. In other cases, as in carbon-monoxide poisoning, the sugar
is probably derived from the proteins, ecause glycosuria boccurs only in
those cases where the poisoned animal has a sufficient quantity of protein
at its disposal (Straub and Rosexsteix^). Protein starvation \\ith a
simultaneously abundant supply of carbohydrates causes this glycosuria to
disappear.
A hyperglucsemia \dth. glycosuria may also be caused by a decreased
ability of the animal body to consume or destroy the sugar. In this case
the sugar must accumulate in the blcod. and the formation of severe cases
of diabetes meUitus is now generally explained by this process.
The inability of diabetics to destro}- or consume the sugar does not
seem to be connected "uith anj- decrease in the oxidative energy- of the
cells. The oxidative processes are not diminished generally in diabetics
' See Dock, Pfliiger's Arch., 5; Bock and Hoffmann, Exp. Studien iiber Diabetes
(Berlin, 1874); CI. Bernard, Le§ons snr le diabete (Paris); T. Araki, Zeitschr. f. physiol.
Chem., 15, 351; Straub, Arch. f. exp. Path. u. Pharm., 3S; Rosenstein, ibid.. 40;
Pfiiiger, Pfliiger's Arch., 96.
300 THE LIVER.
(ScHULTZEN, Nencki and Sieber^), and this has recently been substan-
tiated b}' Baumgarten. This latter investigator made experiments ^\ith
several bodies which on account of their aldehyde nature were closely
related to sugar or were cleavage or oxidation products of the same,
namely, glucuronic acid, d-gluconic acid, c^-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 tliis it must
be remarked that the two varieties of sugar, dextrose and le\'ulose, which
are oxidized with the same readiness, act differently in diabetics. Accord-
ing to KuLZ and other investigators levulose is, contrary to dextrose,
utilized to a great extent in the organism, and may, according to ^Iinkowski,^
even cause a deposit of glycogen in the liver in animals with pancreas
diabetes (see telow). 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 especially
the dextrose suffers diminution, and the explanation of this has teen
sought in the fact that the dextrose is not previously split before com-
bustion.
CO
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 "v^dll be thoroughly explained in a following chapter, this quotient is
greater the more carbohydrates are burnt in the body, and it is correspond-
ingly smaller when protein and fat are chiefly burnt. The investigations
of Leo, Hanriot, Weintraud and Laves,^ and others have shown that
in severe cases of diabetes, in the starving condition the low quotient is
not raised after partaking of dextrose, as in healthy individuals, but that
it is raised after feeding le\ailose, which is also of value to diabetics (Wein-
traud and Laves). The povertj^ of the organs and tissues of diabetics
in glycogen shows that not only is the combustion of the dextrose dimin-
ished, but also the transformation of the same into glycogen, and its valu-
ation as a whole is decreased.
There are also certain investigators who consider that diabetes is due
to an increased production of sugar in the liver — a view which has received
some support in the artificially produced pancreatic diabetes.
The investigations of ^Minkowski, v. Mering, Domenicis, and later
' Schultzen, Berl. klin. Wochenschr., 1872; Nencki and Sieber, Journ. f. prakt.
Chem. (X. F.), 26, 35; Baumgarten, "Ein Beitrag zur Kenntniss des Diabetes mel-
litus," Habilitationsschrift, also Zeitschr. f. exp. Path. u. Therap., 2, 1905.
^ Kiilz, Beitrage zur Path. u. Therap. des Diabetes melHtus (Marburg, 1874), 1;
Weintraud and Laves, Zeitschr. f. physiol. Chem., 19; Haycraft, ibid.; Minkowski,
Arch. f. exp. Path. u. Pharm., 31.
3 See V. Noorden, Die Zuckerkrankheit, .3. Aufl., 1901.
PAI\CRE.\S AND GLYCOLYSIS. 301
of man}' other investigators ^ have shown that a true diabetes of a severe
kind is caused b}' the total or nearly total extirpation of the pancreas of
many animals, especiall}'' dogs. As in man in severe forms of diabetes,
so also in dogs with pancreatic diabetes, an almndant elimination of sugar
talces place even on the complete exclusion of carbohydrates from the
food.
Artificial pancreas diabetes may, at least in cases where the pancreas
has not been completely extirpated, present exactly the same conditions
as diabetes in man, but opinions differ as to the cause of this diabetes.
It is generally accepted that in pancreas diabetes a diminished consumption
of sugar takes place; but there are several investigators who are of another
opinion and who explain this form of diabetes as due at least not entirely
to a diminished combustion of sugar, but to a diseased increase in the
sugar formation. From tliis it follows that the pancreatic gland exerts on
the formation of sugar in the liver a regulating action which is absent on
the extirpation of the gland.
]\Iany important observations show that a close relation exists between
the liver and pancreas diabetes. Pfluger has also shown that especially
in diabetes produced by Saxdmeyer's method (partial extirpation with
subsequent destruction of the remains of the gland in the abdominal cavity,
when the animal remains alive for a longer time than after total extir-
pation) the liver does not lose weight, although the total weight of the
animal diminishes greatly, wliile in starvation ^\'ithout diabetes the hver
loses weight more than the other parts of the body. PFLtJGER concludes
from this that the hver in diabetes works actively and is the most
important seat of production of diabetic sugar.
We do not know how the pancreas acts in the formation or the de-
struction of sugar, and we have essentially two contradictoiy \-iews on
tliis subject. According to one view the action is of a nervous kind, while
the other ^•iew is that we are deaUng \sith an internal secretion of special
bodies wliich in an unknown manner perhaps act upon the nerve centres
and regulate the formation or the destruction of sugar. The assumption
of an internal secretion is rather generally accepted and is based on
the investigations of Minkowski. Hedox, Laxceraux. Thiroloix. and
others 2 upon the action of the subcutaneous transplantation of the gland.
According to these investigations a subcutaneously transplanted piece of
' See Minkowski, Untersuchungen iiber Diabetes mellitus nach Exstirpation des
Pankreas (Leipzig, 1S93); v. Xoorden, Die Zuckerkrankheit (Berlin, 1901), which
contains a very copious index of the literature. In regard to diabetes see also CI.
Bernard, Legons sur le diabete (Paris); "Seegen, Die Zuckerbildung ini Thierkorper
(Berlin, 1890), and Pfliiger, Das Glykogen, 2. Aufl., 1905.
' See Minkowski, Arch. f. exp. Path. u. Pharm., 31; Hedon, Diabete pancreatique,
Travaux de Physiologie (Laboratoire de Montpellier, 1898), and the works on diabetes.
302 THE LIVER.
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 then
subsequently removed, an active elimination of sugar appears immediately.
PFLtJGER has made important objections to the force of proof in these
experiments.
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.^ We are also not
acquainted mth the kind of active substance here formed.
The glycolytic property of the blood as shown by Lupine was con-
si(iered 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 gly-
colysis 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
destenction of sugar we are also obliged to accept a glycolysis in the organs
and tissues. The views 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 view (Stoklasa) considers the glycolysis
as analogous to alcoholic fermentation, where we have processes brought
on by special tissue zymases (see Chapter I).
Another important question is whether one organ can bring about
glycolysis or wheiher a combination of organs is required. Cohnheim
has found that a cell free fluid can be obtained from a mixture of pan-
creas and muscle, which destroys dextrose, while the pancreas alone does
not have this action and the muscle only to a slight extent. The pan-
creas does not contain, according to Cohnheim, a glycolytic enzyme, but
a substance resistant to boiling temperatures, which is soluble in water and
alcohol, and which, like an amboceptor, activates a glycolytic proenzyme
which exists in the muscle fluid, but which is inactive alone and which
retards glycolysis when it exists in excess. De Meyer holds a nearly
similar view, but with this exception, that he does not consider that the
activating substance comes from the muscles but from the leucocytes.
LUPINE 2 has also expressed the opinion that the pancreas does not have
a direct glycolytic action by internal secretion, but more likely by the
glycolysis encouraged by the action of cell protoplasm.
' See Diamare and Kubiabko, Centralbl. f. Physiol., 18, and Diamare, ibid., 19,
Rennie, ibid., 18; Sauerbeck, Vnchow's Arch , 177.
' Cchnheirrj, Zeitbchr. f. phjbiol. Chera., 39, 42, 4.3, and 47; Do Meyer, Arch iniern.
de Physiol., 2, cited from Bicchem Centralbl., 3.
SUGAR FORMATION FROM PROTEIN. 303
The statements of Cohnheim have not been fully confirmed by other
investigators. On the contrar}-, several investigators, Stoklasa and
collaborators, Feixschmidt, Aexheim and Rosexbaum, and Brauxstein,^
could not detect any glycolytic activity either in the pancreas alone or
in muscles and other organs alone (with the exclusion of bacteria). The
liver also belongs to these organs, in which, it must be remarked, the gly-
colytically active substance has been absent in severe cases of diabetes.
CoHXHEiAi's statements have, on the contrary-, been substantiated in part
by Arnheii.i and Rosexbaum and R. Hirsch, who find that the pancreas
has the power of raising the glycolytic action of the liver and the muscles.
On the other hand, Glaus and Embdex have not been able to obtain the
activating action of the pancreas upon muscle-juice, but according to
CoHXHEiM this is probably due to the fact that these investigators added
too large quantities of pancreas, whereby the retarding action came into
effect. No positive conclusions on the mode of action of the pancreas
in sugar destruction or sugar formation can be drawn from these con-
tradictory statements.
Where does the sugar eliminated in diabetes originate? Does it depend
entirely upon the carbohydrates of the food or the store of carbohydrate
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 elim-
inated that it could not possibly be accounted for by the store of glycogen
or other carbohydrate-containing substances in the body. Similar experi-
ments have also been performed later by Pfluger,^ and 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 dis-
pute. It is not possible to enter into an exhaustive and detailed discus-
sion 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 necessan,- to form
ammonium carbonate, is used for the formation of sugar. The actual rela-
' Stoklasa and collaborators, Centralbl. f. Physiol., 17, and Ber. d. d. ehem. Gesellsch.,
36 and 38; Feinschmidt, Hofmeister'e Beitrage, 4; Hirsch, ibid.; Claus and Embden,
ibid., 6; Arnheim and Rosenbaum, Zeitschr. f. physiol. Cbem., 40; Braunstein,
Zeitschr f. klin. Med.. 51.
' Liitbje, Deuls-cb Arch f. klin. Med , 79, and Pfliiger's Arch., 106; Pfliiger,
Pfliiger.- Arrb lOS
304 THE LIVER.
tion between dextrose and nitrogen in the urine, i.e., the quotient D:N, has
been repeatedly determined in various forms of diabetes. In a large number
of cases this has been found to be equal to 2.8 to 3.8. It may undergo
considerable variation, and in certain cases it may indeed be loAver than 1
as well as higher than 8. 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 \dews of Hammarsten such conclusions are mostly
very uncertain. The sugar eliminated by the urine represents the differ-
ence between the total sugar production of the body and the quantity
of sugar burned or utilized. Only under the su])position 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 observa-
tions seem to show that in the different forms of diabetes variable amounts
of the sugar are burned. A sugar formation from fat can be presumed only
when the quotient is specially high.
The property of protein of increasing the elimination of sugar is con-
sidered 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 want to accept in this case that the pro-
tein, but rather the fat, was the material from which the sugar was pro-
duced, still we must admit eitlier 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 ^ has communicated one experiment among others, in
which a dog with pancreas diabetes, whose weight before starvation was
18 kilos, with nineteen days' starvation eUminated an average of 10.4 grams
sugar for the last six days of starvation. By exclusive protein feeding
the quantity of sugar per day could be raised to a maximum of 123.8 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,
wliich 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.
' Deutsch. Arch. f. klin. Med., 79.
SUGAR FORMATION FROM FAT. 30o
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 ven- impor-
tant increase in the acti\ity of the liver, we are opposed by the great
difficulty that, according to known laws of metabolism, the proteins do
not raise the fat metabolism, but rather diminish it. The protein dis-
places a corresponding quantity of fat from the metabolism, and if the
fat was 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 ehmination is much more easily
explained b\" the assumption of a sugar formation from protein than from fat.
The action of monamino-acids upon the carlx)hydrate metabohsm has
also given important ground for the assumption of a sugar formation from
protein. That a deamidation occtirs in the animal body was shown by
the older obsen.-ations of Bai:^l\xx and Blexdeemaxx. Further proofs of
this were furnished b}- the recent investigations of Xeubeeg and Laxg-
STEES". where in feeding experiments with alanine they found abundance of
lactic acid in urine, and finally Laxg ^ has shown that various organs in
antiseptic autolysis have the power of deamidating amides and amino-acids.
As from amino-acids by deamidation it is possible to produce oxyfatty acids
according to the formula — CH.XH2-H20=— CHCOH^-f XH3, it was inter-
esting to test the action of amino-acids upon carbohydrate metabolism.
Several investigations have been carried on with this in \'iew, such as those
of L.\XGSTEix and Xeuberg, R. Cohx and F. Kraus. which have shown
a ver}- probable formation of carbohydrate under the influence of amino-
acids; but the investigations of Embdex and Salomox and of Embdex
and Alm-\gia2 have positively shown in a dog without a pancreas that
the amino-acids can bring about a re-formation of carbohydrate. 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 formation of sugar ^ith amino-acids as inter-
mediar\- bodies as ver}- probable.
If we presume a formation of sugar from fat we must differentiate
between the two components of neutral fats, that is, tetween the glycerine
and the fatty acids. A formation of sugar from glycerine can be con-
sidered as proven from the investigations of Ceemee. and especially those
of Luthje.3 and in what follows we will discuss only the formation of
sugar from the fattv acids.
' Baumann. Zeitichr. f. physiol. Chem.. 4: Blendermarm, ibid.. G; Xeuberg and
Langstein. .\rch. f. (Anat. u.) Physiol., 1903. Suppl.; Lang. Hofmeister's Beitrage, 5.
^ Langstein and Xeuberg. 1. c; Cohn. Zeitschr. f. physiol. Chem.. 2S: F. Ivraus,
Berl. klin. Wochenschr.. 1904; Embden and Salomon, Hofmeister's Beitrage, 5 and
6, and with^Almagia, ibid.. 7.
'Cremer, Sitzungsber. d. Ges. f. Morph. u. Physiol. Munchen, 1902; Luthje,
Deutsch. Arch. f. klin. Med.. SO.
306 THE LIVER.
The formation of sugar from fat seems to occur in the plant kingdom,
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.^
Where 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) metabohsm, then if the observations are otherwise free
from error we can admit of a formation of sugar from fat. Several such
cases of diabetes in man have been published (Rumpf, Rosenqvist, Mohr,
v. NooRDEN, and others), and also in animals (Hartogh and Schumm).^
Although these researches are not fully conclusive, still certain of them
indicate a probable formation 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 material for the formation of sugar (PFLtJGER) ; still this is
not sufficient as a positive proof.
Starting with the quotient D:N, which he sets at 3.67, Magxus-Levy
has calculated the quantity of oxygen necessary for the combustion of the
protein, provided the sugar was formed therefrom, and also the quantity
of carbon dioxide j^roduced, i.e., the respiratory quotient for these cases.
On comparison of these results with the low respiratory quotient observed
in diabetics, he comes to the conclusion that the sugar is derived from the
protein. Pfluger,^ who has made a different calculation, comes to an
entirely different result, and considers that the low values for the respira-
tory quotient in diabetes are positive proof that the sugar is not formed
from the proteins, but from the fats. As the quotient D:N is not an
accurate measure of the quantity of sugar formed, and as we cannot, for
the present, exactly measure the quantity of oxygen necessary for the
formation of sugar from the 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 proteids.
We have no exact 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
' Kaufmann, Arch. f. Physiol. (5), S, where Chauveau's work is cited.
^ Rumpf, Berl. klin. Wochenschr., 1899; Rosenqvist, ibid.; Mohr, ibid., 1901; v.
Noorden, Die Zuckerkrankheit, 3. Aufl., Berlin, 1901; Hartogh and Schumm, Arch. f.
Path. u. Pharm., 4.>. See also the works of O. Loewi, ibid., 47, and Lusk, Zeitschr. f.
Biologie, 42.
^Magnus-Levy, Zeitschr. f. klin. Med., ')('»; Pfliiger, Pfluger's Arch., 108.
BILE AND ITS F0R:MATI0X. 307
has the power of producmg sugar from protein as well as from fat. The
obsen-ations 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 biliar}- fistula, an operation which was first
performed by Schwann in 1S44 and which has been improved lately by
Dastre and Pawlow,i it is possible to study the secretion of the bile. This
secretion is continuous, but with var}-ing intensity. It takes place imder
a ver\' low pressure; therefore an apparently imimportant 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 stagnation and absorption of
the bile by means of the lymphatic vessels (absorption 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.-
The statements 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. Xoel-Patox. MayoRobsox, H.imm-^esten,
Pfaff and Balch. and Beaxd ^ have found a variation between 514 and
1083 c.c. per twenty- f oiu: hours. Such determinations are of doubtful value,
because in most cases it follows from the composition of the collected bile
that the fluid is not the result of a secretion of normal liver bile.
The quantity of bile secreted is. however, as specially shown by St.u)EL-
maxx,^ subject to such great variation even under physiological conditions
that the study of those circumstances which influence the secretion is ven-
difficult and uncertain. The contradictory- statements by different investi-
gators may probably be explained by this fact.
In stan-ation the secretion diminishes. According to LukJ-Ajxow and
' Schwann. Aich. f. (Anat. u.) Physiol., 1S44; Dastre. .\rch. de Physiol. (5), 2;
Pawlow, Ergebnisse der Physiol., 1, Abt. 1.
- In regard to the quantity of bUe secreted in animals see Heidenhain, Die Gallenab-
sonderung. ia Hermann's Handbuch der Physiol., o, and Stadelmann, Der Icterus xmd
seine verschiedenen Formen (Stuttgart, 1S91).
' Ranke. Die Blutvertheilimg imd der Thatigkeitswechsel der Organe (Leipzis.
ISTl); Xoel-Paton, Rep. Lab. Roy. Coll. Edinburgh, 3; Mayo-Robson, Proc. Roy. Soc,
47: Hammarsten, Xova Act. Reg. Soc. Scient. L'psala (3), 16; Pfaff and Balch, Joum.
of Exp. Med.. 1897; Brand, Pfliiger's Arch.. 90.
* Stadelmann, Der Icterus, etc., Stuttgart, 1891.
308 THE LIVER.
AlbertoniV under these conditions the absolute quantity of sohds de-
creases, while the relative quantity increases. After partaking of food the
secretion increases again. The statements are very contradictory 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 statements Heidenhain ^ 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 par-
taking of food. According to Barbera the time when the maximum
occurs is dependent upon the kind of food. With carbohydrate food it
is two to three hours, after protein food three to four hours, and with fat
diet it is five to seven hours after feeding.
According to the older statements, the proteins, of all the various foods,
cause the greatest secretion of bile, while the carbohydrates diminish the
secretion, or at least excite it much less than the proteins. This coincides
with the recent observations of Barbera.^ The authorities are by no
means ageed 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 car-
l^ohydrate feeding. According to Rosenberg olive-oil is a strong chola-
gogue, a statement which, according to other investigators — Mandelstamm,
DoYON and Dufourt^ — is not sufficiently proved.
As Barbera has shown, a close relationship exists between the bile
secretion and the quantity of urea formed, as an increase in the first goes
hand in hand with an increase of the latter. The bile is, therefore, accord-
ing to him, a product of disassimilation, whose quantity rises and falls with
the degree of activity of the liver.
The question whether there exist 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,
' Lukjanow, Zeitschr. f. pliysiol. Chem., 16; Albertoni, Recherches sur la s6cr^tion
biliaire, Turin, 1893.
^ Hermann's Handb., 5, and Stadelmann, Der Icterus, etc.
3 Centralbl. f. Physiol., 12 and 16.
* Barbera, Bull, della scienz. med. di Bologna (7), 5, Maly's Jahresber., 24, and
Centralbl. f. Physiol., 12 and 16; Rosenberg, Pfli'iger'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 Barbara, 1. c.
SECRETIOX OF BILE. 309
olive-oil. etc.; while others, especially the more recent investigators, 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 positively
proven fact by the investigations of several experimenters.^ Sodiimi
salicylate is also perhaps a cholagogue (Stadel:nl\xx, Botox and
Dufouet).
Acids, and especially, imder normal conditions, hydrochloric acid, seem
to be physiological excitants for bile secretion. According to Falloise
and Fleig the acids act upon the duodenum and the upper part of the
jejunum, and the action is brought about by a secretin formation similar
to the action of acids upon the secretion of pancreatic juice (see Chapter
IX). According to Falloise ~ chloral hydrate introduced into the duo-
denima causes a secretion of bile in an analogous manner by the aid of a
special chloral secretin.
The bile is a mixture of the secretion of the liver-cells and the so-called
mucus which is secreted by the glands of the biliary passages and by the
mucous membrane of the gall-bladder. The secretion of the liver, which
is generally poorer in solids than the bile from the gall-bladder, is thin and
clear, while the bile collected in the gall-bladder is more ropy and viscous
on account of the absorption of water and the admixture of ''mucus," and
cloudy because of the admixture of cells, pigments, and the like. The
specific gra\'ity 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, bro^Tiish green, grass-green, or bluish green. Bile obtained from
an executed person immediately after death is golden yellow or yellow
with a shade of bro-^Ti. 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 certain 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
' Schiff. Pfliiger's Arch.. 3. See Stadelmann, Der Icterus, and the dissertations of
his pupils, especially Winteler, '" Experimentelle Beitrage ziu" Frage des Kreislaufes
der Galle" (Inaug.-Diss. Dorpat, 1S92). and Gartner, '•Experimentelle Beitrage zur
Physiol, und Path, der Gallensekretion" (Inaug.-Diss. Jurjew, 1S93); also Stadehuann,
"Ueber den Ivreislauf der Galle." Zeitschr. f. Biologie. 34.
' Falloise, Bull. Acad. Rov. de Belg., 1903: Fleig, ibid.. 1903.
310 THE LIVER.
ropy properties depend, it seems, chiefly on the presence of a nucleoalbumin
similar to mucin (Paijkull). The bile from the animals investigated
by Hammarsten showed a similar behavior. Hammarsten ^ has, on the
contrary, 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,^ does not in man secret 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 phospJmtides,
cholesterin, soaps, neutral fats, urea, ethereal sulphuric acid, traces of con-
jugated glvx;uronic acids and mineral substances, chiefly chlorides, besides
phosphates of calcium, magnesium, and iron. Traces of copper also occur.
Bile-salts. The bile-acids which thus far have best been studied may
be divided into two groups, the glycocholic and taurocholic axid groups. As
found by Hammarsten,^ a, third group of l)ile-acids occurs in the shark and
probably also in other animals. These are rich in sulphur, and like the
ethereal sulphuric acids they split off sulphuric acid on boiling with hydro-
chloric acid. All glycocholic acids contain nitrogen, but are free from
sulphur and can be split with the addition of water into glycocoU (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 (aminoethylsulphonic acid) and a cholic acid. The reason
for the existence of different glycocholic and taurocholic acids depends
on the fact that there are several cholic acids.
The conjugated bile-acid found in the shark, and called scymnol sulphuric add
by Hammarsten, yields as cleavage products sulphuric acid and a non-nitrogenous
substance, scymnol (CgiH^j^Og), 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
older statements (Zaxetti ■*). 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 further on).
All alkali salts of the biliary acids are soluble in water and alcohol, but
insoluble in ether. Their solution in alcohol is therefore precipitated by
ether, and this precipitate, with proper care in manipulation, gives, for
' Paijkull, Zeitschr. f. physiol. Chem., 12; Hammarsten, 1. c, Nova Act. (3), 16,
and Ergebnisse der Physiol., Bd. 4.
2 Maly's Jahresber., 32.
' Hammarsten, Zeitschr. f. physiol. Chem., 24.
^ See Chem. Centralbl., 1903, 1, 180.
BILE ACIDS. 3il
nearly all kinds of bile thus far investigated, rosettes or balls of fine needles
or four- to six-sided prisms (Plattxer's crystallized bile). Fresh human
bile also crj^stallizes readily. The bile-acids and their salts are optically
active and dextrorotatory. The salts of the different bile-acids act some-
what differently towards neutral salts. The alkali salts of the ordinarj- and
best-studied bile-acids from man, ox, and dog are, according to Tengstrom,i
precipitated by ammonium and magnesium sulphates, and also, in pure
form, by sodium nitrate and sodium chloride (added to saturation). Potas-
sium and sodium sulphates do not precipitate them. The alkali salts
cannot be directly precipitated from the bile by NaCl, on account of the
presence of bodies retarding precipitation, among which we find oil-soaps.
The bile-acids are dissolved by concentrated sulphuric acid at the
ordinar}' temperature, forming a reddish-yellow liquid which has a beautiful
green fluorescence. According to Pregl an oxidation with reduction of
the sulphuric acid into sulphur dioxide takes place. The fluorescent
substance has been called dehydrocholan (see below) by Pregl.^ On
carefully warming with concentrated sulphuric acid and a little cane-sugar,
the bile-acids give a beautiful cherrj'-red or reddish-violet liquid. Pet-
tenkofer'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 60-70° C. Then
a 10 per cent solution of cane-sugar is added, drop by drop, continually
stirring with a glass rod. The presence of bile is indicated by the produc-
tion of a beautiful red liquid, whose color does not disappear at the ordinary
temperature, but becomes more bluish violet in the course of a day. This
red liquid shows a spectrum with two absorption-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 bro-^Ti. The reaction fails
if the sulphuric acid contains sulphurous acid or the lower cfecides of nitro-
gen. 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 fact
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). Accord-
• Zeitschr. f. physiol. Chem., 41. ^ Zeitschr. f. physiol. Chem., 45.
312 THE LIVER.
ing to Mylius and v. Udranszky ^ a 1 p, m. solution of furfurol should
be used. Dissolve the bile, which must first be purified 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 c.c. concentrated sulphuric
acid, and cool when necessary, so that the test does not become too warm.
This reaction, when performed as described, will detect 2^ to 3V rnilligram
cholic acid (v. Udranszky). Other modifications of Pettenkofer's
test have been proposed.
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 so, in the bile
of carnivora. On boiling with acids or alkalies this acid, which is anal-
ogous 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 Mijller ^ have prepared first cholic-acid hydrazide, and then,
by the action of nitrous acid upon this, they oljtained the cholic-acid azide,
C23H39O3CO.N3, and finally from this last in alkaline solution with glyco-
coll they synthetically prepared the alkali salt of glycocholic acid, at the
same time splitting off nitrogen.
Glycocholic acid crystallizes in fine, colorless needles or prisms. It is
soluble with difficulty in water (in about 300 parts cold and 120 parts boil-
ing water), and is easily precipitated from its alkali-salt solution by the
addition of dilute mineral acids. It is readily soluble in strong alcohol, but
with great difficulty in ether. The solutions have a bitter but at the same
time sweetish taste. The acid melts at 138-140° C. (Medvedew^). The
salts of the alkalies and alkaline earths are soluble in alcohol and water.
The solution of the alkali salt in water can be salted out by NaCl, but
not by KCl. The salts of the heavy metals are mostly insoluble or soluble
with difficulty in water. The solution of the alkali salts in water is pre-
cipitated by sugar of lead, cupric and ferric salts, and silver nitrate.
Glycocholeic Acid is a second glycocholic acid, first isolated by Wahl-
GREN"* 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 (Hammar-
STEN ^).
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
» Mylius, Zeitschr. f. physiol. Chem., 11; v. Udranszky, ihid., 12.
2 Zeitschr. f. physiol. Chem., 47.
^Centralbl. f. Physiol., 14.
* Zeitschr. f. physiol. Chem., 36.
«/bzd..43.
GLYCOCHOLEIC AND TAUROCHOLIC ACIDS. 313
175-176° C. The alkali salts are soluble in water, have a pure bitter
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, calcium
and magnesium.
The preparation of the pure glycocholic acids may be performed in
several ways. The bile, which has been freed from mucus by means of
alcohol and the alcohol removed by evaporation, may be precipitated by
a solution of lead acetate. The precipitate is then decomposed by a soda
solution and heat, evaporated to dryness, and the residue extracted with
alcohol, which dissolves the alkali glycocholate. The alcohol is distilled
from the filtered solution and the residue dissolved in water; this solution
is now decolorized by animal charcoal and the glycocholic acid precipitated
from the solution by the addition of a dilute mineral acid. The mixture
of the two glycocholic acids is freed from mineral acid by carefully washing
with water, and then is boiled with water, when the glycocholic acid dissolves
and may be obtained from the filtrate as crystals on cooling. The glyco-
choleic acid with some transformed glycocholic acid (paraglycocholic acid)
remains undissolved and may be purified by converting it into the insoluble
barium salt. If we do not care for the obtainment of pure glycocholeic acid
but want only the pure glycocholic acid, then the decolorization with animal
charcoal can be omitted. If the bile is rich in glycocholic acid, we can
treat the mucus-free bile, according to Hf fner's ^ method, with ether and
hydrochloric acid, when the glycocholic acid crystallizes out in abundance.
The reader is referred to more exhaustive works for other methods of prepa-
ration.
Hyoglycocholic Acid, CojH^gNOs, 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 CaCb, BaCb, and MgCb, and may be salted out like a soap by
Na2S04 when added in sufficient quantity. By precipitation with NaCl in such
quantity that the precipitate redissolves on warming, Hammarsten ^ has 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 ^).
The glycocholate in the bile of the rodent 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 C26H45NSO7. On boiling with acids and alkalies it splits into
cholic acid and taurine. Taurocholic acid has also been prepared syntheti-
cally by BoNDi and Mijller, using the same method as they used for glyco-
cholic acid.
* Journ. f. prakt. Chem., 1874.
^ Not published.
^ Zeitschr. f. physiol. Chem., 12 and 13.
314 THE LIVER.
Taurocholic acid can be readily obtained, by the method suggested
by Hammarsten/ 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 sugar of lead. Basic
lead acetate gives, on the contrary, a precipitate which is soluble in boiling
alcohol. The alkali salts are not only precipitated from their solution by
the same neutral salts that precipitate glycocholic acid, but also by potas-
sium chloride, and by sodium and potassium acetates.
Taurocholic acid is most simply prepared from a glycocholic-acid-free
bile or from one very poor in this acid, such as fish- or dog-bile. From
ox-bile it can be prepared by first precipitating the glycocholic acid with
alum and then repeatedly precipitating the filtrate with ferric chloride
(according to Tengstrom). From this filtrate the taurocholate is precip-
itated by saturating with NaCl, the precipitate pressed and freed from
NaCl by dissolving in alcohol, and as a powder, or dissolved in a little alcohol,
is decomposed by alcohol containing hydrochloric acid. The acid is pre-
cipitated from the filtrate by ether. The taurocholic acid can be repeatedly
recrystallized by solution in alcohol containing water and the careful
addition of ether.
Taurocholeic Acid is a second taurocholic acid, detected by Hammar-
STEN in dog-bile and isolated by Gullbring ^ from ox-bile, and has the
formula C26H45NSO6 or C27H47NSO6. 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.
For the preparation of taurocholeic acid it is best to use dog-bile which
is first precipitated by ferric chloride. The precipitate contains the acid,
while the filtrate can be used for the obtainment of taurocholic acid by
saturating with NaCl. The iron precipitate is converted into the alkali
salt by sodium carbonate, and is decomposed by alcohol containing
* Zeitschr. f. physiol. Chem., 43.
* Hammarsten, Zeitschr. f. physiol. Chera., 43; Gullbring, ibid., 45.
CHOLIC ACID. 315
hydrochloric acid, and then precipitated by ether. I'he amorphous acid
which separates is purified from alcohohc solution by precipitation with
ether. In preparing taurochohc acid from ox-bile, the taurocholeic acid,
which is readily soluble in alcohol-ether, remains in the alcohol-ether on
the proper addition of ether. This crude acid as alkali salt is freed from
taurocholic acid by precipitation with ferric chloride, then again converted
into alkali salt, decomposed wdth acid in alcohol, precipitated by ether, and
purified (Gullbrixg).
Cheno-taurocholic Acid. This is the most essential acid of goose-bile and has
the formula C29H4 XSOg. This acid, though Uttle 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 cr}-stallizable taurocholic acid occurs which can be pre-
cipitated from the solution of the alkali salts by the addition of mineral
acids, similar to glycocholic acid (Ha^hlirstex ^).
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 in the urine in icterus, has. according to
Strecker and nearly all recent investigators, the constitution C24H4o05 =
( CHOH
t'2oH3i ■< (CHoOH).,. According to Mylius.^ cholic acid is a monobasic
( COOH
alcohol-acid wdth one secondary and two primary alcohol groups. CuR-
Tius 3 has sho-uTi by preparing the cholamine, C23H39O3XH2, from the
above-mentioned (p. 312) cholic-acid azide, with cholic-acid urethane as an
intermediary step, that the carboxyl group is not immediately connected
with the CHOH group, but is combined with the chief nucleus without
the neighboring secondary alcohol group. On oxidation it first yields
dchydrocholic acid, C24H34O5 (HAMiLiRSTEx). On further oxidation
hilianic acid, C24H34O8 (Cleve), is obtained, or. more correctly, according
to Lassar-Cohx and Pregl, a mixture of bilianic and isohilianic acids.
On oxidation, bilianic acid yields cilianic acid (Lassar-Cohn), whose
formula, according to Pregl, is C20H28O8. On stronger oxidation it yields
cholesterinic acid, which has not been carefully studied, and finally phthalic
acid, as maintained by Senkowski, but not substantiated by Bulnheim
• Not published.
* 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.
' Ibid., 39.
316 THE LIVER.
or Pregl.i On reduction (in putrefaction) cholic acid may yield desoxy-
cholic acid, C24H40O4 (Mylius). On reduction with hydriodic acid and red
phosphoruSj Pregl obtained a product which he considers as a mono-car-
(CH2
boxvlic acid, with the formula C20H31 ] (CH3)2. Senkowski has obtained
( COOH
an acid with the formula C24H40O2, cholylic add, on the reduction of tlie
anhydride .2
As above mentioned, Pregl ^ has obtained, by the action of concentrated
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. Dehydrocholon is
nitrated by nitric acid, while the cholic acid is not. From this behg-vior,
as well as from the determination of the molecular refraction and disper-
sion of both bodies, Pregl finds it probable that cholic acid belongs to
the hydrated carbocyclic compounds.
Cholic acid crystallizes partly in rhombic plates or prisms with 1
molecule of water and partly in larger rhombic tetrahedra or octahedra
with 1 molecule of alcohol of crystallization (Mylius). These crystals
become quickly opaque and porcelain-white in the air. They are quite
insoluble in water (in 4000 parts cold and 750 parts boiling), rather soIu])le
in alcohol, but soluble with difficulty in ether. The amorphous cholic acid
is less insoluble. The solutions have a bitter-sweetish taste. The crys-
tals 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. Accord-
ing to BoNDi and MIjller the melting-point of the perfectly pure acid is
198°. It forms a characteristic blue compound with iodine (Mylius).
The alkali salts are readily soluble in water, but when treated with a
concentrated caustic or carbonated alkali solution may be separated as an
oily mass which becomes crystalline on cooling. The alkali salts are not
readily soluble in alcohol, and on the evaporation of the alcohol they may
crystallize. The specific rotatory power of the sodium salt is (a)D =
+ 31.4°.* The watery solution of the alkali salts, when not too dilute, is
precipitated immediately or after some time by sugar of lead or by barium
chloride. The barium salt crystallizes in fine, silky needles, and it is rather
insoluble in cold, Init somewhat easilv solul^le in warm water. The barium
* Hammarsten, Ber. d. deutsch. chem. Gesellsch., 14; Cleve, Bull. Soc. chim., 35;
Lassar-Cohn, Ber. d. d. chem. Gesellsch., 32; Pregl, Wien. Sitzungsber., Ill, 1902; Sen-
kowski, Monatshefte f. Chem., 17; Bulnheim, Zeitschr. f. physiol. Chem., 25, in which
the literature on cholesterinic acid may be found.
^ Mylius, 1. c; Pregl, Pfliiger's Arch., 71; Senkowski, Monatshefte f. Chem., 19.
* Zeitschr. f. physiol. Chem., 45.
* See Vahlen, Zeitschr. f. physiol. Chem., 21.
CHOLEIC ACID. 317
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/ has the formula C24H40O4. This acid, which
occurs in varjdng but always small quantities in ox-bile, yields dehydro^
choleic 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 con-
taining water melts at 135-140° C. (Latschinoff). The acid dissolves
in water with difficulty and is also relatively difficultly soluble in alcohol.
It has an intensely bitter taste and gives the ]\Iylius iodine reaction for cholic
acid. The specific rotation is (a:)D= +48.87° (Vahlen). The barium
salt which crystallizes from the hot alcoholic solution as spherical aggre-
gations of radial needles is more difficulth" soluble in water than the cor-
responding cholate.
The relation of choleic acid to desoxycholic acid is not kno^n.
According to Latschinoff and Lassar-Cohn both acids are identical,
w^hile Pregl,2 on the contrary, claims that desoxycholic acid is more
readily soluble in water and when anhydrous has a melting-point of 172-
173°. According to the ordinary views the desoxj'-cholic acid is formed
from cholic acid by reduction. Ekbom ^ could not substantiate this
statement. On using perfectly pure cholic acid he was able to regain
nearly quantitatively after the action of metallic sodium on the alcoholic
solution of the acid or of zinc and alkali. By treatment with zinc and acetic
acid a reaction took place, but the product was a mixture of mono- and
diacetyl derivatives. This indicates that desox3'cholic acid is isomeric with
an acid preformed in the bile, either choleic acid or possibly, as Pregl has
shown, an acid isomeric therewith. The observation of Pregl that
desoxycholic acid, like choleic acid, yields dehydrocholeic acid and cholanic
acid as oxidation products, stands in close connection with such an assump-
tion, but makes the formation of desoxycholic acid from cholic acid by
reduction very improbable.
Both cholic acids are best prepared from ox-bile which has been boiled
for twenty-four hours with baryta-water or caustic soda. According to
Mylius,^ boil the bile for twenty-four hours with five times its weight of a
30 per cent caustic-soda solution, replacing the water lost by evaporation.
' Latschinoff, Ber. d. deutsch. chem. Gesellsch., 18 and 20; Lassar-Cohn, ibid., 26,
and Zeitschr. f. physiol. Chem., I". See also Vahlen. Zeitschr. f. physioi! Chem., 23.
' Wien. Sitzungsber., 111. Math. Naturw. Kl. 1902; Latschinoff, 1. "..; Lassar-
Cohn, 1. c. See also Myhus, Ber. d. d. chem. Gesellsch., 19.
^ Unpublished investigation.
* Zeitschr. f. physiol. Chem., 12.
318 THE LIVER.
Now saturate the liquid with CO2 and evaporate nearly to dryness. The
residue is extracted with 9G per cent alcohol and this alcohohc extract
diluted with water until it contains at the most 20 per cent alcohol; it is
then completely precipitated with a BaCl2 solution. The precipitate,
which contains besides fatty acids also the choleic acid, is filtered, and
the cholic acid, contaminated with choleic acid, is precipitated from the
filtrate by hydrochloric acid. After the cholic acid has gradually crys-
tallized out it is repeatedly recrj'stallized from alcohol or methyl alcohol.
According to Boxdi and Mlller,! perfectly pure cholic acid having a
melting-point of 198° can be obtained by boiling the impure acid for four
hours with 10 per cent caustic soda, reprecipitating with hydrochloric acid,
and recrystallizing from .alcohol.
Choleic acid may l)e obtained from the above-mentioned l^arium pre-
cipitate by first converting the barium salt into sodium salt by sodium car-
bonate, then fractionally precipitating the fatty acids by barium acetate,
separating the choleic acid from the filtrate by hydrochloric acid and
recrystallizing several times from glacial acetic acid.
Pregl 2 has suggested a somewhat different but simpler method for
preparing cholic acid and obtaining the desoxycholic acid from ox-bile. In
regard to this as well as other methods of preparation we must refer to
the original communications and to other handbooks.
Fellic Acid, C23H40O4, is a cholic acid, so called by Schotte:^, which
he obtained from human bile, along with the ordinary acid. This acid is
crystalline, is insoluble in water, and yields barium and magnesium salts
which are very insoluble. It does not respond to Pettexkofer's reaction
easily and gives a more reddish-blue color.
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 l)y BaCl2 and in others not. According to the state-
ments of Lassar-Cohx^ three cholic acids may l^e prepared from human
bile, namely, ordinary cholic acid, choleic acid, and fellic acid.
Lithofellic Acid, CjoHagO^, is the acid related to cholic acid which occurs in
the oriental bezoar stones, which is insoluble in water, comparatively easUy solu-
ble in alcohol, but only slightly soluble in ether.*
The hyo-glycocholic and cheno-taurocholic acids, as well as the gh'co-
cholic acid of the bile of rodents, yield corresponding cholic acids. This
seems to be the case also with the glycocholic acid of the hippopotamus-
bile, which stands very close to the pig-bile (Hammarstex ^). In the polar
' Zeitschr. f. physiol. Chem., 47.
"1. c, Wien. Sitzungsber.
^ Schotten, Zeitschr. f. physiol. Chera., 11; Lassar-Cohn, Ber. d. deutsch. chem.
Gesellsch., 27.
* See Jiinger and Klages, Ber. d. deutsch. ch2m. Gesellsch., 28 (older hterature).
' Investigations not published.
BILE PIGMENTS. 319
bear a third cholic acid exists besides cholic and choleic acids. It is called
ursochokic acid, C19H30O4 or C18H28O4 (Hammarsten ^). The bile of
other animals (walrus, sea-dog) contains special cholic acids (Haivimar-
STEN 2),
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 dj'slysin, 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 dyslj'sins with caustic
alkali they are reconverted into the corresponding cholic acids.
The Detection of Bile-acids in Aximal Fluids. To obtain the
bile-acids pure so that Pettexkofer'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,
distil the alcohol, and evaporate to drj'ness. The residue is completely
exhausted with strong alcohol, filtered, and the alcohol entirely evaporated
from the filtrate. The new residue is dissolved in water, and filtered if
necessary, and the solution precipitated by basic lead acetate and am-
monia. 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 Pettexkofer's
test. It is not necessary to wait for crystallization; but one must not
consider the crystals which form in the liquid as being positively crystal-
lized bile. It is also possible for needles of alkali acetate to be formed.
For the detection of bile-acids in urine see Chapter XV.
Bile-pigments. The bile-coloring matters known thus far are relatively
numerous, and in all probability there are still more of them. Most of the
known bile-pigments are not found in the normal bile, but occur either in
postmortem bile or principally in the bile concrements. The pigments which
occur imder physiological conditions are the reddish-yellow bilirubin, the
green biliverdin, and sometimes also urobilin (and urobilinogen) or a closely
related pigment. The pigments found in gall-stones are (besides the
bilirubin and biliverdin) choleprasin, bilijuscin, biliyrasin, bilihumin,
bilicyanin (and choletelin?) . Besides these, others have been noticed
in human and animal bile by various observers. The two above-men-
tioned physiological pigments, bilirubin and biliverdin, are those which
serve to give the golden-yellow or orange-yellow or sometimes greenish
color to the bile; or when, as is most frequently the case in ox-bile, the
' Zeitschr. f. physiol. Chem., 3(5. - Investigations not published.
320 THE LIVER.
two pigments are present in the bile at the same time, they produce the
different shades between reddish brown and green.
Bilirubin. This pigment has the formula C16H18N2O3, or according
to Orndorff and Teeple 1 more correctly C32H36N4O6, and is designated
by the names cholepyrrhix, biliph.ein, bilifulvin, and ilematoidIxN.
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 occurs also in the contents
of the small intestine, in the blood-serum of the horse, in old blood extrav-
asations (as hsematoidin), and in the urine and the yellow-colored tissue
in icterus. It is converted into hydrobilirubin, C32H40N4O7 (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 Vanlair^). 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
hsematinic-acid imide as an oxidation product (Kxjster). On reduction
v\'ith zinc powder or with nascent HI, it yields hgemopyrrol according to
Orndorff and Teeple.^
Bilirubin is sometimes amorphous and sometimes crystalline. The amor-
phous 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 bilirubin in chloroform to
evaporate spontaneously, are reddish-yellow, rhombic plates, whose obtuse
angles are often rounded. On crystallizing from hot dimethylaniline it
forms on cooling broad columns with both ends sharply cut (KiisTER*).
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 glycerine. It is
somewhat more soluble in alcohol. In cold chloroform it dissolves with
difficulty, and is much more readily soluble in warm chloroform. Its solu-
bility varies, and supersaturated solutions are readily formed (Orndorff
and Teeple). The varying solubility of bilirubin in chloroform depends,
' Salkowski's Festschrift, Berlin, 1904.
- Maly, Wien. Sitzungsber., 57, and Annal. d. Chem., 163; Masius and Vanlair,
Centralbl. f. d. med. Wissensch., 1871, 369.
M. c.
* Ber. d. d. chem. Gesellsch., 30 and 35, and Zeitschr. f. physiol. Chem., 47.
BILIRUBIX. 321
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 have
ing different solubilities. In cold dimethylaniline it dissolves in the propor-
t on of 1 : 100, and in hot dimethylaniline much more readily. Its solutions
show no absorption-bands, but only a continuous absorption from the red
to the violet end of the spectrum, and they have, even on diluting greatly
(.1:500 000), in a layer 1.5 cm. thick a decided yellow color. If a dilute
solution of alkali bilirubin in water is treated with an excess of ammonia
and then with a zinc-chloride solution, the liquid is first colored deep orange
and then gradually olive-broA\Ti and then green. This solution first gives
a darkening of the ^■iolet and blue part of the spectrum and then the bands
of alkaline cholecyanin (see below), or at least the binds 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 chloroform by shaking with
dilute caustic alkali (differing from lutein). Solutions 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, Orxdorff and
Teeple.i 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 not one bodv but
several are formed. Biliverdin is also formed from bilirubin by oxidation
under other conditions. A green coloring-matter similar in appearance is
formed by the action of other reagents such as CI, Br, and I. According to
Jolles,^ by the use of Hijbl's iodine solution biliverdin is produced, while
according to others (Thudichum. Maly ^) substitution products of bili-
rubin are formed.
Gmelin's Reaction for Bile-pigments. If one carefully pours under an
aqueous solution of alkali bilirubin nitric acid containing some nitrous acid,
there is obtained a series of colored layers at the juncture of the two liquids
in the following order from above downwards: green, blue, \-iolet, red.
' Ehrlich, Zeitschr. f. anal. Chem., 23; Proscher, Zeitschr. f. physiol. Chem., 39;
Omdorff and Teeple, 1. c.
' Kiist-er, Ber. d. d. chem. Gesellsch., 35; JoUes, Journ. f. prakt. Chem. (N. F.), 59,
and Pfliiger's -\rch., 75.
'Thudichum, Joum. of Chem. Soc. (2), 13, and Journ. f. prakt. Chem. (N. F.),
53; Maly, Wien. Sitzimgsber., 72.
322 THE LIVER.
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 not be present
in the liquid, because, as is well known, it gives a play of colors, in green
or blue, with the acid.
Hammarstex'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 centimetres
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 pro-
duced 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 hiliciianin and Stokvis calls cholecyanin, and which shows a
characteristic absorption-spectrum. The neutral solutions of this color-
ing-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 Jaff6, between
the lines C and E, separated from each other by a narrow space near D. A
third band between h and F is seen with difficulty. The next oxidation
step after these blue coloring-matters is a red pigment, and lastly a yellow-
ish-brown pigment, called choletelin by Maly, which in neutral alcoholic
solutions does not give any absorption-spectrum, but in acid solution
BILIVERDIN. 323
gives a band between b and F. On oxidizing cholecyanin with lead per-
oxide, Stokvis 1 obtained a product which he calls choletelin, which is
quite similar to urmary urobilin, to be discussed later.
Bilirubin is best prepared from gall-stones of oxen, these concretions
being very rich in calcium bilirubin. The finely powdered concrement is
first exhausted with ether and then with boiling water, so as to remove the
cholesterin and bile-acids. In order to remove the mineral constituents
it is better to use 10 per cent acetic acid instead of hydrochloric acid
(KusTER-). A green pigment is now removed by extraction with alcohol,
and the choleprasia is extracted with hot glacial acetic acid. After
washing with water it is dried, and extracted rej^eatedly with boiling
chloroform. The bilirubin separates from the chloroform as crtists. which
are treated once or twice in the above manner. It Ls then extracted ^^■ith
alcohol and precipitated from its chloroform solution by alcohol or crys-
tallized from dimethylaniline.
The chloroform solution which separates from the crusts of bilirubin eon-
tains, according to Kuster. a pigment related tobilirtibin. poorer in nitrogen,
also precipitable by alcohol, and verc- readily soluble in chloroform. This
has been substantiated by Orxdorff and Teeple.^- This pigment, according
to KfsTER. is a transformation product of bilirubin which is rich in chlorine.
The quantitative estimation of bilirubin may be made by the spectro-
photometric method, according to the steps suggested for the blood-
coloring matters.
Biliverdin, C16H18N2O4 or C32H36X4OS. 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 ven,- small
quantities.
Biliverdin is amorphous; at least it has not been obtained in well-
defined crv'stals. 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 dissolved by
alkalies. gi\"ing a brownish-green color, and this solution is precipitated by
acids, as well as by calcium, barium, and lead salts. Biliverdin gives
Huppert's, Gmelix's, and HA^nL^RSTEx'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 sul-
phide, the biliverdin may be reduced to bilirubin (Haycr.ift and
SCOFIELD 4).
' Heinsius and Campbell, Pfliiger's Arch., 4; Stok\-is, Centralbl. f. d. med. Wis-
eensch., 1872, 785; ibid., 1873, 211 and 449; Jaffe, ibid., 186S; Maly, Wien. Sitzungs-
ber., 59.
' Zeitschr. f. physici. Chem.. 47.
' Kuster, Ber. d. d. chem. Gesellsch., So; Omdorff and Teeple. 1. c.
♦Centralbl. f Physiol., 3, 222, and Zeitschr. f. physiol. Chem., 14.
324 THE LIVER.
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 HCl reaction is
obtained, then dissolved in alcohol and the pigment again separated by
the addition of water. Any bilirubin present may be removed by means
of chloroform. Hugounenq and Doyon ^ prepared biliverdin from bili-
rubin by the action of sodium peroxide and a little acid.
Choleprasin is a green pigment isolated by Kijster ^ 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, Kijster could not ob-
serve any formation of hsematinic acid.
Bilifuscin, so named by Stadeler,^ 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 C64Hgf,N70i4. Bilifuscin has been found in gall-stones. Biliprasin is
a green pigment prepared by Stadbler from gall-stones, which is generally con-
sidered as a mixture of biliverdin and bilirubin. Dastre and Floresco,* 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. Bilihurain 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 hippopotJimus, is, according to
Marchlewski, identical ^vith 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 * in the excrement of cows
fed on green grass.
Gmeijn'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-
' Arch, de Physiol. (5), 8.
^ Zeitschr. f. physiol. Chem., 47.
^ Cited from Hoppe-Seyler, Physiol, u. Path. chem. Analyse, 6. Aufi., p. 225.
* Zeitschr. f. physiol. Chem., 31.
« Arch, de Physiol. (5), 9.
• 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.
COMPOSITION OF THE BILE. 325
ingly. If blood-coloring matters are present at the same time, the bile-
coloring matters are first precipitated by the addition of sodium phosphate
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 Hamniarsten's reagent. Bilirubin is detected in blood,
according to Hedexius,^ by precipitating the proteins with alcohol, filtering
and acidifying the filtrate with hydrochloric or sulphuric acid, and boiling.
The liquid becomes of a greenish color. Serum and serous fluids may be
boiled directly with a little acid after the addition of alcohol.
Besides the bile-acids and the bile-pigments, there occur in the bile also
cholcsterin, lecithin, jecorin or other phosphatides (Ham.marstex), palmitin,
stearin, olein, myristic acid (Lassar-Cohx -), soaps, ethereal sulphuric acids,
conjugated glucuronates, diastatic and proteolytic enzymes. Choline and
glycerophosphoric acid, when they are present, may be considered as decom-
position products of lecithin. Urea occurs, though onh' 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.^ The mineral constituents of the bile are,
besides the alkalies, to which the bile-acids are united, sodium and potas-
sium chloride, calcium and magnesium phosphate, and iron — 0.04-0.115
p. m. in human bile, chiefly combined with phosphoric acid (Youxg^).
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 destruc-
tion of blood. According to Beccari ^ the iron does not disappear from
the bile in inanition, and the percentage shows no constant diminution.
The question as to the extent of elimination by the bile of the iron intro-
duced into the body has received various answers. There is no doubt
that the liver has the property of collecting and retaining iron as well as
other metals from the blood. Certain investigators, such as Xo\t and
KuxKEL, are of the opinion that the iron introduced and transitorily
retained in the liver is eliminated by the bile, while others, such as Ham-
' Upsala Lakaref. Forh., 29, and Maly's Jahresber., 24.
' Zeitschr. f. physiol. Chem., 17; Hammersten, ibid., 32, 36 and 43.
' Hammarsten, ibid., 24.
* Journ. of Anat. and Physiol., 5. 158.
^ Novi, see Maly's Jahresber., 20; Dastre, Arch, de Physiol. (5), 3; Beccari, Arch,
ital. de Biol., 28.
326 THE LIVER.
BURGER, Gottlieb, and Anselm,^ deny any such elimination of iron by
the bile.
Quantitative Composition of the Bile. Complete analyses of human bile
have been made by Hoppe-Seyler and his pupils. The bile was removed
as fresh as possible from the gall-bladder of those cadavers the livers of
which were in no sense pathological.
Older and less complete analyses of human bile have been made by
Frerichs and v. Gorup-Besanez.^ 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 IS-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 2.61 .-q ono
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. Hamalirsten, who had occasion to analj-ze the
liver-bile in seven cases of biliar}- 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 3 has observed
still higher figures, more than 40 p. m. in a couple of cases. This investi-
gator 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,^ 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
' Kunkel, Pfliiger's Arch., 14; Hamburger, Zeitschr. f. piiysiol. Chem., 2 and 4;
Gottlieb, zbid., 15; Anselm, ''Ueber die Eisenausscheidung der Galle," Inaug.-Diss.
Dorpat, 1891. See also the works cited in foot-note 3, p. 244.
= See Hoppe-Seyler, Physiol. Chem., 301; Socoloff, Pfliiger's Arch., 12; Trifanow-
ski, ibid., 9; Frerichs m Hoppe-Seyler 's Physiol. Chem., 299; v. Gorup-Besanez, ibid.
* Jacobsen, Ber. d. deutsch. chem. Gesellsch., 6; Hammarsten, Nova Acta Reg.
Soc Scient. Upsala, 16; Brand. Pfliiger's Arch. 90.
'Brand, 1. c ; Bonanni. Biochem. Centralbl., 1; Strauss, Berl. klin. Wochenschr.,
1903.
COMPOSITION OF THE BILE. 327
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.i
Human bile sometimes, but not always, contains sulphur in an ethereal
sulphuric-acid-like combination (Hammarsten, Oerum, Brand). The
quantity of such sulphur may even amount to J-"^ of the total sulphur.
We do not know the nature of these ethereal sulphuric acids. According
to Oerum ^ 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 relationship of taurocholic to glycocholic
acid varied between 1:2.07 and 1:14.36. The bile analyzed by Jacobsen
contained no taurocholic acid.
As an example of the composition of human liver-bile the following
results of three analyses made by Hammarstex are given. The results
are calculated in parts per 1000.3
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 ^ r,.-,^ 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 relationship between potassium and sodium varies
considerably in different samples. Sulphuric acid and phosphoric acid
occur only in very small quantities.
Bagixsky and Sommerfeld "* have 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. leucine.^
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 determining
' See Brand, 1. c; Hammarsten, 1. c.
2 Skand. Archiv f. Physiol., 16.
3 Recent quantitative analyses may be found in Brand, 1. c; v. Zeynek, Wien,
klin. Wochenschr., 1899; Bonanni, 1. c.
^ Verhandl. d. physiol. Gesellsch. zu Berlin, 1894-95.
* Analyses of bile from children may be found in Heptner, Maly's Jahresber., 30.
328 THE LIVER.
the pigments in this case was not quite trustworthy. More exact results
obtained by spectrophotometric methods are on record for dog-bile.
According to Stadelmaxx ^ dog-bile contains on an average 0.6-0.7 p. m.
bilirubin. At the most only 7 milligrams of pigment are secreted per kilo
of body in the twenty-four hours.
In animals the relative proportion of the two acids varies considerably.
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 car-
nivorous mammals, birds, snakes, and fishes. Among the herbivora,
sheep and goats have a predominance of taurocholic acid in the bile. Ox-
bile sometimes contains taurocholic acid in excess, in other cases glyco-
cholic acid predominates, and in a few cases the latter occurs almost alone.
The bile of the rabbit, hare, kangaroo, hippopotamus, and orang-utang
(Hammarsten^) contains, like the bile of the pig, almost exclusively glyco-
eholic acid. A distinct influence on the relative amounts of the two bile-
acids exerted by differences in diet has not been detected. Ritter ^ 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-salts it must be remarked that no exact con-
clusion 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 com-
pounds other than taurocholic acid.*
The phosphorized constituents of bile are not well known; nevertheless^
there is no doubt that bile contains other phosphatides besides lecithin
(Hammarstex). 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 (Ham-
marsten^).
The cholesterin, which, according to several investigators, not only origi-
nates from the liver, but also from the biliary passages, occurs in larger
quantities in the bladder-l)ile than in the liver-bile, and is present to a
' Noel-Paton, Rep. Lab. Roy. Soc. Coll. Phys. Edinburgh, 3; Stadelmann, Der
Icterus.
^ Investigations not published. See Ergebnisse der Physiol., 4.
' Cited from Maly's .Jahresber., 6, 195.
* Hammarsten, Zeitschr. f. physiol. Chem., 32, and Ergebnisse der Physiol., 4.
' Zeitschr, f. physiol. Chera., 36, and Ergebnisse der Physiol., 4.
CHEMICAL FORMATION OF THE BILE. 329
greater extent in the non-filtered than in the filtered bile (Doyon and
DUFOURT ').
The gases of the bile consist of a large quantity of carbon dioxide, which
increases with the amount of alkalies, only traces of oxygen, and a very
small quantity of nitrogen.
Little is known in regard to the 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 Rittee he found a fatty degeneration of the liver-cells, in re-
turn for which, even in excessive fatty infiltration, a normal bile containing pig-
ments 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 ^). Albumin can pass into the bile after the intra-
venous injection of a foreign protein (casein) (GtfRBER and Hallauer), as well
as after poisoning with phosphorus or arsenic (Pilzecker), or after the irrita-
tion of the liver by the introduction of ethyl alcohol or amyl alcohol (Brauer).
Sugar occurs in bile only in exceptional cases.*
The physiological secretion of the gall-bladder is according to Wahl-
gren^ in man a viscous, alkaline fluid with 11.24-19.63 p. m. solids The
mucilaginous properties are not due to mucin but to a phosphorized pro-
tein substance (nucleoalbumin or nucleoproteid).
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.^
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 investi-
gations of the blood of the portal and hepatic veins under normal condi-
tions, have not given any answer to this question. To decide this, therefore,
* Arch, de Physiol. (5), 8.
^ Ritter, Compt. rend., 74, and Journ. de I'anat. et de la physiol. (Robin), 1872;
Hoppe-Seyler, Physiol. Chem., 317.
' Wertheimer and Meyer, Compt. rend., 108; Filehne, Virchow's Arch., 121; Stern,
ibid., 123.
* Giirber and Hallauer, Zeitschr. f. Biologie, 45; Pilzecker, Zeitschr. f. physioL
Chem., 41; Brauer, ibid., 40.
' See Maly's Jahresber., Z2.
'Winternitz, Zeitschr. f. physiol. Chem., 21; SoUmann, Amer. Medicine, 5 (1903)
330 THE LIVER.
it is necessary to extirpate the liver of animals or to isolate it from the
circulation. If the bile constituents are not formed in the liver, or at least
not alone in this organ, but are eliminated only from the blood, then, after
the extirpation or removal of the liver from the circulation, a:i 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 experiments
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
afte • extirpation of the liver, he was able to discover them on tying the
ductus choledochus. The investigations of Ludwig and I leischl i show
that in t' e 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
Avhen 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 older statements of Cloez and Vulpian, as well as Virchow, the bile-
acids also occur in the suprarenal capsule. These statements have not been
confirmed by later investigations of Stadelmann and Beier.^ 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 proved that the bile-pigments may be formed in
other organs besides. the liver, for, as is generally admitted, the coloring-
matter hsematoidin, which occurs in old blood extravasations, is identical
with the bile-pigment bilirubin (see page 320). LATsckENBERGER^ has
also observed in horses, under pathological conditions, a formation of bile-
pigments from the blood-coloring matters in the tissues. Also the occur-
rence of bile-pigments in the placenta seems to depend on their formation
in that organ, while the occurrence of small quantities of bile-pigments in
the blood-serum of certain animals probably depends on an absorption of
these substances.
* Kobner, see Heidenhain, Physiologie tier Absonderungsvorgange, in Hermann's
Handbuch, 5; FleiscU, Arbeiten aus der physiol. Anstalt zu Leipzig, Jahrgang 9.
' Zeitschr. f. physiol. Chem., IS, in which the older literature may be found.
^ See Maly's Jahresber., 16, and Monatshefte f. Chem., 9.
CHEMICAL FORMATION OF THE BILE. 331
Although the bile-pigments may be formed in other organs besides the
liver^ stin it is of first importance to know what bearing this organ has on
the elimination and formation of bile-pigments. In this regard it must be
recalled that the liver is an excretory organ for the bile-pigments circulat-
ing in the blood. Tarchanoff has 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 confirmed lately
by the investigations of Vossius.^
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 pas-
sages, 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. Min-
kowski and Naunyn^ have also found that poisoning with arseniuretted
hydrogen produces a liberal formation of bile-pigments 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 physiological con-
ditions.
In regard to the materials from which the bile-acids are produced, it
may be said with certainty that the two components, glycocoU and taurine,
which are both nitrogenized, are formed from the protein bodies. The
close relationship of taurine to the cystine group of the protein molecule
has been especially shown by the investigations of Friedmann (see Chapter
II), and very recently v. Bergmann ^ has shoT\Ti by feeding do^s with sodium
cholate and cystine that the animal body can transform 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-nitrogenized cholic acid, which was formerly
considered as originating from the fats, nothing is known positively.
The blood-coloring matters are considered as the mother-substances of
the bile-pigments. If the identity of haematoidin and bilirubin was settled
beyond a doubt, then this view might be considered as proved. Independ-
ently, however, of this identity, which is not admitted by all investigators,
the view that the bile-pigments are derived from th^ blood-coloring matters
has strong arguments in its favor. It has been shown by several experi-
• Tarchanoff, Pfliiger's Arch., 9; Vogsius, cited from Stadelmann, Der Icterus.
^ Stern, Arch. f. exp. Path. u. Pharm., IJ); Minkowski and Naunyn, ibid., 21.
^ Hofmeister's Beitrage, 4. See also Wolilgemuth, Zeitsclir. f. physiol. Chem., 40o
332 THE LIVER.
menters that a yellow or yellowish-red pigment can be formed from the
blood-coloring matters, which gives Gmelin's test, and which, though it
may not form a complete bile-pigment, is at least a step in its formation
(Latschenbekger). a further proof of the formation of the bile-pigments
from the blood-coloring matters consists in the fact that hsematin on reduc-
tion yields urobilin, which is identical with hydrobilirubin (see Chapter
XV). Further, hsematoporphyrin (see page 212) 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 obser-
vations of several investigators,^ by 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 hgemoglobin solution,
causing an increased formation of bile-pigments. The amount of pig-
ments 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 ^ observed in the secre-
tion of pigments by the bile an increase of 61 per cent, which lasted for
more than twenty-four hours.
If bilirubin, which contains no iron, is derived from haematin, which con-
tains iron, then iron must be split off. This process may be represented by the
following formula, C32H34N405Fe + H20-Fe = C32H36N406. 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 Kun-
kel; while 100 parts haematin contain about 9 parts iron. Minkowski
and BaserIxN ^ have also found that the abundant formation of bile-pigments
occurring in poisoning by arseniuretted hydrogen does not increase the
quantity of iron in the bile. The quantity apparently does not seem to
correspond with that in the decomposed blood-coloring matters. It follows
from the researches of several investigators ^ that the iron is, at least chiefly,
retained by the liver as a ferruginous pigment or protein substance.
What relationship does the formation of bile-acids bear to the forma-
tion of bile-pigments? Are these two chief constituents of the ])ile derived
simultaneously from the same material, and can we detect a certain connec-
' See Stadelmann, Der Icterus, etc. Stuttgart, 1891.
•" Sec Stade mann ibid.
' Kunkel, Pfluger's Arch., 14; Minkowski and Baserin, Arch. f. exp. Path. u.
Pharm., 23.
* See Naunyn and Minkowski, Arch. f. exp. Path. u. Pliarm., 21; Latschenberger,
1. c.; Neu-Tiann, Virchow's Arch. Ill, and the hterature in foot-note 2 p 2 2.
BILE CONCRETIONS. 333
tion between the formation of biliruljiu 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 produc-
tion of bile-acids. According to Stadelmann the formation of bile-
pigments and bile-acids is duo to a special activity of the cells.
An absorption of bile from the liver and the passage of the bile con-
stituents into the Vjlood 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, especially when
in animals a solution or destruction of the red blood-corpuscles takes
place 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 occurs also in man in severe
infectious diseases. It has also been claimed many times that a transfor-
mation of blood-pigments into bile-pigments occurs elsewhere than in the
liver, namely, in the blood. Such a belief has been made very improb-
able by the important researches of Minkowski and Naunyn, Afanassiew,
SiLBER\L^NN, and especially of Stadelmann, ^ and in some of the above-
mentioned cases, as after poisoning with phosphorus, toluylenediamine, and
arseniuretted hydrogen, it has been disproved by direct experiment.
The icterus is also in these cases hepatogenic ; it depends upon an absorp-
tion 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 gall-
stones contains lime-pigment as chief constituent, another cholesterin,
and the third calcium carbonate and phosphate. The concrements of the
last-mentioned group occur very seldom in man. The so-called cholesterin-
stones are those which occur most frequently in man, while the lime-pig-
ment stones are not found very often in man, but often in oxen.
' The literature belonging to this subject is found in Stadelmann, Der Icterus, etc.,
Stuttgart, 1891.
334 THE LIVER.
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 the}- consist chiefly of calcium bilirubin with little or no bili-
verdin. Sometimes also small black or greenish-black, metallic-looking
stones are found, which consist chiefly of bilifuscin along with biliverdin.
Iron and copper seem to be regular constituents of pigment-stones. Man-
ganese and zinc have also been found in a few cases. The pigment-stones
are generally heavier than water.
The choksterin-stones, whose size, form, color, and structure may vary
greatly, are often lighter than w^ater. 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 waxlike, 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 9S1 p. m. (Ritter ^). The cholesterin-stones also sometimes contain
variable amomits of lime-pigments, which may give them a very changeable
appearance.
Cholesterin, C27H46O (Obermuller), or, as ordinarily given, C27H44O
(Mauthner and Sum a). By the action of concentrated sulphuric acid
or phosphoric acid, and also in other wa3's, hydrocarbons are obtained,
which are called cholesteriline, cholesterone, and cholesterilene. Mauthxer
and SuiDA,2 -^y^q have closely studied these hydrocarbons, have been able
to prepare a crystalline cholesteriline by heating cholesterin with anhydrous
copper sulphate. The hydrocarbons stand, according to Weyl,^ in close
relationship to the terpene group, and the color reactions of cholesterin
as well as the recent investigations on the constitution of this body seem
to substantiate this view^ Very painstaking and thorough investigations
on the constitution of cholesterin have been made, of which we must
especially mention those of Mauthner and Suida, Windaus and Stein,
DiELs and Abderhalden.* Although these researches have not lead to
positive conclusions, still we are justified in concluding that cholesterin prob-
ably consists of a complex of five hydrogenized rings, of which one con-
> Journ. de I'anat. et de la physiol. (Robin), 1872.
^ Obermuller, Arch. f. (Anat. u.)Fhysiol., 1889, and Zeitschr. f. physiol. Chem., 15;
Mauthner and Suida, Wien. Sitzungsber., Math. Nat. Klasse, 103, Abt. 26, which also
contains the older literature.
' Arch. f. (Anat. u.) Physiol, 1886, p. 182.
* Mauthner and Suida, Monatshefte f. Chem., 15, 17, 24; Windaus, "tjber Choles-
terin." Hab.-Schrift, Freiburg i. B., 1903, Ber. d. d. chem. Geselkch., 36, 37, and
39; with Stein, ibid., 37; Diels and Abderhalden, ibid., 36, 37, and 39; G. Stein,
"tJber Cholesterin," Inaug.-Dissert. Freiburg i. B., 1905.
CHOLESTERIN. 335
tains a double bondage and another a secondary alcohol group. Several
facts seem to make it probable that cholesterin stands close to the
hydrogenized retene and hence is a complicated terpene. From this
standpoint, the close relationship between cholesterin and cholic acid is of
great interest.
On reduction of cholesterin, and also of the ketone, cliolesterone, corre-
sponding to cholesterin, by means of metallic sodium in amyl-alcohol solu-
tion, DiELS and Abderhalden obtained two isomeric dihydrocholes-
tcrincs, C27H48O, a- and ^-cholestanol, of which the first seems to be identical
with he dihydrocholesterin obtained by Neuberg and Rauchwerger 1
by the action of sodium in amyl-alcohol solution. The identity of a-choles-
tanol with koprosterin, which will be mentioned below, is considered by
Neuberg as not improbable, while Diels and Abderhalden deny this.
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 abun-
dant 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
appears also 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 exam-
ple, it exists in part as fatty-acid esters in wool-fat, blood, lymph, brain
vemix caseosa, and epidermis formations. Several kinds of cholesterin,
called phytosterines, have been found in the vegetable kingdom.
Cholesterin which has been crystallized from warm alcohol on cooling
and that which is present in old transudates contains 1 molecule of water of
crystallization, melts at 145° C, 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 feeling.
Cholesterin is insoluble in water, dilute acids, and alkalies. It is neither
dissolved nor changed by boiling caustic alkali. It is easily soluble in boil-
ing alcohol and crystallizes on cooling. It dissolves readily in ether,
chloroform, and benzene, and also in the volatile or fatty oils. It is dis-
solved to a slight extent by alkali salts of the bile-acids, better in the pres-
ence of oleic soap (Gerard 2), The solutions in ether and chloroform are
levorotatory.
Among the many compounds of cholesterin studied by Obermuller
the propionic ester C2H5.CO.O.C27H45 is of special interest because of the
' Salkowski's Festschrift, 1904, and Neuberg, Ber. d. d. chem. Gesellsch., 39.
' Compt. rend. soc. biolog., 58.
336 THE LIVER.
behavior of the fused compound on cooUng, and it is used in the detection
of cholesterin. 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
a cholesterin crj'stal, this crystal will shov/ colored rings, first a bright
carmine-red and then violet. This fact is employed in the microscopic
detection of cholesterin. Another test, and one verj^ good for the micro-
scopical detection of cholesterin, consists in treating the crj-stals first with
the above dilute acid and then with some iodine solution. The crystals
wi.l be gradually colored violet, bluish green, and a beautiful blue.
Salkowski's ^ 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 fluor-
escence. If the chloroform solution is poured into a porcelain dish it
becomes violet, then green, and finally yellow.
LiEBERiLA.Nx-BuRCH.A_RD's ^ Reaction. Dissolve the cholesterin in about
2 c.c. chloroform and add first 10 drops of acetic anhydride and then con-
centrated 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 presence of very
little cholesterin the green color may appear immediately,
Neuberg Rauchwerger's ^ Reaction. With rhanmose, or better still
with o-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 h. This
reaction is of interest because it is also given by bile-acids, some camphor
derivatives, abietinic acid, and a hydride of retene. For details of its
performance, see original publication.
Pure, dry cholesterin when fused in a test-tube over a low flame with two or
three drops of propionic anhydride yields a mass which on cooling is first violet,
then blue, green, orange, carmine-red, and finally copper-red. It is best to re-fuse
the mass on a glass rod and then to observe the rod on cooling, holding it against
a dark background (Obermuller).
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.
* Pfliiger's Arch., 6.
' C. Liebermann, Bar. d. deutsch. chem. Gesellsch., 18, 1804; H. Burchard, Bei-
trage zur Kenntnis der Cholesterine. Rostock, 1889.
' Salkowski's Festschrift, 1904.
CHOLESTEP.LX. 337
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 faeces, 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
<)5-96°C. (89-90° according to Hausmanx),i and is dextrorotatory, (a)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 C07H48O, which is formed
in the human intestine by the reduction of ordinary cholesterin. These investi-
gators have found another cholesterin, hippokoprosterin, with the formula C27H54O,
in horses' faeces.
Isocholesterin is a cholesterin, so called by Schulze,^ with the formula
C2«H430H, which occurs in wool-fat and is therefore found to a great extent in
so-called lanolin. It gives the Liebermann-Burcharo reaction, but does not
give Salkowski's reaction. It melts at 138-138.5° C.
Spongosterin is the name givenby Henze ^ 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.
25
Obermijller's reaction is negative. Its specific rotation is («)^ = 19.59°.
The cholesterins belong to the so-called lipoids, which have been men-
tioned in previous chapters (V and VI) and are of the greatest importance
as constituents of the outer envelope of erj'throcytes and the cells in
general. In this regard the cholesterin is of special interest for haemolysis,
in that, as shown by Ransom, it inhibits the hsemolytic action of saponin
and hence it has a certain protective power in the animal body. This
action of cholesterin, as found by Haus\la.nn, is destroyed by replacing
the hydroxyl groups. According to Madsen and Noguchi ^ the combina-
tion of cholesterin and saponin is a loose one.
The so-called cholesterin-stones are best emplo3'ed in the preparation
of cholesterin. The powder is first boiled with water and then repeatedly
boiled with alcohol. The cholesterin which on cooling separates from the
"warm filtered solution is boiled with a solution of caustic potash in alcohol
so as to saponify any fat. After the evaporation of the alcohol the choles-
terin is extracted from the residue with ether, by which the soaps are not
' Bondzynski, Ber. d. deutsch. chera. Gesellsch., 29; Bondzynski and Humnicki,
Zeitschr. f. physiol. Chem., 22; Flint, ibid., 23, and Amer. Journ. Med. Sciences, 1862;
Miiller, Zeitsclir. f. physiol. Chem., 29; Hausmann. Hofmeister's Beitrage, 6.
^ Ber. d. deutsch. chem. Gesellsch., 6; Journal f. prakt. Chem. (N. F.), 25; and
Zeitschr. f. physiol. Chem., 14, 522. See also E. Schulze and J. Barbieri, Journal f.
prakt. Chem. (N. F.),2o, 159. In regard to the formula for isocholesterin, see Darm-
stadter and Lifschiitz, Ber. d. deutsch. chem. Gesellsch., 31, and E. Schulze, ibid., 1200.
^ Zeitschr. f. physiol. Chem., 41.
♦Ransom, Deutsch. med. Wochenschr., 1901; Hausmann, Hofmeister's Beitrage,
6; Madsen and Noguchi, Kgl. Dansk. Vidensk. Selskabs. Forh., 1904.
338 THE LIVER.
dissolved; filter, evaporate the ether, and purify the cholesterin by
recrj'stallization from alcohol-ether. The cholesterin may be extracted
with fat from tissues and fluids by first extracting with ether and then
proceeding as suggested by Ritter.i The essential points in his method
consist in saponifying the fat with sodium alcoholate, removing the alcohol
by evaporating to dryness with NaCl, and finally extracting the dried,
pulverized mass with ether. After evaporating the ether the residue is
dissolved in as little alcohol as possible and the cholesterin precipitated
b}' the addition of water. It is ordinarily easily detected in transudates
and pathological formations by means of the microscope.
* Zeitschr. f. physiol. Chem., 34.
.CHAPTER IX.
DIGESTION.
The purpose of digestion is to separate those constituents of the
food which serve as the nutriment of the body from those which are useless,
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, wliich is essentially dependent upon the physical properties
of the food, con ists 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 absorbed 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 standpoint must there-
fore 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 sublingual and sub-
maxillary glands of many animals), and partly mixed glands (as the sub-
maxillary gland in man). The alveoli of the albuminous glands contain cells
which are rich in proteid but which contain no mucin. The alveoli of the
mucin-glands contain cells rich in mucin but poor in proteid. Cells arranged
in different ways, but rich in proteids, also occur in the submaxillary and
sublingual glands. According to the analyses of Oidtmann ^ the salivary
glands of a dog contain 790 p. m. water, 200 p. m. organic and 10 p. m.
» Cit. from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Anfl., 732. The figiires
tliere given amount to 1010 parts instead of 1000 parts.
339
340 DIGESTION.
inorganic solids. Among the solids we find mucin, proteids, nucleoproteids,
nuclein, enzymes and their zymogens, besides extractive bodies, leucine, xan-
thine bodies, and mineral substances.
The occurrence of a mucinogen has not been proved. On the complete removal
of all mucin E. Holmgren ' 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 differ-
ent secretions by itself and then of the mixed saliva.
The submaxillary saliva in man may be easily collected by introducing
a canula through the papillary opening into Wharton's duct.
The submaxillary saliva has not always the same composition or prop-
erties; 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
fibres in the chorda tympani, and partly on the sympathetic nervous system,
through the fibres 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) con-
sists chiefly in their quantitative constitution; the less abundant sym-
pathetic 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 (Eck-
HARD^). 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,^ 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,"* have also found that the osmotic
pressure of the submaxillary saliva is considerably lower than that of the
blood. The gases of the chorda-saliva have been investigated by PFLtJGER.^
He found 0.5-0.8 per cent oxygen, 0.9-1 per cent nitrogen, and 64.73-85.13
' Upsala Lakaref. Forh. (N. F.), 2; also Maly's Jahresber., 27.
' Cited from Kijhne's Lehrb. d. physiol. Chem., 7
See Maly's Jahresber., 31, 494.
* Zeitschr. f. Biologie, 40.
* Pfliiger's Arch., 1.
PAROTID SALIVA. 341
per cent carbon dioxide — all results calculated at 0° C. and 760 mm. pres-
sure. The greater part of the carbon dioxide ^vas chemically combinetl.
The two kinds of submaxillary secretion just named have not thus
f^r 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 towards litmus. The specific gravity is 1.002-1.003,
and it contains 3.6-4.5 p. m. solids.^ As organic constituents are found
mucin, traces of proteid 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 cor-
puscles, but is otherwise transparent and very ropy. Its reaction is
alkaline, and it contains, according to Heidexhain,- 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 b}' the method
suggested by Bidder and Schmidt, which consists in tying the exit to all
the large salivar}- glands and cutting off their secretion from the mouth.
The quantity of liquid secreted under these circumstances (in dogs) was
so ver\^ 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 epi-
thelium-cells, mucous cells, and salivary corpuscles. The quantity of solids
in the buccal mucus of the dog is, according to Bidder and Schmidt,^
9.98 p. m.
Parotid Saliva. The secretion of this saliva is also partly dependent
on the cereljral nervous system (n. glossopharyngeus) and parti}- 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. Mastication also-
'See Maly, "Chemie der Verdauungssafte iind der Verdauung." in Hermann's
Handb., 5, part II, 18. This article contains also the pertinent literature.
2 Studien d. phyeiol. Instituts zu Breslau, Heft 4.
^ Die Verdauungssafte und der Stoffwechsel (Mitau and Leipzig, 1852), 5
342 DIGESTION.
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 Stexson'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 proteid but no mucin, which is to
be expected from the construction of the gland. It also contains a dias atic
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. Potas-
sium sulphocyanide seems to be present, though it is not a con .ant con-
stituent. KiJLz 1 found a maximum of 1.46 p3r 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^ school has shown, is
greatl}' 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 abundance of
saliva, while with food rich in water the secretion is considerably less and
accommodates itself according 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 condition. By the action of
strong chemical bodies the saliva is secreted in proportion to the strength
of the irritant. The irritants are thereby diluted and the mouth washed
■out at the same time. The partaking of acids brings about the secre-
tion of a thin saliva, poor in mucin, in quantities sufficient to neutarl-
ize the acid, while on the introduction of food the gland i secrete a saliva
rich in mucin and diastatic enzymes.
The mixed buccal saliva in man is a colorless, faintly opalescent, slightly
ropy, easily frothing liquid w'ithout 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
» Zeitschr. f. Biologie, 23.
^ Arch, intemation. de Physiol., 1, 1904. See also Neilson and Terry, Amer. Journ.
of Physiol., 15. Somewhat contradictory statements in regard to the accommoda-
tion of the secretion of the glands to requirements (in man) can be found in Zebrovski,
Pflijger's Arch., 110.
PTYALIN. 343
considerably not only in different individuals but also in the same indi-
vidual during different parts of the day, so that it is difficult to state the
average alkalinity. According to Chittenden and Ely it corresponds to
the alkalinity of 0.8 p. m. Na2C03 solution, or to 0.2 p. m, solution accord-
ing to CoHN. According to Foa the actual alkalinity ("OH-ion concentra-
tion) is always considerably less than that found by titration, and the
reaction determined electrometrically is very nearly neutral. The reac-
tion may also be acid, as found by Sticker to be the case some time after
a meal, but this is not true at least for all individuals. The specific gravity
varies between 1.002 and 1.008, and the quantity of solids between 5
and 10 p. m. According to Cohn i, J =—0.20° on an average and the
amount of NaCl is 1.6 p. m. The solids, irrespective of the form-constitu-
ents mentioned, consist of 'protein, mucin, oxidases,^ two enzymes, ptyalin
and maltase, and mineral bodies. It is also claimed that urea is a normal
constituent of the saliva. The min ral bodies are alkali chlorides, bicar-
bonates 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
he saliva of non-smokers (Schneider and KRiJGER), while from ordinary
smokers the quantity of sulphocyanides may rise to 0.2 p. m. (Fleck-
seder 3),
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 ^ method.
Ptyalin, or salivarj^ diastase, is the amylolytic enzyme of the saliva.
This enzyme is found in human saliva,^ but not in that of all animals,
' Chittenden and Ely, Amer. Chem. Joum., 4, 1883; Chittenden and Richards,
Amer. Joum. of Physiol., 1; Foa, Compt. rend. ^c. biolog., 58; Sticker, cited from
Centralbl. f. Physiol., 3, 237; Cohn, Deutsch. med. Wochenschr., 1900.
^ Bogdanow-Beresowski, cited from Biochem. Centralbl., 2, 653.
'Munk, Virchow's Arch., 69; Schneider, Amer. Journ. of Physiol., 5; Kriiger,
Zeitschr. f. Biologie, 37; Fleckseder, Centralbl. f. innere Med., 1905. lu regard to
the variation in the amoimt of various constituents in saliva see Fleckseder, 1. c, and
Tezner, Arch, intemation. de Physiol., 2.
* Gscheidlen, Maly's Jahresber., 4; Solera, see ibid., 7 and 8; Munk, Virchow's
Arch., 69; Ganassini, Biochem. Centralbl., 2, p. 361.
' In regard to the variation in the quantity of ptyalin in himian 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.
344 DIGESTION.
especially not in the typical carnivora. It occurs not only in adults, but
also in new-bom infants. In opposition to Zweifel's views, Berger ^
claims that it is present not only in the parotid gland of children, but also
in the mucin gland.
According to H. Goldschmidt ^ 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
ran be obtained purest by Cohnheim's^ method, which consists in carry-
ing the enzyme down mechanically with a calcium-phosphate precipitate
and washing the precipitate with water, which dissolves the ptyalin, and
from which it can l)e obtained by precipitating with alcohol. For the
study or demonstration of the action of ptyalin one employs a watery or
glycerine extract of the salivary glands, or simply the saliva itself.
Ptyalin, like other enzymes, is characterized by its action. This con-
sists in converting starch into dextrins and sugar. The process going on
in this conversion may be described as follows: In the first stages soluble
starch or amidulin is formed. From this amidulin, er3-throdextrin and
sugar are produced by hydrolytic cleavage. The erythrodextrin then splits
into a-achroodextrin and sugar. From this achroodextrin by splitting
^-achroodextrin and sugar are formed, and finally this /9-achroodextria
splits into sugar and pachroodextrin. Other investigators explain this
process in another manner (see Chapter III), hence the exact procedure is
not completely clear. Still the results are positive as to the sugar pro-
duced in this process. For a long time it was considered that dextrose was
the sugar formed from starch and glycogen, but Seegen and 0. Nasse have
shown that this is not true. Musculus and v. Mering have shown that
the sugar formed b}^ the action of saliva, amylopsin, and diastase ujDon
starch and glycogen is for the most part maltose. This has been substan-
tiated by Brown and Heron. E. Kulz and J. Vogel ^ have also demon-
strated that in the saccharification 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
1 Zweifel, Untersuchungen iiber den Verdauungsapparat der Neugeborenen (Berlin,
1874); Berger, see Maly's Jahresber., 30, 309.
2 Zeitschr. f . physiol. Chem., 10.
3 Virchow's Arch., 28.
* Seegen, Centralbl. f. d. med. Wissensch., 1876, and Pfliiger's Arch., 19; Nasse
ibid., ll; Musculus and v. Mering, Zcitscl\r. f. physiol. Chem., 2; Brown and Heron,
Liebig's Annal., 199 and 204; Kiilz and Vogel, Zeitschr. f. Biologie, 31
ACTION OF PTYALIN. 345
dextrose is claimed by Tebb, Rohmann, and Hamburger ^ to be only a
product of the inversion of the maltose by the maltase.
The action of ptyalin in various reactions has been the subject of numer-
ous invest igations.2 Natural alkaline saliva is very active, but it is not
so active as when made neutral. It may be still more active under cer-
tain circumstances in faintly acid reaction, and according to Chittenden
and Smith it acts better when enough hydrochloric acid is added to satu-
rate the proteins present than when only neutralized. \Yhen 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.). HyHro-
chloric acid has not only the property of preA'enting 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. 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 experi-
menters have not made use of the different times required to produce equal
chemical changes as a measure of the velocit}', but have taken the quan-
tities of substance changed in equal times. Although the results are some-
what divergent it is possible to deduce the following from them. The
velocity increases, at least under conditions othenvise favorable, with the
amount of enzyme and with an increasing temperature to a little above 40° C.
^Tebb, Journ. of Physiol., 15; Rohmann, Ber. d. deutsch. chem. Gesellsch., 27;
Hamburger, Pflager's Arch. , 60.
2 See Hammarsten, Maly's Jahresber., 1; Chittenden and Griswold, Amer. Chem.
Jomn., 3; Langley, Journal of Physiol., 3; Nylen, Maly's Jahresber., 12, 241; Chit-
tenden and Ely, Amer. Chem. Journ., 1; Langley and Eves, Journal of Physiol., 4;
Chittenden and Smith, Y.ile College Studies, 1, 1885, 1; Schlesinger, Virchow's Arch..
125; Schierbeck, Skand. Arch. f. Physiol., 3; Ebstein and C. Schulze, Virchow's Ari'h.
134; Kubcl. Pfliiger's Arch., 76.
346 DIGESTION.
Foreign substances, such as metallic salts,i 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
Other salts, such as magnesium sulphate, in small quantities (0.25 p. m.)
accelerate, and in larger quantities (5 p. m.) check the action. The presence
of peptone has an accelerating action on the sugar formation (Chittenden
and Smith and others). The accumulation of the products of the amyloiytic
decomposition also checks the action of the saliva. This has been shown by
special experiments made by Sh. Lea.^ He made parallel experiments
with digestions in test-tubes and in dialyzers, and found on the removal of
the products of the amyloiytic decomposition by dialysis that the forma-
tion 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
the bismuth test (see Chapter XV). It is also necessar}^, as a control, to
first t€st the starch-paste and the saliva for the presence of dextrose. The
steps in he transformation of starch into amidulin, erythrodextrin, and
achroodextrin may be shown by testing with iodine.
Maltose occurs in saliva to only a slight extent. It converts maltose
into dextrose. According to Sticker 3 saliva also has the power of splitting
sulphuretted hydrogen from the sulphur oils of radishes, onions, and cer-
tain other vegetables.
The quantitative composition of the mixed saliva must vary considerably,
not only because of individual differences, but also because under varying
conditions there may be an unequal division of the secretion from the
different glands. We give opposite a few analyses of human saliva as
examples of its composition. The results are in parts per 1000.
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 (P2O3) 188.48, and chlorine 183.52.
The quantity of saliva secreted during twenty-four hours cannot be ex-
actly 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 *, 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
* See O. Xasse, Pfliiger's Arch., 11, and Chittenden and Painter, Yale College
Studies, 1, 1885, 52; Kubel, Pfliiger's Arch., 76.
' Joum. of Physiol., 11.
* Miinch. med. >/ochenschr., 43.
♦Bidder and Schmidt, 1. c, 13; Tuczek, Zeitschr. f. Biologie, 12.
COMPOSITION OF THE SALIVA.
347
the average secretion from 1 gram of gland in animals, namely, 14.2 grams
in the horse and 8 grams in oxen. The quantity of secretion per hour
may be 8 to 14 times greater than the entire mass of glands, and there is
probably no gland in the entire body, so far as is known at present — the
kidneys not excepted — whose ability of secretion under physiological con-
*
p
s
m
X
o
a
p
o
■<
1-5
IS
a
o
S
fa
•V
c
si
< a
u
H
a
a
X
z
z
■<
s
a
►J
Water
992.9
7.1
1.4
3.8
995 . 16
4.84
1.62
1.34
0.06
1.82
994.1
5.9
2.13
1.42
0.10
2.19
988.3
11.7
994.7
5.3
3;5-8'4
in
filtered
.saliva.
994 2
Solids
5 8
Mucus and epithelium
2.2
Soluble organic substances .
(Ptyalin of early investigators.)
Sulphocyanides
3.27
0.064
to
0.090
1.4
0.04
Salts
1.9
1.30
2.2
ditions equals that of the salivary glands. A remarkably abundant secre-
tion of saliva is induced by pilocarpine, while atropine, on the contrary,
prevents 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 from
many reasons, especially the following: The salivary glands have,
a specific property of eliminating certain substances, such as potas-
sium 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 in-
creased 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, Laxgley and Fletcher, Novi ^).
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.
' Zeitschr. f. physiol. Chem., 5. The other analyses are cited from Maly, Chemie
der Verdauungssafte, Hermann's Handbuch d. Physiol., 5, part II, 14.
^ Virchow's Arch., 53.
3 Heidenhain, Pfliiger's Arch., 17;. Werther, ibid., 38; Langley and Fletcher,
Proc. Roy. Soc, 45, and especially Phil. Trans. Roy. Soc. London, 180; Novi,
Arch. f. (Anat. u.) Phsyiol., 1888.
348 DIGESTION.
The Physiological Importance of the Saliva. The quantity of water in
the saUva 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 her-
bivora, 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 regu-
lates 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 converting starch
into sugar is not inherent in the saliva of all animals, and even when it pos-
sesses this property the intensity varies in different animals. In man,
whose saliva forms sugar rapidly, a production of sugar from (boiled) starch
undoubtedly takes place in the mouth, but how far this action proceeds
after the morsel has entered the stomach depends upon the rapidit}^ with
which the acid gastric juice mixes with the swallowed food, and also upon
the relative amounts of the gastric juice and food in the stomach. The
large quantity of water which is swallowed with the saliva must be ab-
sorbed 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, convejdng the
dissolved and finely divided bodies, passes into the blood from the intestinal
canal during digestion.
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 carbonate 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 in-
organic constituent is calcium carbonate.
II. The Glands of the Mucous Membrane of the Stomach, and the
Gastric Juice.
Since long ago the glands of the mucous coat of the stomach have been
divided into two distinct classes. Those which occur in the greatest abun-
dance and which have the greatest size in the fundus are called fundus glands,
also rennin or pepsin glands, and the others which occur only in the neigh-
borhood 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 cov-
GASTRIC JUICE. 349
ered throughout with a layer of columnar epithelium, which is generally
considered as consisting of goblet cells that produce mucus by a metamor-
phosis of the protoplasm.
The fundus glands contain two kinds of cells: adelo.morphic or chief
cells, and delomorphic or parietal cells, the latter formerly called
REXxix or pepsin cells. Both kinds consist of protoplasm rich in proteins;
but their relationship to coloring-matters seems to show that the protein
substances of both are not identical. The nucleus must consist chiefly of
nuclein. Besides the above-mentioned constituents, the fimdus glands
contain as more specific constituents several enz3'mes 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 Heidexhaix, independent
of the columnar epithelium of the excretory ducts they take no part worthy
of mention in the formation of mucus, which according to his views is
effected by the epithelium covering the mucous membrane. The pyloric
glands also "seem to contain the zymogens referred to above. Alkali chlo-
rides, alkali phosphates, and calcium phosphates are found in the mucous
coating of the stomach.
LiEBERMANX ^ has obtained an acid-reacting residue on digesting the mucosa
of the stomach with pepsin-h^'drochlorie 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 Beaumoxt on per-
sons with gastric fistula led to the suggestion that gastric fistulas be made
on animals, and this operation was first performed by Bassow - in 1842 on
a dog. Verxeuil performed the same on a man in 1876 with successful
results. Pawlow^^ has recently improved the surger}' of gastric fistula
and has added much to the study of gastric secretion.
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 PA^^'Low and his pupils.
' Pfliiger's Arch., 50.
'Helm, Zwei Krankengeschichten, Wien, 1803. cit. from Hermann's Handbuch,
5. part II, 39; Beaumont, '"The Physiologj' of Digestion," 1833; Bassow, Bull, de
la SCO. des natur. de Moscou, 16, cit. from Maly in Hermarm's Handbuch, 5, 38;
Temeuil, see Ch. Richet, "Du .sue gastrique chez Thomme," etc. (Paris, 1878), 158.
' Pawlow, Die Arbeit der Verdauungsdriisen (Wiesbaden, 1898), where the works
of his pupils are also mentioned. See also Ergebnisse der Physiologic, 1, Abt. 1.
350 DIGESTION.
In order to obtain gastric juice free from saliva and food residues they arranged
besides a gastric fistula also an oesophageal fistula from which the swallowed food
could be withdrawn with the saliva without entering the stomach, and in this
manner an apparent 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 secretion
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 pro-
duce any secretion. Chemical and mechanical irritations of the mucous
membrane of the mouth cause 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 occur-
ring under its influence by the vagus fibres appears earlier than that pro-
duced by chemical irritants, but only after an interval of at least 4| 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
only water and certain unknown extractive substances contained in meat
and meat extracts, in impure peptone, and also, it seems, in milk. Accord-
ing to Herzex and Radzikowski ^ alcohol is also a strong agent in pro-
ducing a flow of juice. Carbonated alkalies have a preventive instead of
an accelerating action on secretion. Bitter substances partaken of in
small amounts a certain time before a meal increase the secretion, while
larger amounts have a retarding action (Bomssow, Strashesko 2). 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 chemical
secretion b}^ 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:
* Pfliiger's Arch., 84, 513.
' Borissow, Arch. f. exp. Path. u. Pharm., 51; Strashesko, see Biochem. Centralbl.,
4c 148.
SECRETION OF GASTRIC JUICE. 351
Acidity.
Digestive Activity.
Duration of Secretion.
1.
Meat.
Bread.
Bread.
2.
Milk.
Meat.
Meat.
3.
Bread.
Milk.
Milk.
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 in-
testine, and in this way, as shown by the investigations of Pa^^xow 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. Ac-
cording to PioxTKOWSKi 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
5 to 6 hours after a meal with fat the secretion of juice is stopped, as just
at this time the soaps are being formed. According to Frouix the food
in the intestine produces a secretion of gastric juice which continues after
the action of the psychic moment has ceased. Leconte^ arrived at sim-
ilar results, and he ascribes less importance to the chemical secretion as com-
pared with the psychic secretion than Pawlow does.
LoNNQUiST^ has made observations upon dogs as to the dependence
of the secretion of gastric juice upon the presence of food or substances
causing flow in the stomach or in the intestine alone, or simultaneously in
both, w'ith the exclusion of the psychical influence. For this purpose he
expsrimented with the stomach, isolated according to Heidexhaix-Paw-
Low, as well as with fistulse in the stomach and intestine, and besides this
he also separated the stomach and intestine from each other by means
of a membrane between the pylorus and the intestine produced by oper-
ative means. An abundant secretion of juice was produced in the stomach
isolated from the intestine by water, alcohol, meat, and meat extracts,
and by the digestive products of egg-albumin. Dilute hydrochloric acid
(0.1-0.5 per cent) or natural gastric juice caused only a faint secretion.
Dilute sodium-chloride or soda solutions (0.25-€.50 per cent) acted some-
what like water; stronger soda solutions (1-1.5 per cent) produced a much
greater secretion of juice. Water or salt solution in the duodenum was
without action. Liquid fat had (reflexly) a strong retarding action, and
> See Biochem. Centralbl., 3, 660.
' Frouin, Compt. rend. soc. biol., 53; Leconte, La Cellule, 1".
^ "Bidrag tils Kaimedomen om mags'aftafsondringen," Akademisk afhandling. Hel-
siHgfors, 1906.
352 DIGESTION.
soda solutions, in the same manner, had a weaker retarding action. Water
as well as alcohol was absorbed from the stomach, and the alcohol acted
for the first half hour as a strong excitant for the flow of juice.
We know very little positively in regard to the gastric secretion in man.
According to the older statements 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 physiological secretion,
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 statements on this subject are unfortu-
nately very uncertain and contradictory, still there is no doubt that in
man, at least, alcohol and meat extracts may be active in causing secre-
tion.
The question as to how far the observations made by Pawlow and his
school can be applied to man is of special interest; still we have only very
few statements on this point at the present time. Hornborg,^ who recently
studied a case of gastric fistula with oesophageal stricture in a boy, could
not observe any special influence of the psychic excitement. The chewing
of indifferent or bad tasting bodies had no action, while on the contrary
the chewing of bodies with a pleasant taste produced a more or less abun-
dant secretion. Umber ^ observed in only one instance, in a case of a
man after gastrotomy, an insignificant truly psychic secretion of gastric
juice; chewing of an indifferent body or of chewing-tobacco brought
about no secretion. After an apparent feeding with meat, with a latent
period of 3 minutes, a secretion of gastric juice rich in HCl and
enzymes occurred. Contrary to the behavior in dogs, the juice secreted
after chewing bread was richer in acid than after chewing meat; the
quantity was on the contrary less. Umber also observed that after the
introduction of a food enema into the rectum a secretion of gastric juice
was produced by reflex action. Cade and Latarjet^ have made observa-
tions on a girl twenty years old w^ho had a blind sac formed by con-
striction, which was analogous to Pawlow's "little stomach." The juice
which flowed from the fistula opening of this blind sac was at least not
always normal, judging from the amount of acid and by its action. In
this person a purely psychic secretion was unquestionably observed by a con-
tinuous recollection of a pleasant sensation of taste, although it was not
especially strong.
From these observations of Hornborg and Umber, as well as from some
> Hornborg, "Bidrag till kannedomen om magsaftafsondringen hos manniskan "
Inaug.-Dissert. Helsingfors, 1903.
'Berlin, klin. Wochenschr., 1905.
'Compt. rend. soc. biolog., 57.
COMPOSITION OF GASTRIC JUICE, 3J3
older observations of Schule, Troller, Riegel, and Scheuer/ we con-
clude that in man the psychic secretion is much less than that produced b}-
the introduction of food or bodies having a pleasant taste. That the prepar-
ation of the food in the mouth has an essential influence upon the secretion
is proven without doubt, but we are not united 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.
The Qualitative and Quantitative Composition of the Gastric Juice. The
human gastric juice, which can seldom be obtained pure and free from
residues of the food or from mucus and saliva, is a clear, or only ver\' faintly
cloudy, and in man nearly colorless fluid of an insipid, acid taste and strong
acid reaction. It contains, as form-elements, glandular cells or their nuclei,
mucus-coripuscles, 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, chief!}' or in large part of hydrochloric acid. Coxtejean 2
has regularly found traces of lactic acid in the pure gastric juice of fasting
dogs. After partaking of food, especially after a meal rich in carboh^'drates,
lacnc acid occurs abundantly, and sometimes acetic and butyric acids. In
new-bom dogs the acid of the stomach is lactic acid, according to Gmelin.3
The quantity of free hydrochloric acid in the gastric juice is, according to
Pawlow and his pupils, in dogs 5-6 p. m.. and in cats an average of 5.20
p. m. HCl. In man (he acidity has been found to vary considerably, but
it is generally calculated as 2-3 p. m. HCl. Accorchng to Verhaegex's
researches there is no doubt that pure human gastric juice from perfectly
healthy persons has a higher acidity. Umber observed after apparent
feeding with bread 3.5 p. m., and Horxborg* found higher results in a
boy with gastric fistula. The juice secreted before taking food contained
3.05 p. m. acid. After taking food the acidity was higher. The acidity
of juice after bread was 3.65-5.11 p. m., with an average of 4.39 p. m., and
the juice after meat 4.01-5.66 p. m., or an average of 4.62 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
' The literature may be found in Umber's work, 1. c.
^ Bidder and Schmidt, Die Verdauungssafte, etc., 44; Richet, 1. c; Contejean, Con-
tributions a I'etude de la physiol. de I'estomac, Theses, Paris, 1892.
' Pfliiger's Arch., 90 and 103.
* See Richet, 1. c; Contejean, 1. c: Verhaegen, "La Cellule," 1896 and 1897; Um-
ber, 1. c; Hornborg, 1. c; and the literature on the estimation of hydrochloric acid
in the gastric contents (p. 375).
354 DIGESTIOX.
substances. The results obtained for the acidity of gastric juice by
physical methods are nearly identical with those obtained by titration
(P. Fraxckel 1).
As chief organic constituent, perfectly fresh gastric juice (of dogs) con-
tains 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 (Nexcki 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. Gastric juice also con-
tains lecithin and chlorine, and yields nucleoproteid. proteose, nuclein bases,
and pentose as cleavage products (Nencki and Sieber-).
The specific gravit}- of gastric juice is low, 1.001-1.010. It is corre-
spondingly poor in solids. Older analyses of gastric juice from man, the dog,
and the sheep were made by C. Schmidt.^ As these analyses refer only
to impure gastric juice they are of little value. The quantity of solids
in saliva-free gastric juice from a dog was 27 p. m., with 17.1 p. m. or-
ganic substance. The quantity of free hydrochloric acid was 3.1 p. m.
Besides these Schmidt found NaCl 1.46; CaCU 0.6; KCl 1.1; NH4CI 0.5;
earthy phosphates 1.9; and FeP04 0.1 p. m. Nencki ^ found 5 milli-
grams sulphocyanic acid per litre of gastric juice of a dog. The pure
gastric juice of another dog contained, according to Nexcki and Sieber,-
an average of 3.06 p. m. solids.
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. Wliile 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 enzj^mes 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 ^ the pepsins found in man and various
higher animals are somewhat different. Enzymes also occur in various
plants and animal organs; although not identical with pepsin, they act i:i
acid reaction and stand to a certain extent between pepsin and trypsin.
* Zeitschr. f. exp. Path. u. Therap., 1.
2 Zeitschr. f. physiol. Chem., 32.
M. c.
*Ber. d. d. chem. Gesellsch., 28.
* Klug, Pfliiger's Arch., 60; Wroblewski, Zeitschr. f. physiol. Chem., 21.
PEPSIN. 355
To this group belongs Glaessner's pseudopepsin, which according to him
is the only peptic enzyme in the pyloric end. Pseudopepsin, whose exist-
ence is disputed by Klug, while others (Reach, Pekelharixg) affirm its
occurrence in the mucous membrane, acts, according to Glaessner, also
in neutral and alkaline reaction and yields trs-ptophane among other cleavage
products. According to Berg.maxx ^ it is identical with erepsin (see
below). Among the enzymes of the mucosa of the stomach belongs the
so-called antipepsin discovered by Weixlaxd,^ which has a retarding
action upon pepsin digestion and, as some claim, prevents the self -digest ion
of the mucous membrane.
Pepsin is as difficult to isolate in a pure condition as other enzymes.
The pepsin prepared by Brucke and Suxdberg gate negative results with
most reagents for proteins, and showed nevertheless a powerful action,
which seems to indicate that it was very pure. Schoumow-Simaxowski,
Nexcki and Sieber, and also Pekelharixg have designated as the true
enzyme the substance containing chlorine, which they obtained by strongly
cooling the gastric juice. That this substance is not an individual, and
hence cannot be pepsin, follows from the investigations of Pekelharixg.
WTiile pepsin, according to Nencki and Sieber. was rich in phosphorus
and contained nucleoproteid, Pekelharixg's pepsin was free from phos-
phorus and yielded no nucleoproteid. Friedexthal and Miva.mota^ have
also shown that the pepsin is still active after the splitting off of the nuclein
complex (and also the protein). As pepsin is readily precipitated with the
proteins and combines therewith, it is difficult to decide whether pepsin is
a protein substance or not, and the question as to the nature of pepsin 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 glycerine.
It is precipitated by alcohol, but only slowly destroyed thereby. In
aqueous solution its action is quickly destroyed on heating to boiling.
According to Bierxacki-^ pepsin in neutral solutions is destroyed by heat-
ing to 55° C. In the presence of 2 p. m. HCl a temperature of 55° C. is
not injurious, and the compound with acid is more resistant than the
free pepsin (Grober ^). Pepsin in acid solution is destroyed b}- heating
to 65° C. for five minutes. On adding peptone and certain salts the pepsin
^ Glaessner, Hofmeister's Beitrage, 1; Ivlug, Pfliiger's Arch., 92; Reach, Hofmeis-
ter's Beitrage, 4; Pekelharing, Arch, des scienc. biolog., St. Petersbourg, 11; Pawlow-
Festband, 1904; Bergmann, Skand. Arch. f. Physiol., 18.
- Zeitschr. f. Biologie, 44.
' 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; Xencki and Sieber, ibid., 32; Friedenthal and Miyamota, CentralbL
f. Physiol.. 15, 785.
* Zeitschr. f. Biologie, 28.
^ Arch. f. exp. Path. u. Pharm., 51.
356 DIGESTION.
may be heated to 70° C. without decomposing. In the dry state it can, on
the contrary, be heated to over 100° C. without losing its physiological
action. The only property which is characteristic of pepsin is that it dis-
solves 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 precipi-
tates of other bodies, such as calcium phosphate or cholesterin. The
rather complicated methods of BrIjcke and Sundberg are based upon
this property. Pekelharing makes use of a prolonged dialysis and pre-
cipitation with 0.2 p. m. HCl.
Very permanent pepsin solutions, from which the enzyme with con-
siderable protein can be precipitated by alcohol, may be prepared by
extraction with glycerine. Solutions having a strong action may also be
prepared l)y making an infusion of the gastric mucosa of an animal in acid-
ified water (2-5 p. m. HCl). 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 dis-
solves. Unboiled fibrin swells up in a solution containing I p. m. HCl,
forming a gelatinous mass, and does not dissolve at ordinary temperature
within a couple of days. Upon the addition of a little pepsin, however,
this swollen mass dissolves quickly at ordinary temperatures. Hard-
boiled-egg albumin, cut in thin pieces with sharp edges, is not perceptibly
changed by dilute acid (2-4 p. m. HCl) at the temperature of the body in
the course of several hours. But the simultaneous presence 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 explanation, 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 tempera-
ture of the body unboiled fibrin is more easily dissolved by acid alone, it is
best always to work with boiled fibrin.
ESTIMATION OF PEPSIX. 357
As pepsin has not thus far been prepared in a positively pure condition,
it is impossible to determine the absolute quantity of pepsin in a liquid. It
is only possible 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, h, \, \, ^, 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, fs, of one series is digested in the same time as tests p=l, ^,1 oi the other
series; hence the first solution contained four times as much pepsin. This method
is not used as often as the following:
Mett's Method. Draw up white of egg m a glass tube 1-2 millimetres in diam-
eter, coagulate it by plunging it into hot water at 95° C, and cut the ends off
sharply; then add two tubes to each test-tube with a few cubic centimetres 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 millimetres. The quantity of pepsin in the
comparative tests is as the square of the millimetres of the albumin-column dis-
solved in the same time. Thus if in one case 2 millimetres of albumin were dis-
solved and in the other 3 millimetres, then the quantity of pepsin is as 4:9. If
the fluid removed from the stomach, which is rich in bodies ha"ving a disturbing
influence upon pepsin digestion, is to be tested, then the liquid must be first prop-
erly diluted with 2-4 p. m. hydrocliloric acid (Xierexsteix and Schiff')-
Objections have been .'aised against these methods from several sides, es-
pecially by Grxjtzxer, but they can be recommended for practical purposes as
being simple and rather accurate. Huppert and E. Schl'tz measure the relative
quantities of pepsin from the amount of secondary proteoses formed under certain
conditions. The proteoses were determined by the polariscope. J. Schutz
determines the total proteose-nitrogen, and Spriggs ■ finds that the change in the
viscosity is a measure of the amount of pepsin.
VoLHARD and LoHLEiN ^ use an acid casein solution for the pepsin determina-
tion, and determine, after precipitation with sodium sulphate, the acidity of the
filtrate of the digested test as weU 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 proportion as the
digestion progresses less substance is precipitated by the sulphate, and the acidity
of the fUtrate becomes correspondingly higher. The increase in acidity in the
different portions varies withia certain limits as the square root of the quantity
of ferment.
GRtJTZNER's* test is based on the use of finely cut fibrin colored with carmine.
The fibrin is first allowed to sweU up in 1 p. m. hydrochloric acid and then about
equal quantities are placed in test-tubes of the same diameter and treated with
15 c.c. of 1 p. m. hydrochloric acid, -\fter the addition of the pepsin solution to
' Mett, see Pawlow, 1. c, 31; Xierenstein and Schiff, Berl. klin. Wochenschr.. 40.
^ Huppert and Schiitz, Pfliiger's Arch., SO; J. Schiitz, Zeitschr. f. physiol. Chem.,
30; Spriggs, ibid., 35.
^ Hofmeister's Beitrage, 7.
* Griitzner, Pfliiger's Arch., 8 and 106. See also A. Kom, • Uber Methoden Pepsin
quantitativ zu bestlmmen," Inaug.-Dis.sert., Tubingen 1902.
358 DIGESTION.
be tested the fibrin dissolves, giving a red color to the solution, and the strength
of the digestion is determined by the depth of the color. For comparison a series
of tubes are used containing carmine solution diluted in known proportions, and
which are arranged so that, for example, when one pepsin solution had a color
corresponding to No. 1, and the other, on the contrary, to No. 4, then the latter
had four times as much fibrin dissolved as the first.
The rabidity 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 relationship has been found
between the strength of various acids and their action in pepsin digestion,
and the statements in regard to the action of different acids are somewhat
contradictory.! Sulphuric acid, it seems, has a weaker action than the
other inorganic acids. The degree of acidity is also of the greatest impor-
tance. 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
vegetable 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. ScHiJTZ,^ whose statements have been confirmed ])y
several others, the digestion products produced in a certain time are, within
certain limits, proportional to the square root of the relative amounts
of pepsin (the ScHiJTz-BoRissow^ rule). The accumulation of products
of digestion has a retarding action on digestion, although, according to
Chittenden and Amerman,^ the removal of the digestion products by means
of dialysis does not essentially change the relationship between the proteo.ses
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 digestion also increases until about 40° C,
when the maximum is reached. According to the investigations of Flaum *
it is probable that the relationship between proteoses and peptones remains
the same, irrespective of whether the digestion takes place at a low^ or high
temperature, as long as the digestion is continued for a long enough time.
* See Wroblewski, Zeitschr. f. physiol. Chem., 21, and especially Pfleiderer, Pfliiger's
Arch., 66, which also gives references to other works; Larin, Biochem. Centralbl.,
1, 484; and A. Pick, Wien. Sitzungsber., M. N. Klasse, 112.
' Zeitschr. f. physiol. Chem., 9.
' Journ. of PhysioL, 14.
* Zeitschr. f. Biologie, 28.
ACTION OF PEPSIN. 359
If the swelling up of the protein is prevented, as by the addition of neutral
salts, such as NaCl, in sufficient amounts, or by the addition of bile to the
acid liquid, digestion can be prevented to a greater or less extent. Foreign
bodies of different kinds produce different actions, in which naturally the
variable quantities in which they are added are of the greatest importance-
Salicylic acid and carbolic acid, and especially sulphates (Pfleiderer),
retard digestion, while arsenious acid promotes it (Chittenden), and hydro-
cyanic 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. j\ and max. J normal salt solutions), J. SchIjtz i found that the
ani.ons 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
he 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 2). 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 nucleoproteids or nucleoalbumins an insoluble residue
of nuclein or pseudonuclein always remains, although under certain cir-
cumstances 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 enclosed 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 peptones. In the digestion
of proteins, substances similar to acid albuminates, parapeptone (Meiss-
ner 3), antialburnate, and antialbumid (KitHxNe), may also be formed.
On separating these bodies the filtered liquid, neutralized at boiling-point,
' Hofmeister's Beitriige, 5.
^ Studies from the Lab. Physiol. Chem. Yale University, 1, 76. See also Chitten-
den and Stewart, ibid., 3, 60.
^ The works of Meissner on pepsin digestion are found in Zeitschr. f. rat. Med., 7,
8, 10, 12, and 14.
360 DIGESTION.
contains proteoses and peptones in the old sense, while the sa-called Kuhne
true peptone and the other cleavage products are obtained only after a
longer and more intense digestion. The relationship between the various
proteoses changes very much in different cases and in the digestion of the
various proteins. For instance, a larger quantity of primary proteoses is
obtained from fibrin than from hard-boiled-egg albumin or from the pro-
teins of meat; and the different proteins, according to the researches of
Klug,^ 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 55° C. (Hasebroek ^). For information in regard to the
different proteoses and peptones which are formed in pepsin digestion the
reader is referred to previous pages (50 to 61).
Action of Pepsin-Hydrochloric Acid on Other Bodies. The gelatine-
forming substance of the connective tissue, of the cartilage, and of the bones,
from which last the acid dissolves only the inorganic substances, is con-
verted into gelatine by digesting with gastric juice. The gelatine is further
changed so that it loses its property of gelatinizing and is converted into
gelatoses and peptone (see page 79). True mucin (from the submaxil-
lary) is dissolved by the gastric juice, yielding substances similar to pep-
tone and a reducing substance similar to that obtained by boiling with
a mineral acid. Mucoids from tendons, cartilage, and bones dissolve,
according to Posxer and Gies,^ in pepsin-hydrochloric acid, but leave a
residue which amounts to about 10 per cent of the original material and
which, as it seems, consists in great part, if not entirely, of a combination of
proteid with glucothionic acid (Chapters VII and VIII). The solution
contains primary and secondary mucoproteoses and mucopeptones. The
former contain glucothionic acid, but the latter do not. Elastin is dissolved
more slowly and yields the above-mentioned substances (page 76). Ker-
atin and the epidermal formations are insoluble. The nucleins are dissolved
with difficulty, and the cell nuclei, therefore, remain in great part undis-
solved 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. Oxyhcemoglobin is changed into hsematin 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
' Pfliiger's Arch., 65.
' Zeitschr. f. physiol. Chem., 11.
^ Amer. Journ. of Physiol., 11.
CHY^IOSIX AXD PARACHYMOSIN. 361
or the simple varieties of sugar. The statements in regard to the ability
of gastric juice to invert cane-sugar are vers' contradictor}-. 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, j'ields finally the same products as the hydrolytic
cleavage with acids, we can say for the present only that this enz\Tne acts like
other catalyzers in very powerfully accelerating a process which would proceed
also without the catalyzers.
Chymosin (rexxin) and Parachymosin. So far two different rennet
enzymes have been obtained from animal stomachs, namely, the enzyme
called rennin (chymosin), which is found in the calf's stomach and has
been known for a long time, and the parach3'mosin discovered by Baxg^
which is the rennet enzyme of the human stomach. This latter occurs in
the gastric juice of man under physiological conditions, but may be absent
under special pathological conditions (Schumburg, Boas. Johxsox', Klem-
perer2). Chymosin is habitually found in the neutral, waten,- 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 (Hammarstex).
We have no knowledge as to whether the rennet enzyme found in different
animals is chymosin or parachymosin. 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. Also antibodies to the rennet enzymes, atitichymosins,
occur in the animal kingdom, as shown by Hamm.arstex and Rodex in
blood-serum, and ma}- be produced in the animal body by the introduction
of rennin into the latter (Morcexroth^).
' Deutsch. med. Wochenschr , 1899. and Pfliiger's .\rch., 79.
^ Schumbiug, Virchow's Arch., 97, A good review of the literature may be foimd
in Szydlowski, Beitrage zur Kenntnis des LabenzjTn nach Beobachtungen an Saug-
lingen, Jahrb. f. Kinderheilkunde (N. F.), S-l. See also Lorcher, Pfliiger'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.
^ See Roden, Upsala Lakaref. Forh.,- 22; Morgenroth, Centralbl. f. Bakter., 26
362 DIGESTION.
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 concentra-
tion. If an active and strong infusion of the gastric mucosa in water
containing 3 p. m. HCl is heated to 37-40° 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 solution 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 specially shown by
FuLD, within certain limits, the coagulation time, T, is equal to a con-
stant, C, divided by the quantity of rennin, L. As shown by Bang,^
this law does not apply to parachymosin.
From the different laws of pepsin and rennin action it follows that the repeated
appearance recently of the view of Pawlow's school, that pepsin and rennin are
the same bodies, cannot be correct. The experiments given by Pawlow and
Parastschuk as proof for this view are unfortunately in error in principle. Also
the investigations published by Sawjalow are not decisive, and the recent work
of Schmidt-Nielsen ^ has given new proofs for the non-identity of the two en-
zymes. According to Nencki and Sieber,- with whom Pekelharing agrees,
the enzyme of the gastric juice forms a gigantic molecule which is able to perform
the different actions at the same time, although each enzyme action is connected
with a certain atomic complex. Such a view might appear plausible, especially as
pepsin and rennin enzyme regularly occur together in the animal kingdom. As
the body which is precipitated from gastric juice by cooling and which forms
the ferment has been shown to be a mixture, and also as the two enzymes and
also the proenzymes have been separated by Glaessner ^ from each other, this
view does not seem to be sufficiently well grounded.
Parachymosin differs from chymosin by being much more resistant
towards acids, but is more readily destroyed by alkalies. Calcium chloride
accelerates the casein coagulation with parachymosin very much more
than with chymosin.
Rennin, like other enzymes, may be carried down by other precipitates
and thus may be obtained relatively pure. It may also be obtained, con-
taminated with a great deal of proteins, by extracting the mucous coat of
the stomach with glycerine.
A comparatively pure solution of rennin may be obtained in the follow-
ing way. An infusion of the mucous coat of the stomach in hydrochloric
acid is prepared and then neutralized, after which it is repeatedly shaken
with new quantities of magnesium carbonate until the pepsin is precipi-
• Fuld, Hofmeister's lieitriige, 2; Bang, 1. c.
^ Pawlow and Parastschuk, Zeitschr. f. physiol. Chem., 42; Sawjalow, ibid., 46;
Schmidt-Nielsen, ibid., 48.
3 Glaessner, Hofmeister's Beitrage, 1; Nencki and Sieber, Zeitschr. f. physiol.
Chem., 32; Pekelliaring, foot-notes, and 3, p. 355.
GASTRIC LIPASE. 303
tated. The filtrate, which sliould act strongly on milk, is precipitated by
basic lead acetate, the precipitate decomposed with very dilute sulphuric
acid, the acid liquid filtered and treated with a solution of stearin soap.
The rennin is carried down by the fatty acids set free, and when these last
are placed in water and removed by shaking with ether, the rennin remains
in the watery solution.
Plastein. As mentioned on page 56, Danilewsky first showed the
power of rennin solutions of causing a partial coagulation of proteoses and
of converting them into so-called plastein. This action, which is also
ascribed to other enzyme solutions (see page 56), has probably nothing to
do with the rennin enzyme, but depends more likely upon another enzyme.
The nature of these enzymes, as well as the manner and importance of the
plastein formation, is still unknown.
Gastric Lipase (stomach steapsin). F. Volhard ^ has made the dis-
covery that the gastric juice has a strong fat-splitting action only when
the fat is in a fine emulsion, as in the yolk of the egg, in milk or in cream.
This action, which is still not undisputed (Inouye), depends upon an enzyme
which can be extracted from the mucosa by glycerine, and whose action,
it seems, follows SchIjtz's law for pepsin, that the quantity is proportional
to the square of the quantity of enzymotic products. This enzyme, which
seems to be produced from a zymogen, does not behave in the same way
W'hen obtained from different animals. The enzyme from the pig stomach
is very sensitive towards acids but less sensitive towards alkalies. That
from the dog and from the human stomach is, on the contrary, sensitive
tow^ards alkalies, while comparatively resistant towards acids (Fromme).
The enzyme is only slowly extracted from the pig stomach by glycerine, and
the extract shows its maximal activity only after several days. The filtered
glycerine extract is inactive (Fromme). Falloise, by experiments on
rabbits and dogs, has shown that gastric lipase is not derived by regurgita-
tion from the intestine, nor does it come from the pancreas by means of
the blood, but it is formed in the stomach. Laqueur, by means of
experiments with the filtered juice obtained from a small Pawlow stomach,
has shown that this juice had a splitting action upon yolk emulsion. Gas-
tric lipase, according to Laqueur,- cannot be pancreas steapsin, and is
also not an intracellular tissue enzyme, but is secreted by the glands.
The question whether the parietal cells principally or the chief cells,
or both, take part in the formation of free acid is somewhat disputed.^
* Volhard, Miinch. med. Wochenschr., 1900, and Zeitschr. f. klin. Med., 42, 43.
See also Stade, Hofmeister's Beitriige, 3; A. Fromme, ibid., 7; A. Zins._er, ibid.; H.
Engel, ibid.; and Inouye, Arch. f. Verdauungskrank., 9.
^ Falloise, Arch. int. de Physiol., 3 (1906); Laqueur, Hofmeister's Beitrage, 8.
^ See Heidenhain, Pfliiger's Arch., IS and 19, and Hermann's Handbuch, 5, part I,
"Absonderungsvorgange"; Klemensiewicz, Wien. Sitzungsber., 71; Frankel, Pfliiger'a
364 DIGESTION.
There can be no doubt that the hydrochloric acid of the gastric juice
originates from the chlorides of the blood, because, as is well known, a
secretion of perfectly typical gastric juice takes place in the stomachs of
fasting or starving animals. As the chlorides of the blood are derived
from the food, it is easily understood, as shown by Cahn,^ that m dogs
after a sufficiently long common-salt starvation the stomach secreted a
gastric juice containing pepsin, but no free hydrochloric acid. On the
administration of soluble chlorides, a gastric juice containing hydrochloric
acid was immediately secreted. On the introduction of alkali iodides or
bromides, KtJLz, Nencki and Schoumow-Simanowski ^ 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 hydro-
chloric acid is not the blood nor the glands, but the interior of the stomach
in the immediate neighborhood of the w^all. The hydrochloric acid is
assumed to be produced from the chlorides of the food, as the semiper-
meable wall is not permeable for the CI ions but is for the Na ions and for
the H 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 CI ions. This theory is
difficult to reconcile with the fact that in dogs with apparent feeding the
empty stomach secretes abmidant juice. Other objections have also been
raised against this by Beneath and Sachs. The secretion of free hydro-
chloric acid from the alkaline blood has been explained in other ways
(Maly, Bunge, L. Schwarz), but as yet no satisfactory theory has been
suggested.^
After a full meal, when the store of pepsin in the stomach is completely
exhausted, Schiff claims that certain bodies, especially dextrin, have
the property of causing a supply of pepsin in the mucous membrane. This
"charge theory," though experimentally tested by several investigators,
has nevertheless not yet been confirmed. On the contrary, the state-
ment of Schiff that a substance forming pepsin, a "pepsinogen" or "pro-
pepsin," occurs in the ventricle has been proved. Langley * has shown
positively the existence of such a sulistance in the mucous coat. This
Arch., 48 and 50; Contejean, 1. c, Chapter II; Kranenburg, Archives Teyler, Series
II, Haarlem, 1901, and Mosse, Centralbl. f. Physiol., 17, 217.
' Zeitschr. f. physiol. Chem., 10.
2 Kulz, Zeitschr. f. Biologic, 23; Nencki and Schoumow, Arch, des sciences bid.
de St. P^tersbourg, 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-
triige, 5.
* Schiff , Letjons sur la physiol. de la digfotion, 18G7, 2; Langley and Edkins,
Journ. of Physiol., 7.
PYLORIC SECRETION. 365
substance, propepsin, shows a comparatively strong resistance to dilute
alkalies (a soda solution of 5 p. m.), which easily destroy pepsin (Langley).
Pepsin, on the other hand, withstands better than propepsin the action of
carbon dioxide, which quickly destroys the latter. The occurrence of a
remiin zymogen, and possibly also of a steapsmogen, m the mucous coat
has been mentioned above.
According to Herzen and his collaborators * we must differentiate between
pepsinogens and bodies accelerating the flow of juice. To the first belong inulin
and glycogen, while alcohol belongs to the latter class of bodies. Dextrin not
only accelerates the flow of juice, but also acts as a pepsinogen, especially as the
latter. Meat extract which has both actions is especially a flow-accelerator.
The pepsinogen action consists in converting the zymogen into pepsin and in
this way increases the quantity of pepsin ; the flow-accelerating substances in-
crease 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 parietal cells were pepsin cells, but since
the investigations of Heidenhain and his pupils, Laxgley and others, the
formation of pepsin has been attributed to the chief cells.^
The Pyloric Secretion. That part of the pyloric end of the dog's
stomach which contains no fundus glands was dissected by Klemexsie-
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 alka-
line, 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 proved by Heidenhain by observations on a permanent
pyloric fistula, and the amount may sometimes be considerable. Conte-
jean has 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 Klemensieavicz is due, according to
CoNTEjEAN, to an abnormal secretion caused by the operation, because
the stomach readily yields an alkaline juice instead of an acid one under
abnormal conditions. The statements of Heidenhain and Klemensie-
wicz have been substantiated by Akermann, while Kresteff, w^ho
operated according to another method, and Schemiakine ^ have come
to the same results. Kresteff found in the juice (of dogs) pepsin, but no
chymosin. Yerhaegen ^ has observed in hiunan beings towards the end
' Pfliiger's Arch., 84.
^ See foot-note 3, p. 364.
^Heidenhain and IClemensiewicz, 1. c. ; Contejean, I. c, Chapter 11, and Skand.
Arch. f. Physiol., 6; Akermann, ibid., 5; Ivresteff, Maly's Jaliresber., 30; Schemia-
kine, Arch, des scienc. biolog. de St. Petersboiirg, 10.
* See the work of Verhaegen, "La Cellule," 1896, 1897.
366 DIGESTION.
of the ventricle digestion a non-fluid acid, which, according to him, originates
in the pyloric region.
The secretion of gastric juice under different conditions may x&iry con-
siderably. The statements concerning the quantity of gastric juice secreted
in a certain time are therefore so unreliable that they need not be taken
into account.
The Chyme and the Digestion in the Stomach. By means of the chem-
ical stimulation caused by the food, a copious secretion of gastric 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, Caxxox, Schemiakixe.^ and others, on the movements
of the stomach, we conclude that this organ consists of two phj-siologically
different parts, the pylorus and the fundus. The greater fundus part,
which serves essentially as a reservoir, may by a rhythmic, strong con-
traction of the muscle, acting like a sphincter between it and the pylorus
part, be separated from the latter, and according to some observers sa
completely so that during contraction almost nothing passes from the
fundus to the pylorus part. Differing from the fundus part, the pylorus
is the seat of ver}- 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. By ver}- instructive investigations on different animals
(frogs, rats, rabbits, guinea-pigs, and dogs) Grutzxer^ has shown, when
the animals are fed with food having different colors, and the stomach
removed after a certain time, and the contents frozen, that the frozen,
sections show a regular stratification of the contents. These layers are
so arranged that the food first taken is found in direct contact with the
mucosa, while the food taken later is 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 onlv the foodstuffs which lie close to the surface of
* Hofmeister and Schiitz, Arch. f. exp. Path. u. Pharm., 20; Moritz, Zeitschr. f.
Biologie, 32; Cannon, Amer. Journ. of Physiol., 1; Schemiakine, 1. c.
» Pfliiger's Arch., 106.
DIGESTION IN THE STO^kUCH. 367
the mucous membrane undergo the process of peptic digestion, and it is
principally this ingesta, which lies on the surface and is laden with pepsin and
mixed with gastric juice, which is 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 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 oljservations on the behavior of fluids or semifluid food. Accord-
ing to Grutzxer, also 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 salivar}- diastase, although
sensitive towards acids, can continue its action for a long time in the con-
tents of the stomach. Caxxox and Day ^ have shown that this is true
by special experiments upon animals, and the occurrence of sugar and
dextrin in the contents of the human stomach has been repeatedh- observ'ed.
In camivora, whose saliva shows nearly no diastatic action, it is a priori
not expected that there should be a diastatic action in the stomach — of
course excluding the action of micro-organisms which may be present.
Still, according to Friedexthal. dogs can readily digest starch, and the
gastric juice of dogs, according to him, contains a diastatic enzyme which
is active even in strong acid reaction. -
The gastric contents which have been prepared in the pylorus part are
passed through the pylorus into the intestine intermittently. This mate-
rial is generally fluid, but it is possible that pieces of solid food ma}- also
occur, and this has been often observed. From Busch's observations on a
human intestinal fistula, it required generally 15-30 minutes after eating
for undigested food to pass into the upper part of the small intestine. In
a case of duodenal fistula in a human being observed by Kuhxe, he saw,
ten minutes after eating, uncurdled but still coagulable milk and small
pieces of meat pass out of the fistula. The time in which the stomach
unburdens itself of its contents depends naturally upon the coarseness or
fineness of the food; essentially, however, it depends upon the reflex action
'Cannon and Day, Amer. Journ. of Physiol., 9; Friedenthal, Arch. f. (.\nat. u.)
Physiol., 1899, .>^uppl.
* See Scheunert and Grimmer. Zeitschr. f. physiol. Chem., 48, on the occurrence of
dias'atic enzjTnes in plant foodstuffs.
368 DIGESTION.
from the stomach or intestine causing an opening or closing of the pylorus,
which action is dependent upon the quantity and character of the food,
the amount of fat, and the degree of acidity in the contents of the
stomach and intestine. The emptying of the food into the small
intestine causes, as shown by Pawlow, a closing of the pylorus by chemo-
reflex in which the hydrochloric acid and the fat take part, and we thus
find in this regard an alternate action between the stomach and duodenum.
The time necessary for the stomach to empty itself must differ considerably
under various conditions. Beaumont ^ found in his extensive observations
on the Canadian hunter St. Martin that the stomach, as a rule, is emptied
1^-5| hours after a meal, depending upon the character of the food.
The time in which different foods leave the stomach depends also upon
their digestibility. In regard to the unequal digestibility in the stomach
of foods rich in proteins, which really form the object of the action of the
gastric juice, a distinction must be made between the rapidity with which
the proteins are converted into proteoses and peptones and the rapidity
with which the food is converted into chyme, or at least so prepared that
it may easily pass into the intestine. This distinction is especially im-
portant from a practical standpoint. When a proper food is to be decided
upon in cases of diminished gastric digestion, it is important to select such
foods as leave the stomach easily and quickly, independently of the diffi-
culty or ease with which their protein is peptonized, and which require as
little action as possible on the part of this organ. From this point of view
those foods, as a rule, are most digestible which are fluid from the start or
may be easily liquefied in the stomach; but these foods are not always
the most digestible in the sense that their protein is most easily peptonized.
As an example, hard-boiled white of egg is more easily peptonized than
fluid white of egg at a degree of acidity of 1-2 p. m. HCl;^ nevertheless
we consider, and justly, that an unboiled or soft-boiled egg is easier to
digest than a hard-boiled one. Likewise uncooked meat, when it is not
chopped very fine, is not more quickly but more slowly peptonized by the
gastric juice than the cooked, but if it is divided sufficiently fine it is often
more quickly peptonized than the cooked.
The greater or less facility with which the different protein foods are
digested in the stomach has been comparatively little studied. The most
complete investigations on this subject are those of Fermi,^ but as they
do not allow of a short discussion we must refer to the original publication.
* Busch, Virchow's Arch., 14; Kiihne, Lehrb. d. physiol. Chem., 53; (Pawlow and)
Serdjukow, Maly's Jahresber., 29; Lintwarew, Biochem. Centralbl., 1, 96. See also
Richet, 1. c; Beaumont, The Physiology of Digestion, 1833.
^ Wawrinsky, Maly's Jahresber., 3.
'Arch. f. (Anat. u.) Physiol., 1901, Suppl.
DIGESTION IN THE STOMACH. 369
The rapidity with which different foods leave the stomach has been
studied by Cannon ^ in the case of cats. 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 carbohy-
drate 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.
As our knowledge of the digestibility of the different foods in the
stomach is slight and dubious, so also our knowledge of the action of other
bodies, such as alcoholic drinks, bitter principles, spices, etc., on the nat-
ural 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 observe only a disturbing action, while other investi-
gators report having found that the alcohol first acts somewhat as a
disturbing agent, but afterwards, when it is absorbed, produces an abund-
ant secretion of gastric juice, and thereby facilitates digestion (Gluzinski,
Chittenden ^). The accelerating action of alcohol upon the flow of gastric
juice has already been mentioned on page 352.
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 point, and
that therefore the work of the stomach could be taken up more or less
by the intestine. This in fact is the case. Thus the stomachs of dogs
and cats have been completely extirpated or nearly so (Czerny, Carvallo
and Pachon), and also that part necessary in the digestive process has
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 being.=.3
^ Amer. Journ. of Physiol., 12.
^ Gluzinski, Deutsch. Arch. f. klin. Med., 39; Chittenden, Centralbl. f. d. med. Wis-
eensch., 1889; Chittenden and Mendel and others, Amer. Journ. of Physiol., 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.)
370 DIGESTION.
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 some-
times found to a considerable extent undigested in the excrements.
In order to judge of the role of the stomach in digestion the amount of
the digestion products in the stomach has been determined. These deter-
minations, partly on man and partly on animals, have led, as is to be
expected, to varying results (Cahn, Ellenberger and Hofmeister,
Chittenden and Amerman). The recent investigations of E. Zunz show
that boiled meat in the stomach of a dog yields chiefly proteoses with small
amounts of simpler cleavage products, and only very little acid albumin is
formed. The quantity of proteose nitrogen was 90 per cent of the nitrogen
of the non-coagulable substances. Tobler, who has studied in dogs with
duodenal fistula, the digestion in the stomach under conditions which
were as nearly physiologically normal as possible has arrived at different
results. After feeding finely chopped meat freed from extractives by water,
he found that about 20 to 30 per cent of the total nitrogen was absorbed in
the stomach, while about 20 per cent left the stomach in an insoluble form
and 50 to 65 per cent in solution. The chief mass of the dissolved protein
(about 80 per cent) consisted of peptones, the remainder of proteoses.
London and Sulima ^ have arrived at different results. They experi-
mented upon dogs, making various fistulse, such as gastric, pyloric, and
duodenal fistulse, and used as food partly hard-boiled egg, which was given
to the dogs in quantities of 200 grams and in as large pieces as possible, and
partly raw white of egg. In both cases, contrary to the experience of
Tobler, the proteins were not absorbed in the stomach. They also found
in the dog with pyloric fistula that after feeding 200 grams boiled protein
the chief portion of the digestion products (about 87 per cent) left the
stomach within the first two hours of digestion. Of the cleavage prod-
ucts the contents of the stomach contained chiefly proteoses, while in
the material which left the stomach in the dog with pyloric fistula, the
peptones prevailed. The proteoses seem to be retained for a longer time in
the stomach, making a further transformation into peptones possible.
As is to be expected, the behavior is not always the same, and important
variations often occur. It is, however, quite generally assumed that no
peptonization of the proteins worth mentioning occurs in the stomach,
Physiol., 1883; Groh^, Arch. f. exp. Path. u. Pharm., 49. In regard to a human
case of Schlatter see Wroblewski, Centralbl. f. Physiol., 11, 665.
' Cahn, Zeitschr. f. klin. Med., 12; Ellenberger and Hofmeister, Arch. f. (Anat. u.)
Phyisol., 1S90; Chittenden and Amerman, Journ. of Physiol., 14; E. Zunz, Hofmeister's
Beitrage, 3. See Reach, ibid., 4. See also Zunz, Annal. de la soc. roy. d. scienc. mdd.
et natur. de Bruxelles, 12 and 13; Tobler, Zeitsclu". f. physiol. Chem., 45; London,
and Sulima, ibid., 4fi.
DIGESTION IX THE STOMACH. 371
and that the protein foods are only prepared in the stomach for the real
digestive processes in the intestine. That the stomach, at least the fundus
part, serves in the first place as a storeroom follows from its shape, and
this function is of special value in certain new-bom animals, for instance
in dogs and cats. In these animals the secretion of the stomach contains
only hydrochloric acid but no pepsin, and the casein of the milk is converted
by the acid alone into solid lumps or a solid coagulum which fills the stomach.
Small portions of this coagulum pass into the intestine only little by little,
and an overburdening of the intestine is thus prevented. In other animals,
such as the snake and certain fishes which swallow their food entire, it is
certain that the major part of the process of digestion takes place in the
stomach. The importance of the stomach in digestion cannot at once be
decided. It varies for different animals, and it may vary in the same
animal, depending upon the division of the food, the rapidity with which
the peptonization takes place, the more or less rapid increase in the amount
of hydrochloric acid, and so on.
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 w^hich lactic acid and other organic acids are formed. According to
CoHX an amount of hydrochloric acid more than 0.7 p. m. completely
arrests lactic-acid fermentation, even under othenvise favorable circum-
stances, and according to Strauss and Bialocour the limit of lactic-acid
fermentation lies at 1.2 p. m. hydrochloric acid united to organic bodies.
The hydrochloric acid of the gastric juice has unquestionably an antifer-
mentative 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, especiall}^
as spores, are unacted upon. The fact that gastric juice can diminish or
retard the action of certain toxalbumins, such as tetanotoxine and diph-
theria toxine, is also of great interest (Nexcki, Sieber, and Schoumowa ^).
Because of this antifermentative and antitoxic action of gastric juice it
is considered that the chief importance of the gastric juice lies in its anti-
septic action. The fact that intestinal putrefaction is not increased on the
extirpation of the stomach, as derived from experiments made on man and
animals 2 does not uphold this view.
* Cohn, Zeitschr. f. physiol. Chem., 14; Strauss and Bialocour, Zeitschr. f. klin.
Med., 2S. See also Kiihne, Lehrb., 57; Bunge, Lehrb. d. Physiol., 4. Aufl., 148 and
1.59; Hirschfeld, Pfliiger's Arch., 47; Nencki, Sieber, and Schoumowa, Centralbl. f.
Bacteriol.. etc., 23. In regard to the action of gastric juice upon pathogenic microbes
we must refer the reader to handbooks of bacteriology.
^ See Carvallo and Pachon, 1. c, and Schlatter in Wroblewski, 1. c.
372 DIGESTION.
Since the hydrochloric acid of the gastric juice prevents the contents
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. Schierbeck ^ has shown that a
part of the carbon dioxide is formed by the mucous membrane of the
stomach. The tension of the carbon dioxide in the stomach corresponds,
according to him, to 30-40 mm. Hg in the fasting condition. It increases
after partaking food, independently of the kind of food, and may rise to
130-140 mm. Hg during digestion. The curve of the carbon-dioxide
tension in the stomach is the same as the curve of acidity in the different
phases of digestion, and Schierbeck has also found that the carbon-dioxide
tension is considerably increased by pilocarpine, but diminished by nico-
tine. According to him, the carbon dioxide of the stomach is a product
of the activity of the secretory cells.
i\fter death, if the stomach still contains food, autodigestion goes on
not only in the stomach, but also in the neighboring organs, during the
slow cooling of the body. This leads to the question, Why does the stomach
not digest itself during life? Ever since Pavy has shown that after tying
the smaller blood-vessels of the stomach of dogs the corresponding 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 cannot be contradicted; but the
reason is not to be found in the neutralization of the acid. The investi-
gations of Fermi and Otte 2 show that the blood circulation acts in an
indirect manner by the normal nourishment of the cell protoplasm, and this
is the reason why the digestive fluids, the gastric juice as well as the pan-
creatic 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 connected closely with the secretion of the antipepsin
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
enzvmes because it is thermolabile, while the antipepsin of Danilewsky,
' Planer, Wien. Sitzungsber., 42; Schierbeck, Skand. Arch. f. Physiol., 3 and 5.
^ Pa\y, Phil. Transactions, 153, fart I, and Guy's Hospital Reports, 13; Otte,
Travaux du laboratoire de I'lnstitut de Physiol, de Liege, 5, 1896, which also con-
tains the literature.
EXAAIINATIOX OF THE GASTRIC COXTENTS. 373
Hansel, and 0. Schwahz ^ is resistant towards heat and can hardly be
considered as an enzymotic body. This is true for at least the thermo-
stabile antipepsin of Schwaez, 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 as well as in
the absorption and in the mechanical work of the stomach may occur. Pepsin
is almost always present, although the amomit may vary considerably, but the
absence of the rennin, as above stated, may occur in many cases. In regard
to the acid, we must remark that the secretion is sometimes uacreased so that
an abnormally acid gastric juice is secreted and at other times it may be dimin-
ished so that little if any hydrochloric acid is formed. A h}-persecretion of
acid gastric juice sometimes occurs. In the secretion of too little hydrochloric
acid the same conditions appear as after the neutralization of the acid contents
of the stomach outside of the organism. Fermentation processes now appear in
which, besides lactic acid, there occur also volatile fatty acids, such as butjTic
and acetic acids, etc., and gases like hydrogen. These fermentation products are
therefore often found in the stomach in cases of chronic catarrh of the stomach,
which may give rise to belching, pjTosis, and other s^Tnptoms.
Among the foreign substances found in the contents of the stomach we have
UREA, or ammonium carbonate derived therefrom in uraemia; blood, which
generally forms a dark-brown mass tln'ough the presence of hsematiii, due to the
action of the gastric juice; bile, which, especially during vomiting, easily finds
its way through the pylorus into the stomach, but whose presence seems to be
without hnportance.
If it is desired to test the gastric juice or the contents of the stomach
for pepsin, fibrin may be employed. If this is thoroughly washed immedi-
ately after beating the blood, well pressed, and placed in glycerine, it may be
kept in serviceal^le condition for an indefinitely long time. The gastric
juice or the contents of the stomach — the latter, if necessaiy, having been
previously diluted with 1 p. m. hydrochloric acid — is filtered and tested
with fibrin at ordinary' temperature. (It must not be forgotten that a
control test must be made with acid alone and another portion of the same
fibrin.) If the fibrin is not noticeably digested within one or two hours,
no pepsin is present, or at most there are only slight traces.
In testing for rennin the liquid must be first carefully neutralized. To
10 c.c. of unboiled, amphoteric (not acid) cow's milk add 1-2 c.c. of the
filtered neutralized liquid. In the presence of rennin the milk should
coagulate to a solid mass at the temperature of the body in the course of
10-12 minutes without changing its reaction. If the milk is diluted too
much by the addition of the liquid of the stomach, only coarse flakes are
obtained and no solid coagulum. Addition of lime-salts is to be avoided,
as in great excess the}' may produce a partial coagulation even in the
absence of typical rennin.
In many cases it is especially important to determine the degree of
* See Hansel, Biochem. Centralbl., 1. p. 404, and 2, p. 326; ^Yeinland, Zeitschr. f.
Biologie, 44; Schwarz, Hofmeister's Beitriige, 6.
374 DIGESTION.
acidity of the gastric juice. This may be done ]:)y 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., HCl. Generally the acidity
is designated by the number of cubic centimetres of N/10 caustic soda
which is required to neutralize the several acids in 100 c.c. of the liquid
of the stomach. An acidity of 43 per cent means that 100 c.c. of the hquid
of the stomach required 43 c.c. of N/10 caustic soda to neutrahze 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 quantities
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 solutiou (Mohr's reagent
has Ijeen modified by several investigators), methylaniline-violet, tro-
p.EOLiN 00, Congo red, malachite-green, phloroglucin-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 solu-
tion 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
OiJNZBURG's test with phloroglucin-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 proved. A negative result does
not eliminate the presence of hydrochloric acid, as the delicacy of these
reactions has a limit, and also the simultaneous presence of protein, pep-
tones, and other l^odies influences the reactions more or less. The reactions
for lactic acid may also give negative results in the presence of compara-
tively large quantities of hydrochloric acid in the liquid to be tested. Sugar,
sulphocyanides, and other bodies may act with these reagents similarly to
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 oxidation with sulphuric acid and manganese dioxide.
The aldehyde is detected by its forming iodoform with an alkaline iodine
solution or by its forming aldehyde mercury with Nessler's reagent.
* Deutsch. med. Wochenschr., 1893, and Miinchener med. Wochenschr, 1893.
EXAMIXATIOX OF THE G.\STRIC COXTE^TS. 375
The quantitative estimation consists in the formation of iodoform with X/10
iodine solution and caustic potash, adchng an excess of hydrochloric acid and
titrating ^\-ith a X/10 sodium-arsenite solution, and retitrating with iodine solu-
tion, after the adrUtion of starch-paste, until a blue coloration is obtained. This
method presupposes the use of ether entirely free from alcohol.
In testing for lactic acid Cro.veir and Croxheim ^ recommend a solu-
tion of ioclij\e in potassium iodide containing aniline. Lactic acid is con-
verted mto iodoform, which with the aniline develops the nauseating odor
of isonitrile. The shaking out of the lactic acid with ether is imnecessarj",
but natm-ally alcohol or acetone must not be added.
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 hychochloric acid. It is a
weJJ-known fact that hydrochloric acid combmes wltn proteins, and a
considera]3le part of the h\'drochloric acid may therefore exist m the con-
tents of the stomach, after a meal rich lq proieixis. m combination with
them. This hydrochloric acid combined with proteins cannot be con-
sidered as free, and it is for this reason that certain investigators consider
such methods as that of Sjoqvist, wliich will be described below, as of little
value. However, it must be remarked that, according to the imanimous
experience of many investigators, the hydrochloric acid combmed with
proteins is physiologically active. Those reactions (color reactions) which
respond only to actual!^' free hydrochloric acid do not show the physiolog-
ically active tiydrochioric acid. The suggestion of determining the "phys-
iologically 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 defiaed
whether one wishes to determine the actually free or the physiologically
active hydrochloric 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
(monophosphates), and partly to both. According to Leo^ one can test
for acid phosphates by calcium carbonate, which is not neutralized there^
with, while the free acids are. If the gastric content has a neutral reaction
after shaking with calcium carbonate and the carbon dioxide is driven out
by a current of air. then it contains only free acid; if it has an acid reaction,
then acid phosphates are present, and if it is less acid than before, it con-
tains both free acid and acid phosphate. It must not be forgotten that
a faint acid reaction may. after treatment with calcium carbonate, also
be due to the protein. This method can Iike"uise be applied in the estima-
tion of free acid.
Various titration methods have been suggested for the estimation of
the free hydrochloric acid, but these cannot yield conclusive results for
the reasons given in a pre^■ious chapter (see estimation of the alkalinity
of the blood-serum, page 190). For this determination physico-chemical
methods are necessar}-. but they have not been used to any great extent
for clinical purposes on account of the difficulty in their manipulation.
> Berlin, klin. Wochenschr., 190.5, p. lOSO.
*CentraibL f. d. med. Wissensch.,lSS9, p. 4S1; Pfliiger's Arch., -tS, and Berlin,
klin. "Wochenschr., 1905, p. 1491.
376 DIGESTION.
As it is not •vnthin the scope of this l^ook to give the various methods for
the quantitative estimation of h\-drochloric acid for clinical purposes \vc
must refer to the various liandbooks for clinical methods, such as those
of V. Jaksch, Eulenburg, Kolle, and Weixtraud, and the work of O.
Reissner,! for details as to the cjualitative and quantitative tests for
hydrochloric acid and lactic acid.
The methods suggested by Leo, Hayem and Winter, Martius and
LfrTTKE, and by Reissxer, as well as the following method of Morxer
and Sjoqvist,2 are used for tlie quantitative estimation of the total hydro-
chloric acid.
The method of K. IVroRXER and Sjoqvist depends upon the following principle :
When the gastric juice is evaporated to chjniess with barium carbonate and then
calcined, the organic acids burn up and give insoluble barium carbonate, while
the hychocliloric acid forms soluble barium chloride. From the quantity of
this the original amount of hydrochloric acid can be calculated. 10 c.c. of the
filtered contents of the stomach are mixed in a small platinum or silver dish with
a knife-point of bariiun carbonate free from clilorides and evaporated to dryness.
The residue is burned and allowed to glow for a few minutes. The cooled carbon
is gently rubbed with water and completely extracted with boiling water and the
filtrate (about §0 c.c.) precipitated by ammonium chromate after the addition of
ammonium acetate and acetic acid and boiling. The carefully collected precipi-
tate is washed and dissolved in water by the aid of a little HCl, treated with KI,
and hydrochloric acid and titrated with hjqjosulphite solution. The reactions take
place as follows- 4HC1 +2BaC03 = 2BaCl2 + 2H2O +2C0.; 2BaCl2+2(XHJoCr04 =
2BaCrO, + 4XH,C1 ; 2BaCrO, + 16HC1 + 6KI = 2BaCl2 + Cvfk + 8H2O + 6KC1 + 3I2 ;
and 3L_. + 6Na2S203 = 6XaI + SXaaS^Ofi. Each cubic centimetre of the hyposul-
phite corresponds to 3 mgm. HCl. Complete directions for the necessary solu-
tions and for the performance of the method may be found in SJOQ\^ST, Zeitschr.
f. klin. Med., 32.
In testing for volatile fatty acids the contents of the stomach should not
be directly distilled, as volatile fatty acids may be formed by the decom-
position of other bodies, such as protein and haemoglobin. The neutral-
ized contents of the stomach are therefore precipitated with alcohol at
ordinary temperature, filtered quickly, pressed, and repeatedly extracted
with alcohol. The alcoholic extracts are made faintly alkaline by soda
and the alcohol distilled. The residue is now acidified by sulphuric or
phosphoric acid and distilled. The distillate is neutralized by soda and
evaporated to dryness on the water-bath. The residue is extracted with
absolute alcohol, filtered, the alcohol distilled off, and this residue dissolved
in a }ittle water. This solution may be directly tested for acetic acid with
sulphuric acid and alcohol or with ferric chloride. Formic acid may be
tested for by silver nitrate, which quickly gives a black precipitate; and
butyric acid is detected by the odor after the addition of an acid. In
regard to the methods for more fully investigating the different volatile
fatty acids, the reader is referred to other text-])Ooks.
'Zeitschr. f. klin. Med., 48.
^ In regard to the methods here mentioned see Reissner, 1. c.
INTESTINAL GLANDS. 377
III. The Glands of the 3Iucous 3Iembrane of the Intestine and their
Secretions.
The Secretion of Brunner's Glands. These glands are partly considered
as small pancreatic glands and partly as mucous or salivary glands. Their
importance in various animals is different. According to Grutzner they
are closely related in dogs to the pyloric glands and contain pepsin. This
also coincides with the observations of Glaessner and of Poxoiviarew,
which differ from each other only in that Ponomarew finds that the secre-
tion is inactive in alkaline reaction and contains only pepsin, while Glaessxer
claims it is active in both acid and alkaline reaction and that it contains
propepsin. According to Abderhalden and Roxa ^ the pure duodenal
secretion of the dog contains a proteol}i:ic enzyme which does not belong
to the trypsm type but rather to the pepsin variety. The statements as
to the occurrence of a diastatic enz^^me in Bruxxer's glands are disputed.
The Secretion of Lieberkuhn's Glands. The secretion of these glands
has been studied by the aid of a fistula in the intestine according to the
method of Thiry and Vella. Very little if any secretion takes place in
fasting animals (dogs) when the mucous membrane is not irritated. In
Iambs Pregl found the secretion continuous. The ingestion of food
causes a secretion, and in lambs increases the secretion already taking
place. Mechanical, chemical, and electrical stimulants act in the same
manner in dogs (Thiry). The secretion is also markedly increased in man
by the local irritation of the mucous membrane (Hamburger and Hekma^).
In the cases observed bj' these experimenters the flow of fluid was greatest
at night as well as between five and eight o'clock in the afternoon, and
was lowest between two and five o'clock in the afternoon. Pilocarpine
does not increase the secretion in lambs, and in dogs it does not seem to
be always active (Gamgee^). Among the chemical excitants we must
specially mention acids and gastric juice, which latter acts by its acidity.
The action of acids seems to be indirect, by means of the secretin which
will be mentioned below. Several salts, NaCl, Na2S04, and others, may
cause an abimdant secretion of fluid into the intestine when injected intra-
venously or subcutaneously, as well as after direct application to the peri-
toneal surface of the intestine. This action can be arrested b}^ the antag-
* Griitzner, Pfliiger's Arch., 12; Glaessner, Hofmeister's Beitrage, 1; Ponomarew,
Biochem. Centralbl., 1, 351; Abderhalden and Rona, Zeitschr. f. physiol. Cliem., 47.
2 Thiry, Wien. Sitzungsber., 50; Vella, Moleschott's Untersuch., 13; Pregl, Pfliiger's
Arch., 61; Gamgee, Physiol. Chem., 2, 410, where Vella and Masloff are quoted;
Kruger, Zeitschr. f. Biologie, 37; Hamburger and Hekma, Journ. de physiol. et de
path, generate, 1902 and 1904.
^ Gamgee, 1. c.
378 DIGESTION.
onistic, inhibiting action of a lime salt (MacCallum ^). 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 abimdant 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 (Rohmann).
Intestinal juice has a strong alkaline reaction towards litmus, generates
carbon dioxide on the addition of an acid, and contains (in dogs) nearly a
constant quantity of NaCl and Na2C03, 4.8-5 and 4-5 p. m. respectively
(GuMiLEWSKi, Rohmann 2). The intestinal juice of the lamb corresponded
to an alkalinity of 4.54 p. m. NaoCOa. It contains protein (Thiry found
8.01 p. m.), the quantity decreasing with the duration of the elimination.
The quantity of solids varies. In dogs the quantity of solids is 12.2-24.1
p. m. and in lambs 29.85 p. m. The specific gravity of the intestinal juice
of the dog, according to the observations of Thiry, is 1.010-1.0107, and
in lambs 1.01427 (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. Ham-
burger and Hekma and Nagano ^ 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 towards litmus. The content of alkali calcu-
lated 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 Boldireff,'* a
lipase which acts especially upon emulsified fat (milk) and is different from
pancreas lipase. The intestinal juice of animals and man also contains an
enzyme, erepsin, discovered by 0. Cohnheim, which does not act ordinarily
upon native proteins, but upon proteoses and peptones, and the juice also
has a faint amylolytic action. The juice, and to a high degree the mucous
coat, contains invertase and maltase, which fact has been recently substan-
tiated by the observations of Paschutin, Brown and Heron, Bastianelli,
» University of California Publications, 1, 1904.
^ Delzenne and Frouin, Compt. rend. soc. biolog., 56; Gumilewski, Pfliiger's Arch.,
39; Rohmann, ibid., 41.
'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.
* Boldireff , Archiv d. sciences biolog. de St. Petersbourg, 11.
INTESTINAL JUICE. 379
and Tebb.^ A lactose-inverting enzyme, a lactase, also occurs, as shown
by RoHMANN and Lappe, Pautz and Vogel, Weinland, and Orban,2 in
new-born infants and young, animals, and also in grown mammals which
were fed upon a milk diet. The lactase is found to a greater extent in
the mucosa than in the juice.
Besides erepsin and the other enzymes mentioned the intestinal mucosa
also contains antienzymes, antipepsin and antitrypsin. (Danilewsky and
Weinland 3), also enterokinase or a mother-substance of the same, and
finally also the so-called prosecretin. These two last-mentioned bodies,
which are closely connected with the secretion of pancreatic juice, will
be discussed in connection with this digestive fluid.
The various enzj-mes are not formed in equal quantities in all parts of
the intestine. Lipase, diastase, and invertase occur, according to Boldi-
keff, all through the intestine, while the kinase occurs only in the upper part
of the intestine (Boldireff, Bayliss and Starling, Delezenne). Ac-
cording to Hekma the kinase occurs in all parts of the intestine, but most
abundantly in the duodenum and the upper part of the jejunum. The
enzymes, according to Falloise, 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 investigations of Ver-
non the behavior of erepsin in different animals is not the same. 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 secretion, according to Bayliss 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 forma-
tion of the enzymes, and the same is true also for the enterokinase, according
to Bayliss and Starling, Hekma, Falloise, and others, which, however,
according to Delezenne,* is formed in the leucocj^es and Beyer's glands.
BoTTAZzi 5 has obtained a very complex protein from the intestinal mucosa,
which is readily soluble in water and alkali but is precipitated by acids. It coagu-
lates at 55° to 56° and probably also contains carbohydrate and considerable
' Paschutin, Centralbl. f. d. med. Wissensch., 1870, 561; Brown and Heron, Annal.
d. Cliem. u. Pharm., 204; Bastianelli, Moleschott's Untersuch. zur Naturlehre, 11
(ihis contains all the older literature). See also Miura, Zeitschr. f. Biologie, 32; Wid-
dicombe, Journ. of Pliysiol., 28; Tebb, ibid., 15.
^ Rohmann and Lappe, Ber. d. d-eutsch. chem. Gesellsch., 28; Pautz and Vogel,
Zeitschr. f. Biologie, 32; Weinland, ibid., 38; Orban, Maly's Jahresber., 29.
^ See foot-note 1, p. 373.
"Boldireff, Arch. d. scienc. biolog. de St. Petersbourg, 11; Bayliss and Starling,
Journ. of Physiol., 29, 30; Hekma, 1. c; Falloise, see Biochem. Centralbl., ■I, p. 153;
Vernon, Journ. of Physiol., 33; Delezenne, Compt. rend. soc. biolog 51 and 56.
' See Biochem. Centralbl., 3, p. 65.
380 DIGESTION.
iron. Intravenous injection of this protein brings about an abundant secretion
of saliva, pancreatic juice, bile, and intestinal juice, and promotes the peristaltic
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 and peptones. In this change mono- as well as di-
amino-acids are produced. Erepsin occurs in the mucous membrane and in
the intestinal juice of man as well as of dogs; the mucous membrane seems
to be richer than the juice (Salaskin, Kutscher and Seemann ^). An
enzyme like erepsin occurs also 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
by 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 auto-
lytic enzymes, if they are not identical, behave somewhat differently from
the intestinal erepsin and are not identical therewith. Enzymes having
an action similar to erepsin occur, according to Vines,^ 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 auto-
lytic enzymes studied so far.
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 charac-
teristic pancreas is absent in certain vertebrates and in certain fishes.
''Cohnheim, Zeitschr. f. physiol. Chem., 33, 35, 36, and 47; Salaskin, ibid., 35;
Kutscher and Seemann, ibid., 35.
' 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 and
19.
PAIsCREAS. 381
Those functions which should be regulated by this organ seem to be per-
formed in these animals by the Uver, which may be rightly called the hepa-
TOPAXCREAS. In man and in most vertebrates the formation of bile and of
certain secretions containing enzymes important for digestion is divided
between the two organs, the liver and the pancreas.
The pancreatic gland is similar in certain respects to the parotid gland.
The secreting elements of the former consist of nucleated cells whose basis
forms a mass rich in proteins, which expands in water and in which two
distinct zones exist. The outer zone is more 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 var\'ing relative size of
the two zones. According to Heidexil.\ix ^ 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 con-
ditions the glandular cells are undergoing a constant change, at one time
consuming from the inner part and at another time growing from the outer
part. The inner granular zone is converted into the secretion, and the
outer, more homogeneous zone, which contains the repairing material, is
then converted into the granular substance. The so-called islands of
Laxgerhaxs 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 consists,
it seems, of micleoproteids, while the globulins and albumins occur only to a
slight extent as compared with the nucleoproteids. Among the compound
proteids is the substance studied and isolated by Uiiber but previously
discovered by Hammarstex^ and called a-proteid. This nucleoproteid
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 on boiling _.3-proteid, so called by H.^i-
M-\rstex, which is much richer in phosphorus than the nucleoproteid.
The native proteid (a) is the mother-substance of guanylic acid; accord-
ing to Umber it dissolves by pepsin digestion without leaving any residue
and yields on tiypsin digestion guanylic acid on one side and proteoses
and peptones on the other. It can be extracted from the gland by a
' Pfluger's .Ajch., 10.
^ See Diamare and Kuliabko, Centralbl. f. Physiol., 18 and 19; Rennie, ibid., IS;
Sauerbeck, Virchow's Arch... 177 Suppl.
^ Umber, Zeitschr. f. klin. Med.., 40 and 43; Hammarsten, Zeitschr. f. physiol.
Chem., 19.
382 DIGESTION.
physiological salt solution and is precipitated by acetic acid. Besides this
compound proteid the pancreas must contain at least one other proteid,
which is the mother-substance of the thymonucleic acid obtainable from
the pancreas.
Besides these protein substances the gland contains also several enzymes,
or more correctly zymogens, which will be discussed later. Among the
extractive bodies, which are probably in part formed by post-mortem
changes and chemical action, we must mention leucine (butalanine),
tyrosine, purine bases in variable quantities/ inosite, lactic acid, volatile fatty
acids, and fats. The mineral bodies vary considerably in quantity not only
in animals and man but also in men and women (Gossmann), The calcium
seems, according to Gossmann, to exist in much greater amount than the
magnesium. According to the investigations of 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 ^ found in a man 17.92 p. m. ash
and 13.05 p. m. in a woman.
Besides the already-mentioned (Chapter VIII) relationship 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,
LuDWiQ, and Heidenhain, and perfected by Pawlow.^ If the operation
is performed with sufficient rapidity and under favorable conditions a
powerfully active secretion may be obtained either immediately after the
operation (temporary fistula) or after some time (permanent fistula).
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, according to Bernstein, Heidenhain, and
others, within the first three hours. According 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, according to Pawlow's school, dependent
* See Kossel, Zeitschr. f. physiol. Chem., 8.
^ Gossmann, Maly's Jahresber., 30; Oidtmann, cited from GoruD-Besanez, Lehr-
buch, 4thEd.,732.
' Bernard, Legons de Physiol.. 2, 190; Ludwig, see Bernstein, Arbeiten a. d. physiol.
. Aristalt zu Leipzig, 1869; Heidenhain, Pfliiger's Arch., 10, 604; Pawlow, Die Arbeit
der Verdauungsdrsiien, Wiesbaden, 1898, and Ergebnisse der Physiologie, 1, Abt. 1.
* Bernstein, 1. c, foot-note 3, Walther, Arch, des sciences biol. de i t.
P^tersbourg, 7.
PA^■CREATIC JUICE. 383
upon the food, and the amount of the three enzymes, diastase, tn-psin, and
steapsin, changes with the variety of food. The observations !R-hich form,
the basis of this view have been somewhat differently explained in the Ught
of recent investigations on the conditions necessary for the conversion of
trs-psinogen into tr\-psin.
Pawlow and his pupils, especially Schepowalxikoff. have shown,
that the above-mentioned (page 379) enterokinase activates the tr}psinogeii
into trvpsin. These observations were later confirmed by others, especially
by Delezen'XE and Frouix, Popielski, Camus and Gley. Batliss and
Starling, and fiuther studied. The pure juice contains only tr}-psinogen
and no trypsin. By mixing with the intestinal juice, or by contact with the
intestinal mucosa, the trypsinogen is converted into trypsin by the kinase.
Enterokinase, which itself has no action upon proteins, has been found in
all higher animals examined. A kinase with a similar action has also been
detected by Delezexxe in the lymph-glands and in impure fibrin, a state-
ment which is contradicted by Batliss and Starling and Hekma. The
enterokinase is made inactive by heat and is therefore considered as an
enzyme. Hamburger and Hekiia. who detected enterokinase in human
intestinal juice, do not con.-ider it an enzyme, because a certain quantity
of intestinal juice will activate only a certain quantity of trypsin (see below).
The above statements concerning the action of a vars'ing diet upon the
enzyme content of the juice have been somewhat changed by the investiga-
tions of Pawlow's school (Lintwarew and others). For instance, a diet of
bread and milk causes the secretion of a large quantity of juice which is
rich in trj'psinogen but contains almost no trj'psin. On gi\"ing meat after
this the juice also contains tr\-psin; after a rich meat diet the secretion
becomes scant and the juice contains only trs'psin but no trypsinogen.
There is here one difference between Pawlow's school and certain
other investigators. AccorcHng to Delezenne and Frouin, Popielski,
Bayliss and Starling, Prym, and others,^ the juice never contains
trypsin but always onlv tr^-psinogen, if it is collected through a canula in
Wirsung's duct, so that contact with the intestinal mucosa is prevented.
Popielski explains the ob.se rvat ions of Pa"«xow's school by tlie fact that
a contact of the juice with the intestinal secretions was not perfectly pre-
vented, and that with one kind of diet a rapid flow of jtiice took place and
with another a slower flow.
It is not clear whether there are also kinases for the other two enzymes.
Pawlow's pupils claim that the diastase is always eliminated as enzyme,
while according to Pozerski a kinase also exists for this zymogen. In
* In regard to the literature on enterokinase, secretin, and secretion of pancreatic
juice, see O. Cohnheim. Biochem. Centralbl., 1, 169. and S. Rosenberg, ibid., 2, 708;
PrjTn, Pfl ger's Arch., 104 and 107.
384 DIGESTION.
regard to steapsin the statements arc somewhat contradictory. Accord-
ing to LiNTWAREW there is secreted with food rich in carbohydrates and
fats a zymogen which is quickly changed into the enzyme by bile or
intestinal juice. With a meat diet the steapsin is secreted already formed.
The specific irritants for the secretion of pancreatic juice are, according
to Pawlow and his collaborators, acids of various kinds — hydrochloric acid
as well as lactic acid — and fats, the latter acting probably by virtue of
the soaps produced therefrom. Alkalies and alkali carbonates have, on the
contrary, a retarding action. It appears that the acids act by irritating
the mucosa of the duodenum. Water, which causes a secretion of acid
gastric juice, likewise becomes indirectly a stimulant for the pancreatic
secretion, but may also be an independent exciter. The psychical moment
may, at least in the first place, have an indirect action (secretion of acid
gastric juice), and the food seems otherwise to have an action on pancreatic
secretion by its action on the secretion of gastric juice.
The most important excitant for the secretion of juice is hydrochloric
acid, but the views are not united as to the manner in which the acid acts
According to Pawlow's school, the acid acts reflexly upon the intestine,
causing a secretion of a juice containing only trypsinogen. That a reflex
action is here produced is not contradicted by the investigations of Popiel-
SKi, Wertheimer and Lepage, Fleig,i and others. According to the
researches of Bayliss and Starling, which have been confirmed by Camus,
Gley, Flei.g, Herzex, Delezenne, and others, a second factor must also
be active here. Bayliss and Starling have shown that a body which
they have called secretin can be extracted from the intestinal mucosa by
a hydrochloric-acid solution of 4 p. m. , and this when introduced into the
blood produces a secretion of pancreatic juice. The secretin, which accord-
ing to Bayliss and Starling ^ is the same in all vertebrates examined, is not
destroyed by heat; it is therefore not identical with enterokinase, and is not
considered as an enzyme. It is formed from another substance, prosecretin,
by the action of acids. According to Delezenne and Pozerski^ secretin
occurs as such in the intestinal mucosa, and. the acid acts only by the
destruction of certain bodies having a retarding action. According to
Popielski secretin action is different from acid action; the secretin
according to him is a peptone, and the secretin action can also be obtained
by Witte's peptone. The statements about secretin and its action are
veiy divergent. It is diflficult to obtain a clear conception of the
^Gentralbl. f. Physiol., 16, 681, and Compt. rend. soc. biol., 55. See also foot-
note l,p. 38.3.
"^ Journ. of Physiol., 29.
^Delezenne and Pozei-ski, Corapt. rend. soc. biol., 56; Popielski, Centralbl. f.
Physiol., 19.
PANCREAS AND ENZYME FORMATION. 385
amount of zymogens or enzymes secreted by the juice under the in-
fluence of the secretin. It seems to be clear that this juice, at least
in many cases, contains only trypsinogen and no trj-psin.
A second means of causing secretion is the fat, which probably c^xy
acts after it has been saponified. Oil-soap introduced directly into the
duodenum brings about a strong secretion of pancreatic juice (Sawitsch,
Babkixe ^), and at the same time a flow of bile, gastric juice, and
the secretion of Bruxxer's glands occurs. The pancreatic juice secreted
imder these circumstances has about the same amount of enzymes as the
juice secreted after partaking of food. We know \ery little as to how the
soaps act. Fleig^ has 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 mtroduced into the
blood brings about a strong secretion of pancreatic juice. This sapo-
krinin, which is derived from a prosapokrinin, is not an enzyme and is not
identical wdth secretin. It dissolves in 60 per cent alcohol and is not
destroyed by boiling. Sapokrinin affects the secretion of pancreatic juice
alone, while the soaps also excite the secretion of bile and gastric juice.
The secretion of pancreatic juice may also be increased by alcohol (Fleig,
GiZELT 3).
The activation of the tr}'psinogen into trypsin may in life be brought
about — as the researches of Herzex, which have been substantiated b}^
Gachet and Pachon, Bellamy, jMendel and Rettger, have sho^Ti — not
only in the intestine, but also in the gland itself. This activation of the
trj^psinogen in the gland itself is caused in a manner still unknown by a
body of unknowm 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,^ 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 ac+ion on a splenec-
tomized dog with permanent pancreatic fistula. The observations of
Herzen that a spleen infusion has a strong activating action upon a
weak pancreas infusion were sibstantiated by Pry:m,^ but he claims that
this is due essentially to micro-organisms.
The conversion of the tr}'psinogen into trj^psin in the removed gland or
^ Arch, des scienc. biolog. de St. Petersboiirg, 11.
Compt. rend. soc. biolog., 55, and Journ. de physiol. et de pathol. g4n., 1904.
« Centralbl. f. Physiol., 19.
* Bellamy, Joum. of Physiol., 27; Mendel and Rettger, Amer. Joum. of Physiol., 7.
A very complete reference to the literature may be foimd in Menia Besbokaia Du
rapport fonctionell entre le pankr6as et la rate, Lausanne, 1901.
^ Pfliiger's Arch., 104 and 10..
386 DIGESTION.
in an infusion under the influence of air and water and also by other bodies
has been known for a long time. According to ^'ERX0N the tr^'psin 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 Starlixg and by Hekma. The ordinary view of
Heidexilun, that the transformation of tr}-psinogen into trj'psin is also
brought about by acids, has been found to be incorrect by Hekma.^ Besides
the enterokinase and the micro-organisms we know for the present of no
agent of organic origin which has the power of activating trypsinogen with
positiveness. According to Delezenne, on the contrary, the pancreatic
juice can be activated by calcium salts, and according to E. Zuxz 2 also by
magnesium and in certain cases by barium, lithium, and strontium salts.
The way in which the tr\^psinogen is converted into trypsin is still un-
knowTi and is the subject of disputed \aews. According to one view, pro-
posed by Pawlow and defended by Bayliss and Starlixg, the tryp-
sinogen is transformed into trj^psin by the action of the kinase. According
to the views of Delezenne, Dastre and Stassaxo, and others,^ the trypsin,
on the contrar}^, is a combination between the kinase and trj-psinogen,
analogous to the hsemolysins, which according to Ehrlich's side-chain
theory are combinations between a complement and an amboceptor.
The reflex formation of lactase after the introduction of milk-sugar
into the intestine, as observed by Weixlaxd, is to be considered as an
intraglandular enzyme formation. 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 hydrol}i;ic ferments in the body;
"c'est I'aliment qui fait le ferment." It has not been determined in what
way the milk-sugar produces this adaptation of the gland. The investiga-
tions of Baixbridge'* seem to show that the milk-sugar causes the pro-
duction of a body in the intestinal mucosa, which is brought to the pancreas
by the blood and there makes the formation of lactase possible. This
special property of the pancreas is denied by Plimmer.^
'Vernon, Journ. of Physiol., 28; Hekma, Kon. Akad. v. Wetenschappen te
Amsterdam, 190.3, and Arch. f. (Anat. u.) Physiol., 1904; BayUss and Starhng, Journ.
of Physiol., 30.
^ Delezenne, Compt. rend. ;-oc. biolog., 59, and Compt. rend., 141; Zmiz, see Biochem.
Centralbl., 5, 69.
' Bayliss and Starling, Journ. of Physiol., 30 and 32, which also cites the other
investigators. See also foot-note 1, p. 383,
* Weinland, Zeitschr. f. Biologie, 38 and 40; Brocard, Journ. de physiol. et de
path, gen., 4; Bainbridge, Journ. of Physiol., 31. Contradictory views are given
by Bierry, Compt. rend., 140, and Compt. rend. soc. biolog., 58, and Pl.mmer, Journ.
of Physiol., 34.
* Journ. of Physiol., 34.
COMPOSITION OF PANCREATIC JUICE. 387
The statements as to the quantity of pancreatic juice secreted in the
twenty-four hours differ very much. According to the determinations
of Pawlow and his collaborators, Kuwschixski, "Wassiliew, and Jablon-
SKY,^ the average quantity (with normally acting juice) from a permanent
fistula in dogs is 21.8 c.c. per kilo in the twenty-four hours.
The pancreatic juice of the dog is a clear, colorless, and odorless alka-
line fluid which when obtained from a temporary fistula is ver}^ rich in
proteins, sometimes so rich that it coagulates like the white of the egg
on heating. Besides proteins the juice contains also the three above-
mentioned enzymes (or their zymogens), amylopsin, trypsin, steapsin,
also an enzyme similar to erepsin, and besides these a rennin, which was
first observed by Kuhxe. Besides the above-mentioned bodies the pan-
creatic juice habitually contains small quantities of leucine, fat, and soaps.
As mineral constituents it contains chiefly alkali chlorides and considerable
alkali carbonate, some phosphoric acid, lime, magnesia, and iron.
The older analyses of the juice from a permanent fistula by C. ScHAncT
are the results of a more or less abnormal secretion, hence we shall give only
the analyses of juices from temporary fistulas on dogs.^ The results are
given in parts per 1000.
a. b.
Water 900.8 884.4
Solids. 99.2 115.6
Organic substance 90.4
Ash 8.8
The mineral constituents consisted chiefly of XaCl, 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 ^ 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. Xa^COs.
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." 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 Glaessxer.3 The secretion was clear, foamed readil}-,
had a strong alkaline reaction even towards phenolphthalein, and contained
globulin and albumin but no proteoses and peptones. The specific gravity
was 1.0075 and the freezing-point depression was J = —0.46-0.49°. The
solids were 12.44-12.71 p. m.. the total protein 1.28-1.74 p. m., and the
* Arch, des sciences de St. Petersbourg, 2, .391. The older statements of Keferstein
and Halhvachs. Bidder and Schmidt, and others may be found in K ilme, Lehrbuch,
114.
^ Cited from Maly in Hermann's Handbucli de ■ Physiol., 5. Theil II, 189.
^ Journ. of Pliysiol., 31.
*Zeitschr. f. physiol. Ciiem., 40. See also EUinger and Kohn, ibid., 45, and the
investigations upon cystic fluids from tlie pancreas by Schumm, ibid., 36, and Murray
and Gies, American Medicine, 4, 1902.
388 DIGESTION.
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 c.c. The quantity of secretion, of ferments, and of
alkalinity was lowest in starvation, but soon rose with the taking of food,
and reached its maximum in about four hours.
Amylopsin or pancreatic diastase, which, according to Korowin and
ZwEiFEL, is not found in new-born infants and does not appear imtil more
than one month after birth, seems, although not identical with ptyalin, to
be nearly related to it. Amylopsin acts very energetically upon boiled
starch, and according to Kijhne also upon unboiled starch, especially at
37° to 40° C, and according to Vernon ^ best at 35° C. It forms, similar
to the action of saliva, besides dextrin, chiefly isomaltose and maltose,
with only very little dextrose (Musculus and v. Mering, Kulz and
VoGEL^). The dextrose is probably formed by the action of the invertin
existing in the gland and juice. The pancreatic juice of the dog contains
in fact, according to Bierry and Terroine,^ maltase, whose action becomes
apparent only after very faint acidification of the juice. According to
Rachford the action of the amylopsin is not reduced by very small quan-
tities of hydrochloric acid, but is diminished by larger amounts. Vernon,
GRtJTZNER, and Vv'achsmaxn '^ 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.)
If the natural pancreatic juice i? not to be obtained, then the gland
may be treated with water or glycerine. This infusion or the glycerine
extract diluted with water (when glycerine has been used which has no
reducing action) may be tested directly with starch-paste. It is safer,
however, to first precipitate the enzyme from the glycerine extract by
alcohol, and wash with this liquid, dry the precipitate over sulphuric acid,
and extract with water. The enzyme is dissolved by the water. The
test for sugar may be performed in the same manner as in the saliva.
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
glycerine, which is an enzymotic process; and secondly, it has also the
property of emulsifying the fats.
'Korowin, Maly's Jahresber., 3; Zweifel, foot-note 1, p. 344; Kiihne, Lehrbuch,
117; Vernon, Journ. of Physiol., 27.
^ See foot-note 4, p. 344.
' See Tebb, Journ. of Physiol., 15; Bierry and Terroine, Compt. rend. soc. bio-
log. 58.
* Rachford, Amer. Journ. of Physiol., 2; Vernon, 1. c; Griitzner, Pfliiger's
Arch., 91.
STEAPSIN. 389
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 coloreel red by alkanet tincture. If such fat is mixed with
perfectly fresh alkaline pancreatic juice or with a freshly prepared infusion
of the fresh gland and treated with a little alkali or with a faintly alkaline
glycerine extract of the fresh gland (9 parts glycerine and 1 part 1 per cent
soda solution for each gram of the gland), and some litmus tincture added
and the mixture warmeel to 37° C, the alkaline reaction ■u ill gradually
disappear and an acid one take its place. This acid reaction depends upon
the conversion of the neutral fats by the enzyme into glycerine and free
fatty acids.
The splitting of the neutral fats may also be shown more exactly by
the following methoel: The mixture of neutral fats (absolutel}- free from
fatty acids) and pancreatic juice or pancreas infusion is digested at the tem-
perature of the body and treated with some soda and repeatedly shaken
with fresh quantities of ether until all the unsplit neutral fats are removed.
Then it is made acid with sulphuric acid, and after the acid liquor has been
shaken with ether, the ether is evaporated, and the residue tested for fatty
acids.
Another simple process for the demonstration of the fat-splitting action
of the pancreatic glands is the following (Cl. Bernard) : A small portion of
the perfectly fresh, fuiely divided gland substance is first soaked in alcohol
(90 per cent). Then the alcohol is removed as far as possible by pressing
between blotting-paper, after which the pieces of gland are covered with
an ethereal solution of neutral butter-fat (which may be obtained by shak-
ing milk with caustic soda and ether). After the evaporation of the ether
the pieces of gland covered with butter-fat are pressed between two w^atch-
glasses and then gently heated to 37° to 4()° C. in this position. After
some time a marked oelor of butyric acid appears.
The action of the pancreatic juice in splitting fats is a process analogous
to that of saponification, the neutral fats being decomposed, by the addition
of the elements of water, into fatty acids anel glycerine according to the
following formula: C3H5.O3.R3 (neutral fat) +3H20 = C3H5.03.H3 (glycerme)
+3(H.0.R) (fatty acid). This depends upon a hydrolytic splitting, which
was first positively proved by Bernard anel Berthelot. The pancreas
enzyme also decomposes other esters, just as it does the neutral fats
(Nexcki, Baas). The fat -splitting enzyme of the pancreas is, according
to Pawt^ow and Bruxo, aided in its action bj'' the bile, anel according
to Engel obeys Schutz-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 Kaxitz ^ have led to the same results.
' Bernard, Ann. de chini. et physique (3), 25; Berthelot, Jahresber. d. Chem.,
1855, 733; Nencki, Arch. f. exp. Path. u. Pharm., 20; Baas, Zeitschr. f. physiol. Chem.,
1-t, 416; Bruno, Arch, des sciences biolog. de St. Petersbourg, 7; Engel, Hofmeister's
Beitrage, 7; Kanitz, Zeitschr. f. physiol. Chem., 46.
390 DIGESTION.
PoTTEViN * found that the pancreas (free from water) coiild form olein from
oleic acid and glycerine 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.
The fatty acids which are sjDlit off by the action of the pancreatic jiiice
combine in the intestine with the alkalies, forming soaps, which have a
strong emulsifying action on the fats, and thus the pancreatic juice becomes
cf great importance in the emulsification and the absorption of the fats.
Trypsin. The action of the pancreatic juice in digesting proteins was
first observed by Bernard, but first proved by Corvisart.^ It depends
upon a special enzyme called by Kuhne trj'psin. This enzyme, as previ-
ously explained, does not occur in the gland as such but as trj'psinogen.
According to Albertoni ^ this zymogen is found in the gland in the last
third of the intra-uterine life. Enzymes more or less like trypsin occur
in other organs and are very widely diffused in the vegetable kingdom,*
in yeast and in higher plants, and are also formed by various bacteria.
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 antitrypsms by immunization are still disputed
Trypsin, like other enzymes, has not been prepared in a piu"e condition.
Nothing is positively kno^ATi in regard to its nature, but as obtained thus far
it shows a variable behavior (KtJHXE, Klug, Levene, Mays, and others).
At least it does not seem to be a nucleoproteid, and trj^psin has also been
obtained which did not give the biuret test (Klug, Mays, Schwarzschild).
Trypsin dissolves in water and glycerine, while Kuhxe's trypsin was insol-
uble in glycerine. It is very sensitive to heat, and even the body tempera-
tiu'e gradually decomposes it (Vernon, Mays). In neutral solution ifc
becomes inactive at 45° C. In dilute soda solution of 3-5 p. m. it is still
more readily destroyed (Biernacki, Vernon ^). The presence of proteid
or proteoses has, to a certain extent, a protective action on heating an
alkaline trj^psin solution, and this has been substantiated by recent investi-
gations of Bayliss and Vernon. The simpler cleavage products have a still
1 Compt. rend., 138.
^ Gaz. hebdomadaire, 1857, Nos. 15, 16, 19, cited from Bunge, Lehrbuch, 4. Aufl.,
185.
3 See Maly's Jahresber., 8, 254.
* In this connection see Vines, Annals of Botany, 16, 17, 18, 19, and Oppenheimer,
Die Fermente, 1900.
' Kiihne, Verh. d. naturh.-med. Vereins zu Heidelberg (N. F.), 1, 3; E3ug, Math
naturw. Ber. avis Ungam, 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 Beitriige, 4,.
TRYPSIN. 391
greater protective action (Vernon ^). Trypsinogen, according to the
unanimous statements of several experimenters, is more resistant towards
alkalies than trypsin. Trypsin is gradually destroyed by gastric juice
and even by digestive hydrochloric acid alone.
The preparation of pure trypsin has been tried by various experimenters.
The most careful work in this direction was done by KtJHNE and Mays.
Various methods have been suggested by Mays, but we cannot enter into
a discussion of them. A very pure preparation can be obtained by making
use of the combined salting out with NaCl and MgS04. 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
glycerine (HEmENHAiN 2). An impure but still very active infusion can
be obtained after a few days b}^ allowing the finely divided gland to stand
with water which contains 5-lU c.c. chloroform per litre (Salkowski) 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 other enzymes, trypsin is characterized by its action, and this
action consists in dissolving protein and in splitting it into simpler products,
mono- and diamino-acids, trj^ptophane, etc., in alkaline, neutral, and indeed
in very faintly acid solutions. This action has been so far considered as
characteristic for trypsin. Recent investigations seem to indicate that
this action is not due to one enzyme alone but to the combined action
of several enzymes.
There is no question that in the pancreas there occurs besides trypsin
also an enzyme similar to erepsin (Bayliss and Starling, Vernon 3).
According to the latter this erepsin has a strong action upon peptone,
and he believes that the 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 380), whose relationship to pancreas erepsin has not
been determined.
The unity of trj'psin 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 gelatine, and he
calls this enzyme glutitiase. This glutinase is much more resistant towards
acids than trypsin, and by proper treatment with acids Pollak ^ was able
to change a pancreas infusion so that it acted upon gelatine and not upon
certain proteins. The correctness of these statements has, indeed, not
* Bayliss, Arch, des scienc. biolog. de St. Petersbourg, 11, Suppl.; Vemon, Joum.
of Physiol., 31.
' Pfliiger's Arch., 10.
' Bayliss and Starling, Joum. of Physiol., 30; Vernon, ibid., 30.
* Hofmeister's Beitrage, 6. Contradictory statements may be found in Ehren-
reich, cited in Biochem. Centralbl., 4.
392 DIGESTION.
been generally accepted; nevertheless, we have here a warning to be care-
ful 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.
As in recent times the unity of trj'psin has been in doubt, the foUovring
statements apply only to the enzyme which we have been in the habit of
calling trypsin.
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 amoimt 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 putrefac-
tion a little thymol, chloroform, or toluene should be added to the liquid.
Tryptic digestion differs essentially from pepsin digestion, irrespective 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. HCl, 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 substances
such as gelatine, this latter body may be used in detecting trj^psin. The
liquefaction of strongly disinfected gelatine is, according to Fermi, ^ a
very delicate test for trypsin or tryptic enzymes. Various suggestions for
the use of gelatine in the trypsin test have been made, but in considera-
tion of the above statements of Pollak in regard to glutinase it is prob-
ably best for the present to discard the use of gelatine in detecting trj^psin.
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.^
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 ScHiJTZ-BoRissow is perfectly applicable to tr^'psin,
and the amount digested is proportional to the square root of the quantity of
ferment. Based upon the investigations of Bayliss, Hedin, and Lohlein,^
this assumption does not seem to have sufficient foundation, and further
' Arch. f. Hygiene, 12 and 55.
2 Weiss, Zeitschr. f. physiol. Chem., 40; Lohlein, Hofmeister's Beitrage, 7.
^ Pawlow, Die Arbeit der Verdauungsdriisen, Wiesbaden, 189S, p. 33; Bayliss,
Arch, des scienc. biolog. de St. P^tersbourg, 11, Suppl.; Hedin, Journ. of Physiol.,
32; Lohlein, 1. c.
ACTION OF TRYPSIN. 393
exp: riments in this direction are verj- desirable, as so far experimenters have
worked with pancreas infusions or commercial trypsin preparations, which
are generally impure mixtures of enzj-mes. Tryptic digestion is also acceler-
ated by an increase of temperature, at least to about 40° C, at which tem-
perature the protein is very rapidly dissolved by the trypsin. The reaction
is also of the greatest importance. Trypsin acts energetically in neutral,
cr still better in alkaline, solutions, and best in an alkalinity of 3-4 p. m.
Na2C03; but the nature of the protein is also of importance. The action
of the alkali depends upon the number of hydroxyl ions (Dietze, Kaxitz),
and according to Kaxitz^ the digestion proceeds best in those solutions
which are 1/70-1/200 normal in regard to hydroxyl ions. Free mineral
acids, even in verv' small quantities, completely prevent the digestion. If the
acid is not actually free, but combined with protein bodies, then the diges-
tion may take place quickly when the acid combination is not in too great
excess (Chittexdex and Cummixs). Organic acids act less disturb ingl}-,
and in the presence of 0.2 p. m. lactic acid and the simultaneous pres-
ence of bile and common salt the digestion may indeed proceed more quickly
than in a faintly alkaline liquid (Lixdberger). The statement of Rach-
FORD and Southgate, that the bile can prevent the injurious action of
the hydrochloric acid, and that a mixture of pancreatic juice, bile, and
hydrochloric acid digests better than a neutral pancreatic juice, could
not be substantiated by Chittexdex and Albro. That bile has an action
tending to aid the trs-ptic digestion has been shown by many investigators
and recently by Bruxo, Zuxtz and Ussow.^
Carbon dioxide, according to Schierbeck,^ has a retarding action in
acid solutions, but it accelerates the tryptic digestion in faintly alkaline
liquids. Foreign bodies, such as borax and potassium cyanide, maj' pro-
mote trj'ptic digestion, while other bodies, such as salts of mercmy, iron,
and others (Chittexdex and Cummixs), or salicylic acid in large quan-
tities, may have a preventive action. According to Weiss "* the halogen
alkali salts disturb trv'ptic digestion only slightl}^ and NaCl seems to have
the strongest action. The sulphates have a much stronger retarding action
than the chlorides. Borax had no influence, while sodium phosphate, on
the contrary, had a strong accelerating action. The nature of the -proteins
is also of importance. Unboiled fibrin is, relatively to most other proteins,
* Kanitz, Zeitschr. f. physiol. Chem. , 3", who also cites Dietze.
^ Chittenden and Cummins, Studies from the Physiol. Chem. Laboratory of Yale
College, New Haven, 1SS5, 1, 100; Lindberger, Maly's Jahresber., 13; Rachford and
Southgate, Medical Record, 1S95; Chittenden and Albro, Amer. Joum. of Physiol., I,
1898; Rachford, Joum. of Physiol., 25; Bruno, 1. c; Zvmtz and Ussow, Arch. f. (Anat.
u.) Physiol., 1900.
^ Skand. Arch. f. Physiol., 3.
M. c.
394 DIGESTION.
dissolved so very quickly that the digestion test with raw fibrin gives an
incorrect idea of the power of trypsin to dissolve coagulated protein bodies
in general. Boiled fibrin is digested with much greater difficulty and
requires also a higher alkalinity: 8 p. m. NagCOs (Vernon ^). The resist-
ance of certain native protein solutions, such as blood-serum and egg-white,
against the action of trypsin is remarkable; a behavior which can be
explained by the occurrence of antitrypsin in these solutions. An accumu-
lation of the products of digestion tends to hinder the trypsin digestion.
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 2), Besides this one obtains from fibrin,
as well as from other proteins, the products previously mentioned in Chapter
II. In trypsin digestion the cleavage may proceed so far that the mix-
ture fails to give the biuret reaction. This does not indicate, as E. Fischer
and Abderhalden have shown, a complete cleavage of the. protein mole-
cule into mono- and diamino-acids, etc. In tryptic digestion, as shown by
Abderhalden and Reinbold,3 using the protein edestin, 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 glycocoU, stubbornly
resist the cleavage action of the trypsin. The polypeptide-like bodies dis-
covered by Fischer and Abderhalden, which are produced in digestion,
and which do not give the biuret reaction, are the atomic complexes which
resist the action of trypsin. These polypeptides contain the pyrrolidine
carboxylic acid and phenylalanine groups of the protein, but also yield
other monamino-acids such as leucine, alanine, glutamic acid, and aspartic
acid. In tryptic digestion no more nitrogen is split off as ammonia than
on hydrolysis with acids (Mochizuki), which is a difference between trypsin
and the autolytic enzymes. Among the above-mentioned products we find
on the autodigestion of the gland other substances, such as oxyphenyl-
ethylamine (Emerson), which is produced from tyrosine by fermentive CO2
cleavage; also uracil (Levene), guanidine (Kutscher and Otori), the
purine bases, which originate from the nuclein bodies, and choline, which
latter is formed from lecithin (Kutscher and Lohmann 4). If putrefaction
is not completely prevented, still other bodies occur which will be con-
sidered later in connection with the putrefactive processes in the intestine.
» Journ. of Physiol., 28.
^ Herrmann, Zeitschr. f. physiol. Chem., 11.
2 Zeitschr. f. physiol. Chem., 11 and 16. See also Chapter II.
•* Fischer and Abderhalden, Zeitschr. f. physiol. Chem., 31); Mochizuki, Hofmeister's
Beitrage, 1; Emerson, ibid., 1; Levene, Zeitschr. f. physiol. Chem., 37; Kutscher and
Lohmann, ibid., 39; Kutscher and Otori, ibid., 13, and Centralbl. f. Physiol., 18.
ACTION OF TRYPSIN. 395
The Action of Trypsin upon other Bodies. The nucleoproteids and nucleins
are so digested that the proteid 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 nuclease (Sachs). A cleavage of nucleic acids with the setting free of
phosphoric acid and purine bases is, according to Iwanoff,^ not brought
about by trypsin. This splitting is first produced by the action of nuclease
or erepsin (see page 380). Gelatine is dissolved and digested by pancreatic
juice. A cleavage with the separation of glycocoU and leucine does not
occur (KtJHNEand Ewald), or only to a trivial extent (Reich-Herzberge^).
The gelatine-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 indis-
tinctly constructed network of collagenous substance (KDhne 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, arc dissolved all but the nuclei, connective tissue, fat-cor-
puscles, 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 chili n and horn substance do not seem to be acted upon
by the enzyme. Oxyhmnoglobin is decomposed by trypsin with the split--
ting off of haematin. Trj-psin has no action upon fats and carbohydrates.
We have the investigations of Gulewitsch, Gonneriviaxn, Schwarz-
scHiLD,3 E. Fischer and Bergell, and Abderhalden ^ upon the action of
trypsin on simply constructed substances of knoiMi constitution, such as
acid amides and several others that give the biuret reaction. An undoubted
cleavage on Curtius's biuret base was first observed by Schwarzschild.
The investigations of Fischer and his co-workers are much more complete
and important. From these it is shown that the pancreatic juice splits a
large number of peptides, as well as di- and tri- or tetrapeptides, while
it is without action upon a large number of others. The structure
of these plays an important role, as, for example, alanyl-glycine,
CH3.CH(NH2).C0.NH.CH2.C00H, is split, while its isomereglycyl-alanine,
NH2.CH2.CO.NH.CH(CH3).COOH, is, on the contrarj^ not split. The
nature of the amino-acids existing in the peptide is also of importance.
• Iwanoflt, Zeitschr. f. physiol. Chem., 39, which also contains the literature; Sachs,
Hid., 46.
^ Kiihne and Ewald, Verh. d. naturh.-med. Vereins zu Heidelberg (N. F.), 1; Reich-
Herzberge, Zeitschr. f. physiol. Chem., 84.
' Hofmeister's Beitrage, 4, where the other works are also cited.
* Fischer and Bergell, Ber. d. d. chern. Gesellsch., Z?t and 37; Fis^^her and Abder-
halden, Sitzungsber. der Kgl. Pr. Akad. d. Wissensch., Berlin, 1905.
396 DIGESTION.
Those dipeptides which contain alanine as acyl — for example, alanyl-
glycine, alanyl-alanine, and alanyl-leucine A — are readily hydrolyzed, while
several dipeptides in which a-aminoljutyric acid or leucine functionates as
acyl are \ery resistant. The number of amino-acid groups is also of
importance, as, for example, triglj'cyl-glycine is not split, while tetraglycyl-
glycine is. In those peptides which are racemic bocUes the hydrolysis takes
place asymmetrically, so that only one half of the racemic body is attacked,
and those active amino-acids result as products whicli are contained in the
natural protein bodies. Tiiis hydrolysis of various pol3-peptides by means
of pancreatic juice is of especially great interest from several points of
\'iew.
Pancreatic rennin is an enzyme found in the gland and inthe juice which coagu-
lates neutral or alkaline milk (Ki^hne and Roberts and others). This enzyme is
not identical with trypsin, and the optimimi of its action lies according to Vernon
between 60° and 65°. According to Halliburton and Brodie ^ casein is con-
verted by the pancreatic juice of the dog into "pancreatic casein," a sub-
stance 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 verj' 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 ofWiRsuNG'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.
Besides the enzymes which have been discussed in connection with the
pancreatic juice, the gland also contains others, among which can be men-
tioned the enzyme which, according to Stoklasa and his collaborators,
occurs chieflv in organs and tissues and which decomposes sugar into alcohol
and carl)on dioxide, like zymase. According to tSiMACEK,^ in the pancreas
the glycolysis by means of alcoholic fermentation, and the hydrolysis of
the disaccharides, arc united together as a specific action, and he has
obtained precipitates from cell-free press-fluid with alcohol and ether
which brought on both actions without bacterial action. The statements
as to the importance of the pancreas for glycolysis are very contradictory,
and we therefore refer the reader to what has been previously stated on
this subject in Chapter VIII, pages 302 and 303.
* Ki'ihne and Roberts, Maly's Jahresber., 9; see also Edkins, Journ. of Physiol., 12
(literature references); Halliburton and Brodie, ibid., 20; Vernon, ibid., 27.
^ Maly's Jahresber., 29, 353.
' Stoklasa, see foot-note 1, p. 303; Simacek, Centralbl. f. Physiol., 17.
CHEMICAL PROCESSES IN THE INTESTINE. 397
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; ^ 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. The bile has, at least in certain animals,
a slight diastatic action, which in itself can hardly be of any great impor-
tance, but which shows that the bile has not a preventive but rather a
beneficial influence on the energetic diastatic action of the pancreatic
juice. Martin, Williams, Pawlow, and Bruno ^ 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 occur habitually
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; still the products formed thereby are not well known.
That cellulose undergoes a fermentation in the intestine by the action of
micro-organisms, producing marsh-gas, acetic acid, and butyric acid, has
been especially shown by Tappeiner; still it is not known to what extent
the cellulose is destroyed in this way.* The extensive experiments of
Ellenberger and his collaborators, and especially the observations of
ScHEUNERT upon the digestion of cellulose, are very' important. Scheu-
NERT^ finds that the alkaline contents of the csecum of the horse, pig, and
^ See Wroblewski and collaborators, Hofmeister's Beitrage, 1.
^Martin and Williams, Proceed, of Roy. Soc, 45 and 48; Bruno, foot-note 1,
p. 389.
3 See foot-note 2, p. 379.
* On the digestion of cellulose see Henneberg and Stohmann, Zeitschr. f. Biologie>
21, 613; V. Knieriem, ibid., 67; Hofmeister, Arch. f. wiss. u. prakt. Thierheilkunde,
11; Weiske, Zeitschr. f. Biologic, 22, 373; Tappeiner, ibid., 20 and 24; Mallevre,
Pfliiger's Arch., 49; Omeliansky, Arch. d. scienc. biol. de St. P^tersbourg, 7; E. Miiller,
Pfliiger's Arch., 83; Lohrisch, Zeitschr. f. physiol. Chem., 4" (literature).
° Ellenberger, Arch. f. (Anat. u.) Physiol., 1906; Scheunert, Zeitschr. f. physiol.
Chem., 48.
398 DIGESTION.
rabbit have the power of dissolving cellulose to a considerable extent. This
power increases as the abundance of micro-organisms increases and vice
versa; but even in the complete absence of these organisms considerable
quantities of cellulose are dissolved. The secretion or the extract of the
csecal mucosa or the csecal glands does not contain a cellulose-dissohdng
enzyme, and the solution of cellulose in the caecum seems therefore to be
entirely connected with the micro-organisms or their products.
The bile has, as shown by Moore and Rockwood ^ and then especially
by PFLiJGER, the property to a high degree of dissolving fatty acids, espe-
cially 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 Rach-
roRD,2 it accelerates the fat-splitting action of the steapsin. According
to V. FuRTH and Schutz ^ the bile-salts are the active constituents of the
bile in this cleavage, and the fatty acids set free can combine with the alkalies
of the intestinal and pancreatic juices and the bile, producing soaps which
are of great importance in the emulsification of the fats.
If to a soda solution of about 1-.3 p. m. Na2C03 is added pure, perfectly
neutral olive-oil in not too large 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 per-
manent emulsion, making the liquid appear like milk. The free fatt}' 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, Loewen-
THAL 4). This emulsifying action of the fatty acids split off by the pan-
creatic 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 363).
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,^ to have no disturbing action
on gastric digestion. According to Boldireff,^ in continuous starvation,
' Proceedings of Roy. Soc, 60, and Journ. of Physiol., 21. In regard to Pfliiger's
work see Absorption.
^ Nencki, Arch. f. exp. Path. u. Pharm., 20; Rachford, Journal of Physiol., 12.
3 Centralbl. f. Physiol., 20.
* Brucke, Wien. Sitzungsber., 61, Abt. 2; Gad, Arch. f. (Anat. u.) Physiol., 1878;
Loewenthal, ibid., 1897.
" Oddi, in Centralbl. f. Physiol., 1, 312; Dastre, Arch, de Physiol. (5), 2, 316.
'Centralbl. f. Physiol., 18, 457.
ACTION OF THE BILE. 399
on feeding fat and food rich in fat. as well as after large amounts of acid,
a mixture of bile, pancreatic juice, and intestinal juice passes readily into the
stomach. After food rich in fat, which retards the secretion of gastric
juice and the motilit}' of the stomach, a digestion due to this alkaline
mixture may take place in the stomach.
Bile itself has no solvent action on proteins in neutral or alkaline reaction,
but still it may exert an influence on protein digestion in the intestine. The
acid contents of the stomach, containing an alnnidance 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 contrarj^, the bile does not disturb
the chgestion of proteins by the pancreatic 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 trypsin is accelerated by the bile. In a dog
killed while digestion is going on, the faintly acid, bile-containing material
of the intestine shows regularly a strong digestive action on proteins.
The precipitate formed on the meeting of the acid contents of the
stomach with the l^ilc easily rcdissolves 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 under faintly acid reaction. Since in
man the excretor}- 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 fer-
mentation and putrefaction processes caused by micro-organisms. These
are less intense in the upper parts of the intestine, but increase in intensity
towards the lower part of the same, 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. Macfadyex, M.
Nencki, and N. Sieber ^ have investigated a case of human anus praeter-
naturalis, in which the fistula occurred at the lower end of the ileum, and
they were able to investigate the contents of the intestine after it had been
exposed to the action of the mucous membrane of the entire small intestine.
The mass was yellow or yellowish brown, due to bilirubin, and had an acid
' Arch. f. exp. Path. u. Pharm., 28.
400 DIGESTION.
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
em]3yreumatic odor recalling that of volatile fatty acids, and only seldom
had a putrid odor resembling that of indol. The essential acid present was
acetic acid, accompanied by fermentation lactic acid and paralactic acid,
volatile fatty acids, succinic acid, and bile-acids. Coagulable proteins,
peptone, mucin, dextrin, dextrose, 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-men-
tioned organic acids.
Further investigations of Jakowsky and of Ad. Schmidt ^ led 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 car-
nivora. In these latter it has been possible to follow the intestinal diges-
tion by investigating the contents of the various parts of the intestine as
well as by forming fistulas. London and Suluia produced fistulac in dif-
ferent dogs in the duodenum, jejunum, and ileum, and could follow the
digestion of boiled egg-white, A complete destruction of the same took
place, so that 99.7 per cent of the protein was dissolved and flowed out of
the fistula at the ileum (2-3 cm. in front of the caecum). The intestinal
contents gave the biuret reaction very faintly, and the dissolved substance
seemed to have been transformed into end-products. Maetzke,^ who
carried on his investigations on a dog with a fistula at the lower end of the
ileum, on feeding meat never found a putrid or faecal odor to the intestinal
contents. The digestion and absorption of the meat as well as of the carbo-
hydrate was also nearly complete. Leucine and tyrosine were looked for
but not found, and the absence of these bodies was explained by the fact
that they were absorbed.
Because of the absorption it is also difficult to state the extent of de-
struction of the proteins in the intestine. Several experimenters who have
investigated the intestinal contents of dogs during the digestion of meat
have detected amino-acids such as leucine, tyrosine, lysine, and arginine
(KuTSCHER and Seemann). glutamic and aspartic acids, alanine (London),
and polypeptides not giving the biuret reaction (Abderhalden^).
> Jakowsky, Arch, des scienc. biol. de St. Petersbourg, 1; Ad. Schmidt, Arch. f.
Verdauungskr., 4.
» London and SuUma, Zeitschr. f. physiol. Chem., 40; Maetzke, Beobachtungen
an Hunden mit Anus prseternaturahs, Inaug. -Dissert. Breslau, 1905.
' Kutscher and Seemann, Zeitschr. f. physiol. Chem., 34; Abderhalden, ibid., 44;
London, ibid., 47.
DIGESTION IN THE INTESTINE. 401
The digestion and absorption of proteins in the stomach and small
intestine may be nearly complete, but this is not always so. In experi-
ments with raw egg-white Loxdox and Sulima reobtained about 73 per
cent of the coagulable protein from the ileum fistula, and in the entire
intestine from the pylorus to the csecum only about 12 per cent of the
food substance was absorbed. Also in milk-feeding a considerable part
of the protein passes into the large intestine (Berlatzki i).
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
nimierous investigators, especially Nexcki, Baumaxx^, Brieger, H. and
E. Salkowski, and their pupils. The products which are formed in the
putrefaction of proteins are (in addition to proteoses, peptones, amino-acids,
and ammonia) imlol, skatol, paracresol, phenol, phenylpropionic add, and
phenylacetic acid, also paraoxyphenylacetic acid and hydroparocumaric
acid (besides paracresol. produced in the putrefaction of tyrosine), volatile
fatty acids, carbon dioxide, hydrogen, marsh-gas, methylmercaptan, and
sulphuretted hydrogen. In the putrefaction of gelatine neither tjTOsine
nor indol is formed, while glycocoll is produced instead.
Among these products of decomposition a few are of special interest
because of their beha^-ior within the organism and because after their
absorption the}' pass into the urine. A few, such as the oxyacids, pass
unchanged into the urine. Others, such as phenols, are directly trans-
formed into ethereal sulphuric acids by spithesis, and are eliminated as
such by the urine; on the contrary, others, such as indol and skatol, are
only converted into ethereal sulphuric acids after oxidation (for details see
Chapter XV). The quantity of these bodies in the urine varies also with
the extent of the putrefactive processes in the intestine; at least this is
true for the ethereal sulphuric acids. Their quantity increases in the urine
■uith a stronger putrefaction, and the reverse takes place, nameh', a disap-
pearance from the urine, or a great reduction in quantity, as Baumaxx,
Harley and Goodbody - have shown b}^ experiments on dogs, when the
intestine was disinfected liy various agents.
Among the above-mentioned putrefactive products in the intestine the
two following, indol and skatol, should be especially noted.
' See Biochem. Centralbl., 2.
^Baumann, Zeitsclir. f. physiol. Chem., 10; Harley and Goodbody, Brit. Med.
Joum., 1899.
402 DIGESTION.
CH
Indol, C8H7N = C6H4 CH, and Skatol, or methyl-indol,
\ /
NH
C.CH3
^ X
C9H9N = C6H4 ^CH, are two bodies which stand in close relationship
\ /
NH
to the indigo substances and are formed in variable quantities from pro-
tein compounds under different conditions. Hence they occur habitually
in the human intestinal canal, and, after oxidation into indoxyl and skat-
oxyl respectively, pass, at least partly, into the urine as the corresponding
ethereal sulphuric acids and also as glucuronic acids.
These two bodies have been prepared synthetically in many ways.
Both may be obtained from indigo by reducing it with tin and hydro-
chloric acid and heating this reduction product with zinc-dust (Baeyer ^).
Indol may be formed from skatol by passing it through a red-hot tube.
Indol suspended in water is in part oxidized into indigo-blue by ozone
(Nencki 2).
Indol and skatol crystallize in shining leaves, and their melting-points
are 52° and 95° C. respectively. Indol has a peculiar excrementitious
odor, w^hile 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 crys-
tallizing 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 acid a red liquid and
then a red precipitate of nitroso-indol nitrate (Nencki). It is better first
to add two or three drops of nitric acid and then a 2 per cent solution of
potassium nitrite, drop by drop (Salkowski^). 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 nitroprussidc and alkali
(Legal's reaction). On acidifying with hydrochloric acid or acetic acid
' Annal. d. Chem. u. Pharm., 140, and Suppl., 7, 56; also Ber. d. deutsch. chem.
Gesellsch., 1.
* Ber. d. deutsch. chem. Gesellsch., 8, 727, and ibid., 722 and 1517.
' Zeitschr. f. physiol. Chem., 8, 447. In regard to newer reactions for indol and
skatol, see Steensma, ibid., 47.
GASES OF THE INTESTINE. 403
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. Skatol dissolves in concentrated hydrochloric acid with a violet
coloration. On warming skatol with sulphuric acid a beautiful purple-red
coloration is obtained (Ciamiciax and Magnanini ^).
For the detection of indol and skatol in, and their preparation from,
excrement 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 picrate precipitate is then distilled with ammonia. The
two bodies are obtained from the distillate by repeated shaking with ether
and evaporation of the several ethereal extracts. The residue, containing
indol and skatol, is dissolved in a very small quantity of absolute alcohol
and treated with 8-10 vols, of water. Skatol is precipitated, but not the
indol. The further treatment necessary for their separation and purifica-
tion will be found in other works.^
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 fermentation 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 372, on the
composition of the gases of the stomach. Nitrogen is habitualh' 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, parti}'
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
intestinal juices by their neutralization through the hydrochloric acid of
the gastric juice and by organic acids formed in the fermentation. Hydro-
gen occurs ir largest quantities after a milk diet, and in smallest quantities
after a purely meat diet. This gas seems to be formed chiefly in the
butyric-acid fermentation of carbohydrates, although it may occur in
large quantities in the putrefaction of proteins under certain circumstances.
» Ber. d. d. chem. Gesellsch., 21, 1928.
" For quantitative, colorimetric determinations of indol in fseces, see Einhorn and
Huebner, Salkowski's Festschrift, Berlin, 1904.
404 DIGESTION.
There is no doubt that the methylmercaptan and sulphuretted hydrogen
which occur normally in the intestine originate from the proteins. The
marsh-gas undoubtedly originates in the putrefaction of proteins. As
proof of this Ruge ^ found 26.45 per cent marsh-gas in the human intestine
after a meat diet. He found a still greater quantity of this gas after a
vegetable (leguminous) diet; this coincides with the observation that
marsh-gas may be produced by a fermentation of carbohydrates, but
especially of cellulose (Tappeiner 2). Such an origin of marsh-gas, especially
in herbivora, is to be expected. A small part of the marsh-gas and carbon
dioxide may also arise from the decomposition of lecithin (Hasebroek^).
Putrefaction in the intestine not only depends upon the composition 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. Glycocholic 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 fseces. In the foetus, 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 man in
the small but in the large intestine.
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 protein which undergo putrefaction. That the secretions
rich in proteins are destr03^d in putrefaction in the intestine follows from
the fact that putrefaction may also continue during complete fasting.
From the observations of Mxjller^ 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 elimina-
tion, which at first decreased so that it was nearly minimum, increased
again from the fifth day of starvation, and on the eight or ninth day it
was three to seven times as much as in man under ordinary circumstances.
In doo-s, on the contrary', the elimination of indican during starvation is
considerable, but the phenol 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
* Wien. Sitzungsber., 44. ^ Zeitsch. f. Biologie, 20 and 24.
s Zeitschr. f. physiol. Chem., 12. •• Berlin, klin. Wochenschr., 1887.
PUTREFACTION IN THE INTESTINE. 405
in digestion. The putrefaction may be of benefit to the organism in so
far as the formation of such products as proteoses, peptones, and perhaps
also certain amino-acids is concerned. The question has indeed been
asked (Pasteur), is digestion possible ^YitllOut micro-organisms? Nuttal
and Thierfelder have shown that guinea-pigs removed from the uterus
of the mother by Caesarian section could with sterile air digest well and
assimilate sterile food (milk or crackers) in the complete absence of bac-
teria in the intestine, and developed normally and increased in weight.
ScHOTTELius ^ has arrived at other results by experiments with hens.
The chickens, hatched under sterile conditions, kept in sterile rooms and
fed with sterile food, had continuous hunger and ate abundantly, but
soon died, in about the same time as a starving chicken. On mixing with
the food of other chickens, at the proper time, a variety of bacteria from
hen faeces, 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 ma}-, by
the formation of further cleavage products, involve a loss of valuable mate-
rial to the organism, and it is therefore important that putrefaction in the
intestine be kept within certain limits. If an animal is killed while diges-
tion in the intestme is going on, the contents of the small intestine give
out a peculiar but not putrescent odor. Also the odor of the contents of
the large intestine is far less offensive than a putrefying pancreas infusion
or a putrefying mixture rich in protein. From this one may conclude that
putrefaction in the intestine is ordinarily not nearly so intense as outside
of the organism.
It seems thus to be provided, under physiological conditions, that
putrefaction shall not proceed too far, and the factors which here come
mider consideration are probably of different kinds. Absorption is un-
doubtedly one of the most important of them, and it has been proved by
actual observ^ation that the putrefaction increases, as a rule, as the absorp-
tion 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 putrefaction (Hirsch-
ler2). It has been shown by Pohl, Bierxacki, Rovighi, Wixterxitz,
ScHMiTZ, and others ^ that milk and kephir have a specially strong pre-
ventive action on putrefaction. This action is not due to the casein, but
chiefly to the lactose and also in part to the lactic acid.
1 Nuttal and Thierfelder, Zeitsclir. f. physiol. Chem., 21 and 22; Schottelius, Arch
f. Hygiene, 34 and 42.
^ Hirschler., Zeitsclir. f. phj^siol. Chem., 10; Zimnitzki, ibid., 39 (literature).
^ Schmitz, ibid., 17, 401, which gives references to the older Uterature, and 19. See
also Salkowski, Centralbl. f. d. med. Wiss., 1893, 467, and Seelig. Virchow's Arch., 146
(literature).
406 DIGESTION.
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, LixdbergerI). There
is no question that the free bile-acids have a strong preventive action on
putrefaction outside of the organism, and it is therefore difficult to deny
such an action in the intestine. Notwithstanding this the anti-putrid
action of the bile in the intestine is not considered by certain investigators
(VoiT, RoHMANX, HiRscHLER and Terra Y, Landauer and Rosenberg 2)
^s of great importance.
Biliary fistulas have been established so as to study the importance of
the bile in digestion (Schwann, Bloxdlot, Bidder and Schmidt,^ and
others). As a result it has been observed that with fatty foods an imper-
fect absorption of fat regularly takes place and the excrements contain,
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 consideral^ly increased after the operation,
otherwise the animal will become very thin, and indeed die with symptoms
of starvation. In these cases the excrements have the odor of carrion, and
this was considered a proof of the action of the bile in checking putrefac-
tion. The emaciation and the increased want of food depend, naturally,
upon the imperfect al^sorption 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 proteins and fats be increased, then
the latter, which can be only very incompletely absorbed, accumulate
in the intestine. This accumulation of the fats in the intestine only
renders the action of the digestive juices on proteins more difficult,
and thus increases the amount of putrefaction. This explains the ap-
pearance of fetid faeces, 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 putre-
faction. 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 con-
* Maly and Emich, Monatshefte f. Chem., 4; Lindberger, foot-note 2, p. 393.
^ Voit, Beitr. ziir Biologie, Jubilaumschrift, Stuttgart, 1882; Rohmann, Pfliiger's
Arch., 29; Hirschler and Terray, Maly's Jahresber., 20; Landauer, Math. u. Naturw.
Ber. aus Ungarn, 15; Rosenberg, Arch. f. (Anat. u.) Physiol., 1901.
^ Schwann, Midler's Arch. f. Anat. u. Physiol., 1844; Blondlot, cited from Bidder
and Schmidt, Verdauungssafte, etc., 98.
PUTREFACTION IX THE INTESTINE. 407
ditions 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 carboh3''drates,
which are capable of checking putrefaction, can, so to speak, undertake
the anti-putrid action of the bile. But as there are also cases (in dogs
with biliary fistula) where the intestinal putrefaction is not increased with
exclusive meat diet,^ it is maintained that the absence of bile in the intes-
tine, even by exclusive carbohydrate food, does not always cause an in-
creased putrefaction.
Although the question as to the manner in which the putrefactive
processes in the intestine under physiological conditions are kept within
certain limits cannot be answered positively, still it may be asserted that
the acid reaction of the upper parts of the intestine, and the absorption of
water in the lower parts, 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. After
the investigations and observations of Kast, Stadelmaxn, Wasbutzki,
BiERNACKi and ]\Iester had proved that an increased putrefaction in the
intestine occurred when the quantitj^ of hydrochloric acid in the gastric
juice was diminished or deficient. Schmitz 2 has lately shown in man that
on the administration of hydrochloric acid, producing a hj-peracidity of the
gastric juice, the putrefaction in the intestine may be checked. The ques-
tion arises whether the reaction in the small intestine is alwa3's acid and
whether the acidity is strong enough to prevent putrefaction. In this
connection it must be recalled that the acidity of the contents of the small
intestine is not due to hydrochloric acid, but chiefly to organic acids, acid
salts, and free carbon dioxide. There are several statements as to the reac-
tion of the intestinal contents, although they are somewhat contradictory,
by Moore and Rock wood, Moore and Bergin, ]\Iatthes and Mar-
QUARDSEX, I. MuxK, Nexcki and Zaleski, Hemmeter.3 From these
statements one can conclude that the reaction may vaiy not only among
different animals, but also in the same animals under different 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 XaHC03 and free COo, and finally that in certain
* See Hirschler and Terray , 1. c.
^ Zeitsclir. f. physiol. Chem., 19, 401, which includes all the pertinent literature.
^ Moore and Rockwood, Journ. of Physiol., 21; IMoore and Bergin, Amer. Journ.
of Physiol., 3; Matthes and Marquardsen, Maly's Jahresber., 28; ]*Iunk, Centralbl. f.
Physiol., 16; Nencki and Zaleski, Zeitschr. f. physiol. Chem., 2"; Hemmeter, Pfl;,ger's
Arch., 81.
408 DIGESTION.
animals the intestinal contents are alkaline throughout. The question
how, under these conditions, putrefaction is excluded, cannot be explained.
It is possible, as Bienstock admits, that the explanation lies in an antag-
onistic bacterial action and that the carbohydrates, especially lactose,
which retard putrefaction, form a good nutritive media for those bacteria
which destroy the putrefactive producers or retard their development.
Perhaps also, according to the experience of Conradi and Kurpjuweit,^
the autotoxines produced by the intestinal bacteria may, by their antiseptic
action, keep the putrefactive processes in the intestine within bounds.
Excrements. 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,^ was 333 grams, with 75 grams
of solids. With a strictly meat diet the excrements are scanty, pitch-like,
and black. The scanty excrements in starvation have a similar appear-
ance. A large quantity of coarse bread yields a great amount of light-
colored excrement. In these cases the faeces 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 faeces
(ScHiERBECK ^). If there is a large proportion of fat, it takes a lighter,
clay-like appearance. The decomposition products of the bile-pigments
seem to play only a small part in the normal color of the f seces.
The constituents of the faeces arc of different kinds. In the excrements
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 excrements contain 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 putrefac-
tion or of digestion, such as skatol, indol, volatile fatty acids, purine
bases, lime, and magnesia soaps. Occasionally, also, parasites of different
* Bienstock, Arch. f. Hygiene, 39; Conradi and Kurpjuweit, Munch, med. Wochen-
schr., 1905.
2 Zeitschr. f. Biologie, 25, 264.
^ Arch. f. Hygiene, 51.
* In regard to the purine bases in feces, see Hall, Journ. of Path, and Bacteriol., 9;
Schittenhelm, Arch. f. kiln. Med., SI; Schittenhehn and Kriiger, Zeitschr. f. physiol.
Chem., 45.
EXCREMENTS. 409
kinds occur; and lastly, the excrements contain micro-organisms of various
species.
That the mucous membrane of the intestine by its secretion and by
the abundant quantity of detached epithelium contributes essentially to
the formation of excrement follows from the discovery first made by
L. HeRxMaxx and substantiated by others/ that a clean, isolated loop of
intestine collects material similar to faeces. Human faeces seem to consist
in greater part of intestinal secretion and only in a smaller part of residue
from food on a meat or milk diet. Many foods produce a large quantity
of faeces chiefly by causing an abundant secretion.^
The reaction of the excrements 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 excrements by Brieger,
and so named b}' 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 faeces an abnormal color. The excrements are colored black b}'
bismuth, yellow by rhubarij, and green by calomel. This last-mentioned
color was formerly accounted for by the formation of a little mercur}-
sulphide, but now it is said that calomel checks the putrefaction and the
decomposition of the bile-pigments, so that a part of the bile-pigments
passes into the faeces as biliverdin. In the yolk-yellow or greenish-yellow
excrements of nursing infants one can detect bilirubin. Neither bilirubin
nor Ijiliverdin seems to exist in the excrements of mature persons under
normal conditions. On the contrary, there is found stercohilin (Masius
anel Vanlair), which is identical with urobilin (Jaffe ^). Bilirubin may
occur in pathological cases in the faeces of mature persons. It has been
observeel in a crystallized state (as haematoidin) in the faeces of children
as well as of grown persons.
The absence of bile (acholic faeces) causes the excrements to have, as
above stated, a gray color, due to large quantities of fat; this may, however,
be partly attributed to the absence of bile-pigments. In these cases a
large quantity of crystals has been observed which consist chiefly of mag-
nesium soaps or sodium soaps. Hemorrhage in the upper parts of the
"Hermann, Pfli'iger's Arch., 46. See also Ehrenthal, ibid., 48; Berenstein, ibid.,
53; Klecki, Centralbl. f. Physiol., 7; 736, and F. Voit, Zeitschr. f. Biologie, 29; v.
Moraczewski, Zeitschr. f. physiol. Cheni., 25.
^ In regard to the constitution of faeces with various foods, see Hammerl, Kermauner,
Moeller, and Prausnitz, Zeitsclir. f. Biologie, 35, and Poda, Micko, Prausnitz and
Miiller, ibid., 39.
^ See bile-pigments, Chapter VIII, and vu-obilin, Cliapter XV.
410 DIGESTION.
digestive tract yields, when it is not very abundant, a dark-brown excre-
ment, due to hsematin.
ExcRETiN, so named by Marcet,' is a crystalline body occurring in human
excrement, but which, according to Hoppe-Seyler, is perhaps only impure
cholesterin (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 excrements, their
quantitative analyses are of little value and therefore will be omitted.^
Meconium is a dark brownish-green, pitchy, mostly acid mass without
any strong odor. It contains greenish-colored epithelium cells, cell-detritus,
numerous 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 enz3^mes,
calcium and magnesium phosphates. Sugar and lactic acid, soluble
protein bodies and peptones, also leucine and tyrosine and the other pro-
ducts of putrefaction . occurring in the intestine, are absent. Meconium
may contain imdecomposed taurocholic acid, bilirubin and biliverdin, but
it does not contain any stercobiline, which is considered as proof of the
non-existence of putrefactive processes in the digestive tract of the foetus.
In medico-legal cases it is sometimes necessary to decide whether spots
on linen or other substances are caused l^y meconium. In such cases the
following conditions exist: The spot caused by meconium has a brownish-
green color and can be easily separated from the material because, on
account of the ropy property of the meconium, it is difficult to wet through.
When moistened with water it does not develop any special odor, but on
warming with dilute sulphuric acid it smells somewhat fetid. It forms
with water a slimy, greenish-yellow liquid containing brown flakes. The
solution gives with an excess of acetic acid an insoluble precipitate of
mucin; on boiling it does not coagulate. The filtered, watery extract
responds to Gmelin's, but still better to Huppert's reaction for bile-
pigments. The liquid precipitated by an excess of milk of lime gives a
nearly colorless filtrate, which after concentration shows Pettenkofer's
reaction.
The contents of the intestine under abnormal conditions are perhaps less the
subject of chemical analysis than of an inspection and microscopical investiga-
tion or bacteriological examination. On this account the question as to the
properties of the contents of the intestine in different diseases cannot be thor-
oughly treated here.^
* Annal. de chim. et de phys., 59.
^ In regard to these analyses as well as to the faeces under abnormal conditions
and to the pertinent literature, see Ad. Schmidt and J. Strassburger, Die Fgeces des
Menschen, etc., Berlin, 1901 and 1902.
' See Schmidt and Strassburger, 1. c.
INTESTINAL CONCRKMENTS. 411
Appendix.
JNTESTINAL CONCREMENTS.
Calculi occur xevy seldom- in the human intestine or in the Litestine of
camivora, 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, espe-
cially ammonium-magnesium phosphate or magnesimn phosphate, and
these salts usually form the chief constituent of the concrements. In man
they are sometimes oval or round, yellow, yellowish gray, or brownish gray,
of variable size, consisting of concentric layers and containing chiefly
ammonium-magnesium phosphate and calcimn phosphate, besides a small
quantity of fat or pigment. The nucleus ordinaril}- 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 -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 calciiun and magnesium phosphate
(about 70 per cent), oat-bran (15-18 per cent), soaps and fat (about 10
per cent). Concretions which contain ven,^ much fat (about 74 per cent)
occaiiionally 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 hea^y
(as much as 8 kilos) and consist in great part of concentric layers of ammo-
nium-magnesium phosphate. Another variety of concrements which
occurs in horses and cattle consists of gray-colored, often verj- 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, consisting of matted hairs and
plant-fibres, and termed hair-balls. The so-called ".egageopiL/E.'' which
probably originate from the axtilopus rupicapra, belong to this group,
and are generally considered as nothing else than the hair-balls of cattle.
The so-called oriental bezoar-stone belongs also to the intestinal concre-
ments, and probably originates from the intestinal tract of the capra
^GAGRUS and axtilope 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 con-
tains as chief constituent lithofellic acid, C20H36O4, which is related to
cholic acid, and besides this a bile-acid, lithobilic acid. The others are
nearly blackish brown or dark green, ven,' glossy, consisting of concentric
layers, and do not melt on heating. They contain as chief constituent
412 DIGESTION.
ellagic acid, a derivative of gallic acid, of the formula CuHgOg, which,
according to Graebe,i is the dilactone of hexaoxydiphenyldicarboxylic acid
and which gives a deep-blue color with an alcoholic solution of ferric chlo-
ride. This last-mentioned bezoar-stone originates, to all appearances,
from the food of the animal.
Ambergris is general!}' 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 boihng alkalies. It dissolves in alcohol, ether, and oils.
VI. Absorption.
The problem of digestion consists in part in separating the valuable con-
stituents of the food from the useless ones and dissolving or transforming
them into forms which are adapted for the processes of absorption. In
discussing the absorption processes we must treat of the form into which
the different foods are changed before absorption, of the manner 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 proteins
are absorbed from the intestinal canal, it is of interest to leam whether the
animal body can, perhaps, also utilize such protein a^ is introduced intra-
venously, subcutaneously, or into a body-cavity, i.e., evading the intestinal
canal, or, as Oppexheimer calls it. parenteral.
Since the first investigations of Zuxtz and v. Merixg on this subject
several expsrimenters, such as Neumeister. Friedexth.u. and Lew^ax-
DowsKY, MuxK and Lewaxdowsky, Oppexheimer. Mexdel and Rock-
wood, and others,^ have sho^ATi, 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.
If the animal body can assimilate parenterally introduced protein, then
the question arises, whether it can also take up undigested protein from the
intestinal canal and utilize it. In this regard we have the observations of a
large number of investigators, such as Brucke, Bauer and Voit. Eich-
» Ber. d. d. chem. Gesellsch., 3(5.
^ Zuntz and v. Me:ing, Pfliiger's Arch., 32; Neumeister, Verh. d. phys.-med.
Gesellsch. zu W rzburg, 1889, and Zeitschr. f. Biologie, 2"; Friedenthal and Lewan-
dowsky, Arch. f. (Anat. u.) Physiol., 1899; Munk and Lewandowsky, ibid., 1899, Suppl.;
Oppenheimer, Hofmeister's Beitrage, i; Mendel and Rockwood, Amer. Joum. of
Physiol., 12.
ABSORPTION OF PROTEINS. 413
HORST, CzERXY and Latschexberger, ^'oIT and Friedlaxder,^ who
have shown that non-pcptonized protein can be absorbed from the intestine.
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 oval-
bumin or seralbumin and 69 per cent of alkali albuminate (dissolved in
alkali) were absorbed. Mexdel and Rockwood, on the contrary, in
experiments with casein and edestin in the living intestinal loop, could
prove only the slightest absorption on excluding digestion as completely
as possible, while the corresponding proteoses were abundantly absorbed.
It is difficult to decide in these experiments as to how far the proteins
were taken up in an actually unchangeel or partly modified form. The
alimentary albuminuria obsen-ed repeatedly after the introdtiction of
large quantities of protein into the intestinal canal speaks for an absorption
of undigesteel protein uneler certain circumstances. To deciele this question
the biological method, using the precipitine reaction, has l^een made use
of, and AscoLi and ^'igxo.^ using this method, claim to have shown the
passage of non-mocUfied protein into the blood and lymph. Based upon
many investigations on this subject we can consider it possilile that under
certain circiunstances, as on flooding the intestmal canal with protein,
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-modifieel 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 the same, and the next question is whether the
proteins are chiefly absorbed as proteoses or peptones or as simpler atomic
complexes.
This question cannot be answered for the present. Investigations on
the con+^nts of the stomach anel intestine have shoT\7i the presence of
proteoses and peptones as well as non-biuret-giving atomic complexes and
amino-acids. The results of the investigations of Schmidt-Mulheim.
Ellexberger and Hofmeister, Ewald and Gumlich. Zuxz. Reach,
Kutscher and Seemaxx, Abderhaldex. Glaessxeb, and others 3 have
* Briicke, '\\'ien. Sitzungsber., a9; Bauer and Voit, Zeitschr. f. Biologic, o; Eich-
horst, Pfliiger's Arch., 4; Czerny and Latschenberger, Virchow's Arch., 59; Voit and
Friedlander, Zeitschr. f. Biologic . 33.
2 Zeitschr. f. physiol. Chem., 39.
^Schmidt-Midheim. Arch. f. (Anat. u.) Physiol., 1879; Ellenberger and Hofmeister,
ibid., 1890; Ewald and Gumlich, Berlm. klin. Wochensclir., 1S90; E. Zunz, Hof-
meister's Beitriige, 3; Reach, ibid., 4; Zmiz, Aimal. de la soc. roy. d. scienc. de Bru-
xelles, 13; Kutscher and Seemann, Zeitsclir. f. physiol. Chem., 34 and 35; Abderhalden,
ibid., 44; Glaessner, Zeitsclir. f. klin. Med., 52.
414 DIGESTION.
given, as was to be expected, contradictory and variable results, and
as the absorption runs more or less parallel with digestion the quantities
of the various products found in the intestinal canal cannot give any positive
conclusions as to the amounts produced.
The proteoses, as well as the peptones, have been repeatedly found in
the stomach and the intestine, and therefore the question has been raised
for a long tims how these bodies are absorbed and how they are introduced
in the tissues. The generally accepted view is that they do not pass into
the blood through the lymphatics, but through the intestinal epithelium,
and this view is based essentially on the two following conditions: On
completely isolating the chyle from the blood circulation, the protein
absorption from the intestine is not impaired (Ludwig and Schmidt-
MtJLHEiM) ; and on a diet rich in protein the quantity thereof in the chyle
(in man) was not noticeal^ly increased (Munk and Rosensteix). Asher
and Barbera ^ have shown in experiments on a clog that the quantity of
protein in the lymph was slightly increased after partaking of considerable
protein. This experiment does not disprove the assertion of a\IuxK that
the blood-vessels form nearly the exclusive exit of the proteins from the
intestinal tract.
After a diet rich in proteins neither proteoses nor peptones are found
in the blood or the chyle. Nor are they ^^resent in the urine; and the
absence of these bodies in the blood after digestion cannot be explained by
the statement that they, like the proteoses (peptones) injected subcutane-
ously or directly into the blood, are quickly eliminated through the kidneys
(Plosz and Gyergyai, Hofmeister, Schmidt-Mulheim^). It might be
supposed that the proteoses (peptones) formed in digestion are retained by
the liver, and that this is the reason why they are not found in the blood.
This explanation does not seem to be sufficient. Neumeister has inves-
tigated the portal blood of rabbits into whose stomachs large quantities
of proteoses and peptones had been introduced, without finding traces of
the bodies in question.
He has also shown that when the liver of a dog is supplied with
portal blood to which peptone is added (ampho-peptone), this is not
retained by the liver. Shore has arrived at similar results in regard to
the importance of the liver, and has also shown that the spleen cannot
transform peptone. Peptone seems to pass neither into the blood nor
the chylous vessels, and the following ol:>servation of Ludavig and Sal-
' Sclimid1>Miilheim, Arch. f. (Anat. u.) Physiol., 1877; Munk and Rosenstein, Vir-
chow's Arch,, 123; Asher and Barbera, Centralbl. f. Physiol., 11, 403; Munk, ibid.,
11, 585. See also Mendel, Amer. Journ. Physiol., 2.
' Plosz and Gyergyai, Pfliiger's Arch., 10; Hofmeister, Zeitschr. f. physiol. Chem.,
5; Sclimidt-M ilheim, Arch. f. (Anat. u.) Phy.siol., 1880.
ABSORPTION OF PROTEINS. 415
viOLi bears out this assumption. These investigators introduced a peptone
solution into a double-Ugatured, isolated piece of the small intestine, which
was kept alive by passing defibrinated blood through it, and observed that
the peptone disappeared from the intestme, but that the blood passing
through did not contain any peptone, Cathcart and Leathes ^ with their
own experiments as a basis, give another interpretation of Salvioli's obser-
vations, namely, by the statement that the disappearance of the peptone
from the loop of the intestine depends upon a hydrolysis of the same. On
the other hand, they also found that no peptone was taken up by the
circulating blood.
It must be remarked in connection with this view that, according to
Embden and Knoop, and Langstein, proteoses sometimes occur in blood-
serum, and also that Nolf ^ has found, after abundant absorption of
proteoses from the intestine, a small amount in the blood. This occurrence
of proteoses in the blood is not contradictory to the view that the chief
quantity of proteoses and peptones does not pass from the intestine into
the blood as such.
Many observations indicate that the proteoses and peptones are trans-
formed in some way in the intestine or intestinal wall, and a retransforma-
tion of proteoses into protein is considered most plausible.
Certain investigators, such as v. Ott, Nadine Popoff, and Julia
Brinck,^ are of the opinion that the proteoses and peptones are transformed
into seralbumin before they pass into the walls of the digestive tract. This
transformation is brought about by means of the epithelium-cells, as also
by the vital activity of a fungus called by Julia Brinck Micrococcus
restituens. No positive proofs have been presented to support this view.
The view that the transformation of the proteoses and peptones takes
place after they have been taken up by the mucous membrane has better
foundation. According to the observations of Hofmeister,'* the walls
of the stomach and the intestine are the only parts of the body in which
proteoses (peptones) occur constantly during digestion, and the fact that
proteoses (peptones) at the temperature of the body disappeared after a
time from the excised but apparently still living mucous coat of the stom-
ach, also confirm this.
This disappearance of proteoses is considered by Hofmeister as a
transformation into ordinary protein. For such a transformation of pro-
' Neumeister, Sitzungsber. d. phys.-med. Gesellsch. zu Wiirzburg, 1889, and Zeitsclir.
f. Biologic, 24; Shore, Journ. of Physiol., 11; Salvioli, Arch. f. (Anat. u. (Physiol..
1880, Suppl.; Cathcart and Leathes, Journ. of Physiol., 33.
^ See Chapter VI, foot-note 1, p. 183.
^ V. Ott, Arch. f. (Anat. u.) Physiol., 1883; Popoff, Zeitschr. f. Biologic, 25; Brinck,
ibid., 453.
* Zeitschr. f. physiol. Chem., 6, and Arch. f. exp. Path. u. Pharm., 19, 20, and 22.
416 DIGESTION.
teoses in the mucosa of the stomach, Glaessner i has suggested new
experimental evidence, while the Hofmeister school (Embden and Knoop)
consider the regeneration of peptone into coagulable protein in the intes-
tine as not proved.
According to Hofmeister the leucocytes, which are increased during
digestion, play an important part in the transformation of the proteoses
and peptones. They may in the first place take up the proteoses (pep-
tones) and be the means of transporting them to the blood, and secondly
by their growth, regeneration, and increase may stand in close relationship
to the transformation and assimilation of the bodies. Heidenhain, who
considers that the transformation of peptones into protein in the mucous
membrane is positively settled, does not attribute so great an importance
to the leucoc3rtes in the absorption of the peptones, chiefly on the groimd
of comparative estimation of the quantity of absorbed peptones and leuco-
cytes. He considers it as more probable that the reconversion of the
peptones into protein takes place in the epithelium layers. This view is
further corroborated by the investigations of Shore.^
On account of the discovery of erepsin by CoHxXHeim, the theorv^ as to
the absorption of proteins has taken another direction. There seems to
be a tendency to lean towards the view that the proteoses and peptones
are split in the intestine, or in the intestinal mucosa, into simpler bodies
which do not give the biuret test and from which the proteins are regen-
erated. The question whether the active agent is erepsin or trypsin
is only of secondary importance, as both of these enzymes split the pro-
teoses and peptones alike.
According to the investigations of the Hofmeister school on pepsin
digestion, and of Fischer and Abderhalden on tr^^psin digestion (see
Chapter II), the disappearance of the biuret test does not indicate a com-
plete cleavage of the proteins into amino-acids, since peptoids or poly-
peptides occur; consequently it is for the present not possible to say to what
extent the proteins are broken dowTi in the intestinal canal, and how far the
amino-acids and more complex atomic groups not giving the biuret reaction
are produced. It is just as difficult to state with positiveness, although
feeding experiments with this in view have been carried out, how far a
regeneration of protein from such abiuret peptides or from amino-acids
is possible.
The possibility of keeping an animal for a certain time in nitrogenous
equilibrium with abiuret digestion products was first demonstrated by Loewi.
He fed dogs with an abiuret digestion mixture of pancreas tissue and kept
them in nitrogenous equilibrium for more than a month. Henderson
* Hofmeister's Beitrage, 1.
^ Heidenhain, Pfluger's Arch., 43; Shore, 1. c.
ABSORPTION OF PROTEINS. 417
and Dean were also able in a bitch to observe nitrogenous equilibrium
for at least a few days by feeding the abiuret products of the acid cleavage
of meat, while Lesser, on the contrary, could not bring the animal in
nitrogenous equilibrium by using fibrin digested with trypsin. These
negative results not only confront the positive results of Loewi but also
the observations of Abderhalden and Rona, as well as of Hexriques
and Hansen,! and there is no doubt that mice, rats, and dogs can be kept
for at least a certain time in nitrogenous equilibrium with abiuret digestion
products consisting in great part of monamino-acids. Of special interest
is, no doubt, the fact that in the experiments of Abderhalden and Rona,
as well as of Henriques and Hansen, the abiuret products obtained from
casein by pancreatic digestion could protect the animal from loss of nitro-
gen, while the products obtained by acid hydrolysis of casein or a mixture
of amino-acide corresponding to casein (Abderhalden and Rona) could
not do this. Remarkable is the observ^ation of Henriques and Hansen
that the products (monamino-acids?) not precipitable by phosphotungstic
acid could also cover the nitrogen loss. It is hardly possible to draw any
positive conclusions from the above experiments as to the ability of the
animal body to regenerate proteins by synthesis from abiuret digestion
products. It is just as difficult to say whether and to what extent a
synthesis of protein from the simple cleavage products takes place in the
intestinal wall. Cathcart and Leathes found that when peptone or
end-products of pancreatic digestion were absorbed by the intestinal loop
the amount of nitrogenous substances in the blood not precipitated by
tannic acid regularly increased, which seems to indicate that these simple
cleavage products were taken up by the blood.
The extent of the protein absorption is dependent essentially upon the
kind of food introduced, since as a rule the protein substances from an
animal source are much more completely absorbed than from a vegetable
source. As proof of this the following observations are given: In his experi-
ments on the utilization of certain foods in the intestinal canal of man Rub-
ner 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 in-
troduced. 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 conditions are quite different with vegetable food, as
shown by the researches of Meyer, Rubner, Hultgren and Lander-
gren, who made experiments with various kinds of rj^e bread and found
' Loewi, Arch. f. exp. Path. u. Pharm., 4S. See also Henderson and Dean, Amer.
Journ. of Physiol., 9; Lesser, Zeitschr. . f . Biologie, 45; Abderhalden and Rona,
Zeitschr. f. physiol. Chem., 42, 44, and 47; Henriques and Hansen, ibid., 43.
418 DIGESTION.
that the loss of nitrogen through the faeces amounted to 22-48 per cent.
Experiments with other vegetable foods, and also the investigations of
Schuster, Cra:\ier, Meinert, Mori.^ and others on the utilization of foods
with mixed diets, have led to similar results. With the exception of rice,
wheat bread, and certain verj^ finely divided vegetable foods, it is found
in general that the nitrogen deficit by the faeces increases with a larger
quantity of vegetable material in the food.
The reason for this is manifold. The large quantity of cellulose fre-
quently present in vegetable foods impedes the absorption of proteins.
The greater irritation produced by the vegetable food itself or by the organic
acids formed in the fermentation in the intestinal canal causes a more
violent peristalsis, which drives the contents of the intestine 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.
In speaking of the functions of the stomach we stated that after the
removal or excision of this organ, an abundant digestion and absorption
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 extirpation of
the gland by Minkowski and Abelmann, Sandmeyer, V. Harley, after
destroying the gland by Rosenberg, and also in man after closing the
pancreatic duct by Harley and Deucher.^ In all these different cases such
discrepant figures have been obtained for the utilization of the proteins —
between 80 per cent after the apparently complete exclusion of pancreatic
juice in man (Deucher) and IS per cent after extirpation 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. This is
not to be wondered at, because one would expect that in such cases the
other digestive fluids undergo variation, and indeed to various degrees in
the different cases. Zunz and Mayer 3 have also 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
» Rubner, Zeitsclir. 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, "tjber Massennahrung," Berlin, 1885; Kell-
ner and Mori, Zeitschr. f. Biologie, 25.
2 Abelmann, "Uber die Ausnitzung der Nahrungsstoffe nach Pankreasexstirpa-
tion" (Inaug.-Dissert. Dorpat, 1890), cited from Maly's Jahresber., 20; Sandmeyer,
Zeitschr. f. Biologie, 31; Rosenberg, Pfl: ger's Arch., 70; Harley, Joum. of Pathol,
and Bacteriol., 1895; Deucher, Correspond. Blatt f. Schweiz. Aerzte, 28.
^ Mem. de I'Acad. roy. de medic, de Belg., 18.
ABSORPTION OF CARBOHYDRATES. 419
that in this case the demolition of the protein in the stomach goes further
than in a normal animal.
The carbohydrates are, it seems, chiefl}' absorbed as monosaccharides.
Dextrose, levulose, and galactose are probably absorbed as such. The
two disaccharides, saccharose and maltose, ordinarily undergo an inversion
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, on the contrary, if the lactase formation is not excited by
milk food, the sugar is not inverted or only to a slight extent (Voit and
LusK, Weinland, PoRTiER, RoHMANN and Nagano), and it probably is ab-
sorbed as such in these animals if it does not undergo fermentation, or, as
RoHMANX and Nagano ^ assumed, if it is not transformed in the intes-
tinal mucosa in some unknown way. An absorption of non-inverted car-
bohydrates is not improbable, and according to Otto and v. Merixg^ the
portal blood contains besides dextrose a dextrin-like carbohydrate after
a carbohydrate diet. A part of the carbohydrates is destroyed by fermen-
tation in the intestine, with the formation of lactic and acetic acids and
other bodies.
The different varieties of sugars are absorbed with var^dng degrees of
rapidity, but as a general thing absorption occurs very quickly. This ab-
sorption takes place more quickly in the upper part of the intestine than in
the lower part (Roh^l^xx, Laxxois and Lepixe, Rohmaxx and Nagaxo^).
It is generally admitted that the simpler sugars are more quickly absorbed
than the disaccharides, while the statements as to the absorption of the
disaccharides differ somewhat (Hedox, Albertoxi, Waymouth Reid,
Rohmaxx and Nagaxo). There seems to be no doubt that lactose is
absorbed more slowly than the two other disaccharides. According to
the extensive experiments of Rohmaxx and Nagaxo, saccharose is
absorbed more quickly than maltose. Nagaxo '^ contends that the pentoses
are absorbed more slowly than hexoses.
On the introduction of starch even in very considerable quantities into
the intestinal tract no dextrose passes into the urine, a condition which
probably depends in thi? case upon the absorption and assimilation 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 b}' the urine takes place, and this elimination of sugar is called
* Voit and Liisk, Zeitschr. f. Biologie, 2S; Rolimann and Nagano, Pfliiger's Arch..
95, which contains the references to the Hterature.
^ Otto, see Maly's Jahresber., 17; v. Mering, Arch. f. (Anat. u.) Physiol., 1877.
^ Lannois et Lepine, Arch, de Physiol. (3), 1; RShmann, Pfliiger's Arch., 41; sea
also foot-note 1.
* In regard to the Hterature on the absorj^tion of sugars, see foot-note 1.
420 DIGESTION.
alimentary glycosuria. In these cases the assimilation of the sugar and the
absorption do not occur at the same time, hence the liver and the remaining
organs do not have the necessary time to fix and utilize the sugar. This
glycosuria may also in part be due to the fact that the introduction of
considerable quantities of sugar forces this substance to be absorbed not
only in the ordinary way through the blood-vessels to the liver (see below),
but also in part by passing into the blood circulation through the
lymphatic vessels, thus evading the liver.
That quantity of sugar to which we must raise the ingested substance in
order to produce an alimentary glycosuria gives, according to Hofmeister,i
the assimilation limit for that same sugar. This limit is different for various
kinds of sugar; and it also varies for the same sugar not only in different
animals, but also in chfferent members of the same species, as also in the
same individual under different circumstances. In general it can be said
that in regard to the ordinary varieties of sugar, such as dextrose, levulose,
saccharose, maltose, and lactose, the assimilation hmit is highest for dex-
trose and lowest for lactose. It must be admitted that with an over-
abundant 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 also disaccharides 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 measurable quantities, but are chiefly taken up by the
blood in the capillaries of the villi and in this way pass into the mass of
the Ijlood. These investigations have been confirmed by observations of
I. MuNK and Rosenstein ^ on human l^eings.
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 cpitheUum. Ordinarily these capillaries
find the necessary time for the removal of the water and the solids dis-
solved in it. But when a large quantity of liquid, such as a sugar solution.
> Arch. f. exp. Path. u. Pharm., 25 and 26.
^ For the Uterature 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, foot-note
3, p. 293. See also Bkimenthal, Zur Lehre von der Assimilationsgrenze der Zucker-
arten, Inaug.-Dissert. 1903, Strassburg.
'v. Mering, Arch. f. (Anat. u.) Physiol., 1877; Munk and Rosenstein, Virchow's
Arch., 123.
* Pfhjger's Arch., 43, Suppl.
ABSORPTIOiN OF CARBOHYDRATES. 421
is introduced into the intestine at once, this is not possible, and in these
cases a part of the dissolved bodies passes into the chylous vessels and the
thoracic duct (Ginsberg and Rohmann i).
The introduction of larger quantities of sugar into the intestine at one
time can readily cause a disturbance with diarrhceal evacuations of the
intestine. If the carbohydrate is introduced in the form of starch, then
very large quantities may be absorbed without causing any disturbance,
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 HuLTGREisr and Landergren,^
with rye bread the utilization of carbohydrates was less complete, and
the loss in a few c-ases 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 carl^ohydrates 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 ])odies are al^sorbed 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 absorption was not
impaired, while in others it was, on the contrary, rather diminished, and
with dogs devoid of pancreas it has been found that the absorption was
decreased to 50 percent of the starch partaken (Rosenberg, Cavazzani^).
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).
The experimental evidence thus far obtained for this assumption is not
very conclusive.^
^ Ginsberg, Pfliiger's Arch., 44; Rohmann, ibid., 41.
2 Rubner, Zeitschr. f. Biologic, 15 and 19; Constantinidi, ibid., 23; Hultgren and
Lander gr en, 1. c.
^ Cavazzani, Centralbl. f. Physiol., 7. See foot-note 1, p. 419; also Lombroso,
Hofmeister's Beitrage, 8.
^ Munk, Virchow's Arch., SO. See also v. Walther, Arch., f. (Anat. u.) Physiol.,
1890; Minkowski, Arch. f. exp. Path. u. Phann., 21; Frank, Zeitschr. f. Biologic, 36;
Moore, see Biochem. Centralbl., 1, 741- Frank and Ritter, Zeitschr. f. Biologic, 47.
422 DIGESTION.
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 glycerine 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 emul-
sification of neutral fats formed synthetically. This doubt has greater
warrant in that Frank i has shown that the fatty-acid ethyl ester is abun-
dantly taken up by the chyle 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 contradicts 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 emul-
sion in the intestine so long as it is acid. This difficulty is not too serious,
as the reaction is often due to only carbonic acid and bicarbonates and
also as found by KiJHXE and recently shown by Moore and Krumbholz,^
the proteins have a preserving action upon fat emulsions. The older views
as to fat absorption were that the 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.^
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
* Zeitschr. f. Biologie, 3(5.
^Kiihne, Lehrb. der physiol. Chem., 122; Moore and Krumbholz, Joum. of Phy-
siol, 22.
^ In regard to the newer literature on fat absorption, see the works of Pfi ger,
Pfi ger's .^rch., SO, §1, 82, 85, 88, 89, and 90, where the work nf other investigators is
cited and discussed.
ABSORPTION OF FATS. 423
soaps is secreted back 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-men-
tioned bodies — each alone as well as different mixtm-es of these — for the
various fatty acids, and has closely studied the mode of action of the bile.
From his investigations he has arrived at the conclusion that no imsplit fat
is absorbed, that all fats, before their absorption, must first be split into
glycerine and fatty acids, and that the bile, on account of its solvent
power for soaps and fatty acids, is sufficient for the aljsorption of large
quantities of fat eaten. The object of the formation of an emulsion is,
according to this view, that the fat in this condition forms such a large
surface for the action of the steapsin or the fat -splitting agents.
The possibility that all the fat must be first split and that no unsplit
fat is absorbed is, according to these researches, not to be denied. It is
the opmion 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 con-
clusion must be left for fmther investigations.
The next question is whether all the fat or the greater part of the same
passes into the blood throtigh the lymphatics and the thoracic duct.
According to the researches of Walther and Frank i on dogs, it seems
that only a small part of the fats, or at least of the fatty acids fed passes
into the chylous vessels; but these obser^-ations can hardly be applied to
the absorption of neutral fats, or to the absorption in man imder normal
circumstances. Muxk and Rosexsteix,^ in their investigations on a
girl with a lymph fistula fotmd 60 per cent of the 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
foimd 37 per cent of the introduced body as neutral fat in the chvle.
The completeness with which fats are absorbed depends, imder normal
conditions, essentially upon the kind of fat. In this regard it is known,
especially from the investigations of Muxk and Arxschixk,^ that the
varieties of fat with high melting-points, such as mutton-tallow and espe-
cially 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 upon the rapidity of absorption, as ^Iuxk and Rosexstein
found that solid mutton-fat was absorbed more slowly than fluid lipanin.
The extent of absorption in the intestinal tract is, under physiological con-
1 Walther. Arch. f. (Anat. u.) Physiol., 1900; Frank, ibid., 1892.
2 Virchow's Arch., 123.
' Munk. Virchow's Arch.. 80 and 95; Arnschink. Zeitschr. f. Biologie. 26.
424 DIGESTION.
ditions, very considerable. In the case of a dog investigated by Voit it
was found that out of 350 grams of fat (butter) partaken, 346 grams were
absorbed from the intestinal canal, and according to the investigations of
RuBNER 1 the human intestine can absorb over 300 grams of fat per diem.
The fats are, according to Rubner, much more completely absorbed when
free, in the form of butter or lard, than when enclosed in cell-membranes,
as in bacon.
Claude Bernard showed long ago with exiDeriments 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 intes-
tine 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 2 has performed
the reverse experiment on dogs. He tied the ductus choledochus and
adjusted a biliary fistula so that the bile flowed into the intestine below
the mouth of the pancreatic passages. On killing the animal after a meal
rich in fat the chylous vessels were first found milk-white below the dis-
charge of the biliary fistula. From this Dastre draws the conclusion
that a comliined action of the bile and pancreatic juice is important in the
absorption of fats — a conclusion which stands in good accord with the
experience of many others.
Through numerous observations of many investigators, such as Bidder
and Schmidt, Voit, Rohmaxn, Fr. Muller, I. Muxk,^ and others, it has
been shown that the exclusion of the bile from the intestinal tract dimin-
ishes the absorption of fat to such an extent that only one seventh to
about one half of the quantity of fat ordinaril}^ absorbed undergoes al:)Sorp-
tion. In icterus with entire exclusion of the bile, a considerable decrease
in the absorption of fat is noticed. As under normal conditions, so also in
the absence of bile in the intestine, the lower-melting parts of the fat are
more completely absorbed than those which have a high melting-point.
I. MuNK found in his experiments on dogs with lard and mutton-tallow
that the absorption of the high-melting tallow was reduced twice as much
as the lard on the exclusion of the bile from the intestine.
We also learn from the investigations of Rohmann and I. Munk that
in the absence of bile the relationship between fatty acids and neutral fats
is changed, namely, about 80-90 per cent of the fat existing in the faeces
consists of fatty acid, while under normal conditions the faeces contain
1 part neutral fat to about 2-2^ parts free fatty acids. It is not possible
* Voit, Zeitschr. f. Biologie, 9; Rubner, ibid., 1.').
2 Arch, (le Physiol. (5), 2.
^F. Muller, Sitzungsber. der phys.-med. Gesellscli. zu Wurzburg, 1885; I. Munk,
Virchow's Arch., 122. See also foot-notes 2 and 3, p. 406.
ABSORPTION OF FATS. 425
to state how this increased quantity of fatty acids in the fat of the faeces
is produced upon the exclusion of the bile from the intestine.
There is no doubt that the bile is of great importance in the absorption
of fats. Still there is also no doubt that rather considerable quantities of
fat may be absorbed from the intestine in the absence of Ijile. What rela-
tion 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, Rosen-
berg, Hedon and Ville, and also on man by Fr. Muller and Deucher.i
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 no absorption, took place. The con-
ditions may be somewhat different in the different cases; but it is certain
that the absence of pancreatic juice from the intestine essentially affects
the fat absorption. It is also just as certain that the absorption of fat is
most abundant in the simultaneous presence of bile as well as pancreatic
juice in the intestine. A little fat may still be absorbed even in the absence
of these two fluids, as shown by the investigations of HiiDON and Ville
and CUNNINGHAM.2
The reason for the fact that the fat absorption is diminished in the
absence of bile from the intestine must be sought for in the above-mentioned
role of this fluid. It is more difficult to state why the absence of pan-
creatic 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 fseces 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 lipase or by micro-organisms or
otherwise may produce a cleavage of the fat in these cases. The imperfect
^ Miiller, "Unters. iiber den Icterus," Zeitschr. f. klin. Med., 12; Hedon and Ville,
Arch, de Physiol. (5), 9; Harley, Journ. of Physiol., 18, Joum. of Pathol, and Bacteriol.,
1895, and Proceed. Roy. Soc, 61. In regard to the other authors see foot-note 2,
p. 418.
^ Hedon and Ville, 1. c. ; Cunningham, Journ. of Physiol., 23.
426 DIGESTION.
fat absorption after the extirpation of the pancreas can possibly he explained
by the removal of a considerable part of the alkalies necessary for the
formation of the emulsion and for the solution of the fatty acids, but as
Sandmeyer found in dogs deprived of their pancreas that the fat absorption
was raised by giving chopped pancreas with the fat, this can hardly be
a sufficient explanation.
The 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 impor-
tance in the absorption of such salts.
The soluble constituents of the digestive secretions may, like other
dissolved bodies, be absorbed, as is demonstrated by the passage of pepsin
into urine; the enzymes may also be absorbed. The occurrence of uro-
bilin in urine attests the absorption of the bile-constituents under physio-
logical conditions despite the fact that the occurrence of very small traces
of bile-acids in the urine is disputed. The absorption of bile-acids by the
intestine seems to be positively proved by other observations. Tap-
PEiNER 1 introduced a solution of bile-salts of a known concentration into
an intestinal knot and after a time investigated the contents. He found
that in the jejunum and the ileum, but not in the duodenum, an absorption
of bile-acids took place, and further that of the two bile-acids only the
glycocholic acid was absorbed in the jejunum. Further, Schiff long ago
expressed the opinion that bile undergoes an intermediate circulation, in
such wise that it is absorbed from the intestine, then carried to the liver
by the blood, and lastly eliminated from the blood by this organ. Although
this view has met with some opposition, still its correctness seems to be
established by the researches of various investigators, and more recently
by Prevost and Binet, and specially by Stadelmann and his pupils.^
After the introduction of foreign bile into the intestine of an animal the
foreign bile-acids appear again in the secreted bile.
How does the removal of large portions of the various parts of the
intestine affect absorption? Harley^ has been able to perform a partial
extirpation of the large intestine and in another instance a complete extir-
pation. This last condition increased the faeces considerably, especially
because of the large increase in the water (fivefold). Fats and carbohy-
drates were absorbed just as completely as in the normal. The absorp-
tion of the proteins, on the contrary, was reduced to only 84 per cent as
compared to 93-98 per cent in normal dogs. After extirpation the fseces
' Wien. Sitzungsber., 77.
^ Schiff, Pfliger's Arch., 3; Prevost and Binet, Compt rend., 106; Stadelmann,
see foot-note 1 , p. 309.
^ Proceed. Roy. Soc, 64.
ABSORPTION IX DIFFERENT PARTS OF THE INTESTINE. 427
sometimes did not contain any m-obilin or only traces thereof, while bile-
pigments existed in large amounts.
Erlanger and Hewlett ^ found that dogs, from which 70-83 per cent
of the total length of the jejunum and ileum had been removed, could be
kept alive like other animals if only the food was not too rich in fat. When
the food contained large amounts of fat then 25 per cent was evacuated
by the fseces as compared to 4-5 per cent in the normal animal. Under
these same conditions the amount of nitrogen in the faeces was increased
to twice the normal amount.
After the exclusion of the colon in rabbits, Bergmann and Hultgren ^
could find no definite action upon the availability of the cellulose and
also no diminution in the utility of the other constituents of the food could
be observ-ed. Zuntz and Ustjanzew 3 also found that the removal of the
caecum had no influence on the utilization of nitrogen; but in regard to
other points they arrived at different results. They fomid, namely, that
the caecum of the rodent is of great importance for the digestion of crude
fibre and the pentosanes. 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 pentosanes 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
the views are disputed. 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 inves-
tigations on this subject, we must refer to larger works ^ and to text-books
on physiology for further information.
* Amer. Journ. of Physiol., 6.
^Skand. Arch. f. Physiol, U.
^ Verhandl. d. physiol. Gesellsch. zu BerHn, 1904-1905.
* See Hober, Physikalische Cliemie der Zelle, Leipzig, 1906, and I. Munk, Ergeb-
nisse der Physiologie, I, Abt. 1; Hamburger, Osmotischer Druck und lonenlehre,
Bd. 2, Wiesbaden, 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 gelatine-yielding
fibrils, which, like the cells, are imbedded in an interstitial or intercellular
substance. The fibrils consist of collagen. The interstitial substance con-
tains chiefly mucoid {tendon-mucoid) , besides serglobidin and seralbumin,
which occur in the parenchymatous fluid (Loebisch ^).
The connective tissue also often contains fibres or formations consisting
of elastin, sometimes in such great quantities that the connective tissue
is transformed into elastic tissue. A third variet}^ of fibres, the reticular
fibres, also occurs, and according to Siegfried these consist of reticulin.
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 con-
tains the fibrils of the connective tissue together with the cells and the
elastic substance.
The so-called tendon mucin is not true mucin, but a mucoid, which,
as first shown b}' Levexe 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 glucoproteids,
contain 2.2-2.33 per cent sulphur, as shown by the analyses of Chitten-
den 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 sulphate
* Zeitschr. f. physiol. Chem., 10.
^ Levene, ibid., 31 and 39; Cutter and Gies, Amer. Joum. of Physiol., 6; Chittenden
and Gies, Joum. of exp. Med., 1.
428
CONNECTIVE TISSUES. 429
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 fomid of the greatest tech-
nical importance in the preparation of leather. In regard to the collagens,
gelatines, elastins, and reticulins, see pages 75 to 81.
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 lunbilical cord is the most accessible material for the investigation
of the chemical constituents of the gelatinous tissues. The mucin occiuring
therein has been described on page C7. C. Th. Morxer ^ has found a
mucoid in the vitreous humor which contains 12.27 i>er cent nitrogen and
1.19 per cent sulphur.
Yomig connective tissue is richer in mucoid than old. Halliburton ^
found an average of 7.66 p. m. mucoid in the skin of verj^ 3'oung 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
ciuantity of mucoid is also increased.
The connective tissue and also the elastic tissue are richer in water and
poorer in sohds in yoimg animals as compared with f ull-growTi animals. This
may be seen from the following analyses of the Achilles tendon (Buerger
and GiEs) and of the ligamentum nuchse (Vaxdegrift and Gies ^).
Achill
es tendon.
Calf.
Ox.
Water
. 675.1 p. m.
628.7 p.m.
Solids
. 324.9 "
371.3 "
Organic bodies . . . .
. 318.4 "
366.6 "
Inorganic bodies . .
. 6.1 "
4.7 "
Fat
10.4 "
Proteid
9 9 "
ilucoid
12.83 "
Elastin
16.33 "
Collagen
315 88 "
Extractives, etc . . .
8.96 "
Ligament.
Calf.
Ox.
651.0 p. m.
575.7 p. m
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 fotmd by him in the crj-stalline lens of
the ox, namely, 0.5814 gram per kilo of dried substance. In man he found
* Zeitschr. f. physiol. Cliem., 18, 250.
' Mucin in Myxoedema : Further Analyses. King's College Collected Papers No. 1,
1893.
^ Buerger and Gies, Amer. Joum. of Piiysiol.. 6; Vandegrift and Gies, ibid., 5.
* PflLiger's .\rch., S4 and S9.
430 TISSUES OF THE CONNECTIVE SUBSTANCE.
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 found also 0.403 gram FeoOa, 0.693 gram MgO, 3.297
grams CaO, and 3.794 grams P2O5 for every kilo of dried substance,
n. Cartilage.
Cartilaginous tissue consists 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 former \'iew^s, 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. Morner,i have shown that the matrix of
the cartilage consists of a mixture of collagen with other bodies.
The tracheal, thyroideal, cricoidal, and ar5i:enoidal cartilages of full-
grown cattle contain, according to Morner, four constituents in the matrix
namely, chondromucoid, chondroitin-sulphuric acid, collagen, and an albu-
minoid.
Chondromucoid. This body, according to Morner, has the composi-
tion 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 w^hen boiled with hydrochloric acid. Chondro-
mucoid is decomposed by dilute alkalies and yields alkali albuminate,
peptone substances, chondroitin-sulphuric acid, alkali sulphides, 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 bj^ acetic acid in great excess and by small
quantities^ of mineral acids. The precipitation may be retarded by neutral
salts or b,v chondroitin-sulphuric acid. The solution containing NaCl and
acidified with HCl is not precipitated by potassium ferrocyanide. Precipi-
tants for chondromucoid are alum, ferric chloride, sugar of lead, or basic
lead acetate. Chondromucoid is not precipitated by tannic acid, and it
* Morochowetz, Verhandl. d. naturh. med. Vereins zu Heidelberg, 1, Heft 5; Morner,
Skand. Arch. f. Physiol., 1.
CHONDROITIN-SULPHURIC ACID. 431
may by its presence prevent the precipitation of gelatine 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. Morxer 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. Morxer has also found it in the ox-kidney
and in human urine as a regular constituent. According to Krawkow,
who found it in the cervical ligament of the ox, it combines with proteid,
forming amyloid (see page 69), which explains the occurrence of this body
in amyloid-degenerated livers, as observed by Oddi.^ The identity of the
ethereal sulphuric acid occurring in liver amyloid with chondroitin-sul-
phuric acid does not seem to be quite clear, according to the researches of
MoNERY. According to Levexe,^ 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 C18H27NSO17, according to
Schmiedeberg.3 As primary products this acid yields ©n cleavage sulphuric
acid and a nitrogenous substance, chondroitin, according to the following
equation :
CisHoyNSOiT +H20 = H2S04 + Ci8H27NOi4.
Chondroitin, which is similar to gum arable and which is a monobasic acid,
yields acetic acid and a new nitrogenous substance, chondrosin, as cleavage
products, on decomposition with dilute mineral acids:
Ci8H27NOi4 + 3H20 = 3C2H402 + Ci2H2iNOii.
Chondrosin, which is also a gummy substance soluble in water, is a mono-
basic acid and reduces copper oxide in alkaline solution even more strongly
than dextrose. It is dextrogyrate and represents the reducing substance
obtained by previous investigators in an impm'e form on boiling cartilage
with an acid. The products obtained on decomposing chondrosin with
barium hydrate tend to show, according to Schmiedeberg, that chondro-
sin contains the atomic groups of glucuronic acid and glucosamine. This
assumption does not seem to have sufficient foundation. According to
Ogler and Neuberg,'* chondrosin does not give the orcin test nor does
»C. Morner, 1. c, and Zeitschr. f. physiol. Chem., 20 and 23: K. Morner, Skand.
Arch. f. Physiol., 6; Krawkow, Arch. f. exp. Path. u. Pharm., 40; Oddi, ihid., 33.
2 Mon^ry, Compt. rend. soc. biol., .54; Levene, Zeitschr. f. physiol. Chem., 39.
^ Arch. f. exp. Path. u. Pharm., 28.
* Zeitschr. f. physiol. Chem., 37.
432 TISSUES OF THE CONNECTIVE SUBSTANCE.
it yield furfurol. It contains neither glucuronic acid nor glucosamine, and
on cleavage with baryta it yields, besides a carbohydrate complex which
has not been studied, an oxyamino-acid having the formula CeHisOeN;
also a hexosamine acid or tetraoxyaminocaproic acid.
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 arable.
Nearly all of its salts are soluble in water. The neutralized solution is
precipitated by tin 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 gelatine or proteid.
Chondromucoid and chondroitin-sulphuric acid may be prepared, accord-
ing to MoRNER, by extracting finely cut cartilage with water, which dis-
solves the preformed chondroitin-sulphuric acid besides some chondro-
mucoid. In this watery extract, the chondroitin-suliDhuric acid prevents
the precipitation of the chondromucoid by means of an acid. If 2-4 p. m.
HCl is added to this watery extract and warmed on the water-bath, the
chondromucoid gradually separates, while the chondroitin-sulphuric acid
and the rest of the chondromucoid remain in the filtrate. If the cartilage,
which has been lixiviated with water, at the temperature of the body, is
extracted with hydrochloric acid of 2-3 p. m. until the collagen is con-
verted into gelatine and dissolved, the remaining chondromucoid may be
removed from the insoluble residue by dilute alkali and precipitated from
the alkaline extract by an acid. It may be purified by repeated solution
in water with the aid of a little alkali, and precipitation with an acid, and
then finally by extraction with alcohol and ether.
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 separated
from the filtrate by H2S. If further purification is necessar}^ the acid is
precipitated with alcohol, the precipitate dissolved in water, this solution
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.
ScHMiEDEBERG 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 ^ vol.) and the clear filtrate treated with absolute alcohol and
some ether. The precipitate, consisting chiefly of a combination or a
COLLAGEN AND ALBUMINOID OF CARTILAGE. 433
mixture of chondroitin-sulphuric acid and gelatine peptone (peptochondrin),
is first washed with alcohol and then with water. It is then dissolved in
alkaline water and the basic alkali compound precipitated from this
solution by the addition of alcohol, whereby the gelatine-peptone alkali
remains in solution. The precipitate is purified by repeated solution in
alkaline water and precipitated by alcohol. To obtain chondroitin-sul-
phuric 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 for Oddi's method.
The collagen of the cartilage gives, according to Morner, a gelatine which
contains only 16.4 per cent N and which can hardly be considered identical
with ordinary gelatine.
In the above-mentioned cartilages of full-grown animals the chondroitin-
sulphuric acid and chondromucoid, perhaps also the collagen, are found
surrounding the cells as romid balls or lumps. These balls (Morner's
chondrin-halls) , which give a blue color with methyl-violet, lie in the meshes
of a trabecular structure, which is colored when brought in contact with
tropaeolin.
The albuminoid 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 is more similar to elastin, but differs
from this substance by containing sulphur. This albuminoid gives the
color reactions of the protein bodies.
The preparation of cartilage gelatine and the albuminoid may be per-
formed 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 gelatine, while the albuminoid remains
undissolved (contaminated by the cartilage-cells). The gelatine may be
purified by precipitating with sodium sulphate, which must be added to
saturation in the faintly acidified solution, redissolving the precipitate in
water, dialyzing well, and precipitating with alcohol.
According to Morner, no albuminoid is found in young cartilage, but
only the three first -mentioned constituents. Nevertheless the young carti-
lage contains about the same amounts of nitrogen and mineral substances
as the old. The cartilage of the ray {Raja hatis Lin.), which has been
investigated by Lonnberg,^ contains no albuminoid and only a little
chondromucoid, but a large proportion of chondroitin-sulphuric acid and
collagen.
• Maly's Jahresber., 19, 325.
434 TISSUES OF THE CONNECTIVE SUBSTANCE.
According to Pfluger and Handel,' glycogen occurs to a slight extent
in all matrices, and of these it is richest in the cartilage. Tendons, liga-
ment um nuchse, and cartilage of the ox contained 0.06, 0.07, and 2.17 p. m.
glycogen respectively (Handel).
Hoppe-Seyler found in fresh human rib-cartilage 676.7 p. m. water,
301.3 p. m. organic and 22 p. m. inorganic substance, and in the cartilage
of the knee-joint 735.9 p. m. water, 248.7 p. m. organic and 15.4 p. m.
inorganic substance. Pickardt found 402-574 p. m. water and 72.86 p. m.
ash (no iron) in the laryngeal cartilage of oxen. The ash of cartilage con-
tains considerable amounts (even 800 p. m.) of alkali sulphate, which
probably does not exist originally as such, but is produced in great part by
the incineration of the chondroitin-sulphuric acid and the chondromucoid.
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 according to Bunge ^
the amount of Na and CI is greatest in young animals. In 1000 parts of
cartilage dried at 120° C, Bunge found 91.26 parts NagO 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.
The Cornea. The corneal tissue, which is considered by many investi-
gators to be related to cartilage in a chemical sense, contains traces of
proteid and a collagen as chief constituent, which C. Morner^ 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 sub-
stance. The globulins found by other investigators in the cornea are not
derived from the matrix, according to Morner, but from the layer of
epithelium. According to Morner, Descemet's membrane consists of
membranin (page 69), which contains 14.77 per cent N and 0.90 per cent S.
In the cornea of oxen His * found 758.3 p. m. water, 203.8 p. m. gelatine-
forming substance, 28.4 p. m. other organic substance, besides 8.1 p. m.
soluble and 1.1 p. m. insoluble salts.
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.
' Pfl :ger's Arch., i)2; Handel, ibid.
2 Hoppe-Seyler, cited from Kiihne's Lehrbuch d. physiol. Chem., 387; Pickardt,
Centralbl. f. Physiol., Ci, 735; Bunge, Zeitschr. f. physiol. Chem., 28.
3 Zeitschr. f. physiol. Chem., IS.
■•Cited from Gamgee, Physiol. Chem., 1880, 451.
BONE. 435
The cells have not been closely studied in regard to their chemical con-
stitution. On boiling with water they yield no gelatine. They contain no
keratin, which is not usually present in the bony structure (Herbert
Saoth ^).
The ruatriz of the bony structure contains two chief constituents,
namely, an organic substance, and the so-called bone-earths, lime-salts,
enclosed in or combined with it. K 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 con-
sidered as identical with the collagen of the connective tissue. It also
contains, as Hawk and Gies ^ have shown, mucoid and albuminoid. After
the removal of the lime-salts by hydrochloric acid of 2-5 p. m. these experi-
menters were able to extract the mucoid by one-half saturated 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 ol3tained
the albuminoid as an insoluble- residue.
The osseomucoid on boiling with hydrochloric acid yielded a reducing
substance and sulphuric acid, 1.11 per cent sulphur appearing in this
form. The osseomucoid stands close to the chondro- and tendon mucoid
in elementar}' composition, as may be seen from the following analyses:
o
31.40 (Hawk and Gies)
31.28 (C. Morner)
30.60 (Chittexdex and Gies)
28.01 (C. Morner)
The osseoalbuminoid is insoluble in 2 p. m. hydrochloric acid and in 5 p. m.
Na2C03, but dissolves in 10 per cent KOH with the formation of albumin-
ates. The composition of chondro- and osseoalbuminoid is as follows:
c
Osseoalbuminoid 50. 16
Chondroalbuminoid 50 . 46
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
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
' Zeitschr. f. Biologic, 19. ' Amer. Joum. of Physiol., 5 and 7.
C
H
N
S
Osseomucoid
. 47.43
6.63
12.22
2.32
Chondromucoid. .
. 47.30
6.42
12.58
2.42
Tendon mucoid. .
. 48.76
6.53
11.75
2.33
Corneal mucoid. .
50.16
6.97
12.79
2.07
H
N
S
0
7.03
16.17
1.18
25 . 46 \ Hawk and
7.05
14.95
1.86
25 . 68 / Gies
436 TISSUES OF THE CONNECTIVE SUBSTANCE.
sulphate occurring in the boue-ash are derived, according to Morner,i
from the chondroitin-sulphuric acid. According to Gabriel, potassium
and sodium are essential constituents of bone-earth, and this has been
substantiated by Aron.^
The opinions of investigators differ somewhat 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 (CaCl2,3Ca3P208). If
we eliminate the magnesium, the chlorine, and the fluorine, the last, accord-
ing to Gabriel, occurring only as traces, the remaining mineral bodies form
the combination 3(Ca3P208)CaC03. According to Gabriel the simplest
expression for the composition of the ash of bones and teeth is (Ca3(P04)2 +
Ca5HP30i3 + Aq), in which 2-3 per cent of the Ume is replaced by magnesia,
potash, and soda, and 4-6 per cent of the phosphoric 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 different animals.
As an example of the composition of bone-earth we here give the analyses
of Zalesky.^ The figures represent parts per thousand.
Man. Ox. Tortoise. Guinea-pig.
Calcium phosphate, Ca3P,0g 838 . 9 860 . 9 859 . 8 873 . 8
Magnesium phosphate, MgjPPg 10.4 10.2 13.6 10.5
Calcium combined with CO,, Fl, and a... 76.5 73.6 63.2 70.3
CO, " 57.3 62.0 52.7
Chlorine 1.8 2.0 1.3
Fluorine^ 2.3 3.0 2.0
Some of the CO2 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:
Man. Ox. Elephant.
&T &. Femur. Femur.
Calcium phosphate 874.5 878.7 857.2 900.3
Magnesium phosphate 15.7 17.5 15.3 19.6
Calcium fluoride 3.5 3.7 4.5 4.7
Calcium chloride 2.3 3.0 3.0 2.0
Calcium carbonate 101.8 92.3 119.6 72.7
Iron oxide .^ 1.0 1.3 1.3 1.5
The quantity of organic substance in the bones, calculated from the loss
of weight in burning, varies somewhat between 300 and 520 p. m. This
' Zeitschr. f. phy.siol. Chem., 23.
^Gabriel, ibid., 18, which also contains the pertinent literature; Aron, Pfliiger's
Arch., 106.
' Hojjpe-Seyler, Med.-chem. Untersuch., p. 19.
'' The statements as to the quantity of fluorine are contradictory; see Harms,
Zeitschr. f. Biologic, 38; Jodblauer, ibid., 41.
*Compt. rend., 114.
BONE. 437
variation may in part be explained by the difficulty in obtaining the bony
substance entirely free from water and partly by the very variable amount
of blood-vessels, nerves, marrow, and the like in different bones. The
unequal amounts of organic substance found in the compact and in the
spongy parts of the same bone, as well as in bones at different periods
of development in the same animal, depend probably upon the varying
quantities of these above-mentioned tissues. Dentin, which is compara-
tively pure bony structure, contains only 260-280 p. m. organic substance,
and Hoppe-Seyler ^ 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. The yellow marrow contains chiefly fat, which consists of olein, pal-
mitin, and stearin, and which differs from the fat of the other parts of the body
by having a higher acetyl equivalent (Zink ^). Protein has been found especially
in the so-called red marrow of the spongy bones. According to Forrest, the
protein consists of a globulin coagulating at 47-50° C. and a nucleoalbumin
with 1.6 per cent phosphorus (Halliburton ^), besides traces of albumin. Besides
this the marrow contains so-called extractive bodies, such as lactic acid, hypo-
xanthine, and cholesterin, but mostly bodies of an unknown character.
The diverse quantitative composition of the various bones of the skele-
ton depends probably on the varying quantities of other tissues, such as
marrow, blood-vessels, etc., which they contain. The same reason explains,
to all appearances, the larger quantity of organic substance in the spongy
parts of the bones as compared with the more compact parts. Schrodt ^
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 vertebras. In birds the tubular
bones are richer in mineral substances than the fiat bones (DiJRiNG), and
' Physiol. Chem., 102-104.
2 See Chem. Centralbl., 1897, I, 2%.
^ Forrest, Journ. of Physiol., 17 ; Halliburton, ibid., 18.
* Cited from Maly's Jahresber., (J.
438 TISSUES OF THE CONNECTIVE SUBSTANCE.
the greatest quantity of mineral bodies has been found in the humerus
(HiLLER, During i).
We do not possess trustworthy statements in regard to the composition
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. Graffen-
BERGER ~ 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 of birds contain, as a rule, somewhat more water than those of mam-
malia, and the bones of fishes contain the largest quantity of water. The bones
of fishes and amphibians contain a greater amount of organic substance. The
bones of pachyderm.s and cetaceans contain a large proportion of calcium carbo-
nate; those of granivorous birds always contain silicic acid. The bone-ash of
amphibians and Tishes 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 contradic-
tory. The attempts, also, to substitute other alkaline earths or alumina for
the lime of the bones have given contradictory results.^ On feeding suffi-
cient calcium and phosphorus in the food Aron ^ 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 con-
clusion in regard to the growth or metabolism in the bones.
Under pathological conditions, as in rachitis and softening of the bones,
an ossein has been found which does not give any typical gelatine on boiling
with water. Othen\'ise pathological conditions seem to affect chiefly the
quantitative composition of the bones, and especially the relationship
between the organic and the inorganic substance. In exostosis and osteo-
sclerosis 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
^ Hiller, cited from Maly's Jahresber., 14; Diiring, Zeitschr. f. physiol. Chem., 23.
2 Voit, Zeitschr. f. Biologie, 16; Brubacher, ibid., 27; Graffenberger in Maly's
Jahresber., 21.
^ See H. Weiske, Zeitschr. f. Biologie, 31.
* Pfl'ger's Arch., lOG.
BONE. 439
animals contradictor}'- results have been obtained. In young, undeveloped
.animals Erwix Voit ^ produced, by lack of lime-salts, a change similar to
rachitis. In fuU-growTi 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 (Heitzmaxn, Heiss, Bagixsky^). 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
diminished. On feeding continuously for a long time with a food which
yielded an acid ash (cereal grains), Weiske has observed a diminution in
the mineral substances of the bones in full-grown herbivora.^ A few inves-
tigators 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 •* found lactic acid in the cyst-
like, altered bony substance in osteomalacia.
Well-known investigators have disputed the possibility of the lime-
salts being washed from the bones in osteomalacia by means of lactic
acid. They have given special prominence to the fact that the lime-salts
held in solution by the lactic acid must be deposited on neutralization of
the acid by the alkaline blood. This objection is not very important, as
the alkaline blood-serum has the property to a high degree of holding earthy
phosphates in solution, which fact can be easily proved. The investiga-
tions of Levy ^ contradict the statement as to the solution of the lime-salts
by lactic acid in osteomalacia. He has found that the normal relationship
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 occiu^ in the same quantitative relationship as the
carbonate, and according to Le\"y, 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
figiu'es refer to the dried substance. According to Brubacher, rachitic bones
are richer in water than the bones of healthy childi'en, and poorer in mineral
1 Zeitschr. f. Biologie, 16.
^ Heitzniann, Maly's Jahresber., 3, 229; Heiss, Zeitschr. f. Biologie, 12; Baginsky,
Virchow's Arch., 87.
' See Maly's Jahresber., 22; also Weiske. Zeitschr. f. physiol. Chem., 20, and
Zeitschr. f. Biologie, 31.
* Cited from v. Gorup-Besanez, Lehrh. d. physiol. Chem., 4. Aufl.
* Zeitschr. f. physiol. Chem., 19.
440 TISSUES OF THE CONNECTIVE SUBSTANCE.
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 ' found a larger quantity of magne-
sium than calcium in a bone. The ash contained 417 p. m. phosphoric acid, 222
p. m. lime, 269 p m. magnesia, and 86 p. m. carbon dioxide. Other investigators
have on the contrary found considerably more calcium than magnesium.
The tooth-structure is nearly 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 gelatine 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 epithelium forma-
tion containing a large proportion of lime-salts. Corresponding to its
character and origin, the organic substance of the enamel does not yield
any gelatine. 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 rela-
tive amounts of calcium and phosphoric acid are, according to the analyses
of Hoppe-Seyler, about the same as in bone-earths. The quantity of
chlorine according to Hoppe-Seyler is remarkably high, 0.3-0.5 per cent,
while Bertz^ found that the ash of enamel was free from chlorine and
that dentin was very poor in chlorine.
Carnot,^ 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.
According to Gabriel the amount 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.* The same investigator found that the amount of phosphates 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.
' Chabrie, Les phenomenes cliim. de rossification, Paris, 1895, 65.
^ See Maly's Jahresber., 30.
^ Compt. rend., 114.
* See foot-note 4, p. 436.
FATTY TISSUE. 441
IV. The Fatty Tissue.
The membranes of the fat-cells withstand the action of alcohol and
ether. They are not dissolved by acetic acid nor by dilute mineral acids,
but are dissolved by artificial gastric juice. They may possibly consist 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 (see Chapter I).
The less water the fatty tissue contains the richer it is in fat. Schulze
and Reixecke ^ found in 1000 parts
Water. Membrane. Fat.
Fatty tissue of oxen 99.7 16.6 883.7
" "sheep 104.8 16.4 878.8
" "pigs 64.4 13.6 922.0
The fat contained in the fat-cells consists chiefly of triglycerides of
stearic, palmitic, and oleic acids. Besides these, especially in the less solid
kinds of fats, there are glycerides of other fatty acids (see Chapter IV).
In all animal fats there are besides these, as Fr. Hofmaxx - has shoT\Ti,
also free, non-volatile fatty acids, although in very small amounts.
Human fat is relatively rich in olein, the quantity in the subcutaneous
fatty tissue being 70-80 per cent or more.^ In new-bom infants it is poorer
in oleic acid than in adults (Kxopfelila.cher, 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 incUviduals of the same species of animals is rather variable, a
fact which is probably dependent upon the food. AccorcUng to the
researches of Hexriques and Haxsex 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 Wixkler.* In animals with a thick subcutaneous
fat deposit the outer layers, according to Hexriques and Haxsex'^, are
richer in olein than the inner layers. The fat of cold-blooded arimals is
especially rich in olein. The fat of domestic animals has. according to
* Annal. d. Cliem. u. Pharm.. 142.
^ Ludwig-Festschrift, 1874, Leipzig.
' See Jaeckle, Zeitschr. f. physiol. Chem., 36 (literature).
* Knopfelmacher, Jahrbuch f. Kinderheilkunde (X. 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. .\rch. f.
Path. u. Pharm., 48.
442 TISSUES OF THE CONNECTIVE SUBSTANCE.
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 variation. The fat
of lipoma seems, according to Jaeckle, to be poorer in lecithin than other
fats.
The properties of fats in general, and the three most important varieties
of fat, have already been considered in a previous chapter, hence the forma-
tion of the adipose tissue is of chief interest at this time.
The formation of fat in the organism may occur in various ways. The
fat of the animal body may consist partly of fat absorbed from the food
and deposited in the tissues, and partly of fat formed in the organism
from other bodies, such as proteins or carbohydrates.
That the fat from the food which is absorbed in the intestinal canal may
be retained by the tissues has been shown in several ways. Radziejewski,
Lebedeff, and Munk have fed dogs with various fats, such as linseed-oil,
mutton-tallow, and rape-seed-oil, and have afterwards found the adminis-
tered fat in the tissues. Hofmann starved dogs until they appeared to
have lost their fat and then fed them upon large quantities of fat and only
little proteins. When the animals were killed, he found so large a quantity
of fat that it could not have been formed from the administered proteins
alone, but the greater part must have been derived from the fat of the
food. Pettenkofer and Voit arrived at similar results in regard to the
behavior of the absorbed fats in the organism, though their experiments
were of another kind. Munk has found that on feeding with free fatty
acids, these are deposited in the tissues, not, however, as such; but they
are transformed by synthesis with glycerine into neutral fats on their pas-
sage from the intestine into the thoracic duct. The connection between
the fat of the food and of the body has also been shown by oth*^rs, espe-
cially by Rosenfeld. Coroxedi and Marchetti and especially Winter-
NiTZ 1 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 disputed, and
many other explanations of the formation of this substance have been
offered. According to the experiments of Kratter and K. B. Lehmann,
' Coronedi and Marchetti, cited by Winternitz, Zeitschr. f. physiol. Chem., 24.
A review of the literature on fat formation may be found in Rosenfeld, Fettbildung,
in Ergebnisse der Physiologic, 1, Abt. 1.
FORMATION OF FAT. 443
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 recently that in
the formation of adipocere the fat itself takes part, in that tlie olein decom-
poses with the formation of solid fatty acids; still it must be considered
that lower organisms undoubtedly take part in its formation. The pro-
duction 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 forma-
tion 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 phos-
phorus, 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 Polimaxti, who claims to
have sho-uTi the formation of fat from proteins in phosphorus poisoning,
that we cannot consider the formation of fat as conclusively proved.
Recent investigations of Athaxasiu. Taylor. Schwalbe, and others,
especially of Rosexfeld.^ have made it probable that in these instances no
new formation of fat from protein took place, but rather a fat migration
(Rosexfeld).
Another more direct proof for the formation of fat from proteins has
been given by Hofmaxx. He experimented with fly-maggots. A num-
ber of these were killed and the quantity of fat determined. The remainder
were allowed to develop in blood whose proportion of fat had been pr3\'i-
ously 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- 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 constituents of the blood and their decompo-
sition products, and that these serve as food for the maggots.
As a more convincing proof of fat formation from proteins, the investi-
gations of Pettexkofer and Voit are often quoted. These investigators
fed dogs with large quantities of meat containing the least possible propor-
tion 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 organism splits into a nitrogenized and a non-nitrog-
enized part, the former changing into the nitrogenized final product,
'Bauer. Zeitschr. f. Biologie, 7; Leo, Zeitschr. f. physiol. Chem., 9; Polimanti,
Pfl ger's Arch., 70; Pfluger. ibid.. 51 (literature on the formation of fat from protein)
and 71; Athanasiu, ibid., 7-t; Taylor, Joum. Exp. Medicine, -l; see also foot-note 1,
Oi
See Rosenfeld, Fettbildung, Ergebnisse der Physiologie, 1, Abt. 1.
444 TISSUES OF THE CONNECTIVE SUBSTANCE.
urea, and like products, and the latter, 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 investigations,
which vrere continued for a series of years, were subject to such great
defects that they are not conclusive as to the formation of fat from pro-
teins. 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 relationship of nitrogen to carbon in
meat poor in fat was assumed by Voit to be as 1: 3.68, while according to
PFLtJGER 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 recalcu-
lation of the figures using these coefficients, Pfutger 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 denied by Pfluger. On
feeding a dog on meat poor in fat (containing a known quantity of ether
extractives, glycogen, nitrogen, water, and ash), Kumagawa^ 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 pro>
tein.
Several French investigators, especially Chauveau, Gautier, and
Kaufmann, consider the formation of fat from proteins as positively proved.
Katjfmann has recently substantiated this view b}^ a method which will
be spoken of in detail in Chapter XVIII, in which he studied the nitro-
gen 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 inter-
mediate step, can take place. The possibility of a direct fat formation
from proteins without the carbohydrate as intermediary must also be
generally admitted, although such a formation has not been conclusively
proved.
According to Chauveau and Kaufmann, in the direct formation of fat
* See Rosenfeld, Fettbildung, Ergebnisse der Physiologie, 1, Abt. ].
* Kaufmann, Arch, de physiol. (5) 8, where the worLs of Chauveau and Gautier
are cited.
FORMATION OF FAT. 445
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 tak-
ing 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 deficient in carbon.
The formation oj jot 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 proved, but also that it was improbable. The imdoubt-
edly great influence of the carbohydrates on the formation of fat as ob-
served and proved by Liebig was explained by the statement that the
carbohydrates were consumed instead of the absorbed fat or that derived
from the proteins, hence they have a sparing action on the fat. Bv means of
a series of nutrition experiments with foods especially rich in carbohydrates
Lawes and Gilbert, Soxhlet, Tscherwinsky, Meissl and Stromer (on
pigs), B. Schultze, Chaniew^ski, E. Voit and C. Lehmaxn (on geese), I.
MuNK and Rubner and Lummert ^ (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 as 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 CHo; hence a reduction must occur.
Analogous to Nexcki's view as to the butyric-acid fermentation, when
lactic acid is formed from the sugar and from this CO2H2 and acetaldeliyde
(C2H4O) are produced, and from this latter, by the union of two molecules,
butyric acid is formed, so Magnus-Levy 2 attempts to explain the forma-
tion of fat in the animal body from carbohydrates by synthesis from
aldehyde and reduction. He considers that the process proceeds in the
following way: (a) 9C3H603 = 9C2H40 + 9H2 + 9C02 and (6) 9C2H4O + 7H2
= CisH3602 (stearic acid) +7H2O.
After feeding with very large quantities of carbohydrates the relation-
ship between the inspired oxygen and the expired carbon dioxide, i.e., the
CO
respiratoiy quotient -j^, was found greater than 1 in certain cases (Han-
' Lawes and Gilbert, Phil. Transactions, 1859, part 2; Soxhlet, see ^laly's Jahresber.,
11, 51; Tscherwinsky, Landwirthsch. Versuchsstaat, 29 (cited from Maly's Jahresber.,
13); Meissl and Stromer, Wien. Sitzungsber., SS, Abt. 3; Schultze, Maly's -Jahresber.,
11, 47; Chaniewski, Zeitschr f. Biologic, 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. Biologic, 22; Lummert,- Pfliiger's Arch.. "1.
• Arch. f. (Anat. u.) Physiol., 1901.
446 TISSUES 01 THE CONNECTIVE SUBSTANCE.
RIOT and RiCHET, Bleibtreu, Kaufmann, Laulanie ^). 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 oxgen.
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 accu-
mulation is quickly exliausted; and it is very probable that the lipase is
of importance here, as Loevenhart^ has found that all over the body
where fat is deposited in large amounts lipase also occurs in considerable
amounts. There is perhaps not one of the various tissues that decreases
so much in starvation as the fatty tissue. The organism, then, possesses
in this tissue a depot where there is stored during proper alimentation a
nutritive substance of great importance in the development 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.
' Hanriot and Riehet, Annal. de Chim. et de Phys. (6), 23; Bleibtreu, Pfliiger's
Arch., 56 and 85; Kaufmann, Arch, de Physiol. (5), 8; Laxilanie, ibid., 791.
^ 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 be satisfied
at the present time with a few microchemical reactions in the investi-
gation of the chemical composition of the muscular fibres.
Each muscle-tube and each muscle-fibre consists of a sheath, the sar-
COLEMMA, which scems to be composed of a substance similar to elastin,
and containing a large proportion of 'protein. This last, which in life pos-
sesses the power of contractility, has in the inactive muscle an alkaline
reaction, or, more correctly speaking, an amphoteric reaction with a pre
dominating action on red litmus paper. Rohmann has found that the
fresh, inactive muscle shows an alkaline reaction with red lacmoid, and an
acid reaction with brown turmeric. From the behavior of these coloring-
matters with various acids and salts 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.!
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, such as dilute hydro-
' The various statements in regard to the reaction of the muscles and the cause
thereof are conflicting. See Rohmann, Pfli ger's Arch., 50 and Ho; Heffter, Arch. f.
exp. Path. u. Pharm., 31 and 38. These references contain the pertinent literature.
447
448 MUSCLES.
chloric acid, soda solution, or gastric juice, they swell greatly and break
up into "Bowal^n's disks." B}^ the action of alcohol, chromic acid,
boiling water, or m general such reagents as cause a shrinking, the fibres
split longitudinally into fibrils; and this behax-ior shows that several
chemically different substances of various solubilities enter into the con-
struction of the muscular fibres.
The protein mj'osin 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 Daxilew^sky, confirmed by
J. HoLMGREx,! mvosin may be completely extracted from the muscle with-
out changing its structure, by means of a 5 per cent solution of ammonium
chloride, which fact contradicts the above view. Danilewsky 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 contams 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
KiJHXE, a spontaneously coagulating liquid, the muscle-plasma, which
coagulates quickly, separating a protein body, myosin, and yielding also
a serum. That liquid which is obtained by pressing the living muscle is
called jnuscle-plasma, while that obtained from the dead muscle is called
muscle-seruryi. These two fluids contain different protein bodies.
Muscle-plasma was first prepared by Kuhxe 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 com-
mon-salt solution of 5-6 p. m. Then the muscles are quickly cut and
immediately thoroughly frozen so that they can be groimd 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 cooliug or freezing is not necessary. It is sufficient to extract
1 Danilewsky, Zeitschr. f. physiol. Chem., 7; J. Holmgren, ]\Ialy's Jahresber., 23.
2 See Krhne, Untersuchungen i.ber das Protoplasma (Leipzig, 1864), 2; Hallibur-
ton, Journ. of Physiol., 8; v. Furth, Arch. f. exp. Path. u. Pharm., 36 and 3"; Hof-
meister's Beitrage, 3, and Ergebuisse der Physiologic, 1, Abt. 1; Stewart and Soll-
mann, Journ. cf Physiol., 24.
PROTEINS OF THE MUSCLE. 449
the muscle free from blood, as above directed, with a 6 p. m. common-
salt solution.
Muscle-plasma forms a yellow to brownish-colored fluid with an alkaline
reaction. It is somew^hat different 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. FtJRTH, 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 question 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 Kuhxe and v. Furth the reac-
tion remains alkaline during coagulation, while according to Halliburtox,
Stewart and Soll.maxx, it becomes acid. According to the older \dews
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 nomenclature,
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 neces-
sar}^ 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 Kuhxe, 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 Kuhxe also gives to the mother-substance of the plasma-clot, and
this mother-substance foi-ms, according to certain investigators, the chief
mass of contractile protoplasm. The statements as to the occurrence of
myosin in other organs besides the muscles require further proof. The
quantity of myosin in the muscles of different animals varies, according
to Danilewsky,! between 30 and 110 p. m.
Myosin, as obtained from dead muscles, is a globulin whose elementan,'
composition, according to Chittexdex and Cummixs,^ 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
' Zeitschr. f. physiol. Chem., 7.
^ Studies from the Physiol. Chem. Laboratory of Yale College, New Haven, 3, 115.
450 MUSCLES.
>jXiLyosin separates as fibres, or if a myosin solution with a minimum quantity
of alkali is allowed to evaporate on a microscope-slide to a gelatinous mass,
doubly refracting myosin may be obtained. Myosin has the general prop-
erties 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 MgS04, in a solution containing 94 per cent of the salt with its water of
crystallization (Halliburton). The precipitated myosin becomes insoluble
readily. 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 Chitten-
den 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. The filtered extract is then treated with magnesium sulphate in
substance until 100 c.c. of the liquid contains 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 c.c, of the liquid
holds 94 grams of the salt in solution. The myosin 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, accord-
ing to Danilewsky,^ in extracting the muscle with a 5-10 pf:>r cent ammo-
nium-chloride solution, precipitating the myosin from the filtrate by
strongly diluting with water, ard redissolving the precipitate in ammonium-
chloride solution, and the myosin obtained from this solution is repre-
cipitated either by diluting with water or by removing the salt by dialysis.
Musculin,2 called paramyosinogen by Halliburton, and myosin
by V. FiJRTH, is a globulin which is characterized by its low coagulation
temperature, about 47° C, which may vary in different species of animals
(45° in frogs, 51° C. in birds). It is more easily precipitated than myosin
by NaCl or MgS04 (50 per cent salt, including; water of crystallization).
According to v. FDrth it is precipitated by ammonium sulphate with a
concentration of 12-24 per cent salt. If the dead muscle is extracted with
water a part of the musculin goes into solution and may be precipitated
therefrom by carefully acidifying. It separates from a dilute salt solution
on dialysis. Musculin readily passes into an insoluble modification which
* Zeitschr. f. physiol. Chem., 5, 158.
2 As we have up to the present no conclusive basis for the identity of the globulins
called myosin and paramyosinogen, and also as the use of the name myosin for the last-
mentioned substance may readily cause confusion, the author does not feel justified
in dropping the old name musculin (Nasse).
PROTEINS OF THE MUSCLE. 451
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
temperature and has other precipitating properties for neutral salts than
the older substance called myosin, it is difficult to concede to this view.
Myoglohulin. After the separation of the musculin and the myosin from the
salt extract of the muscle by means of MgS04 the myoglobulin may be precipitated
by saturating the filtrate with the salt. It is similar to serglobulin, but coagu-
lates at G3° C. (Halliburton). Myoalhumin, or muscle-albumin, seems to be
identical \\'ith seralbumin (seralbumin a, according to Halliburton), and prob-
ably 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 aJl 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 " muscle-stroma."
According to D.ixilewsky the amount of such stroma substance is con-
nected 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.
According to J. Holmgren. ^ this stroma substance does not belong to
either the nucleoalbumin or the nucleoproteid group. It is not a gluco-
proteid, as it does not yield a reducing substance when boiled with dilute
mineral acids. It is very similar to the coagulable proteins and dissolves
in dilute alkalies, forming an albuminate. The elementary composition of
this substance is 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, occur also
among the stroma substances. When the muscles are previously extracted
with water the stroma substance also contains a part of the myosin hereby
made insoluble. To the proteins insoluble in water and neutral salts
belongs the nucleoproteid detected by Pekelharing, which occurs as
traces and is soluble in faintly alkaline water, and which originates probably
from the muscle nuclei. According to Bottazzi and Ducceschi - the heart
muscle is richer in nucleoproteid 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 -oluble
and has a greater aptitude to precipitate than other acid albumins, seems
not to occur preformed in the muscles. Heubner's ^ mytolin is modified muscle-
proteid, chiefly myosin, which has lost a part of its sulphur by the action of
alkali.
' See foot-note 1, p. 44S.
2 Pekelharing, Zeitschr. f. physiol. Chem., 22; Bottazzi and Ducceschi, Centralbl
f. Physiol., 12.
' Arch. f. exp. Pathol, u. Pharm., iiS.
452 MUSCLES.
Proteins of the Muscle-plasma. As above stated, myosin was ordinarily
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 fer77ient," which is related
"to fibrin ferment but not identical with it, has also found that a solution
of purified myosin, in dilute salt solution (5 per cent MgS04), and suffi-
ciently diluted with water, coagulates after a certain time, and at the
same time becomes acid, and a typical myosin-clot separates. This coagu-
lation, which is accelerated by warming or by the addition of myosin fer-
ment, is, according to Halliburton, a process analogous to the coagulation
of the muscle-plasma. According to this same investigator, myosin when
dissolved in water by the aid of a neutral salt is reconverted into myosino-
gen, 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 forms also 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. FDrth) and myogen.
Musculin (Nasse) = paramyosinogen (Halliburton) = myosin (v.
FtJRTH) 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
par cent, of the proteins of rabbit muscle-plasma. It does not separate
from its solutions on dialysis and is not a true globulin, but a protein sui
generis. It coagulates at 55-65° C. and is precipitated in the presence of
26-40 per cent ammonium sulphate. Myogen solutions 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. ]\Iyogen passes spontaneously, especially with higher tempera-
tures as well as in the presence of salt, into an insoluble modification,
myogen fibrin. A protein, coagulating at 30-40° C, soluble myogen fibrin,
is produced as a soluble intermediate step. This substance occurs to a
considerable extent in native frog-muscle plasma. It does not always
occur in the muscle-plasma of warm-blooded animals, and when it does
it is present only to a slight extent. It can be separated by precipitating
with salt or by diffusion. Halliburton's assumption as to the action of
PROTEINS OF THE MUSCLE PLASMA. 453
a special myosin ferment has not sufficient basis, according to v. Furth,
nor has the often-admitted analogy with the coagulation of the blood.
The difference between the musculin and the myogen in their becoming
insoluble is that the musculin passeg into myosin fibrin without any sol-
uble 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 Sollmanx admit of only two soluble proteins in the
muscles. One is the paramyosinogen, which is the same as v. Furth's
myosin +the soluble myogen fibrin. The other they call myosinogen,
which corresponds to v. Furth's myogen or to Halliburton's myosinogen
+ myoglobulin. It is an atypical globulin which coagulates at 50-60° C.
The paramyosinogen as well as the myosinogen are readily converted
into an insoluble modification, myosin. The myosin of the above investi-
gators is the same as v. Furth's myosin filjrin +myogen fibrin, and cor-
responds, it seems, also to myosin mixed with paramyosinogen (Halli-
burton). Stewart and Sollmann differ from Halliburton in con-
sidering that paramyosinogen al^o 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 (four different things are designated by
the name myosin) that it is extremely difficult to give any correct review
of the various notions.^ Thorough investigations on this subject are very
necessary.
Myoproteid is a proteid found by v. Fijrth in the plasma from fish-
muscles. It does not coagulate on boiling, is precipitated by acetic acid,
and considered as a compound proteid by v. Furth.
In connection with v. Furth's work, Przibram has carried on investigations
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' found somewhat
more musculin and less myogen in the muscle-juice than in the normal muscle.
Muscle-jrigments. There is no question that the red color of the muscles,
even when completely freed from blood depends in part on haemoglobin.
' For these reasons the author is not sure whether he has understood and correctly
pven the work of the different investigators.
* Przibram, Hofmeister's Beitrage, 2; Steyrer, ibid., 4.
454 MUSCLES.
K. MoRNER has sho-v\Ti that muscle-haemoglobin is not quite identical with
blood-hgemoglobin. The statement of Mac^Iuxn, that in the muscles
another pigment occurs which is allied to haemochromogen and called myo-
hcematin by him, has not been substantiated, at least for muscles of higher
animals (Levy and Morner i). MacMuxx 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 (Hedix and Row-
LAXD 2) have also been found, and the hydrol}'tic 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
1-4 p. m. in the fresh muscles containing water, also the purine bases,
hypoxanthine and xanthine, besides guanine and carnine, but chiefly hypo-
xanthine. The purine bases probably do not occur as such but as complex
combinations. The quantity of nitrogen as purine bases amounts, accord-
ing to BuRiAX 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 hypo-
xanthine. In the embryonic ox-muscles, Kossel ^ found more guanine
than hypoxanthine. The purine bases are produced in the muscles them-
selves, and their production, which also takes place while at rest, is greatly
increased during work (Burian^).
Among, the apparently habitually occurring nitrogeneous extractives,
we should also mention phosphocarnic acid as well as inosinic acid, which
is perhaps allied to it, carnosinc, carnitine, and perhaps also other bodies
which have recently been found in meat extract and which will be mem-
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,
* See MacMunn, Phil. Trans, of Roy. Soc, 177, part 1, Journ. of Physiol., 8, and
Zeitschr. f. physiol. Chem., 13; Le\^, ibid., 13; K. Morner, Nord. Med. Archiv, Fest-
band, 1897, and Maly's Jahresber., 27.
* Zeitschr. f. physiol. Chem., 32.
^ Burian and Hall, Zeitschr. f. physiol. Chem., 38; Kossel, ibid., 8, 408.
*Ibid., 43.
CREATINE. 455
according to Poulsson, Schmidt and Korndorfer.' Uric acid, urea, tavrine,
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 ^ have shown that it varies 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. Ihe reports are very contradictory in regard
to the occurrence of ui'ea in the muscles of higher animals. According to the
investigations of Kaufmann and Schondurff, confirmed by Brunton-Blaikie,^
urea is a regular constituent of the muscles, although M. Nencki and Kowarsk]
dispute this.
The xanthine bodies, with the exception of carnine, have been treated
on pages 159-162, and therefore among the extractive bodies we will first
consider the creatine.
/NH2
Creatine, C4H9N3O2, (HN)C< , or methylguanidine-
\N(CH3).CH2.COOH
acetic acid, occurs in the muscles of vertebrate animals in variable amounts
in different species; the largest quantity is found in birds. It is also found
in the brain, blood, transudates, amniotic fluid, and sometimes also in the
urine. Creatine may be prepared synthetically from cyanamide and
sarcosine (methylglycocoU). On boiling vnth baryta water it decomposes
^with the addition of water and yields urea, sarcosine, and certain other
products. Because of this behavior several investigators consider creatine
as a step in the formation of urea in the organism. On boiling with acids,
creatine is easily converted, with the elimination of water into creatinine,
C4H7N3O, which occurs in urine, and which has also been found in the
muscles of the dog by Monari ^ (see Chapter XV), and probably is a regular
constituent of the muscles.
Creatine cn,'stallizes in hard, colorless, monoclinic prisms which lose
their water of crv^stallization at 100° C. It i=; soluble in 74 parts of water
at the ordinary temperature and 9419 parts absolute alcohol. It dissolves
more easily with the aid of heat. Its waterj^ 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 solution of creatine in water is not prc-
* See Limprlcht, Amial. d. Chem. u. Pharm., 127, and Thesen, Zeitschr. f. physiol,
Chem., 24; Poulsson, Arch. f. exp. Path. u. Pharm., 51; Schmidt and Korndorfer.
ihid., 51.
2 Zeitschr. f. Biologie, 21; see also M. Henze, Zeitschr. f. physiol. Chem., 43;
Mendel, Hofmeister's Beitrage. 5; Kelly, ibid.. 5.
^Kaufmann, Arch, de Physiol. (.5), 6; Schbndorff, Pfliiger's Arch., 62; Nencki
and Kowarski, Arch. f. exp. Path. u. Pharm., 36; Brimton-Blaikie, Journ. of Physiol.,
123. Supplement.
*Maly's Jahresber., 19, 296.
456 MUSCLES
cipitated by basic lead acetate, but gives a white, flaky precipitate with
mercurous nitrate if the acid reaction is neutraUzed. ^'\llen 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 crystalUzes readily
(Jaffe 1).
The preparation and detection of creatine is best performed by the
following method of Neubauer,^ which was first used in the preparation
of creatine from muscles: Finely cut meat is extracted with an equal \\ eight
of water at 50° to 55° C. for 10-15 minutes, pressed, and extracted again
with water. The proteins are removed from the united extracts as far as
possible by coagulation at boiling heat, the filtrate precipitated by the care-
ful 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 crystalUzes in a few days, is collected on a filter, washed
with alcohol of 88 per cent, and purified, when necessary, by recrystalliza-
tion. The quantitative estimation of creatine is performed according to the
same method.
Gamine, C6H8N4O3+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 may be transformsd into hypoxanthine by oxidation.
Carnine has been obtained as a white crystalline mass. It dissolves
with difficulty in cold water, but more readily in warm. It is insoluble in
alcohol and ether. It dissolves in warm hydrochloric acid and yields a salt
crystallizing in shining needles, which gives a double compound with
platinum chloride. Its watery solution is precipitated by silver nitrate*
but this precipitate is dissolved neither by ammonia nor by warm nitric
acid. 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 1 y the follouing 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 sulphide 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.
* Ber. d. d. chem. Gesellsch., 35.
^Zeitschr. f. physiol. Chem., 2 and 6.
'Weidel, Annal. d. Chem. u. Pharm., 158; Krukenberg and Wagner, Sitzungsber,
d. Wiirzb. phys.-med. Gesellsch., 1883; Pouchet, cited from Neubauer-Huppert,
Analyse des Harnes, 10. Aufl., 335.
BASES AND PHOSPHOaiRXIC ACID. 457
Camosine, CgHj^X^Oj, has been isolated by GuLET^^TSCH and Amir.\zdibi ^
from meat extracts. It is a base which is perhaps related to arginine, and 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.
Carnitine, €71155X03 (?), is another base isolated by GuLE■^^^TSCH and Krimberg ^
from meat extracts, has a strong alkaline reaction, and is very readily soluble
in water. It gives a crystalline chloroplatinate, as well as salts with'HCl and
HXO3 which are very readily soluble in water. The HXO3 salt, which is also
crystalline, is strongly levorotatory.
From LiEBiG's extract of beef Kutscher has recently isolated a series of
new bodies, namely, ignotine, CyHj^ y^ fi^jCarnomuscarine, neosmrjCoHnXU^, novaine,
C7H17XO2, viethylguanidine (also found by Gulewitsch), and a crvstallizable
chloroplatinate, Ci8HasX„05.2HCl.PtCI„ of a body which he calls ohUtine. Zuxz ^
has also been able to isolate from fresh muscles the three hexone bases, leucine,
aspartic acid, and glutamic acid. He has not decided whether these bodies exist
preformed in the muscles.
The base musculamine, isolated by Etard and Vila on the hydrolysis of veal,
is nothing but cadaverine, according to Posternak.*
Inosinic acid has been discussed on page 155. We must also include among
the nitrogenous extractives those bodies which were first discovered by Gautier ^
and which occur only in very small cjuantities, namely, the leucomaines, xantho-
creatinine, CJiioSfi, crusocreatinine, CjHgX^O, arnphicrcatine, C9H19X7O4, and
psetidoxantMne, C4H.X5O.
In the analysis of meat and for the detection and separation of the various
extractive bodies of the same we make use of the systematic method as suggested
by Gautier,' for details of which the reader is referred to the original article.
Phosphocamic acid ' 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 relationship to the
nucleins, and as it yields peptone (carnic acid), it is designated as a nucleon by
Siegfried. Phosphocamic acid may be precipitated as an iron compound,
carnijcrrine, from the extract of the muscles free from proteins. The quantity of
phosphocamic acid, calculated as carnic acid, can be determined by multipl^-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. Mijller 1-2 p. m. in the muscles of adults and a maxi-
mum of 0.57 p. m. in those of new-born infants. According to Cavazzaxi nucleon
occurs to a much greater extent in oysters, namely, an average of 3.725 p. m. It
also occurs, as he and Manicardi found, in the plant kingdom. Phosphocamic
acid has not been prepared in the pure state and possesses on this account a
• Zeitschr. f. physiol. Chem., 30.
^ Kutscher, Zeitschr. f. Unters. d. Nahrungs- u. Genussmittel, 10, and Centralbl. f.
Physiol., 19; Zunz, reference, ibid., 18; Gulewitsch, Zeitschr. f. physiol. Chem., 47.
* Etard and Vila, Compt. rend., 135; Postemak, ibid., 135.
5 See Maly's Jahresber., 16, 523.
'>7imi.,22, 335.
' In regard to carnic acid and phosphocamic acid, see the works of Siegfried, Arch,
f. (Anat. u.) Physiol., 1894, Ber. d. deutsch. chem. GeseUsch., 2S, and Zeitschr.
f. physiol. Chem., 21 and 28; M. MiJller, t6tJ., 22; Kriiger, iWrf., 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.
458 MUSCLES.
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 CaCL and XH^. The acid is precipitated as carnifer-
rine by ferric chloride from the filtrate while boiling.
The non-nitrogenous extractive bodies of the muscles are inosite, glyco-
gen, sugar, and lactic acid.
I-osite, CeH 206+H20=C6H6(OH)6+H20. This body, discovered by
ScHERER, is not a carbo'aydrate, but a hexahydrox}'benzene (]\Iaquexxe ^).
With hydriodic acid it yields benzene and tri-iodophenol. Inosite is found
in the muscles, liver, spleen, leucocytes, kidneys, suprarenal capsule, lungs,
brain, testicles, and in the urine in pathological cases, and as traces in
normal urine. It is found verj^ widely distributed in the vegetable king-
dom, especially in the unripe fruit of green beans (Phaseolus ^nllgaris), and
therefore it is also called phaseomaxnite. According to Wixtersteix a
phosphorized compound occurs in the vegetable kingdom which yields
inosite as a cleavage product. This compound is, according to Posterxak ,2
probably ox\methylphosphoric acid, which also yields inosite on decom-
position by condensation.
Inosite cry^stallizes in large, colorless, rhomljic crystals of the monoclinic
system, or, if not pure and if only a small quantity crj'stallizes, it forms
groups of fine cr}^stals similar to cauliflower. It loses its water of crystalliza-
tion at 110° C, also if exposed to the air for a long time. Such exposed
crystals are non-transparent and milk-white. The crj'stals melt at 225° C.
when dr}^ Inosite dissolves in 7.5 parts of water at ordinary' temperature,
and the solution has a sweetish taste. It is insoluble in strong alcohol and
in ether. It dissolves cupric hydrate in alkaline solutions, but does not
reduce on boiling. It gives negative results with ]\Ioore's test and with
Bottger-Almex's bismuth test. It does not ferment with beer-yeast,
but may undergo lactic- and butyric-acid fermentation. The la^'tic acid
formed thereby is sarcolactic acid according to Hilger, and fermenta-
tion lactic acid according to Vohl.^ Inosite is oxidized into rhodizonic
acid by an excess of nitric acid, and the following reactions depend upon
this behavior:
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 drjTiess, a beautiful rose-red residue is
' Bull, de la see. chim. (2j, 47 and 48; Compt. rend., 104.
^ W^interstein, Ber. d. d. chem. Gesellsch., 30; Postemak, Contribution a I'etude
chim. de rassiniilation chlorophyllienne, Revue generale de Botanique, 12 (1900).
^ Hilger, Aunal. d. Chem. u. Pharm., 160; Vohl, Ber. d. d. chem. Gesellsch., 9.
INOSITE AND GLYCOGEN. 459
obtained (Scherer's inosite test). If we evaporate an inosite solution to
incipient dryness and moisten the residue with a little mercuric nitrate
solution, there is obtained a yellowish residue on drying, v.luch becomes a
beautiful red on strongly heating. The coloration disappears on cooling,
but it reappears on gently warming (Ga: lois' inosite test).
To prepare inosite from a liquid or from a watery extract of a tissue,
the proteii s 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 ciystals 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, crj^stals of
inosite separate within twenty-four hours. The crj^stals thus obtained,
as also those which are obtained from the alcoholic solution directly, are
recrystallized by redissolving in very little boiling water and addin • 2-4
vols, of alcohol.
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 greater amount in the muscles of the
extremities than in those of the rump. Sch()xdorff has found a maxi-
mum of 37.2 p. m. in the dog-muscle. The statements as to the quantity
of glycogen in the heart differ somewhat; although the heart is considered
as somewhat poorer in glycogen than the other muscles, still this difference
is not very great and can be explained by the ready disappearance of
glycogen from the heart after death, as well as after starvation and after
strong work (Boruttau, Jensen, Kisch 2). 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 partaking
of food. As stated in Chapter VIII, work, starvation, and lack of carbo-
hydrates in the food cause the glycogen to disappaar earlier from the liver
than from the muscles.
The sugar of the muscles, of which only traces occur in the living muscle,
and W'hich is probably formed after the death of the muscle from the mus-
cle-glycogen, is, according to the investigations of Panormoff, in part
dextrose, but consists chiefly of maltose (Osborne and Zobel ^) with some
dextrin.
> Bohm, Pfliiger's Arch., 23, 44; Schoniorff, ibid., 99.
^ Boruttau, Zeitschr. f. physiol. Chem., 18; Jensen, iW(/., 35; Kisch, Hofmeister'3
Beitrage, 8.
^Panormoff. Zeitschr. f. physiol. Chem., 17; Osborne and Zobel, Journ. of Phy-
siol., 29.
460 MUSCLES.
Lactic Acids. Of the oxypropionic acids with the formula CsHeOs
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 isomeres, is of importance. These three
ethylidene lactic acids are the ordinary, optically inactive fermentation
LACTIC ACID, the dextrorotatory paralactic or s-1rcolactic acid, and
the LEVOLACTic ACID obtained by Schardinger by the fermentation of
cane-sugar by means of a special bacillus. This levolactic acid, which
has also been detected by Blachstein in the cul ure of Gaffky's typhoid
bacillus in a solution of sugar and peptone, and which is formed by vari-
ous vibriones, need not be described here.^
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 occur-
rence of fermentation lactic acid in the brain and other organs has recently
been disputed by Moriya.2 During digestion this acid is also found in
the contents of the stomach and intestine, and as alkali lactate in the chyle.
The paralactic acid is, at all events, the true acid of meat extracts, and this
alone has been found with certainty in dead muscle. The lactic acid which
is found in the brain, spleen, lymphatic glands, thymus, thyroid gland,
blood, bile, pathological transudates, osteomalacious bones, in perspira-
tion in puerperal fever, in the urine after fatiguing marches, in acute
yellow atrophy of the liver, in poisoning by phosphorus, and especially
after extirpation of the liver seems always 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 kid-
neys 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 elimi-
nated I y the urine in animals with extirpated livers is increased with pro-
tein 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
'See Schardinger, Monatshefte f. Chem., 11; Blachstein, Arch, des sciences biol.
de St. P^tersbourg, 1, 199; Kuprianow, Arch. f. Hygiene, 19, and Gosio, ibid., 21.
^ Heintz, Annal. d. Chem. u. Pharm., 157, and Gscheidlen, Pfliiger's Arch., 8,
171; Moriya, Zeitschrift f. physiol. Chem., 43.
LACTIC ACID. 461
of air deficient in oxygen, or by any other means, a considerable elimina-
tion of lactic acid (besides dextrose and also often albumin) takes place
through the urine, an observation which has been confirmed by Saito
and KATSUYA^L\.^ As a scarcity of oxygen, according 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 proteia destruction and in part to a diminished oxidaion.
Araki has not drawn such a conclusion from his experiments, but he
considers the abundant formation of lactic acid to be due to a cleavage of
the sugar formed from the glycogen. He found that in all cases where
lactic acid and sugar appeared in the urine the quantity of glycogen in
the liver and muscles was always diminished. He also calls attention to
the fact that dextrolactic acid may be formed from glycogen, as directly
observed by Ekuxixa,^ and also to the numerous observations on the
formation of lactic acid and the co::sumption of glycogen in muscular
activity. Without denying the possibility of a formation of lactic acid
from protein, he states that with lack of o :ygen we have to deal with an
incomplete combustion of the lactic acid derived by a cleavage of the sugar.
Hoppe-Seyler 3 also positively defends the view as to the formation of
lactic acid from carbohydrates. He was of the view 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 w^ater. The formation of lactic acid in the absence of free
oxygen and in the presence of glycogen or dextrose is, according to Hoppe-
Seyler, ver}' probably a function of all living protoplasm. In the anaero-
bic metabolism of the animal cells, according to the recent investigations
on alcoholic fermentation in the tissues (see Chapters I and ^^II), carbon
dioxide and alcohol are formed from the sugar, with lactic acid as an inter-
mediary step; but even if this view be correct and when the cells, as Stok-
LASA^ and his collaborators have shown, contain a lactic-acid-forming
enzyme, it is not known what kind of lactic acid is here produced. Accord-
ing to MoRiSHiMA an increase in the lactic acid in the liver occurs after
death, probably from the liver glycogen, but this acid is chiefly fermenta-
tion lactic acid. Asher and Jackson ° experimented by transfusing blood
(with and without the addition of sugar) through the lower extremities of
' Gaglio, Arch. f. (Anat. u.) Physiol., 1886; Minkowski. Arch. exp. Path. u. Pharm.,
21 and 31; Araki, Zeitschr. f. physiol. Chem., 15, 16, 17, and 19; Saito and Katsuyama,
ibid., 32.
2 Joum. f. prakt. Chem. (N. F.), 21.
^ Virchow's Festschrift, also Ber. d. deut.sch. chem. Gesellsch., 25, Referatb., 685.
* Simacek, Centralbl. T. Physiol., 1"; Stoklasa, Jelinek, and Cemy, ibid., 16.
^Morishima, Arch. f. exp. Path. u. Pharm., 43; Asher and Jackson, Zeitschr. f.
Biologic, 41.
462 MUSCLES.
dogs, and neither in these experiments nor in those where the larger-organs
(liver and abdominal \nscera) 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 forma-
tion of lactic acid from carbohydrates, still, on the other hand, we have
recent investigations that make such an origin for lactic acid ver}^ proba-
ble. Thus Embden 1 has found, on percolating blood through a sur\dving
liver rich in glycogen, that lactic acid was formed, and also that thi^ acid
was produced in abundance when blood rich in sugar was transfused through
a glycogen-free liver, while, on the contrary, blood poor in sugar led to
only a very inconsiderable formation of lactic acid. The investigations of
A. R. Mandel and Lusk^ 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 bringing about a phlor-
hizm diabetes in the animal. Phosphorus intoxication caused no lactic-
acid formation in a phlorhizin- diabetic dog. Although it is difficult to
give a satisfactory 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 considered
as the material from which the lactic acid is formed in the body. In a
previous chapter (VIII) we mentioned the fonnation of lactic acid in the
animal body by a d lamination of alanine, and this gives us an indication
of a lactic acid formation from protein. Phosphocamic 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 syrupswhich mix in all pro portions
with water, alcohol, or ether. The salts are soluble in water, and most of
them also in alcohol. The two acids are differentiated from each other by
their different optical properties — paralactic acid being dextrogyrate, while
fermentation lactic acid is optically inactive — also by their different solu-
bilities and the different amounts of water of crystallization 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 crystalli-
zation, corresponding to the formula Zn(C3H503)2 + 3H20. The zinc salt
of paralactic acid dissolves in 17.5 parts of water at the above tempera-
ture and contains ordinarily 12.9 per cent water, corresponding to the
formula Zn(C3H503)2 + 2H20. The calcium salt of fermantation 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
» Centralbl. f. PhyaioL, 18, 832. * Amer. Journ. of Physiol., 16.
LACTIC ACIDS. 463
water and contains 24.83 or 26.21 per cent ( = 4 or 4| molecules) water of
crystallization. Both calcium salts crystallize, not unlike tyrosine, in spears
or tufts of very fine microscopic needles. Hoppe-Seyler and Araki^
who have closely studied the optical properties of the lactic acids and
lactates, consider the lithium salt as best suited for the preparation and
quantitative estimation of the lactic acids. The lithium salt contains
7.29 per cent Li. For further information as to the salts and specific
rotation of the lactic acids see Hoppe-Seyler-Thierfelder's Hand-
buch, 7. Aufl., 1903.1
Lactic acids may be detected in organs and tissues in the following
manner: After complete extraction with water, the protein is removed by
coagulation at boiling temperature and the addition of a small quantity of
sulphuric acid. The liquid is then exactly neutralized while boiling with
caustic baiyta, and then evaporated to a syrup after filtration. The residue
is precipitated with absolute alcohol, and the precipitate completely ex-
tracted with alcohol. The alcohol is entirely distilled from the united alco-
holic extracts, and the neutral residue is shaken with ether to remove the
fat. The residue is dissolved in water and phosphoric acid added, and tlie
solution repeatedly shaken with fresh quantities of ether, which dissolves
the lactic acid. The ether is now distilled from the united ethereal ex-
tracts, the residue dissolved in water, and this solution carefully warmed on
the water-bath to remove the last traces of ether and volatile acids. A
solution of zinc lactate is prepared from this filtered solution by boiling
with zinc carbonate, and this is evaporated until crj'stallization commences,
and then is allowed to stand over sulphuric acid. An analysis of the salts
is necessary in careful work. According to Heffter.^ in muscles not having
undergone rigor mortis the lactic acid can be extracted more easily by alco-
hol than by water.
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 con-
siderable quantit}^ of fat in the muscle-fibres is found only in fatty degen-
eration. A part of the muscle-fat can be readily extracted, while another
part can be extracted onl}- with the greatest difficulty. This latter part,
it is claimed, exists finely divided in the contractile substance itself and is
richer in free fatty acids, standing, according to Zuntz and Bogdanow,^
in close relationship to the activit}' of the muscles because it is consumed
during work. LecitJtin 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 decomposition of the lecithin. The
' See also E. Jungfleisch, Compt. rend., 139, 140, and 142.
2 Arch. f. exp. Path. u. Phama., 38.
5 Arch. f. (Anat. u.) Physiol., 1897.
464 MUSCLES.
amount of lecithin is not considerable. In normal dog-heart, as free from
fat as possible, Rubo.v ^ found that the lecithin amounted to 7.5-8.5 per
cent of the drj' substance; for the striated muscle the amount of lecithin
was rather constant, namely, 5.08 per cent. The ether extract of the heart
of the dog contained 60-70 per cent lecithin.
The Mineral Bodic: 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 pre-
dominating salt. Chlorine is found in such insignificant quantities that it
is perhaps derived from a contamination with blood or lymph. The quan-
tity of magnesium is, as a rule, considerably greater than that of calcium.
Iron occurs only in very small amounts. Schmey 2 found variations be-
tween 0.0129 p. m. (rabbits) and 0.0793 p. m. (liuman), calculated on the
fresh muscle substance. The heart-muscle was comparatively richer in
iron, 0.06-0.109 p. m.
The importance of the various mineral bodies for the function of the
muscjes has been studied by several experimenters (Loeb, Lingle, Howell,
Overton, Laxgendorff and Hueck, and others ^). Further proof as to the
pre\'iously discussed ion action of the electrolytes 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, in the contraction and in the
fatigue of the muscle (I'leart); still these investigations have not led to con-
cordant results, so that we are not yet clear on the action of these ions.
Nevertheless it seems to be established that the combined action of various
ions is a necessity for the normal 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 contained about 7 p. m. NaCl, besides small
amounts of CaClg (0.2 p. m.), KCl (0.1 p. m.), and NaHCOa (0.1 p. m.).
* Arch. f. exp. Path. u. Pharm., 52.
2 MacaUum, Joum. of Physiol., 32; Schmey, Zeitschr. f. physiol. Chem., 39-
^ Loeb, Amer. Joum. of Physiol., 3, and Pfliger's Arch., 80, 91; Lingle, Amer.
Journ. of Physiol., 4 (also references to literature); Overton, Pfluger's Arch., 92 and
105; Langendorfif and Hueck, ibid., 96.
PERMEABILITY OF THE MUSCLES. 465
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. ^ The different sheaths of the
muscles, the sarcolemma and perimysium internum, offer no very great
resistance to the diffusion of the most soluble crs^stalloid compounds, while
the muscle-fibres, on the contrarv- (exclusive of the sarcolemma), are almost
if not entirely impervdous to most inorganic compounds and to many
organic compounds. The muscle-fi'jres themselves are actually semiper-
meable structures which are permeable for water but not for the molecules
or ions of sodium chloride and of potassium phosphate. 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
havirg 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 metabohsm 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 contrar}^, derivatives can be pre-
pared from these bodies which pass into the cells verj' readily, and Over-
ton finds that it is not impossible that the organism in part makes use of
a similar artifice in order to regulate the concentration of the nutritive
bodies within the protoplasm.
Rigor Mortis of the Muscles. If the influence of the circulating oxA'gen-
ated blood is removed from the muscles, as after the death of the animal
or by ligature of the aorta or the muscle-arteries (Stenson's test), rigor
mortis sooner or later takes place. The ordinary rigor appearing under
these circumstances is called the spontaneous or the 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 momen-
tarily 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 coagulation of
* Pfliiger's Arch., 92. See also Hober, ibid., 106, and Hamburger, Osmotischer
Druck und lonenlehre, Bd. 3.
466 MUSCLES.
certain proteins, and its occurrence at lower temperatures in cold-blooded
as compared with warm-blooded animals is due, according to v. FtJRTH,
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. 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 chloro-
form, 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 pro-
duced 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 ampho-
teric reaction becomes stronger, w^hich is explained by most investigators
by the assumption of a formation of lactic acid. There is hardly any doubt
that this increase in acidi; y may at least in part be due to a transformation
of a part of the diphosphate into monophosphate by the lactic acid. The
statements in regard to the presence or absence of free lactic acid in the
rigor-mortis muscle are contradict ory.i Besides the formation of acid, the
chemical processes which take place in rigor of the muscles are the follow-
ing: 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 se3m to be
a direct oxidation product, but a product of the cleavage processes. Her-
mann 2 claims that carbon dioxide is produced in the removed muscle,
even in the absence of oxygen, when it passes into rigor mortis. In con-
nection with this view we must call attention to Folin's^ observations
that no protein coagulation took place in rigor under special conditions.
As many investigators admit of an increased formation of lactic acid on
the appearance of rigor mortis, the question arises, from what constituents
of the muscle is this acid derived? The most probable explanation is that
the lactic acid is produced from the glycogen, as certain investigators, such
* It is impossible to enter into the details of the disputed statements as to the reac-
tion of the muscles, etc. We shall only refer to the works of Rohmann, PflTiger's Arch.,
50 and 55, and Heffter, Arch. f. exp. Path. u. Pharm., 31 and 38. These works con-
tain also the researches of the older investigators more or less completely.
2 Untersuchungen iiber den Stoffwechsel der Muskeln, etc., Berlin, 1867.
3 Amer. Journ. of Physiol., 9.
METABOLISM L\ THE MUSCLES. 467
as Nasse and Werther, have obsen-ed a decrease iii the quantity of
glycogen in rigor of the muscle. On the other side, Bohm ^ has observed
cases in which no consumption of glycogen took place in rigor of the muscle,
and he has also found that the quantity of lactic acid produced is not pro-
portional to the quantity of glycogen. It is therefore possible that the
consumption of glycogen and the formation of lactic acid in the muscles
are two processes independent of each other, and, as above stated in regard
to the formation of paralactic acid, the lactic acid of the muscle may be
considered as a decomposition product of protein. The origin of the carbon
dioxide is also not to be sought for in the decomposition of the glycogen or
dextrose. Pfluger and Stixtzixg - have found that in the muscle a sub-
stance occurs which evolves large quantities of car';on 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 connec-
tion 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 tliey relax again and
become softer. This is in part produced by the strong acid dissoh-ing the
myosin-clot and m part Ijy autolx-tic px"ocesses (Vogel^).
Metabolism in the Inactive and Active Muscles. It is admitted by a
nimiber of prominent investigators, Pfltger and Colasanti, Zuntz and
RoHRiG.-* and others, that the metabolism in the muscles is regulated by
the nen'ous system. "\Mien at rest, when there is no mechanical exertion,
there exists a condition which Zuxtz 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 nen^ous system by cutting through the spinal cord or the
muscle-ner\-es. The possibility of reducing the chemical tonus ol the
muscles in various ways offers an important means of decidmg 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 activity, and lastly the
total exchange of material, the receipts and expenditures of the organism,
have been investigated under these two conditions.
'Nasse, Beitr. z. Physiol, der kontrakt. Substanz, Pfiuger's Arch., 2; Werther,
ibid., 46; Bohm, ibid., 23 and 46.
2 Pfl iger's Arch., IS.
^ R. Vogel, Unters. iiber Muskelsaft, Deutsch. Arch, f . klin. Med., 1902.
* See the works of Pfluger and his pupils in Pfl^iger's Arch., 4, 12, 14, 16, and IS;
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.
468 . MUSCLES.
By investigations according to these several methods it has been found
that the active 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 mate-
rial 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 quan-
tity 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 results also from the fact that removed
blood-free muscles when placed in an atmosphere devoid of oxygen can
labor for some time and also yield carbon dioxide (Hermann i).
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, Chan-
DELON, Vay,2 and others). In activity this consumption of glycogen
is increased, and it has been positively proved by the researches of several
investigators (Nasse, Weiss, Kulz, Marcuse, Manche, Morat and
DuFOUR ^) that the quantity of glycogen in the muscles in activity decreases
quickly and freely. As shown by the researches of Chauveau and Kauf-
]vl\nn, Quinquaud, Morat and Dufour, Cavazzani, and especially those
of Seegen,"* the sugar is removed from the blood and consumed during
activity. According to Seegen a very abundant formation of sugar
' 1. c. In regard to gas exchange in removed muscles, see also J. Tissot, Arch, de
Physiol. (5), 6 and 7, and Compt. rend., 120.
^ Chandelon, Pfliiger's Arch., 13; Vay, Arch. f. exp. Path. u. Pharm., 34, which
also contains the pertinent literature.
^ Nasse, Pfl ger's Arch., 2; Weiss, Wien. Sitzungsber., 64; Kiilz, in Ludwig's
Festschrift, Marburg, 1890; Marcuse, Pfl iger's Arch., 39; Manch^, Zeitschr. f. Biolo-
gic, 2.'>; Morat and Dufour, Arch, de Physiol. (5), 4.
^Chauveau and Kaufmann, Compt. rend., 103, 104, and 105; Quinquaud, Maly's
Jahresber., 10; Morat and Dufour, 1. c; Cavazzani, Centralbl. f. Physiol., 8; Seegen,
"Die Zuckerbildung im Thierkorper," Berlin, 1890, Centralbl. f. Physiol., S, 9, and
10; Arch. f. (Anat. u.) Physiol., 1895 and 1896; Pfliiger's Arch., 50.
ACTIVITY AND FORMATION OF ACID. 469
takes place in the liver, and correspondingly the blood of the hepatic vein
is much richer in sugar than that in the portal vein; and this sugar of the
blood is, according to him, the source of heat formation and mechanical
activity. It is nevertheless true that important objections have been
presented against a few of these investigations, and a sugar formation,
according to Seegex's idea, has been denied by several investigators,
and recently by Ztjntz and Mosse; but still there can exist hardly any
doubt that sugar is consumed in muscular activity. A direct proof for this
has recently been given by Joh. Muller.^ In experiments on surva\Tiig
cats' hearts which were percolated with a salt solution containing sugar,
he could detect an undoubted consumption of sugar which was quite con-
siderable.
The amphoteric reaction of the inactive muscles is changed during
activity to an acid reaction (Du Bois-Reymoxd and others), and the acid
reaction increases to a certain point with the work. The quickly contract-
ing pale muscles produce, according to Gleiss,^ more acid during acti-vity
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
Waerex, who found less lactic acid in the tetanized muscle than when at
rest. MoxAEi also found a decrease in the quantity of lactic acid during
activity, and according to Heffter the quantity of lactic acid in the muscle
is diminished in tetanus produced by poison. Contrar}' to these investiga-
tions, Marcuse and Werther have been able to prove the formation of
lactic acid during activity; still the statements are ven,' contradictor}-.
Other observations indicate a formation of lactic acid during acti\'ity.
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, lactic acid is not produced regularly during
work, but only when insufficient oxygen is supplied. Zillesex ^ has also
found that on artificially cutting off the oxygen from the muscles during
life more lactic acid was formed than under normal conditions.
1 Mosse, Pfluger's Arch., 63; Zuntz, Centralbl. f. Thysiol., 10, and Arch. f. (Anat.
u) Fhysiol., 1896, 538. See also Schenck, Pfluger's Arch., 61 and 65; Miiller,
Zeitschr. f. allgem. Physiol., 3.
2 Pfluger's Arch., 41.
^Astaschewsky, Zeitschr. f. physiol. Chem., 4; Warren, Pfliiger's Arch., 24;
Monari, Maly's Jahresber., 19; Heffter, Arch. f. exp. Path. u. Pharm., 31; Marcuse,
1. c; Werther, Pfluger'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.
470 MUSCLES.
It is evident that the experiments with the muscles in situ — in other
words, with muscles through which blood is passing — cannot yield any con-
clusion to the above question, as the lactic acid formed during work may
j)erhaps be removed by the blood. The following objections can be made
against those experiments in which lactic acid has been found after mod-
erate work in the blood or the urine, as also especially against the experi-
ments with removed active muscles, namely, that in these cases the supply
■of ox3'gen to the muscles was not sufficient, and that the lactic acid formed
thereby is not, in accordance with the views of Hoppe-Seyler, a perfectly
normal process. The question as to the formation of lactic acid in the
active muscle under perfect physiological conditions is still an open one,
although several observations make it seem to be very probable.
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
not present as nucleons is diminished and the quantity of phosphates
is increased. Tliis stands in accord with Weyl and Zeitler's ^ observa-
tions that the active muscle contains more phosphoric acid than the inac-
tive muscle. As in the dead muscle, so in the active muscle, the some-
what stronger acid reaction is in part due to a greater quantity of mono-
phosphate.
The amount of protei .s in the removed muscles is, according to the
older investigators, decreased by work. The correctness of this statement
is, however, disputed by other investigators. The older statements in
regard to the nitrogenous extractive bodies of the muscle in rest and in
activity are likewise uncertain. According to the recent researches of
MoNARi^ the total quantity of creatine and creatinine is increased by
work, and indeed the amount of creatinine is especially augmented by an
excess of muscular activity. The creatinine is formed essentially from
the creatine. In excessive activity Monari also found xanthocreatinine
in the muscle, and the quantity was one-tenth that of the creatinine. The
purine bases are, according to Burian,^ increased during work, due to a
greater formation (see above, page 454). 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 *).
Attempts have been made to solve the question relative to the behavior
'Siegfried, Zeitschr. f. physiol. Chem., 21; Macleod, ibid., 28; Weyl and Zeitler,
ibid., 6.
^'Maly's Jahresber., 19, 296.
^ Zeitschr. f. physiol. Chem., 43.
^ Arch. f. (Anat. u.) Physiol., 1845.
NITROGENOUS METABOLISM IN THE MUSCLES. 471
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. AMiile formerly it was held with Liebig that the
elimination of nitrogen by the urine was increased by muscular work, the
researches of several experimenters, especially those of Voit on dogs and
Pettenkofer and Voit on men, have led to quite different results. They
have shown, as has also lately been confirmed by other investigators,
especially I. Munk and Hirschfeld,i that during work no increase or
only a very insignificant increase m 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 proteins
during or after work. There are for example the observ^ations of Flint
and of Pavy on a pedestrian, v. Wolff, v. Funke, Kreuzhage, and
Kellxer on a horse, and Dunlop and his collaborators on working human
beings, and of Krum^l\cher, Pfluger, Zuxtz and his pupils,^ and others.
The researches on the elimination of sulphur during rest and activity also
belong to this categoiy. The elimination of nitrogen and sulphur runs
parallel with the metabolism of proteir.s in resting and active persons, and
the quantity of sulphur excreted by the urine is therefore also a measure
of the protein catabolism. The older researches of Exgelmanx, Flint,
and Pavy, as well as the more recent ones of Beck and Benedict,^ and
Dunlop and his collaborators, show an increased elimination of sulphur
during or after work, and this indicates an increased protein metabolism
because of muscular activity.
That an increased destruction of protein is not necessarily produced by
work follows from the observations of Caspari, Bornstein, Kaup, Wait,
A. LoEW^', Atwater and Benedict,^ that a retention of nitrogen and a
deposition of protein occur during work. The contradictory^ 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 de-
pendent upon man}' conditions, such as the quantity and composition of
the food, the condition of the adipose tissue of the body, the action of the
' Voit, Untersuchungen uber den Eiiifluss des KcchsaLzes, des Kaffees und der
Muskelbewegungen auf den Stoffwechsel Ol^inchen, 1860), and Zeitschr. f. Biologie, 2;
J. Munk, Arch. f. (Anat. u.) Physiol., 1890 and 1896; Hirschfeld, Virohow's Arch., 121.
2 Flint, Journ. of Anat. and Physiol., 11 and 12; Pa\y, The Lancet, 1876 and 1877;
V. Wolff, V. Funke, Kellner, cited from Voit, Hermann's Handb., 6, 197; Dunlop,
Nool-Paton, Stockman, and Maccadam, Journ. of Physiol., 22; Krummacher, Zeitschr.
f. Biologie, 33; Pfl ger, Pfi ger's Arch., 50; Zuntz, Arch. f. (Anat. u.) Physiol., 1894.
^ Engelmann, Arch. f. (Anat. u.) Physiol., 1871 ; Beck and Benedict, Pfitiger's Arch.,
54, and also foot-note 2.
* Caspari, Pfl ger's Arch., 83; Bornstein, ibid.; Kaup, Zeitschr. f. Biologie, 43;
Wait, U S Depart. Agricult. BulletinSO (1901); Atwater and Benedict, ibid., Bull.
69 (1899); Loewj, Arch. f. (Anat. u.) Physiol., 1901.
472 MUSCLES
work upon the respiratory mechanism, etc., all of which have an influence
on the results of the experiments.
Recently Steyrer ^ has found that the muscle juice of a continuously tetanized
muscle was somewhat poorer in musculin and correspondingly richer m myogen
than the juice from a similar non-tetanized muscle. We cannot draw any con-
clusions 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. According to
the recent investigations of Zuntz and Bogdanow,^ the fat belonging to
the muscle-fibres and which is difficultly extracted takes part in work.
Besides these there are several researches by Voit, Pettenkofer and Voit,
J. Frentzel,3 and others which make an increased destruction of fat
during work probable.
If the results of the investigations thus far made of the chemical proc-
esses going 0.1 in the active and inactive muscle were collected together, 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 con-
siderably more than the absorption of oxygen. The respirator}^ quotient^
CO
-^^, is found to be regularly raised during work ; yet this rise, which will
be explained in detail in a following chapter on metabolism, can hardly be
conditioned on the kind of processes going on in the muscle during activity
with a sufficient supply of oxygen. In work a consumption of carbo-
hydrates, 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 consumption of fat has occa-
sionally 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 elimina-
tion of nitrogen as a direct consequence of muscular exertion has thus
far not been positively proved.
In close connection with the above-mentioned facts there is the 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
Hofmeister's Beitrage, 4. ^ Arch. f. (Anat. u.) Physiol., 1897.
^ Pfl iger's Arch., 68.
SOURCE OF MUSCULAR ENERGY. 473
WisLiCENUS ^ climbed the Faulhorn and calculated the amount of mechan-
ical force expended in the attempt. With this they compared the mechan-
ical equivalent transformed in the same time from the proteins, calculated
from the nitrogen eliminated with the urine, and found that the work
really performed was not by any means compensated by the consumption of
protein. It was therefore proved by this that proteins alone cannot be the
source of muscular activity, and that this depends in great measure on the
metabolism of non-nitrogenous substances. Many other observations have
led to the same result, especially the experiments of Voit, of Pettenkofer
and Voit, and of other mvestigators, whose observations show that while
the elimination of nitrogen remains unchanged, the elimination of carbon
dioxide during work is ver}- considerably increased. It is also generally
considered as positively proved that muscular work is produced, kt least in
greatest part, by the cataboUsm of non-nitrogenous substances. Never-
theless there is no warrant for the statement that muscular activity is pro-
duced 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 Pflltger^ are of great interest in this connection.
He fed a bulldog for more than seven months with meat which alone did
not contain sufficient fat and carbohj^lrates even for the production 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. PflCger 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 contrar}% believe
that the muscles first draw on the supply of non-nitrogenous nutriments,
and according to Seegen, Chauveau, and Laulanie ^ the sugar is ir.deed
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 collaborators 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
■ Vierteljahrsschr. d. Zurich, naturf. Gesellsch., 10, cited from Centralbl. f. d.
med. Wiss., 1866, 309.
' Pfliger's Arch., 50.
^ See Seegen, foot-note 4, page 468. The works of Chauveau and his collaborators
are found in Compt. rend., 121, 122, and 123; Laulanie?, Arch, de Physiol. (5), 8.
474 MUSCLES.
muscular work, a definite expenditure of force must require about 30 per
cent more energy wdth fatty food than it does with carbohydrates; but this
is not the case. The investigations of Zuntz, (together with) Loeb, Heine-
MANN, Frextzel 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 investigations of Atwater and Benedict ^ have also led to
similar results as to the fats being a source of muscular energy. The law
of the substitution of the foodstuffs, according to their combustion equiva-
lents, is also true for muscular work, and fat correspondingly acts with its
full amount of energy without* previously being transformed into sugar.
The question which of the foodstuffs the muscle prefers is dependent upon
the relative quantities of the same at the disposal of the muscle. A direct
substitution of the body material by the bodies supplied as food does not
take place in the muscular activity in the ordinary nutritive condition.
According to Johansson and Koraen - the CO2 excretion produced by
certain work is not influenced by the supply of foodstuffs (protei or sugar).
Siegfried considers, as above stated, the phosphocarnic acid as a source of
energy. According to his and KRiJGER's ^ researches, phosphocarnic acid, which
yields on cleavage, among other bodies, carbon dioxide, occurs in part preformed
in the muscle, and in part as a hypothetical aldehyde compound of the same — a
compound which forms phosphocarnic acid on oxidation. Siegfried therefore
makes the suggestion that in the resting muscle, which requires more oxygen
than exists in the carbon dioxide ehminated, this reducing aldehyde substance is
gradually oxidized to phosphocarnic acid, which is used in the acti^^ty of the
muscle with the splitting off of carbon dioxide.
Quantitative Composition of the Muscle. A large number of analyses
have been made of the flesh of various animals for purely practical purposes,
in order to determine the nutritive value of different varieties of meat; but
there are no exact scientific analyses with sufficient regard to the quantity of
different protein bodies and the remaining muscle constituents, that is,
these analyses are incomplete or of little value.
To give the reader some idea of the variable composition of muscje-
substance the following summary is presented, chiefly obtained from K. B.
Hofmann's ■' book, although it does not correspond to the present demands.
The figures are parts per 1000.
' 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-
ni.sse der Physiologie, 3.
^Skand. Arch. f. Physiol., 13.
2 Zeitschr. f. physiol. Chem., 22.
* Lehrbuoh d. Zoochemie (Wien, 1876), 104.
QU.\XTITATI\^E COMPOSITION OF MUSCLES.
475
Muscles of
Maniiuals.
Solids 217-255
Water 745-783
Organic bodies 208-245
Inorganic bodies 9-10
Myosin 35-106
Stroma substance (Da^'ii.ewsky) 78-161
Creat ine 2
Xanthine bodies 1 . 3-1 . 7
Inosinic acid (barium salt) 0.1
Protic acid ■ —
Taurine 0.7 (horse)
Inosite 0.03
Glycogen 4-37
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
Muscles of
Birds.
Muscles of
Cold-blooded
Animals.
225-282
200
717-773
800
217-263
180-190
10-19
10-20
29.8-111
29.7-87
88.0-184
70.0-121
3.4
2.3
0.7-1.3
—
0.1-0.3
—
—
7.0
—
1.1
3-5
In this table, which has little value because of the variation in the
composition of the muscles, no results are given as to the estimates of fat.
O-uing to the variable quantity of fat in meat and the incompleteness of the
older methods of estimation it is hardly possible to quote a positive aver-
age for this substance. After most careful efforts to remove the fat from
the muscles without chemical means, it has been fomid thr.t a variable
quantity of intermuscular fat. which does not really belong to the muscular
tissue, always remauis. The smallest quantity of fat in the muscles from
lean oxen is 6.1 p. m. according to Grouvex. and 7.6 p. m. according to
Petersen. This last observer also found regularly a smaller quantity of
fat, 7.6-8.6 p. m., in the fore quarters of oxen, and a greater amount,
30.1-34.6 p. m., in the hind quarters of the animal, but this could not be
substantiated by Steil.i A small quantity of fat has also been fomid in
the muscles of uild animals. B. Konig ard F.-vrwick fomid 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 respectively 100, 164, and 329 p. m. fat.^
» See Steil, Pfliiger's Arch,, 61.
^ In regard to the literature and complete statements on the composition of flesh
of various animals, see Konig, Chemie der menschUchen Nahrungs- und Genussmittel,
5. Aufl.
476 MUSCLES.
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 alone upon the
amount of fat, 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 towards 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 independently of the
amount of fat is strikingly shown by comparing the muscles of different
species of animals. In cold-blooded animals the muscles generally have
a greater quantity of water, in birds a lower. The comparison of the flesh
of cattle and fish shows verj^ strikingly the different amounts of water
(independent of the quantity of fat) in the flesh of different animals.
According to the analysis of Almen,i 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 elementar}^ 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
HuppERT^ this quantity may var}^ 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 va uo and free from fat
and with the glycogen deducted was as follows: C 49.6; H 6.9; N 15.3;
0 + S 23.0; and ash 5.2 per cent. Kohler found as an average for water
and fat -free beef C 49.86; H 6.78; N 15.68; 0 + S 22.3 per cent, which are
very similar results. This investigator has also made similar analyses of
the flesh of various animals and has determined the calorific value of
the ash- and fat -free dried meat substance. This value was, per gram of
substance, 5.599-5.677 Cal. The relationship of the carbon to nitrogen,
which Argutinsky calls the "flesh quotient," is on an average 3,54:1.
From Kohler's analyses the average for beef is 3.15:1 and for horse-flesh
3.38:1. 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
> Nova Act. Reg.Soc. Scient. Upsal., Vol. extr. ord., 1877; alsoMaly's Jahresber., 7.
^ Voit, Zeitschr. f. Biologie, 1; Huppert, ibid., 7; Nowak, Wien. Sitzungsber., 64,
Abt. 2.
NON-STRIATED MUSCLES. 477
cent other soluble bodies. Frextzel and Schreuer ^ find that about 7.74
per cent of the total nitrogen belongs to the nitrogenous extractives.
There exist complete investigations by Katz ^ as to the quantity of min-
eral constituents of the muscles from man and animals. The variation in
the different elements is considerable. Pork is much richer in sodium as
compared ^^ith potassium than other kinds of meat. The quantity of mag-
nesium 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 (Du Bois-
Reymond) when at rest. During acti\dty they are acid, which is inferred
from the observations of Bernstein, who found that the almost continually
contracting sphincter muscle of the Anodonta is acid during life. The
smooth muscles may also, according to Heidenhaix and KfHNE, pass into
rigor mortis and thereby become acid. A spontaneous but slowly coagulat-
ing plasma has also been obsers-ed in several cases.
In regard to the proteins of the smooth muscles we have the older
statements of Heidenhain and Hellwig;^ but they were first carefully
studied according to newer methods by Munk and Velichi.'* These experi-
menters have prepared a neutral plasma from the gizzard of geese, accord-
ing to V. Fl'Rth's method. This plasma coagulated spontaneously at the
temperature of the room, although slowly. It contained a globulin, pre-
cipitated by dialysis, which coagulated at 55-60° C. and also showed cer-
tain similarities with Kuhne's myosin. A spontaneously coagulating
albumin, which differed from myogen (v. Furth) by coagulatini at 45-50°
C, and which passes by spontaneous coagulation into the coagulated mod-
ification without a soluble intermediate product, exists in still greater
quantities in this plasma. Alkali albuminates do not occur, but a nuclco-
•proteid is found, which exists in about five times the quantity as compared
with striated muscles. Nucleon is, according to Panella,^ a normal con-
stituent of smooth muscles and occurs in larger amounts than in striated
muscles.
* Argutinskj', 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.
2 Pfliiger's Arch., 63. See also Schmey, Zeitschr. f. physiol. Chem., 39.
^ Du Bois-Reymond in Nasse, Hermann's Handb., 1 , 339; Bernstein, iftid. ; Heiden-
hain, ibid., 340, with Hellwig, ibid., 339; Ki.hne, Lehrbuch, 331.
* Munk and Velichi, Centralbl. f. Physiol., 12.
* Maly's Jahresber., 34.
478 MUSCLES.
Recent investigations of Bottazzi and Cappelli, Vincent and Lewis
Vincent, and v. Fi'-rth/ some on the muscles of warm-blooded and some
on those of lower animals, have led to somewhat contradictory results, but
they substantiate, as a whole, the observations of Munk and Velichi.
Besides the nucleoproteids the smooth muscles contain two bodies corre-
sponding in coagulation temperature to musculin and myosinogen (myogen,
V. Fl'rth), but they are not identical therewith. Hamoglohin occurs in the
smooth muscles of certain animals, but is absent in others. In the smooth
muscles (in certain varietie of animals) freatine, creatinine, taurin:, inosite,
glycogen, and lactic acid have been found. The mineral constituents show
the remarkable fact that the sodium compounds exceed the potassium
com o ui Is.
Hexze found abundance of taurine in the muscles of octopods, 5 p. m., but
no creatine, which, according to Fremy and Valenciennes,^ occurs in the muscles
of cephalopods. He also found no glycogen and no paralactic acid, but, on the
contrary, small amounts of fermentation lactic acid. The muscles of octopods
are richer in mineral bodies than the muscles of vertebrates, and are nearly twice
as rich in sulphur as these.
' Bottazzi, Centralbl. f. Physiol., 15; Vincent and Lewis, Journ. of Physiol., 26;
Vincent, Zeitschr. f. physiol. Chem., 34; v. F rth, ibid., 31.
^ Henze, ibid., 43; Fremy and Valenciennes, cited from Kuhnc's Lehrbuch, p. 333.
CHAPTER XII.
BRAIX AND XERVES.
Ox account of the difficulty in making a mechanical separation and
isolation of the different tissue-elements of the central nervous organ and
the nerv^es, 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 ner^'e-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 investigations of modem times,
still it must be admitted that this subject is as yet one of the most obscure
and complicated in physiological chemistry.
Proteins of d fJerent kinds have been shown to be chemical constituents
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 pro-
teins which are insoluble in water and neutral salt solutions, and which
resemble the stroma substances of the muscles and cells, while other pro-
teins are soluble in water and neutral salt solutions. Among the latter
we find chiefly nucleoproteids and globulins. The nucleoproteid found by
Halliburton and also by Levexe^ in the gray substance contains 0.5
per cent phosphorus and coagulates at 55-60°. Levexe obtained adenine
and guanine but no hypoxanthhie as cleavage products. According to
Halliburtox there are two globulins, namely, the neuroglobulin a, which
coagulates at 47° or at 50-53° in the caf.e of birds, and the neuroglobulin /?,
whose coagulation temperature is 70-75°, but which varies somewhat in dif-
ferent animals. In the frog still another protein body occurs, which coag-
ulates at a still lower temperature, about 40°. It must be remarked that
the coagulation temperature of a-globulin corresponds with the tempera-
ture of the first heat contraction of tlie nerves of different classes of animals
(Halliburtox).
Just as there are lecithin-albumins, compounds of proteid \\ith lecithin, so
' Halliburton, On the Chemical Physiology of the Animal Cell, King's College,
London, Physiological Laboralorj-, Collected Papers No. 1, 1893, and Ergebnisse der
Physiologic, 4; Levene, Arch, of Neurology and Psychopalhology, 2 (1899).
479
480 BRAIN AND NERVES.
according to Ulpiani and Lelli ^ there exists an analogous compound in the
brain which is a combination between a protagon-Uke substance and a pseudo-
nuclein.
There does not seem to be any doubt that the proteins belong chiefly
to the gray substance of the brain and to the axis-cylinders. The same
remark also applies to the nuclein, which v. Jacksch ^ found in large quan-
tities in the gray substance. Neurokeratin, which was first detected by
KtJHNE, and which partly forms the neuroglia, and as a double sheath
envelops the outside of the nerve-medulla under Schwann's sheath and
the inner axis-cylinders, occurs in the nerves, but chiefly, or according to
Koch entirely, in the white substance (Kuhne and Chittenden, Baum-
STARK ^).
The phosphorized substance protagon must be considered as one of the
chief constituents, perhaps the only constituent (Baumstark), of the white
substance. This last-mentioned substance, if we keep for the present to
the most carefully studied protagon — because there are perhaps several
different protagons — yields as decomposition products lecithin, fatty acids,
and a nitrogenous substance, cerebrin. It is difficult to state whether this
last body also exists preformed in the brain. At least an allied substance,
cerebron, occurs preformed in the brain. That lecithin also is pre-existent
in the brain and nerves can hardly be doubted. The investigations thus
far made have not shown decisively whether it is more abundant in the
gray or the white substance; according to Koch it is much more abundant
in the white substance. Fatty acids and neutral fats may be prepared from
the brain and nerves; but as these may be readily derived from a decom-
position of lecithin and protagon, 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 constitu?nts of the real nerve-substance. Cholesterin seems
chiefly, and according to Koch perhaps entirely, to occur in the white
substance. Besides these substances the nerve-tissue, especially the white
substance, contains doubtless a number of other constituents not well
known, and among which are several containing phosphorus. Thudi-
chum,* who has made thorough investigations of the brain and has described
a great number of brain constituents, has given the name phosphatides to
all substances of the brain containing the phosphoric-acid radical. Those
phosphatides which contain only one phosphoric-acid radical are called
monophosphati4es, those with two such radicals diphosphatides. The
1 Cited from Chem. Centralbl., 1902, 2, 292.
2Pfl:iger's Arch., 13.
^ Koch, Amer. Journ. of Physiol., 11; Kiihne and Chittenden, Zeitschr. f. Biologie
26; Baumstark, Zeitschr. f. physiol. Chem., 9.
* Thudichum, Die chemische Konstitution des Gehims des Menschen und der Tiers,
Tubingen, 1901.
PROTAGON. 481
monophosphatides can contain one, two, or more nitrogen atoms in their
molecule, while there are also nitrogen-free monophosphatides. Irrespective
of the relation between phosphorus and nitrogen, certain phosphatides
differ from the lecithins by not yielding any glycerophosphoric acid. These
investigations of Thudichum are without doubt of great importance, but
as they have not been repeated we cannot enter into a discussion of the
bodies described by him.
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, fat, and impure cholesterin, and they depend
upon a decomposition of the constituents of the medulla. According
to Gad and Heymans ^ myeline is lecithin in a free condition or in loose
chemical combination.
The extractive bodies seem to be almost the same as in the muscles.
One finds creatine, which may, however, be absent (Baumstark), xanthine
bodies, inosite, choline, paralactic acid (Moriya), phosphocarnic acid, uric
acid, jecorin (according to Baldi,^ in the human brain), and the diamine
neuridine, C5H14N2, discovered by Brieger^ and which is most interesting
because of its appearance in the putrefaction 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 nerv'e-substance protagon
and the cerebrins or cerebrosides must be specially described.
Protagon. This body, which was discovered by Liebreich, is a nitrog-
enized and phosphorized substance whose elementary composition, accord-
ing to Gamgee and Blankenhorn, is C 66.39, H 10.69, N 2.39, and P 1.068
per cent. Baumstark and Ruppel obtained the same figures, while Lieb-
reich found an average of 2.80 per cent N and 1.23 per cent P. Kossel
and Freytag, who obtained still higher figures for the nitrogen, namely,
3.25 per cent, and somewhat lower figures for the phosphorus, 0.97 per
cent, foimd some sulphur, an average of 0.51 per cent, regularly in the
protagon. Ruppel also found some sulphur, but in such small quantity
that he considered it as a contamination. Cramer has prepared by an
^Arch. f. (Anat. u.) Physiol., 1890.
■ Baldi, Arch. f. (Anat. u.) Physiol., 1887, Suppl.; Moriya, Zeitschr. f. physiol.
Cheni., 43.
^ Brieger, Ueber Ptomaine, Berlin, 1885 and 1886.
482 BRAIN AND NERVES.
essentially different method a protagon which contained sulphur but had
in other respects the same composition as that analyzed by Gamgek
and Blankexhorx. Posxer and Gies obtained, in a very extensive
investigation, fractions which had variable compositions. These last
investigators, as well as Thudichum, Lesem, Worxer and Thierfelder,
and KocH,i are therefore of the opinion that protagon does not exist as a
chemical indi\adual, but is a mixture, essentially of cerebrins, lecithin,
and cephalin. The somewhat variable elementary composition also indi-
cates the fact that the protagon as ordinaril}' obtained is not a homogene-
ous substance. On the contrarj^, the assumption that protagon is only a
mixture of cerebrins and lecithin-like bodies is, according to Hammarstex,
very improbable. That a mixture of amorphous or very difficultly crys-
taUizable substances produces so readily such a beautifully crystalline
substance as protagon, which can be recrj'stallized as often as one wishes,
contradicts the ordmary chemical experience. What seems more probable
is that the so-called protagon is a crj^stalline substance which can be purified
from other substances, perhaps its own decomposition products, with the
ver}' greatest difficult3\
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 bar\'ta-water protagon yields the decomposition products of lecithin,
namely, fatty acids, glycerophosphoric acid, and choline. Kossel and
Freytag found indeed three cerebrosides, namely, cerebrix, kerasin
(liomocerebrin), and excephalix.
On boiling with dilute mineral acids protagon yields among other sub-
stances a reducing carbohydrate. On oxidation with nitric acid protagon
yields higher fatty acids.
Protagon appears, wiien drj^, 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 balls or groups of fine crystalline
needles. It decomposes on heating even below 100° C. It is hardly
soluble in cold alcohol or ether, but dissolves, at least when freshly precipi-
tated, in ether on warming. It swells in little water and partly decomposes.
With more w^ater it swells to a gelatinous or pasty mass, which with much
water yields an opalescent Hquid. On fusing with saltpetre and soda,
alkali phosphates are obtained.
" Gamgee and Blankenhorn, Zcitschr. f. physiol. Chem., 3; Baumstark, 1. c;
Ruppel, Zeitschr. f. Biologic, 31; Liebreich, Annal. d. Chem. u. Pharm., 134; Kossel
and Freytag, Zeitschr. f. physiol. Chem., 17; Womer and Thierfelder, ibid., 30; Lesem
and Gies, Amer. Journ. of Thysiol., 8; Thudichum, 1. c; Cramer, Joum. of Physiol.,
31; Posner and Gies, Journ. of Biolog. Chem., 1.
CEREBRIN. 483
Protagon is prepared in the following way: An ox-brain as fresh as
possible, with the blood and membranes carefully removed, is ground fine
and then extracted for several hours -^dth alcohol of 85 vols, per cent at
45° C, filtered at the same temperature, and the residue extracted with
warm alcohol until the filtrate does not yield a precipitate at 0° C. The
several alcoholic extracts are cooled to 0° C. and the precipitates united and
completely extracted with cold ether, wiiich dissolves the cholesterin and
lecithin-like bodies. The residue is now strongly pressed between filter-
paper and allowed to dr}- over sulphuric acid or phosphoric anliydride. It
is now pulverized, digested with alcohol at 45° C, filtered, and slowiy
cooled to 0° C. The crystals which separate may be purified when necessary
by recr\-stallization.
The same steps are taken when one wishes to detect the presence of pro-
tagon.
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 Thudichu:m has embraced under the name cerehrosides. The cere-
brosides are nitrogenous substances free from phosphorus, which yield a
reducing variety of sugar (galactose) on boiling with dilute mineral acids.
On fusing with potash or by oxidation with nitric acid they jield higher
fatty acids — palmitic or stearic acids. The cerebrosides isolated from the
brain are cerebrin, kerasin, encephalin, and cerebron, but it must be re-
marked that there is no doubt but 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.
Cerebrin. Under this name W. Muller i first described a nitrogenous
substance, free from phosphorus, wiiich he obtained by extracting with
boiling alcohol a brain-mass which had been pre\iously boiled with bar}i:a-
water. Following a method essentially the same, but differing somewhat,
Geoghegax 2 prepared from the brain a cerebrin with the same properties
as Muller's, but containing less nitrogen. According to Parous ^ the
cerebrin isolated by Geoghegan, as well as by Muller, consists of a mix-
ture of three bodies, "cerebrin," "homocerebrin," and "encephalin." Kos-
sel and Freytag isolated two cerebrosides from protagon which were
identical with the cerebrin and homocerebrin of Parous. According to
these investigators, the two bodies phrenosin and kerasin, as described
b}" Thudichu:m, seem to be identical with cerebrin and homocerebrin.
Cerebrin, according to Parous, 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.
' Annal. d. Chem. u. Pharm., 105.
^ Zeitschr. f. physiol. Chem., 3.
^ Parcus, Ueber einige neue GehimstoSe, Inaug.-Diss. Leipzig, 1881.
484 BRAIN AND NERVES.
In the dry state it forms a pure white, odorless, and tasteless powder. On
heating it melts, decomposes gradually, smells like burnt fat, and bums
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 balls or grains on microscopical
examination. Cerebrin forms a compomid 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, according to Thierfelder,i galactose.
Kerasln (according to Thudichum), or homocerebrin (according to
Parous), has the following composition: C 70.06, H 11.60, N 2.23, and
O 16.11 per cent. Encephalin has the composition C 68.40, H 11.60,
N 3.09, and 0 16.91 per cent. Both bodies remain in the mother-liquor
after the impure cerebrin has precipitated from the warm alcohol. These
bodies have the tendency of separating as gelatinous masses. Kerasin is
similar to cerebrin, but dissolves more easily in warm alcohol and also in
warm ether. It may be obtained as extremely fine needles. Encephalin
is, according to Parous, a transformation product of cerebrin. In the
perfectly pure state it crystallizes in small lamellae. It swells into a pasty
mass in warm water. Like cerebrin and kerasin, it yields a reducing sub-
stance (probably galactose) on boiling with dilute acid.
The cerebrins are generally prepared according to MtJLLER's method.
The brain is first stirred with baryta-water until it appears like thin milk,
and then it is boiled. The insoluble parts are removed, pressed, and
repeatedly boiled with alcohol, which is filtered while boiling hot. The
impure cerebrin which separates on cooling is freed from cholesterin and
fat by means of ether and then purified by repeated solution in warm
alcohol. According to Parous this repeated solution in alcohol is con-
tinued until no gelatinous separation of homocerebrin or encephalin takes
place.
According to Geoghegan's method the brain is first extracted with cold
alcohol and ether and then boiled with alcohol. The precipitate which
separates on the cooling of the alcoholic filtrate is treated with ether and
then boiled with baryta-water. The insoluble residue is purified by re-
peated solution in boiling alcohol.
The cerebrin may also be obtained from other organs by employing the
above methods. The quantitative estimation, when such is desired, may
be performed in the same way.
KossEL and Freytag prepare cerebrin from protagon by saponifying it
in methyl alcohol solution with a hot solution of caustic baryta in
methyl alcohol. The precipitate is filtered off and decomposed in water by
carbon dioxide and the cerebrin or cerebroside extracted from the insoluble
residue with hot alcohol.
' Zeitschr. f. physiol. Chem., 14.
CEREBRON AND CEPHALIN. 485
Whether the above-described cerebrins are chemical individuals or
mixtures, i.e., impure sub tances, is still undecided. The purest cerebrin
or cerebroside thus far investigated is undoubtedly Thierfelder's cerebron,
and there is hardly any doubt also that Muller's cerebrin consisted essen-
tially of cerebron.
Cerebron. This cerebiin, isolated by Thierfelder and Worner and
then especially studied by Thierfelder, was first isolated by Gamgee and
called 'pseudocerehrin by him. Thudichum's ^ phrenosin seems to be im-
pure cerebron. Cerebron can be prepared directly from the brain without
saponification with barj-ta, 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 C48H93NO9; it melts at 212°, dissolves in warm alcohol, and
separates out on cooling. From proper solvents (acetone 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 need e- and leaf -shaped crj-stals
gradually form. Cerebron also yields galactose, and it can be split by
acids, best in methyl alcohol containing sulphuric acid, into galactose,
a base called sphingosin by Thudichum, and cerebronic acid (Thudichum's
neurostearic acid). The cleavage takes place, according to Thierfelder,
as follows: C48H93N09 + 2H20 = C25H5o03 (cerebronic acid)+Ci7H35N02
(sphingosin) + C6H12O6 (galactose). The cerebronic acid consists of snow-
white crs'stals which are soluble in alcohol and in ether. They melt at
99-100° and give a crystalline methyl ester which melts at 65°. Sphin-
gosin is a base which does not form marked crj'stals and which is insoluble
in water and ether, but gives a sulphate which is soluble in chloroform and
in warm alcohol. According to Thierfelder and Kitagaw^a, sphingosin
is not a unit substance.^
Cephalin is a phosphatide whose formula, based upon the investigations
of Thudichum and Koch^ is probably C42H82NPO13. The views of these
two investigators as to the constitution of this body, which is difficult to
purify, differ ver}' considerably. According to Thudichum, on cleavage it
yields neurine, glycerophosphoric acid, stearic acid, and a specific fatt}' acid,
cephalic acid. According to Koch it contains on the contrar}^, only orie
methyl group attached to nitrogen, and is therefore probably dioxj^stearvd-
monomethyl lecithin. 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. It is obtained
' Thierfelder and Worner, Zeitschr. f. physiol. Chem., 30; Thierfelder, ibid., 43, 44,
and 4G; Gamgee, Text-book of Physiol. Chem., London, ISSO; Thudichum, 1. c.
^Zeitschr. f. physiol. Chem., 4S.
' Thudichum, 1. c; Koch, Zeitschr. f. physiol. Chem., 30.
486 BRAIN AND NERVES.
from the brain after dehydration with acetone by extractin'j; with ether and
precipitating the concentrated ethereal extract with alcohol. The cephalin
is perhaps identical with the myeline substance isolated by Zuelzer ^ from
the brain.
Bethe ^ has prepared the following decomposition products from the brain
of the horse after treatment with CuCL' and alkali : aminocerebrinic-acid glucoside,
Ci^HgiOsN, 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, CHi^No, is a non-poisonous diamine discovered by Brieger, and
which was obtained by him in the putrefaction of meat and gelatine, 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 consist, perhaps, of the same
substance as certain prostatic calculi, but they have not been closely investigated.
Quantitative Composition of the Brain. The quantity of water is greater
in the gray than in the white substance, and greater in new-born or young
individuals than in adults! The brain of the foetus contains 879-926 p. m.
water. According to the observations of Weisbach 3 the quantity of
water in the several parts of the brain (and in the medulla) varies at differ-
ent 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.
'"X "~^ '"X fiT" 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. U 838.4 847.8 839.5
Gvri 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 have also been made of the ox-brain by
Petrowsky,^ and of the brain of a horse by Baumstark. In the analysis
of Petrowsky the protagon has not been considered, and all organic phos-
phorized substances were calculated as lecithin. On these groimds these
> W. Koch, Zeitschr. f. physiol. Chem., 36; Zuelzer, ibid., 27.
2 Arch. f. exp. Path. u. Pharm., 48.
3 Cited from K. B. Hofmann's Lehrb. d. Zoochemie (Wien, 1876), 121.
* Pfluger's Arch., 7.
COMPOSITION OF THE BRAIN. 487
analyses are not of much value from a certain standpoint. In Baumstaek'8
analyses the gray and the white substance could not be sufficiently sepa-
rated, and these analyses, on this account, show partly an excess of white
and partly an excess of gray substance; nearly one-half of the organic
bodies, chiefly consisting of bodies soluble in ether, could not be exactly
analyzed. Neither of these analyses gives sufficient explanation of the
quantitative composition of the brain.
The analyses made up to the present time give, as above stated, an
unequal division of the organic constituents in ihe gray and white sub-
stance. In the analyses of Petrowsky the quantity of proteins and gela-
tine-forming substances in the gray matter was somewhat more than one-
half, and in the white about one-quarter of the solid organic substances.
The quantity of cholesterin in the white was about one-half, and in the
gray substance about one-fifth of the solid bodies. A greater quantity of
soluble salts and extractive bodies was found in the gray subtance than
in the white (Baumstark). The following analyses of Baumstark give
the most important known constituents of the brain calculated in 1000
parts of the fresh, moist substance. A represents chiefly the white, and
B chiefly the gray substance.
A. B.
Water 695.35 769.97
Solids 304.65 230.03
Protagon 25.11 10.80
Insoluble protein and connective tissue 50 .02 60 .79
Cholesterin, free 18.19 6.30
" combined 26.96 17.51
Nuclein 2.94 1.99
Neurokeratin 18 .93 10 .43
Mineral bodies 5.23 5.62
The remainder of the solids probably consists chiefly of lecithin and
other phosphorized bodies. Of the total amount of phosphorus 15-20
p. m. belongs to the nuclein, 50-60 p. m. to the protagon, 150-160 p. m.
to the ash, and 770 p. m. to the lecithin and the other phosphorized organic
substances.
As shown by the above analysis Bauivistark differentiated between free
and combined cholesterin. He believed that a part of the cholesterin in
the brain occurred in the combined state, perhaps as an ester; this view
has been found to be incorrect by the recent investigations of BtJNZ. He
obtained from the brain neither esters of cholesterin with higher fatty
acids nor other compounds of cholesterin which split on saponification.
Tebb 1 has also found only free cholesterin.
The analysis of the brain of an epileptic made by Koch ^ is of very
great interest. We cannot enter into a discussion of his method of analysis.
» B:;nz, Zeitschr. f. physiol. Chem., 46; Tebb, Joum. of Physiol., 34.
^ Amer. Journ. of Physiol., 11; Koch and Woods, Joum. of Diol. Chem., 1.
488 BRAIN AND NERVES.
In order to make the figures found comprehensible, it is perhaps necessary
to call attention to a few points. The two cerebrins, phrenosin and kerasin,
were calculated from the quantity of galactose split off. The quantity of
phosphatides, designated by Koch as lecithans, was determined from the
quantity of methyl groups split off by hydriodic acid below 240° plus the
quantity of true lecithin calculated from the quantity of methyl groups
split off at about 300°. The difference between the quantity of lecithin
and the total quantity of lecithans gave the amount of cephalin and myelin.
The nature of the sulphurized substance is unknown. As the protagon,
according to Koch, is a mixture of various substances, no results as to
the quantity is given. The other figures require no explanation.
Corpus Cort€X
Callasuin (prefrontal).
Water 67.97 84.13
Protein 3.20 5.00
Nucleoproteids 3.70 3.00
Neurokeratin 2.70 (Chittenden) 0 .40 (Chittenden)
Extractives (water-soluble). 1.51 1.58
Lecithins 5.19 3.14
Cephalin and myelin 3.49 0.74
Phrenosin and kerasin 4 . 57 1 . 55
Cholesterin 4.86 0.70
Sulphurized substance 1 .40 1 .45
Mineral bodies 0.82 0.87
As the cerebrosides occur chiefiy in the myelin sheath, Koch, starting
from the amount in the investigated part of the brain, attempts to calculate
the amount of the anah'zed 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 100 parts of the dry substance.
Corpus Gray Substance
Callosum. Al'^^ f[°™ ,
white substance).
Protein 10.00 21.70
Nucleoproteids 11.56 9.66
Neurokeratin 8 . 40 —
Extractives 4.75 5.92
Lecithins 16.22 7.67
Cephalin and myelin 10 . 91 —
Phrenosin and kerasin 14 . 29 —
Cholesterin 15 . 20 —
Sulphurized substance 4 . 37 5 . 43
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 protagon. Mott and Halliburton ^
have also shown that in degenerative diseases of the nervous system the
quantity of substances containing phosphorus diminishes and that in these
'Noll, Zeitschr. f. phy.siol. Chem., 27; Mott and Halliburton, Philos. Transact.,
Ser. B, 191 (1899) and 194 (1901).
TISSUES AND FLUIDS OF THE EYE. 489
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.
The quantity of neurokeratin in the nerves and in the different parts of
the brain has been carefiUly determined by Kuhxe and Chittendex.i
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.
m 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. According to Griffiths,^
neurochitin replaces neurokeratin in insects and Crustacea, the quantity of
the first being 10.6-12 p. m.
The quantity of mineral constituents in the brain amounts to 2.95-7.08
p. m. according to Geoghegax. He fomid in 1000 parts of the fresh,
moist brain 0.43-1.32 CI; 0.956-2.016 PO4; 0.244-0.796 CO3; 0.102-
0.220 SO4; 0.01-0.098 Feo(P04)2; 0.005-0.022 Ca; 0.016-0.072 Mg; 0.58-
1.778 K; 0.450-1.114 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 Cahx 3). The mineral bodies
consist of 422 p. m. Na2HP04 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 ^ observed in 1876 that the layer of rods in the
retina during life had a purplish-red color which was bleached by the action
of light. KtJHNE ^ showed later that this red color might remain for a long
time after the death of the animal if the eye was protected from daylight
or investigated by a sodium light. Under these conditions it was also
possible to isolate and closely study this substance.
' Zeitschr. f. Biologie, 26.
' Compt. rend.. 115.
^Zeitschr. f. physiol. Chem., 5.
* Monatsschr. d. Kgl. Preuss. Akad., 12. Nov., 1876.
^ The investigations of Klihne 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 . Biologie, 32.
490 BRAIN AND NERVES.
Visual red (Boll) or visual purple (Kuhxe) has become known mainly
by the investigations of Kijhne. The pigment occurs chiefly 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 new-
born rabbits, no visual purple has been found in the rods.
A solution of visual purple in water which contains 2-5 per cent crys-
tallized bile, 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 corresponding to the in-
tensity of the light. It passes from red and orange to yellow. Red light
bleaches the visual purple slowly; the ultra-red light does not bleach it at
all. A solution of visual purple shows no special absorption-bands, but
only a general absorption which extends from the red side, beginning at
D and extending to the G line. The strongest absorption is found at E.
KoETTGEN and Abelsdorf ^ have shown that there are, in accordance with
Kijhne' s views, two varieties of visual purjDle, 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. KtJHNE has also found that the retina of the
eye of the frog becomes bleached when exposed for a long time to strong
sunUght, 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-epithelium 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 Kijhne, to be possible in the retina which has been completely
removed. On account of this property of the visual purple of being bleached
' Centralbl. f. Physiol., 9; also Maly's Jahresber., 25, 351.
VISUAL PURPLE AND VITREOUS HUMOR. 491
by light during life we may, as Kuhxe has shown, under special conditions
and by obser\-ing special precautions, obtain after death, by the action of
intense light or more continuous Ught, 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 hsemoglobm the solution of \'isual purple in cholates is
precipitated by saturating with magnesium sulphate, washing the precipi-
tate ^^ith a saturated solution of magnesium sulphate, and then dissohdng
in water by the aid of the cholates simultaneously precipitated.^
The Pigments of the Cones. In the inner segments of the cones of birds, rep-
tiles, and fishes a small fat-globule of var\ang color is found. Kijhne ■ 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 Kxjhxe and Mays,^
contains iron, dissolves in concentrated caustic alkalies or concentrated sulphuric
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. Mokxer, of a gelatme-
formmg substance. The fluid contains a httle proteid and a mucoid,
hyalomucoid, which was first shown by Morxer, 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 httle urea — according to Pic.ajid 5 p. m., according
to Rahl^l\xx 0.64 p. m. Pautz "* found besides some urea paralactic
acid, and, in confirmation of the statements of Chabbas, Jesxer, and Kuhx,
also glucose in the vitreous humor of oxen. The reaction of the vitreous
humor is alkaline, and the quantity of sohds amoimts 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 264.
' Kiihne, Zeitschr. f. Biologie, 32.
^ Kiihne, Die nichtbestandigen Farben der Netzhaut, Untersuch. aus dem physiol.
Institut Heidelberg, 1 , 341.
ns^uhne, ibil., 2, 324.
*M6mer, Zeitschr. f. physiol. Chem., IS; Picard, cited from Gamgee, Physiol.
Chem., 1, 454; Rahlmann, Maly's Jahrcsber., G; Pautz, Zeitschr. f. Biologie, 31. A
complete review of the literature wQl also be found here.
492 BRAIN AND NERVES.
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 memhranins. 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 hydrochloric 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 of the lens contains
14.10 per cent N and 0.83 per cent S, and is a little less soluble than that
from Descemet's membrane.
The 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 Morner has
given the name albumoid. It dissolves readily in very dilute acids or
alkalies. Its solution in caustic potash of 0.1 per cent is very similar to an
alkali-albuminate solution, but coagulates at about 50° C. on nearly com-
plete neutralization and the addition of 8 per cent NaCl. Albumoid has
the following composition: C 53.12, H 6.8, N 16.62, and S 0.79 per cent.
The lens-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 albumoid in the entire lens amounts on an average
to about 48 per cent of the total weight of the proteins of the lens.
The Soluble Protein consists, exclusive of a very small quantity of albu-
min, of two globulins, a- and (i-crystallin. These two globuUns differ from
each other in this manner: a-crystallin contains 16.68 per cent N and 0.56
per cent S; /?-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 diffi-
culty and less completely by acetic acid or carbon dioxide. These globu-
lins are not precipitated by an excess of NaCl at either the ordinary tem-
perature or 30° C. Magnesium or sodium sulphate in substance precipi-
tates 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 inwards; /?-crystallin, on the contrary,
from within outwards.
Zeitschr. f. physiol. Chem., 18. This contains also the pertinent literature.
CRYSTALLINE LENS. 493
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 the crystalbumin, with a specific rotatory power of («)/=— 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)y=— 80.2°, which he called crystalfibrin.
The lens does not seem to contain any protein bodies which coagulate
spontaneously like fibrinogen. That cloudiness which appears after death
depends, according to KIjhne, upon the unequal changing of the concen-
tration of the contents of the lens-tubes. This change is produced 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 cloudiness in diabetes has
been attributed by some to the removal of water. The views on this sub-
ject are, however, contradictory.
The average results of four analyses made by Laptschinsky ^ 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 2.3
In cataract the amount of proteins is diminished and the amount of
cholesterin increased.
The quantity of the different proteins in the fresh moist lens of oxen is
as follows, according to Morner^:
Albumoid (lens-fibres) 170 p. m.
/?-Crystallin 110 "
(v-Crystallin 68 "
Albumin 2 "
The corneal tissue has been previously considered (page 434). The
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 ^ they contain 982 p. m. water, 18 p. m. solids
with 5 p. m. albumin and 13 p. m. NaCl.
' Pfliiger's Arch., 13.
M. c.
' Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 401.
494 BRAIN AND NERVES.
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 i;: very
similar to mucin.
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 albu-
minate (?), and an albuminous body related to Rovida's hyaline substance;
also leucine, tyrosine, creatine, xanthine bodies, cholcsterin, lecithin, inosite,
and fat. In regard to the occurrence of glycogen the statements are some-
what contradictor}-. Dareste ^ found in the testes of birds starch-like
granules, which were colored blue with difficulty by iodine.
In the autolysis of the testes Levene ^ found tvTosine, alanine, leucine,
aminovalerianic acid, aminobutj-ric 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
protei -s, has a neutral or faintly alkaline reaction and a peculiar specific
odor. Soon after ejection semen becomes gelatinous, as if it were coagu-
lated, but afterwards becomes more fluid. "VMien diluted with water white
flakes or shreds separate (Henle's fibrin). According to the anah'ses of
Slow-tzoff,3 human semen contains on an average 96. S p. m. solids with
9 p. m. inorganic and 87.8 p. m. organic substance. The amount of pro-
tein substances was, on an average, 22.6 p. m. and 1.69 p. m. of bodies solu-
ble in ether. The protein substances consist of nucleoproteids, traces of
mucin, albumin, and a substance similar to proteose (found earlier by
Posner). According to Cavazzaxi ^ 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.
' Compt. rend., 7-t.
* Amer. Journ. of Physiol., 11.
' Zeiti?chr. f. physiol. Chem., 35.
* Po.sner, Berl. klin. Wochenschr., 1888, No. 21, and Centralbl. f. d. med. Wissensch.,
1890; Cavazzani, Biochem. Centralbl., 1, o02, and Centralbl. f. Physiol., 19.
495
496 ORGANS OF GENERATION.
The semen in the vas deferens differs chiefly from the ejected semen in
that it is without the pecuUar 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 nucleopro-
teids, besides a substance similar to fibrinogen and to mucin (Stern ^), and
mineral bodies, especially NaCl. Besides this it contains an enzyme vesic-
ulase (see below), lecithin, choline (Stern), and a crystalline combination
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 iden-
tical with the Charcot-Leyden crystals found in the blood and in the
lymphatic glands in leucaemia (Th. Cohn, B. Lewy 2). They are, according
to Schreiner,^ as above stated, a combination of phosphoric acid with a
base, spermine, C2H5N, which he discovered.
Spermine. The views 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 ethyleneimine ; 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 chlo-
ride, platinic chloride, potassium-bismuth iodide, and phosphotungstic acid.
Spermine has a tonic action, and according to Poehl * 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.
According to Bocarius,^ this reaction is due to choline.
Camus and Gley^ have found that the prostate fluid in certain rodents
has the property of coagulating the contents of the seminal vesicles. This prop-
erty is due to a special ferment substance {vesiculase) of the prostate fluid.
'Iversen, Nord. med. Ark., C; also Maly's Jahresber., 4, 358; Stern, Biochem.
Ccntralbl., 1,748.
2 Th. Cohn, Centralbl. f. allg. Path. u. path. Anat., 10 (1899); B. Lewy, Centralbl.
f. d. med. Wis.sen.sch., 1899, 479.
3 Annal. d. Chem. u. Pharm., 194.
* Ladenburg and Abel, Ber. d. deutsch. chem. Ge.sellsch., 21; Majert and A. Schmidt,
ibid., 24; Poehl, Compt. rend., 115, Berlin, kliri. Wochenschr., 1891 and 1893, Deutsch.
med. Wochenschr., 1892 and 1895, and Zeitschr. f. klin. Med., 1894.
^ In regard to Florence's sperm reaction, see Posner, Berl. klin. Wochenschr.
1897, and Richtcr, Wien. klin. Wochenschr., 1897; Bocarius, Zeitschr. f. physiol.
Chem., 34.
•Compt. rend, de see. biolog., 48, 49.
SPERMATOZOA. 497
The spermatozoa show a great resistance to chemical reagents in general.
They do not dissolve completely in concentrated sulphuric acid, nitric acid,
acetic acid, nor in boiling-hot soda solutions. They are soluble in a boiling-
hot caustic-potash solution. They resist putrefaction, and after drj-ing 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 (|) of potassium phosphate.
The spermatozoa show well-knoviii movements, but the cause of this is
not known. These movements may continue for a very long time, as under
some conditions they may be observed for several days in the body after
death, and in the secretion of the uterus longer than a week. Acid Hquids
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.
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-meduUated nerv^es or the axis-cylinders. In the
various kinds of animals investigated, the head contains nucleic acid, which
in fishes is partly combined ^vith protamines and partly wdth histones. In
other animals, such as the bull and boar, protein-like substances occur with
the nucleic acid, but no protamine.
Our knowledge of the chemical composition of spermatozoa has been
greatly enhanced by the important investigations of Miescher i 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 '^ith alcohol-ether contain on an average 960 p. m. protamine
nucleate, which nevertheless is not uniform, but is so di\dded that the outer
layers consist of basic protamine nucleate, while the inner layers, on the
contrary, consist of acid protamine nucleate. Besides the protamine
nucleate there are present in the heads, although to a very slight extent
unkno^\Tl organic substances. The unrij^e salmon spermatozoa, while
'See Miescher, "Die histochemischen und physiologischen Arbeiten von Friedrich
Miescher, gesammclt und herausgegeben von seinen Freunden," Leipzig, 1897.
498 ORGANS OF GENERATION.
developing, also contain nucleic acid, but no protamine, with a protein
substance, " alhuminose," which probably is a step in the formation 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.
Spennatin is a name which has been given to a constituent similar to alkali
albuminate, but it has not been closely studied.
Prostatic concrements are of two kinds. One is very small, generally oval in
shape, with concentric layers. In young but not in older persons they are colored
blue by iodine (Iversen ')• 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 liciuid. The corpora lutea are colored yellow by an
amorphous pigment called lutein. Besides this another coloring-matter
sometimes occurs which is not soluble in alkali; it is crystalUne, but not
identical with bilirubin or ha?matoidin; but it may be identified as a lutein
by its spectroscopic behavior (Piccolo and Lieben, KiJHNE and Ewald ■*).
The cysts often occurring in the ovaries are of special pathological
interest, and these may have essentially different contents, depending
upon their variety and origin.
The serous cysts (Hydrops folliculorum Graafii), which are formed
by a dilation of the Graafian follicles, contain a serous liquid which has a
specific gravity of 1.(305-1.022. A specific gravity of 1.020 is less frequent.
Generally the specific gravity is lower, 1.005-1.014, with 10-40 p. m. solids.
As far as is known, the contents of these cysts do not essentially differ from
other serous liquids.
The proliferous cysts (myxoid cysts, colloid cysts), which are devel-
oped 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 somewhat
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
» Zeitschr. f. physiol. Cheni., 23. ' Nord. med. Ark., 6. ' See Chapter VI, p. 216.
COLLOID. 499
into long threads, and as this mass hi the different cysts is more or less
diluted mth serous liquids their contents may have a variable consistency.
In still other cases the small cysts may also contain a thin, watery fluid.
The cobr of the contents is also variable. Sometimes they are bluish
white, opalescent, and again they are yellow, yellowish browii, or yellowish
with a shade of green. They are often colored more or less chocolate-
brown or red-brown, due to the decomposed 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, epitheliuin-cells, cholesterin crystals, and
colloid corpuscles — large, circular, highly refractive formations.
Though the contents of the proliferous cyst may have a variable compo-
sition, still it may be characterized in typical cases by its slimy or ropy
consistency; by its grayish-yellow, chocolate-browTi, 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, metalhumin, and paralbumin as characteristic con-
stituents of these cysts.
Colloid. This name does not designate any particular chemical sub-
stance, but is given to the contents of tumors with certain physical proper-
ties similar to gelatine 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 Pfannen-
STiEL 1 such a colloid is designated /?-pseudomucin. Sometimes a colloid is
found which, when treated with a verj' dilute alkali, gives a solution similar
to a mucin solution. Colloid is very closely related to mucin and is con-
sidered 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 w^as C 47.27, H 5.86, N
8.40, S 0.79, P 0.54, and ash 6.43 per cent. A colloid found by Wurtz 2
in the lungs contained C 48.09, H 7.47, N 7.00, and 0( + S) 37.44 per cent.
Colloids of different origin seem to be of varsing composition.
»Aich. f. Gynak.,38.
'Panzer, Zeitschr. f. physiol. Chem., 28; Wurtz, see Lebert, Beitr. zur Kenntnis
des Gallertkrebses, Virchow's Arch., 4.
500 ORGANS OF GENERATION.
Metalhumin. This name Scherer ^ gave to a protein substance found
by him in an ovarial fluid. The metalbumin was considered by Scherer
to be an albuminous body, but it belongs to the mucin group, and it is for
this reason called pseudomucin by Hammarsten.^
Pseudomucin. This body, which, like the mucms, gives a reducing
substance when boiled with acids, is a mucoid of the following composition:
C 49.75, H 6.98, N 10.28, S 1.25, 0 31.74 per cent (.Hammarsten). 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 prop-
erty. 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 ^ 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 O 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 paramudii.
The pseudomucin as well as colloid are mucoid substances, and the
carbohydrate obtained from them is glucosamine (chitosamine), as espe-
cially shown by Fr. Muller, Neuberg and Heymann."* From pseudo-
mucin Zangerle ^ obtained 30 per cent glucosamine, and Neuberg and
Heymann have shown that the glucosamine is the only carbohydrate
regularly taking part in the structure of these substances. Still there are
also statements 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.
» Verb. d. physik.-med. Gesellsch. in Wiirzburg, 2, and Sitzungsber. der physik.-
med. Gesellsch. in Wiirzburg fiir 1864-1865; Wiirzburg med. Zeitschr., 7, No. 6.
^ Zeitsohr. f. physiol. Chem., G.
3K. Mitjukoff, Arch. f. Gyniikol., 49.
"Muller, Verh. d. Naturf. Gesellsch. in Basel, 12, part 2; Neuberg and Heymann,
Hofmeister's Beitrage, 2. See also Leathes, Arch. f. cxp. Path. u. Pharm., 43.
*Miinch. med, Wochenschr., 1900.
PSEUDOMUCIN. 501
As hydrolytic cleavage products of pseud omucin Otori ^ has obtained,
besides carbohydrate derivatives such as levulinic acid and humus sub-
stances, leucine, tyrosine, glycocoll, aspartic acid, glutamic acid, valerianic
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.
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 ven,^ small amounts of pseudomucin. The procedure
is as follows: The protein is removed by heating to boiling with the addition
of acetic acid; the filtrate is strongly concentrated and precipitated by
alcohol. The precipitate, a transformation product of pseudomucin,
is carefully washed wdth 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 pres-
ent, 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-men-
tioned case, first add acetic acid to the solution 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 HCl and warmed on the
water-bath until the liquid is deep browii 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
serglohulin 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
solids, w^hich constitute the chief mass, are protein bodies and pseudomucin.
The intraligamentary, papillary cysts contain a yellow, yellowish-
green, or bro^vnish-green liquid which contains either no pseudomucin or
ver}^ 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 con-
taining 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 opal-
escent 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 constit-
uent; protein is sometimes absent, and when it does occur the quantity is
' Zeitschr. f. physiol. Chem., 42 and 43.
502 ORGANS OF GENERATION.
very small. The principal part of the solids consists of salts and extrac-
tive 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.'^
E. LuDwiG and R. v. Zeynek ' have recently 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.
The colloid from a uterine fibroma analyzed by Stollmann ^ 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 constituents 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 centre of the yolk {latehra), and form-
ing a layer between the yolk and yolk-membrane, there occur 'protein,
nuclein, lecithin, and potassium (Liebermann ■*). The occurrence of gly-
cogen is doubtful. The yolk-membrane consists of an albuminoid 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'"'), purine bases (Mesernitzki ^),
glucose in very small quantities, and mineral bodies. The occurrence of
cerebrin and of granules similar to starch (Dareste '^) has not been posi-
tively proved.
Several enzymes have teen found in the yolk, especially a diastatic
enzyme (Muller and Masuyama), a glycolytic enzyme (Stepanek) which
in the absence of air brings about an alcohoUc fermentation of sugar and
* Kemiske Studier over Ovariecystevsedsker, etc., Koebenhavn, 1884. See also
Maly's Jahresber., 14, 459.
2 Zeitschr. f. physiol. Chem., 23.
' American Gynecology, March, 1903.
♦Pfliiger's Arch., 43.
^Ueber Ptomaine, Berlin, 1885.
^Mesernitzki, Biochem. Centralbl., 1, 739.
'Compt. rend., 72.
OVOVITELLIN. 503
in the presence of air forms Ccarbon dioxide and lactic acid, and finally a
proteolytic (Wohlgemuth), a lipolytic, and a chromolytic (?) enzyme. ^
Ovovitellin. This body, which is generally considered as a globulin, is
in reality a nucleoalbumin. The question as to what relationship other
protein substances which are related to ovovitelUn, 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 investi-
gation.
The ovo\dtellin 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 2). Ac-
cording to Osborne and Campbell, the so-called ovovitellin is a mixture
of various vitellin-lecithin combinations, with 15-30 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,
O 23.24 per cent. These figures differ somewhat from those obtained by
Gross 3 for vitellin prepared by another method (precipitation with
(NH4)2SO.i), namely, C 4SX)1, 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.
Gross found in vitellin a globulin coagulathig at 76-77° C. in a solution
containing hydrochloric acid.
On the pepsin digestion of ovovitellin, Osborne and Campbell obtained
a pseud on uclein with varying amomits of phosphorus, 2.52-4.19 per cent.
BuNGE ^ prepared a pseudonuclein by digesting ihe yolk witJi gastric juice,
and his pseudonuclein, according to him, is of great importance m the
formation of the blood, and on these grounds he called it hcvmatogen. 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 solution
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 precipi-
tated from its salt solution by diluting with water, and when allowed to
' MiiUer and Masuyama, Zeitschr. f. Biologie, 30; Stepanek, Centralbl. f. Physiol.,
18, 188; Wohlgemuth in Salkowski's Festschrift and Zeitschr. f. physiol. Chem., 44.
' Med. chem. Untersuch. 216.
' Osborne and Campbell, Connecticut Agric. Exp. Station, 23d Ann. Report, New
Haven. 1900; Gross, Zur Ke^mtn. d. OvoviteUins. Inaug.-Diss. Stra^ssburg, 1899.
^ Zeitpchr. f. physiol. Chem., 9, 49. See also Hugo_:nenq and Morel, Compt. rend.,
140 and 141.
504 ORGANS OF GENERATION.
stand some time in contact with water the vitellin is gradually changed,
forming a substance more like the albumir ates. The coagulation tempera-
ture 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 ^ has also split off glucosamine
from the yolk and has identified it as norisosaccharic 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 repeatedly redis-
solving in dilute common-salt solutions and precipitating with water.
Ichthulin, which occurs in the eggs of the carp and other fishes, is, according
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 used to be considered as a vitelHn. According to Walter
it yields a pseudonuclein on peptic digestion; and this pseudonuclein gives a
reducing carbohydrate on boiling with sulphuric acid. Ichthulin has the follow-
ing composition: C 53.42, H 7.63, N 15.63, O 22.19, S 0.41, P 0.43. It also con-
tains 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^ from perch eggs had a similar behavior and was very readily
changed by a little hydrochloric acid so that it was converted into a typical
pseudonuclein. The codfish ichthulin yielded a pseudonucleic acid with 10.34
per cent phosphorus, but this acid still gave the protein reactions.
The yolk also contains albumin, besides vitellin and the above-mentioned
globulin.
The fat of the yolk of the egg is, according to Liebermann, 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 diglycerides, or
upon a quantity of fatty acid deficient in carbon (Liebermann). In the
lecithin, or more correctly in the lecithin mixture of the yolk, Cousin finds
also linolic acid besides the three ordinary fatty acids. The composition
of yolk fat is dependent upon the food, as Henriques and Hansen ^ have
shown that the fat of the food passes into the egg.
' Ber. d. d. chem. Gesellsch., 34.
nValter, Zeitschr. f. physiol. Chem., 15; Levene, ibid., 32; Hammarsten, Skand.
Arch. f. Physiol., 17.
* Cousin, Compt. rend., 137; Henriques and Hansen, Skand. Arch. f. Physiol., 14.
LUTEIN. 505
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-matters, which
also occur in the vegetable kingdom (Thudichum), and whose relationship
to the vegetable pigments, the xanthophyll group, has recently been shown
by ScHUxcK,^ have been called luteins or lipochromes.
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. Tlie luteins ^^ithstand 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^ has found two pigments free from
iron in the eggs of a water-spider (Maja squinado) — one a red (vitelloriihin) and
the other a j-eUow pigment {vitellolutein) . 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 Poleck,^
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 fhid phosphoric acid and
lime the most abimdant, and then potash, which is somewhat greater in
quantity than the soda. These results are not, however, quite correct : first,
because no dissolved phosphate occurs in the yolk (Liebermaxx), and
secondly, in burning, phosphoric and sulphuric acids are produced, and these
drive away the chlorine, which is not accotmted 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 Paeke,'* to 471.9 p. m, and
528.1 p. m. respectively. Among the solids he found 156.3 p. m. protein,
3.53 p. m. soluble and 6.12 p. m. insoluble salts. The c^uantity of fat,
•Thudichum, Centialbl. f. d. med. Wissensch.. 1869; Schunck, see Chem. Cen-
tralbl., 1903, 2, 119.5.
* Monatshefte f. Chem., 2.
^ Cited from v. Gorup-Besanez, Lehrbuch d. physiol. Chem., 4. Aufl., 740.
^ Hoppe-Seyler, Med. chem. L'ntersuch., Heft 2, 209.
506 ORGANS OF GENERATION.
according to Parke, is 228.4 p. m.; the lecithin, calculated from the amount
of phosphorus in the organic substance of the alcohol -ether extract, was
107.2 p. m. and the cholesterin 17.5 p. m.
The white of the egg is a faintly yellow albuminous fluid inclosed in a
framework of thin membranes; and this fluid is in itself very liquid, but
seems viscous because of the presence of these fine membranes. That sub-
stance which forms the membranes, and of which the chalaza consists, seems
to be a body nearly related to horn substances (Liebermann).
The white of the egg has a specific gravity of 1.045 and always has an
alkaline reaction towards 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.^ 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^ called
" tatalbmyiin."
The protein substances of the whit-e of egg are all glucoproteids, as they
all yield glucosamine. According to the solution and precipitation prop-
erties they are similar to the globulins, albumins or proteoses. The repre-
sentatives of the first two groups, which until recently were considered
as tine proteins, are ovoglohulin and ovalbumin. The proteose-like body
is ovomucoid.
Ovoglobulin separates in part on diluting the egg-white vnih 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 sohition 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 cpite possible
that the globulin is a mixture. That portion which readily becomes
insoluble seems to be identical with Eichholz's glucoproteid or Osborne
and Campbell's ovomucin. Langstein obtained 11 per cent of glucos-
amine 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 determinations of Osborne and
Campbell. In regard to the probable occurrence of several globulins
in the white of the egg there are the statements of Corin and Berard as
well as of Langstein,^ but they have not led to any positive conclusions.
' Cited from v. Gorup-Besanez, Lehrbuch, 4. Aufl., 739.
' Pfluger's Arch., 31, 33, and 39.
'Langstein, Hofmeister's Beitrage, 1; Eichholz, Journ. of Physiol., 23; Osborne
and Campbell, Connecticut Agric. Kxp. Station, 23d Ann. Report, New Haven, 1900;
Dillner, Maly's Jahresber., 15; Corin and Berard, ibid., IS.
OVALBUMIN. 507
Ovalbumin. The so-called albumin of the egg-white is undoubtedh'^
a mixture of at least two albumin-like glucoprotcid-^. The views differ
considerably in regard to the number of these compound proteids (Bond-
ZYNSKi and Zoja, Gautier, Bechamp, Corin and Berard, Panormoff,
and others). Since Hofmeister has been able to prepare ovalbumin in a
crystaUine form, and since Hopkins and Pinkus ^ have shown that not
more than one-half of the ovalbumin can be obtained in such a form,
OsBORXE and Campbell have isolated two different ovalbumins or chief
fractions; the crystallizable the}" call ovalbumin and the non-cr}stallizable
conalhumin. 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 crj'stal-
lizable ovalbumin is also disputed. According to Boxdzynski and Zoja,
cr^'staUizable ovalbumin is a mixture of several albumins ha\'ing somewhat
different coagulation temperatures, solubiliti:^?, and specific rotations,
while Hofmeister and Laxgsteix on the contrary believe that cry^-
tallizable ovalbumin is a unit. The statements 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. According to the consistent
analyses of Osborxe and Campbell and of Laxgsteix, the conalbumin con-
tains 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. Laxg-
steix ^ obtained 10-11 per cent glucosamine 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 rotation is lower. It is made quickly insoluble
by alcohol and is precipitated by a sufficient quantity of HCl, but dissolves
in an excess of acid with greater difficulty than the seralbumin. The
products isolated by Abderhalden and Pregl^ on the hydrolysis of
ovalbumin do not show anyihing of special interest.
In preparing crj'stalline ovalbumin mix, according to Hofmeister, the
beaten white of egg free from foam with an equal volume of a saturated
ammonium-sulphate solution, filter off the globulin, and allow the filtrate
to 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
* Hofmeister, Zeitschr. f. physiol. Chem., 14, 16, and 24; Gabriel, ihid., 15; Bond-
zynski and Zoja, ibid., 19; Gautier, Bull. Soc. chim.,14; Bechamp, 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.
^Zeitschr. f. physiol. Chem., 31.
'/?)Z(i.,46.
508 ORGANS OF GENERATION.
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 crys-
tallize again on spontaneous evaporation. (See also page 507, foot-note 1,
for the Hopkins and Pixkus method.)
Conalbumin can be removed from the filtrate, after the complete crys-
tallization of the ovalbumin, by removing the sulphate by means of dialysis
and coagulating by heat.
Gautier ' 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 consid-
ered by him as a pseudopeptone and then later studied by Salkowski, is,
according to C. Th. Morner,^ a mucoid with 12.65 per cent nitrogen and
2.20 per cent sulphur. On boiling with dilute mineral acids it yields a
reducing substance. Ovomucoid exists in hens' 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 do\\Ti by alcohol, but sodium
chloride, sodium sulphate, and magnesium sulphate give no precipitates
either at the ordinaiy temperature or when the salts are added to saturation
at 30° C. Its solutions are not precipitated by an equal volume of a satu-
rated solution of ammonium sulphate, but are precipitated on adding more
salt thereto. The substance is not precipitated on boiling, but the part
which has become insoluble in cold water which has been dried is dissolved
by boiling water. Zaxetti 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.^
Ovomucoid may be prepared by removing all the proteins by boiling
with the addition of acetic acid and then concentrating the filtrato and
precipitating with alcohol. The substance is purified by repeated solution
in water and precipitation with alcohol.
According to Panormow ' 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.
' Compt. rend., 13.^.
2 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's Beitriige, 3 (literature).
3Zanetti, Chem. Centralbl., 1898, 1; Seemann, cited from Langstein, Ergebnisse
derPhy.siol.,1, Abt. 1,86.
* See Biochem. Centralbl. 5
SHELL MEMBRANE AND EGG SHELL. 509
The mineral bodies of the white of the egg have been analyzed by Poleck
and Weber.i They found in 1000 parts of the ash: 276.6-284.5 grams
potash, 235.6-329.3 soda, 17.4-29 Ume. 17-31.7 magnesia, 4.4-5.5 iron
oxide, 238.4-285.6 chlorine, 31.6-4S.3 phosphoric acid (P2O5), 13.2-26.3
sulphmic acid, 2.8-20.4 silicic acid, and 96.7-116 grams carbon dioxide.
Traces of fluorine have also been found (Nickles^). The ash of the white
of the egg contahis, as compared •v\ith 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 73), 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 verj^ small amounts of
magnesium carbonate and earthy phosphates.
The diverse coloring of birds' eggs is due to several different coloring -matters.
Among these we find a red or reddish-brown pigment called " oorodein " by Sorby,^
which is perhaps identical with haematoporphyrin. The green or blue coloring-
matter, Sorby's oocyan, seems, according to Liebermanx * and Krukenberg,^
to be partly hiliverdin and partly a blue derivative of the bile-pigments.
The eggs of birds have a space at their blunt end filled \\ith gas; this
gas contains on an average 18.0-19.9 per cent oxygen (Hufxer ^).
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 care-
fully 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 '').
The white of the egg of cartilaginous and bony fishes contains only traces of
true albumin, and the cover of the frog's egg consists, according to Giacosa, of
mucin. The eggs of the river-perch contain, according to Hammarsten,^ mucin
in the envelope in the unripe state and only mucinogen in the ripe state. The
crystalline formations (ijolk-sphcrules, or dotterpldttchen) which have been observed
in the egg of the tortoise, frog, ray, shark, and other fishes, and which are de-
scribed by Valexciexxes and Fremy^ under the names emydin, ichthin, ichthidin,
and ichthidin, seem, as above stated in connection with ichthulin, to consist chiefly
of phosphoglucoproteids. The eggs of the river-crab and the lobster contain the
same pigment as the shell of the animal. This pigment, called cyanocrystallinf
becomes red on boiling in water.
' Cited from Hoppe-Seyler, Physiol. Chem.. 778.
^ Compt. rend., 43.
^ Cited from Krukenberg, Verh. d. phys.-chem. Gesellsch. in Wiirzburg, 17.
* Ber. d. deutsch. chem. Gesellsch., 11.
»1. c.
•Arch. f. (Anat. u.) Physiol., 1892.
1 Zeitschr. f . Biologie, 43.
* Giacosa, Zeitschr. f. physiol. Chem., 7,- Hammarsten, Skand. An-h. f. Physiol., 17.
'Cited from Hoppe-Seyler' s Physiol. Chem., 77.
510 ORGANS OF GENERATION.
C. i\IoRNER * has isolated a substance which he calls j^crcaylobulin 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 glucoproteids, such as
ovomucoid and ovarial mucoids, and polysaccharides, such as glycogen, gum
tragacanth or quince-seed gum, and starch-paste, and of being precipitated by
them.
In fossil eggs (of aptenodytes, pelecanus, and hali^us) in old guano
deposits, a yellowdsh white, silky, laminated compound has been found which
is called guanovulit, (NHJlSOi +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 yomig animals. One finds, therefore,
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 lecithin in the yolk, which seems 'habitually to occur in the
developing cell. The occurrence of glycogen is doubtful, and the carbo-
hydrates are perhaps represented by a very small amomit of sugar and
glucoproteids. On the contrary, the egg contains a large proportion of fat,
which doubtless is important as a source of supply of nourishment and
in maintaining respiration for the embtyo. The cholesterin and the lutein
can hardly have a direct influence on the development of the embr}^o.
The egg also seems to contain the mineral bodies necessar}^ for the develop-
ment of the young animal. The lack of phosphoric acid is com]5ensated
by an abundant amount of phosphorized organic substance, and the
nucleoalbumin containing iron, from which the hsematogen (see page 5C3)
is formed, is doul^tless, as Bunge claims, of great importance in the forma-
tion of the haemoglobin containing iron. The silicic acid necessary for the
development of the feathers is also found in the egg.
During the period of incubation the egg loses weight, chiefly due to
loss of water. The quantity of solids, especially the fat and the proteins,
diminishes, and the egg gives off not onl}^ carbon dioxide, but also, as
LiEBERMANN ^ has shown, nitrogen or a nitrogenous substance. The loss
is compensated by the absorption of oxygen, and it is found that during
incubation a respiratory exchange of gases takes place.
As Bohr and Hasselbai.ch 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 foetus. Calculated upon 1 kilogram weight for one hour it is, from the
ninth day on, about tlie same as in the full-groAvn hen. Hasselbalch^
' Zeitschr. f. physiol. Chem., 40.
* Pfliiger's Arch., 43.
'Bohr and Plasselbalch, Maly's Jahresber., 29; Hasselbaleh, Skand. Arch. f.
Physiol., 13.
INCUBATION OF THE EGG. 511
has also sho^^-n that the fertilized hen's egg not only gives off nitrogen the
tirst five or six hours of incubation, but also some oxygen, and that we
are here dealing with an oxygen production which runs parallel with the
cell-di\asion. It is not kno'UTi whether this oxygen formation connected
with the life of the cell is a fermentative or a so-called vital process.
\Miile the quantity of drj' substance in the egg during this period always
decreases, the quantity of mineral bodies, protein, and fat always increases
in the embr^^o. The increase in the amount of fat in the embrj'O depends,
according to Liebermann, 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 remain michanged during incubation. The
yolk and white together contain the necessary quantity of lime for devel-
opment.
The most complete and careful chemical mvestigation on the develop-
ment of the embrv^o of the hen has been made by Liebermaxn. From his
researches we may quote the folloAiNing: In the earlier stages of the devebp-
ment, tissues verj- rich in water are formed, but upon the continuation of
the development the quantity of water decreases. The absolute quantity
of the bodies soluble in water increases with the devek)pment, while their
relative quantity, as compared "^ith 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 albuminoids soKible in water grows contin-
ually and regularly in such a way that their absolute quantity increases,
while their relative quantity remains nearly unchanged. Liebermann
found no gelatine in the embrj'O of the hen. The embrj^o does not contain
any gelatine-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 embrj'o when
about six days old, but then disappears. The quantity of haemoglobin
shows a continual increase compared with the weight of the body. Lieber-
mann found that the relationship of the haemoglobin to the body weight
was 1 :728 on the eleventh day and 1 :421 on the twenty-first day.
By means of Berthelot's thermometric methods Tangl has deter-
mined the chemical energy present at the beginning and end of the develop-
ment of the embrj'o 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 1 gram of ripe or nearly ripe hen's embr\o
(Plymouth egg) was equal to 658 calories. Tliis energ}' originated chiefly
from the fat. Of the total chemical energ}- utilized, two-thirds was used
for the construction of the embryo- and one-third transformed into other
forms of energy as work of development. Still more recent researches of
512 ORGANS OF GENERATION.
Bohr and Hasselbalch ^ show that none of the transformed chemical
energy is used in the construction of the embr}'o, as it leaves the egg almost
entirely as heat.
By their investigations on the development of the trout egg Tangl and
Farkas- have found that the loss in weight of each egg which had an aver-
age weight of 88 milligrams was 4.9 milligrams during the 42 days of
incubation, of which 4.11 milligrams was water and 0.722 milligram dry
substance with 0.367 milligram C. The eggs loose no nitrogen 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 develop-
ment was 6.68 gram-calories.
The tissue of the placenta has not thus far been the subject of detailed chemical
investigation. It contains a protein which coagulates at 60-65° (Bottazzi and
Delfino), also glycocoll and a proteolytic as well as a diastatic enzyme (Ascoli,
Raineri, Bergell, and Liepmann 0- In the edges of the placenta of bitches
and of cats a crystallizable orange-colored pigment (bilirubin?) has been found,
and also a green amorphous pigment, whose relationship to biliverdin is not
kno^ATi.''
From the cotyledons of the placenta in rimiinants a white or faintly rose-colored
creamy fluid, the uterine milk, can be obtained by pressure. It is alkaline in
reaction, but becomes acid quickly. Its specific gra\dty 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 ^\ath 9-10 p.m.
protein bodies and 6-7 p. m. ash.
The amniotic fluid in women is thin, whitish, or pale yellow; sometimes
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 ordinar}^ 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 GrIixbaum have also
found levulose. The human amniotic fluid also contains some urea, uric
'Tangl, Pfliiger's Arch., 93; Bohr and Hasselbalch, Skand. Arch. f. Physiol., 14.
2 Pfluger's Arch., 104.
^Bottazzi and Delfino, Centralbl. f. Physiol., 18, 114; Ascoli, ibid., Ifi; Raineri,
Biochem. Centralbl., 4, 428; Bergell and Liepmann, Munch, med. Wochenschr., 1905.
*See Etti, Maly's Jahresber., 2, 287, and Preyer, Die Blutkristalle, Jena, 1871.
AMNIOTIC FLUID. 513
a/yid, and allantoin. The quantity of these may be mcreased in hydramnion
(Prochowxick, IIarxack), 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 Prochowxick, 0.16 p. m. In the fluid in hydramnion
Prochowxick and Harxack 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 concentration of the amniotic fluid
is somewhat lower than that of the blood, which is no doubt due to a dilu-
tion by the foetal urine (Zaxgemeister and Meissl i).
»Weyl, Arch. f. (.\nat. u.) Physiol., 1S76; Bondi, Centralbl. f. Gynakol., 1903;
Prochownick. Arch. f. Gyniik., 11, also Maly's Jahresber., 7, 155; Harnack, Berlin,
klin. Wochenschr., 1888 No. 41; Zangemeister and Meissl, Munch, med. Wochenschr.,
'1903; Giirber and Grunbaum, Und., 1904.
CHAPTER XIV.
MILK.
The chemical constituents of the mammary glands have been little
studied. The cells are rich in protein and nucleo-proteids. Among the latter
we have one that yields pentose and guanine, but no other purine base, on
boiling with dilute mineral acids. This com'pound proteid, 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. Besides this proteid we have at least one
other, as Maxdel and Levexe and Loebisch^ have isolated a nucleic acid
from the mammary gland, which, like the thymonucleic acids, yielded ade-
nine, guanine, thymine, and cytosine. This nucleic acid also gave the pen-
tose reactions and yielded abundance of levulinic acid. Besides this nucleic
acid. Maxdel and Levexe ~ isolated from the glands aglucothionic acid ^^ith
2.65 per cent S and 4.38 per cent N. We cannot state what relation these
substances bear to that constituent of the gland foimd by Bert, which on
boiling with dilute mineral acids yielded a reducing substance. A similar
substance, wiiich acts perhaps as a step towards the formation of lactose,
has also been observ^ed 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 the 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 a:e found considerable amounts of purine bases. The mam-
mary glands contain also a proteolytic enzyme which, according to Hilde-
nuAXDT,3 occurs to a much greater extent in the active gland as compared
with the inactive one.
' Odenius, Maly'.s Jahre.sber., 30; Mandel and Levene, Zeitschr. f. physiol. Chem.^
46; Loebisch, HofmeLster's Beitrage, 8.
^ Zeitschr. f. physiol. Chem., 4.5.
^ Bert, Compt. rend., 98; Thierfelder, Pfliiger's Arch., 34, and Maly's Jahresber.
13; Hildebrandt, Hofmeister's Beitrage, 5.
514
COW'S MILK. 515
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 inves-
tigated, namely, cow's milk, and then of the essential properties of the
remaining important kinds of milk.^
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 towards
litmus. The extent of the acid and alkaline part of this amphoteric reac-
tion has been determined by different investigators, especially Thorxer,
Sebelien, and Courant.^ The results differ somewhat with the indicators
used, and moreover the milk from different animals, as well as that from
the same animal at different times during the lactation period, varies
somewhat. Courant has 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 c.c. milk
had the same alkaline reaction toward blue lacmoid as 41 c.c. N/10
caustic soda, and the same acid reaction toward phenolphthalein as
19.5 c.c. N/10 sulphuric acid. The actual reaction of cow's milk, which
follows from the electrometric estimation, is, on the contrary, according to
FoA,-*^ 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.
Perfectly fresh amphoteric milk does not coagulate on boiling, but forms
a pellicle consisting of coagulated casein and lime-salts, which rapidly re-
forms after being removed. 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
* 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.
^ Thorner, Maly's Jahresber., 22; Sebelien, ibuL; Courant, Pfliiger's Arch., 50.
'Compt. rend. Soc. biolog. (58), 59, 51.
516 MILK.
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 with-
out 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.
Li the spontaneous souring of milk we generally consider the formation of lactic
acid as the most essential product, but a formation of succinic acid may also take
place, and in certain bacterial decompositions of milk, succinic acid and no lactic
acid is formed. The materials from which these two acids are formed are lactose
and lactophosphocarnic acid. Besides the lactic acids, the optically inactive
as well as the dextro and levo acids, and succinic acid, volatile fatty acids, such
as acetic acid, butyric acid, and others, may be formed in the bacterial decompo-
sition of milk.
Milk sometimes undergoes a peculiar kind of coagulation, being converted
into a thick, ropy, slimy mass (thick milk). This conversion depends upon a
peculiar change in which the milk-sugar is made to undergo a slimy transforma-
tion. This transformation 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 saUcyUc 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,i L06-5.75 millions in 1 c.mm., and
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.
* On the Conditions Influencing the Number and Size of Fat-globules in Cow's Milk,
"Wisconsin Exp. Station, C, 1892.
MILK GLOBULES. 517
According to the obsen^ations of Ascherson/ drops of fat, wiien
dropped in an alkaline protein solution, are covered \\ith a fine albuminous
coat, a so-called haptogen-membrane. As milk on shaking ^^'ith 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 ver>' 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 memljrane
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 ver}' little acetic acid, or when it is coagulated by
rennet — the fat can be verj' easily extracted by ether, the theor\' of a
special albuminous membrane for the fat-globule has been generally aban-
doned. The obsen,'ations of Quincke ^ on the behavior of the fat-globules
in an emulsion prepared "uith gum have led, at the present time, to the
conclusion that each fat-globule in the milk is surrounded by a stratum of
casein solution held by molecular attraction, and this prevents the globules
from miiting with each other. Everything that changes the physical
condition of the casein in the milk or precipitates it must necessarily help
the solution of the fat in ether, and it is in this way that the alkalies,
acids, and rennet act.
V. Storch has sho-^ii, in opposition to these views, that the milk-glol>
ules 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 seems to all appearances to be
identical ^ith the so-called "stroma substance" detected by R.u)ex-
HAUSEN and Daxilewsky. 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 ^-iew that
the fat-globules probably have a membrane, which according to him is a
ver}' labile formation of variable composition. Droop-Richmoxd and
BoNNEMA,^ on the other hand, present several reasons in opposition to
Storch's view. If Storch's observation 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 bod}' forming
a membrane or stroma of the fat-globules becomes very probable.
Tlie milk-fat which is obtained imder the name of butter consists
chiefly of olein and palmitin. Besides these it contains, as triglycerides,
' Arch. f. Anat. u. Physiol., 1840.
^Pfluger's Arch., 19.
' V. Storch, see Maly's Jahresber., 2"; 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.
518 MILK.
myristic acid, stearic acid, small amounts of lauric acid, arachidic acid, and
dioxystearic acid, besides butyric acid and caproic acid, traces of caprylic
acid and capric acid. It must not be accepted that triglycerides of volatile
fatty acids occur, but rather mixed triglycerides of volatile and non-volatile
fatty acids (Riegel). Milk-fat also contains a small quantity of lecithin and
cholestcrin and a yellow coloring-matter. The quantity of volatile fatty
acids in butter is, according to Duclaux, on an average about 70 p. m.,
of which 37-51 p. m. is butyric acid and 30-33 p. m. is caproic acid. The
non-volatile fat consists of jo— /o olein, and the remainder is chiefly palmitin.
The composition of butter is not constant, but varies considerably under
different circumstances.^ According to Lemus^ 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 lactoglohulin, have been closest studied and are well char-
acterized. The milk-plasma contains two carbohydrates, of which the
one, lactose, is of great importance. It also contains extractive bodies,
traces of urea, creatine, creatinine, orotic acid, hypoxanthine (?), lecithin,
cholestcrin, citric acid (Soxhlet and Henkel^), and lastly also mineral
bodies and gases.
Casein. This protein substance, which thus far has been detected posi-
tively 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 nas the following composition:
C 53.0, H 7.0, N 15.7, S 0.8, P 0.85, and 0 22.65 per cent. Its specific
rotation is, according to Hoppe-Seyler, somewhat variable; in neutral
solution it is {a)j)=—80°; its faintly alkaline solution has a stronger rota-
tion, namely, -97.8 to~111.8°, in a solution of N/lO-N/5 NaOH (Long 4).
The question whether the casein from different kinds of milk is identical
or whether there are several different caseins is still disputed.
Casein when dry appears like a fine white powder, which has no meas-
urable solubility in pure water (Laqueur and Sackur). Casein is only
very slightly soluble in the ordinary neutral-salt solutions. According to
* 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.
^ See Maly's Jahresber., 34.
'■' Cited from F. Soldner, Die Salze der Milch, etc., Landwirth.sch. Ver.suchsstation,
35, Separatabzug, 18.
* Hoppe-Seyler, Handl). d. physiol. u. pathol. chem. Analyse, 7. Aufl., 368; Long,
Journ. Amer. Chem. Soc. 27.
CASEIN. 519
Arthus it dissolves rather easily in a 1 per cent solution of sodium fluoride,
ammoniiun or potassium oxalate. It is at least a tetrabasic acid, whose
equivalent weight is 1135 according to Laqueur and Sackur,^ and whose
molecular weight is four or six times this. The salts are split hydrolytically.
It dissolves readily m water ^\ith 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 m solution, but is probably only swollen as in milk, and the Kquid
contains at the same time a large quantity of calcium phosphate without
any precipitate or any suspended particles being visible. Tlie casein solu-
tions containing Ume are opalescent and have on warming the appearance
of milk deficient in fat (which is also true for the salts of casein with the
alkaline earths). Therefore it is not impossible that the white color of the
milk is due partly to the casein and calcium phosphate. Soldxer has
prepared two calcium compounds of casein ^ith 1.55 and 2.36 per cent
CaO, and these compounds are designated di- and tricalcium casein by
COURAXT. 2
According to Laqueur .^ who has determined the electrical conduc-
tivity and the internal friction of casein solutions, all casein-salt solu-
tions consist of a mixture of casein ions (with different amomits of H
which can be split off electrolytically) and unsplit casein (produced by
hydrolysis). By the gradual addition of alkali to the casein he found no
sharp distinguishing point and therefore proposes to drop the names mono-,
di-, and tricasein.
Casein solutions do not coagulate on boiling, but solutions of casein-
lime are covered, like milk, with a pellicle. They are precipitated by very
little acid, but the presence of neutral salts retards the precipitation. A
casein solution containing salt or ordinars^ milk requires, therefore, more
acid for precipitation than a salt-free solution of casein of the same concen-
tration. The precipitated casein dissolves very easily again m a small
excess of hydrochloric acid, but less easily in an excess of acetic acid. The
combination between casein and acid, and especially the combination with
lactic acid, which has been carefully studied by Laxa,-* are, like other
protein and acid compounds, precipitated by neutral salts. Tliese acid
solutions are precipitated by mineral acids in excess. Casein is precipitated
' Laqueur and Sackur, Hofmeister's Beitrage, 3; M. Arthus, Theses presentees
a la faculte des sciences de Paris, 1893.
- Soldner, Die Salze aer MUch, etc.; Couraut, 1. c. In regard to the salts of casein
see the investigations of Soldner, Maly's Jahresber., 25, and J. Rohmann, Berlin, klin.
Wochenschr. , 1895. See also Raudnitz, Ergebnisse der Physiol., 2, Abt. 1.
^ Hofmeister's Beitrage, 7.
' Milchwirtsch. Centralbl., 1905, Heft 12.
520 MILK.
from neutral solutions or from milk by common salt containing calcium or
magnesium sulphate in substance, without changing its properties. Metallic
salts, such as alum, zinc sulphate, and copper sulphate, completely pre-
cipitate the casein from neutral solutions.
On drying at 100° C, casein, according to Laqueur and Sackur, decom-
poses 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 some-
what 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 great 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 therefore an action on
casein even in the absence of lime-salts. These last are only necessary
for the coagulation or the separation of the curd, and the process of coagu-
lation is hence a two-phase process. The first phase is the transformation
of the casein by the rennin, the second is the visible coagulation caused by
the lime-salts. This fact, which was first proved by Hammarsten, was
later confirmed by Arthus and Pages and recently closely studied by
FuLD, Spiro, and Laqueur.^
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. According to Courant, 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.
Milk or casein solutions may indeed be precipitated without rennin by the
addition of a sufficiently large amount of calcium chloride. We are not
quite clear as to the importance of the lime-salts for the rennin coagulation,
and the views are still somewhat variable on this question. The same is
true for the chemical processes going on in rennin coagulation. If one
makes use of a pure solution of casein and as pure rennin as possible, then
after coagulation it is always found that the filtrate contains very small
^ See Maly's Jahresber., 2 and 4; also Hammarsten, Zur Kenntnis des Kaseins und
der Wirkung des Labfermentes, Nova Acta Reg. Soc. Scient. Upsala, 1877, Fest-
schrift; Zeitschr. f. physiol. Chem., 22; Arthus et Pages, Arch, de Physiol. (5), 2, and
M^m. Soc. biol., 43; Fuld, Hofmeister's Beitrage, 2, and Ergebnisse der Physiol., 1,
Abt. 1, where a good review of the literature may be found; Spiro, Hofmeister's
Beitrage, fi and 7, with Reichel, ibid., 7 and 8; Laqueur, ibid., 7.
CASEIN. 521
amounts of a proteid, the whey-proteid, which has other properties and a
lower content of nitrogen (13.2 per cent N, Koster ^) than the casein.
The chief portion of the casein, sometimes given as more than 90 per cent,
separates on coagulation as a body, the paracasein (or curd), which is
closely related to casein. The question whether a cleavage of the casein
takes place here is still unsettled. The paracasein 2 is not further changed
by the rennet enzyme ; it is much more readily precipitated by CaCl2 than
a casein solution of the same concentration, and the precipitation limits for
saturated ammonium- sulphate 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, according to him, less
than that of the casein solutions and indeed even to 20 per cent.
In the processes of rennet coagulation we may, as Reichel and Spiro
have sho'VMi, have a diminution of the action by an apparent consumption
of the ferment. This diminution is indeed, as the above investigators
found, not caused by the process of rennet coagulation and is therefore
not to be considered as a consumption of the enzyme. It depends upon a
division of the rennin between the curd and the whey taking place accord-
ing to a constant factor.
Fresh, unchanged milk does not, as is kno'mi, 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, according to Laxa, that the paracasein is transformed into casein
again by the lactic acid. But as a precipitation of the paracasein from the acid
solution is perhaps a pepsin action, the transformation of the paracasein into
casein by the lactic acid must not be considered as proved. As the commercial
rennet extracts may contain also other enzymes besides rennin, the formation of
proteoses in rennin coagulation, as observed by E. Petry,' must not be con-
sidered as a rennin action without further study.
In the digestion of casein mth pepsin-hydrochloric acid primarily a
phosphorized proteose is formed, from which then the pseudonuclein is
split off (Salkowski). The quantity thus split off is verj^ variable, as
shown by the researches of Salkowski, Hahx, Moraczewski, Sebeliex, and
Zaitschek."* The amount of phosphorus in the pseud onucleins obtained also
■ See Maly's Jahresber., 11.
^ It has been recently proposed to de.signate the ordinary ca.sein as caseinogen and
the curd as casein. Although such a propcsition is theoretically correct, it leads in
practice to confusion. On this account the author calls the curd paracasein, according
to Schulze and Rose (Landwirthsch. Versuchsstat., 31). A sununary of the literature
on the casein coagulation may be found in E. Fuld, Ergebnisse der Physiol., 1; Raud-
nitz, ibicl., 2; and Laqueur, Biochem. Centralbl., 4, 344.
'Laxa, I.e.; Petry, Wien. klin. Wochenschr., 1906.
^Salkowski, Zeitschr. f. physiol. Chem., 2"; Salkowski and Hahn, Pfliigcr's Arch.,
59; Salkowski, ibuL, 63; v. Moraczewski, Zeitschr. f. physiol. Chem., 20; Sebelien,
ibid.. 20; Zaitschek, Pfliiger's Arch., 104.
522 MILK.
varies considerably. According to Salkowski the quantity of pseudo-
nuclein split off is dependent upon the relationship between the casein and
the digestion fluid, e.g., the quantity of the pseudonucleins diminishes as
the pepsin-hydrochloric acid increases. In the presence of 5UU 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!).
From the products of peptic digestion of casein, after the separation of the
pseudonuclein, Salkowski ^ has isolated an acid rich in phosphorus. He considers
this a ■paranucleic acid. It is soluble in water, insoluble in alcohol, levorotatory,
and has the following composition: C 42.51-42.96, H 6.97-7.09, 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 puiity, it
is not an acid comparable to the nucleic acids.
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 reprecipi-
tating 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 con-
taminated with traces of fat is purified by treating with alcohol and ether.
Lactoglobulin was obtained by Sebelien from cow's milk by saturating
it with NaCl in substance (which precipitated the casein) and saturating
the filtrate with magnesium sulphate. As far as it has been investigated
it had the properties of serglobulin; the globulin isolated by Tiemann^
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.
Its composition is, according to him, 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 it
crystaUizes according to Wichmann ^ in forms similar to ser- or ovalbumin.
It coagulates, according to the concentration and the amount of salt in
solution, at 72-84° C. It is similar to seralbumin, but differs from it in
having a considerably lower specific rotatory power: (a)D= — 37°.
'Salkowski, 1. c; Biffi, Virchovv's Arch., 152; Alexander, Zeitschr. f. physiol.
Chem., 25.
^ Zeitschr. f. physiol. Chem., 32.
s/6w/.,25.
* Sebelien, Zeitschr. f. physiol. Chem., 9; Wichmann, ibid., 27.
LACTALBUMIX AXD ENZYMES. 523
The principle of the preparation of lact albumin is the same as for the
preparation of seralbumin from serum. The casein and the globulin are
removed by MgS04 in substance and the filtrate treated as previously
stated (page 182).
The occurrence of other proteins, such as proteoses and peptones, in milk has
not been positively proved. These bodies are easily produced as laboratory
products from the other proteins of the milk. Such a laboratorj^ product is
Millon's and Comaille's lactoprotein, which is a mixture of a little casein with
changed albimiin, and proteose ' which is formed by chemical action. In regard
to opalisin, see Human ^lilk, p. 531.
Milk also contains, according to Siegfried,^ a nucleon related to phos-
phocarnic acid, and which yields fermentation lactic acid (instead of para-
^actic acid) and a special carnic acid, orylic acid (instead of muscle camic
acid), as cleavage products. Lactophosphocamic 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
catalase, oxidases, peroxidases, and reductases, but the statements as to their
occurrence in the milk from different animals are not unanimous. An
amylolytic enzyme w^hich 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 deeom-
poses the lactose into lactic acid, alcohol, and CO2. occurs, according to
Stoklasa^ 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
found in these two kinds of milk, as well as certain others, a proteolytic
enzyme which they call galactase and 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 Z.\itschek
and V. SzoNTAGH, but on the other hand Vaxdevelde, de Waele, and
Sugg * confirm the occurrence of a proteolytic enzyme in milk.
Orotic acid, C,5HiiX204.2H20, is the name given by Biscaro and Belloxi ^
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 knoma. It j-ields urea on treatment
with potassium permanganate.
' See Hammar.«ten. Maly's Jahresber. , 6, 13.
^ Zeitschr. f. phy.siol. Chem., 21 and 22.
3 See Chem. Centralhl., 190.5, 1, 107.
^ Babcock and Russel, Centralhl. f. Bakt. u. Parisitenkunde (II), 6, and Maly's
Jahresber., 31; Zaitschek and v. Szontagh, Pflijger's Arch., 104; Vandevelde de
Waele, and Sugg, Hofmeister's Beitrage,o.
5 See Chem. Centralbl., 1905, 2, 6-3.
524 MILK.
Lactose, milk-sugar, C12H22O11 + H2O. 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. LevuUnic
acid is formed, besides formic acid and humin substances, by the stronger
action of acids. By the action of alkalies, amongst 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,i 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. At 170° to 180° C. it is converted into a
brown amorphous mass, lactocaramel, CgHioOs. 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 sweet-
ish 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 (q;)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
according to E. Fischer 2 the milk-sugar is first split into dextrose and
galactose by an enzyme, lactase, existing in the fungus. The preparation
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.
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-
lactosazone, C24H32N4O9. 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 with anhydrous oxalic acid to 100° C.
It differs from dextrose and maltose by its solubility and crystalline form,
but especialh^ by its not fermenting A\ith 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 dis-
solved in 4 c.c. of pyridine and 6 c.c. of absolute alcohol and viewed through
a layer 10 centimetres long (Neuberg^).
» Bull. Soc. chim. (.3), 13. = Ber. d. d. chem. Gesellsch., 27. » Ibid., 32.
ANALYSIS OF MILK. 525
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 fiUrate evaporated to a syrup. The cr>-stals
which separate after a certain time are recr^-staUized from water after
decolorizing with animal charcoal. A pure preparation may be obtained
from the commercial milk-sugar by repeated recrystallization. The C[uan-
titative estimation of milk-sugar may be performed either by the polar-
istrobometer or by means of titration with Fehlixg's solution. Ten c.c.
of Fehlixg's solution correspond to 0.0676 gram of milk-sugar in 0.5-1.5
per cent' solution after boiling for six minutes. (In regard to Fehlixg's
solution and the titration of sugar see Chapter XV.)
RiTTHAUSEN has 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. Laxdwehr considers
this as anunal gum, and Bechamp ^ as dextrin.
The mineral bodies of milk will be treated in connection ^\ith its quan-
titative composition.
The methods for the quantitative analysis of milk are verj- 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 asbestos.
The evaporation is first done on the water-bath and finished in a current
of carbon dioxide or hydrogen not above 100° C.
The inineral 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 accoimt of the burning of phosphorized
bodies, such as casein and lecithin. We must therefore, according to Sold-
NER, 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 Ritthausex's
method is employed, namely, the precipitation of the milk ^^•ith copper sul-
phate according to the modification suggested by Muxk.^ He precipitates
all the proteins by means of cupric hydrate at boiling heat, and determines
the nitrogen in "the precipitate by means of Kjeldahl's method. This
modification gives more exact results.
The older method of Puls and Stexberg, in which the precipitant is
alcohol, is too complicated and not sufficiently reliable. Sebeliex has sug-
gested a very good method. Three to four grams of milk are diluted -^ith
an equal volume of water, a little common-salt solution added, and the pro-
teins 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 foimd when multiplied by 6.37 (casein and
lactalbumin contain both 15.7 per cent nitrogen) gives the total quantity
' Ritthausen, Journ. f. prakt. Chem. (X. F.), 15; Landwehr, foot-note 1, p. 67;
Bechamp, Bull. Sec. chim. (3) 6.
' Ritthausen, 1 c; I. Munk, Virchow's Arch., 134.
526 MILK.
of proteins. This method, which is readily performed, gives very good
results. I. MuxK 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.
SiMOX 1 has found that the precipitation •s\-ith tannic acid, also \\ith phos-
photungstic acid, is the simplest and most accurate. The objection to
this and other methods in which the proteins are precipitated is that per-
haps other bodies (extractives) may be carried down at the same time (Cam-
ERER and Soldxer2). It is not kno'^n to '^hat 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 Muxk's anal3-ses about i^ of the total nitrogen belongs to the
extractives in cow's milk, and tx 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 accordmg to HiJFNER (urea, etc.) in 100
grams of cow's milk.
To determine the casein and albumin separately we may make use of
the method first suggested by Hoppe-Seyler and Tolmatscheff,^ in
which the casein is precipitated by magnesium sulphate. According to
Sebelien the milk is diluted ^\ith its o^\'n volume of a saturated mag-
nesium-sulphate solution, then saturated with the salt in substance, and
the precipitate then filtered and washed vAih. a saturated magnesium-
sulphate solution. The nitrogen is determined in the precipitate by Kjel-
dahl's method, and the quantity of casein ( + giobulin) 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 MgS04, after diluting with
water, the nitrogen determined by Kjeldahl's method and the result mul-
tiplied by 6.37.
Schlossmaxx^ suggests an alum solution, which precipitates the casein,
in order to separate the casein from the other proteins, the albumin can
be precipitated from the filtrate by tannic acid. The nitrogen in the pre-
cipitate is determined b}^ the Kjeldahl's method. This method has
recently been tested by Simox and he recommends it highly.
The fat is gravimetrically determined by thoroughly extracting the
dried milk with ether, evaporating the ether from the extract, and weighing
the residue. The fat may be determined by aerometric means by adding
alkali to the milk, shaking with ether, and determining the specific gravity
of the fat solution by means of Soxhlet's apparatus. In determining the
amount of fat in a large number of samples the lactocrit of De Laval may
be used \v\ih success. The milk is first mixed \\i\h an equal volume of a
mixture of glacial acetic and concentrated sulphuric acid, warmed 7-8
minutes on the water-bath, and the mixture poured in graduated tubes,
which are placed in the centrifugal machine at 50° C. The height of the
'Puis, Pfliisier's Arch., 13; Stenberg, IVIaly's Jahresber., 7; Sebelien, Zeitschr. f.
physiol. Chem., 13; Simon, ibid., 33.
2 Zeitschr. f . Biologie, 33 and 3fi.
^ Hoppc-Seyler, Med. chem. Untersuch., 272.
* Zeitschr. f. physiol. Chem., 22.
QUANTITATIVE COMPOSITIOX OP COW'S MILK. 527
layer of fat gives its quantity. The numerous and verj- exact analyses of
NiLSOX ^ have shown that \\ith milks containing small quantities of fat,
below 1.5 per cent, the older corrections are unnecessary, and that this
method gives excellent results if we use lactic acid treated with 5 per cent
hydrochloric acid instead of the above mixture of glacial acetic acid and
sulphuric acid. 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 ^^■ith alcohol, which must be evaporated
from the filtrate, or by diluting with water, and remo\ing the casein by
the addition of a little acid, and the lactalbumin by coagulation at boiling
heat. The sugar is determined by titration with Fehlixg's or Knapp's
solution (see Chapter X\). The principle of the titration is the same as
for the titration of sugar in the urine: 10 c.c. of Fehlixg's solution corre-
spond to 0.0676 gram of milk-sugar; 10 c.c. of Kxapp's solution correspond
to 0.0311-0.0310 gram of milk-sugar, when the saccharine liquid contains
about i-1 per cent of sugar. In regard to the modus operandi of the titra-
tion we must refer the reader to more complete works and to Chapter XV,
Instead of these volumetric determinations other methods of estima-
tion, such as Allihx's method, the polariscope method, and others, may
be used. In calculating the analysis or in determining the soUds it is of
importance to remember, as suggested by Caterer and Soldxer, that the
milk-sugar in the residue is anhydrous. Many other methods for deter-
mining the milk-sugar have been suggested and recommended.
The quantitative composition of cow's milk is naturally xevy variable.
The average obtained by Koxig- 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
The quantity of mineral bodies in 1000 parts of cow's milk is, according
to the analyses of Soldxer, as follows: K2O 1.72, Na20 0.51, CaO 1.98,
MgO 0.20, P2O5 1.82 (after correction for the pseud onuclein), CI 0.98 grams.
Buxge3 foimd 0.0035 gram Fe203. According to Soldxer the K, Na,
and CI are foimd in the same quantities in ^^'hole 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 -uith the casein;
the remainder is found united with the phosphoric acid as a mixture of
dicalcium and tricalcium phosphates which is kept dissolved 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 (Soldxer).
The gases of the milk consist chiefly of CO2. besides a little X and
* See Maly's Jahresber., 21.
' Chemie der menschlichen Xahrungs- unci Genussmittel, 4. Aufl.
3 Zeitschr. f. Biologie, 10.
528 MILK.
traces of O. Pfluger i 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 cahdng 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 diam-
eter with abundant fat-granules and fat-globules. The fat of colostrum
has a somewhat higher melting-point and is poorer in volatile fatty acids
than the fat from ordinary milk (Nilson^). 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.^ The composition of colostrum is very variable.
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
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 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 yellowdsh 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.
' Pfliiger's Arch., 2.
' Nilson, 1. c.
^See Sebelien, Maly's Jahresber., 18, and Tiemann, Zeitschr. f. physiol. Chem.,
25. See also Simon, ibid., 33; Winterstein and Strickler, ibid., 47.
HUMAN MILK. 529
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. According to Beil '
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 different amomits 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 older
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 cjuantity of
nucleon was about the same as in woman's milk. Thcf Cjuantity of fat was 15 p. m.,
and the sugar was 50-60 p. m. Reindeer milk is characterized, according to
Werenskiold,^ 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 compo-
sition 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 :^
Milk of the Water. Solids. Proteins. Fat. Sugar. Salts.
Dog 754.4 245.6 99.1 95.7 31.9 7.3
Cat 816.3 183.7 90.8 33.3 49.1 5.8
Goat 869.1 130.9 36.9 40.9 44.5 8.6
Sheep 835.0 165.0 57.4 61.4 39.6 6.6
Cow 871.7 128.3 35.5 36.9 48.8 7.1
Horse 900.6 99.4 18.9 10.9 66.5 3.1
Ass 900.0 100.0 21.0 13.0 63.0 3.0
Pi? 823.7 167.3 60.9 64.4 40.4 10.6
Elephant 678.5 321.5 30.9 195.7 88.4 6.5
Dolphin 486.7 513.3 437.6 4.6
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. Courant
found between the tenth day and the fourteenth month after confinement
practically constant results. The alkalinity, as well as the acidity, was a
little lower than in childbed. One hundred c.c. of the milk had the same
average alkalinity as 10.8 c.c. N/10 caustic soda, and the same acidity
' Studien iiber die Eiweissstoffe des Kumys und Kefirs, St. Petersburg, 1886
(Ricker).
^ Zaitschek, 1. c; Schlossmann, Zeitschr. f. physiol. Chem., 22; Ellenberger, Arch,
f. (Anat. u.) Physiol., 1899 and 1902; Werenskiold, Maly's Jahresber., 25.
^ Details in regard to the milk of different animals may be found in Proscher
Zeitschr f. physiol, Chem., 24; Ahderhalden, ibid., 27.
530 MILK.
as 3.6 c.c. N/10 acid. The relationship between the alkalinity and the
acidity in woman's milk was as 3:1, and in cow's milk as 2.1:1. The
actual reaction determined electrometrically is, according to Foa,i 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^ 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° C. 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,^ 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 jDroteins or in the more accurately determined casein.
A number of older and younger investigators'* claim that the casein from
woman's milk has other properties than that from cow's milk. The essen-
tial differences are the following: The casein from woman's milk is pre-
cipitated with greater difficulty with acids or salts; it does not coagulate
uniformly in the milk after the addition of rennet; it maybe precipitated
by gastric juice, but dissolves completely and easily in an excess of the
same; the casein precipitate 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 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. MtJLLER, Rubner and Heubner ^).
*Compt. rend. Soc. biolog., .^8.
'See Maly's Jahresber., 34.
' Ruppel, Zeitschr. f. Biologie, 31; Laves, Zeitschr. f. physiol. Chem., 19.
*See Biedert, Untersuchungen iiber die chemischen Untcrschiede der Menschen-
und Kuhmilch (Stuttgart), 1884; Langgaard, Virchow's Arch., fi.^; Makris, Studien
iiber die Eiweisskorper der Frauen- und Kuhmilch, Inaug.-Di.ss. Strassburg, 1876.
* Miiller, Zeitschr. f. Biologie, .39; Rubner and Heubner, ibid., 37.
HUMAN :\IILK. 531
The question as to whether the above-mentioned differences depend on
a decided difference in the two caseins or only on an unequal relationship
between the casein and the salts in the two kinds of milk, or upon other
circumstances, has not been decided as yet. According to Szoxtagh 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 has found the following for the composi-
tion of casern from woman's milk: C 52.24, H 7.32, X 14.97, P 0.6S, S 1.117
per cent. According to Kobrak ^ woman's casein yields some pseudonu-
clein, 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 Ijetween a nucleoalbumin
and a basic protein.
Woman's milk also contains lactalbumin, tesides the casern, and a
protein substance, verv^ 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 disputed as in many other cases.
Xo positive proof as to the occurrence of proteoses and peptones in fresh
milk has been given.
Even after those differences are eliminated which depend on the imjjer-
fect analytical methods employed, the quantitative composition of wonian's
w'lk is variable to such an extent that it is impossible to give any average
results. The recent analyses, especially those made on a large numter
of samples by Pfeiffer, Adriaxce, Camerer and Soldxer ,2 have posi-
tively sho^\'n that woman's milk is essentially poorer in proteins but richer
in sugar than cow's milk. The quantity of protein varies tetween 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 con-
siderably, but ordinarily amounts to 30-40 p. m. The quantity of sugar
should not be below 50 p. m., but maj^ rise to even 80 p. m. About 60
p. m. may be considered as an average, but it should loe 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 be-
tween 2 and 4 p. m.
* Szontagh, Maly's Jahresber., 22; Zaitschek, 1. c; Wrohle^vsky, "Beitrage zur
Kenntnisdes Frauenkaseins" (Inaug.-Diss. Bern, 1894), and "Ein neiier eiweis-fartiger
Bestandteil der Milch," Anzeiger der Akad. d. "Wiss. in Krakau, 1898; Kobrak,
Pfliigcr's Arch., SO.
-Pfeiffer, Jahrb. f. Kinderheilkunde, 20, also ^^aly'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, 83 and 30. In regard
to the composition of woman's milk, see also Biel, Maly's Jahresber., 4; Christenn,
ibid..'; Mendesde Leon, iWt?., 12; Cerber, Bull. Soc. chim., 23; Tolmatscheff, Hoppe-
Seyler's Med.-chem. Untersuch., 272.
532 MILK.
From a quantitative standpoint, the most essential differences between
woman's and cow's milk are as follows: As compared with the quantity
of albumin, the quantity of casein is not only absolutely but also relatively
smaller in woman's milk than in cow's milk, while the latter is poorer in
milk-sugar. Human milk is richer in lecithin, at least relatively to the
amount of protein. Burow found 0.49-0.58 p. m. lecithin in cow's milk
and 0.58 p. m. in woman's milk, which corresponds to 1.40 per cent for the
first milk and 3.05 per cent for the second, calculated on the percentage of
protein. According to Koch 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. According to Wittmaack 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 nearly entirely 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
P205:N, according to Schlossma.nn,i is equal to 1:5.4 in human milk
and 1 :2.7 in cow's milk. Woman's milk is poorer in mineral bodies, espe-
cially lime, and it contains only one-sixth of the quantity of lime as com-
pared 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 claimed to be also poorer in citric acid (Scheibe ^),
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 (Siebee ^).
This reaction consists in treating 5 c.c. of woman's milk ^vith- 2.5 c.c. 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. According to them
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 ■• found nearly twice this amount, namely, 0.23 p. m.
In regard to the quantity of mineral bodies in woman's milk we have
' B irow, Zeitschr. f. physiol. Chcm., 30; Koch, ibid., 47; Wittmaack, ibid., 22;
Siegfried, ibid., 22; Valenti, Biochem. Centralbl., 4; Schlossmann, Arch. f. Kinderheil-
kunde, 40.
^ Maly's Jahresber., 21.
8 Zeitschr. f. physiol. Chem., 30.
* Rubner, Zeitschr. f. Biologic, 30; Camerer and Soldner, i6vi., 39; Schondorff,
Pfliiger's Arch., 81.
HUMAN MILK. 533
the analyses of several investigators, especially of Bunge (analyses A and
B) and of Soldner and Camerer (analysis Cy. Bunge analyzed the
milk of a woman, fourteen days after delivery, whose diet contained very
little common salt for four days previous to the analysis {A), and again
three days later after a daily addition of 30 grams of NaCl to the food {B).
The figures are in 1000 parts of the milk:
A. B. C.
KjO 0.780 0.703 0.884
Nap 0.232 0.257 0.357
CaO 0.328 0.343 0.378
MgO 0.064 0.065 0.053
FePs 0.004 0.006 0.002
PA 0.473 0.469 0.310
CI 0.438 0.445 0.591
The relationship of the two bodies potassium and sodium, to each other
may, according to Bunge, vary considerably (1.3-4.4 equivalents of potash
to 1 of soda). By the addition of salt to the food the quantity of sodium
and chlorine in the milk increases, while the quantity of potassium de-
creases. 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 ^ find about
the same amount, namely, 10-20 milligrams Fe203 = 3.5-7 milligrams iron
in 1000 grams human milk.
The gases of woman's milk have been investigated by KiJLZ.^ He found
1.07-1.44 c.c. of oxygen, 2.35-2.87 c.c. of carbon dioxide, and 3.37-3.81
c.c. of nitrogen in 100 c.c. of milk.
The proper treatment of cow's milk by diluting it with water and by
certain additions in order to render it a proper substitute for woman's milk
in the nourishment of children cannot be determined before the difference
in the protein bodies of these two kinds of milk has been completely studied.
The colostrum has a higher specific gravity, 1.040-1.060, a greater
quantity of coagulable proteins, and a deeper j^ellow color than ordinary
woman's milk. Even a few days after deliver}^ 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 ^ and the recent investigations of
Pfeiffer, V. and J. Adriance, Camerer and Soldner on the changes in
the composition of milk after deliver}^ It follows, as a unanimous result
' Bunge, Zeitschr. f. Biologie, 10; Camerer and Soldner, ibid., 39 and 44.
'De Lange, Maly's Jahresber., 27; Jolles and Friedjung, Arch. f. exp. Path, u,
Pharm., 46: Camerer and Soldner, Zeitschr. f . Biologie, 46.
^Zeitschr. f. Biologie, 32.
" See Hoppe-Seyler Physiol. Chem., 734.
634 MILK.
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
quickl}^ and then more gradually diminishes as long as the lactation con-
tinues, 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.
Adrian'CE (120 analyses), increases rather quickly the first days and then
only slowly until the end of lactation. The analyses of Pfeiffer, Camerer
and SoLDNER also show an increase in the quantity of milk-sugar.
The two mammary glands of the same woman may yield somewhat different
milk, as sho\\ai 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
contains less casein than that of brunettes, a difference which Tolmatscheff ^
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. Accorchng to Vernois and Becquerel, 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 ciualitative stand-
point the same constitution as milk, but may show important differences and
variations from a Cjuantitative point of view. Schlossberger and Hauff,
GuBLER and Quevenne, and v. Genser ^ have made analyses of this milk and
give the followmg 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 respresentatives of the
three chief groups of organic nutritive substances — proteins, carbohy-
drates, and fat; and all milk seems to contain without doubt also some
lecithin and nucleon. The mineral bodies in milk must also occur in proper
proportions, and on this point the experiments of Bunge on dogs are of
' Sourdat, Compt. rend., 71; Brunner, Pfliiger's Arch., 7.
^ l'Heritier, cited from Hoppe-Seyk-r, Physiol. Chem., 738; Vernoi.s and Bec-
querel, Du lait chez la femme dans I'etat de sante, etc. (Paris, 1853); TolmatsehetT,
Hoppe-Seyler, Med .-chem. Untersuch., 272.
'Schlossberger and Hauff, Annal. d. Chem. u. Pharm., 9(5; Gubler and Quevenne,
cited from Hoppe-Seyler's Physiol. Chem., 723; v. Gen.ser, ibid.
MILK AND FOOD. 535
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. Buxge ^ found in 1000 parts of the ash the following results
{A represents results from the new-boni dog, and B the milk from the bitch) .v
A. B.
KjO 114.2 149.8
Nap 106.4 88.0
CaO 295.2 272.4
Ma;0 18.2 . 15.4
Fe^O, 7.2 1.2
P2O3 394.2 342.2
CI 83.5 169.0
Buxge explains the fact that the milk-ash is richer in potash and poorer
in soda than the new-bom 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 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 Hugounexq, de Laxge, Camerer and Soldxer^
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 to
the milk. As an example the following analyses are given (of Camerer and
Soldxer). (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.
K„0 78 314
Na,0 91 119
CaO 361 164
MgO 9 26
¥e,Oi 8 6
PA 389 135
CI 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 coincide.
Bunge ^ 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 relativel}' poorer in phosphates and
' Zeitschr. f. physiol. Chem., 13.
^ Hugounenq, Compt. rend., 128; de Lange, Zeitschr. f. Biologic, 40; Camerer
and Soldner, ibid., 39, 40, and 44.
^ Bunge, "Die zunehmende Unfahigkeit der Frauen ihre Kinder zu stillen," Miin-
chen, 1900, cited by Camerer, Zeitschr. f. Biolo<rip, 40.
536 MILK.
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 second is prominent.
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 relationship 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
relationship exists also, as shown by the researches of Proscher, and espe-
cially of Abderhalden,' between the quantity of protein in the milk and
the rapidity of development of the sucking young. The amount of protein
is greater in the milk the quicker the animal develops.
The influence of the food on the composition of the milk is of interest
from many points of view and has been the subject of many investigations.
From these we learn that in human beings as well as in animals an insuffi-
cient diet decreases the quantity of milk and the quantity of solids, while
abundant food increases both. From the observations of Decaisne 2 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 somewhat increased.
Food rich in proteins increases the quantity of milk, and also the solids
contained, especially the fat, according to most statements. 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 ^ 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 .^ In carnivora, as shown by Ssubotin,^
the secretion of milk-sugar proceeds uninterruptedly on a diet consisting
1 Proscher, Zeitschr. f. physiol. Chem., 24; Abderhalden, ibid., 27; Pages, Arch,
de Physi )1. (5), 7.
=> Cited from Hoppe-Seyler, 1. c, 739.
^ See Maly's Jahresber., 2G. See also Rasch, Ergebnisse der Physiologie, 2, Abt. 1.
^In regard to the literature on the action of various foods on woman's milk, see
Zalesky, "Ueber die Einwirkung der Nahrung auf die Zusammcnsetzung und Nahr-
haftigkeit der Frauenmilch," Berlin, klin. Wochenschr. , 188S, which also contains the
literature on the importance of diet on the composition of other kinds of milk. In
regard to the rxtensive literature on the influence of various foods on the milk pro-
duction of animals, .see Konig, Chem. d. menschl. Nahrungs und GenussmitteJ, 3. Aufl.,
1, 298. See also Maly's Jahresber., 29, 30, 31, and Morgen, Beger and Fingerlmg,
Landw. Versuchsl., 01.
6 Centralbl. f. d. med. Wissensch., 1866, 337.
CHEMISTRY OF MILK SECRETION. 537
exclusively of lean meat. 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 Commaille ^ 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 some-
times a peculiar sharp after-taste, although not always.^
Chemistry of Milk-secretion. That the constituents which occur actu-
ally dissolved in milk pass into the secretion not alone by filtration or diffu-
sion, but more likely are secreted by a specific secretory activity of the
glandular elements, is shown by the fact that milk-sugar, which is not
found in the blood, is to all appearances formed in the glands themselves.
A further proof lies in the fact that the lactalbumin is not identical \^ ith
seralbumin; and lastly, as Bunge ^ has shown, the mineral bodies secreted
by the milk are in quite different proportions from those in the blood-
serum.
Little is kno-wn 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 originated
probably from mistaking an alkali albuminate for casein. Better founded
is the statement that the casein originates from the protoplasm of the
gland-cells. There does not seem to be any doubt that the protoplasm 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 *
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 forming a nucleoalbu-
min, the casein. The untenableness of this view has been shown by Lo-
BiscH, and the investigations of Hildebrandt ^ 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.
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 showii that after feeding
iodized casein, the milk-fat of goats contained a little iodine, which indicates
1 Cited from Konig, 2, 235.
' See Beck, Maly's Jahresber., 25.
'Lehrbnch d. physiol. und pathol. Chem., 3. Aufl., 93.
^Hermarm^s Handbuch, 5, TeU 1, 380.
^Basch, Jahrb. f. Kinderheilkunde, 1-898; Hildebrandt Hofmeister's Beitrage, 5;
Lobisch, ibid., 8.
538 MILK.
that the iodized milk-fat could also have a different origin. As a con-
tamination of the casein fed with iodized fat was not excluded in these
experiments, they do not seem to modify the proof of the investigations of
WixTERXiTZ and others (Caspaei, Paraschtshuk i). The abundant quan-
tities of iodized fat which were eliminated with the milk in these cases with-
out doubt depend, at least in great part, upon the iodized fat of the food,
hence it cannot be said that all of the milk-fat containing iodine was
unchanged iodized fat of the food. The investigations 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. Hexriques and Haxsex 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 equiva-
lent and a higher melting-point, from which they also concluded that a
transformation of the food-fat in the glandular cells is possible. The
experiments of Gogitidse 2 with soaps also speak for the fact that the
mammary glands have the property of forming fats by S}nithesis from their
components. As a formation of fat from carbohydrates in the animal
organism is at the present day considered as positively proved, it is likewise
possible that the milk-glands also produce fats from the carbohydrates
brought to them by the blood. It is a well-known fact that an animal
gives off for a long time, daily, considerably more fat in the milk than it
receives as food, and this proves that at least a part of the fat secreted by
the milk is produced from proteins or carbohydrates, or perhaps from both.
The question as to how far this fat is produced directly in the milk-glands,
or from other organs and tissues, and brought to the gland by means of
the blood, cannot be decided.
The origin of milk-sugar is not known. Miixxz 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 pro-
duct 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 observa-
' Winternitz, Zeitschr. f. physiol. Chem., 24; Jantzen, Centralbl. f. Physiol., 15;
Caspari, Arch. f. (Anat. u.) Physiol., 1899, Supplbd. and Zeitschr. f. Biologic, 46;
Paraschtschuk, Chcm. Centralbl., 1903, 1.
'Spampani and Daddi, Maly's Jahresber., 2(5; Henriques and Hansen, ibifl, 29;
Gogitidse, Zeitschr. f. Biologic, 45 and 46. See also Basch Ergebnisse d. Physiol., 2,
Abt. 1.
CHEMISTRY OF MILK SECRETION. 539
tioiis of Bert and Thierfelder ^ that a mother-substance of the milk-
sugar, a saccharogen, occurs in the glands cannot give further explanation as
to the formation of milk-sugar, as the nature of this mother-substance is still
imkno'^ii. As the animal bod}' has undoubtedly the power of converting
one variety of sugar into another, the origin of the milk-sugar can be
sought simply m the dextrose introduced as food or formed in the bod}'.
Certain observations indicate such an origin, among others those of
PoRCHER,2 ^.}^Q fovmd that dextrose appeared in the urine after delivery
when the mammary glands of the goat had previously been 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.
The passage of foreign substances into the milk stands in close connec-
tion with the chemical processes of milk secretion.
It is a well-kno-^-n 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 mto the organism of the nursing child by
means of the milk.
Among these substances may be mentioned opium and moi-phine, 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 c^uantities as to have any direct
action on the nursing child. '^ 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,
mercur}^, 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 * have observed
indeed a markedl}' abnormal composition, but no positive conclusion can be
derived therefrom.
The changes in cow's milk in disease have been little studied. In tuberculosis
of the udder Storch ^ found tubercle bacilli in the milk, and he also noted that
the milk became more and more diluted, during the disease, with a serous licjuid
similar to blood-serum, so that the glands finally, instead of yielding milk, gave
only blood-serum or a serous fluid. Hussox ^ found that milk from murrain
' Miintz, Compt. rend., 102; Bert and Thierfelder, foot-note 3, p. 514.
^Compt. rend., 138 and 141.
^ See Klingemann, Virchow's Arch., 126, and Rosemann, Pfliiger's Arch., 78.
^Schlossberger, Annal. d. Chem. u. Phami., 90; Joly and Filhol, cited from
v. Gonip-Besanez, Lehrb., 4. Aufl., 438.
^ See Bans;, Om Tuberkulose i Kopns Yver og om tuberkulos Malk. Xord. med.
Arkiv, 10, and also Maly's Jahresber., 14, 170; Storch Maly's Jahresber., 14.
^ Compt. rend., 7.3.
540 MILK.
cows contained more proteins but considerably less fat and (in severe cases) less
sugar than norma] 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.
CHAPTER XV.
URINE.
Urine is the most important excretion of the animal organism; it is the
means of eUminating the nitrogenous metaboUc 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 quali-
tatively by the appearance of foreign bodies in the excretion. IMoreover 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 introduced into the organism
are absorbed and chemically changed. In this respect especially urinary
analysis has furnished very important particulars in regard to the nature of
the chemical processes taking place within the organism, and it is therefore
not only an important aid in diagnosis to the physician, but it is also of
the greatest importance to the toxicologist and the physiological chemist.
In studying the secretions and excretions the relationship must be sought
between the chemical structure of the secreting organ and the chemical
composition of its secreted products. Investigations with respect to the
kidneys and the urine have led to very few results from this standpoint.
Although the anatomical relation of the kidneys has been carefully studied,
their chemical composition has not been the subject 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 con-
stituents of the kidneys known at the present time can, therefore, have only
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 nucleoproteid. The globulin coagulates at about 52° C, and the
nucleoproteid contains 0.37 per cent jDhosphorus. According to L. Leiber-
MANN the kidneys contain a lecithalbumin, and he ascribes to this bod}^ a
special importance in the secretion of acid urines. The kidneys also contain
541
542 URINE.
according to Lonnberg, a mucin-like substance. This substance yields no
reducing body on boiling with acids and belongs chiefly to the papillae, and
is, according to Loxnberg, a nucleoalbumin (nucleoproteid?). The cor-
tical substance is richer in another nucleoalbumin (nucleoproteid) unUke
mucin. It has not been decided what relationship this last substance
bears to Halliburton's nucleoproteid. The nucleic acid obtained by
Mandel and Levene from beef kidneys yielded guanine, adenine, thy-
mine, and cytosine on cleavage. According to Morner ^ chondroitin-
sulphuric acid occurs as traces. jMandel and Levene ^ have also obtained
glucothionic acid from the kidneys. Fat occurs only in very small amounts
in the cells of the tortuous urinary passages. Among the extractive bodies
of the kidneys one finds imrine 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 ^
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 occurs gener-
ally 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 physio-
logical 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, 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 warm-
1 Halliliurton, 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.
phy.siol. Cheni., 47; Morner, Skand. Arch. f. Physiol., 6.
^Zeitschr. f. physiol. Chem., 45.
^ Cited from v. Gorup-Besanez, Lehrbuch, 4. Aufi., 732.
REACTION. 543
ing. 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 sedimen-
tum lateritium. Urine has a salt}" and faintly bitter taste produced by
sodium chloride and urea. The odor of urine is peculiarly aromatic; the
bodies which produce this odor are unkno^^^l.
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 bro\\Ti in stronger concentration.
As a rule the intensity of the color corresponds to the concentration, but
under pathological conditions exceptions occur such as is 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 mav
become less acid or neutral, while the reverse occurs when an herbivore 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 equiva-
lents. This depends upon the fact that in the physiological combustion of
neutral substances (proteins and others) within the organism, acids are pro-
duced, chiefly sulphuric acid, but also phosphoric and organic acids, such as
hippuric, uric, and oxalic acids, aromatic oxyacids, and others. From this it
follows that the acid reaction is not due to one acid alone. The ordinary
view that the acid reaction is due chiefly to dihydrogen phosphates is
therefore not true. 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.
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 iuice containino-
hydrochloric acid is secreted, the urine may be neutral or even alkaline .^
The statements of various investigators are rather contradictor^^ in re2;ard
1 Contradictory statements are found in Linossier, Maly's Jahresber., 27.
544 URINE.
to the time of the appearance of the maximum and minimum of the acid-
ity, which ma}' in part be explained by the varying individuahty and
conditions of Ufe of the persons investigated. It has not infrequ2ntly 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 urme has not been positively determined.
According to Hoffmann, Ringstedt, Oddi and Tarulli, and Vozarik
muscular work raises the degree of acidity, but Aoucco ^ claims that it
decreases it. Abundant perspiration reduces the acidity (Hoffivl\nn) .
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 wliich are burnt up with difficulty are 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 spht off from the proteins or their decomposition
products, and this excess of acid combines therewith, forming ammonium
salts, which pass into the urine. In herbivora such a combination of the
excess of acid with ammonia does not seem to take place, or not to the same
extent, and therefore herbivora soon die when acids are given. This is
true at least for rabbits, while according to Baer - this power of increasing
the elimination of ammonia exists also in the goat, monkey, and pig, hence
no definite difference in this regard exists between herbivora and carnivora.
Nevertheless the degree of acidity of human urine 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 alkaU salts of vegetable
acids — tartaric acid, citric acid, and malic acid — as are easily burnt into
carbonates 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 alkaUne 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-organ-
isms.
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 g-^^ntle heat. If the
alkaline reaction is due to ammonia, the paper becomes red again; but if
it is caused by fixed alkalies, it remains l)lue.
' Hoffmann, see Maly's Jahresber., 14; Ringstedt, ibid., 20; Oddi and Tarulli,
ihid., 24; Aducco, ibid., 17; Vozarik, Pfliiger's Arch., 111.
^ See Winterberg, Zeitsehr. f. physiol. Chem., 2.5. and T. Baer, Arch. f. exp. Path. u.
Pharm., h\.
DETERMINATION OF THE ACIDITY. 545
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, all the older methods suggested for the estimation of his
portion of the phosphoric acid are not suited for acidity determinations.
We now determine the acidity simply by acidimetric methods, titrating
with N/10 caustic alkali, using phenolphthalein as an indicator (Naegeli,
HoBER. FoLix). 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 depends upon 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, ac-
cording to FoLiN, by the addition of neutral potassium oxalate, which
precipitates the lime, and in this way the disturbing action of the ammo-
nium salts is also inliiljited. Perfectly acccurate results are not obtained
by tliis method, but it is the best of those which have been suggested.
It is performed as follows: 25 c.c. of urine are placed in an Erlenmeyer
flask (about 200 c.c. capacity), treated with 1-2 drops of h percent phen-
olphthalein solution, and shaken with 15-20 grams of powdered potassium
oxalate and immediately titrated with N/10 caustic soda with constant
shaking until a pronounced pale-rose color appears. VozXrik ^ 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
to about 1.5-2.3 grams in man in the 24 hours.
By titration we learn the amount, of hydrogen present which can be
substituted by a metal, i.e., the acidity in the ordinaiy older sense, but not
the true acidity, the ion acidity, which is given by the concentration of the
hydrogen ions of the urine. For similar reasons, as indicated pre\-iouslv in
treating of the alkalinity of the blood-serum (page 191), the ion acidity
cannot be determined by titration, wliile it can be determined accordino-
to the principle of the electrometric gas-chain method as there given. Such
estimations have been made by v. Rhorer and by Hober.^ For normal
urine v. Rhorer found as a minimum 4x10"*^, as a maximum 76x10"^
and as an average 30x10"''. Hober found 4.7X10"^, 100 X"^, and
49X10"'', respectively. On an average the urine contains therefore 30-50
grams of hydrogen ions in 10 million liters, and as in the same quantity
of purest water there is contained in round numl:)ei-s 1 gram of hvdrogen
ions, the urine contains, therefore. 30-50 times as many hydrojren ions as
^Naegeli, Zeitschr. f. physiol. Chem., 30; Hober, Hofmeister's Beitrage, 3- Folin
Amer. Journ. of Physiol., 9; Vozarik, 1. c.
^ V. Rhorer, Pfliiger's Arch., 86; Hober, 1. c.
546 URINE.
the water. From Hober's investigations it also follows that no direct
relationship 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 limits for the freezing-point depression has
been found by a number of investigators to be J = 0.87°- 2.71° C.^ After
partaking of considerable water it may be markedly lower; and on diminished
supply of water it may be consideralily higher.
According to Bugarsky a certain relationship exists between the freezing-
J
point depression and the specific gravity, namely, -= constant = 75. This
equation, where s represents the specific gravit}^, has no general application, and
according to Steyrer ^ is only approximate for normal urines. The validity
of the relationship found by Bugarsky between the electrical conductivity
and the ash content of the urine, seems also to require further proof.
The specific gravity of urine, which is dependent upon the relationsliip
existing between the quantity of water secreted and the solid urinary con-
stituents, especially the urea and sodium chloride, may vary considerably,
but is generally 1.017-1020. After drinking large quantities of water it
may fall to 1.002, while after profuse perspiration or after drinking very
little water it may rise to 1.0S5-1.040. In new-l^orn infants the snecific
gravity is low, 1.007-1.005. The determination of the specific gravity is
an important means of learning the a^'erage amount of solids eliminated
from the organism in the urine, and on this account the determination
becomes of true value only when at the same time- the quantity of urine
voided in a given time is determined. The different portions of urine
voided in the course of the twenty-four hours are collected, mixed together,
the total quantity measured, and then the specific gravity taken.
The determination of the specific gravity is most accurately o]:)tained with
the pycnometer. For ordinary cases the specific gravity may be deter-
mineci with sufficient accuracy by means of areometers. The areometers
found in the trade, or n riff meter:--, are graduated from 1.000 to 1.040; for
exact ol)servations 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 remo^■ed with a glass rod or filter-
paper. The cylinder, which should he about four-fifths fidl. must be wide
enough to allow the urinometer to swim freely in the liquid without touch-
ing the sides. The cylinder and urinometer shoidd both be drv or j^reviously
washed with the urine. On reading:, the eye is brought on a level with the
■See Strauss, Zeitschr. f. klin. Med., 47.
^ Bugarsky. Pfliijrer's Arch., 68; Steyrer, Hofmcister's Beitrage, 2.
ORGANIC PHYSIOLOGICAL CONSTITUENTS. 547
lower meniscus — which occurs when the surface of the liquid and the lower
limb of the meniscus coincide; the readinj:; is then made from the point
where this curved line coincides wi;h the scale ot the urinometer. If the
eye is not in the same hoii/.ontal plane with the convex line of the
meniscus, but is too hi,s;h or too low, the surface of the lic[uid assumes the
shape of an ellipse, and the reading in this position is incorrect. Before
readino;, 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 ever}' three degrees above the normal temperature one unit ot the last
order is added t') the reading, and for ever\' three degrees below the nor-
mal temperature one unit (as above) is subtracted from the specific g^a^dty
observed. For example, when a urinometer graduated for 15° C. shows
a specific 2:ra\ntv of 1.017 at 24° C, then the specific gravitv at 15" C.=
1.017 + 0.003=1.020.
When great exactitude is required, as, for instance, a determination to
the fourth decimal point, we make use of a urinometer constructed by
LoHxsTEiN.i JoLLES ^ has also devised a small urinometer for the deter-
mination of the specific gravity of small amounts of urine, 20-25 c.c. The
specific gravit}^ may also be determined by the West^^h.al hydrostatic
balance.
n. Organic Physiological Constituents of Urine.
+ X'TT
Urea, Ur, C0N2H4 = C0<*^f;-, has been synthetically prepared in sev-
iN rl2
eral wa3'S, especially, as Wohlee showed in 1828, by the metameric trans-
formation 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, agrinine, other amino-
acids, and polypeptides.
Urea is found most abundantly in the urine of carnivora and man, but in
smaller quantities in that of herbivora. 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 perspira-
tion 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.-^
and bile * of sharks. Urea is also found in certain tissues and organs of
mammals, especially in the liver and spleen, 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.
' Pfliigpr's Arch , ."iO; Chem. Centralbl., 1895, 1, and 1896, 2.
MVien. med. Pressc, 1897, No. 8.
^ V. Schrocder, Zeitschr. f. physiol. Chem., 14.
* Hammarsten, ihtd., 24.
548 URINE.
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 slimination 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 great-
eat after an exclusive meat diet, and lowest, indeed less than during starva-
tion, after the consumption of non-nitrogenous substances, since these
diminish the metaljolism of the proteins of the body.
If the consumption of the proteins of the body is increased, then the
elimination of nitrogen is correspondingly increased. This is found to be
the case in fevers, after poisoning with arsenic, antimony, phosphorus, and
other protoplasmic poisons, and when there is a diminished supply of
oxygen — as in severe and continuous dyspnoea, poisoning with carbon
monoxide, hemorrhage, etc. In these cases it used to be considered that
the rise in the excretion of nitrogen was due to an increased elimination of
urea, because no exact difference was made between the quantity of urea
and of total nitrogen in the urine. Recent researches have conclusively
demonstrated the untrustworthiness of these observations. Since PFLtJGER
and Borland have shown that 16 per cent of the total nitrogen of the urine
exists under physiological conditions in other compounds, not urea, atten-
tion has been called to the relationship of the different nitrogenous con-
stituents of the urine to each other, and it has been found, under patho-
logical conditions, that this relationship may vary considerably, especially
in regard to the urea. We have numerous determinations by different
investigators, such as Borland, E. Scrultze, Camerer, Voges, Morner
and Sjoqvlst, Gumlicr, Bodtker,i and others, on the relationship 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:
> Pflijger and Bohland, Pfliiger's Arch., 38 and 43; Bohland, ibid., 43; Schultze,
ihid., 45; Camerer, Zeitschr. f. Biologic, 24, 2", and 28; Voges, Ueber die Mischung
der stickstoffhaltigen Bestandtheile im Ham, etc. (Inaiig.-Diss. Berlin, 1892) cited
from Maly's Jahresber., 22; K. Morner and Sjoqvist, Skand. Arch. f. Physiol., 2.
See also Sjoqvist, Nord. med. Arkiv. 1892, No. 36, and 1894, No. 10; Gumlich, Zeitschr.
f. physiol. Chem., 17; Bodtker, see Maly's Jahresber. , 26.
NITROGEN OF THE URINE. 549
A. B.
Per Cent. Per Cent.
Urea 84-91 73-76
Ammonia 2-5 7 . 8-9 . 6
Uric acid 1-3 3.0-8.5
Remaining nitrogenous substances (extractives).. . . 7-12 7.3-14.7
The variable relationship between uric acid, ammonia, and urea nitrogen
in children and adults is remarkable, since the urine of children is consider-
ably richer in uric acid and ammonia, and considerably poorer in urea, than
the urine of adults. The absolute quantity of urea nitrogen in adults
amounts to about 10-16 grams per day. In disease the proportion of the
nitrogenous substances may be markedly changed, and a decrease in the
quantity of urea and an increase in the quantity of ammonia have been
observed in certain diseases of the hver. 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
passages, the ehmination of urea is considerably diminished.
Recently by means of Pfaux idler's ^ method, by precipitating the urine
with phosphotungstic acid and closely studying the precipitate as well as
the filtrate, it has been possible to learn further about the division of the
nitrogen of the urine. We determine a, the total nitrogen; 6, the nitrogen
of the phosphotungstate precipitate ; and c, the nitrogen in the filtrate from
the phosphotungstate precipitate. This last contains the urea, hij^puric
acid, and other bodies whose nitrogen is ordinarily designated as monamino-
acid nitrogen. The urea nitrogen is especially determined. The Ijodies
precipitated by phosphotungstic acid are not all known ; but uric acid and
purine bases, ammonia, creatinine, pigments, diamino-acids, diamines and
ptomaines (if they occur), sulphocyanides, carbamic acid, urine mucoid,
and proteid belong to this group. Of these bodies, ammonia, uric acid,
creatinine and jxirine bases are specially determined.
The urea nitrogen is always the greatest part of the total nitrogen, but
otherwise the division of the nitrogen undergoes considerable variation.
According to v. Jacksch^ 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 sufficient. Very
great variations seem to occur not only in the healthy indi\'idual, but also
and to a greater degree in diseased conditions.^
' Pfaundler, Zeitschr. f. physiol. Chem., 30.
^Zeitschr. f. klin. Med., 50.
'See Satta, Hofmeister's Beitrage, 6, which also gives the literature, and Erben,
Zeitschr. f. Heilkunde, 25.
550 URINE.
Formation of Urea in the Organism. The experiments to produce urea
directly from proteins by oxidation have led to the formation of some guan-
idine, but 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 trv^ptic 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 II). 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 in-
terest since Kossel and Da kin have discovered the presence of an enzyme,
arginase, in the liver and other orgaas, which has the power of splitting
arginine with the formation of urea. Thompson^ 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 subcutaneously has in liis experi-
ments 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 w^hole of the nitrogen of the
arginine introduced. In these cases, wdthout mentioning that the arginine
seemed to raise the nitrogen catabolism, probably also urea was formed
from the ornithine. This can be explained Ijy a deamidation of the orni-
thine 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 creatine; still such an origin of urea in the animal body
has not thus far been proved.
The amino-acids are considered as special mother-substances of urea.
By the researches of Schultzex and Nencki and Salkowski wath leucine
and glycocoll, those of Stolte with several amino-acids, and those of v.
Knieriem with asparagine. 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, and glycocoll or
leucine, as also the researches of Ascoli,^ have led to similar results, but
it must be remarked that we have no proof as to the identity of the newly
* Kossel and Dakin. Zeitschr. f. physiol. Chem., 41; Thompson, Journ. of Physiol.,
32 and 33.
^ Schultzen and Nencki, Zeitschr. f. Biologic, 8; v. Knieriem, ibid., 10; Salkowski,
Zeitschr. f. physiol. Chem., 4; Salaskin, ibid., 25; Loewi, ibid., 2o; Stolte, Hofmeistcr's
Beitrage, 5; Richet, Compt. rend., 118, and Compt. rend. Soc. biol., 49; Ascoli,
Pfluger's Arch., 72.
FORMATION OF UREA. 551
formed substance with urea. Xothing can be stated in regard to the extent
of formation of amino-acids in the physiological destruction of proteins in
the animal body, with the exception of those formed in the intestinal diges-
tion. The possibihty of such a formation of urea is beyond dispute. As
sho\^-n by Abderhaldex ^ with Teruughi and Babkix, the polypeptides,
like the amino-acids, can also be converted into urea in the animal body.
Nothing positive can be said in regard to the manner in which this
formation of urea occurs; but it is admitted that it is partly a formation
from ammonia and partly from carbamic acid.
The possibilit}- of a formation of urea from ammonia has been positively
shown. Thus the researches of v. Kxieriem. Salkowski. Feder, I. Muxk,
CoRAXDA, SchmiedeberCt and Fr. Walter. Hallervordex, and Pohl
and }>^iJxzER,2 on the behavior of ammonium salts in the animal body and
the ehmination of the ammonia imder various conditions, have shown
that not only ammonium carbonate, but also those ammonium salts wliich
are burnt into carbonate in the organism, are transformed into urea by
carnivora as well as herbivora. v. Schroeder.^ by irrigating the sur\-i\-ing
dog's liver with blood treated with ammonium carbonate or ammonium
formate, has shown that the formation of urea takes place, at least in part,
in this organ. Nexcki, Pawlow, Zaleski and Salaskix * 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 Hver retains in great part the ammonia thus supplied. The formation
of urea from ammonia in the liver is a positively proved fact, and the urea
formation from ammonium carbonate is to be considered as a synthesis
with the elimination of water.
The assumption of a spUtting off of ammonia from amino-acids is not
difficult of conception, as now, especially from the investigations men-
tioned in Chapter VIII. we know T\-ith positiveness that deamidation of
amino-acids does take place in the animal bod}'. The ammonia spUt off
finds in the blood and tissues the carbon dioxide necessary- for the formation
of carbonate, and to all appearances the conditions are also suitable for
the formation of carbamate.
Important observations have been made wliich give support to the ^iews
of ScHULTZEX' and Nexcki.^ namely, that the amino-acids are transformed
'Zeitschr. f. physiol. Chem., 4".
''v. Knieriem, Zeitschr. f. Biolotrie, 10; Feder, ibiyl., 13; Salkowski, Zeitschr. f.
Biologie, 1; Munk, ibid., 2; Coranda, Arch. f. exp. Path. u. Pharm., 12; Scjimiede-
berg and Walter, ibid., 7; Hallerv'orden, ibid., 10; Pohl and Miinzer, Arch. f. exp.
Path. u. Phann., 43.
^ Arch. f. exp. Path. u. Pharm., 15. See al.^o Salomon, Virchow's Arch., 97.
* Arch, des sciences bid. do St. Petersbourg, 4; see also Chapter \'T, p. 241.
* Zeitschr. f . Biologie, 8.
552 URINE.
into urea with carbamic acid as an intermediate step. Drechsel has
shown that the amino-acids yield carbamic acid by oxidation in alkaline
fluid outside of the organism, and he obtained urea from ammonium car-
bamate by passing an alternating electric current through its solution,
i.e., by alternate oxidation and reduction. Drechsel has also been
able to detect small quantities of carbamates in blood, and later in con-
junction with Abel he detected carbamic acid in alkaline horse's urine.
Drechsel therefore accepts the formation of urea from ammonium car-
bamate, and according to him the alternating oxidation and reduction take
place in the following way:
H4N.O.CO.NH2 + 0 = H2N.O.CO.NH2 + H20
Ammonium carbamate
H2N.O.CO.NH2 + H2 = H2N.CO.NH2 + H2O.
Urea
Abel and Muirhead 1 have later observed an abundant eUmination of
carbamic acid in human and dog's urine after the administration of large
quantities of milk of lime, and the probaljility of the regular appearance of
this acid in normal acid-reacting human and dog's urine has been demon-
strated by M. Nencki and Hahn.^ These last-mentioned investigators
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. In this case the portal vein is directly connected with the inferior
vena cava, and communication is thus established between the two, so that
the blood of the portal vein flows directly into the vena cava, without
passing through the liver. 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 symptoms also appear
after the introduction of carbamate into the stomach, while the introduc-
tion of carbamate into the stomach of a normal dog had no action. As
these observers also found that the urine of the dog on w^hich the operation
was made was richer in carbamate than that of the normal dog, they con-
cluded that the symptoms were due to the non-transformation of the
ammonium carbamate into urea in the liver, and they consider the amnio-
' Drechsel, Ber. d. siichs. 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.
'' Hahn, Massen, Nencki et Pawlow, La fi.stule d' Eck de la veine cave inf^rieure et
de la veine porte, etc Arch, des sciences biol. de St, Petersbourg, 1, No. 4, 1892.
'' Rothberger and Winterberg, Zeitschr f. exp. Path, u Therap . 1, have found that
the phenomena of meat poisoning and the carbamic-acid intoxication are not identical.
FORMATION OF UREA. 553
nium carbamate as the substance from which the urea is derived in the
mammahan liver.
The view as to the formation of urea from ammonium carbamate does
not contradict the above statement as to the transformation of the carbonate
into urea, since we can imagine that the carl^onate is first converted into
carbamate with the expulsion of a molecule of water, and that this then is
transformed into urea with the expulsion of a second molecule of water.
F. HoFMEiSTER ^ has found in the oxidation of different members of the
fat series, as well as in amino-acids and proteins, that urea was formed
in the presence of ammonia, and he therefore suggests the possibility that
urea may be formed by an oxidation-synthesis. According to him, in the
oxidation of nitrogenous substances a radical CONH2, containing the
amide group, unites at the moment of formation with the radical NH2
remaining on the oxidation of ammonia, forming urea.
Besides the above-mentioned theories as to the formation of urea, there
are others which will not be given, because the only theory which has thus
far been positively demonstrated is the formation of urea from ammonium
compounds and amino-acids in the liver.
The liver is the only organ in which, up to the present time, a formation
of urea has been directh" detected '? 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 experi-
ments, 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 com-
pounds, a simultaneous increase in the elimination of ammonia is to be
expected.
The extirpation and atrophy experiments made on animals by different,
methods by Nexcki and Hahx, Slosse. Liebleix. Nexcki and Pawlow
Salaskin and Zaleski ^ have shown that sometimes a rather marked in-
crease of ammonia and a diminished elimination of urea takes place after
the operation, but also that there are cases in which, irrespective of the
pronounced atrophy, an alnmdant formation of urea occurs, and no appre-
' Arch. f. exp. Path. u. Pharni., 37.
' In regard to the investigations of Prevost and Dumas, Meissner, Volt, Grehant.
Gscheidlen and Salkowski, and others, on the role of the kidneys in the formation of
urea, see v. Schroeder, Arch. f. exp. Path. u. Pharm., 15 and 19, and Voit, Zeitschr.
f. Biologie, 4.
•* 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. Pdters-
bouTg. o. See also v. .Meister, Maly's Jahresbr., 25; Salaskin and Zaleski, Zeitschr. f.
physiol. Chcm., 29.
554 URINE.
ciable, if any, change in the proportion of ammonia to the total nitrogen
and urea is observed. After shutting out tlie organs of the posterior part
of the body, especially the hver and kidneys, from the circulation, Kauf-
MANN ^ 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 ^ on human beings
with cirrhosis of the liver, acute yellow atrophy of the liver, and phosphorus
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 further investigation must
be instituted before it will be possible to assume a reduced ability of the
organism to produce urea. An increased elimination of ammonia may, as
shown by ^Iijnzer in the case of acute phosphorus poisoning, be dependent
upon the formation of abnormally large quantities of acids, caused by ab-
normal metaliolism, and these acids require a greater quantit}"^ of ammonia
for their neutralization according 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 Hver has been especially shown by Salaskin and
Zaleski.3
• 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 investigation
can yield further information as to the extent and importance of the forma-
tion of urea in the liver from ammonium compounds.
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 salt-
peter. It melts at 132° C. At ordinary tem]ieratures it dissolves 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, decom-
' Compt. rend. soc. biol., 46, and Arch, de Physiol. (5), 6.
^ See Hallervorden, Arch, f . exp. Path. u. Pharm., 12; Weintraud, ibid., 31 ; Miinzer
and Winterberg, ibid., 33; Sta 'elman'i, Deiitsch. Arch, f . klin. Med., 33; Fawitzki,
ibid., 45; Miinzer, ibid., 52; Frankel, Berlin, klin. Wochenschr., 1878; Richtcr, ibid.,
1896; Morner and Sjoqvist, Skand. Arch. f. Physiol., 2, and Sj6.-]vist, Nord- l]ed.
Arkiv, 1892; Gumlich, Zeitschr. f. physiol. Chem., 17; v. Noorden, Lehrb. d. Pathol,
des Stoffwechsels, 2. Aufl., Bd. 1, 104.
^Zeitschr. f. physiol. Chem., 29.
PROPERTIES OF UREA, o55
poses, gives off ammonia, and leaves finally a non-transparent white residue
which, among other substances, contains cyanuric acid and biuret, which
latter dissolves in water, giving a beautiful reddish-violet Uciuid with copper
sulphate and alkali {biuret reaction). On heating with baryta-water or
caustic allcah. also in the so-called alkaline fermentation of urine caused by
micro-organisms, urea sphts 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 alkaUne solution of
sodium hypobromite decomposes urea into nitrogen, carbon dioxide, and
water according to the equation
CON2H4 + SNaOBr = 3NaBr + CO2 + 2H2O + Ng.
With a concentrated solution of furfurol and hydrochloric acid urea
in substance gives a coloration passing from yellow, green, blue, to violet,
and then beautiful purple-violet after a few minutes (Schiff's reaction).
According to Huppert ^ the test is best performed by taking 2 c.c. of a
concentrated furfurol solution, 4-6 drops of concentrated hydrochloric acid,
and adding to this mixture, which must not be red, a small crj'stal of urea.
A deep violet coloration appears in a few minutes.
Urea forms cr3-stanine compounds with many acids. Among these the
one with nitric acid and the one with oxahc acid are the most important.
Urea Nitrate, C0(NH2)2-HN03. On crj-stalhzing cjuickly tliis com-
pound forms thin rhombic or six-sided overlapping tiles, or colorless
plates, with an angle of 82°. When crj'stalhzing slowly, larger and
thicker rhombic pillars or plates are obtained. Tliis compound is rather
easily soluble in pure water, but is considerably less soluble in water con
taining nitric acid; it may be obtained by treating a concentrated solutiori
of urea with an excess of strong nitric acid free from nitrous acid. On
heating this compound it volatilizes without leaA^ng 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 j^laced 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 cr}'stals must be identified as m-ea nitrate by
heating and by other means.
Urea Ox-\late, 2. CO (NHo) 2 -1120204. This compound is more spar-
ingly 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 sokition of urea.
' Huppert-Xeubaucr, Analyse dcs Hams, 10. Aufl., 296.
556 URINE.
Urea also forms combinations with mercuric nitrate in variable propor-
tions. If a ver}' 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 combines also with salts, forming mostly
crystalUzable combinations, as, for instance, with sodium chloride, with
the chlorides of the heav}^ metals, etc. An alkaline but not a neutral
solution of urea is precipitated with 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.^ With phenylhj^drazine,
urea in strong acetic acid gives a colorless crystalline compound of phenyl-
semicarbazid, CcHsNH.NH.C0NH2, which is soluble with difficulty in cold water
and melts at 172° C. (Jaffe ').
The method of preparing urea from urine is in the main as follows: Con-
centrate the urine, which has been faintl}' acidified with sulphuric acid, at a
low temperature, add an excess of nitric acid, at the same time keeping the
mixture cool, press the precipitate well, decompose it in water with freshly
precipitated barium carbonate, dry on the water-bath, extract the residue
with strong alcohol, decolorize when necessary with animal charcoal, and
filter while warm. The urea which crystallizes on cooling is purified by
recrystallization from warm alcohol. A further quantity of urea may be
obtained from the mother-liquor by concentration. 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. But as Liebig's method
for the estimation of urea is really a method for determining the total
nitrogen, and as the physician has not always at hand the apparatus and
utensils necessary for a Kjeldahl determination, he often makes use of
this method ; hence both wnll be given in detail.
Kjeldahl's method consists in transforming all the nitrogen of the
organic substances into ammonia by heating with a sufficiently concentrated
sulphuric acid. The ammonia is distilled off after supersaturating with
alkali and the ammonia collected in standard sulphuric acid. The follow-
ing reagents are necessary' :
1. Sulphuric Acid. Either a mixture of equal volumes of pure concen-
trated and fuming sulphuric acid or else a solution of 200 grams phosphoric
^ See Tollens and his pupils, Bcr. d. doutsch. chem. Gescllsch., 29, 2751; Gold-
schmidt, ibid. ,2^, and Chcm. Centralbl., 1897, 1, 33; Thoms, ibid., 2, 144 and 737.
^ Zeitschr. f. physiol. Chem., 22.
ESTIMATION OF UREA. 557
anhydride in 1 liter of pure concentrated sulphuric acid. 2. Caustic soda free
from nitrates, 30-40 per cent solution. The quantity of this caustic-soda
solution necessary to neutralize 10 c.c. of the acid mixture must be deter-
mined. 3. Metallic mercury or pure yellow mercuric oxide. (The addition of
this facilitates the destiTiction 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. N/5 sulphuric acid and N/5 caustic-
soda solution.
In performing the determination 5 c.c. of the carefully measured and
filtered urine is placed in a long-neck Kjeldahl flask, a drop of mercury or
about 0.3 gram of mercuric oxide added, and then treated with 10-15 c.c.
of the strong sulphuric acid. The contents are heated very carefully,
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
coohng the contents are transferred to a voluminous distilling-flask, care-
fully 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 solu-
tion which has previously been treated with 30-40 c.c. 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, and the
regurgitation of the acid is prevented by having a Ijulb Ijlown on the exit-
tube. Not less than 25-30 c.c. of the standard acid is used for every 5 c.c.
of urine, and on completion of the distillation the acid is retitrated with
N/5 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.
Liebig's method is based upon the fact that a dilute solution of mer-
curic nitrate under proper conditions precipitates all the urea from its
solution, forming a compound of constant composition. As indicator, a
soda solution or a thin paste of sodium bicarbonate is used. An excess of
mercuric nitrate produces herewith a yellow or yellowish-brown compound,
while the compound of urea and mercury is white. PFLtJuER ^ has given
full particulars for this method; therefore we will describe Pflijger's
modification of Liebig's method.
Pfliiger, and Pfliiger and Bohland, in I Auger's Arch. 21, 36, 37, and 40.
558 URINE.
As phosphoric acid is also precipitated by the mercuric-nitrate solution, this
must be removed from the urine by the addition of a baryta solution before titra-
tion. Pfluger also suggested that the acidity produced by the mercury solution
be neutralized during titration by the addition of a soda solution. The liquids
necessary for the titration are the following:
1. M crcuric-nitratc Solution. This solution is calculated for a 2 per cent urea
solution, and 20 c.c. of the first should correspond to 10 c.c. of the latter. Each
cubic centimeter of the mercury solution corresponds to 0.01 gram urea. As a
small excess of HgO is necessary in the urine to cause the final reaction (with
alkali carbonate or bicarbonate) to appear, each cubic centimeter of the mercury
solution must contain 0.0772 instead of 0.0720 gram HgO. The mercury solution
contains therefore 77.2 grams HgO in 1 liter.
The solution may be prepared from pure mercury or mercuric oxide by dis-
solving in nitric acid. The solution, freed as completely as possible from an
excess of acid, is diluted by the careful addition of water, stirring meanwhile,
until it has a specific gravity of 1.10, or a little higher, at 20° C. The solution
is standardized with a 2 per cent solution of pure urea which has been dried over
sulphuric acid and the operation conducted as will be described later. If the
solution is too concentrated, it is corrected by carefully adding the necessary
amount of water, avoiding the precipitation of the basic salt, and titrating again.
The solution is correct if 19.8 c.c. of it, added at once to 10 c.c of the urea solution
and the quantity (11-12 c.c. or more) of normal soda solution necessary to nearly
completely neutralize the liquid, gives the final reaction when exactly 20 c.c. of
the mercury solution has been employed.
2. Baryta Solution. This consists of 1 vol. of barium nitrate and 2 vols, of
barium-hydrate solution, both saturated at the ordinary temperature.
3. Normal Soda Solution. This solution contains 53 grams of pure anhydrous
sodium carbonate in 1 liter of water. According to PFLiJGER a solution having
a specific gravity of 1.053 is sufficient. The amount of this soda solution neces-
sary to completely neutralize the acid set free during the titration is determined
by titrating with a pure 2 per cent urea solution. To facilitate operations a table
can be made showing the cjuantity of soda solution necessary when from 10 to
35 c.c. of the mercmy solution is used.
Before the titration the following must be considered. The chlorides of
the urine interfere with the titration in that a part of the mercuric nitrate
is transformed into mercuric chloride, which does not precipitate the urea.
The chlorides of the urine are therefore removed by a silver-nitrate solu-
tion, which also removes any bromine or iodine compounds which may
exist in the urine. If the urine contains proteid in noticeable amounts, it
must be removed by coagulation and the addition of acetic acid, but care
must be taken that the concentration and the volume of the urine are not
changed during these operations. If the urine contains ammonium car-
bonate in noticeable quantities, caused by alkaline fermentation, this titra-
tion method cannot be applied. The same is true of urine containing
leucine, tyrosine, or medicinal preparations precipitated by mercuric
nitrate.
In cases where the urine is free from proteid er sugar and not specially
poor in chlorides, the quantity of urea, and also the approximate quantity
of mercuric nitrate necessary for the titration, may be learned from the
specific gravity. A specific gravity of 1.010 corresponds to about 10 p. m.,
ESTIMATION OF UREA. 559
a specific gravity of 1.015 generally somewhat less than 15 p. m., and a
specific gravity of 1.015-1.020 about 15-20 p. m. urea. With a specific
gravity higher than 1.020 the urine generally contains more than 20 p. m,
of urea, and above this point the amount of urea increases much more
rapidly than the specific gravity, so that with a specific gravity of 1.030 it
contains over 40 p. m. urea. Fever-urines with a specific gravity above
1.020 sometimes contain 30-40 p. m. urea, or even more.
Preparation for the Titration. If a large amount of urea is sus-
pected from a high specific gravity, the urine must first he diluted with a
carefully measured quantity of water, so that the amount of urea is re-
duced below 30 p. m. In a special portion of the same urine the amount of
chlorides is determined by one of the methods w^hich will 1 )e given later, and
the number of cubic centimeters of silver-nitrate solution necessarj- is
noted. Then a larger quantity of urine, say 100 c.c, is mixed with one-
half or, if this is not sufficient to precipitate all the sulphuric and phos-
phoric acids, with an equal volume of the baryta solution; it is then allowed
to stand a little while, and the precipitate is filtered through a dried filter.
From the filtrate containing the urine diluted with water a proper quan-
tity, corresponding to about 60 c.c. of the original urine, is measured, and
exactly neutralized with nitric acid added from a l^urette, so that the exact
quantity employed is known. The neutralized mixture of urine and barj^ta
is treated with the proper quantity of silver-nitrate solution necessary to
completely precipitate the clilorides, which were ascertained by a previous
determination. Tlie mixture, containing a known volume of urine, is now
filtered through a dried filter into a flask, and from the filtrate an amount
is measured off corresponding to 10 c.c. of the original urine.
Execution of the Titration. Nearly the whole quantity of the mer-
curic-nitrate solution, which is judged from the specific gravity of the urine
to be the minimum amount required, is added at once, and immediately
aftei-wards the quantity of soda solution necessary, as indicated by the
table. If the mixture becomes yellowish in color, then too much mercury
solution has been added and another determination must be made. If the
test remains white, and if a drop taken out and placed on a glass plate
with a dark background and stirred with a drop of a thin paste of sodium
bicarbonate does not give a yellow color, the addition of mercury solution is
continued by adding repeatedly at first J and later tV c.c, and testing
after each addition in the following way : A drop of the mixture is placed
on a glass plate with a dark background beside a small drop of the bicar-
bonate paste. If the color after stirring the two drops together is still
white after a few seconds, then more mercury- solution must be added; if,
on the contrarj% it is yellowish, then — if not too much mercurj' solution
has been added by inattention — the result to xV c.c. has been found. B}^
this approximate determinatio, which is sufficient in many cases, we haven
560 URINE.
fixed the minimum amount of mercury solution necessary to add to
the quantity of urine in question, and we now proceed to the final deter-
mination.
A second quantity of the filtrate, corresponding to 10 c.c. of the
original urine, is filtered, and the. same quantity of mercury solution
added at one time as was found necessary to produce the final reaction,
and immediately after the corresponding amount of soda solution, which
must not indicate the end of the reaction. Then continue adding the
mercury solution xV <'-c. at a time without neutralizing with soda, until
a drop taken out and mixed with the soda solution gives a yellow color-
ation. If this final reaction is obtained after the addition of 0.1-0.2 c.c,
then the titration may be considered as finished. If, on the contrary, a
larger quantity is necessary, the addition of the mercury solution must
be continued until a final reaction is obtained with simple carbonate, and
the titration repeated again, adding the quantity of mercury solution used
in the previous test at one time, and also adding the corresponding amount
of soda solution. If then the end reaction is obtained by the addition of
i^Tj c.c, the titration may be considered as finished.
If in each titration a quantity of filtrate containing urine and baryta
correspcnding to 10 c.c. of the original urine is used, then the calculations
are ver\' simple, since 1 c.c. of mercuric-nitrate solution corresponds to
0.01 gram of urea. As the mercuiy solution is made for a 2 per cent urea
solution, and as the filtrate of urine and liaryta generally contains less
urea (if the quantity of urea is above 2 per cent, it is easy to avoid any mis-
take by diluting the urine at the Ijeginning of the operation), a mistake
occurs here which can be corrected in the following way, according to
Pfluger: To the measured volume of the filtrate from the urine (the
filtrate with bars'ta after neutralization with nitric acid, precipitation vnth
silver nitrate and filtration) the quantity of normal soda solution employed
is added, and from this sum the volume of mercury solution used is sub-
tracted. The remainder is then multiplied by 0.08, and the product sub-
tracted from the number of cubic centimeters of mercury solution used.
For example, if the filtrate (urine and baryta + nitric acid + silver nitrate)
measured 25.8 c.c, and the number of cubic centimeters of soda solution
used in the titration was 13.8 c.c, and of the mercury solution 20.5 c.c, we
have then 20.5-|(39.6-20.5)X0.081 = 20.5- 1.53= 18.97, and the corrected
quantity of mercur\^ solution is therefore 18.97 c.c. If the cubic centi-
meters of the filtrate (in this case 25.8 c.c.) correspond to 10 c.c. of the
original urine, then the amount of urea is 18.97X0.01 = 0.1897=18.97 p. m.
urea.
Besides the urea other nitrogenous constituents of the urine are precipi-
tated by the mercury solution. In the titration we really do not obtain
the quantity of urea, but, as PFLtJGER has shown, the total quantity of
ESTIMATION OF UREA. 561
nitrogen in the urine expressed as urea. As urea contains 46.67 per cent
N, the total quantity of nitrogen in the urine may be calculated from the
quantity of urea found. The results obtained by this calculation corre-
spond well, according to Pfluger, with the results found for the total
nitrogen as determined by Kjeldahl's method.
Glassivl\nx ^ has recently suggested a modification of the Liebig-
PFLiJGER titration method which consists in precipitating the urea with
an excess of mercuric-nitrate solution and then determining the excess
of mercuric nitrate in the filtrate by means of ammonium sulphocyanide.
Among the methods suggested for the special estimation of urea, that of
Morner-Sjoqvist, in combination with Folix's method, is perhaps the
most tiTistv.orthy and readily performed. For this reason only this method
will be given in detail, while we must refer to sj^ecial works for the other
methods, such as Buxsex's method with its many modifications as sug-
gested by Pfluger, Bohlaxd and Bleibtreu.^
Principle oj M orner-Sjoqvist' s Method.^ According to this method the
nitrogenous constituents of the urine, with the exception of urea, ammonia,
hippuric acid, creatinine, and traces of allantoin, are precipitated by a mix-
ture of alcohol and ether after the addition of a solution of bariun chloride
and barium hydrate or in the presence of sugar with solid barium hydiate.
The urea is determined in the concentrated filtrate, after dri\-ing off the
ammonia, by Kjeldahl's nitrogen estimation. Because of the slight error
due to the presence of liippuric acid and creatinine, several modifications
have been suggested by Salaskix and Zaleski and by Brauxsteix *
These errors are best prevented, according to ^Iorxer, by the use of
FoLix's method.
Principle of Folin's Method.^ On heating urea with hydrochloric acid
and cr}'stalUne magnesium chloride, which melts in its water of cr}-stalUza-
tion at 112-115° C. and then boils at about 150-155° C, the urea is com-
pletel}" 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 pre-
viously existing in the urine must be specially determined.
Determination of Urea hy the Morner-Sfoqvist and Folin Method.^ Five
c.c. of the urine are treated with 1.5 grams of powdered barium hydroxide,
and when as much of this is dissob-ed as possilde by gentlv mixing, it is
»Ber. d. d. chem. Gesellsch., 39.
2 Pfliiger's Arch., 38, 43, and 44.
^Skand. Arch. f. Physiol., 2, and Monier, ibid., 14, where the recent literature
may also be found.
* Braunstein, Zeitschr. f. physiol. Chem., 31; Salaskin and Zaleski, ibid., 28.
5/&;(/.,32, 36, and37.
' See Morner, Skand. Arch. f. Physiol., 14.
562 URINE.
precipitated by 100 c.c. of the alcohol and ether mixture (^ vol. ether). On
the iollowing 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 hquid is treated with
2 c.c. of hydrochloric acid of sp. gr. 1.124 (for 5 c.c. urine), and carefully
transferred to a flask of 200 c.c. capacit}', and evaporated to dryness on
the water-ljath. Then add 20 grams of cr\'staUine magnesium chloride to
the contents of the flask and 2 c.c. 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 cooUng it is diluted to about | 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 CO 2 and cooling, the acid is retitrated. Corrections must be made for
the ammonia of the urine and for that contained in the magnesium 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^ according to ]\1orxer, othenvise the humin sub-
stances produced from the sugar take up and retain nitrogen.
Knop-Hufner's method ^ is based on the fact that urea, by the action of
sodium hypobromite, spHts into water, carbon dioxide (which dissolves in the
alkali), and nitrogen, whose volume is measured (see page 555). This method
is less accurate than the preceding ones, and therefore in scientific work it is
discarded. It is of value to the physician and for practical j^urposes, because
of the ease and rapidity ■nith which it may be performed, even though it may
not give very accurate results. For practical piu-poses a number of different
apparatuses 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, Schoxdorff has proposed a method where the
proteins and extractives are first precipitated by a mixture of phospho-
tungstic acid and hydrochloric acid, and then the filtrate made alkahne
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 diox-
ide 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 (PFi.iJGER's Arch., 62). See also Hoppe-Seyler-
Thierfelder's Handbuch, 7. Aufl.
Urein is the name given by Ovid Moor to a product which he obtained by
extracting urine, which had been evaporated to a syrujD, 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 but that urein is a mixture of sub-
stances. According to MoOR,^ the amount of urea in the urine is only about
' Knop, Zeitschr. f. analj^l. Chem., 9; Hiifncr, Journ. f. prakt. Cheni. (N. F.), 3.
In regard to the extensive literature, see Huppert-Neubauer, 10. Aufl., 304, and follow-
ing.
2 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.
CREATININE. 563
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 mth the urea cannot
be denied a 'priori. From the investigations published so far it must be said
that Moor's assertions are not sufficiently groimded.'
\H
Carbamic Acid, CH3N02=CO <qjj-. This acid is not known in the free state,
but only as salts. Ammonium carbamate is produced by the action of dry ammo-
nia on dry carbon dioxide. Carbamic acid is also produced by the action of
potassium permanganate on protein and several other nitrogenous organic bodies.
The occcurrence of carbamic acid in human and animal urines has already
been considered in connection with the formation of urea. The calciiun salt,
which is soluble in water and ammonia but insoluble in alcohol, is the most
important 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, MACLEonand Haskixs 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.^
Carbamic-acid ethylester (urethane), as shown by Jaffe,^ maj' pass, by the
mutual action of alcohol and urea, into the alcoholic extract of urine when one
is working ^v-ith large quantities.
.NH CO
Creatinine, C4H7X3O, or NH : q/ I , is generally considered as
\X(CH3).CH2
the anhydride of creatine (see page 455) found in the muscles. It occurs
in human urine and in that of certain mammaUa. It has also been found
in ox-blood, milk, though in veiy small amounts, and in the flesh of certain
fishes.
.loHNSOx'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.^
The quantity of creatinine in human urine is, in a grown man voiding a
normal quantity of urine in the course of a da}-, 0.6-1.3 grams (Xeubauer),
or on an average 1 gram. Johnson 5 found 1.7-2.1 grams per day, and
similar results have been obtained by Hoogexhuyze and Verploegh.^
The quantity of creatinine vsith a diet free from meat is, according to
FoLEN'.'^ somewhat variable for different individuals, but is constant for
'See Kubiabko, Maly's Jahresber., 31, 41.5; Erben, Zeitschr. f. physiol. Chem.
38; Folin, ibid., 37; Gies, Journ. Amer. Chem. See, 25; Haskins, Anier. Journ. of
Physiol., 12; Lippich, Zeitschr. f. phy,siol. Chem., 48.
^ Nolf , Zeitschr. f. physiol. Chem., 23; Macleod and Haskins, Amer. Journ. of
Physiol., 12.
^Zeitschr. f. physiol. Chem., 14.
*S. Johnson, Proceed. Roy. Soc, 42, 43; Chem. News, 55; Toppelius and Pern-
merehne, Arch. f. Phann., 234; Woemer, Arch. f. {Axia.i. u.) Physiol., 1898.
* Huppert-Neubauer, Hamanalyse, 10. Aufl., 387.
* Zeitschr. f. physiol. Chem., 4(5.
' Amer. Journ. of Physiol. 13; af. Klercker, Hofmeister's Beitrage, 8.
564 URINE.
the same person. He found the quantity never below 1 gram and often
between 1.3 and 1.7 grams. Nurslings also eliminate creatinine, although
the quantity is only small (Hoogenhuyze and Verploegh). The quantity
of creatinine is dependent upon the food in so far as it is increased l)y meat
diet, but otherwise, according to Folin, it is not dependent upon the food.
The creatinine is, according to him, the product of the endogenous metabo-
lism of the cells, and its quantity does not dejDend, as was also shown later
by Klercker, upon the quantity of protein food introduced and catabo-
lizcd. The eUmination of creatinine, therefore, does not run parallel with
the elimination of urea and is not correspondingly greater with food very
rich in protein than with food very poor in protein.
The statements as to the behavior of the creatinine elimination with
work are very contradictory .^ 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.
Little is known about the behavior of creatinine in diseases. In cases
with an increase in metabolism the quantity is said to rise, while in other
cases, as in ansemia and cachexia mth reduced metabolism, the quantity
is lessened.
Creatinine crystalhzes 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 statements
in regard to its solubilities differ widely.- It is more soluble in warm alcohol
and nearly insoluble in ether. In alkaline solution creatinine is converted
into creatine ver}^ easily on warming.
Creatinine gives an easily soluble crystalline compound with hydro-
chloric acid. A solution of creatinine acidified with mineral acids gives
cr}'stalline precipitates with phosphotungstic and phosphomolybdic acids
even in very dilute solutions (1:10 000) (Kerner, Hofmeister^). It is
precipitated, like urea, by mercuric-nitrate solution and also by mercuric
chloride. On treating a dilute creatinine solution with sodium acetate and
then with mercuric chloride a precipitate of glassy globules having the
composition 4 (C4H7N30.HCl.HgO)3HgCl2 separates on standing some time
(Johnson). Among the compounds of creatinine, that with zinc chloride,
creatinine zinc chloride, (C4H7N30)2ZnCl2, is of special interest. This com-
* The literature on this subject may be found in Hoogenhuyze and Verploegh, I. c.
^ See Huppert-Neubauer, 10. Aufi. and Hoppe-Seyler-Thierfolder's Handbuch,
Aufl.
^ Kerner, Pfliiger's Arch., 2; Hofmeister, Zeitschr. f. physiol. Chem., 5.
CREATININE. 565
bination 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 compound, hence they must
not be present; this, however, may be prevented by an addition of sodium
acetate. In the impure state, as ordinarily obtained from urine, creati-
nine zinc chloride forms a sandy, yellowish powder which under the micro-
scope appears as fine needles forming concentric groups, mostly complete
rosettes or yellow balls or tufts, or grouped as brushes. On slowly crys-
tallizing or when very pure, more sharply defined prismatic crystals are
obtained. The compound is sparingly soluble in water.
Creatinine acts as a reducing agent. JMercuric oxide is reduced to-
metallic mercurj^, and oxaHc acid and methylguanidine (methyluramine)
are formed. Creatinine also reduces cupric hydrate in alkaline solution^
forming a colorless soluble compound, and only after continued boiling
with an excess of copper salt is free subxoide of copper formed. Creatinine
interferes with Trommer's test for sugar, partly because it has a reducing
action and partly by retaining the copper suboxide in solution. The com-
pound -with copper suboxide is not soluble in a saturated soda solution,
and if a Uttle creatinine is dissolved in a cold saturated soda solution and
then a few drops of Fehling's reagent added a white flocculent compound
separates after heating to 50-60° C. and then cooling (v. ^Iaschke's ^
reaction). An alkahne bismuth solution (see Sugar Tests) is not reduced
by creatinine.
If we add a few drops of a freshly prepared verj' 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's ^ reaction) . If the cold yellow
solution is neutralized and treated with an excess of acetic acid a crystalline
precipitate of a nitroso-compound (C4H6N4O2) of creatinine separates on
stirring (Kramm 3). If, on the contrary, the yellow solution is treated with
an excess of acetic acid and heated, the solution becomes first green and then
blue (Salkowski ^) ; finally a precipitate of Prussian blue is obtained. If
a solution of creatinine in water (or urine) is treated with a watery solution
of picric acid and a few drops of a dilute caustic-soda solution, a red colora-
tion lasting several hours occurs immediately at the ordinarj^ temperature,
which turns yellow on the addition of acid (Jaffe's ^ reaction). Acetone
gives a more reddish-yellow color. Dextrose gives with this reagent a red
coloration only after heating.
* Zeitschr. f. analyt. Chem., 17.
^ Ber. d. deut.sch. chem. Gesellsch., 11,
' Centralbl. f. d. med. Wissensch., 1897.
* Zeitschr. f. physiol. Chem., 4.
Uhid., 10.
566 URINE.
In preparing creatinine from urine the creatinine zinc chloride is first
prepared according to Neubauer's ^ method. One hter or more of urine is
treated with milk of lime until alkaline and then CaCl2 solution is added
until all the phosphoric acid is precipitated. The filtrate is evaporated to a
syrap after faintly acidifying with acetic acid and this is treated while still
warm with 97 per cent alcohol (about 200 c.c. for each liter of urine). After
■about twelve hours it is filtered and the filtrate treated first with a little
.sodium acetate and then ^^■ith an acid-free zinc chloride solution of a specific
gravity of 1.20 (aliout 2 c.c. for each liter of urine.) After thorough stir-
ring it is allowed to stand forty -eight hours and the precipitate is collected
on a filter and washed with alcohol. The creatinine zinc chloride is dis-
solved in hot water, boiled with lead oxide, filtered, the filtrate decolorized
by animal charcoal, evaporated to dryness, and the residue extracted with
strong alcohol (which leaves the creatinine undissolved) . The alcoholic ex-
tract is evaporated to the point of crystallization, and the crystals purified
by recrj^stallization from water.
Creatinine may also be prepared from urine by precipitating with a
mercuric-chloride solution according to either Maly's or Johnson's ^
process.
The best method for preparing creatinine is the following, suggested by
FoLiN.^ The creatinine is first precipitated as the double picrate of creati-
nine 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 con-
verted into the doul)le zinc-chloricle salt and this last treated with moist
lead hydroxide. Alter the removal of the lead the solution contains a mix-
ture of f-reatinine and creatine, which last is completely transformed into
creatinine by heating tor 48 hours with normal sulphuric acid. After exact
neutralization with barium-hydrate solution it is concentrated to the point
of crystallization.
The quantitative eMiwation of creatinine may be performed according to
Neubauer's method for the preparation of creatinine, or more simply ]iy
Salkowski's 4 modification 01 this method. 240 c.c. of the urine freed from
])roteid (Ijy l^oiling with acid) and from sugar (l\y lermentation with yeast)
are made alkaline with milk of lime, and precipitated by CaClo and made up
to 300 c.c: 250 c.c. (=200 c.c. urine) of this are measured off, neutralized
or made only faintly acid with acetic acid and evaporated to aboiit 20 c.c,
then thoroughly stirred with an equal volume of al^solute alcohol, and
completely transferred to a 100, c.c. flask which contains some alcohol,
the residue in the dish being washed with alcohol. On thorough shaking
and cooling, the flask is filled up to the 100-c.c. mark with al)solute alcohol
and allowed to stand twenty-four hours. 80 c.c. (=160 c.c. urine) of
the filtrate are collected in a beaker and treated with 0.5-1 c.c. of zinc-
chloride solution, and the covered beaker is left standing in a cool place
* Ann. d. Chom. u. Pharm., 119.
^Maly, Annal. d. Chem. u. Pharm,, 159; Johnson, Proceed. Roy. See, 43.
^ Zeitschr. f. physiol. Chem., 41.
* Zeitschr. f. physiol. Chem., 10 and 14.
CREATININE AND XANTHOCREATININE. 567
for two or three daj-s. The precipitate is collected on a small dried and
weighed filter, using the filtrate to wash the crystals Irom the beaker.
After allowino- the crystals to completely drain off, they are washed with
a little alcohol until the filtrate gives no reaction for chlorine, and dried at
100° C. 100 parts of creatinine zinc chloride contain 62.44 parts of creati-
nine. As the precipitate is never quite pure, the quantity of zinc must be
carefully determined, in exact experiments, by evaporating with nitric acid,
heating, washing the oxide of zinc with water (to remove any NaCl), drj'ing,
heating, and weighing. 22.4 parts zinc oxide correspond to 100 parts
creatinine zinc-chloride. Instead of weighing, the nitrogen can be deter-
mined by Kjeldahl's method and the creatinine calculated from this.
FoLiN 1 has suggested a colorimetric method for determining creatinine
which is based upon Jaffe's picric-acid reaction and is as follows: 10 c.c.
of the urine are treated in a graduated flask of 500 c.c. capacity with 15 c.c.
of a 1.2 per cent solution of picric acid and 5 c.c. of a 10 per cent NaOH
solution. After shaking and allowing to stand for 5 minutes it is diluted
with water to 500 c.c. and mixed. This solution is now compared in a
DuBOSCQ colorimeter with a ^ normal potassium-bichromate 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 c.c. picric-acid solution and 5 c.c. NaOH solution
and dilution to 500 c.c. The calculations are simjile. 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 quantitv of creatinine
8 1
in 10 c.c. of the urine will be =;7^X 10, or 11.25 milligrams. This method
is not only simple, but also, according to Folin, Hoogenhuyze and Ver-
PLOEGH, gives much more trustworthy results than Neubauer's method.
In regard to other methods, see the works of Kolisch and Gregor.^
Xanthocreatinine, CgHjoN^O. This body, which was first prepared from meat
extract by Gautier, has been found by Monari in dog's urine after the injection
of creatinine into the abdominal cavity, and in human urine after several hours
of exhausting marching. According to Colasanti it occurs to a relatively greater
extent in lion's urine. Stadthagex ^ 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 chloricle, which
crystallizes in fhie needles. Xanthocreatinine has a poisonous action.
HN— CO
Uric Acid, Ur, C5H4N4O3, 2, 6, 8-trioxypurine = OC C— NH\
I II >C0, has
HN— C— NH/
^ Z:^itschi. f. phyiol. Chc-ni., 41.
2 Kolisch, Centralbl. f. innere Med., 1895; Gregor, Zeitschr. f. physiol. Chem., 31.
3 Gautier, Bull, de racad. de med. (2) 15, and Bull, de la .-oc. chira. (2), 48: Monari.
Maly's Jahresber., 17; Colasanti, Arch. ital. d. Biologie, 15, Fasc. 3; Stadthagen,
Zeitschr. f. klin. Med., 15.
568 URINE.
been prepared 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 acid (E. Fischer
and Tullner), and finally E. Fischer and Ach ^ have prepared uric acid
from pseudouric acid by heating with oxalic acid to 145° C.
On strongly heating uric acid it decomposes with the formation of
urea, hydrocyanic acid, cyanuric acid, and ammonia. On heating with
concentrated hydrochloric acid in sealed tubes to 170° C. it splits into
glycocoll, carbon dioxide, and ammonia. By the action of oxidizing agents
splitting and oxidation take place, and either monoureides or diureides
are produced. By oxidation with lead peroxide, carbon dioxide, oxalic
acid, urea, and allantoin, which last is glyoxyldiureide, are produced (see
below) . By oxidation with nitric acid in the cold, urea and a monoureide,
the mesoxalyl urea, or alloxan, are obtained, C5H4N403 + 0 + H20=
C4H2N2O4+ (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, C4H5N3O4.2 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,^ uric acid is quantitatively split hereby into urea and carbon
dioxide, according to the equation
C5H4N403 + 2H20 + 30 = 3C02+2CO(NH2)2.
Uric acid occurs most abundantly in the urine of birds and of scaly
ampliibians, in which animals the greater part of the nitrogen of the urine
appears in this form. Uric acid occurs frequently in the urine of carniv-
orous mammalia, but is sometimes absent; in urine of herl^ivora it is habitu-
ally 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 Ijirds), and in the brain. It haljitually occurs in the blood
of birds. Traces have been found in human l^lood under normal con-
" Horbaczewski, Monatshefte f. Chem., 6 and S; Behrend and Roosen, Ber. d. d.
chem. Gesellsch., 21; Fischer and Tiillner, ibid., 35; Fischer and Ach, ibid., 28.
^ See Sundwik, Zeitschr. f. physiol. Chem., 20 and 41; also Behrend, Annal. d. Chem.
u. Pharm., 333.
' See Chem. Centralbl., 1903, where the other investigators are cited, and Centralbl.
f. Physiol., 19.
URIC ACID. 569
ditions. Under pathological conditions it occurs to an increased extent
in the blood, as in pneumonia and nephritis, but especially in leuca-mia and
sometimes also in arthritis. Uric acid also occurs in large quantities in
''chalk-stones," certain urinary calcuU, 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 considerably
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 in-
creased, 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, l)ut the investigations of Hirschfeld, Rosenfeld and
Orgler, Sivex, Burian and Schur,^ and many others have positively
proved 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 statement that the elimination of uric
acid is smaller with a vegetaljle diet than with an animal diet, when the
quantity may be 2 grams or more per twenty-four hours, is explained by
this.3
The statements in regard to the influence of other circumstances, as
also of different substances, on the elimination of uric acid are rather con-
tradictory. This is in part due to the fact that the older investigators
used an inaccurate method (Heintz), and also that the extent of uric-acid
elimination is dependent in the first place upon the individuality. Thus
the statements in regard to the action of drinking-water * and of alkalies ^
are very contradictory. Certain medicines, such as quinine and atropine,
diminish, while others, such as pilocarpine and also, as it seems, salicyhc
acid, 6 increase the elimination of uric acid.
' Philos. Trans. Roy. Soc, 186, B, 661.
2 See the extensive review of the literature in Wiener, "Die Hamsaure," in Ergeb-
nisse der Physiologie, 1, Abt. 1, 1902.
^ J. Ranke, Beobachtungen und Versuche i'lber die Ausscheidung der Hamsaure,
etc. (Miinchen, 1858); Mares, Centralbl. f. d. med. Wissensch., 1888; Horbaczewski,
Wien. Sitzungsber., 100, Abt. 3, 1891. In regard to the action of various diets the
reader is referred to the above-cited authors, and especially to A. Hermann, .\rch. f.
klin. Med., 43, and Camerer, Zeitschr. f. Biologic, 33, and Folin, Amer. Journ. of
Physiol., 13.
* See Schondorff, Pfliiger's Arch., 46, which contains the pertinent literature.
^ See Clar, Centralbl. f. d. med. Wissen&ch., 1888; Haig, Journ. of Physiol., 8; and
A. Hermann, Arch. f. klin. Med., 43.
" See Bohland, cited from Maly's Jahresber., 26; Sclireiber and Zaudy, ibid., 30.
570 URINE.
Little is known with positi\eness in regard to the elimination of uric
acid in disease. In acute diseases with crises the eUmination of uric acid is
increased after the crisis, wliile the older statements that the uric acid is
habitually increased in fevers has been contradicted by many. The state-
ments in regard to the elimination of uric acid in gout and nephritis are also
uncertain and contradictory. In leucaemia the ehmination is increased
absolutely as well as relatively to the urea, and the relationship between
the uric acid and urea (total nitrogen recalculated as urea) may be even
1:9, while under normal conditions, according to different investigators,
it is 1:10 to 66 to 100. ^
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 investigators, and we
are sure that uric acid can Ije produced from purine bodies either outside
or inside the animal body, and also that food rich in nucleins (especially
the thymus gland) increases the ehmination of uric acid and purine bases
(alloxuric bases ^). 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 conse uent destruction of leucocytes, has
been nearly generally discarded. A.t 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
deri\ed trom the nucleins of the destroyed cells of the body and in part
irom the nucleins or free purine bases introduced with the food. It is there
fore possible to admit with Burian and Schur ^ of a double origin for the
uric acid as well as the urinar}' purines (all ]:)urine bodies ot the urine, in-
cluding the uric acid), namely, an endogenous and an exogenous origin.
Burian and Schur attempted to determine the quantity of endogenou.s
urinary purines by feeding with sufficient food, but as free as possil^le from
purine bodies, and they found that tliis quantity was constant for every
indi\idual, while it was variable for different persons. The observations
of SivEX, RocKWooD,* and others have also led to the same results. Other
' In regard to the extensive literature on the elimination of uric acid in disease
we must refer to special works on internal diseases.
^ 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.
5 PHuger's Arch., 80, 87, and 94.
* Amer. Joum. of Physiol., 12.
FORMATION OF URIC ACID. 571
investigators, such as Sciireiber and "Waldvogel, Loewi, and Folix,^
have arrived at somewhat different results, or they draw differentde ductions
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 onh' ver}- 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 their 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 (Horbaczew^ski, Spitzer and
Wiener 2), recently Schittexhelm, Buriax, Jones and Partridge,^ by
more careful investigations have shown that enzymes of different lands
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 b}^ Burian, the uric acid is formed. The
deamidizing enzymes seem to be present in most organs, yet there exists,
in tliis regard, a marked difference between certain animals; thus guanase
occurs in the ox-spleen but not in the pig-spleen (Jones and Winterintz) .
The oxidase occurs especially in the spleen (though not in the dog spleen,
according to Schittex'^helm) and liver, but also in the muscles and lungs.
Still, as ScHiTTEXHELM 4 has especially shown, a verj- marked difference
exists in animals, and the activity of the organs of various animals requires
a very thorough investigation.
Jones and Austrl^n ^ have carried on investigations on the occurrence
in different organs of pigs, dogs, and rabbits, of enzymes taking part in the
nuclein metabolism. The occurrence of these enz^^mes in the liver is of
special interest. In the above-mentioned animals and in the ox they found
the following: The l^eef-liver contains guanase, adenase, and xanthine
oxidase, and produces uric acid from guanine as well as from adenine.
Guanase is absent from the pig-liver, while adenase and xanthine oxidase
are present. In these animals the liver forms uric acid from adenine but
not from guanine. The rabbit-liver does not contain any adenase and
hence uric acid is formed only from guanase. wliile the dog-liver, on the
contrar}', which contains guanase but neither adenase nor xanthine oxidase,
cannot form uric acid from <ruanine nor from adenine.
' Schreiber and Waldvogel, .\rch. f. exp. Path. u. Phann., 42; O. Loewi, ibid., 44
and 4o: Folin, Amer. Journ. of Physiol., 13.
' See foot-note 2, page .570.
^ Schittenhelm, Zeitschr. f. physiol. Chem., 42, 43, 45, and 46; Burian, ibid., 43-
Jones and Partridge, ibid.. 42; .Jones and Wintemitz ibid., 44; Jones, ibid., 45.
* Zeitschr. f. physiol. Chem., 46.
' Zeitschr. f. physiol. Chem., 48.
572 URINE.
In birds the conditions are different, v. Mach ^ has shown that in these
animals a part of the uric acid may be formed from the purine bodies. The
ehief quantity of uric acid, however, is undoubtedly formed in birds by
synthesis.
The formation of uric acid in birds is increased by the administration
of ammonium salts (v. Schroder), and urea acts in a similar manner in
these animals (^Ieyer and Jaffe). Minkowski observed in geese with
extirpated livers a ver\' significant decrease in the elimination of uric acid,
while the elimination of ammonia was increased to a corresponding degree.
This indicates a participation of ammonia in the formation of uric acid in
the organism of birds ; and as ^Iinkowski has also found after the extirpa-
tion of the liver that considerable amounts of lactic acid occur in the urine,
it is probable that the uric acid in birds is produced in the liver by syn-
thesis, perhaps from lactic acid and ammonia; although, as Salaskin and
Zaleski and Lang have shown, after the extirpation of the liver primarily
an increase in the formation of lactic acid occurs and this causes an in-
crease in the eUmination of ammonia (neutralization ammonia) . The direct
proof for the uric-acid formation from ammonia and lactic acid in the
hver of birds has been given by Kow'alewski and Salaskin ^ 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. Of these
leucine, glycocoU, and aspartic acid increase the elimination of uric acid in
birds (v. Knieriem^), but whether they are first decomposed with the
splitting off of ammonia is still unknown.
The possibilit}' of a formation of uric acid from lactic acid has been
shown in another manner by Wiener,* namely, by feeding birds with urea
and lactic acid and different non-nitrogenous substances, oxy-, keto-, and
dibasic acids of the aUphatic series. The dibasic acids, with a chain of 3
carbon atoms or their ureides, showed themselves most activ^e as uric-acid
formers, and Wiener is therefore of the opinion that the active substances
must first be converted in'o 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.
' Arch. f. exp Path. u. Pharm., 24.
* V. Schroder, Zeitschr. f. physiol. Chem., 2; Meyer and Jaff^, 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.
' Zeitschr. f. Biologie. 13.
* 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. 573
Among the substances tested, only tartronic acid and its ureide, dialuric acid,
have shown themselves active in the experiments with the isolated organs, and
Wiener therefore also considers that the other acids must be first converted into
tartronic acid by oxidation or reduction. From lactic acid, CH3.CH(0H).C00H,
we first obtain tartronic acid, C00H.CH(0H).C00H, which by the attachment
of a urea residue forms dialuric acid, CO<ytt pQ>CHOH, and from this, by the
attachment of a second urea residue, uric acid is formed.
We cannot give any positiv^e answer as to the question w^hether uric acid
is formed by synthesis also in man and other mammalia. Wiener has
in part 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 feed-
ing lactic acid and dialuric acid to man. According to Burian ^ 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 contraiy they accelerate the enzymotic
oxidation of purine bases and hence, according to Burian, this explains,
perhaps, the increase, in uric-acid elimination.
The liver seems to be the organ in birds where the synthetical formation
of uric acid occurs, and the fact that it was possible for ^Minkowski ^ 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 con-
sider the liver as at least one of the organs taking part in the work, as
shown by W^iener's investigations. The liver, spleen, and muscles are
considered as the most important organs for the oxidative uric-acid forma-
tion from nucleins and purine bases, but it must not be forgotten that these
organs in various animals have a somewhat different behavior in this re-
gard.
Uric acid w^hen introduced into the mammalian organism is, as first
shown by Wohler and Frerichs for the dog and later substantiated by
several experimenters,^ in great part destroyed and more or less com-
pletely changed into urea. This does not seem to be the same for all ani-
mals. In rabbits, according to Wiener, the uric acid is destroyed with
the formation of glycocoU as an intermediate step. The statements are
very contradictory in regard to carnivora. According to an older view,
which has received support by the recent investigations of Salkoavski. a
part of the uric acid introduced into dogs is eliminated as allantoin, which
• Zeitschr. f. physiol. Chem., 43.
2 1. c.
'Wohler and Frerichs, Annal. d. Chem. u. Pharm., Go. See also Wiener, Ergeb-
nisse der Physiologie, 1, Abt. 1.
571 URINE.
is also true according to Mendel and Brown for cats. The correctness
of tliis \iew is denied by Wiener, Pohl and Poduschka, but the recent
investigations of ^Mendel and White i give further proofs of its correct-
ness. The possibihty of a uric-acid destiTiction in man, with allantoin as
an intermediary step, cannot, for the same reasons, l)e denied.
The demoUtion of uric acid seems to be possible, according to the nu-
merous researches of Chassevant and Richet, Ascoli, Jacoby, W^iener,
Schittenhelm, Burian, Almagia and Pfeiffer,^ in several organs, such
as the liver, kidneys, muscle, and bone-marrow, although its behavior
differs in various animals.
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 admit, therefore, that a part of the
uric acid formed in the body is destroyed in a similar manner to that intro-
duced from without. Burian and Schur ^ have indeed suggested a factor,
the so-called "integral factor," with which the quantity of uric acid elimi-
nated in the twenty-four hours must be multiplied in order to find the
quantitv of uric acid formed during this time. According to them, car-
nivora eliminate unchanged about -gV- 3V of the uric acid introduced into
the circulation, rabbits about \, and man 4.
Properties and Reactions of Uric Acid. Pure uric acid is a white, odor-
less, and tasteless powder consisting of very^ small rhombic prisms or plates.
Impure urio 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 micro-
scope, and these sometimes appear as spools because of the rounding of
their obtuse angles. The plates are sometimes six-sided, irregularly devel-
oped; 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 cr\''stallization, as when the urine de-
posits a sediment or when treated with acid, large, invariably colored cr\'S-
tals separate. Examined with the microscope these crystals always appear
vellow or yellowish brow^n in color. The most common type is the whet-
stone shape, formed by the rounding off of the obtuse angles of the rhoml)ic
•Wiener, Arch. f. exp. Path. ii. Pharm., 40 and 42, and Ergebnisse der Physiologie,
1 Abt. 1; Pohl, Arch. f. exp. Path. u. Pharm., 48; Poduschka, ib-id., 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, Jbid., 12.
2 Chassevant et Richet, Compt. rend. Soc. biolog., 49; Ascoli, Pfliiger's Arch., 72;
Jacoby, Virchow's Arch., 157; Wiener, Arch. f. exp. Path. u. Pharm., 42, and Centralbl.
f. Physiol., 18; Schittenhelm, Zeitschr. f. physiol. Chem., 43 and 45; Burian, ibid.,
43; Almagia, Hofmeister's Beitrage, 7; Pfeiffer, ibid., 7.
s Pfliiger's Arch., 87.
PROPERTIES OF URIC ACID. 575
plate. The whetstones are generally connected together, two or more
crossing each other. Besides these forms, rosettes of prismatic crj'stals,
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 glycerine, but very insoluble in cold water, in 39 480 parts at
18° C. (His and Paul). At this temperature, according to them, 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 according to Smale the monophosphate has only a slight sol-
vent action. According to RiJDEL ^ urea is an imjwrtant solvent, but this
statement has not been confirmed by the observations of His and Paul.
Uric acid is not only dissolved by alkalies and alkali carbonates, but also by
several organic bases, such as ethylamine and propylamine, piperidine and
piperazine. Uric acid dissolves without decomposing in concentrated
sulphuric acid. It is completely precipitated from the urine by picric
acid (Jaffe^). XJric acid gives a chocolate-brown precipitate with phos-
photungstic acid in the presence of hydrochloric acid.
Uric acid is dibasic and correspondingly forms two series of salts, neu-
tral and acid. Of the alkali urates the neutral potassium and lithium salts
are most easily soluble and the ammonium salt dissolves with difficultv.
The acid alkali urates are very^ insoluble and separate as a sediment (sedi-
mentum loteritium) from concentrated urine on cooling. The salts with
alkaline earths are very insoluble.
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 purjole-red (ammonium
purjuirate or murexide) on the addition of a little ammonia. If, instead of
the ammonia, we add a little caustic soda (after cooling), the coior becomes
deeper blue or 1:)luish violet. This color disappears quickly on warminff,
differing from certain xanthine 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 droj)s
' His, Jr., and Paul, Zeitschr. f. physiol. Chem., 31; Smale, Centralbl. f. Physiol.,
9; Riidel, Arch. f. exp. Path. u. Phann., 50.
2 Zeitschr. f. physiol. Chem., 10.
576 UIUNE.
of concentrated sulphuric acid and commercial benzene (containing thio-
phene) a beautiful blue coloration is obtained (Dt^nk;es' reaction i).
Uric acid does not reduce an alkaline solution of bismuth, while, on the
contrary, it reduces an alkaline cupric-hydrate solution. In the presence of
only a little copper salt we obtain a white precipitate consisting 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 gelati-
nous 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).
The precipitation of free uric acid from its alkaU salts by means of
acids can be prevented to some extent by the presence of thymic acid or
nucleic acid (Goto 2), It is questionable whether this is of any physio-
logical importance.
Preparation of Uric Acid from Urine. Filtered normal urine is treated
with 20-30 c.c. of 25 per cent hydrochloric acid for each liter of urine.
After forty -eight hours collect the crystals and purify them by redissolving
in dilute alkali, decolorizing with animal charcoal and re]3recipitating with
hydrochloric acid. Large quantities of uric acid are easily obtained from
the excrements of serpents by boiling them 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; dis-
solve the separated and washed acid potassium urate in caustic potash, and
precipitate the uric acid in the filtrate by addition of an excess of hydro-
chloric acid.
Quantitative Estimation of Uric Acid in the Urine. As the older method
suggested by Heintz, even after recent modifications, gives inaccurate
results, it will not l)e considered here.
Salkowski and Ludwig's^ method consists in precipitating by silver
nitrate the uric acid from the urine previously treated with magnesium
mixture, and weighing the uric acid obtained from the silver precipitate.
Uric acid determinations by this method are often performed according to
the suggestion of E. Ludwig, which requires tne following solutions:
' Journ. de Pharm. et de Chim., 18. Cited from Maly's Jahresber., 18.
2 Zeitschr. f. pliysiol. Chem., 30.
^ 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. 577
1. An AMMONiACAL siLVER-xiTRATE SOLUTION, which Contains in 1 liter 26
grams of silver nitrate and a quantity of ammonia sufficient to redissolve com-
pletely the precipitate produced by the first addition of ammonia. 2. Magne-
sia MiXTLRE. Dissolve 100 grams <if 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. Dissolv elO 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 hych"ogen and then mixed with the other half.
The concentration of the three solutions is so arranged that 10 c.c. of
each is sufficient for 100 c.c. of the urine.
100-200 c.c, according to concentration, of the filtered urine freed
from protein (by boiling after the addition of a few drops of acetic acid) is
poured into a beaker. In anotlier vessel mix 10-20 c.c. of the silver solu-
tion with 10-20 c.c. of the magnesia mixture and add ammonia, and when
necessars' also some ammonium chloride, until the mixture is clear. This
solution is added to the urine wliile stirring, and the mixture allowed to
stand quietly for half an hour. The precipitate is collected on a filter,
waslied 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 c.c. of the alkali-sulphide solution, which has
previously been diluted with an equal volume of water, and allow this solu-
tion to flow through the above filter into the beaker containing the silver
precipitate; wash with boiling water, and warm the contents of the beaker
on a water-bath for a time, stirring constantly. After cooling, filter into a
porcelain dish, wash the filter with boiling water, acidify the filtrate with
hydrochloric acid, evaporate it to about 15 c.c, add a few drops more of
hydrochloric acid, and allow it to stand for twenty-four hours. The uric
acid which has cr\'stallized is collected on a small weighed filter, washed
wdth w^ater, alcohol, ether, and carbon clisulphide, dried at 100-110° C, and
w^eighed. For each 10 c.c 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 substituted (Ludw^g). Too intense or too long continued heat-
ing with the alkali sulphide must be prevented, otherwise a part of the
uric acid may be decomposed.
Salkowski deviates from this procedure by precipitating the urine first
with a magnesium mixture (50 c.c. to 200 c.c. urine), filling up to 300 c.c,
and filtering. Of the filtrate, 200 c.c. is precipitated by 10-15 c.c. of a
3 per cent silver-nitrate solution. The silver precipitate is shaken with 200-
300 c c 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 concentratecl to a few cubic
centimetres, treated with 5-8 drops of hydrochloric acid, and allowed to
stand until the next day.
Hopkins' method is based on the fact that the uric acid is completely
precipitated from the urine as ammonium urate on saturating with am
monium 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 modi-
fications of this method have been worked out by Folin, Folin and Schaf-
578 URINE.
FEE, WoRNER, and JoLLES.i The last named converts the uric acid
into urea by oxidation with potassium permanganate in sulphuric-acid
solution and then determines the quantity of this by sodium hypobromite.
Of these methods we shall descril^e only that suggested by Folin-Schaffer.
Folin-Schaffer Method. Treat 300 c.c. urine with 75 c.c. of a solution
containing 500 grams of ammonium sulphate, 5 grams of uranium acetate,
and 60 c.c. 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 c.c. of the fil-
trate (corresponding to 100 c.c. of the vu'ine) and add 5 c.c. of concentrated
ammonia. After twenty -four hours the precipitate 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 by water (total 100 c.c.) into a flask,
treated with 15 c.c. of concentrated sulphuric acid, and titrated at 60-63°
C. with N/20 potassium-permanganate solution. Each cubic centimeter
of this solution corresponds to 3.75 milligrams uric acid. Because of the
solubility of the ammonium urate a correction of 3 milUgrams must be
added for exevy 100 c.c. of the urine.
In regard to the numerous other methods for estimating uric acid, we
must refer to special w^orks on the subject, and especially to Huppert-
Neubauer .
Purine Bases (Alloxuric Bases). The alloxuric bases (purine bases)
found in human urine are xanthine, guanine, hypoxanthine, adenine, yara-
xanthine, heteroxanthine, episarkine, epiguanine, 1-methylxanthine, and car-
nine. The occurrence of guanine and camine (Pouchet) is, according to
Kruger and Salomon ,2 not positive Ij^ shown. The quantity of these
bodies in the urine is extremely small and varies in different individuals.
Flatow and Reitzexstein ^ found 15.6-45.1 milligrams in the urine voided
during twenty -four hours. The quantity of alloxuric bases in the urine is
increased regularly after feeding with nucleus nucleins or food rich in nu-
cleins, and after free destruction of leucocytes. The quantity is especially
increased in leucsemia. We have a number of observations on the elimina-
tion of these bodies in different diseases, l)ut they are hardly trustworthy on
account of the inaccuracy of the methods used in the determinations. It
must also be remarked that the three alloxuric bases, heteroxanthine, para-
xanthine, and 1-methylxanthine, which form the chief mass of the alloxuric
bases of the urine, are derived, according to the investigations of Albanese,
BoNDZYNSKi and Gottlieb, E. Fischer, ]\I. Kriiger and G. Salomon, and
•Hopkins, Journ. of Path, and Bact., 1893, and Proceed. Roy. Soc, 52; Folin,
Zeitsehr. f. physiol. Chem., 24; Folin and Schaflfer, ibid., 32; Worner, ibid., 29; Jolles,
ibid., 29, and Wien. med. Wochenschr., 190.3.
'Zeitsehr. f. physiol. Chem., 24; Pouchet, "Contributions a la connaissance des
mati^res extractives de I'urine." These Paris, 1880. Cited from Huppert-Neubauer,
333 and 335.
' Deutsch. med. Wochensclir., 1897.
PURINE BASES. 579
Schmidt! from the theobromine, caffeine, and theophylhne which occur
in the food. "With the purine bases \xe must also differentiate between
those of endogenous and those of exogenous origin.- As the four true
nuclein bases and carnine have been treated in Chapters V and XI, it only
remains to describe the special urinarj' purine bodies.
HX— CO
I I
Heteroxanthine, C6H6X402 = 7-monomethvlxanthine, OC C.X.CH,, was first
detected in the urine by Salomon. It is identical with the monomethvLxanthine
which passes into the ui'ine 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 f]592 [^arts at 1S° C). It is readily soluble in amnionic, and alkaUes.
The crystalline sodium salt Ls 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 hydiochloric
acid by water. Heteroxanthine is precipitated by copper sulphate and bisul-
phite, mercuric chloride, basic lead acetate and ammonia, and by silver nitr.ate.
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 \Yeidet/s reaction, accordins; to Fischer (see Chapter V).
CH3.X— CO
i-Methylxanthine, CeH^X^O, , OC C.XH , was first isolated from the
1 ^! ^CF
HX-C.X.^- ^'
urine and studied by Kpuger, and then by Krt'ger and Salomon.^ 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 ])lates. The chloride is decomposed into the base and hydrochloric
acid by \s-ater. 1-methylxanthine gives crystalline double salts vdth platinum
and gold. It is not precipitated by ba.sic lead acetate, nor when pure by basic
lead acetate and ammonia. With ammonia and silver nitrate it gives a gela-
tinous precipitate. The siivr-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 oi caustic soda. It gives Weidei.'s reac-
tion (according to Fiscuek) beautifully.
^ .Aibancse, Ber. d. d. chem. Gesellsch., 32; Arch. f. exp. Path. u. Pharm., 35;
Bondzynski and Gottlieb, ibid., 36, and Ber. d. deutsch. chem. GeseUsch., 28; E.
Fischer, ibid., 30, 2405; Kriiger and Salomon. Zeitschr. f. physiol. Chem., 26: Kriiger
and Schmidt, Ber. d. d. chem. Gesellsch.. 32, and Arch. f. exp. Path. u. Pharm.. 4o.
- See Burian and Schur. foot-note 3, page 570, and Kaufmann and Mohr. Deutsch.
Arch. f. klin. Med., 74.
' Salkowski's Festschrift, 1904.
* Kriiger, Arch. f. (Anat. u.) Physiol., 1894; Kriiger and Salomon, Zeitschr. f
physiol. Chem., 24.
580 URINE.
CH3.X— CO
I I
Paraxanthine, C7H8NP2 = 1.7-dimethylxanthine, OC C.X.CH3, urotheo-
HX— C.N^^"
bromine (Tiiudtchum), was first isolated from the urine by Thudichum and Sat.o-
MON.' It crystallizes beautifully in six-sided plates or in needles. The sodium
compound crystallizes, in rectangular plates oi- 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
chloroi:)latinate crystallizes very beautifully. ?^Iercuric chloride precipitates it only
when added in excess and after a long time. The siher-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 ^ in pigs' and dogs' urine,
as well as in urine in leucaemia. Balke gives C^HoXgO 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. Episarldne does
not o-ive the xanthine reaction with nitric acid nor Weidel's reaction. With
hydrochloric acid and potassium clilorate 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 epiiruanine.
IIX—CO
I I
Epiguanine, CuH^XsO = 7-methylguanine, H2N.C C.X.CH3, was first pre-
II II NflH
pared from the urine by Kruger.^ 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 shming 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 anmionia give a gelatinous precipitate.
It responds to the xanthine test with nitric acid and alkali. According to
Fischer it acts like episarkine ^^ith Weidel's test.
In preparing alloxuric bases from the urine, the fluid is supersaturated with
ammoiiia and precipitated by a silver-nitrate solution. The precipitate is then
decomposed with sulphuretted hydrogen. The boiling-hot filtrate is evaporated
to ch-yncss 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 in-
stead of precipitating with silver solution we desire to precipitate, according to
Krijger and Wulff, with copi)er suboxide, the urine may l)e heated to boiling
and immediately are added, successively, 100 c.c. of a 50 per cent sodium-bisul-
phate solution and 100 c.c. of a 12 per cent copper-sulphate solution for every
liter of urine. The thoroughly washed precipitate is decomposed with hydro-
chloric acid and sulphuretted "hydrogen. The uric acid remains in great part
'Thudichum, "Grundziige d. anal. med. klin. Chemie" (Berlin, 1886); Salomon,
Arch. f. (.\nat. u.) Physiol., 1882, and Ber. d. deutsch. chem. Gesellsch., 16 and 18.
2 Balke, "Zur Kenntniss der Xanthinkorper" (Inaug.-Diss., Leipzig, 1893); Salo-
mon, Zeitschr. f. physiol. Chem., 18.
3 Arch. f. (Anat. u.) Physiol., 1894; Kriiger and Salomon, Zeitschr. f. physiol.
Chem., 24 and 26.
ESTIMATION OF ALLOXURIC BASES. 581
on the filter. Further details in regard to the treatment of the solution of the
hydrochloric-acid compounds may be found in Kuuger and Salomon.^
Quarditatioe Estimation of Allozuric Bases according to Salkowski.^
400 to 600 c.c. of the urine free from protein is first precipitated by mag-
nesia mixture and then by a 3 per cent silver-nitrate solution as de-
scribed on page 577. The thoroughly washed silver preicpitate is decom-
posed by sulphuretted hydrogen after being suspended in 600-800 c.c.
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 A\ith 20-30 c.c. of hot 3 per cent sulphuric
acid and allowed to stand twenty-four hours; the uric acid is filtered off,
washed, the filtrate made ammoniacal, and the xantiiine bodies precipi-
tated again by silver nitrate, the precipitate collected on a small chlorine-
free filter, washed thoroughly, dried, carefully inciiierated, the ash dis-
solved in nitric acid, and titrated with ammonium saij)hocyanide accord'
ing to Volhajrd'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
alloxuric bases or to 0.7381 gram alloxuric b-awes. By this method the
uric-acid and alloxuric bases can be simultaneously determined in the same
portion of urine .^
Malfatti ^ determines the nitrogen of the allo>airic 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
Kjeldahl determination.
The nitrogen of the alloxuric bases is also determined as the difference between
the uric-acid nitrogen and the total nitrogen of the alloxuric bodies of the silver
precipitate (Camerer, Arnstein ^). Salkovvski 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 Aknstein * this can readily be done by
boiling the precipitate in water with seme 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 alloxuric bases in the urine is but little known, the
quantity of nitrogen of the alloxuric bases is always calculated as a certain
alloxuric base, for example xanthine (Camerer), and the quantity so found
used as a measure for the alloxuric bases.
According to a new method of Kruger and Schmid ' the uric acid and
the purine bases are precipitated as a cuprous compound by copj^er-sulphate
solution 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
' Zeitschr. f. physiol. Chem., 26, and also Hoppe-Seyler-Thierf elder's Handbuch,
7, Aufl., 154.
^ Pfliiger's Arch., ('9.
^ In regard to the details we refer the reader to the original paper.
* Centralbl. f. innere Med., 1897.
5 Camerer, Zeitschr. f. Biologie, 26 and 28; Arnstein, Zeitschr. f. physiol. Chem., 23.
" Salkowski, 1. c; Arnstein, CentralU. f. d. nied. Wissensch., 1S9S.
'Zeitschr. f. physiol. Chem., 45' and Hoppe-Seyler-Thierfelder's Handbuch,
7. Aufl., 435.
582 URINE.
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 ' for clinical purposes.
Oxaluric Acid, CgH^Np, = (CON2H3).CO.COOH. This acid, whose relation
to uric acid and urea has been spoken of above, does not always occur in the
urine, and then only in traces as the ammonium salt. This salt is not directly
precipitated by CaClj and NHg, but on boiling it is decomposed into urea and
oxalate. In preparing oxaluric acid from urine the latter is filtered through
animal charcoal. The oxalurate retained by the charcoal may be obtained by boil-
ing with alcohol.
COOH
Oxalic Acid, C2H2O4, or -^^^t occurs under physiological conditions
COOH
in very small amounts in the urine, about 0.02 gram in twenty-four hours
(FuRBRiNGER 2). According to the generally accepted view it exists in
tlie urine as calcium oxalate, which is kept in solution by the acid phos-
phates present. Calcium oxalate is a frequent constituent of urinary sedi-
ments and occurs also 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 ;^ and as many vegetables and fruits, such as cabbage, spinach,
asparagus, sorrel, apples, grapes, etc., contain oxalic acid, it is possible that
a part of the oxalic acid of the urine originates directly from the food.
That oxalic acid may be formed in the animal body as a metabolic product
from proteins or fats follows from the observations of Mills and Luthje,*
who found in dogs on an exclusively meat and fat diet, as also in starvation,
that 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,^ 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). Gelatine and gelatine-yielding tissues seem to
increase the excretion of oxalic acid, which stands in accord with the
observations of Kutscher and Schenk 6 that on the oxidation of gelatine
oxamic acid is produced from the glycocoll and this then decomposes
readily into ammonia and oxalic acid. After feeding nucleins no constant
' Niemilowicz, Zeitschr. f. physiol. Chem., 35; Gittelmacher-Wilenko, ibid., 86;
Hall, Wien. kl.n. Wochenschr., 16.
^ Deutsch. Arch. f. klin. Med., IS. See also Dunlop, Journ. Path, and Bacterid., 3.
^ In regard to the behavior of oxalic acid in the animal body, see pages 629 and 6C0.
^ Mills, V;rchow's Arch., 99; Liithje, Zeitschr. f. klin. Med., 35.
^ Reale and Boeri, Wien. med. Wochenschr., 1895; Terray, Pfliiger's Arch., 65;
Salkowski, Berl. klin. Wochenschr., 1900.
'Zeitschr. f. physiol. Chem., 43.
OXALIC ACID AND ALLAXTOIN. 583
increase in the elimination of oxalic acid lias been observed. ^ The pro-
duction of oxalic acid due to an incomplete combustion of the carbo-
hydrates has also been suggested. The work of Hildebraxdt and P.
JMayer seems to indicate this under abnormal conditions. In alimentary
glycosuria or diabetes Luzzato 2 could not observe any rise in the elimi-
nation of oxalic acid. "We have no grounds for the assumption that oxalic
acid is produced under physiological conditions by an incomplete com-
bustion of carbohydrates. We cannot exclude the possibility of the for-
mation of oxalic acid from the oxidation of uric acid in the animal body,
yet we have no positive proof of such a formation.-^
Oxalic acid is best detected and quantitatively determined according
to the method suggested by Salkowski: Shaking out the oxahc acid from
the acidified urine by means of ether and then proceeding as foUo-vrs accord-
ing to AuTEXRiETH and Barth : -*
The twenty-four-hour urine is precipitated l:)y CaCU and ammonia
in excess. After 18-20 hours the precipitate is collected (the filtrate must
be clear) and dissolved in a Uttle hydrochloric acid and shaken out 4-5
times vith 1.50-200 c.c. ether (containing 3 per cent absolute alcohol).
The united ethereal extracts are filtered through a dr\- filter and distilled
after the addition of about 5 c.c. of water. The liquid, if necessary-, is
decolorized with animal charcoal and precipitated with CaClo and am-
monia, mado acid after a certain time with acetic acid, and finally the
oxalate is collected, washed, burned to CaO, and weighed.
.XH.CH.HN V
Allantoin (Glyoxyldiureide), C4H6N4O3 , 0C<^ | /CO, oc-
^XH.CO HoX/
curs in the urine of chiklren within the first eight days after birth, and in very
small amounts also in the urine of adults (Gusserow, Ziegler and Her-
mann). It is found in rather abundant quantities in the urine of pregnant
women (Gusserow). Allantoin has also been found in the urine of suck-
ing calves CW^ohler), in urine of oxen (Salkowski), and sometimes in the
urine of other animals (^Ieissxer). It is also found in the amniotic fluid
and, as first shown by Vauquelix and Lassaigxe,^ in the allantoic fluid
of the cow (hence the name). Allantoin is formed, as above stated, by the
oxidation of uric acid outside of the animal body, hence a similar formation
' See Stradomsky, Virchow's -Vrch., 1G3; Mohr and Salomon, Deutsch. Arch. f.
klin. Med., 70; Salkowski, 1. c.
^ Hildebrandt, Zeitschr. f. physiol. Chem., 35; P. Mayer, Zeitschr. f. klin. Med., i'-
Luzzato, Salkowski's Festschrift, 1904.
^ See "Wiener, Lrgebnisse der Physiol., 1, Abt. 1.
^ Salkowski, Zeitschr. f. physiol. Chem., 29; Autenrieth and Barth, ibid., 35.
5 Ziegler and Hermann, see Gusserow, Arch. f. Gynjikol, 3 — both cited from Huppert-
Neubauer, Harn-Analj^se, 10. Aufl., 377; Wohler, Annal. d. Chem. u. Pharm., 70;
Salkowski, Zeitschr. f. physiol. Chem., 42; Meissner, Zeitschr. f. r.-.t. Med. (3), 31; Las-
saigne, Annal. de Chim. et Phys., 17.
584 URINE.
of allantoin is assumed in the animal organism (see page 573). According
to PoDUSCHKA and ]\Iinkowski,i allantoin introduced into dogs appears
almost entirely in the urine, while in man only a small portion of the in-
gested substance is eliminated by the kidneys. In carnivora the excretion
of allantoin is considerably increased, according to ^^Iinkowski, Cohn,
Salkowski, and ^Iendel and Brown,^ after feeding thymus or pancreas.
According to Mendel and White, on the intravenous injection of urates
an abundant elimination of allantoin takes place in dogs and cats. A
strong allantoin excretion is also found in dogs after poisoning with hy-
drazine (BoRissow), hydroxy lamine, semicarbazide, and aminoguanidine
(PoHL^). He also obtained allantoin in the autolysis of the intestinal
mucosa, hver, thymus, spleen, and pancreas. As no allantoin exists in
the organs of normal starving dogs, and as Pohl has found it in the liver
and, as traces, also in the other organs after poisoning with hydrazine, he
claims that the allantoin is formed in the nuclein destruction produced
by the death of the cell-nuclei.
Allantoin is a colorless substance often cr}^stallizing in prisms, difficultly
soluble in cold water, easily soluble in boiling water, and also in warm
alcohol, but not soluble in cold alcohol or ether. A wateiy allantoin solu-
tion gives no precipitate with silver nitrate alone, but by the careful addi-
tion of ammonia a white flocculent precipitate is formed, C4H^gN403,
which is soluble in an excess of ammonia and which consists after a certain
time of ver}^ small, transparent microscopic globules. The dry precipitate
contains 40.75 per cent silver. A water}- allantoin solution is precipitated
by mercuric nitrate. On continued boiling allantoin reduces Fehling's
solution. It gives Schiff's furfurol reaction less rapidl}- 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
Loewy's^ method, which consists of the following: The faintly acidified
urine is precipitated with a mercurous-nitrate solution, the filtrate treated
with H2S, 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 filtrate evap-
orated to dryness. The residue is extracted with hot water and then the
solution precipitated with mercuric nitrate. The precipitate is collected
and decomposed by H2S. From the evaporated filtrate the allantoin
^ Arch. f. exp. Path. u. Pharm., 44; Minkowski, ibid., 41.
2 Minkowski, 1. c, and Centralbl. f. innere Med., 1898; Cohn, Zeitschr. f. physiol.
Chem., 25; Salkowski, Centralbl. f. d. med. Wissenseh., 1898; Mendel and Brown,
Amer. Journ. of Physiol., 3.
^ Mendel and White, Amer. Journ. of Pliysiol., 12; Borissow, Zeitschr. f. physiol.
Chem., 19; Pohl, Arch. f. exp. Path. u. Pharm., 46.
* Arch. f. exp. Path. u. P. arm., 44.
HIPPURIC ACID. 585
ctystallizes out. This method can be used for the quantitative determina-
tion of allantoin.
or* n TT
Hippuric Acid (Benzoyl-amino acetic acid) , C9H9NO0,
^^ y. 9 9 ... HN.CH2.COOH.
This acid decom;.oses into benzoic acid and glycocoll on boihng with
mineral acids or alkaUes, and also by 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: C6H5COOH4-NH2.CH2.COOH =
C6H5.CO.NH.CH2.COOH + H2O. This acid may be synthetically pre-
pared from l)enzamide and monochloracetic acid, C6H5.CO.NH2 + CH2CI.
COOH = C6H5.CO.NH.CH2.COOH + HCl, and in various other ways, but
most simply from glycocoll and benzoyl chloride in the presence of alkali.
Hippuric acid occurs in large amounts in the urine of herbivora, but
only in small quantities in that of carnivora. The quantity of 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 vegetables
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. Nothing 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 theisubstituted benzoic acids are converted into hippuric acid and sub-
stituted hippuric acids within the body. Moreover, those bodies are trans-
formed into hippuric acid which by oxidation (toluene, cinnamic acid,
hydrocinnamic acid) or by reduction (quinic acid) are converted into ben-
zoic acid. The question of the origin of hippuric acid is therefore connected
with the question of the origin of benzoic acid ; the formation of the second
component, glycocoll, frcta the protein substances in the body is unques-
tionable.
Hippuric acid is found in the urine of starving dogs (Salkoavski). also
in dog's urine after a diet consisting entirely of meat (^Ieissner and
Shepard, Salkowski, and others i). 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. Amono- the
products of the putrefaction of protein outside of the body Salkowski has
found phenylpropionic acid, C6H5.CH2.CH2.COOH, which is oxidized in
the organism to benzoic acid and eliminated as hippuric acid after combin-
ing with glycocoll. Phenylpropionic acid seems to be formed from the
aminophenylpropionic acid, which is derived from several protein substances.
The supposition that the phenylpropionic acid is jiroduced from tvrosine bv
' Salkowski, Ber. d. dcutsch. chem. Gesellsch., 11; Meissncr and Shepard, Unter-
such. iiber das Entstehen der Hippursaure im tliierischen Organisnius. Hanover 1866
586 URINE.
putrefaction in the intestine has not been substantiated by the researches
of Baumann, Schotten, and Baas.i The importance of putrefaction in
the intestine in producing hippuric acid is evident from the fact that after
thoroughly disinfecting the intestine of dogs with calomel the hippuric acid
disappears from the urine (Baumann 2).
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 intestines of these animals, but it is especially due to the large quantity
of substances in the plant-food from which benzoic acid can be formed.
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
followed by a diminution in the second, and that, for example, quinic acid produces
a diminution in the excretion of uric acid corresponding to the increased forma-
tion of hippuric acid (Weiss, Lewin), cannot be considered as sufficiently proved *
(Hupfer).
The kidneys may be considered in dogs as special organs for the syn-
thesis of hippuric acid (Schmiedeberg and Bunge^). 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.
As the thorough investigations of Wiechowski teach, the synthesis of
hippuric acid does not stand in any direct relationship to the extent of
protein metabolism; it varies, on the contrary, with the duration of circu-
lation of benzoic acid and the quantity of glycocoll present in the body.
The amount of the latter in intermediary metabolism is so great that in
rabbits, on the administration of benzoic acid, more than one half of the
total urine nitrogen may exist as glycocoll. ]Magnus-Levy s found in
rabbits and sheep up to 27.8 per cent of the total nitrogen as hippuric-acid
' E. and H. Salkowski, Ror. d. deutsch. chem. Gesellsch., 12; Baumann, Zeitschr.
f. physiol. Chem., 7; Schotten, ibid., 8; Baas, ibid., 11.
' Ibid., 10, 131.
3 Weiss, Zeitschr. f. physiol. Chem., 25, 27, 3S; Lewin, Zeitschr. f. klin. Med., 42;
Hupfer, Zeitschr. f. physiol. Chem., 37. See also Wiener, "Die Harnsiiure," Ergeb-
nisse der Physiol., 1, Abt. 1.
'' Arch. f. exp. Path. u. Pharm., (»; also A. Hoffmann, ibid., 7, and Kochs, Pfliiger's
Arch., 20; Bashford and Cramer, Zeitschr. f. pliysiol. Chem., 35.
^ Wiecliowski, Hofmeister's Beitrage, 7 (literature); A. Magnus- Levy, Miinch.
med. Wochenschr., 1905.
PROPERTIES AND REACTIONS OF HIPPURIC ACID. 587
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. We cannot explain how this aljundant formation and eUmination
of glycocoll as hippuric acid is brought about.
Properties and Reactions of Hippuric Acid. This acid cr}stallizes in
semi-transparent, long, four-sided, milk-white, rhombic prisms or columns,
or in needles by rapid cr}-stallization. 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 liciuid
which crystaUizes on cooling. On continuing to heat it decomposes, pro-
ducing a red mass and a sublimate of l)enzoic 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 crj^stalline form and its insolubility in petroleum
ether. Hippuric acid and lienzoic acid both give Lucke's reaction, namely,
they generate an interse odor of nitrol^enzene when evaporated to dr\'ness
with nitric acid and when the residue is heated with sand in a glass tube.
Hippuric acid forms ciystalHzable salts, in most cases, with bases. The
comljinations with alkalies and alkaline earths are soluble in water and
alcohol. The silver, copper, and lead salts are soluble w^ith 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 l^y the addition of an excess of hydrochloric acid. The
cr}'stals are pressed, dissolved in milk of lime by boiling, and treated as
above; the hippuric acid is precipitated again from the concentrated fil-
trate by hydrochloric acid. The cr}'stals are purified by recrs'stalUzation
and decolorized, when necessarj^, by animal charcoal.
The quantitative estimation of hippuric acid in the urine may be per-
formed by the following method, (Bunge and Schmiedeberg i): The urine
is first made faintly alkaline with soda, evaporated nearly to dr}-ness. and
the residue thoroughly extracted with strong alcohol. After the eva]3ora-
tion 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 re-
peatedly washed with water, which is removed by means of a separator}'
funnel, then evaporated at a medium temperature and the dr\' residue
* Arch. f. exp. Path. u. Pharm., 6. Tn regard to other methods, such as Blumen-
thal as well as Pfeiffer-, Bloch and Riecke. see Maly's Jahresber., 30 and 32. See also
Wiechowski, 1. c.
588 URINE.
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, CioHuNOg =CeH,.CH2.CO.NH.CH2.COOH. This acid, which
is produced in the animal body by a combination of glycocoll with the phenyl-
acetic acid, CHg.CHj.COOH, formed in the putrefaction of the proteins, has-
been prepared from horse's urine by Sai^kowski,' but it probably also occurs in
human urine.
Benzoic Acid, CyHr.Oj or CrHj.COOH, is found in rabbit's urine and sometimes,
though in small amounts, in dog's urine (Weyl and v. Anrep). According to
Jaarsveld and Stokvis and to Kronecker it is also found in human urine in
diseases of the kidneys. The occurrence of benzoic acid in the urine seems to
be due to a fermentative decomposition of hippuric acid. Such a decomposition
may very easily occur in an alkaline mine or in one containing proteid (Van db
Velde and Stokvis). In certain animals — pigs and dogs — the kidneys, accord-
ing to ScHMiEDEBERG and MINKOWSKI,' Contain a special enzyme, Schmiede-
berg's histozym, which s^Dlits the hippuric acid with the separation of benzoic
acid.
Ethereal Sulphuric Acids. In the putrefaction of proteins in the intes-
tine, phenols, whose mother-substance is considered to be tyrosine, and also
indol and skatol are produced. These phenols directly, and the two last-
named bodies after they have been oxidized respectively into 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 sub-
stances— indoxyl- and skatoxyl-suljyhuric acids. To this group belong also
the pyrocatechin-sidphuric acid, which occurs only in very small amounts in
human urine, and hydroquinone-sulphuric acid, which appears in the urine
after poisoning with phenol, and under physiological conditions perhaps
other ethereal acids occur which have not been isolated. The ethereal
sulphuric acids of the urine were discovered and specially studied by Bau-
MANN.^ The quantity of these acids in human urine is small, while
horse's urine contains larger quantities. 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 relationship
of the sulphate-sulphuric acid A to the conjugated sulphuric acid B in
' Zeitschr. f. physiol. Chem., 9.
^Weyl and v. Anrep, Zeitschr. f. physiol. Chem., 4; Jaarsveld and Stokvis, Arch,
f. exp. Path. u. Pharm., 10; Kronecker, ibid , 16; "Van der Velde and Stokvis, ibid.,
17; Schmiedeberg, ibid., 14, 379; Minkowski, ibid., 17.
3Pfliiger's Arch., 12 and 13.
PHENOL AND P-CRESOL-SULPHURIC ACID. 589
health is on an average as 10:1. It undergoes such great variations, as
found by Bau^l^xx and Herter.^ and after them by many other investi-
gators, that it is hardly possible to consider the average figures as normal.
After taking phenol and certain other aromatic substances, as well as -when
putrefaction within the organism is general, the eUmination of ethereal
sulphuric acid is greatly increased. On the contrar\-, it is diminished when
the jDutref action in the intestine is reduced or prevented. For this reason it
may be greatly diminished by carbohydrates and exclusive milk diet.^ The
intestinal putrefaction and the elimination of ethereal sulphuric acid have
also been diminished in some cases by certain therapeutic agents which
have an antiseptic action; still the statements are not unanimous.^
Great importance has been given to the relationship between the total
sulphuric acid and the conjugated sulphuric acid, or between the conjugated
sulphuric acid and the sulphate-.sulphuric acid, in the study of the intensity
of the putrefaction in the intestine under different conditions. Several
investigators, F. Muller, Salkowski. and v. Noordex.^ consider cor-
rectly that tliis relationship is only of secondaiy value, and that it is more
correct to consider the absolute value. It must be remarked that the abso-
lute 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, C6H5.O.SO2.OH and
C6H4<qtV' "■ . These acids are found as alkaU salts in human urine,
in which also orthocresol has been detected. The quantity of cresol-sul-
phuric 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 quantity of phenols which
are separated from the ethereal-sulphuric acids of the urine amounts to 17-51
milhgrams in the twenty -four hours (:\Iuxk). The methods for the quanti-
tative estimation used heretofore give, according to Ru.mpf. as well as Koss-
LER and Pexxy.^ such inaccurate results that new determinations are ver>'
desiral)le. .After a vegetable diet the quantity of the.se ethereal-sulphuric
M-. d. Velden. Viichow's Arch., 70; Herter, Zeitschr. f. physiol. Chem., 1.
2 See Hirscliler, Zeitschr. f. physiol. Chem., 10; Biernacki, Deutsch. Arch. f. khn.
Med., -49; Rovighi, Zeitschr. f. physiol. Chem., 16; "Winternitz, ibid., and Sclmiitz,
ibid., 17 and 19.
^ See Baumann and Morax, Zeitschr. f. physiol. Chem., 10; Steiff, Zeitschr. f.
khn. Med., 16; Ro\-igVi. 1. c, Stem, Zeitschr. f. Hygiene, 12; and Bartoschewitsch,
Zeitschr. f. physiol. Chem., 1"; Mosse, ibid.. 23.
*Muller, Zeitschr. f. khn. Med., 12; v. Xooiden, ibid., 17; Salkowski, Zeitschr.
f physiol. Chem., 12.
^Munk, Pfliiger's Arch., 12; Rumpf, Zeitschr. f. physiol. Chem., 16; Ivossler and
Penny, ibid., 17.
690 URINE.
acids is greater than after a mixed diet. After the ingestion of carbolic acid,
which is in great part converted by sj-nthesis within the organism into phe-
nol-sulphuric acid, also into pyrocatechin- and hydroquinon-sulphuric acid,^
or when the amount of sulphuric acid is not sufficient to combine
with the phenol, it forms phenyl-glucuronic acid ^ the quantit}^ of phenols
and ethereal-sulphuric acids in the urine is considerably increased at the
expense of the sulphate-sulphuric acid.
An increased elimination of phenol-sulphuric acids occurs in active
putrefaction in the intestine with stoppage of the contents of the intestine,
as in ileus, diffused peritonitis with atony of the intestine, or tuberculous
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 bee<n oljserved in a few
other cases of diseased conditions of the body.^
The alkali salts of phenol- and cresol-sulphuric acids crystallize in white
plates, similar to mother-of-pearl, which are rather freety soluble in water.
They are soluble in boiling alcohol, but only slightly soluble in cold alcohol.
On boiling with dilute mineral acids they are decomposed into sulphuric
acid and the corresponding phenol.
Phenol-sulphuric acids have been synthetically prepared by Baumann
from ]:>otassium pyrosvilphate and potassium phenolate or p-cresolate. For
the method of their preparation from urine, which is rather complicated,
and also for the known ])henol reactions, the reader is referred to other
text-l)ooks. The quantitative estimation of these ethereal-sulphuric acids
was usualty performed by weighing the phenol which was separated from
the urine as tribromphenol. At the present time the following method is
employed :
KossLER and Penny's Method with Neuberg's^ Modification. The
liquid containing phenol is treated with N/10 caustic soda until strongly
alkaline, warmed on the water-l^ath 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 hypo-
iodite. which latter forms tri-iodophenol with the phenol according to the
following equation:
CeHsOH + 3NaI0 = CeHoIg.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
' See Baumann, Pfliiger's Arch., 12 and 13, and Baumann and Preusse, Zeitsehr.
f. physiol. Chem., 3, 156.
^ Sf^bmiedeberg, Arch. f. exp. Path. u. Pharm., 14.
^ See G. Hoppe-Seyler, Zeitsehr. f. physiol. Chem., 12, (this contains also all
references to the literature on this subject ; Fedeli, Moleschott's Untensuch., 15.
* Koss'.er and Penny, Zeitsehr. f. physiol. Chem., 1"; Neuberg, ibid., 27.
PYROCATECHIN AND HYDROQUINONE. 591
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 miUigrams 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 pre-
cipitation with lead and distilled again (Neuberg). For details, see
Neuberg, 1. c, and Hoppe-Seyler-Thierfelder's Handbuch, 7. Aufl.
The methods for the separate determination of the conjugated sulphuric
acid and the sulphate-sulphuric acid will be spoken of later in connection
with the determination of the sulphuric acid of the urine.
Pyrocatechin-sulphuric Acid. This acid was first found in horse's urine in
rather large quantities by Baumann. It occurs in human urine only in the
very smallest amounts, and perhaps not constantly, but it is present
abundantly in the urine after taking phenol, pyrocatechin, or protocatechuic
acid.
With an exclusively meat diet this acid does not occur in the urine, and it
therefore must originate from vegetable food. It probably originates from the
protocatechuic acid, which, according to Preusse, passes in part into the urine
as pyrocatechin-sulphuric acid. This acid may also perhaps be formed by the
oxidation of phenol within the organism (Baumann and Preusse ').
Pyrocatechin, or o-I^ioxybenzene, CHJOH)^, was first observed in the urine
of a child (Ebstein and J. Mult.er). The reducing body alcapton, first found
by Bodeker - in human urine and which was considered for a long time as iden-
tical with pyrocatechin, is in most cases probably homogentisic acid or urolcucic
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 oxygen of the air. If very dilute ferric chloride is treated with tartaric acid
and then made alkaline with ammonia, and this added to a watery solution
of pjTocatechin, we obtain a violet or cherry-red liquid which becomes green
on adding excess of acetic acid. Pyrocatechin is precipitated by lead acetate.
It reduces an ammoniacal silver solution at the ordinary temperature, and re-
duces alkaline copper-oxide solutions with heat, but does not reduce bismuth
oxide.
A urine containing pyrocatechin, if exposed to the air, especially when alkaline,
quickly becomes dark and reduces alkaline copper solutions when heated. In
detecting pyrocatechin in the urine it is concentrated when necessary, filtered,
boiled with the addition of sulphuric acid to remove the phenols, and repeatedly
shaken after cooling with ether. The ether is distilled from the several ethereal
extracts, the residue neutralized with barium carbonate and shaken again with
ether. The pyrocatechin which remains after evaporating the ether may be
purified by recrystallization from benzene.
Hydroquinone, or p-Dioxybenzene, CH^COH),, 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'.
Hydroqinnone does not occur as a normal constituent of urine, but only after
the administration of hydroquinone; and according to Lewin,^ it may be found
' Baumann and Herter, Zeitschr. f. physiol. Cheni., 1; Preusse, ibid., 2; Baumann
ibid., 3.
2 Ebstein and Miiller, Virchow's Arch., 62; Bodeker, Zeitschr. f. rat. Med. (3)^ 7.
^ Vircliow's Arch., 92.
592 URINE.
in the urine of rabbits as an ethereal-sulphuric acid, being a decomposition
product of arbutin. . , , , .
Hydroquinone forms rhombic crystals which are readily soluble in water,
alcohol, and ether. It melts at 169° C. Like pyrocatechin, it easily reduces
metallic oxides. It acts like pjTOcatechin with alkalies, but is not precipitated
with lead acetate. It is oxidized into quinone by ferric cliloride and other oxidiz-
ing agents, and quinone can be detected by its peculiar odor. Hydroquinone-
sulphuric acid is detected in the m'ine by the same methods as pyrocatecliin-
sulphuric acid.
CH
/\
Indoxyl-sulphuric Acid, C8H7NS04 = HC C— C.O.SOgCOH), also called
I II II
HC C CH
X/\/
CH NH
URINE INDICAN, formerly called uroxaxthine (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 Vv'hich can be separated
from the urine is considered as a measure of the quantity of indoxyl-sul-
phuric acid (and indoxyl-glucuronic acid) contained in the urine. This
amount, according to Jaff^;,^ 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 arid is derived, as previously mentioned (page 401),
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 considerabty increased (Jaffe, Baumann and
Brieger, and others). It is also increased by the introduction of orthoni-
trophenylpropioUc acid in the animal organism (G. Hoppe-Seyler^).
Indol is formed by the putrefaction of proteins. The putrefaction of se-
cretions rich in protein in the intestine explains also the occurrence of indican
in the urine during starvation. Gelatine, on the contrary, does not increase
tlie elimination of indican.
An abnormally increased elimination of indican occurs in those diseases
where the small intestines are obstructed, causing an increased putrefaction
and thus producing an al^undance 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 confirmed recently by
Ellinger and Prutz.^ They removed an intestinal loop in dogs and
replaced it in a reversed position, the distal end of the loop being attached
1 Pfliiger's Arch., 3.
^ Jaff6, 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 Ilervieux,
Joum. de Physiol., 7.
' Jaffe, Virchow's Arch., 70; Ellinger and Prutz, Zeitschr. f. physiol. Chem., 38.
INDOXYL-SULPHURIC ACID. 593
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 obstruction in the small intes-
tine caused an increased elimination of indican, wliile an obstruction in the
large intestine showed no such action.
The putrefaction of proteins in other organs and tissues besides the in-
testine may also cause an increase in the indican of the urine. Certain
investigators. Blumexthal. Rosenfeld, and Lewix. claim to have shown
that an increased excretion of indican can be l:)rought about also \\ ithout
putrefaction by an increased destruction of tissue in starvation and also
after i)hlorhizin poisoning; but these statements are vehemently opposed
by other investigators, such as P. Mayer, Scholz. and Ellixger. and are
improbable. The indol. it seems, is not formed from the tryi^tophane
(indolaminopropionic acid) as intermediary" step in the demolition of the
proteins in the animal body, but rather from the ])utrefaction of the tri'pto-
phane in the intestine. Gextzex' ^ has also shown that tryptophane intro-
duced subcutaneously or per os into the body does not lead to an indican-
uria, but only when it is exposed to bacterial decomposition in the large
intestine. The statements as to the eUmination of indican after oxalic-
acid poisoning are somewhat contradictoiy. After poisoninc; with oxalic
acid Harxack and v. Leyex found an increased indican elimination, and
MoRACZEWSKi believes he has- proved a certain parallelism between the
quantity of indican and the quantity of oxalic acid in diabetes. Scholz -
obtained, on the contrary, no increase in the excretion of indican after
oxalic-acid poisoning.
An increased eUmination of indican has been observ-ed in manv dis-
eases.^ and in these cases the quantity of phenol eliminated is also gener-
ally increased. A urine rich in phenol is not always rich in indican.
The potassium salt of indoxyl-sulphuric acid, wliich was prepared pure
by Bau^l^xx and Brieger from the urine of a dog fed on indol. has since
been prepared synthetically V)y Baumaxx and Thesex.-* by fusing phenyl-
glycine-orthocarboxylic acid with aDcaU and then from tliis producing the
>BIumenthal. -\rch. f. (Anat. u.) Physiol., 1901, Suppl., and 1902, with Rosenfeld,
Charite annaleu. 27. and Hofmeister's Reitrage. o: Lewin, Hofmeister's Beitrage. 1;
Mayer. Arch. f. (.\nat. u.) Physiol., 1902. Zeitschr. f. klin. Med., 47. and Zeitsclir. f.
physiol. Chem., 29, 32; Scholz, ibid.. 3S; Ellinger, ibid., 39; Gentzen. "ITjer die Vors-
tufen des Indols bei der Eiweissfaulnis im Thierkorper," Inaug. -Dissert. Koncisberg,
1901.
2 Hamack, Zeitschr. f. physiol. chemie, 29; Scholz, 1. c: Moraczewski. Centralbl.
f. innere Med., 1903.
3 See Jaffe. Pfliiger's Arch., 3; Senator, Centralbl. f. d. med. Wissensch.. 1S77;
G. Hoppe-Seyler. Zeitschr. f. physiol. Chem.. 12 (contains older literature); also
Berl. klin. Wochenschr., 1S92.
* Baumann with Brieger, Zeitschr. f. physiol. Chem., 3; with Thesen. ibid. 23.
594 URINE.
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, ])ut in the presence of oxidizing reagents is converted into
indigo-blue: 2C8H7NO + 20=Ci6HioN202 + 2H20. The detection of indi-
can is based on this last fact.
For the rather complicated preparation of indoxyl-sulphuric acid as the
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, wliich also serves as an approximate test for the quan-
tity of indican, is sufficient.
JaffiS-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
bichromate, alkali chlorate, and hydrogen peroxide (the latter suggested
by Porcher and HervieuxI). With Obermayer's reagent the test is
performed as follows:
The acid urine (if alkaline it must l;)e acidified with acetic acid) (Ellin-
ger) is precipitated with Ijasic lead acetate, 1 c.c. for every 10 c.c. of
the urine. 20 c.c. of the filtrate are treated in a test-tube with an equal
volume of pure concentrated hydrochloric acid (specific gra\dty 1.19)
which contains 2-4 grams ferric chloride to the liter, and 2-3 c.c. 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 pro-
duced, whose formation has been explained in various ways. The quantity
of indigo red l^ecomes 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, and Ellinger^).
According to Ellinger the source of the indigo-red formation may be the
isatin that is produced by the superoxidation of the indoxj^ by the action of
the reagent, and this isatin forms indigo red with the indoxyl in the hydrochloric-
acid solution. Maillard, on the contrarj', 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 l)y comparison with a
solution of indigo in chloroform of known strength (ICrauss and Adrian) .
' Jaffe, Pfliiger's Arch., 3; Obermayer, Wien. klin. Wochenschr., 1890; Porcher
and Hervieux, Zeitschr. f. physiol. Chem., 39.
^ Rosin, Virchow's Arch., 123; Bouma, Zeitschr. f. pliysiol. Chem., 27, 30, 82,
39; Wang, ibid., 25, 2", 28; Ellinger, ibid., 3S and -11; Maillard, Bull. soc. chim., Paris
(.3), 29, and Compt. rend., 36; also L'indoxyle urinaire et les couleurs qui en derivent,
Paris, 190.3, and Zeitschr. f. physiol. Chem., 41.
SKATOXYL-SULPHURIC ACID. 595
Wang and others convert the indigo into indigo-sulphonic acid by con-
centrated siilphuriq acid and titrate with potassium permanganate. There
is still doubt as to the surest and most trustwortlw method for the deter-
mination of indican, and especially as to the question how the indigo resi-
due is to be washed (see Wang, Bou>l\, Ellinger, and Salkowski ^), and
for this reason we shall refer only to the works cited above.
Because of the difficulty arising from the production of indirul)in in
addition to indigotin, Bouma has recommended the conversion of all the
indoxyl into indirubin by boiling the urine with hydrochloric acid contain-
ing isatin. The indiruljin can l^e taken up by chloroform and deter-
mined by titration with potassium permanganate and sulphuric acid after
purification of the chloroform residue. Oerum ^ 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-
giucuronic acid (Schmiedeberg). Such an acid has been found in the urine
of animals after the administration of the sodium-salt of o-nitro-phenyl-
propiohc acid (G. Hoppe-Seyler). Porcher and Hervieux ^ have ob-
tained 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 W.\ng * have recently observed such
cases.
CH
/\
HC C— C.CH3
Skatoxyl-sulphuric Acid, C9H9NS04= | || || , has not
HC C C.O.SOoOH
CH NH
been positively prepared as a constituent of normal urine, but Otto has
once prepared its alkali salt from dialjetic urine. Perhaps skatoxyl occurs
in normal urine as a conjugated glucuronate (Mayer and Neuberg ^), and
it is believed that the urine contains a skatol-chromogen from which red and
^eddish-^•iolet 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
w^hich is formed by putrefaction in the intestine, and wliich is then conju-
gated 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
• lirauss, Zeitsclir. f. physiol. Chem., IS; Adrian, ibid., 19; Wang, ihid., 25; Sal-
kowski, ibid., 42.
^ Bouma, Zeitsclir. f. physiol. Chem., 32; Oerum, ibid., 43.
3 Schmiedeberg, Arch. f. exp. Path. u. Pharm., 14; G. Hoppe-Seyler, Zeitschr. f.
physiol. Cliem., " and S; Porcher and Her^-ieux, Journ. de Physiol., 7.
■'Grober, Miinch. med. Wochenschr. , 1904; Wang. Salkowski's Festschrift, 1904.
*Otto, Piiuger's Arch., 33; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29.
596 URINE.
acid, while skatol gives only a small quantity (IMester^). The state-
ments are somewhat contradictory on this subject and the behavior is
somewhat unsettled. According to Staal the chromogen of the skatoj
red is neither a conjugated sulphuric acid nor a conjugated glucuronic acid.
The potassium salt of skatoxyl-sulphuric acid is crystalline; it dissolves
in water, but with difficulty in alcohol. A watery solution becomes deep
violet with ferric chloride, and red with concentrated nitric acid. The salt
is decomposed by concentrated hydrochloric acid with the separation of a
red precipitate. The nature of this red coloring-matter produced by the
decomposition of skatoxyl-sulphuric acid is not well known; neither has the
relationship existing between this and other red coloring-matters in the
urine been decided. On distillation with zinc-dust the skatol-chromogen
yields skatol.
Urines containing skatoxyl are colored dark red to violet by Jaffe's indi-
can 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 hydro-
chloric acid. The coloring-matter which yields skatol with zinc-dust may
be removed from the urine by ether. Urines rich in skatoxyl darken from
the surface downward when allowed to stand in the air, and may become
reddish, violet, or nearly black. Rosin is of the opinion that no skatol-
chromogen exists in human urine, and that the observations made hereto-
fore were due to a confusion with indigo red or urorosein. It cannot be
disputed that derivatives of skatol occur in the urine, while the recent in-
vestigations of Staal, Grosser, Porcher, and Hervieux - indicate that
skatol-redand urorose in are identical or at least closely related pigments.
Only the formation of skatol by distillation with zinc powder can be con-
sidered as a positive proof as to the skatol nature of a pigment.
Salkowski ^ has demonstrated that the occurrence of skatol-cnrhoxylic acid
(indol acetic acid), CgHg.N.COOH, in normal urine is probable. This is also a
product of jnitref action. When introduced into the animal body this acid re-
appears unchanged in the urine. With hydrochloric acid and very dilute ferric-
chloride solution it gives an intense violet color to the solution. This test
responds with a watery solution containing 1 : 10 000 of skatol-carboxylic acid.
Aromatic Oxyacids. In the putrefaction of proteins in the intestine,
'paraoxyphemil-acetic acid, C6H4(OH).CH2COOH, and yaraoxyphenyl-'pro-
-pionic acid, C6H4(OH).C2H4.COOH, are formed from tyrosine as an inter-
mediate 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
' Brieger, Bar. d. deutsch. chem. Gesellsch., 12, and Zeitschr. f. physiol. Chem.,
4, 414; Mester, ibid., 12.
* Rosin, Virchow's Arch., 123; Staal, Zeitschr. f. i^liysiol. Chem., 4G; Grosser,
ibid., 44; Porcher and Hendeux, Compt. rend., 138, and Journ. de Physiol., 7.
3 Zeitschr. f. physiol. Cliem., 9.
P-OXYPHEXYLACETIC AND HOMOGENTISIC ACIDS. 597
same conditions as the phenols, especially in acute phosphorus poisoning,
in which the increase is considerable. A small portion of these oxyacids
is combined with sulphuric acid.
Besides these two oxyacids wliich regularly occur in human urine we
sometimes have other oxyacids in urines. To these belong homogentisic
acid and uroleucic acid, the first of which forms the specific constituents of
the urine in most cases of alcaptonuria, oxymandelic acid, found by Schultzex
and E.IESS in urine in acute atrophy of the liver, oxyhydroparacoumaric acid,
found by Blenderm.\nn in the urine on feeding rabbits with tyrosine, gallic
acid, which, according to Bau^l\nx,i sometimes appears in horse's urine,
and kynurenic acid (oxyquinoUncarboxylic acid), which up to the present
time has been found only in dog's urine. Although all these acids do not
belong to the physiological constituents of the urine, still they will be
treated in connection with these.
Paraoxyphenylacetic A ci d, CgHgOs = CeH^ < ^^ ^^qqjj ^^^
p-Oxyphenylpropionic Acid (Hydroparacoumaric Acid), C9Hio03 =
C6H4<^jj^^jj^QQQjj, are crystalline and are soluble both 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 Millox's reagent.
To detect the presence of these oxyacids proceed in the following way (Bau-
MAXX): Warm the urine for a while on the water-bath with hydrochloric acid
in order to drive off the volatile phenols. After cooling shake three times with
ether, and then shake the ethereal extracts with dilute soda solution, which dis-
solves the ox3-acids, 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 evaporated
the residue dissolved in a little water, and the solution tested with Millox'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), C8H804=
/0H(1)
CeHss— OH (4) . This acid, which was discovered by Marshall ^ and
^CHo.COOHCo)
by him called glycosuric acid, was isolated in larger quantities by Wolkow
and BAU^L\xx in a case of alcaptonuria and carefully studied by them.
They called it homogentisic acid because it is a homologue of gentisic acid,
and they showed that the peculiar properties of so-called alcaptonuric urine
in this case were due to this acid. This acid has later been found in mam-
* Schultzen and Riess, Chem. Centralbl., 1869; Blendermann, Zeitschr. f. phjrsiol.
Chem., 6, 267; Baumann, ibid., G, 193.
* The Medical News, Philadelphia, January 8, 1887.
598 URINE.
cases of alcaptonuria by Embden, Garnier and Voirin, Ogden, Garrod,
and many others. Glycosuric acid, isolated from alcaptonuric urine by
Geyger/ seems to be identical with homogentisic acid.
The quantity of acid eliminated is increased by food rich in protein. On
the ingestion of tyrosine by persons with alcaptonuria, Wolkow and
Baumann 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 - have given a
direct proof that homogentisic acid can also be formed from phenylalanine.
Tyrosine and phenylalanine are quantitatively converted into homogentisic
acid in alcaptonuria (Falta). Dil^romtyrosine, on the contrary, yields
as little homogentisic acid as bromine or iodine derivatives of protein
bodies (Falta). According to the investigations of Langstein and AIeyer,
and especially of Falta, different proteins yield varying quantities of
homogentisic acid in alcaptonuria, and accordingly larger amounts in pro-
portion as the protein is rich in tyrosine and phenylalanine.
Wolkow^ and Baumann explain the formation of homogentisic acid
from tyrosine by an aljnormal fermentation in the upper parts of the in-
testine, but this view has now been generally rejected. Homogentisic acid
is burnt in the healthy organism, and in consonance with the views of Falta
and Langstein alcaptonuria is considered as an anomaty in the metabolism.
O. Neubauer and Falta ^ found in experiments with different aromatic
substances that the aromatic a-oxyacids as well as the a-amino acids de-
rived from the protein bodies, are converted into homogentisic acid in the
organism of alcaptonurics. It can be admitted with Falta that the phenyl-
alanine in the body by deamidation is converted into phenyl-a-lactic acid,
C6H5.CH2.CHOH.COOH, from which by taking up two hydroxyl groups
dioxyphenyl-a-lactic acid (uroleucic acid), (OH)2C6H3-CH2.CHOH.COOH,
is formed, and then from this by oxidation dioxyphenylacetic acid (homo-
gentisic acid), (OH)2C6H3.CH2.COOH, is produced. Tyrosine also is sup-
posed to undergo an analogous transformation whereby a removal of OH
groups in the para position must be admitted and in both cases the homo-
gentisic acid formed is under normal conditions further destroyed by a
^ Wolkow and Baumann, Zeitschr. f. physiol. Cliem., 15; Embden, ibid., 17 and
^8; Garnier and Voirin, Arch, de Physiol. (5), l; Ogden, Zeitschr. f. physiol. Chem.,
20; Geyger, cited from Embden, 1 c, 18.
^ 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.
' Zeitschr. f. physiol. Chem., 42.
HOMOGENTISTIC ACID. 599
rupture of the benzene ring. The deniohtion of the tyrosine and the
phenylalanine according to this view takes place in normal organisms by
way of the alcaptonic acids, and the metabolic anomaly in alcaptonuria
consists in that the demolition stops at this point and the property of the
organism in alcaptonuria of rupturing the benzene ring is absent.
Garrod/ who has observed several cases of alcaptonuria, has also tabu-
lated about forty cases of alcaptonuria which he finds in the literature.
From this he shows that the anomaly of the protein metaljolism occurs
oftener in males than in females, and also that blood relationship of the
parents (first cousins) predisposes to alcaptonuria.
On fusing homogentisic acid with alkali it yields gentisic acid (hydro-
quinone-carboxylic acid) and hydroquinone. When introduced into the
intestine of the dog a part is converted into toluhydroquinone, which is
eliminated in the form of an ethereal sulphuric acid. Homogentisic acid
has also been prepared synthetically by Baumaxx and Fraxkel,^ starting
with gentisic aldehyde.
Homogentisic acid crystallizes with 1 mol. of water in large, trans-
parent prismatic crystals, wliich become non-transparent at the tempera-
ture of the room with the loss of water of crystallization. They melt at
146.5-147° C. They are- soluble in water, alcohol, and ether, but nearly
insoluble in chloroform and benzene. Homogentisic acid is optically in-
active and non-fermentable. Its watery solution has the properties of so-
called alcaptonuric urine. It becomes greenish brown from the surface
downward on the addition of very little caustic soda or ammonia with
excess 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 bis-
muth solutions. It gives a lemon-colored precijiitate with Millox'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 Ave obtain the amide of diben-
zoylhomogentisic acid, which melts at 204° C, and v.hich can be used in
the isolation of the acid from the urine, and also for its detection (Ortox and
Garrod). Among the salts of this acid must be mentioned the lead salt
containins: water of crvstallization and 34.79 ]ier cent Pb. This salt melts
at 214-215° C. ' ^
In order to prepare the acid, heat the urine to 1 -oiling, add 5 grams of
lead acetate for everv 100 c.c, filter as soon as the lead acetate has dis-
solved, 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 l)y H2S. After the spontaneous evaporation of
' Med. chinirg. Transact., 1899 (where all known cases are tabulated); also The
Lancet, 1901 and 1902; Garrod and Hele, Journ. of Physiol., 33.
2 Zeitschr. f. phyaol. Chem., 20.
600 URINE.
the ether the acid is obtained in nearly colorless crystals (Orton and
Garrod ^).
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. As regards
details of this method the reader is referred to the works of Baumann, C. Th.
MoRNER and Mittelbach, Garrod and Hurtley. Deniges^ has suggested
another method.
Uroleucic acid, C9H10O5, is, according to Huppert, probably a dioxyphenyl-
lactic acid, CB, (OH)2.CH2.CH(OH).COOH._ This acid was first prepared by Kirk
from the urine of children with alcaptonuria, which also contained homogentisic
acid. Langstein and Meyer ^ have also found a small amount of this acid in
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.
Oxymadelic acid, CHsO^, paraoxyphenylglycolic acid, HO.C„H,.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 COH
HC C C.COOH,
Kynurenic acid (;--oxy-^-quinolincarboxyhc acid), CicH7N03= I || |
HC C CH
CH N
has only been found thus far in dogs' urine; its quantity is increased by meat
feeding. According to the observations of Glaessner and Langstein, the mother-
substance seems to be contained among the products of pancreatic digestion
which are soluble in alcohol and precipitable by acetone. Ellinger ■* has
recently been able to show positively that tryptophane is the mother-substance
of this acid. By the introduction of tryptophane in the organism he has shown
the formation of a kynurenic acid not only in dogs but also in ral)bits. 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 CO, and kynurin. On evaporation to dry-
ness on the water-bath with hydrochloric acid and potassium chlorate a reddish
residue is obtained which becomes first brownish green and then emerald-green
on adding ammonia (Jaffe's reaction ^).
'Orton and Garrod, Journ. of Physiol., 27; Garrod, ibid., 23.
^ Mittelbach, Deutsch. Arch. f. klin. Med., 71 (which contains the work of Baumann
and Morner); Garrod and Hurtley, Journ. of Physiol., 33; Denig?s, Chem. Centralbl.
1897, 1, 338.
3 Huppert, Zeitschr. f. physiol. Chem., 23; Kirk, Brit. Med. Journ., 1886 and 1888;
Langstein and Meyer, 1. c.
* Glaessner and Langstein, Hofmeister's Beitrage, 1 ; Ellinger, Ber. d. d. chem.
Gesellsch., 37, 1804, and Zeitschr. f. physiol. Chem., 43. The older literature on
ky::urenic acid may be found in Josephsohn, Beitrage zur Kenntnis der Kynurensaure
ausscheidung beim Hunde, Inaug.-Dissert., Konigsberg, 1898.
' Zeitschr. f. physiol. Chem., 7. In regard to kynurenic acid, see also Huppert-
Neubauer, 10. Aufl., and Mendel and Jackson, Amer. Journ. of Physiol., 2; Mendel
and Schneider, ibid., 5; Camps, Zeitschr. f. physiol. Chem., 33.
URINARY PIGMENTS. UROCHROME. 601
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 hoemato^
porphyrin as a regular constituent. Uroerythrin also is of frequent occur-
rence in normal urine, but not always. Finally, the excreted urine when
exposed to the action of light regularly contains a yellow pigment, urobilin,
which is derived from a chromogen, urobilinogen, by the action of light
(Saillet) and air (Jaffe, Disque,^) and others. Besides this chromogen,
urine contains various other bodies from which coloring-matters may be
produced by tlie action of chemical agents. Humin substances (perhaps
in part from the carbohydrates of the urine) may l^e formed by the action
of acids (v. Udranszky) without regard to the fact that such substances
may sometimes originate from the reagents used, as from impure amyl
alcohol (v. Udranszky ^). To these humin bodies developed by the action
of acid in normal urine when exposed to the air must be added the urophain
of Heller, the various uromelanins and other bodies described by different
investigators (Plosz, Thudichum, Schunk^). Indigo-blue (uroglaucin of
Heller, urocyanin, cyanurin, and other coloring-matters of older investi-
gators ^) is split off from the indoxyl-sulphuric acid or indoxyl-glucoronic
acid. Red coloring-matter may be formed from the conjugated indoxyl
and skatoxyl acids, and urohodin (Heller), urorubin (Plosz), urohcematin
(Harley), and perhaps also iirorosein (Nencki and Sieber ^) probabh'
have such an origin.
We cannot discuss more in detail the different coloring-matters obtained
as decomposition products from normal urine. Htematoporphyrin has
already been referred to in a previous chapter (VI) and will best be de-
scribed 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 ^ had previously given the same name to a less pure
pigment isolated by himself. According to Garrod urochrome is free from
iron, but contains nitrogen. It stands, it seems, in close relationship to
urobilin, as Garrod has obtained a urobilin-like pigment by the action of
impure aldehyde on urochrome, and Riva '^ claims that urobilin yields a
» Jaffe, Centralbl. f. d. med. Wisaensch. 1868 and 1869, and Virchow's .^Lrch., 47;
Disque, Zeitschr. f. physiol. Chem., 2; Saillet, Revue de medecine, 17, 1897.
^ V. Udranszky, Zeitschr. f. physiol. Chem., 11, 12, and 13.
^ Plosz, Zeitschr. f. physiol. Chem., 8; Thudichum, Brit. Med. Journ., 201, and
Journ. f. prakt. Chem., 104; Schunk, cited from Huppert-Neubauer, 10. Aufl., 509.
* See Huppert-Neubauer, 161.
* In regard to this and other red pigments, see Huppert-Neubauer, 593 and 597 ;
Nencki and Sieber, Journ. f. prakt. Chem. (2), 26.
'Garrod, Proceed. Roy. Soc, 55; Thudichum, 1. c.
' Garrod, Journ. of Physiol., 21 and 29; Riva, cited from Huppert-Neubauer, 524.
602 URINE.
body similar to urochrome on careful oxidation with permanganate. Accord-
ing to Garrod urobilin can be converted into urochrome by evaporating its
aqueous solution containing some ether on the water-bath. The fact that
urochrome can be transformed into urobilin ])y means of active acetaldehyde
may be used, according to Garrod, as a means of detecting urochrome.
Urochrome is, according to Garrod, amorphous, brown, very readily
soluble in water and ordinary alcohol, but less soluble in absolute alcohol.
It dissolves Init slightly in acetic ether, amyl alcohol, and acetone, while it
is insoluble in ether, chloroform, and benzene. Urochrome is precipitated
by lead acetate, silver nitrate, mercuric acetate, phosphotungstic and phos-
phomolybdic 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 readliy decomposed, with the formation
of brown substances, by the action of acids. According to Klemperer,'-
urochrome contains 4.2 per cent nitrogen.
Urochrome can be prepared according to a rather complicated method
which is Ijased 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 OR the salt solution, which contains the urochrome and which can
be used for the further preparation of the latter (see Garrod, 1. c.)-
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.
The urochrome can l)e quantitatively estimated, according to Klem-
perer, by a colorimetric method, using a solution of tnie yellow G. If
0.1 gram of this dye is dissolved in 1 liter of water and 5 c.c. of this solu-
tion diluted to 50 c.c. with water, then this solution has the same color and
shade as a 0.1 per cent urochrome solution. The urine must be diluted
with water until it has the same depth of color. The comparison is per-
formed in vessels with parallel walls.
Urobilin is the pigment first isolated from the urine by Jaff^i.^ and
which is characterized by its strong fluorescence and by its absorption-
spectnim. Various investigators have prepared from the urine by different
methods pigments w'hich differed slightly • from each other but behaved
essentially like Jaff^i's urobilin. Thus different urobilins have been sug-
gested, such as normal, febrile, physiological, and pathological urobilins.^
'■ Berlin, klin. Wochenschr., 40.
^ Centralbl. f. d. med. Wissensch., 1868 and 1869, and Virchow's Arch., 47.
^ 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, Pfliiger's Arch., 61.
UROBILIN. 603
The possibility of the occurrence of different urobiUns in the urine cannot
be denied; but as urobiUn is a readily changeable body and difficult to
purify from other urinary ])igments, the question as to the occurrence of
different urobilins must still he considered open. According to Saillet ^
no urobilin exists originally in human urine, but only the mother-substance
of the same. urob^Uinogen, from which the uroljilin 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
reduction. ^Ialy obtained his hydrolnlirubin by the reduction of bilirubin
wdth sodium amalgam, and Disque obtained a prodvict which is still more
similar to urobilin, while Stokvis prepared by the oxidation of cholecyanin
with a little lead peroxide a choletelin which acted ver}^ much like urobilin.
Hoppe-Seyler, Le Nobel, Nencki and Sieber have obtained urobilinoid
bodies by the reduction of ha^matin and ha:'matoporphyrin with tin or zinc
and hydrochloric acid, while jMacjMunn ^ obtained by the oxidation of
heematin with hydrogen peroxide in alcohol containing sulphuric acid a
pigment Vv'hich 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 hydrobilimbin,
but according to the researches of Hopkins and Garrod ^ this view is not
correct, because, irrespective of other small differences, each body has
an essentially distinct composition. Hydrobihrubin contains C 64.68,
H 6.93, N 9.22 (jMaly), w^hile urinary urobilin, on the contrary, contains
C 63.46, H 7.67, N 4.09 per cent. The urobilin from faeces, stercohilin,
has the same composition as urinar}^ urobilin with 4.17 per cent nitrogen.
Urinary urobilin mav not be identical with hydrobihrubin, but this does
not exclude the possibility that urobilin, according to the generally ad-
mitted view, is derived from bilirubin (although not by simple reduction
and taking up water) in the intestine. Several ph}'siological as well as
clinical observations ^ speak for this view, among which we must mention
the regular appearance in the intestinal tract of stercobilin, undoubtedly
derived from the bile-pigments and having the same composition as urinary
urobilin, the absence of urobilin in the urine of new-born infants as well as
* Revue de m^decine, 17, 1897.
^ Maly, Ann. d. Chem. u. Pharm., 163; Disqu^, Zeitschr. f. physiol. Chem., 2;
Stolcvis, Centralbl. f. d. med. Wissensch., 1873, 211 and 449; Hoppe-Seyfer, Ber. d.
deutsch. chem. Gesellscli., 7; Le Nobel, Pfli'iger's Arch., 40; Nencki and Sieber,
Monatshefte f. Chem., 9, and Arch. f. exp. Path. u. Pharm., 24; MacMunn, Proc. Roy.
Soc, 31.
^ Journ. of Physiol., 22.
*See Fr. Miiller, Scliles. Gesellsch. f. vateri. Kultur, 1892; D. Gerhardt, "Ueber
Hydrobilimbin und seine Bezieh. zum Ikterus" (Inaug.-Diss., Berlin, 1889); Beck,
Wien. klin. Wochenschr., 1895; Harley, Brit. Med. Journ., 1896.
604 URINE.
on the complete exclusion of bile from the intestine, and also the increased
eUmination of urobihn with strong intestinal putrefaction. On the other
hand there are investigators who, basing their opinion on clinical observa-
tions, deny the intestinal origin of urobilin and claim that the urobilin is
derived from a transformation of the bilirubin elsewhere than in the intes-
tine, by an oxidation of the bile-pigment or by a transformation of the
blood-pigments.^ The possibility of a different mode of formation of uri-
nary urobihn 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 physio-
logical conditions.
The quantity of urobilin in the urine under physiological conditions is
very variable. 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 Jaff]&, Disqu6, Gerhardt, G. Hoppe-Seyler,^ and
others. The quantity is increased in hemorrhage and in such diseases
where the blood-corj^uscles are destroyed, as is the case after the action of
certain bood-poisons, such as antifibrine and antipyrine. It is also in-
creased 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 be different, 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 solu-
bility is augmented by the presence of neutral salts. It may be com-
pletely precipitated from the urine by saturating with ammonium sulphate,
especially after the addition of sulphuric acid (MiiHU^). 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-alcohohc)
solution; alkali solutions remove the urobilin from the chloroform. The
neutral or faintly alkaline solutions are precipitated by certain metallic
salts (zinc and lead), but not by others, such as mercuric sulphate. Uro-
bilin 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
' 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.
^ In regard to the Hterature on this subject we refer the reader to D. Gerhardt,
"Ueber HydrobiHrubin und seine Beziehungen zum Ikterus" (BerHn, 1889), and
also G. Hoppe-Seyler, Virchow's Arch., 124.
' Journ. de Pharm. et Chim., 1878, cited from Maly's Jahresber., 8.
UROBILIN. 605
which may be mistaken for the biuret test, by the action of copper sulphate
and alkah.i
Neutral alcoholic urobilin solutions are in strong concentration brownish
5^ellow, 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 h and F, which borders on F, 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 ammoniacal solution of the pig-
ment it becomes red and shows a beautiful green fluorescence. This solu-
tion, as also that made alkaline with fixed alkalies, shows a darker and more
sharply defined band, d, between h and F, almost midway between E and F.
If a sufficiently concentrated solution of urobilin alkali is carefully acidi-
fied with sulphuric acid it becomes cloudy and shows a second band exactly
at E and connected with 7- by a shadow (Garrod and Hopkins, Saillet^).
Urobihnogen is colorless or is oni}^ slightly colored. Like urobilin it is
precipitated from the urine by saturating with ammonium sulphate. Ac-
cording to Saillet it may be extracted by acetic ether from urine acidi-
fied with acetic acid. It dissolves also 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 Neu-
BAUER and Bauer,3 gives the Ehrlich reaction with dime thy lamiclo-
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 filtered alcoholic solution
is diluted with water, saturated wHh 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, Avhich is washed with
water and boiled with alcohol, is decomposed by alcohol containing sul-
phuric acid, the filtered alcoholic solution is mixed wdth J vol. chloroform,
diluted with water, and shaken repeatedly, but not too energetically. The
urobilin is taken up l^y the chloroform. This last is washed once or twice
with a little water and then distilled, leaving the uroljilin. The pigment
may be precipitated directly from the urine rich in uroliilin by ammonia
and zinc chloride, and the precipitate treated as above described (Jaffe).
' See Salkowski, Berlin, klin. Wochenschr., 1897, and Stokvis, Zeitschr. f, Biologie,
34.
' Garrod and Hopkins, Journ. of Physiol., 20; Saillet, 1. c.
' Neubauer, cited from Centralbl. f. Physiol., 19, 145; Bauer, cited from Biochem.
Centralbl.. 4, .350.
006 URINE.
The method suggested by IMehu (precipitation with ammonium sul-
phate) has been modified by Garrod and Hopkins in that they first re-
move the uric acid by saturating with ammonium chloride and then satu-
rating the filtrate with ammonium sulphate. The precipitated urobilin is
thus made purer than by saturating with the sulphate directly. The
urobilin is extracted from the dried precipitate by a great deal of water,
rei^recipitated by ammonium sulphate, and this procedure rej^eated several
times if necessar}'. The dried precipitate finally obtained is dissolved in
absolute alcohol. In regard to small details, and to a second method sug-
gested by these experimenters, we refer to the original work.^
Saillet extracts the urobilinogen from the urine by shaking with acetic
ether, using a kerosene-oil light.^
The color of the acid or alkaline solution, the beautiful fluorescence of
the ammoniacal solution treated with zinc chloride, and the absorption-
bands of the spectrum, all serve as means of detecting urobilin. In fever-
urines the urobilin may be detected directly or after the addition of ammo-
nia and zinc chloride l)y its spectrum. It may also sometimes 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 Schlesingf:r ^ it can be readily detected if the urine is pre-
cipitated by an equal volume of a 10 per cent solution of zinc acetate in
absolute alcohol. Disturbing bodies are here precipitated and the filtrate
gives the fluorescence directly, and also the spectrum. Grimbert'* has
given another comparatively simple method.
In the quantitative estimation of urobilin we proceed as follows, accord-
ing to G. Hoppe-Seyler: ^ 100 c.c. of the urine is acidified with sulphuric
acid and saturated with ammonium sulphate. The precipitate is collected
on a filter after some time, washed with a saturated solution of ammonium
sulphate, and repeatedly extracted with equal parts of alcohol and chloro-
form after pressing. The filtered solution is treated with water in a sepa-
ratory funnel until the chloroform separates well and becomes clear. The
chloroform solution is evanorated on the water-bath in a weighed beaker,
the residue dried at 100° C, and then extracted wdth ether. The ethereal
extract is filtered, the residue on the filter dissolved in alcohol, and trans-
ferred to the beaker and evaporated, then dried and weighed. According
to this method G. Hoppe-Seyler found 0.08-0.14 gram of urobiUn in one
day's urine of a healthy person, or an average of 0.123 gram.
Urobilin may also be determined spcctrophotometrically according: to Fr.
MtJLLER or Saillet.^ Saillet found that the limit for the perceptil^ility of
' Joiirn. of Physiol., 20.
^ In regard to this and other methods, we must refer the reader to special works.
'Deutsch. med. Wochenschr., 1903.
<See Cliem. Centralbl, 1904, 1, 1023.
5 Virchow's Arch., 124.
'Fr. Miiller, see Huppert-Neubauer, 861; Saillet, 1. c.
UROERYTHYRIN. 607
the absorption-bands of an acid-urobilin solution lies in a concentration of
1 milligram of urobilin in 22 c.c. 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 dilu-
tion. The freshly voided urine, shielded from light, is acidified with acetic acid,
completely extracted in kerosene-oil light with acetic ether, and the dissolved
urobilinogen oxidized to urobilin with nitric acid. On the addition of ammonia
and shaking with water the urobilin passes into the watery solution. This is
acichfied with hydi'ochloric acid and diluted until the above limit is reached.
Uroerythrin is the pigment which often gives the l)eautiful red color to
the urinars^ sediments {sedimentum lateritium). It also frequently occurs,
although onl}' in ven- 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, circulatoiy disturbances of the liver, and in
many other pathological conditions.
Uroerv'thrin, which has been especially studied by Zoja, Riva, and
Gaerod,^ has a pink color, is amorjjhous, and is verv quickly destroyed by
light, especially when in solution. The best solvent is amvl alcohol; acetic
ether is not so good, and alcohol, chloroform, and water are even less valu-
able. The yery dilute solutions show a pink color; but on greater con-
centration they become reddish orange or bright red. They do not fluoresce
either directly or after the addition of an ammoniacal solution of zinc chlo-
ride; 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 h. Concentrated sulphuric acid
colors a uroer^-thrin 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
HER^^EUx2 claim that uroer^-thrin is a skatol pigment.
In preparing uroerythrin according to Garrod, the sediment is dissolved
in water at a gentle heat and satm'ated 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 ha'matoporph^Tin remaining
being removed by shaking with chloroform : the precipitate is then faintly acidi-
fied with acetic acid and shaken with chloroform, which takes up the uroerythrin.
The chloroform is evaporated in the dark at a gentle heat.
Volatile fatty adds, such as formic acid, acetic acid, and perhaps also but^Tic
acid, occur under normal conditions in human urine (v. Jaksch), also in that of
^ Zoja, Arch. ital. di clinica med., 1S9.3. and Centralbl. f. d. med. "Wissensch. , 1892;
Riva, Gaz. med. di Torino, Anno 43, cited from Maly's Jahresber., 24; Garrod, Journ.
of Physiol., 17 and 21.
^ Journ. de Physiol., 7.
608 URINE.
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, O.OOS-0.009 gram per twenty-four hours; accord-
ing to V. RoKiTANSKY, 0.054 gram; and according to Magnus-Levy, 0.060 gram.
The quantity is increased by exclusively farinaceous food (Rokitansky), in
fever and in certain diseases, while in others it is diminished (v. Jaksch, Rosen-
feld). Large amounts of volatile fatty acids are produced in the alkaline fer-
mentation of the urine, and the quantity is 6-15 times as large as in normal urine
(Salkowski ')• Non-volatile fatty acick have been detected as normal con-
stituents of urine by K. Morner and Hybbinette.^
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), and especially abundant in eclampsia
(Zweifel). 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 de-
creased in any way, and this probably explains the occurrence of lactic acid in
the urine after epileptic attacks (Inouye and Saiki). Minkowski ^ has shown
that lactic acid occui's in the urine in large quantities on the extirpation of the
liver of birds.
Glycerophosphoric acid occurs as traces in the urine,* 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 investigations
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 proved by the investigations of Baumann
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 carbohydrate (animal gum), as shown by Landwehr, Weden-
ski, and Baisch. The quantity of carbohydrates eliminated under normal
conditions in the twenty-four hours' urine and determined by the benzoyla-
* 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.
2 Skand. Arch. f. Physiol., 7.
^ Colasanti and Moscatelli, Moleschott's Untersuch., 14; Schultzen and Reiss,
Chem. Centralbl., 1869; Zweifel, Arch. f. Gynakol, 76; Araki, Zeitschr. f. physiol.
Chem., 15, 16, 17. 19. See also Irisawa, ibid., 17; v. Terray, Pfli'iger's Arch., 65;
Schutz, Zeitsclu'. f. physiol. Chem., 19; li.ouye and Saiki, ibid., 37; Minkowski, Arch,
f. exp. Path. u. Pharm., 21 and 31.
* See Pasqualis, Maly's Jaliresber., 24.
CONJUGATED GLUCORONATES. 609
tion method; which is perhaps not sufficiently trustworthy, varies consid-
erably between 1.5 and 5.09 grams. ^
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, according to Salkowski,- a nitrogenous carbohydrate which has strong
reducing action upon alkaline copper solutions after cleavage with hydrochloric
acid.
Besides traces of sugar and the reducing substances previously men-
tioned, uric acid and creatinine, the urine contains still other bodies of this
character. These latter are partly conjugated compounds of glucuronic
acid, CeHioOy, 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.
Several new methods for the determination of the reducing power of the urine
have been suggested.*
Conjugated glucoronates occur, as indicated by Fluckiger and first
positively shown by Mayer and Neuberg,^ in very small amounts in nor-
mal 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 glucuronates perhaps
sometimes the urine contains the urea glucuronic acid, the ureidoglucu-
ronic acid prepared synthetically by Neuberg and Neimann.^
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, turpen-
tine, morphine, and many other substances. The elimination of glucuronic
acid may be markedly increased in severe disturbances of the respiration,
severe dyspnoea, in diabetes mellitus, and by the direct introduction of large
amounts of dextrose. According to P. Mayer, as stated on page 122, in the
oxidation of dextrose a part of it forms glucuronic acid, hence it is to be
' 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.
^ Berlin, klin. Wochensclir., 1905.
^ Fliickiger, Zeitschr. f. physiol. Chem., 9. See also Huppert-Neubauer, page 72.
* See Rosin, Miinch. med. Wochenschr., 46; Niemilowicz, Zeitschr. f. physiol. Chem.,
36; Niemilowicz with Gittelmacher-Wilenko, ibid., 36, and Hdlier, Compt. rend., 129.
^ Fliickiger, 1. c; Mayer and Neuberg, Zeitschr. f. physiol. Chem., 29.
• Zeitschr. f. physiol. Cliem., 44.
610 URINE.
expected that the glucuronic acid can in part be derived from the dex-
trose. As a conjugation of the glucuronic acid with other bodies, such
as aromatic atomic complexes, prevents the combustion of this acid in
the animal body, it ought to follow that after the introduction of such
an atomic complex in the body during a glycosuria a corresponding
reduction of the glucose elimination would take place with the increased
excretion of conjugated glucuronates. In order to prove this possibility
O. LoEWi ^ fed dogs with camphor during phlorhizin 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.^ Normally rabbits convert nearly all the
camphor introduced into conjugated glucuronic acid. According to Mayer
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 quanti-
ties of glucuronic acid. By the simultaneous administration of camphor
and dextrose while starvation is going on, the elimination of glucuronic
acid rises again to the same height as it was before the starvation period.
This shows that the sugar had conjugated itself with the camphor as glucu-
r.onic acid. Hildebrandt ^ has also made experiments showing that glucu-
ronic acid can very likely be formed from sugar. The observations of
M ayer are not substantiated by the recent investigations of Fenyvessy,*
and the statements on this question are contradictory.
The conjugated glucuronic acids are formed, based upon the investi-
gations of SuNDWiK, Fischer and Piloty,5 by a combination tagkin place
first between the conjugator and the dextrose by means of the aldehyde
group, and then the end alcohol group, CH2OH, is oxidized to COOH. The
conjugated glucuronic acids at least in most cases seem to be constructed
after the glucoside type, a view which has received further support by
the synthesis of phenolglucuronic acid and euxanthonglucuronic acids by
Neuberg and Neimann.6 Based upon their cleavage (as far as they have
been investigated) by kephir lactase and emulsion, but not by yeast lactase
(Neuberg and Wohlgemuth '7), the conjugated glucuronic acids must
' Arch. f. exp. Path. u. Pharm., 47.
"Zeitschr. f. klin. xMed., 47.
'" Arch. f. exp. Path. u. Pharm., 44.
* See Maly's Jahresber., 34.
*E. Sundwik, Akademische Abhandlung Helsingfors, 1886; see also Maly's
Jahresber., 10, 76; Fischer and Piloty, Ber. d. d. chem. Gesellsch., 24.
' Zeitschr. f. physiol. Chem., 44.
' See Neuberg, Ergebnisse der Physiologic, Bd. 3, Abt. 1, 444.
ORGANIC COMBINATIONS CONTAINING SULPPIUR. 611
belong to the ,i9-series of glucosides. The ureidogkicuronic acid is still not
constructed upon the glucoside type, but according to the aldehydimine
type, H,N.C0.N.CH.(CH0H)4C00H. The reducing urochloralic acid can
hardly be built upon the glucoside type.
According to the body with which they are conjugated the glucuronates
show different behavior; they all rotate the plane of polarization to the
left, while the glucuronic acid itself is dextrorotatory. On taking up
water they spUt into glucuronic acid and the conjugated group. They are
precipitated by basic lead acetate or by basic lead acetate and ammonia.
Most of the conjugated glucuronic acids do not have a reducing action. A
few reduce copper oxide and certain other metallic oxides in alkahne solu-
tion and hence cause errors in the investigation of the urine for sugar. As
the detection of conjugated glucuronic acids is connected with the tests for
sugar in the urine, we will treat of this in connection with these tests.
Organic combinations containing sulphur of unknown kind, which may
in small part consist of sulphocyanides, 0.04 (Gscheidlen)-O.II 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 acids (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 ^ 19-24 per cent of the total sulphur. In starvation,
according to Fr. Mijller, 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^)
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, is dependent to a less degree than the sul-
phate excretion upon the extent of the protein metaboUsm. The relation-
ship between the neutral and acid sulphur depends in the first place upon
the extent of the sulphuric-acid excretion. According to Harnack and
Kleine 5 the relationship of the oxidized sulphur to the total sulphur
' Gscheidlen, Pfli'iger's Arch., 14; Munk, Virchow's Arch., 69.
^ Ibid., 58, and Zeitsclir. f. physiol. Chem., 9.
^Stadthagen, Virchow's Arch., 100; Lepine, Compt. rend., 91 and 97; Harnack
and Kleine, Zeitschr. f. Biologic, 37.
^ Fr. Miiller, Berl. klin. Wochenschr., 1887; Reale and Boeri, Maly's Jaliresber., 24;
Harnack and Kleine, 1. c; Presch, Virchow's Arch., 119; Yvon Arch de Physiol
(5), 10.
^Benedict, Zeitschr. f. klin. Med., 36; Harnack and Kleine, 1. c; Folin, Amer.
Journ. of Physiol., 13.
612 URINE.
changes always in the same way as the relationship of the nitrogen of the
urea to the total nitrogen. The more unoxidized sulphur is eliminated
the more abundant are the nitrogen compounds, not urea, in the urine — a
Statement which coincides with recent observations showing that the
neutral sulphur originates chiefly from the oxyproteic acid, the alloxy-
proteic acid, 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. Accord-
ing to the investigations of W. Smith,' it is probable that the most unoxidizable
part of the neutral sulphur occurs as sulpho-acids. An increased elimination of
neutral sulphur has been observed in various diseases, such as pneumonia, cysti-
nuria, 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. 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 oxidiz able part of the neutral
sulphur Is determined by oxidation with bromine or potassium chlorate and
hydrochloric acid (Lepine, Jerome^).
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 '). 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.^ Hyposulphites occur constantly in cat's urine and,
as a rule, also in dog's urine.
Antoxyjjroteic acid is & nitrogenous SLCid containing sulphur which Bond-
ZYNSKi, 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 spUt off by alkali. This acid
is soluble in water, is dextrorotatory, and is precipitated only from con-
centrated solution by phosphotungstic acid. It does not give the protein
color reactions, but gives Ehrlich's diazo-re action (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 precipi-
tated by mercuric nitrate and acetate, and by this last reagent even from
solutions strongly acidified wnth acetic acid. Basic lead acetate does not
precipitate the pure acid.
Oxyproteic acid is the name given by Bondzinski and Gottlieb ® to a
» L6pine, 1. c; Smith, Zeitschr. f. physiol. Chem., 17.
- Jerome, Pfliiger's Arch., 60.
3Fr. Miiller, Berlin, klin. Woclienschr., 1887; Salkowski, ibid., 1888.
* Heffter, Pfluger's Arch., 38; Salkowski, ibid., 39; Presch, Virchow's Arch., 119.
* Zeitschr. f. physiol. Chem., 46.
« Centralbl. f. d. med. Wissensch., 1897, No. 33.
ANTOXYPROTEIC AND ALLOXYPROTEIC ACIDS. 613
nitrogenous acid containing sulphur and whicli they prepared from human
urine, which has recently been further studied by Bondzynski, Dom-
BROWSKI 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 phosphotungstic acid, hence
it leads to an error in the Pfluger-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 antoxyproteic acid.
The acid which is found in large quantities especially in the urine of
dogs poisoned with phosphorus (Bondzynski and Gottlieb) is considered
like the preceding acids as an intermediary oxidation product of the pro-
teins, and oxyproteic acid seems to represent a higher state of oxidation or
a demolition of the proteins than the antoxyproteic acid.
The acid called uroproteic acid by Cloetta is probably a mixture of several
bodies, according to the recent investigations of Bondzynski, Dombrowski, and
Panek. The same applies also to the barium oxyproteate prepared by Pregl '
from the urine.
AUoxy prottic acid is a third acid related to the above, which was first
isolated by Bondzynski and Panek ^ 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 0, 37.23 per cent, based upon»new investigations. The free
acid is soluble in water. It gives neither the biuret reaction nor Ehrlich's
reaction, and is not precipitated by phosphotungstic acid. Differing from
the other acids, it is precipitated by basic lead acetate and its salts are only
slightly soluble in alcohol.
The preparation of the three above-mentioned acids is based in part
upon the fact that alloxyproteic acid alone is precipitated b}" basic lead
acetate and that the two other acids can be precipitated from the filtrate
by mercuric acetate, the antoxyproteic acid in acetic acid solution, and the
oxyproteic acid in neutral solution. The preparation is nevertheless very
tedious and complicated and therefore we must refer to the original works ^
for details.
Uroferric acid is an acid isolated by Thiele '^ from the urine, according
to Siegfried's method for preparing pure peptone. It also contains
' Cloetta, Arch. f. exp. Path. u. Pharm., 40; Pregl, Pfluger's Arch., 75.
' Ber. d. d. chem. Gesellsch., 36.
^ Zeitschr. f. physiol. Chem., 46.
*Ibid., 37.
614 URINE.
sulphur, 3.46 per cent, and has the formula CssHseNgSOig. The acid forms
a white powder which is readily soluble in water, saturated ammonium-
sulphate solution, and methji alcohol. It is soluble with difficulty in abso-
lute 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 ^^Iillon'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 is {a)o= -32.5°. On cleavage it yields melanine sub-
stances, sulphuric acid, aspartic acid, but no hexone bases. The existence
of this acid is disputed by Bondzynski, Dombrowski and P.\nek.
Abderhalden and Pregl i have shown that human urine normally
contains compounds which stand perhaps in close relationship to the poly-
peptides 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 glycocoU, also leucine, alanine, glutamic acid, phenyL
alanine, and probably also aspartic acid. The relationship between these
polypeptide-like bodies and the above-mentioned proteic acids and to the
uroferric acid has not been investigated.
Amino-acids may, when they are introduced in large amounts into the body,
also pass in part into the urine. This has been shown for r-alanine by R. Hirsch
for the dog, and by Plaut and Reese for dog and man, and for /--leucine by
Abderhaldex and Samuely ^ in rabbits. Embden and Reese, Forssner, Abder-
halden and ScHiTTENHELM, and Samuely ^ were able, by means of the naph-
thaline sulphochloride method, to detect glycocoll in normal human urine, and
this glycocoll must occur in the urine in a combination which is readily split
by alkali, .\lthough we have numerous investigations, other amino-acids besides
glycocoll could not be detected in normal human urine, while, on the contrary,
in pathological conditions other amino-acids have been found several times.
The amino-acid fraction of the urine seems to be increased in starvation and in
high altitudes (Loewy ■*).
Organic combinations containing phosphorus (glycerophosphoric acid, phospho-
carnic acid (Rockwood), etc., which ^neld phosphoric acid on fusing with salt-
peter and caustic alkali, are also found in urine (Lepine and Eitmonnet, Oertel).
With a total elimination of about 2.0 grams total P0O5, Oertel found on an
average about 0.0.5 gram PgOj as phosphorus in organic combination. According
to Symmers 5 the organic combined phosphoric acid may in many pathological
' Zeitschr. f. physiol. Chem., 46.
' R. Hirsch, Zeitschr. f. exp. Path. u. Therap., 1; Plaut and Reese, Hofmeister's
Beitriige, 7; Abderhalden and Samuely, Zeitschr. f. physiol. Chem., 4".
^ Embden and Reese, Hofmeister's Beitriige, 7 ; G. Forssner, Zeitschr. f . physiol.
Chem., 47; Abderhalden and Schittenlielm, ibid., 47; Samuely, ibid., 47.
* Deutsch. med. Wochenschr., 1905.
^ Rockwood, Arch. f. (Anat. u.) Physiol., 1895; Oertel. Zeitschr. f. physiol. Chem.,
26, wliich citas the other Avorks. See also Keller, Zeitschr. f. pliysiol. Cliem., 29;
Maridel and Oertel, X. Y. Univ. Bull. Med. Sciences, i; Symmers, Journ. of Path, and
Bact., 10.
PTOMAINES AND LEUCOMAINES. 615
conditions be 25-50 per cent of 'he 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 pejmn (Brucke and others), which, according to ]\Iatthes,
undoubtedly originates from the stomach, and a diastatic enzyme (Cohnheim and
others) and trypsin^
Mucin. The nubecula consists, as shown by K. Morxer,^ of a mucoid which
contains 12.74 per cent N and 2.3 per cent S. This mucoid, which apprarently
originates in the urinary passages, may pass to a slight extent into solution in
the urine. In regard to the nature of the mucins and nucleoalbumins otherwise
occurring in the urine we refer the reader to the pathological constituents of the
urine.
Ptoviaines and leucomaines, or poisonous substances of an unknown kind,
which are often described as alkaloidal substances, occur in normal m-ine (Pouchet,
Bouchard, Aducco, and others). Under pathological conditions the quantity
of these substances may be increased (Bouchard, Lepine and Guerix, Villiers,
Griffiths, Albu, and others). Within the last few years the poisonous proper-
ties 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 ui'ines have not the
same action. In order to be able to compare the toxic power of the urine under
different conditions, Bouch-VRD determines the urotoxic coefficiext, which is
the weight of rabbit in kilos that is killed by the quantity of m'ine excreted in
twenty-four hours by 1 kUo of the person experimented upon.^
Baumaxx and v. Udraxszky have shown that ptomaines may occur in the
urine under pathological conditions. Tliey demonstrated the presence of the two
ptomaines discovered and first isolated by Brieger — putresciue, C^KjjN, (tetra-
methylendiamine), and cadaverine, CsHuX, (pentamethylendianiine)— in the urine
of a patient suffering from cystinuria and catarrh of the bladder. Cadaverine
has later been fomid by Stadthagex and Brieger in the luine in two cases of
cystinuria. Brieger, v. Udraxszky and Baumaxx, and Stadthagex have
shown that neither these nor other diamines occur under physiological condi-
tions, while DoMBROWSKi, on the contrary, found cadaverine besides another
ptomaine with the formula CjHuXOo in normal urines, and Kutscher and Loh-
MANN * have found neurine. The occurrence in normal urine of any " urine
poison " is denied by certain investigators, such as Stadthagex, Beck, and v. d.
Bergh.^ The poisonous action of the urine, according to them, is due in part
to the potassium salts and in part to the sum of the toxicity of the other normal
urinary constituents (urea, creatinme, etc.), which have very little poisonous
action individually. The occurrence of special urine poisons under normal
conditions is difficult to deny on account of numerous statements on this subject,
although the chemical laature of these substances is still not sufficiently known.
Many substances have been observed in animal urine which are not found in
human ui'ine. To these belong the above-described kynurenic acid, urocanic acid,
' In regard to the literature on enzymes in the urine, see Huppert-Neubauer, 599 ;
Matthes, Arch. f. exp. Path. u. Pharm., 49.
2 Skand. Arch. f. Physiol., 6.
^ A complete bibliography on the ptomaines and leucomaines of the urine is found
in Huppert-Neubauer, 403.
* Baumann and Udranszky, Zeitschr. f. physiol. Chem., 13; Stadthagen and Brieger,
Virchow's Arch., 115; Dombrowski, Arch, polonais. d. sciences bid., 1903; Kutscher
and Lohmann, Zeitsclir. f. physiol. Chem., '48.
* Stadthagen, Zeitschr. f. kliu. Med., 15; Beck, Pfliiger's Arch., 71; v. d. Bergh,
Zeitschr. f. klin. Med., 35.
616 URINE
also found in dog's urine and which seems to stand in some relationship to the
purine bases; damaluric acid and damolic acid (according to Schotten,' probably
a mixture of benzoic acid with volatile fatty acids), obtained by the distillation
of cow's urine; and lastly lithuric acid, fomid in the urinary concrements cf
certain animals.
III. Inorganic Constituents of Urine.
Chlorides. The chlorine occurring in the urine is undoubtedly combined
with the bases contained in this excretion; the chief part is in combination
with sodium. In accordance with this, the quantity of chlorine in the
urine is generally expressed as NaCl.
The question as to whether a part of the chlorine contained in the urine
exists as organic combinations, as considered by Berlioz and Lepinois, is
still disputed .2
The quantity of chlorine combinations in the urine is subject to con-
siderable variation. In general the quantity from a healthy adult on a
mixed diet is 10-15 grams of NaCl per twenty -four hours. The quantity of
common salt in the urine depends chiefly upon the quantity of salt in the
food, with which the elimination of chlorine increases and decreases. The
free drinlving 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.'*
The quantitative estimation of chlorine in the urine is most simply per-
formed by titration with silver-nitrate solution. The urine must not con-
tain either proteid (w^hich 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
' Zeitschr. f. physiol. Chem., 7.
'Berlioz and Lepinois, see Chem. Centralbl., 1894, 1, and 1895, 1; also Petit and
Terrat, ibid., 1894, 2, and Vitali, ibid., 1897, 2; Viele and Moitessier, Maly's Jahresber.,
31; Meillere, ibid.; Bruno, ibid., 452.
^ Zeller, Zeitschr. f. physiol. Chem., 8; Kast, ibid., 11; Vitali, Chem. Centralbl.,
1899, 2.
* On the elimination of chlorine in disease, see Albu and Neuberg, Physiol, u. Pathol,
des Mineralstoffwechsels, Berlin, 1906.
CHLORIDES. 617
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 othei'^N'ise excellent titration method of ]\Iohr, according to Avhich
we titrate with silver nitrate in neutral liquids, using neutral potassium
chromate as an indicator, cannot be used directly on the urine in careful
work. Organic urinaiy 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 ]je destroyed.
For this purpose evaporate to drs-ness 5-10 c.c. of the urine, after the
addition of 1 gram of chlorine-free soda and 1-2 grams chlorine-free salt-
peter, and carefully fuse. The mass is dissolved in water, acidified faintly
wdth nitric acid, and then neutralized exactly witli 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 CI or
0.01 gram XaCl. This last-mentioned solution contains 29.075 grams of
AgNOs in 1 liter.
Freuxd and Toepfer, as well as Bodtker.^ have suggested modifica-
tions of Mohr's method.
Volhard's ^Method. Instead of the preceding determination, Vol-
hard's method, which can be performed directly on the urine, maj^ 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 b}' 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
CI. 2. A saturated solution at the ordinary^ temperature of chlorine-free
iron alum or ferric sulphate, 3. Chlorine-free nitric acid of a specific
graA^ty of 1.2. 4. A potassium-sulphocyanide solution which contains 8.3
grams KCNS per liter, and of which 2 c.c. corresponds to 1 c.c. of the
silver-nitrate solution.
About 9 grams of potassium sulphocyanide is dissolved in water and diluted
to 1 liter. The quantity of KCXS contained in this solution is determined by the
silver-nitrate solution in the follo'ning way: Measure exactly 10 c.c. of the silver
solution and treat it with 5 c.c. of nitric acid and 1-2 c.c. of the ferric-salt solu-
tion and dilute vaih water to about 100 c.c. 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 fomad in the solu-
^Freund and Toepfer, see Maly's Jahresber., 22; Bodtker, Zeitschr. f. phyhiol
Chem.. 20.
618 URINE.
tion by this means indicates how much it must be diluted to be of the proper
strength. Titrate once more with 10 c.c. of AgNOg solution and correct the sul-
phocyanide solution by the careful addition of water until 20 c.c. exactly corre-
sponds to 10 c.c. of the silver solution.
The determination of the chlorine in the urine is performed by this
method in the following way Exactly 10 c.c. of the urine is placed in a
flask which has a mark corresponding to 100 c.c. and which is provided
with a stopper; 5 c.c. of nitric acid is added; dilute with about 50 c.c. of
water and then allow exactly 20 c.c. of the silver-nitrate solution to flow
in. Close the flask with the stopper and shake well, remove the stopper
and wash it with distilled water into the flask, and fill the flask to the
100-c.c. mark with distilled water. Close again with the stopper, carefully
mix by shaking, and filter through a dry filter. Measure ofT 50 c.c. of
the filtrate by means of a dry pipette, add 3 c.c. of ferric-salt solution, and
allow the sulphocyanide solution to flow in until the liquid above the
precipitate has a permanent red color. The calculation is very simple.
For example, if 4.6 c.c. of the sulphocyanide solution was necessary to
produce the final reaction, then for 100 c.c. of the filtrate (=10 c.c. urine)
9.2 c.c. of this solution is necessary. 9.2 c.c. of the sulphocyanide solution
corresponds to 4.6 c.c. of the silver solution, and since 20—4.6=15.4 c.c.
of the silver solution was necessary to completely precipitate the chlorine
in 10 c.c. of the urine, then 10 c.c. contains 0.154 gram of NaCl. The
quantity of sodium chloride in the urine is therefore 1.54 ])er cent, or 15.4
p. m. If we always use 10 c.c. for the determination, and always 20 c.c.
of AgNOs solution, and dilute with water, to 100 c.c, 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 c.c. of the
filtrate. The quantity of NaCl p. m. therefore under these circumstances
= 20— R, and the percentage of NaCl = '" .
If it is necessary to destroy the organic urinary constituents before titration,
this can best be performed, according to Dehn,' by evaporating the urine (10 c.c),
after the addition of a small amount of sodium peroxide, to dryness on the water-
bath then faintly acidifying with nitric acid and then titrating according to
VoLHARD. Incineration is umiecessary.
For the approximate estimation of chlorine in the urine Ekehorn has
made use of Volhard's titration method ])y using for the determination
a glass tube closed at one end and divided into half cubic-centimeters
and called the chlorometer. The reagents necessary are: (a) a mixture
of 20 c.c silver-nitrate solution (according to Volhard), 5 c.c nitric acid
and water to 100 c.c; (b) 40 c.c. sulphocyanide solution (according to
Volhard) and 60 c.c. of a ferric alum, chlorine-free and saturated at the
ordinary temperature. The silver-nitrate solution, of which each cul)ic
centimeter corresponds to 0.002 gm. NaCl, is equivalent to the iron sulpho-
cyanide solution. First 2 c.c. of the urine is placed in the graduated tube
and then 0.5 c.c. sul])hocyanide 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 c.c is sub-
^ Zcitschr. f. physiol. Chcm., 44.
PHOSPHATES. 619
tracted from the silver solution for the 0.5 c.c. of the sulphocyanide; the
tube is so graduated that the quantity of NaCl in the urine in parts per
thousand is read off directly on the tube. The difference between these
results and those obtained by Volhard's titration method amounts only,
according to C. Th. Morner/ to 0.25 to at most 0.5 p. m.
The approximate estimation of chlorine in the urine (which must be
free from proteid) 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 droj) .sinks to the bottom as a rather
compact cheesy lump. In diminished c^uantities of chlorides the precipi-
tate is less compact and coherent, and in the presence of very little chlorine
a fine white precipitate or only a cloudiness or opalescence is oljtained.
Phosphates. Phosphoric acid occurs in acid urines partly as dihydrogen,
MH2PO4, and partly as monohydrogen M2HPO4, phosphates, both of
which may be found in acid urines at the same time. Ott 2 found that on
an average 60 per cent of the total phosphoric acid was di- and 40 per
cent was monohydrogen phosphate. The total quantity of phosphoric acid
is very variable and depends on the character and the quantity of food. The
average quantity of P2O5 is in round numbers 2.5 grams, with a variation of
1-5 grams per day. A small part of the phosphoric acid of the urine orig-
inates 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 in-
creased ; still it is undecided to what extent the excretion of phosphoric acid
is a measure of the absorption and decomposition of these bodies.^ 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 phos-
phates in proportion to the quantity of lime and magnesium phosphates.
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 quan-
tity excreted by the urine is small. This is true especially of herbivora,
in which the kidneys are the chief organs for the excretion of alkali phos-
phates. In man, according to Ehrstrom, the content of lime in the food
seems to play no important role, as in his experiments aljout one-half of
the phosphoric acid taken as CaHP04 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, l;)ut also upon the relative
' Ekehorn, Hygiea, Stockholm, 1906; Morner, Upsala Lakaref. Forh. (N. F.), 11.
^ Zeitschr. f. physiol. Chem., 10.
^ 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; Roh-
mann and Steinitz, Pfliiger's Arch., 72; Loewi, Arch. f. exp. Path. u. Pharm., 41
and 45.
620 URINE.
amounts of the alkaline earths and the alkali salts of the food. In car-
nivora, in which phosphate injected subcutaneously is eliminated by the
intestine (Bergman x), the urine is habitually poor in phosphates. ^
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 relationship 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 rela-
tionship between nitrogen and phosphoric acid in the urine can be kept
nearly constant. Thus on feeding with an exclusive meat diet, as ob-
served by Voit2 in dogs, when the nitrogen and phosphoric acid (P2O5)
of the food exactly reappeared in the urine and fseces, the relationship was
8.1:1. In starvation, as shown by the compilation of R. Tigerstedt,^
the phosphorized constituents of the body are destroyed to a much greater
extent than when food is given ver}' poor in phosphorus. In starvation
this relationsliip 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. ]Munk4 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 w^ould also be augmented. This is not
the case, and indeed we have observed cases with an increased elimination
of alloxuric l^odies with a diminution in the phosphoric-acid excretion.
Cases of leucaemia have been observed in which the phosphoric-acid excre-
tion 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 occurs also in inflamma-
tory and renal diseases. The earthy phosphates of the urine sometimes
have the tendency of precipitating either spontaneously or after warming,
and this has been called phosphaturia. We are dealing here with a dimin-
ished acidity and, it seems, Avith a diminished excretion of phosphoric acid
' Ehrstrom, Skand. Arch. f. Physiol., 14; Bergmann, Arch. f. exp. Path. u. Pharm.,
47.
^ Physiologie des allgemeinen Stofifwechsels und der Ernahrung in L. Hermann's
Handbuch, fl, Thl. 1, 79.
' Skand. Arch. f. Physiol., 16.
* Preysz, see Maly's Jahresbcr., 21; Olsavszky and Kkig, Pfliiger's Arch., 54;
Munk Arch. f. (Anat. u.) Physiol., 1895.
ESTIMATION OF PHOSPHATES. 621
and an increased elimination of lime, or at least an essentially different
relationship between the phosphoric acid and the alkaline earths of the urine,
as compared with the normal (Paxek, Iwanoff, Soetber and Krieger).^
Quantitative Estimation of the Total Phosphoric Acid in the Urine. This
estimation is most simply performed by titrating with a solution of ura-
nium acetate. The principle of the titration is as follows: A warm solu-
tion of phosphates containing free acetic acid gives a whitish-yellow pre-
cipitate of uranium phosphate with a solution of a uranium salt. This
precipitate is insoluljle in acetic acid, but dissolves in mineral acids, and
on this account there is always added, in titrating, a certain quantit}^ 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 cor-
responds to 0.007 gram P2O5 and which conains 20.3 grams of uranium
oxide per liter. 20 c.c. of this solution corresponds to 0.100 gram P2O5.
2. A solution of sodium acetate. 3. A freshly prepared solution of potas-
sium ferrocyanide.
The uranium solution is prepared from uraniimi 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 b}^ titrating
with a solution of sodium phosphate of known strength (10.085 grams crystallized
salt in 1 liter, which corresponds to 0.100 gram PoO^ in 50 c.c). 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 c.c. of the uranium solution corre-
sponds exactly to 50 c.c. of the above phosphate solution.
The sodium-acetate solution should contain 10 grams sodium acetate and
10 grams cone, acetic acid in 100 c.c. For each titration 5 c.c. of this solution
is used with 50 c.c. of the urine.
In performing the titration, mix 50 c.c. of filtered urine in a beaker
with 5 c.c. 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 potassium-
ferrocyanide solution. If the amount of uranium solution added has not
been sufhcient, the color will remain pale j-ellow 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 c.c. of
the urine. The calculation is so simple that it is imnecessary to give an
example.
' Panek, see Maly's Jahresber., 30, 112; Iwanoff, Biochem Centralbl , 1, 710;
Soetber and Krieger, Deutsch. Arch. f. klin. Med., 72; Campani, Biochem. Centralbl.,
3, 616; Tobler, Arch. f. exp. Path. u. Pharm., 52.
622 URINE.
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 or with alkalies, we first determine the total phosphoric
acid in a portion of the urine and then remove the earthy phosphates in
another portion by ammonia. The precipitate is collected on a filter,
washed, transferred into a beaker with water, treated with acetic acid, and
dissolved by warming. This solution is now diluted to 50 c.c. with water,
and 5 c.c. sodium-acetate solution added, then titrated with uranium solu-
tion. 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. As the. sulphuric acid chiefly
originates from the proteins, it follows that the elimination of sulphuric
acid and the elimination of nitrogen nms nearly parallel, and the relation-
ship N:H2S04 is about 5:1. A complete parallelism can hardly be ex-
pected, 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 variation as compared with the
large proportion of nitrogen contained therein. In general the elimination
of nitrogen and sulphuric acid under normal and under diseased conditions
seem to nm rather parallel. Sulphuric acid occurs in the urine partly pre-
formed (sulphate-sulphuric acid) and partly as ethereal, sulphuric acid.
The first is designated as ^- and the other as 5-sulphuric acid.
The quantity of total sulphuric acid is determined in the following way,
but at the same time the precautions described in other works must be
observed. 100 c.c. of filtered urine is treated with 5 c.c. of concentrated
hydrochloric acid and boiled for fifteen minutes. While boihng i)recipitate
with 2 c.c. of a saturated BaCU solution, and warm for a little while until
the l)arium 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 BaCU from
the urine acidified with acetic acid, then decomposing the ethereal-sul-
phuric acid by boiUng after the addition of hydrochloric acid, and finally
POTASSIUM, SODIUM AND AMMONIA. 623
determining the sulphuric acid set free as barium sulphate. A still better
method is the following, suggested by Salkowski;!
2U0 c.c. of urine is precipitated by an ec^ual volume of a barium solution
which consists of 2 vols, barium hydrate and 1 vol. barium-chloride solu-
tion, both saturated at the ordinary temperature. Filter through a dry
filter, measure off 100 c.c. of the filtrate which contains only the ethereal-
sulphuric acid, treat with 10 c.c. 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-sul])huric acid.
FoLiN 2 has suggested a method for estimating the sulphate-sulphuric
acid as well as the ethereal-sulphuric acid, and also the total sulphur, which
is somewhat different from the ordinary methods.
Nitrates occur in small quantities in human urine (Schonbein), and they
probably originate from the drinking-water and the food. According to Weyl
and Citron,^ 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 Salkowski,^ 3-4
grams K2O and 5-6 grams Na20, with an average of about 2-3 grams
K2O and 4-6 grams Na20. 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 potassium. 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 performed by the gravi-
metric methods as described in works on quantitative analysis. In the
determination of the total alkalies recently new methods have been de-
vised by Pribram and Gregor, and for the potassium alone a method by
AuTENRiETH and Bernheim.5
Ammonia. Some ammonia is habitually found in human urine and in
that of carnivora. As above stated (page 551), on the formation of urea
from ammonia, this quantity may represent the small amount of ammonia
* Baumann, Zeitschr. f. physiol. Chem., 1; Salkowski, Virchow's Arch., 79.
^ Journ of Biol. Chem , 1, and Amer. Journ. of Physiol., 13.
' Schonbein, Journ. f. prakt. Chem., 92; Weyl, Virchow's Arch., 96, with Citron,
ibid., 101.
' Ibid., 53.
' Pribram and Gregor, Zeitschr. f . analyt. Chem., 38; Autenrieth and Bernheim,
Zeitschr f. physiol. Chem., 37.
€24 URINE.
which, is excluded from the synthesis to urea by being combined with
acids formed in excess by combustion and not united with tlie 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 liy the urine is about 0.7 gram
NH3 per day (Neubauer), corresponding to 4.6-5.6 per cent of the total
nitrogen of the urine according to Camerer, Jr. As above stated, all the
ammonia of the urine is not represented by the residue which has eluded
synthesis iinto urea by neutralization with acids, becaues, as shown by
Stadelmann and Beckmann,^ ammonia Ls ehminated by the urine even
during the continuous administration of fixed alkalies.
Ammonia exists on an average of about 0.90 milligram in 100 c.c. of
human blood, and in different amounts in all the tissues thus far investi-
gated.2 According to Nencki and Zaleski ^ it is abundantly formed in
the cells of the digestive glands, the stomach, the pancreas, and the intes-
tinal mucosa (of dogs) at 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 554), 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,* 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 unequal behavior of different
animals towards acidosis has been discussed in the previous pages.
Acids formed in the destruction of proteins in the body act on the elim-
ination 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
' 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.
^ See Salaskin, Zeitschr f physio! Chem., 25, 449, and foot-notes 1 and 2, page
241.
' Arch des science biol. de St Petersbourg, 4, and Salaskin, J. c. See also Nencki
and Zaleski, Arch. f. exp Path, u Pharm., 37.
* John, 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, and the works
cited on page 5.'j4.
CALCIUM AND MAGNESIUM. 625
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 produced, wliich
pass into the urine combined with ammonia.^ Other observations also
indicate that the .elimination of ammonia by the urine is increased on insuf-
ficient or diminished supply of alkahes or alkaline earths.
The detection and quantitative estimation of ammonia used to be performed
according to the method suggested by Schlosixg. 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 X/10 sulphuric acid.
After the absorption of the ammonia the quantity is determined by titrating the
remaining free sulphmic acid with a X/10 caustic-alkali solution. This method
gives low results, and in exact work we must proceed as suggested by Bohland.*
The recent methods for estimating the ammonia are all based upon the
distillation of the ammonia, after the addition of lime, magnesia, or alkaU
carbonate, at low temperatures either by the aid of vacuum (Nenxki and
Zaleski, Wurster, Kruc;er, Reich, and Schittexhelm, and Schaffer)
or by the aid of a current of air (Folin) and then collecting it in a standard
acid.
According to the methods suggested by KRiJGER, Reich and Schitten-
helm^ 25 c.c. of the urine is placed in a distillation-flask with about 10
grams of NaCl and 1 gram of Na2C03, 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 absorlTed in X/10 acid
contained in a Peligot tube siuTounded by ice-water, and when the
distillation is finished the acid is retitrated. making use of rosohc acid as
indicator. In regard to details, see the original pubhcations. Schaffer 's
method is practically the same.
Calcium and magnesium occur in the urine chiefly as phosphates.
The quantity of earthy phosphates eUminated daily is somewhat more
than 1 gram, and of this amount | is magnesium and J calcium phos-
phate. This statement, as found by Renw^^ll and Gross,^ 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 solubihty of the first, among which
the calcium salt CaHP04 is especially insoluble, is particularly augmented
by the presence in the urine of dihydrogen alkali phosphates and sodium
chloride COtt^). The quantity of alkahne earths in the urine depends
' On the elimination of ammonia in disease, see the recent works of Rumpf, Vir-
chow's Arch., 134; Hallervorden, ibid.
'Pfliiger's Arch., 43, 32
' Zeitschr. f physiol. Chem., 89; Schaffer, Amer. Journ. of Physiol., 8, which con-
tains the Uterature.
* Renwall, Skand. Arch. f. Physiol , 16; Gross, Biochem. Centralbl., 4, ISO.
^Zeitschr. f. pliysiol. Chem., 10
626 URINE.
on the composition of the food. The Ume-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 the absorption of the same. 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. Nothing
is known with positiveness in regard to the constant and regular change
in the elimination of calcium and magnesium salts in disease/ and in these
conditions the excretion is chiefly dependent upon the diet, and upon the
formation and introduction of acid.
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
statements in regard to the iron present seem to show that the quantity is very
variable, from 1 to 11 milligrams per liter of urine (^Iagnier, 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 ^ 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 is liable to great variation.
The circumstances which under physiological conditions exercise a great
influence are the follo^^'ing: the blood-pressure, and the rapidity of the
blood-current in the glomeruli; the quantity of urinar}^ 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 othen\ase removed;
and it is decreased by a diminished ingestion of water or by a greater loss
1 See Albu and Neuberg, 1. c.
^Kunkel, cited from Maly's Jahresber., 11; Giacosa, ibid., 16; Robert, Arbeiten
des Pharm. Inst, zu Dorpat, 7; Magnier, Ber, d. deutsch. chem. Gesellsch., 7; Gott-
lieb, Arch. f. exp. Path. u. Pharm., 26; Jolles, Zeitschr. f. anal. Chem , 36; Hoffmann,
Zeitschr. f. anal Chem., 40; Neumann and Mayer, Zeitschr. f. physiol. Chem., 37.
QUANTITY AND QUANTITATIVE COMPOSITION OF URINE. 627
of water in other ways. Ordinarily in man just as much water is eliminated
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 con-
siderably increased. Diminished introduction of water or increased elimi-
nation of water by other ways— as in ^iolent diarrhoea or vomiting, or in
profuse perspiration — greatl}^ diminishes the amount of urine excreted.
For example, the urine may sink as low as 500-400 c.c. per day in intense
summer heat, while after copious draughts of water the elimination of
3000 c.c. of urine has been ol)served 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 calculated as 1500 c.c. for
healthy adult men and 1200 c.c. for women. The minimum elimination
occurs during the early morning, between 2 and 4 o'clock; the maximum,
in the first hours after waking and from 1-2 hours after a meal.
The quantit}' 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
li^'ing is regular. Therefore the percentage of soUds in the urine is natu-
rally in inverse proportion to the quantit}- of urine. The average amount
of solids pert wenty-four hours is calculated as 60 grams. The quantity
may be calculated with approximate accuracy from the specific gra^'ity
if the second and third decimals of this factor be multiplied b}' Haser's
coefficient, 2.33. The product gives the amount of solids in 1000 c.c.
of urine, and if the quantity of urine eUminated in twenty -four hours be
measured, the quantity of solids in twent3--four hours may be easil}- calcu-
lated. For example, 1050 c.c. of urine of a specific gravity 1.021 was elimi-
nated in twenty-four hours; therefore the quantity of solids excreted was
48 9x 1050
21x2.33 = 48.9 and " — =51.35 grams. Long i has made a new
determination of the coefficient for a specific gra\'ity taken at 25° C. and
finds that it is equal to 2.6, which corresponds nearly 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 gra^'ity of the first is
2.15 and the last only 1.32, so it is easy to undei*stand, when the relative
proportion of these two bodies essentially de\'iates from the normal, why
the above calculation from the specific gra^'ity 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
wdth a greater elimination, and a \ery considerable excretion of urine
' Joum. Amer. Chem. Soc, 25.
628 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-
His), in which there is a copious excretion with a ven- high specific gravity-
due to the sugar. In cases where very little urine is excreted {oliguria),
e.g., during profuse perspiration, in diarrhoea, and in fevers, the specific
gra^4ty of the urine is as a rule xery high; the percentage of solids is also
high and the urine lias a dark color. Sometimes, as for example in certain
cases of albuminuria, the urine may have a low specific gravity notwith-
standing the oUgTiria, and be poor in solids and light in color.
In certain cases it is interesting to know the relationship between the
carbon and the nitrogen, or the quotient C/N. This factor may vary
between 0.7 and 1; as a mle, it amounts on an average to 0.87, but changes
according to the nature of the food and is higher alter a diet rich in carbo-
hydrates than after food rich in fat (Pregl, Tangl, Langstein and Steinitz,
and others ^).
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 figiu'es for the quantities of
the most important constituents which are eliminated during the course of
twenty-four hours in a volume of 1500 c.c. of urine. These figures apply
only to a diet which corresponds to Voit's standard figures, namely 118
grams protein, 56 grams fat, and 500 grams carbohydrate per day, and to
a man of average weight.
Daily quantity of solids = 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 (H^SO,) ... 2.5 "
Creatinine 1.5 " Phosphoric acid (P.,05). . . . 2.5 "
Hippuric acid 0.7 " Potash (ICO) * 3.3 "
Remaining organic bodies. . 2.1 " Ammonia (NH3) 0.7 "
Magnesia (MgO) 1 f. ^ ,,
Lime (CaO) / "•*
Remaining inorganic bodies 0.2 "
Urine contains on an average 40 p. m. solids. The quantity of urea is
about 20 p. m., and common salt about 10 p. m.
The physico-chemical methods are being used in urinary analysis even
to a greater extent than in the analysis of other animal fluids. A great
number of cry^oscopic determinations but fewer conductivity determinations
have been made. A constant relationship between the values found by
phj'sico-chemical methods and the analytical methods has been sought,
for example, between the freezing-point depression and the specific gravity
* Pregl, Pfliiger's Arch , 75, which contains the older literature. Tangl, Arch. f.
(Anat. u.) Physiol., 1899, Suppl.; Langstein and Steinitz, Centralbl. f. Physiol., 19.
CASUAL URINARY CONSTITUENTS. 629
or 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 diagnostic 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 use-
fulness of the various constants and relations which are based upon theo-
retical considerations, the views are unfortunateh' still too divergent.
V. Casual Urinary Constituents.
The casual appearance in the urine of medicinal agents or of urinary con-
stituents resulting from the introduction of foreign substances into the
organism is of practical importance, because such compounds may inter-
fere in certain urinar>' investigations; they also afford a good means of
determining whether certain substances have been introduced into the
organism or not. From this point of \aew a few of these bodies will be
spoken of in a following section (on the pathological urinarv^ constituents).
The presence of these foreign bodies in the urine is of special interest in
those cases in which they serve to elucidate tlie chemical transformations
which certain substances undergo within the organism. As inorganic
substances generally' leave the body unchanged, ^ they are of very Httle
interest from this standpoint; but the changes which certain organic sub-
stances undergo when introduced into the animal body may be studied b}'
the transformation products as found in the urine.
The bodies belonging to the fatty series undergo, though not without
exceptions, a combustion leading towards 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 carbonates
and render the urine neutral or alkaline, may act in this manner. The
volatile fatty acids poor in carbon are less easily oxidized than those rich in
carbon, and they therefore pass unchanged into the urine in large amounts.
This is especially true of formic and acetic acids (Schotten, Grehant and
QuiNQUAUD^). The statements in regard to oxahc acid are contradictory.
In birds, according to Gaglio and Giunti, it is not oxidized. In mammals
it is in great part oxidized, according to Giuxti, while Gaglio and Pohl
' In regard to the behavior of certain of these bodies, see Heffter, Die Ausscheidung
korperfremden Substanzen im Harn, Er^ebnisse d. Physiol., 2, Abt. 1.
^ Scliotten, Zeitschr. f. physiol. Chem., 7; Grehant and Quinquaud, Compt.-rend.,
104.
630 URINE.
claim that it is not destroyed. Marfori and Giuxti claim that in human
beings oxalic acid is in great part oxidized, although the recent investiga-
tions of Salkowski, Pierallini, Stradomsky, Klemperer and Trit-
SCHLER 1 seem to show that the acid is only in part destroyed in the animal
body. In order to exactly determine that portion of the ingested oxalic
acid which is absorbed and excreted by the urine or oxidized in the body, it
must necessarily be known whether or not a portion of the acid is destroyed
in the intestine and is therefore not absorbed. Tartaric acids act differ-
ently, according to Brion;^ thus in dogs the levotartaric acid is nearly
entirely consumed, while a little more than 70 per cent of dextrotartaric
acid is burnt. Racemic acid is oxidized to a still less extent in the animal
body. Succinic and malic acids are completely combustible, according to
PoHL.3 Examples of the different behavior of stereoisomeric substances
have already been given on page 109.
The acid amides appear not to be altered in the body (Schultzen and
Nencki^). 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 550) for leucine, glycocoll, and aspartic
acid, they are decomposed w'ithin 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 glyceric 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
inactive acids are so changed and transformed that the component foreign
to the body is more or less abundantly excreted, while that occurring in
the body protein is oxidized (Schittenhelm and Katzenstein, Wohlge-
muth 5) . In connection with the amino-acids it is to be recalled that
according to the observations of Abderhalden and Bergell ^ glycyl-
glycine introduced subcutaneously in rabbits appeared in the urine as gly-
cocoll.
Various amino acids show a somewhat different behavior. Sarcosine
^Gaglio, Arch. f. exp. Path. u. Pharm., 22; Giunti, Chem. Centralbl, 1897, 2;
Marfori, Maly's Jahresber., 20 and 27; Pohl, Arch. f. exp. Path. u. Pharm., 37; Sal-
kowski, Berl. kHa. Wochenschr., 1900; Pierallini, Virchow's Arch., 160; Stradomsky,
ibid., 163; Klemperer and Tritschler, Zeitschr. f. klin. Med., 44.
^ Zeitsclir. f. physiol. Chem., 25.
^ Polil, Arch. f. exp. Path. u. Pharm., 37, which also contains the statements on
the intermediary products formed in the oxidation of the fatty bodies.
* Zeitschr. f . Biologic, 8.
* Schittenhelm and Katzenstein, Zeitschr. f. exp. Path., 2, cited from Biochem.
Centralbl., 5; Wohlgemuth, Ber. d. d. chem. Gesellsch., 38.
' Zeitschr. f. physiol. Clem., 39.
CASUAL URIXARY CONSTITUENTS. 631
(methylglycocoll), (CH3)NH.CH2.COOH, which is not readily burnt,
passes therefore in great part unchanged mto the urine , but perhaps also
passes in small part into the corresponding uramino-acid. methijlhydantoic
acid, NH2.CO.N(CH3).CH2.COOH (Schultzex i). Likewise taurine,
aminoeth} Isulphonic acid, which acts somewhat differentl}' in different
animals (Salko wski 2) , passes in human beings, at least in pait, into the
corresponding uramino-acid, taurocarhamic acid. NH2.CO.NH.C2H4.SO2.OH.
A part of the taurine also appeai-s 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 hyposulphurovs 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 thiosulphate) (Blum, Abderhaldex and Samuely^).
The nitriles, including hydrocyanic acid, pass, according to Laxg, into
sulphocyanide combinations, and this sulphocyanide apparenth- 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 convert the alkah-cyanides
readily into sulphocyanides. The alkali sulphocj^anides when ingested
are nearly quantitatively eliminated in the urine, according to Pollak.^
By substitution with halogens, bodies othenvise readily oxidizable are
converted into difficultly oxidizable ones. While the aldehydes are readily
and completely burnt like the primary' and secondarv' alcohols of the fatty
series, the halogen substituted aldehydes and alcohols are, on the contrar}',
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.^
By conjugation mith sulphuric acid, the alcohols which are othen\'ise
readily oxidizable may be guarded against combustion, and consequently
the alkali salt of ethylsulphuric acid is not burnt in the body (Salkowski ^).
The organic combinations containing sulphur act somewhat differently.
W. Smith states that the sulphur of the thio-acids, like thiogly colic acid.
' Ber. d. deutsch. chem. Gesellsch., 5. See also Baumami and v. Mering, ibid., 8,
584, and E. Salkowski, Zeitschr. f. physiol. Chem., 4, 107.
^ Ber. d. deutsch. chem. Gesellsch., 6, and Virchow's Arch.. 58.
^ Blum, Hofmeister's Beitrage, o; Abderhalden and Samuely, Zeitschr. f. physiol.
Chem., 46.
^ Lang, Arch. f. exp. Path. u. Pharm., 34; Pascheles, ibid.; Pollak, Hofmeister's
Beitrage, 2.
^ See Hamack and Griindler, Berlin, klin. Wochenschr., 1S83; Zeller, Zeitschr. f.
physiol. Chem., 8; Kast, ibid., 11; Binz, Arch. f. exp. Path. u. Pharm., 28; Zeehuisen,
Maly's Jahresber., 23.
' Pfliiger's .-Vrch., 4.
632 URINE.
CH2.SH.COOH, is in part oxidized to sulphuric acid, and according to
GoLDMANX the same result occurs with aminothiolactic acid (cysteine)
and the sulphur of the thio-alcohols (ethyl mercaptans). On the
contrar}^, ethylsulphide, sulphonic and sulpho acids in general (Salkowski,
Smith i) are not changed into sulphuric acid. Oxyethylsulphonic acid,
HO.C2H4.SO2.OH, which is in part oxidized to sulphuric acid, is an ex-
ception (Salkowski).
Conjugation with glucuronic acid occurs, according to the investigations
of SuNDViK and especially of O. Neubauer, in many substituted as well
as non-substituted alcohols, aldehydes, and ketones. Chloral hydrate,
C2CI3OH -I- H2O, passes, after it has been converted into trichlorethyl-
alcohol bv a reduction, into a levogyrate reducing acid, urochloralic acid
or trichlorethylglucuronic acid,C2Cl3H2.C6H407 (Musculus and V. Mering).
Of the primar}^ alcohols investigated by Neubauer 2 (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
relativelv large quantities. Secondaiy alcohols produced conjugated glucu-
ronic acids, and indeed to a greater extent than the primary alcohols,
especially in rabbits. The ketones are reduced in part into secondary alco-
hols and are partly excreted as the conjugated acid. This could be shown
for acetone with rabbits Imt 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 prob-
able.
The fact that benzene may be oxidized outside of the body into carbon
dioxide, oxalic acid, and volatile fatty acids has been known for a long
time; and as in these cases a rupture of the benzene ring must take place,
so also, it must be admitted, when aromatic substances undergo a com-
bustion in the animal body, a splitting of the benzene nucleus with the
formation of fatty bodies must be the result. If this does not occur, then
the benzene nucleus is eliminated with the urine as an aromatic compound
of one kind or another. As the benzene nucleus can protect a substance
belonging to the fatty series from destruction when conjugated with it.
' Smith, Pfliiger's Arch., 53, 55, 57, and Zeitschr. f. physiol. Chem., 17; Salkowski,
Virchow's Arch., 66; Pfliiger's Arch., 39; Goldmann, Zeitschr. f. physiol. Chem., 9;
also Baumann and Kast, ibid., 1-t.
^ Sundvik, Maly's Jahresber., 16; Musculus and v. Mering, Ber. d. deutsch. chem.
Gesellsch., 8; also v. Mering, ibid., 15; Zeitschr. f. physiol. Chem., 6; Kiilz, Pfliiger's
Arch., 28 and 33; 0. Neubauer, Arch. f. exp. Path. u. Pharm., 46.
CASUAL URINARY CONSTITUENTS. 633
which is the case with the glycocoU of liippuric 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-sulphuric
acids are examples of this kind.
The difficulty in deciding whether the benzene ring itself is destroyed
in the body lies in the fact that we do not know all the different aromatic
transformation products wliich may be produced by the introduction of
any such substance into the organism, and wliich 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. Schottex, Baum.\xn, and others
have found that certain amino-acids, such as phenylamino-propionic
add, 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. Kxoop ^ phenyl-a-lactic acid and phenyl-
oi-ketopropionic acid (phenyl pyroracemic acid) have a similar beha\ior,
while Juv.^lta's statement that phthaHc acid is destroyed in the animal
body is denied by E. Pribra:m.2 The benzene derivatives vary in behavior
according to the position of the substitution, for, as found by R. Cohx,^
among the di-derivatives the ortho-compounds are more readily destroyed
than the corresponding meta- or para-compounds.
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 (Schultzex and Naunyn), and this is then further
in part oxidized into dioxyhenzenes (Baumaxx and Preusse). Naphtha-
lene appears to be converted into oxy naphthalene, and probably a part
also into dioxynaphthalene (Lesnik and ^I. Nexcki). The hydrocarbon
with an amino- or imino-group may also be oxidized by a substitution of
hydroxyl for hydrogen, especially when the formation of a derivative in
the para-position is possible (Klixgenberg). For example, aniline,
C6H5.XH2, passes into paraminophenol, which latter passes into the
urine as its ethereal-sulphuric acid, H2N.C6H4.O.SO2.OH (F. ^Iuller).
Acetanilid is in part converted into acetyl paraminophenol (Jaff:^ and
' 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., 11; F. Iviioop, Der Abbau aromatischer Fett-
sauren im Tierkorper, Habilit.-Schrift, Freiburg, 1904.
' Juvalta, Zeitschr. f physiol. Chem., 13; Pribram, Arch. f. exp. Path. u. Pharm. 51.
• Zeitschr. f. physiol. Chem., 1".
634 URINE.
HiLBERT, K. Morner), and carhazol into oxycarbazol (Klingen-
BERG ^).
An oxidation of the side chdin may occur by the hydrogen atoms being
replaced by hydroxy!, as in the oxidation of indol and skatol into indoxyl
and skatoxyl. An oxidation of tlie side chain may also take place with the
formation of carboxyl; thus, for example. foZwene, C6H5.CH3 (Schultzen
and Naunyn), ethyl-henzene, C6H5.C2H5, and ipropylhenzene, C6H5.C3H7
(Nencki and Giacosa^), 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 ben-
zene nucleus and the carboxyl, is not oxidized, but is eliminated after con-
jugation with glycocoll as phenaceturic acid (Salkowski^). Phenylamino-
acetic acid, CeHs.CHNHo.COOH is in part converted into mandelic acid
(phenylglycollic acid), CeHs.CHOH.COOH, and in great part is eliminated
as such (ScHOTTEN, Knoop •*) . 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^ have ]3roposed 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 has shown by experiments with several acids, such as phenyl-
butyric acid, phenyl-a-lactic acid and others that this rule does not hold
good. The phenylbutyric acid, C6H5CH2.CH2.CH2.COOH, is not oxidized
in the animal body into benzoic acid, but into phenylacetic acid, and the
phenyl-a-lactic acid, C6H5.CH2.CH(OH).COOH, is decomposed nearly en-
tirely and only a small residue is eliminated unchanged. Knoop has, on the
contrary, made it very probable that, at least for the saturated, normal fatty
acids wnth phenyl substituted at the end, on their oxidation they follow
the rule that the carboxyl group produced from the body stands in the /3
position to the original carboxyl. This explains, for example, the forma-
tion of phenylacetic acid from phenylbutyric acid, and benzoic acid from
phenylvalerianic acid, C6H5.CH2.CH2.CH2.CH2.COOH, for in the last-
' Schultzen and Naunyn, Reichert and 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.
Wochen.schr. , 1S87; Jaffe and Hilbert, Zeitschr. f. physiol. Chem., 12; Morner, ibid.,
13; Klingenberg, " 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.
' Zeitschr. f. physiol. Chem., 4.
''■Ibid.. 7 and 9.
* Ibid., 8.
'Ibid., 7.
CASUAL URINARY CONSTITUENTS. 635
■mentioned case phenylpropionic acid must first be produced and then
benzoic acid from this. Exceptions to this rule are the propionic acids
substituted in the a-position, i.e., phenylalanine, phenyl-a-lactic acid, and
phenyl-a-ketopropionic acid, 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 nearly 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 carbox}^. Thus xylene, CoR^iClis)-,, is oxidized
into toluic acid, C6H4(CH3)COOH (Schultzen and Naunyn); fuesitylene,
C6H3(CH3)3. into mesitylenic acid, C6H3(CH3)2.COOH (L. Nencki); cymene.
(CH3)2CH.C6H4.CHo, into cumic acid (M. Nencki and Ziegler^); and
vanillin, OH.C6H3<pttqS into, vanilUnic acid (Y. Kotake),^
Reductions may also occur and examples of this kind are the conver-
sion, as observed by E. MEYER,^of nitrobenzene, C6H5NO2, or ol nitro phenol,
HO.C6H4.NO2 into aminophenol, HO.C6H4.NH2, and also the behavior
of m-nitrobenzaldehyde in the animal body as mentioned below.
Syntheses of aromatic substances with other atomic groups occur fre-
quently. 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 ■* more correctly
to Keller and Ure. All the numerous aromatic substarices 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^ benzoic acid does not pass into hippuric acid in
birds, but into another nitrogenous acid, ornithuric acid, C19H20N2O4. This
acid yields as splitting products, besides benzoic acid, ornithine, a body
which has been spoken of on page 97. Not only are the oxybenzoic ocids
and the substituted benzoic acids conjugated with glycocoll, forming corre-
sponding hippuric acids, but also the above-mentioned acids, toluic, mesity-
lenic, 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
> L. Nencki, Arch. f. exp. Path. u. Pharm., 1; Nencki and Ziegler, Ber. d. deutsch.
chem. Gesellsch., 5. See also O. Jacobsen, ibid., 12.
^ Zeitschr. f. physiol. Chem., 46.
'Ibid., 46.
" Die Ausscheidung korperfremder Substanzen im Harn. Ergebnisse der PiiysioJ., 4,
252.
^ Ber, d. d. chem. Gesellsch., 10 and 11,
636 URINE.
acid (Bertagnini, Baumann, Herter, and others), while Baumann and
Herter ^ find it only very probable for m-ox^iDenzoic acid. The oxy-
benzoic acids are also in part eliminated as conjugated sulphuric acids,
which is especially true for w-oxybenzoic acid. The three aminolienzoic
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,^ that ?^2-aminobenzoic acid passes in part into
uvaminobenzoic acid, H2N.CO.HN.C6H4.COOH. It is also in part elimi-
nated as aminohippuric acid.
The behavior of the halogen-substituted compounds of toluene varies
in different animals according to Hildebrandt's experiments. In dogs
they are converted into the corresponding substituted hippuric acid. In
rabbits o-bromtoluene is completely changed to hippuric acid, the m- and
p-bromtoluene only partly. The three chlortoluenes are converted in rab-
bits into the corresponding benzoic acid and are eUminated as such and
not as hippuric acid.
The substituted aldehydes are of special interest as substances which
may undergo conjugation with glycocoU. According to the investigations
of R. CoHN 3 on this subject o-nitrohenzaldehyde when introduced into a
rabbit is only in a verj^ small part converted into nitrobenzoic acid, and
the chief mass, about 90 per cent, is destroyed in the body. According
to SiEBER and Smirnow* m-mitrobenzaldehyde passes in dogs into m-nitro-
hippuric acid, and according to Cohn into urea-m.-nitrohippurate. In
rabbits the behavior is quite different. In this case not only does an oxida-
tion of the aldehyde into benzoic acid take place, but the nitro-group is
also reduced to an amino-group, and finally acetic acid attaches itseK to
this with the expulsion of water, so that the final product is m-acetyl-
aminobenzoic acid, CH3.CO.NH.C6H4.COOH. This process is analogous
to the beha-vior of furfurol, and the reduction does not take place in the
intestine, but in the tissues. The 2?-nitrobenzaldehyde acts in rabbits in
part like the m-aldehyde and passes in part into p-acetylaminohenzoic 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^ 2>nitrobenzaldehyde yields only urea p-nitrohip-
purate in dogs. The above-mentioned pyridine-carhoxylic acid, formed from
' Zeitschr. f. physiol. Chem., 1, where Bertagnini's work is also cited. See also
Dautzenberg, Maly's Jahresber., 11, 231.
-Salkowski, Zeitschr. f. physiol. Chem., 7; Cohn, ibid., 17; Hildebrandt, Hof-
meister's Beitrage, 3.
^ Zeitschr. f. physiol. Chem., 17.
* Monatshefte f. Chem., 8.
CASUAL URINARY CONSTITUENTS. 637
methy [pyridine (a-picoline) passes into the urine after conjugation with
glycocoll as oi-pyridinuric acid}
To those substances which undergo a conjugation with glycocoll Ijelongs
also furjurol (the aldehyde of pyromucic acid), which, when introduced into
rabbits and dogs, as shown by Jaffe and Cohx.2 is fust oxidized into pyro-
mucic acid and then eUminated as pyromucuric acid. C7H7X4O, after
conjugation with glycocoll. In birds this behavior is different, namely,
the acid is conjugated with another substance, ornithine, C5Hi2N202, which
is a diamino valerianic acid, forming pyromucinornithuric acid.
Furfurol also undergoes conjugation with glycocoll in other forms in
mammals. Thus Jaffe and Cohn found that it is in part combined with
acetic acid, forming furfuracrylic acid, C4H30.CH:CH.COOH, which passes
into the urine coupled with glycocoll as furjuracryluric acid.
It has not been proved 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
by Jaffe and Le\^,^ is conjugated with glycocoll in the body (rabbits)
and eUminated as thiophenuric acid.
Another ver}^ important synthesis of aromatic substances is that of
the ethereal-sulphuric acids. Phenols and chiefly the hydroxylated aromatic
hydrocarbons and their derivatives are voided as ethereal-sulphuric acids,
according to Baumann, Herter and others.^
A conjugation of aromatic acids with sulphuric acid occurs less often.
The two pre\aously-mentioned aromatic acids, p-oxyphenylacetic and p-oxy-
phenylpropionic acid, are in part eliminated in this form. Gentisic acid
(hydroquinone-carboxylic acid) also increases, according to Likhatscheff.s
the quantity of ethereal-sulphuric acid in the urine, and according to RosT
the same occurs, contrarv- to the older statements, with gallic acid (trioxy-
benzoic acid) and tannic acid.^
While acetophenone (phenylmethylketone) . C6H5.CO.CH3, as shown by
M. Nexcki, is oxidized to benzoic acid and eliminated as hippuric acid,
the aromatic oxyketones with hydroxjd gTOups, such as resacetophenone,
* In regard to the extensive literature on glycocoll conjugations we refer the reader
to O. Kiihling, Ueber Stoffwechselprodukte aromatischer Korper. Inaug.-Diss.,
Berlin, 1SS7.
2 Ber. d. d. Chem. Gesellsch , 20 and 21.
^ Le\'y, Ueber das Verlialten einiger Thiophenderivate, etc., Inaug.-Diss., Konigs-
berg, 1889; Jaffe and Levy, Ber. d. d. chem. Gesellsch., 21.
* In regard to the hterature, see O. Kiihling, 1. c.
^Zeitschr. f. physiol. Chem., 21.
* In regard to the behavior of gallic and tarmic acids in the animal body, see C.
Momer, Zeitschr. f. physiol. Chem., 16, wliich also contains the older literature; also
Harnack, ibid., 21, and Rost, Arch. f. exp. Path. u. Pharm., 3S, and Sitzungsber. d.
Gesellsch. zur Beford. d ges. Naturmss. 201 Marburg, 1898.
638 URINE.
G6H3(OH)(OH)(CO.CH3), paraoxijpropiophenone, C6H4(OH)(COCH2.CH3),
12 3 4
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 (Nexcki and RekoW'SKI^). 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 , wliich
last is protected from combustion, occurs rather often. The phenols, as
above stated (page 590), 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 pre\dous oxidation and hj'dration. Thus Hildebr.\ndt and Fromm
and Clemens - have shown that the cyclic terpenes and campfiors, by oxida-
tion or hydration, or in certain cases by both, are converted into hydroxyl
derivatives when the body in question is not previous^ hydroxylized, and
that these hydroxyl derivatives are eliminated as conjugated glucuronic
acids. Conjugated glucuronic acids are detected in the urine after the intro-
duction of various substances, e.g., therapeutic agents into the organism,
namely, terpenes, horneol, menthol, camphor (camphoglucuronic acid was
first observed by Schmiedeberg), naphtJialene, oil of turpentine, oxyquino-
lines, antipyrine, and many other bodies.^ Orthonitrotoluene in dogs passes
first into o-nitrobenzyl alcohol and then into a conjugated glucuronic acid,
uronitrotoluolic acid (Jaffe).* The glucuronic acid split off from this
conjugated acid is levogyrate and hence is not identical but only isomeric
wdth the ordinary glucuronic acid. Ditnethylaminohenzaldehyde, according
to Jaffe, is converted in part into dimethylaminobenzoglucuronic acid
in rabbits. The same conjugated glucuronic acid is also produced, accord-
ing to HiLDEBRAXDT,5 from p-dimethyltoluidine , which is first changed into
p-dimeihylaminohenzoic acid. Indol and skatol seem, as above stated
(page 595), to be eUminated in the urine partly as conjugated glucuronic
acids.
A synthesis in which compounds containing sulphur, iJiercapturic acids, are
formed and eliminated after conjugation with glucuronic acid, occurs when
' Arch. d. scienc. bid. de St. Petersbourg, 3, and Ber. d. deutsch. chem. Gesell-
sch., 27.
' Hildebrandt, Arch. f. exp. Path. u. Pharm., 45, 46; Zeitschr. f. physiol. Chem.,
36; with Fromm, ib/d., 33; and with Clemens, ibid., 37; Fromm and Clemens, /6id., 34.
^ See O. Kiihling, 1. c, which gives the literature up to 1887; also E. Kiilz, Zeitsclir.
f. Biologic, 27; the works of Hildebrandt, Fromm and Clemens, see foot-note, 2;
Brahm, Zeitschr. f. physiol. Chem., 28; Fen}^-essy, ibid., 30; Bonanni, Hofmeister's
Beitrage, 1; Lawrow, Ber. d. d. chem. Gesellsch., 33.
■• Zeitschr. f. physiol. Chem., 2.
'Jaffe, Zeitschr. f. physiol. Chem., 43; Hildebrandt, Hofmeister's Beitrage, 7.
CASUAL URINARY CONSTITUENTS. 639
chlorine and bromine derivatives of benzene are introduced into the organism
of dogs (Bau]vl^*x and Pretjsse, Jaffe). Thus chlorhenzene combines
with cysteine, forming chlorphenylmercapturic acid, C11H12CISXO3. The
important inxestigations of FRIED^L\xx ^ show that the phen3-lthiolactic acid
which forms the foundation of the mercapturic acids belongs to the .j-series,
and in this way the direct chemical connection of this body with the pro-
tein-cystine (a:-amino-^5-thiolactic acid) is established. Fried^lvnx has
also been able to convert cysteine into bromphenylmercapturic acid.
Pyridine, C5H5X, which does not combine either with glucuronic acid
or with sulphuric acid after pre\ious oxidation, shows a special beha\dor.
It takes up a methyl group as found by His and later confirmed by Cohn ^
and forms an ammonium combination, methylpyridylammonium hydroxide,
HO.CH3.XC5H5.
Several alkaloids, such as quinine, morphine, and strychnine, may pass
into the urine. After the ingestion of turpentine, balsam of copaiva, and
resins, these may appear in the urine as resin acids. Different kinds of
coloring-matters, such as alizarin, crysophanic acid, after rhubarb or senna,
and the coloring-matter of the blueberry, etc., may also pass into the urine.
After rhubarb, senna, or santonine the urine assumes a yellow or greenish-
yellow color, which is transformed into a beautiful red by the addition
of alkali. Phenol produces, as above mentioned, a dark-brown or dark-
green color which depends mainly on the decomposition products of hydro-
quinone and humin substances. After naphthalene the urine has a dark
color, and several other medicinal agents produce a special coloration.
Thus after antipyrine it becomes yellow or blood-red. After balsam of
copaiva the urine becomes, when strongly acidified with hydrochloric acid,
gradually rose- and purple-red. After naphthalene or naphthol the urine
gives with concentrated sulphuric acid (1 c.c. 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 ^^I. Xexcki.^ After turpentine the urine
may have a pecuUar odor similar to that of \dolets.
* Baumann and Preusse, Zeitschr. f. physiol. Chem., 5; Jaflfe, Ber. d. deutsch.
chem. Gesellsch., 12; Friedmann, Hofmeister's Beitrage, 4.
- His, Arch f . exp. Path. u. Pharm., 22; Cohn, Zeitschr. f. physiol. Chem., 18.
^ Arch. f. exp. Path. u. Pharm., 28.
640 URINE.
VI. Pathological Constituents of Urine.
Proteid. The appearance of slight traces of proteid in normal urines
has been repeatedly observed by many investigators, such as Posnee,
Plosz. v. Noorden. Leube, and others. According to K. ^Iorxer ^ pro-
teid regularly occui-s as a normal urinary constituent to the extent of 22-78
milligrams per liter. Frequently traces of a substance similar to a nucleo-
albumin, which is easily mistaken for mucin, and whose nature will be
treated of later, appear in the urine. In diseased conditions proteid
occurs in the urine in a variety of cases. The albuminous bodies wliich
most often occur are serglobulin and seralbumin. Proteoses (or pep-
tones) 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 precipitate is produced on boiUng, this
may consist of proteid, or of earthy phosphates, or of both. The mono-
hydrogen calcium phosphate decomposes on boiling, and the normal phos-
phate may separate out. The proper amount of acid is now added to the
urine, so as to prevent any mistake caused by the presence of earthy phos-
phates, 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 c.c. 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 centi-
meter of the boiling-hot urine.
On using acetic acid, when the quantity of proteid is very small, and
especiallv when the urine was originally alkaline, the proteid may some-
times remain in solution on the addition of the above quantity of acid.
If, on the contrar}^ 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 combination between it and the
proteid is formed which is soluljle 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
* Skand. Arch. f. Physiol., 6 (hteiature).
PROTEID. 641
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 ib on these grounds that the heat test,
although it gives very good results in the hands of experts, is not recom-
mended 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 acidifj-ing another portion
with acetic acid at the ordinary' temj^jerature. Mucin and nucleoalbumin
substances similar to mucin are hereby precipitated. If in the perform-
ance of the heat and nitric -acid test a precipitate fu'st 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 telow). In a urine rich in urates a precipitate
consisting of uric acid separates on cooling. This precipitate is colored and
granular, and is hardly to be mistaken for an proteoses or proteid precipitate.
Heller's test is performed as follows (see page 41) : The urine is ver}'
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. ^^ ith this
test a red or reddish-violet transparent ring is always obtained ■uith normal
urine; it depends upon the indigo coloring-matters and can hardly be mis-
taken for the white or whitish proteid ring, and this last must not be mis-
taken 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 rea-
son two simultaneous rings may exist in urines which are rich in urates and
do not contain ver}^ 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 deli-
cacy 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 ver\^ rich in urea a ring-like separation of urea nitrate may also
appear. This ring consists of shining cr^'stals, and it does not appear
in urine pre\'iously diluted. A confusion with resinous acids, Avhich 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 contaias tme 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 con-
tain 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 ff albumin
may be detected without difficulty. Still the student must not be satisfied
642 URINE.
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 meta phosphoric acid (see page 41) is very convenient
and easily performed. It is not quite so delicate and positive as Helllr's
test. The proteoses are also precipitated by this reagent.
Reaction with Acetic Acid and Potasstiun Ferrocyanide. Treat the urine
first with acetic acid until it contains about 2 per cent, and then add drop
by drop a potassium-ferrocyanide solution (1:20), carefully avoiding an
excess. This test is very good, and in the hands of experts it is even more
delicate than Heller's". In the presence of ver}- small quantities of pro-
teid it requires more practice and dexterity than Heller'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 infiuence.
This reagent also precipitates proteoses.
Spiegler*s Test. Spiegler recommends a solution of 8 parts mercuric
chloride, 4 parts tartaric acid, 20 parts gl3^cerine, and 200 parts water as a very
delicate reagent for proteid in the urine. A test-tube is half filled with this-
reagent and from a pipette the urine is allowed to flow upon its surface drop by
drop 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 1:3.50,000. .Jolles ' 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 XaCl, and 500 c.c. water.
Rocpi'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.^
As every normal urine contains traces of proteid, it is apparent that
very dehcate reagents are to be used only with the greatest caution. For
ordinary cases Heller's test is sufficiently delicate. If no reaction Is
obtained with this test within 2^ to 3 minutes, the urine tested contains
less than 0.003 per cent of proteid, and is to be considered free from proteid
in the ordinary' sen.?e.
The use of precipitating reagents presumes that the urine to be investi-
gated is perfectly clear, especially in the presence of only very little pro-
teid. 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, H does not seem to be of any
importance (Grutzxer. Schw'eissinger^).
The different color reactions cannot be directly used, especially in deep-
colored urines which contain only little proteitl. The common salt of the
urine has a disturbing action on ^Iillon's reagent. To prove more posi-
tively the presence of proteid, the precipitate ol^tained in the boiling test
' Spiegler. Wien. klhi. Wochenschr., 1S92, and Centralbl. f. d. klin. Med., 1S93;
Jolles. Zeitschr. f. physiol. Chem., 21.
^ Pliarmaceut. Centralbl., 1889, and Zeitschr. f. phy.-iol. Chem., 29.
^ .Ifdles, Zeitschr. f. anal. Chem., 21); CJriitzner, Chem. Cenitalbl., 1901, 1; Schweis-
sineer. ibid.
DETECTION OF GLOBULIN AND ALBUMIN. 643
may be filtered, washed, and then tested with Millox'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 potassium-ferrocyanide test. In
using the heat test alone the proteoses may be easily overlooked, but these
are detected, on the contrar}-, 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 Furbrixger's
gelatine capsules, which contain mercuric cUoride, sodium chloride, and citric
acid; and Geissler's albumin-test papers, which consist of strips of filter-paper
which have been dipped in a solution of citric acid and also mercuric-chloride and
potassium-iodide solution and then dried.
If the presence of proteid has been positively proved in the urine by
the above tests, it then remains necessarj^ to determine its character.
The Detection of Globulin and Albumin. In detecting serglobuUn 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 sul-
phate. In both cases a white, flocculent precipitate is formed in the
presence of glubulin. In using ammonium sulphate with a urine rich in
urates a precipitate consisting of ammonium urate may separate. This
precipitate does not appear immediately, but only after a certain time, and
it must not be mistaken for the globulin precipitate. In detecting ser-
albumin heat the filtrate from the globuUn precipitate to boihng-point, or
add about 1 per cent acetic acid to it at the ordinary temperature.
For the detection and also for the quantitative estimation of the various
globulins (fibringlobulin, euglobulin, and pseudoglobulin) Oswald ' has proposed
the fractional precipitation with ammonium sulphate. It is still a question
whether this method, which is not quite reliable, can be used in urine investi-
gations.
Proteoses and peptones have been repeatedly found in the urine in
different diseases. ReUable reports are at hand on the occurrence of pro-
teoses in the urine. The statements in regard to the occurrence of pep-
tones date from a time when the conception of proteoses and peptones
was different from that of the present day, and in part they are based upon
investigations using untrustworthy methods. According to Ito - true
^ Miinch. med. VVochenschr., 1904.
^ In regard to the literature on proteoses and peptones in urine, see Huijpert-
Neubauer, Ham-Analyse, 10. Aufl., 466 to 492; also A. Stoffregen, Ueber das Vorkom-
nien von Pepton im Ham, Sputum und Eiter (Inaug.-Diss., Dorpat, 1891); E. Hirsch-
feldt, Ein Beitrag zur Frags der Peptonurie (Inaug.-Diss., Dorpat, 1892); and espe-
cially Stadelmann. Urtersuchungen iiberdie Peptonurie. Wiesbaden, 1894; Ehrstrom^
Bidrag till kannedomen om Albumosurien, Helsingfors, 1900; Ito, Deutsch. Arch,
f. klin. Med., 71.
644 URINE.
peptones are sometimes found in the urine in cases of pneumonia; what
has been designated as urine peptone seems to have been chiefly deutero-
peptones.
In detecting the proteoses the proteid-free urine, or urine boiled with
addition of acetic acid, is saturated with ammonium sulphate, which precipi-
tates the proteoses. 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 (Salkow^ski, Stokvis i). A small quantity of the
proteid may remain in solution after coagulation and tliis may be precipi-
tated by the ammonium sulphate and be mistaken for proteoses. The
coagulable proteid may be completely precipitated by saturating with
ammonium sulphate in boiling solution; but according to Devoto^ small
quantities of proteose may be formed from the proteid by heating for a long
time with the salt. On heating for a short time no such formation of
proteose takes place, and the proteids are completely coagiilated.
For these reasons Bang^ has suggested the following method for the
■detection of proteoses in the presence of coagulable proteid. The urine is
heated to boiling with ammonium sulphate (8 parts to 10 parts urine)
and boiled for a few seconds. The hot liquid is centrifuged for ^ to 1 min-
ute and separated from the sediment. The urobilin is removed from
this by extraction \^dth alcohol. The residue is suspended in a Uttle water,
heated to boiling, filtered, whereby the coagulable proteid is retained
on the filter, and any urolDilin still present in the filtrate is shaken out
with chloroform. The watery solution, after removal of the chloroform,
is used for the biuret test. For clinical purposes this method is very ser-
A^iceable.
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, wanned
again until the blue color disappears, cooled, and finally tested with copper
£!ulphate. This method has been recently somewhat modified by V. Aldor
and Cerny.'* In regard to other more complicated methods we refer to
Huppert-Neubauer.
.MoRAWiTZ and Dietschy ^ 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 re»moval of the urobilin by alcohol and extracting with water, the
biuret test may be applied.
If the proteoses have been precipitated from a larger portion of urine
by ammonium sulphate, this precipitate is tested for the presence of dif-
ferent proteoses for the reasons given in Chapter II. The following serves
'Salkowski, Berlin, klin. Wochenschr., 1897; Stokvis, Zeitschr. f. Biologie, 34.
^ Zeitschr f. physiol. Chem , 15.
^ Deutsch med. Wochenschr., 1898.
•• Salkowski, Centralbl. f.d. med. Wissensch., 1894; v Aldor, Berl. klin. Wochenschr.,
S6; 6erny, Zeitschr. f. analyt. Chem., 40.
*Arch. {. exp. Path. u. Pharm., 'yi.
ESTIMATION OF PROTEID L\ URIXE. 645
as a preliminar}' 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 tliis salt. In the presence of onl}- proto-
proteose the urine gives Heller's test, is precipitated even in neutral
solution on saturating with NaCl, but does not coagulate on boiling. Tlie
presence of heteroproteose is shown by the urine behaving like the above
with NaCl and nitric acid, but shows a difTerence on heating. It gradually
becomes cloudy on warming and separates at about 60° C. a sticky precipi-
tate wliich 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 Bexce-Joxes
proteid, which occurs in the urine in rare cases in disease with changes in
the spinal marrow. It gives a precipitate on heating to 40-60° C, which on
further heating to boiling dissolves again more or less completely, depending
upon the reaction and upon the amount of salt present. It does not sepa-
rate 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 ciystals (Grutterixk and de Graaff, ^Magx'US-
Levy 1). This body shows a somewhat different behavior in the various
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, Abderhaldex 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
primar}' as well as secondaiy proteoses on peptic digestion (Grutterixk
and DE Graaff). and yields the same hydrolytic cleavage products as the
other proteids (Abderhaldex and Rostoski).
Quantitative Estimation of Proteid in Urine. Of all the methods pro-
posed thus far, the coagulatiox 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 l^een
pre\iously heated on the water-bath, to completely separate the proteid so
that the filtrate will not respond to Heller's test. Then coagulate
20-50-100 c.c. 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 -^ath alcohol and ether, dr^^ and weigh, incinerate and
weigh again. In exact determinations the filtrate must not give Hel-
ler's test.
The separate estimation of globulixs and albumixs is done by care-
fully neutralizing the urine and precipitating -^nth MgS04 added to satura-
tion (Hammarstex) . or simply by adding an equal volume of a saturated
' Magnus-Le\-y, Zeitschr. f. physiol. Chem.. 30 (literature); Grutteriiik and de
Graaff, ibid., 34 and 46; Moitessier, Conipt. rend. soc. biolog., o"; Abderhalden and
Rostoski, Zeitschr. f. physiol. Cliem., 46.
646 URINE.
neutral solution of ammonium sulphate (Hofmeister andPoHLi). The
precipitate consisting of globuhn 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 quan-
tity of albumin is calculated as the difference between the quantity ot
globulin and the total proteids.
Approximate Estimation of Proteid in Urine. Of the methods sug-
gested for this purpose none has been more extensively employed than
Esbach's.
Esbach's^ Method. The acidified urine (with acetic acid) is poured
into a specially graduated tube to a certain mark, and then the reagent
(a 2 per cent citric-acid and 1 per cent picric-acid solution in water) is added
to a second mark, the tube closed with a rubber stopper and carefully
shaken, avoiding the production of froth. The tube is allowed to stand
twenty-four hours, and then the height of the precipitate on the gradu-
ation 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 con-
taining a medium quantity of proteid (Christensen and Mygge^). This
method is only to be used in a room in which the temperature may be kept
nearly constant. The directions for its use accompany the apparatus.
Other methods for the approximate estimation of proteid are the optical
methods of Christensen and Mygge, of Roberts and Stolnikow as modified
by Brandberg, with Heli,er'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.
Nudeoalhumin 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 substances
occur in the renal and urinary passages; still in most cases this nucleo-
albumin, as shown by K. ^Morner,^ is of an entirely different kind.
Every urine, according to iMorner, contains a little proteid and in
addition substances precipitating proteid- If the urine freed from salts by
' Hammarsten, Pfliiger's Arch., 17; Hofmeister and Pohl, Arch, f exp. Path. u.
Pharm., 20.
■ In regard to the literature on this method and the numerous experiments to deter-
mine its value, see Huppert-Neubauer. 10 Aufl., 853.
'Christensen, Virchow's Arch., 115.
* Skand. Arch. f. Physiol., 6.
DETECTION OF NUCLEOALBUMIXS. 647
dialysis is shaken with chloroform after the addition of 1-2 p. m. acetic
acid, a precipitate is obtained which acts Uke a nucleoalljiimin. 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 ^\•hich precipi-
tates proteids. The most important of these proteid-precipitating sub-
stances is chrondroitin-sulphuric acid and nucleic acid, although the latter
appears to a much smaller extent. TaurochoUc acid may in a few instances,
especially in icteric urines, be precipitated. The substances isolated by
different investigators from urine by the addition of acet'c acid and called
''dissolved mucin" or " nucleoalbumin" are considered by ]\1orxer to be
a combination of proteid cliiefiy witli clirondroitin-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. Tliis happens to a still greater extent in cases where the proteid
as well as the proteid-precipitating substance is eliminated to an increased
extent.
Detection of so-called Nucleoalbumins. When a urine becomes cloudy
or precipitates on the addition of acetic acid, and when it gives a more
typical reaction with Heller's test after the dilution of the urine than
before, one is justified in making tests for mucin and nucleoalbumin. As
the salts of the urine interfere considerably with the precipitation of these
substances by acetic acid, they must first be removed by dialysis. As large
a quantity of urine as possible is dialyzed (with the addition of chloroform)
until the salts are removed. Then acetic acid is added imtil it contains
2 p. m., and the mixture allowed to stand. The precipitate is dissolved in
water by the aid of the smallest possible quantit}^ of alkali and precipitated
again. In testing for chrondroitin-sulphuric acid a part is warmed on the
w^ater-bath with about 5 per cent hydrochloric acid. If positive results are
obtained on testing for sulphuric acid and a reducing substance, then chon-
droproteid 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.
NucleoMstonc. In a case of pseudoleucaemia A. .Jolles found a phosphorized
protein substance which he considers as identical with nucleohistone. Histone is
<>laimed to have been found in some cases by Krehl and Matthes, and by Kolisch
and BuRiAN.'
' JoUes, Ber. d. deutsch. chem. Gesellsch., 30; Krehl and Maithes, Deutsch. Arch.
f. kh'n. Med,, 51; Kolisch and Burian, Zeitschr. f. klin. Med., 29.
648 URINE.
Blood and Blood-coloring Matters. The urine may contain blood from
hemorrhage in the kidneys or other parts of the urinar>' passages (H^iL\-
turia). In these cases, when the quantity of blood is not ver>' 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, hsemoglobin, or, and indeed quite often,
methaemoglobin (h.emoglobixuria). 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 trans-
fusion of blood, and also in the periodic appearance of hsemoglobinuria
with fever. In hsemoglobinuria the urine may also have an abundant
grayish-brown sediment rich in proteid which contains the remains of the
stromata of the red blood-corpuscles. In animals hsemoglobinuria may
be produced by many causes which force free hsemoglobin into the plasma.
To detect blood in the urine, we make use of the microscope, the spec-
troscope, the guaiacum 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 contrarj', 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 urinarj' passages.
These formations are called hlood-casts.
The spectroscopic investigation is naturally of ver\^ 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.
Guaiacum Test. Mix in a test-tube equal volumxes of tincture of guaia-
cum and old turpentine which has become strongly ozonized by the action
of air under the influence of light. To this mixture, which must not have
the slightest blue color, add the urine to be tested. In the presence of blood
or blood -pigments, first a bluish-green and then a 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 Liebermaxx ' this reaction is brought about
by the blood pigments acting as catalysators upon the organic peroxides
existing in the turpentine, accelerating the decomposition of these and the
• Pfluger's Arch., 104.
HiEMATOPORPHYRIN. 649
active oxygen taken up by the guaiaconic acid which is oxidized to guaiac
blue (guaiaconic acid ozonide). Urine containing pus, even when no blood
is present, gives a blue color with these reagents ; but in this case the tincture
of guaiacum alone, without turpentine, is colored blue by the urine (Vitali ^).
This is at least true for a tincture that has been exposed for some time to
the action of air and sunUght. The blue color produced by pus differs
from that produced by blood-coloring matters by disappearing on heating
the urine to boiling. A urine alkaUne by decomposition must first be
made faintly acid before performing the reaction. The turpentine should
be kept exposed to sunlight, while the tincture of g-uaiacum must be kept
in a dark glass bottle. These reagents to be of use must be controlled
by a Uquid containmg blood. With positive results, however, this test is
not absolutely decisive, tecause 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.
Heller-Teichmann's Test. If a neutral or faintly acid urine contain-
ing blood is heated to boiling, one always obtains a mottled precipitate
consisting of proteid and hiematin. If caustic soda is added to the boiling-
hot test, the liquid l:)ecomes clear and turns green when examined in thin
layers (due to hsematin alkali), and a red precipitate, appearing green by
reflected light, re-forms, consisting of earthy phosphates and haematin.
This reaction is called Heller's blood-test. If this precipitate is col-
lected after a time on a small filter, it may he used for the hsemin test (see
page 213). 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 hsemin crystals. If, on the contra^v^ the
amount of phosphates is very small, then first add a little CaCl2 solution tcv
the urine, heat to boiling, and add simultaneously with the caustic potash
some sodium-phosphate solution. In the presence of only ver}' small
quantities of blood, first make the urine very faintly alkaline with am-
monia, add tannic acid, acidify with acetic acid, and use this precipitate in
the preparation of the hsemin crv^stals (Struve^).
O. and R. Adler ^ have recommended leucomalachite green or benzidine in
the presence of peroxide and acetic acid as especially sensitive reagents for blood.
We have no great experience thus far as to the mode of use of these reagents
in urine investigations.
Haematoporphyrin. Since the occurrence of hsematoporphyrin in the
urine in various diseases has been made verv probable by several investi-
gators, such as Neusser, Stokvis, ]\1acMunn, Le Nobel, Copeman, and
others,'* Salkowski has positively shown the presence of this pigment
in the urine after sulphonal intoxication. It was first isolated in a pure
* See Maly's Jahresber., IS.
^ Zeitschr. f. anal. Chem., 11.
^ Zeitschr. f. phy.sioI. Chem., 41.
^ A very complete index of the literature on hsematoporphyrin in the urine may be
found in R. Zoja, Su qua'che pigmento di alcune urine, etc., in Arch. Ital. di. clin.
Med., 189.3.
650 URLXE.
crystalline state by Hammarsten ^ from the urine of insane women after
sulphonal intoxication. According to Garrod and Saillet^ traces of
haematoporphyrin (Saillet's urospectrin) occur regularly 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 quan-
tities in the urine after the lengthy use of sulphonal.
Urine containing haematoporphyrin is sometimes only slightly colored,
while in other cases, as for example, after the use of sulphonal, it is more or
less deep red. In these last-mentioned cases the color depends, in greatest
part, not upon the haematoporphyrin, but upon other red or reddish-brown
pigments which have not been sufficiently studied.
In the detection of small quantities of haematoporphyrin proceed as
suggested by Garrod. Precipitate the urine with a 10 per cent caustic-
soda solution (20 c.c. for every 100 c.c. of urine). The phosphate precipi-
tate containing the pigment is dissolved in alcohol-hydrochloric acid (15-20
c.c.) 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 Ciiloroform, which
takes up the pigment, and test this solution with the spectroscope.
In the presence of larger quantities of haematoporphyrin the urine is
first precipitated, according to Salkowski, with an alkaUne barium-
chloride solution (a mixture of equal volumes of barium-hydrate solution,
saturated in the cold, and a 10 per cent barium-chloride solution), or, accord-
ing to Hammarsten,^ with a barium-acetate solution. The washed pre-
cipitate, which contains the haematoporphyrin, is allowed to stand some
time at the temperature of the room with alcohol containing hydrochloric
or sulphuric acid and then filtered. The filtrate shows the characteristic
spectrum of haematoporphyrin in acid solution and gives the spectrum of
alkaline haematoporphyrin after saturation with ammonia. If the alcoholic
solution is mixed with chloroform and a large quantity of water added and
carefully shaken, sometimes a lower layer of chloroform is obtained which
contains veiy pure haematoporphyrin, while the upper layer of alcohol and
water contains the other pigments besides some haematoporphyrin.
Other methods which have no advantage over this one of Garrod have been
suggested by Riva and Zoja as well as Saillet.^
Baumstark ^ found in a case of leprosy two characteristic coloring-matters
in the urine, " urorubroha;matin " and " urofuscohsematin," which, as their names
indicate, seem to stand in close ralationship to the blood-coloring matters. Uro-
ruhrohcematin, C68H94N8Fe202«, contains iron and shows in acid solution an absorp-
tion-band in front of D and a broader one back of D. In alkaline solution it
^ Sa'kowski, Zeitschr. f . physio 1. Chem., lo; Hammarsten, Skand. Arch, f . Physiol., 3.
-Garrod, Joum. of Physio!., 13 (contains review of literature) and 17; Saillet,
Revue de Medecine, Ifi.
^Salkowski, 1. c; Hammarsten, 1. c.
* Riva and Zoja, Maly's Jahresber., 24; Saillet, i. c. See also Nebeithau, Zeitschr.
f. physiol. Chem., 27.
' Pfliiger's Arch., 9.
PUS. 651
shows four bands — behind D, at E, beyond F, and behind G. It is not soluble
either in water, alcohol, ether, or cliloroform. It gives a beautiful brownish-red
non-dichroitic liquid with alkalies. Urofuscohcematin, CsgHioeXgOon, which is free
from iron, shows no characteristic spectrum; it dissolves in alkalies, producing
a brown color. It remains to be proved 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. M()rxer 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 ^).
Urorosein, so named by Xenxki,^ is a urinary coloring-matter occurring in
various diseases, but which is not a constituent of normal urine. The pigment
does not occur preformed in the urine, but first makes its appearance after the
addition of mineral acids. It is readily soluble in water, dilute mineral acids,
ethyl and amyl alcohol, and can be removed from the acid urine by shaking with
the latter. It differs from indigo red in the following: Alkalies immediately
decolorize a urorosein solution, but not an indigo-red solution. Urorosein is
removed from its amyl-alcohol solution by shaking with dilute alkali, while indigo
red is not. If the acid urine is shaken with cliloroform, indigo red is taken up,
but not urorosein. Urorosein is soon decomposed by light and shows a sharply
defined absorption-band between D and E. The red pigment appearing in urines
rich in skatol after the addition of hydrochloric acid differs from urorosein by
being insoluble in water, but readily soluble in ether and chloroform. The state-
ments in regard to the properties of skatol-red are somewhat chvergent, and it is
therefore difficult to state a positive difference between urorosein and skatol-red.
Pus occurs in the urine in various inflammatory affections, especially
in catarrh of the badder and in inflammation of the peh-is of the kidneys
or of the urethra.
Pus is best detected by means of the microscope. 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 wa}" 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 tliis means into a sUmy
tough mass.
The pus-corpuscles swell up in alkaUne urines, dissolve, or at least are
so changed that they cannot be recognized under the microscope. The
urine in these cases is more or less shmy or fibrous, and the proteid can be
precipitated in large flakes by acetic acid, so that it might possibly be mis-
taken for mucin. The closer investigation of the precipitate produced by
acetic acid, and especially the appearance or non-appearance of a reducing
substance after boihng it with a mineral acid, demonstrates the nature of
the precipitated substance. Urine containing pus always contains proteid.
' K Murner. Zeitschr. f. physiol. Chem.. 11; v. Jaksch. ibid., 13.
- Nencki and Sieber, Joum. f. prakt. Chem. (X. F.), 2G.
652 URINE.
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 Mackay
and V. Udranszky and K. ]\Iorner ^ they do not. Pathologically they
are present in the urine in hepatogenic icterus, although not invariably.
Detection of Bile-acids in the Urine. Pettenkofer's test gives the
most decisive reaction; but as it gives similar color reactions with other
bodies, it must be supplemented by the spectroscopic investigation. The
direct test for bile-acids is easily performed after the addition of traces of
bile to a normal urine. But the direct detection in a colored icteric urine
is more difficult and gives very misleading results; the bile-acid must there-
fore 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 precipi-
tate consisting of the alkali salts of the biUarj'^ acids is used in performing
Pettenkofer's test.
Haycraft has suggested a reaction for clinical jDurposes which consists in
sprinkling flowers of sulphur upon the urine. In icteric urine the powder quickly
sinks to the bottom, while in normal urine it remains on the surface. The vp,lue
of this test is stUl 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
urobihn in icteric urine see p. 604.
Detection of Bile-coloring Matters in Urine. ^lany tests have been pro-
posed for the detection of these substances. Ordinarily we obtain the best
results either with Gmelin's or wath 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 Uquid 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 he formed wiiich is surrounded by
'Cited from Huppert-Neubauer, Ham- Analyse, 10. Aufl. 229.
BILE-PIG MEXTS. 653
colored rings which appear yellowish reel,, ^'iolet, blue, and green from
within outward. Tliis modification is veiy deUcate, and it is hardly possi-
ble 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 dehcate than Rosexbach's modification.
Huppert's Reaction. In a dark-colored urine or one rich in indican
good results are not always obtained with Gmelix'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 CaCU solution, and then
with a solution of soda or ammonium carbonate. The precipitate which
contains the bile-coloring matters is filtered, washed, dissolved in alcohol
which contains 5 c.c. of concentrated hydrochloric acid in 100 c.c. (I. Muxk),
and heated to boiUng when the solution becomes green or bluish green.
According to Xakayama ^ this reaction is more deUcate on using a mixture
of ferric chloride, acid, and alcohol.
Hammarstex's React ioji. For ordinaiy cases it is sufficient to add a
few drops of urine to about 2-3 c.c. of the reagent (see page 322). when the
mixture immediately after shaking turns a beautiful green or bluish green,
which color remains for several days. In th€ presence of only vers* small
quantities of bile-pigments, especially when blood or other pigments are
simultaneously present, pour about 10 c.c. of the acid or nearly neutral
(not alkaline) urine into the tube of a small centrifugal machine and add
BaClo solution and centrifuge for about one minute. The liquid is decanted
off and the sediment stirred with about 1 c.c. of the reagent and centri-
fuged again. A beautiful green solution is obtained, which may be changed
by the addition of increased quantities of the acid mixture to blue, ^'iolet.
red, and reddish yellow. The gree^i color may be obtained in the presence
of 1 part bile-pigment in 500,000-1,000,000 parts urine. In the presence
of large amounts of other pigments calcium chloride is better suited than
barium chloride.
Bouma2 has suggested the use of alcohol containing ferric chloride
and hydrochloric acid instead of the above-mentioned acid mixture. He
has also worked out a colorimetric method of quantitative estimation of
bilinibin in urine by means of this reagent.
The veiy delicate reaction suggested by Jolles is unfortunately not
ser^'iceable on account of the formation of froth, especially in the presence
of proteid and blood-pig-nients ; but he has changed it by centrifuging the
urine with chloroform and barium chloride and suspending the chloroform-
barium residue in alcohol; after which he treats it with a solution of iodine
and mercuric chloride in alcohol containing hydrochloric acid.^ The color
becomes green or bluish green. This test seems to be good.
Stokvis's reaction is especially valuable as a control test in those cases
in which the urine contains only xevy Uttle bile-coloring matter together
with larger quantities of other coloring-matters. The test is performed as
follows: 20-30 c.c. of urine is treated with 5-10 c.c. of a solution of zinc
* Munk, Arch. f. (.Ajiat. u.) Physiol., 1S9S; Xakayama, Zeitschr. f. physiol.
Chem., 36
2 Deutsch. med. Wochenschr., 1902 and 1904.
' Deutsch. Arch, f . k'in. Med., 78.
654 URINE.
acetate (1:5). The precipitate is washed on a small filter with water and
then dissolved in a little ammonia. The new filtrate gives, either directly
or after it has stood a short time in the air until it has a peculiar brownish-
green color, the absorption-bands of bilicyanin (see page 322). This reac-
tion is unfortunately not sufficiently delicate.
^lany other reactions for bile-coloring matters in the urine have been
proposed; but as those above mentioned are sufficient, it is perhaps only
necessary to give here a few of the other reactions without entering into
details.
Smith's Reaction. Pour carefully over the uiine some tincture of iodine,
whereby a green ring appears between the two liquids. The urine may also be
shaken with the tincture of iodine until it has a green color.
Ehrlich's Test. First mix the urine with an equal volume of dilute acetic
acid and then add drop by drop a solution of sulphodiazobenzene. The acid
mixture becomes dark red in the presence of bilirubin, and this color becomes
bluish \'iolet on the addition of glacial acetic acid. The sulphodiazobenzene is
prepared by mixing 1 gram of sulphanilic acid, 15 c.c. of hydrochloric acid, and
0.1 gram of sodium nitrite ; this solution is diluted to 1 liter with water. This
test is not successful and positive when directly applied, if the urine is rich in other
pigments.
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 addi-
tion 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 608), quite positively proved. If
sugar appears in the urine in constant and especially in large quantities, it
must be considered as an abnormal constituent. In a previous chapter
several of the principal causes of glycosuria in man and animals were men-
tioned, and the reader is referred to Chapters 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 considerable
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 ajjpear 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 nde very
low, while their al^solute daily quantity is increased. The urine is pale.
SUGAR LN URIXE. 655
but of a high specific gravity, 1.030-1.040 or even higher. The high spe-
cific gravity depends upon the quantity of sugar present, which varies in
different cases, but may reach 10 per cent. The urine is therefore charac-
terized 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 medic-
inal 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 oj stigar in the urine is ordinarily, in the presence of not
too small quantities, a very simple task. The presence of only ver}- small
quantities may make its detection sometimes ver\' 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 frequentl}' emplo^-ed and are especially recom-
mended are as follows:
Tromm]:r'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 116. This test may lead to very great mistakes in urines poor in sugar,
especially wiien they have at the same time normal or increased amounts of
physiological constituents, and therefore it cannot be recommended to
physicians or to persons inexperienced in such work. Normal urine con-
tains reducing substances, such as uric acid, creatinine, and others, and
therefore a reduction takes place in all urines on using this test. A separa-
tion 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 sulfoxide in normal urines, or a j^eculiar yellowish
red liquid due to finely di\-ided cuprous hydrate. 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 positi\e 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 cojjper suboxide
in solution, the investigator may easily overlook small quantities of sugar
that may he present.
Trommer's test may of course be made positive and useful, even in the
presence of very small amounts of sugar. l)y using the modification su/:-
gested by Worm Duller. As this modification is rather compUcatcd
and requires much practice and exactness, it is probably rarely employed
by the busy physician. The following test is to be preferred.
Almi&x's bismuth test, which recently has been incorrectly called Nylax-
DEr's test, is performed with the alkaline-bismuth solution prepared as
above described (page 116). For each test 10 c.c. of urine is taken and
treated with 1 c.c. of the ])ismuth solution and boiled for a few minutes.
In the presence of sugar the urine l^ecomes dark vcllow or vellowish
656 URIiXE.
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 veiy little sugar the test does not become black or dark
brown, but simply deej^er-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 l^e used without disadvantage. In a urine poor in sugar only
1 c.c. of the reagent for every 10 c.c. of the urine must be employed.
Small amounts of proteid may retard this reaction and reduce the deli-
cacy of the test. Large quantities of proteid may, however, give rise to an
error b}^ forming bismuth sulphide, and therefore it must always be first
removed. The statement of Bechhold that mercury compounds in the urine
disturb the test has not been substantiated by Zeidijtz i on properly })er-
forming 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, moreover, readily performed, and on this
account is to be recommended to the physician.
The bumping and ejection of the fluid can be readily prevented by heating
over a very small flame after the test has been brought to a boil and by gently
shaking the contents of the not too narrow test-tube. The recommendation
of heating for a longer time in the water-bath, fifteen minutes or more, is to be
discarded, as the delicacy of the test is thereby so much increased that it gives a
reaction with a physiological sugar content of 0.02 per cent.
When the amount of sugar in the urine is not less than 0.1 per cent a
positive reaction is obtained if the test is boiled for 2-3 minutes and then
allowed to stand quietly for 5 minutes. The phosphate precipitate is then
black or nearly Ijlack. In detecting smaller quantities of sugar— O.05
per cent, the test as a rule must be boiled longer — about 5 minutes.
The value of this test hes in the fact that it positively detects small
quantities of sugar — 0.1 per cent or somewhat less. Equally with
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 therapeutic
agents, such as rhubarb, senna, antipyrine, salol, turpentine and others,
the bismuth test gives positive results. From this it follows that we should
never be satisfiecl with this test alone, especially when the reduction is not
very great. When this test gives negative results the urine can be con-
sidered from a clinical standpoint as free from sugar, and when it gives
positive results other tests must be applied. Among these the fermentation
test and the polarization test are of special value.
The question in what degree the use of therapeutic agents affects the Worm-
Ml'iLLER test has been only slightly investigated. That normal urines some-
times give the bismuth test is a common experience, and these urines according
to the experience of Hammarsten and certain other observers, as a rule also
give the WoRM-MtJLLER test when properly performed. There does not seem to
be any doubt that in many of these cases it is due to an increased quantity
of physiological sugar in the urine. Further investigations on this point are
very desirable.
'Bechhold, Zeitschr. f. physiol. Cliem., 46; Zeidlitz, unpublished investigations.
SUGAR IN URINE. 657
Fermentation Test. On using this test the process must vaty aci-ord-
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 b}^ 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
Avhich the yeast has been added into a Schrotter's gas-burette or a Lohn-
STEix's saccharimeter (see below). As the fermentation proceeds, the
carbon dioxide collects in the upper part ot the tube, while a corresponding
quantity of liquid is expelled below. As a control in this case two similar
tests must be made, one with normal urine and j'east to learn the quantity
of gas usually developed, and the other with a sugar solution and yeast
to determine the activity of the yeast.
If, on the contrary-, only a faint reduction with the bismuth test is
found, no positive conclusion can be drawn from the absence of any carbon
dioxide or the appearance of a ver\' insignificant quantity. The urine
absorbs considerable amounts of carbon dioxide, and in the presence of
only small amounts of sugar the fermentation test as above performed
may lead to negative or inaccurate results. In this case proceed in the
folloT\-ing wa}': Treat the acid urine, or urine which has been faintly
-acidified -uith a little sulphuric acid, with yeast whose acti^^ty has been
tested by a special test on a sugar solution, and allow it to stand 24-30 hours
iit about 30°. Then test again with the bismuth test, and if the reaction
noAv 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 taken that the urine
be acid before as well as after fermentation. If the reaction becomes alka-
line 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 fermentation.^
If a good polariscope is at hand it must not be forgotten to control the
results of the fermentation bv determining the rotation before and after
fermentation. The phenylhydrazine test also, in many othen\-ise 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 c.c. of the urine
two knife-points of phenylhydrazine hydrochloride and three knife-points
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
Avater-bath. It is then placed in a beaker of cold water. If the quantity
of sugar present is not too small, a yellow cr}-stalline precipitate is now
obtained. If the precipitate appears amorphous, there are found, on
looking at it under the microscope, 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 cr\-stals are
' On the perfonnance of the fermentution test and certain sources of error, see
Salkowski, BerHu. klin. Wochenschr., 1905 (Ewald-Festnummer), and Pfliiger, Pfltiger's
Arch, 105.
658 URINE.
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 i). In doubtful cases where certainty
is desired, prepare tlie 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 precipitated as crystals by the
addition of benzene, ligroin, or ether. If the characteristic yellow crystal-
line 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 2 is simple, practical, and
at the same time sufficiently delicate. 5 c.c. of the urine is treated with
2 c.c. of acetic 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 c.c. After quickly cooling warm again and then allow it to
cool slowly. After 5-10 minutes beautifulh^ formed crystals are obtained
even in the presence of only 0.02 per cent sugar. According to the exjjeri-
ence of Hammarsten tliis modification, even in the presence of 0.1 per cent
sugar in concentrated urines, does not always give a positive reaction.
The value of the phenylliydrazine test has l^een 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 not heated in the water-bath for too
short a 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 ^ and Ham-
marsten's experience. This test only shows a non-physiological quan-
tity of sugar when a rather abundant crystallization is obtained from a
small quantity of urine (about 5 c.c). Too great a delicacy of test is not
to be recommended.
Rubner's test is performed as follows: The virine is precipitated by
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, su]:)stances, such as the conjugated glucuronic acids. In the
presence of only very little sugar the value of this test depends on the deli-
cacy of the instrument and the dexterity of the observer. As a urine which
shows no rotation or is actually faintly levorotator\^ may contain 0.2 per
' Rosenfeld, Deutsch. ir.ed. Wochenschr., 1888; Geyer, cited from Roos, Zeitschr.
f. physiol. Chem., 15.
• Arcli. f. (Anat. u.) Plij^siol., 1899, Suppl. See also Margulies, Berlin, klin. Woclien-
schr., 1900.
^ Hirschl, Zeitschr. f. pliysiol. Clieni., 14; Kistermann, Deutsch. Arch: f. klin.
Med., oO; Roos, 1. c; Holmgren, Maly's Jah;esber., 27.
SUGAR IN URINE. 659
cent sugar or perhaps even more, this test must be combined with the fer-
mentation test if we are seeking ver}- small amounts of sugar. The sugar
in these cases can be detected only by the use of a very accurate and deli-
cate instrment. Tliis method is in man}' cases not serviceable for the
physician.
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 am-
moniacal basic lead acetate, wash this precipitate with water, decompose it
with H2S when suspended in water, concentrate the filtrate, treat it with
strong alcohol until it is SO vol. per cent, filter when necessary-, and add
an alcoholic caustic-alkaU solution. Dissolve the precipitate consisting of
saccharates in a little water, precipitate the potash by an excess of tartaric
acid, neutralize the filtrate with calcium carbonate in the cold, and filter.
The filtrate may be used for testing with the polariscope as well as for the
fermentation, bismuth, and phenylhydrazine tests. The presence of dex-
trose may be detected by this same process in animal fluids or tissues from
which the proteids have been removed by coagxilation or by the addition
of alcohol.
In the isolation of sugar and carbohydrates from the luine the benzoic-
acid esters of the same may be prepared according to Baumaxx's method.
The urine is made alkaline ^^ith caustic soda to precipitate the earthy phos-
phates, the filtrate treated with 10 c.c. of benzoyl chloride and 120 c.c.
of 10 I3er cent caustic-soda solution for ever}- 100 c.c. of the filtrate (Reix-
BOLD^), and shaken vuitil the odor of benzoyl chloride has disappeared.
After standing sufficiently long the ester is collected, finely divided, and
saponified with an alcoholic solution of sodium ethylate in the cold accord-
ing to Baisch's method ,2 and the various carbohydrates separated according
to his suggestion.
To the physician, who naturally wants simple and quick methods, the
bismuth test is especially to be recommended. If this test gi\-es 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 controlled by
other tests, especially by the fermentation test.
Other tests for sugar, as, for example, the reaction with orthonitrophenyl-
propiolic acid, picric acid, diazobenzeiie-sulphonic acid, are superfluous. The
reaction with n-naphthol, which is a reaction for carbohydrates in general, for
glucuronic acid and mucin, may, because of its extreme delicac}', 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
may be suspected of great quantities of carbohydrates. 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. Udraxsky, Luther,
Roos and Treupel,^ have investigated this test in regard to its applicabilit}^
as an approximate test for carbohydi-ates in the luine.
Quantitative Determination of Sugar in the Urine. The urine for such
an estimation must fisrt be tested for proteid, and if any be present it must
l^e removed by coagulation and the addition of acetic acid, care l^eina: taken
' Pfliiger's Arch . 91.
^ Zeitschr. f. physiol. Chem., 19.
^ See Roos and Treupel, Zeitschr. f. phj-siol. Chem., 15 and 16.
660 URINE.
not to increase or diminish the original volume of urine. The quantity of
sugar may be determined by titration' with Fehlixg's or Knapp's solu-
toin. by fermextatiox, by polarizatiox, and in other ways.
The titration liquids not only react with sugar, but also with certain
other reducing substances, and on this account the titration methods give
rather high results. When large quantities of sugar are present, as in typi-
cal diabetic urine, which generally contains a lower percentage of normal
reducing constituents, this is indeed of little account; but when small quan-
tities of sugar are present in an othenvise normal urine, the mistake may,
on the contrar\% be important, as the reducing power of normal urine may
correspond to 5 p. m. dextrose (see page 609). In such cases the titration
procedure must be employed in connection with the fermentation method,
which will be described later. It is to be remarked that in typical diabetic
urines with considerable quantities of sugar the titration with Fehlixg's
solution is just as reliable as with Knapp's solution. When the urine on
the other hand, contains only little sugar with normal amounts of physiolog-
ical constituents, then the titration with Feeling's solution is more difficult,
in certain cases indeed almost impossible, and the results become ver\^
uncertain. In such cases Knapp's method gives good results, according to
Worm Muller and his pupils.^
The titration with Fehling's solution depends on the power of sugar
to reduce copper oxide in alkaline solutions. For this there was formerly
employed a solution which contained a mixture of copper sulphate, Rochelle
salt, and sodium or potassium hydrate (Fehling's solution) ; but as such
a solution readily changes, use is made of a copper-sulphate solution and
an alkaline Rochelle-salt solution prepared separateh^ and the two solutions
mixed in equal volumes before using.
The concentration of the copper-sulphate solution is such that 10 c.c. of
this solution is reduced by 0.0.5 gram of dextrose. The copper-sulphate
solution contains 34.6.5 grams of pure, cr}\stanized, non-efflorescent copper
sulphate in 1 liter. The sulphate is crj^stalUzed from a hot saturated solu-
tion by cooling and stirring, and the crj^stals are separated from the mother-
liquor and pressed between blotting-paper until dr\^ The Rochelle-salt
solution is prepared by dissolving 173 grams of the salt in 350 c.c. of water,
adding 600 c.c. of a caustic-soda sohition of a specific gravity of 1.12, and
diluting with water to 1 liter. According to Worm AIullfr, these three
liquids — Rochelle-salt solution, caustic soda, and water — should be sepa-
rately boiled before mixing together. For each titration mix in a small flask
or porcelain dish exactly 10 c.c. of the copper-sulphate solution and 10
c.c. of the alkaline Rochelle-salt solution and add 30 c.c. of water.
The urine, freed from proteid, is diluted with water before the titration,
so that 10 c.c. of the copper solution requires between 5 and 10 c.c. of
the diluted urine, which corresponds to between 1 per cent and J per cent
of sugar. A urine of a specific gravity of 1.030 may be diluted five times;
on3 more concentrated, ten times. The urine so diluted is poured into a
burette and allowed to flow into the boiling copper-sulphate and Rochelle-
salt solution until the copper oxide Is completely reduced. This has taken
place when, immediately after boiling, the blue color of the solution disap-
pears. It is very difficult and requires some practice to exactly determine
> Pfliiger's Arch., 16 and 23; Otto, Journal f. prakt. Chem. (N. F.), 26.
SUGAR IX UPJXE. 6G1
this point, especially when the copper suboxide settles with difficulty. To
determine whether the color has disappeared, allow the copper suboxide
to settle a little below the meniscus formed l^y the surface of the liquid.
If this layer is not h\\ie, the operation is repeated, adding 0.1 c.c. less of
urine; and if, after the copper suboxide has settled, the liquid has a blue
color, the titration may be considered as completed. Because of the diffi-
culty in oljtaining tliis point exactly another end-reaction has been sug-
gested. Tliis consists in filtering immediately after boiling a small portion
of the titrated mixture through a small filter into a test-tube wMch con-
tains a little acetic acid and a few drops of potassium-ferrocyanide solution
and water. The smallest quantity of copper is shown by a red coloration.
If the operation is quickly conducted so that no oxidation of the suboxide
into oxide takes place, this end-reaction is of value for urines which are rich
in sugar and poor in urea and which have been strongly diluted with water.
In urines poor in sugar which contain the normal amount of urea and which
have not been considerably diluted, a considerable quantity of ammonia
may be formed from the urea on boiling the alkaUne liquid. This ammonia
dissolves the suboxide in part, which then easily passes into oxide; besides
the dissolved suboxide gives a red color with potassium ferrocyanide. In
just those cases in which the titration is most difficult this end-reaction
is the least reUable. Practice also renders it unnecessary, and it is
therefore best to depend simply upon the appearance of the liquid.
To facilitate the settUng of the copper suboxide and therebv clearing
the liquid, Munk ^ has suggested the addition of a little calcium-chloride
solution and boiUng again. A precipitate of calcium tartrate is produced
which carries down the suspended copper suboxide with it. and the color of
the liquid can then be seen more readily. This artifice succeeds in many
cases, but unfortunately there are urines in which the titration wtih Fehling's
solution in no way gives exact results. In those cases in which onlv small
quantities of sugar exist in a urine rich in physiological constituents it is
best to dissolve a ver}- exactly weighed quantity of pure dextrose or dex-
trose-sodium chloride in the urine. The urine can now be stronglv diluted
with water" and the titration l^ecomes successful. The difference' between
the sugar added and that found by titration gives the reducing power of the
original urine calculated as dextrose.
The necessarj^ conditions for the success of the titration under all cir-
cumstances are, according to Soxhlet,^ the following: The copper-sul-
phate and Rochelle-salt soluti^^n must, as above, be diluted to 50 c.e. with,
water; the urine should contain only between 0.5 and 1 per cent of sugar,
and the total quantity of urine required for the reduction must be added
to the titration liquid at once and boiled with it. From this last con-
dition it follows that the titration is dependent upon minute details, and
several titrations are required for each determination.
It is best to give here an example of the titration. The proper amount
of copper-sulphate and Rochelle-salt solution and water (total volume = 50
c.c.) is heatecl to boiling in a flask; the color must remain blue. The urine
diluted five times is now added to the boiling-hot liquid. 1 c.c. at a time;
after each addition of urine boil for a few seconds and look for the appear-
ance of the end-reaction. If one finds, for example, that 3 c.c. is too little,
* Virchow's Arch., 105. ^ Journ. f. prakt. Chem. (X. F.), 21.
662 URINE.
but that 4 c.c. is too much (the Uquid becoming yellowish), then the urine
has not been sufficiently diluted, for it should require between 5 and 10 cc.
of the urine to produce the complete reduction. The urine is now diluted
ten times, and it should require between 6 and 8 c.c. for a total reduction.
Now prepare four new tests, which are boiled simultaneously to save time,
and add at one time respectively 6, 6^, 7, and 7^ c.c. of urine. If it is
found that between 6^ and 7 c.c. are necessary to produce the end-reac-
tion, then make four other tests, to which add respectively 6.6, 6.7, 6.8,
and 6.9 c.c. of urine. If in this case the liquid is still somewhat bluish
with 6.7 c.c. and completely decolorized with 6.8 c.c, the average figure
6.75 c.c. is considered as correct.
The calculation is simple. The 6.75 c.c. used contains 0.05 gram of
sugar, and the percentage of sugar in the dilute urine is therefore (6.75 :0.05 =
5
100:a-) = — = = 0.74. But as the urine was diluted with ten times its vol-
6.75
5X 10
ume of water, the undiluted urine contained ^ „^ =7.4 per cent. The
' 0.75
general formula on using 10 c.c. of copper-sulphate solution is therefore
5 X w
— ; — , in which n represents the number of times the urine has been diluted,
k
and k the number of cubic centimeters of the diluted urine employed for
the titration.
The TITRATION ACCORDING TO Knapp 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 mer-
curic cyanide and 100 c.c. of caustic-soda solution of a specific gravity
of 1.145 per liter. When the titration is performed as described below (ac-
cording to WoRM-MiJLLER and Otto), 20 c.c. of this solution should cor-
respond 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 de-
termined by a preliminary test. To determine the end-reaction as de-
scribed below, the test for the excess of mercury is made with sulphuretted
hydrogen.
In performing the titration allow 20 c.c. of Knapp's solution to flow
into a flask and dilute vvith 80 c.c. of water, or when the urine contains
less than 0.5 per cent of sugar use only 40-60 c.c. After this heat to
boiling and allow the dilute urine to flow gradually into the hot solution,
at first 2 c.c, then 1 c.c, then 0.5 c.c, then 0.2 c.c, and lastly 0.1 c.c.
After each addition let it boil h minute. When the end-reaction is approach-
ing, the liquid begins to clarify and the mercury separates with the phos-
phates. 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
SUGAR Ix\ URINE. 663
acid, and tested with sulphuretted hydrogen (OttoI). 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 decomposing for a long time (Worm MiIllp^r and his pupils 2).
The views of the various investigators on the value of this titration method
are nevertheless somewhat contradictoiy.
The titration according to Pavy consists in adding a boiling ammoniacal
solution of copper sulphate to the urine until it is decolorized, when the
suboxide formed is dissolved by the ammonia into a colorless solution.
The admission of air must be completely excluded. In regard to the per-
formance of this highly recommended method we must refer to the works
of Pavy, Kumagawa and Suto, and Sahli.^
Besides the above-described methods there are various others. K. B.
Lehmann uses an excess of copper salt and retitrates with potassium iodide
and hyposulphite. The sugar can also be determined according to Allihn,
and especially according to Pfluger's modification of this 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 ordinaiy cases, is that of Roberts. This con-
sists in determining the specific gravity of the urine before and after fer-
mentation. 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 sub-
stantiated since by several other investigators (Worm ^Iuller and others).
If the urine, for example, has a specific gravity of 1.030 before fermentation
and l.OOS after, then tlie quantity of sugar contained therein was 22X0.23
= .5.06 per cent.
In performing this test the specific gra\dty must be taken at the same
temjjerature before and after the fermentation. The urine must be faintly
acid, and when necessarv^ it should be acidified with a Httle hydrochloric
acid or sulphuric acid. The activity of the yeast must, when necessar\', be
controlled by a special test. Place 200 c.c. of the urine in a 400 c.c. flask,
add a piece of compressed yeast the size of a pea. and subdi^^de the veast
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 fermenta-
tion is ordinarily ended, but this must be verified by the bismuth test.
After complete fermentation filter through a drv^ filter, bring the filtrate to
Ihe proper temperature, and determine the specific gravitv.
' Journal f. prakt. Chem., 26.
2 Pfluger's Arch., 16 and 23.
' Favy, The Physiology of the Carbohydrates, London, 1894; Kumagawa and
Suto, Salkowski's Festschrift, 1904; SahH, Deutsch. med. Wochenschr., 1905. In
regard to other methods, see Huppert-Ne'ubauer. Analyse des Harnes.
* Lehmann, Arch. f. Hygiene, 30; Pfliiger, Pfluger's Arch., 66.
664 URINE.
If the specific gravity l^e determined with a good pyknometer supplied
with a thermometer and an expansion-tube, this method, when the quan-
tity of sugar is not less than 4-5 p. m., gives, according to ^\ orm Duller.
very exact results, but this has been disputed by Budde.^ For the physi-
cian the method in this form is not quite serviceable. Even when the
specific gravity is determined by a delicate urinometer which can give the
density to the fourth decimal, quite exact results are not obtained, because
of the ordinary errors of the method (Buddk); but the errors are usually
smaller than those which occur in titrations made by unskilled hands.
When the c^uantity of sugar is less than .5 p. m. these methods cannot
be used. Such small amounts cannot, as already mentioned, be determined
by titration directly, because the reducing power of normal urine corre-
sponds to 4-5 p. m. of sugar. In such cases, according to Worm ^Julij:r,
it is better first to determine the reduction 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 difference found between
the two titrations calculated as sugar gives the true quantity of the latter.
The determination of the sugar by fermentation can be so performed
that the loss in weight due to the CO2 can be estimated or the volume of
the gas measured. For this last purjDOse Lohxsteix^ has constructed a
special fermentation saccharometer, of which his "precision saccharometer''
is to be recommended. Based upon Lohxstein's instrument, Wagner ^^
has constructed a ' fermentation saccharo-mano meter," which has certain
advantages over Lohnstein's apparatus.
Estimation 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 levorotatory^ substances such as proteids, 5-oxybutyric
acid, conjugated glucuronic acids, the so-called Leo's sugar, and less often
cystine, all of which areunfermentable. The proteid is removed by coagu-
lation, and the others are detected by the polariscope after complete fer-
mentation. 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 instrument and with suffi-
cient practice very exact results can be ol:)tained by this method. The
value of this procedure consists in the rapidity with which the determina-
tion can l)e made. In using instruments specially constiTicted for clinical
purposes the accuracy is less than with the less expensive fermentation
test. Un^er such circumstances, and as the estimation by means of polari-
zation can he 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. Levog^^rate urines containing sugar have been observed bv
several ol)servers. although the nature of the sugar was not well known to
the earlier oljservers. In recent years several positively authentic cases
> Roberts, Edinburgli Med. Joum., 18G1, and The Lancet, 1, 1862; Worm-Miiller,
Pfliiger's Arch., 3S and 37; Budde, ibid., 40, and Zeitschr. f. physiol. Chem , 13 See
also Huppert-Neubauer, 10. Aufl., and Lohnstein, Pfliiger's Arch., 62.
^Berlin, klin. Wochenschr., 3o, and Allg. med Central-Ztg , 1899.
3 Miinch. med. Wochen.schr. , 190.5.
LACTOSE. 665
of le"VTilosiiria have been described, and also cases of diabetes have been
found where levulose exists in the urine besides dextrose.
Le\'ulose may be detected as follows: The urine is levorotatorj- and
the levorotatory substance ferments with yeast. The urine gives the ordi-
nary reduction tests and the ordinar}- phenylglucosazone. With methyl-
phem-lhydrazine it gives the characteristic le\-ulose methylphenvlosazone,
and it also gives Seliwaxoff's reaction on heating after the addition of
an equal volume of hydrochloric acid and a Httle resorcin. With this test
it must be remarked that too lengthy or too strong heating must not be
applied, since other carbohydrates may also give the reaction (see page
119 and the works of Rosix and Umber). After heating and cooling it
can be neutralized with soda and shaken out with amyl alcohol. This
removes a red pigment which gives a band in the spectrum tetween E and h
on stronger concentration; also a band in the blue at F (Rosix).i
Laiose is a substance named by Huppert and found by Leo • in diabetic urines
in certain cases, and which he considers as a sugar. It is levogjTate, 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 Sixety and F. Hofmeistek.
and this has been substantiated by other investigators. After the ingestion
of large quantities of milk-sugar some lactose may be found in the urine
(see Chapter IX on absorption). Laxgsteix and Steixitz have observed
the passage of lactose and also of galactose ^ into the urine of nurslinos
with diseases of the stomach. The passage of lactose into the urine is
called lactosuria.
The positive detection of this sugar in the urine is difficult, because
it is, like dextrose, dextrogA-rate and also gives the usual reduction tests.
If urine contains a dextrog}'rate, non-fermentable sugar which reduces
bismuth solutions, then it Is ver\' probable that it contains lactose. It
must be remarked that the fermentation test for lactose is. according to
the experience of Lu.sk and ^'oit,-^ best performed by using pure cultivated
yeast (saccharomyces apiculatus). This yeast only ferments tl^e dextrose,
while it does not decompose the milk-susar. If, according to Voir. Rub-
ner's test is performed without heating to boiling, but only to 80° C, the
color becomes yellow or brown in the presence of lactose, instead of red.
The most positive means for the detection of this sugar is to isolate the
• Umber, Salkowski's Festschrift, Berlin, 1904; Rosin, ibid., and Zeitsclir. f. physiol.
Chem., 38.
' Vircliow's Arch., 107.
3 Hofmeister, Zeitschr. f. physiol. Chem., 1, which also contains the pertinent
literature. See also Lemaire, ibid., 21 : Langstein and Steinitz, Hofniei.ster's Beitrage, 7.
* Carl Voit, Ueber Die Glycogenbildung nach Aufnahme verschiedener Zuckeraten,
Zeitsclir. f. Biologie, 28.
666 URINE.
sugar from the urine. This may be done by the following method, suggested
by F. Hofmeister:
Precipitate the urine with sugar of lead, filter, wash with water, unite the
filtrate and wash-water, and precipitate with ammonia. The liquid filtered
from the precipitate is again precipitated by sugar of lead and ammonia until the
last filtrate is optically inactive. The several precipitates with the exception
of the first, which contains no sugar, are united and washed with water. This
precipitate is decomposed in the cold with sulphuretted hydrogen and filtered.
The excess of sulphuretted hydrogen is driven off by a current of air; the acids
set free are removed by shaking with silver oxide. Now filter, remove the
soluble silver by sulphuretted hydrogen, treat with barium carbonate to unite
with any free acetic acid present, and concentrate. Before the evaporated residue
becomes syrupy it is treated with 90 per cent alcohol until a flocculent precipi-
tate is formed which settles quickly. The filtrate from this when placed in a
desiccator deposits crystals of lactose, which are purified by recrystallization,
■decolorizing with animal charcoal and boiling with 60-70 per cent alcohol.
Pentoses. Salkowski and Jastrowitz first found in the urine of per-
sons addicted to the morphine habit a variety of sugar which was a pen-
tose and yielded an osazone which melted at 159° C. Since this several
other cases of pentosuria have been observed, and according to Kulz and
VoGEL small amounts of pentose also occur in the urine of diabetics, as
also in the urine of dogs with pancreatic or phlorhizin diabetes.^
The pentose isolated by Neuberg from the urine in chronic pentosuria
was i-arabinose. In alimentar}^ pentosuria the /-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 Blumen-
THAL and also by v. Jaksch.^
A urine containing pentose reduces bismuth as well as copper solutions^
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 dex-
trose small amounts of pentose may also undergo fermentation. The
preparation of the osazone serves in the detection of pentoses; this com-
pound 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
l3e employed (see page 111). 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 c.c. of the urine is mixed
with an equal volume of HCl 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 precipita-
tion of a bluish-green pigment is in itself significant.
'In regard to the literature, see foot-note 1, page 110. See also Bkmienthal,
"Die Pentosurie," Deutsche IClinik, 1902.
» Blumenthal, Deutsche KUnik, 1902; v. Jaksch, Centralbl. f innere Medizin, 1906.
CONJUGATED GLUCURONIC ACIDS. 667
BiAL 1 uses as reagent 30 }3er cent hydrochloric acid, which contains
1 gram of orcin and 25 drops of a ferric-chloride solution (62.9 per cent of
the crj-stalline salt) in 500 c.c. of the acid. 4-5 c.c. of the reagent is
heated to boiling and then a few drops (not more than 1 c.c.) of the urine
is added to the hot but not boiling liquid. In the presence of pentose the
Hquid turns a beautiful green. The usefulness of Bial's reagent is ques-
tioned by several experimenters. The delicacy is not ver^- great and the
possibility of confounding with other carbohydrates is not excluded.
Lepixe and Boulud^ have shown the presence of maltose in cases of
diabetes. After boiling with hydrochloric acid the specific rotation dimin-
ishes, while the reducing power increases in such urines.
Conjugated Glucuronic Acids. Certain conjugated glucuronic acids
such as menthol- and turpentine-glucuronic acid may spontaneously decom-
pose in the urine, and in this case they may readik lead to a confusion vrith
pentoses. The urine should l^e always as fresh as possible for these exami-
nations.
A confusion of the glucuronic acids which have a reducing power on
copper or bismuth solutions with dextrose and le\'ulose can be prevented
by the fermentation test. They may also be distinguished from dextrose
by their optical behavior, as the conjugated glucuronic acids are levo-
g}-rate. On boiling with an acid dextrorotatory glucuronic acid is pro-
duced and the levorotation is changed to dextrorotation.
The conjugated glucuronic acids, hke the pentoses, give the phloro-
glucin-hydrochloric-acid test. On the contrar\- they do not give the orcin
test directly, but only after cleavage with the setting free of glucuronic
acid. On using Bial's reagent no mistaking for pentoses occurs, although
this statement requires further substantiation. The pentoses may also
be isolated and identified by their osazones. 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 boihng with the
acid. A further proof is that suggested by v. Alfthax.^ 500 c.c. of the
urine is benzoylated and the ester obtained saponified with sodium ethylate.
The free and conjugated glucuronic acid is thus obtained as sodium com-
pounds, insoluble m alcohol, while the pentoses, if present, remain in the
alcoholic filtrate. We have no suflBcient 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, boiUng with dilute sulphuric acid in order to spUt the
conjugated acid, and then after neutrahzing A\-ith soda prepare the charac-
* Deutsch. med. Wochenschr., 1903; ' Compt. rend., 132.
5 Arch. f. exp. Path. u. Pharm., 4", * Zeitschr. f. physiol. Chem., 29.
6G8 URINE.
teristic bromphenylhydrazine compound of glucuronic acid (see page 123)
with p-bromphenylhydrazine hydrochloride and sodium acetate.
Inosite occurs in the urine in albuminuria and in diabetes mellitus, but
only rarely and in small quantities. Inosite is also found in the urine after
the excessive drinking of water. According to Hoppe-Seyler ^ traces of
inosite occur in all normal urines.
In detecting inosite the proteid is first removed from the urme. Then con-
centrate the urine on the water-bath to j of its original volume and precipitate
with sugar of lead. The filtrate is warmed and treated with basic lead acetate as
long as a precipitate is formed. The precipitate formed after twenty-four hours
is washed with water, suspended in water, and decomposed with sulphuretted
hydrogen. A little uric acid may separate from the filtrate after a short time.
The liquid is filtered, concentrated to a syrupy consistency, and treated while
boiling with 3-4 vols, alcohol. The precipitate is quickly separated. After
the addition of ether to the cooled filtrate, crystals separate after a time, and
these are purified by decolorization and recrystallization. With these crystals
perform the tests mentioned on page 459.
Acetone Bodies (acetone, acetoacetic acid, /9-oxy butyric 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.^ According to v.
Jaksch and others acetone is a normal urinary constituent, though it may
occur only in very small amounts (0.01 gram in twenty -four hours).
In regard to the origin of these bodies it was previously considered that
they were produced by an increased destruction of protein. One of the
various reasons for this was the increase in the eUmination of acetone and
acetoacteic acid during inanition (v. Jaksch, Fr. Muller^). This stands
also in good 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 diseases
with abstinence and cachexia, where the body protein is largely destroyed.
The formation of acetone bodies from protein is also indicated by the fact
that acetone has been obtained as an oxidation product from gelatine and
protein (Blumenthal and Neuberg, Orgler*). On the other hand, no
parallelism exists between the acetone bodies and the nitrogen excretion in
' Handbuch d. physiol. u. pathol. chem. Analyse, 6. Aufl., 196.
' In regard to the extensive literature on acetone bodies the reader is referred to
Huppert-Neubauer, Ham-Analyse, 10. Aufl., and v. Noorden's Lehrb. d. Pathol, des
Stoffwechsels. Berlin, 1906.
^ V. Jaksch, Ueber Acetonurie und Diaceturie. Berlin, 1885; Fr. Mi'iller, Berieht
iiber die Ergebnisse des an Cetti ausgefiihrten Hungerversuches. Berlin, klin. Wochen-
schr., 1887.
^ Blumenthal and Xeuberg, Deutsch. med. Wochenschr., 1901; Orgler, Hofmeis-
ter's Beitrage, 1.
ACETONE BODIES. 669
diabetics, and the fact that in man no certain relationship exists between the
acetone elimination and the nitrogen and sulpliur excretion seem to show
that the acetone bodies are not entirely derived from the proteins. In
man the excretion of acetone does not increase with the rise in the quan-
tity of protein, and an increase in the latter above the average causes a
diminution in the elimination of acetone (Rosexfeld, Hirschfeld, Fr.
YoiT^). At the present time the tendency is more and more to the view
that the acetone bodies do not originate from the proteins but from the
fats; if they are not the only source, they are at least the most important.
It is generally accepted that in man the carbohydrates have a strong
influence on the elimination of acetone bodies, namely, the exclusion of
carbohydrates from the food or the diminution in their amount or their
assimilation may lead to more or less increased elimination of acetone
bodies. This behavior may occur in diabetes as well as in starvation and
in the above-mentioned diseased conditions. With abundant supply of
carbohydrates the acetone bodies are markedly diminished or may dis-
appear entirely, and a similar retarding action has been found by Satta^
to be brought about by other bodies, such as glycerine, tartaric acid, lactic
acid, and citric acid. The increased excretion of acetone with carbohydrate
starvation occurs also in healthy individuals with a fatty diet, or on the
supply of sufficient calories in other ways (alimentar\' acetonuria).
If we do not accept the formation of acetone bodies from proteins,
then we must admit such a formation from the fats. As proof of this
there are certain cases of diabetes with strong eUmination of acetone bodies
(^-oxybutyric acid) where the quantity of protein transformed was too
small to account for the acetone bodies OIAGxus-LE^•Y) . The free elimi-
nation 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
relationship has been found between the fat consumed and the acetone
bodies eliminated. Certain investigators (Geelmuydex, Schwarz. Wald-
VOGEL^) have also observed an increase in the acetonuria on partaking
of fatty food.
There is no doubt that the fats bear a certain relationship to the ace-
tone bodies, and that they are probably in part the source of the same.
It has not been proved, on the contrary, that the fats are the only or the
most important source of the acetone bodies, and to all appearances we must
' Hirschfeld, Zeitschr. f. klin. Med., 28; Geelmuyden, see Maly's Jaliresber., 26,
and Zeitschr. f. physiol. Chem., 23 and 26; Rosenfeld, Centralbl. f. innere Med., 16;
Voit, Deutsch. Arch. f. khn. Med., 66.
' Hofmeister's Beitrage, 6.
^Magnus-Levy, Arch. f. exp. Path lu -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.
670 URINE.
consider the proteins equally with the fats as the source of these bodies.
The researches of Embden and his coworkers are of special interest in this
connection. After Embden and Kalberlah showed that the liver was an
acetone-forming organ Embden, Salomon and Schmidt i showed by experi-
ments with removed livers that butyric acid, oxybutyric acid, leucine,
tyrosine, and in fact all aromatic bodies (such as tyrosine, phenylalanine,
phenyl-a-lactic acid and homogentisic acid) which contain a benzene
nucleus which can be burnt in the body, may be transformed into acetone
in the liver.
In drawing conclusions as to the origin of the acetone bodies it must
not be forgotten that the conditions in man are distinctly different from
those in carnivora (Geelmuyden, Fr. Voit). In dogs the elimination of
acetone bodies is not increased in starvation, but is reduced; it is aug-
mented with increased quantities of meat, runs parallel with the nitrogen
excretion and is not diminished by carbohydrates (Fr. Voit).
/CH3
Acetone, CqHbO, dimethylketone = CO^ , occurs, as above stated,
in ver}^ small amounts in normal urine. In diabetes it may give a poma-
ceous or fruit odor to the urine as well as to the expired air.
Irrespective of the alimentary acetonuria derived from the food, there
occurs an increased elimination of acetone, as above stated, in many dis-
eases, as also after nervous lesions, certain intoxications, and after admin-
istration of phlorhizin or extirpation of the pancreas (v. ^^Iering and Min-
kowski, Azemar2).
Acetone is a thin, water-clear liquid, boiling at 56.3° C. and possessing a
pleasant odor of tmit. It is lighter than water, Avith wliich it mixes in all
proportions, also with alcohol and ether. The most important reactions
for acetone are the following.
Lieben's Iodoform Test. When a waterj^ solution of acetone is treated
with alkali and then with some iodo-potassium-iodide solution and gently
warmed a yellow precipitate of iodoform is formed, which is known by its
odor and by the appearance of the crystals (six-sided plates or stars) under
the microscope. This reaction is ver}- delicate, but it is not characteristic"
oi acetone. Gunning's modi-fication of the iodoform test consists in using
an alcoholic solution of iodine and ammonia instead of the iodine dissolved
in potassium iodide and alkaU hydrate. In this case, besides iodoform, a
]:>lack precipitate of iodide of nitrogen is formed, but this gradually dis-
ai")pears on standing, leaving the iodoform visible. This modification has
' Hofmeister's Beitrage, 8.
^ Az^niar, Ac^tonurie experimentale." Travaux de physiologic, 1898 (labora-
toiie de M. le piofesseur E. Hedon, Montpellier).
ACETO ACETIC ACID. 671
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 milli-
gram of acetone in 1 c.c.
Reynolds's mercuric-oxide test is based on the power of acetone to dis-
solve- freshly precipitated HgO. A mercuric-chloride solution is precipi-
tated by alcoholic caustic potash. To this add the liquid to Ije 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 Guxxixo's test. Aldehydes also dissolve
appreciable quantities of mercuric oxide.
Legal's Sodium Nitroprusside Test. If an acetone solution is treated
with a few drops of a freshly prepared sodium-nitroprusside solution and
then with caustic-potash or soda solution, the liquid is colored rubv-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 cannot be mistaken
for acetone. If ammonia is employed instead of the caustic alkah (Le
Nobel), the reaction takes place with acetone but not with aldehvde.
Pexzoldt's indigo test depends on the fact that orthonitrobenzaldehyde
in alkaUne 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 mav
be dissolved with a blue color by shaking with chloroform. 1.6 miUigrams
acetone can be detected bj- this test.
Bela v. Bitto's * reaction is based on the fact than on adding a solution of
metadinitrobenzene made alkaline with caustic potash to acetone, a violet-red
color is produced wMch becomes cherry -red on acidifA'ing \\-ith an organic acid or
metaphosphoric acid. Aldehyde gives a similar \'iolet-red color which becomes
yellowish red on acidification. Creatinine does not give this reaction. From-
MER - has suggested the following method for detecting acetone: Treat 10 c.c.
of the urine ^^^th 1 gram potassium hydrate and add 10-12 drops of an alkaline
solution of salicyl-aldehyde. On warming a purple-red coloration is obtained in
the presence of acetone.
CH3
Acetoacetic acid, C4Hb03, acetylacetic acid, diacetic acid=A,xT •
COOH
This acid has not been observed as a physiological constituent of the urine.
It occurs in the urine chiefly under the same conditions as acetone. Like
' Annal. d. Cheni u. Pharm.. 2()9. - Berlin, kh'n. 'Wochenschr. 1905.
672 URINE.
acetone the acetoacetic acid occurs often in children, especially in high
fevers, acute exanthema, etc. Diacetic acid decomposes readily into
acetone. According to Araki ^ it is probably produced as an intermediate
product in the oxidation of |9-oxybutyric acid in the organism. The three
bodies appearing in the urine, acetone, acetoacetic acid, and /3-oxybutyric
:acid, tand in close relationship to each other.
This acid is a colorless, strongly acid liquid which mixes with water, •
alcohol, and ether in all proportions. On heating to boihng with water,
and especially with acids, this acid decomposes into carbon dioxide and
acetone, and therefore gives the above-mentioned reactions for acetone.
It differs from acetone in that it gives a violet-red or brownish-red color
^v'ith a dilute ferric-chloride solution. For the detection of this acid we
make use of the following reactions w^hich may be applied directly to the
urine.
Gerhardt's Reaction. Treat 10-15 c.c. of the urine -^-ith 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 saHcylic acid, phenol,
sulphocyanides). A portion of the urine shghtly acidified and boiled does
not give this reaction on account of the decomposition of the acetoacetic
acid.
Arnold and Lipliawsky's Reaction. 6 c.c. of a solution containing
1 gram of p-arainoacetophenone and 2 c.c. of concentrated hydrochloric acid
in 100 c.c. of water are mixed with 3 c.c. of a 1 per cent potassium-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 c.c. of this mixture
(according to the quantity of acetoacetic acid in the urine), add 15-20 c.c.
HCl of sp. gr. 1.19, 3 c.c. of chloroform, and 2-4 drops of ferric-chloride
Solution and mix without shaking. In the presence of acetoacetic acid the
■chloroform is colored violet or blue (otherwise only yellowish or faintly
red). This reaction is more delicate than the preceding test and reacts
with 0.04 p. m. acetoacetic acid. Large amounts of acetone (but not the
quantity occurring in urines) give this reaction according to Allard.^
BoNDi and Schwarz's^ Reaction. 5 c.c. of the urine is titrated drop,
by drop with iodine-potassium iodide solution until the color is orange-red
Then w'arm gently and when the orange-red color has disappeared add the
' Zeitschr. f. physiol. Chem., 18.
=> Arnold, Wien. klin. Wochenschr., 1899, and Centralbl. f. innere Med., 1900;
Lipliawsky, Deutsch. med. Wochenschr., 1901; Allard, Berl. klin. Wochenschr., 1901.
^Wien. kiln. Wochenschr., 1906.
/?-OXYBUTYRIC ACID. 673
iodine solution again until the color remains permanent on warming. Then
boil, when the irritating vapors of iodo-acetone will attack the eyes. Ace-
tone does not give this reaction.
Detection of Acetone and Acetoacetic Acid in the Urine. Before testing
for acetone test for acetoacetic acid; as this acid gradually decomposes on
allo\\ing the urine to stand, the specimen must be as fresh as possible. In
the presence of acetoacetic acid the urine gives the above-mentioned tests.
In testing for acetone in the presence of acetoacetic acid make the urine
sUghtly allcaUne and shake in a separator)^ funnol with ether free from
alcohol and acetone. Remove the ether and shake it with water, which
takes up the acetone, and test for acetone in the watery solution.
In the absence of acetoacetic acid the acetone may be tested for directly
in the urine; this may be done by Penzoldt's test. This test, which is
only approximate, is of value only when the urine contains a considerable
amount of acetone. For a more accurate test we distill at least 250 c.c.
of the urine faintly acidified with sulphuric acid, care being taken to have
a good condensation. ]\lost of the acetone is contained in the first 10-20
c.c. of the distillate. A better result may be obtained by distilling a large
quantity of urine until about ro 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. ^ 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 distil-
lation.
The quantitative estimation of acetone in the urine is done by convorting
it first into iodoform. The urine is acidified with acetic acid (according to
HupPERT, 1-2 c.c. 50 per cent acetic acid for ever\^ 100 c.c. urine) and
distilled. The quantity of acetone in the distillate is best determined
according to ^Iessinger and Huppert's method by determining volu-
me trically the quantity of iodine used in the formation of iodoform. In
regard to this method and its execution the reader is referred to Huppert-
Neubauer.^
CH3
/3-Oxybutyric Acid, C4H803 = CHOH. The occurence of this acid in
CH2
COOH
the urine w^as first positively shown by ]\Iinkowski, KiJLZ, and Stadel-
MANN.4 It occurs especially in severe cases of diabetes, when it may form
*See also Salkowski, Pfliiger's Arch., 56.
* Hofmeister's Beitrage, 8.
' Hamanalyse, 760, and also Geelmuyden, Zeitschr. f. anal. Chem., 35, and Vaubel,
Chem. Centralbl., 1905, 1, 1617.
* Minkowski, Arch. f. exp. Path. u. Pharm., IS and 19; Stadelmann, ibid., 17;
KiUz, Zeitschr. f. Biologie, 20 and 23.
674 URINE.
the largest portion of the acetone bodies (Magnus-Levy, Geelmuyden) . It
has also been observed in scarlet fever, measles, in scurvy, and in diseases
of the brain with abstinence. It seems to be always accompanied with
acetoacetic acid.
The /3-oxy butyric acid 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)u= —24.12° for solutions of 1-11 per cent and has
a disturbing action upon the determination of sugar by means of the polari-
scope. It is not precipitated by basic lead acetate or by ammoniacal lead
acetate, neither does it ferment. On boiling with water, especially in the
presence of a mineral acid, this acid decomposes into a-crotonic acid,
which melts at 71-72° C, and water: CH3.CH(OH).CH2.COOH = H20 +
CHs.CH-.CH.COOH. It yields acetone on oxidation with a chromic-acid
mixture.
Detection of ^-Oxyhutyric Acid in the Urine. If a urine is still levo-
gyrate after fermentation with yeast, the presence of oxy butyric acid is
probable. A further test may be made, according to Kulz, by evaporating
the fermented urine to a symp and, after the addition of an equal volume
of concentrated sulphuric acid, distilling directly without cooling, a-cro-
tonic acid is produced which distills over, and, after collecting in a test-
tube, crystals which melt at -|-72° C. separate on cooUng. If no crystals
are obtained, shake the distillate with ether, evaporate, and test the melt-
ing-point of the residue which has been washed with water. According
to Minkowski the acid may be isolated as a silver salt.^
The Quantitative Estimation may be performed as follow^s, according
to Bergell:^ 100-300 c.c. of the sugar-free urine or fermented urine
is made slightly alkaline with sodium carbonate and concentrated to a
syrup. This, on cooling, is rubbed with synipy phosphoric acid (keeping
it cool), anhydrous copper sulphate (20-30 grams), and fine sand, and the
dry mass thoroughly extracted with anhydrous ether in an extraction
apparatus. The residue after the evaporation of the ether is dissolved
in water and decolorized, if necessary, with animal charcoal, and the quan-
tity of the acid calculated from the polarization. Other methods have been
suggested by Darmstadter, Boekelman and Bouma, and Magnus-Levy.^
Ehrlich's * Urine Test. Mix 250 c.c. of a solution which contains 50 c.c.
HCl and 1 gram of sulphaiiilic acid in one liter with 5 c.c. 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 thereby, or orange after the addition of ammonia (aromatic oxyacids may
' Arch. f. exp. Path. ii. Pharm., 18, 35; Zeitschr. f. anal. Cliem., 24, 153.
2 Zeitschr. f. physiol. Chem., 33.
' Darmstadter, ibi/L, 37; Boekelmann and Bouma, see Maly's Jahresber., 31;
Magnus-Levy, Arch f. exp. Path u. Pharm. 45.
* Ehrlich, Zeitschr. f. kUn. Med.. 5. See also Clemens, Deutsch. Arch. f. klin.
Med., 63 (literature).
CYSTINE. 675
sometime, after a certain time give red azo bodies which color the upper layer of
the phosphate sediment). In pathological urines there sometime.:! occur.-? (and this
is the charactpristic Hinzo reaction) a primary yellow coloration, with a very
marked secondary r^^d coloration on the addition of ammonia,, and the frotli is
also tinged with red. The upper layer ot ilie sedlineat udcom^s giejni./a. Tae
body which gives this reaction is unknown, but it occurs especially in the urine
of typhoid patients (Ehrlich). Opinions differ in regard to the significance of
this reaction. The fact that the antoxyproteic acid gives this reaction as above
stated (page 612) is of interest.
Another urine test suggested by Ehrlich consists in adding a hydrochloric
acid containing 2 per cent dimethylaminobenzaldehyde to the urine; normal urines
are colored faintly red, while certain pathological urines become cherry-red.
The cause of this reaction is not sufficiently known; according to Neubaubr ' it
appears to be connected with the urobilinogen.
Rosenbach's urine test, which consists in adding nitric acid drop by drop
to the boiling-hot urine and obtaining a claret-red coloration and a bluish-red
foam on shaking, depends upon the formation of indigo substances, especially
indigo red.^
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, poi.soning \vith 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 by
the older methods in urine, especially in acute yello^v atrophy of the liver,
in acute phosphoiiis-poisoning, and in severe cases of typhoid and smallpox.
Since the use of /^-naphthalene sulphochloride has been used in the detection
of amino-acids these bodies have not only been repeatedly found in normal
urine (glycocoU, see page 614.) but also in pathological urines. Besides an
increased amount of glycocoU in certain cases of gout (Alex. Ignatowski)
and the finding of tyrosine and leucine in cystinuria (Abderhalden and
Schittenhelm) and in certain other cases, Abderhaldex and Barker ^
have also found phenylalanine (besides glycocoU, tyrosine, and leucine) in
the urine in dogs after phosphorus poisoning.
Cystine (see page 92). Baumann and Goldmaxn* claim that a sub-
> See Proscher, Zeitschr. f. physiol. Chem., 31, and Clemens, Deutsch. Arch. f.
klin. Med., 71; Neubauer, Centralbl. f. Physiol ID, 145.
^ S.e Rosin, Virchow's Arch., 123.
" ^'gnatowski, Zeitschr. f. physiol. Chem. 42; Abderhalden and Schitten'.ielm, ibid.
•15; Abderhalden and Bark-cr, ibid. 42.
* Baumann, Zeitschr. f, physiol. Cnem.' 8. In regard to the literature on cystine
see Brenzinger, tbid., 16; Baumann and Goldmann, ibid., 12; Baumann and v.
676 URINE.
stance similar to cystine occurs in very small amounts in normal urine.
This substance occurs in large quantities in the urine of dogs after poison-
ing with phosphorus. Cystine itself is only found with positiveness, and
even then ver\' rarely, in urinaiy calculi and in pathological urines, irom
which it may separate as a sediment. Cystinuria occurs oftener in men
than in women. Baumann and v. Udraxszky found in urine in cystinuria
the two diamines, cadaverine (pentamethylendiamine) and -putrescine (tetra-
methylendiamine), wliich are produced in the putrefaction of proteins.
These two diamines were also found in the contents of the intestine in
cystinuria, while under normal conditions they are not present. Hammar-
STEX therefore considers that perhaps some connection exists between
the formation of diamines in the intestine, by the peculiar i3utrefaction
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, although
sometimes those having cystinuria can quantitatively destroy the cystine
introduced. 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
bv Cammridge and Garrod ^ 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 92 and 93.
Cystine is easily prepared from cystine calculi by dissohdng them in
alkali carbonate, precipitating the solution with acetic acid, and redissolv-
ing the precipitate in ammonia. The cystine crystallizes on the spontane-
ous evaporation of the ammonia. The cystine dissolved in the urine is
detected, in the absence of proteid and sulphuretted hydrogen, by boiling
with allvali and testing with a lead salt or sodium nitropmsside. To isolate
cystine from the urine, acidify the urine strongly with acetic acid. The
precipitate containing cystine is collected after twenty -four hours and
dio-ested with hydrochloric acid, which dissolves the cystine and calcium
oxalate, leaving the uric acid undissolved. Filter, supersaturate the filtrate
with ammonium carbonate, and treat the precipitate with ammonia, which
dissolves the cystine and leaves the calcium oxalate. Filter again and pre-
cipitate 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 b}' dissoh-ing in ammonia
and precipitating with acetic acid and then further tested. Traces of dis-
solved cN'stine may be detected by the production of benzoyl-cystine, ac-
cording to Baumaxx and Goldman.
Udranszky, iibid., 13; Stadthagen and Brieger. Berlin, klin. Wochenschr., 1889; Cam-
midge and Ciarrod, Journ of Path, and Bacteriol. 1900 (literature on diamines in thj
urine and feces).
' Joum. of Path, and Bacteriol . YM 0.
URINARY SEDIMENTS AND CALCULI. 677
VII. Urinary Sediments and Calculi.
Urinan^ sediment is the more or less abundant deposit which is found
in the urine after standing. This deposit maj- consist partly of organized
and partly of non-organized constituents. The first, consisting of cells of
various kinds, yeast-fungi, bacteria, spermatozoa, casts, etc., must be
investigated by means of the microscope, and the following only applies
to the non-organized deposits.
As previously mentioned (page 543), the urine of healthy indi\aduals
may sometimes, even on voiding, be cloudy on account of the phosphates
present, or become so after a little while because of the separation of urates.
As a mle, urine just voided is clear, and after cooUng shows only a faint
cloud (nubecula) which consists of urine mucoid, a few epithelium-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 eon-
sisting of uric acid or urates, and sometimes also calcium-oxalate crj'stals,
in which yeast-fungi and bacteria are often to be seen. This change, which
the earlier investigators called "acid fermentation of the urine," is
generally considered as an exchange of the dihydrogen alkali phosphates
with the urates of the urine. ^lonohydrogen phosphates besides acid urates
or free uric acid or a mixture of both, according to conditions,^ are hereby
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 "alic^line fermentation," which consists in the
decomposition of the urea into carbon dioxide and ammonia b}- means of
lower organisms, micrococcus urese, bacterium ureae, and other bacteria.
MuscuLUs 2 has isolated an enzyme from the micrococcus urea? 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 carbohydrates of the urine
(Salkowski^). 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 crA'stals, sometimes covered with prismatic cr>'stals of alkali urate;
dark -colored spheres of ammonium urate, cr\-stals of calcium oxalate, and
' See Huppert-Neubauer 10. Aufl., and A. Ritter, Zeitschr. f. Biologie, 35.
^ Musculus, Pfluger's Arch , 12.
^ Salkowski, Zeitschr. f. physiol. Chem., 13.
678 URINE.
sometimes cn'stallized calcium phosphate are also found. Cn-stals of
ammonium-magnesium phosphate (triple phosphate) and spherical ammo-
uium urate are specially characteristic of alkahne 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.
Won-organized Sediments.
Uric Acid. This acid occurs in acid urines as colored crj^stals wliich are
identified partly by theii' form and partly by their property of gi\ing the
murexid test. On warming the urine they are not dissolved. On the
addition of caustic alkali to the sediment the cr\'stals dissolve, and when a
drop of this solution is placed on a microscope-slide and treated ^x\ih. a drop
of hvdrochloric acid small crj-stals 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 microscopic
cr}'stals of uric acid separate on the addition of hydrochloric acid. Crys-
talline alkali urates occur vers- rarely in the urine, and as a rule only in
such as have tecome neutral but not alkaline by alkaline fermentation.
The cr\'stals are somewhat similar to those of neutral calcium phosphate;
they are not dissolved by acetic acid, however, but give a cloudiness there-
with due to small cr}'stals of uric acid.
Ammonium urate may indeed occur as a sediment in a neutral urine
which at first w^as strongly acid and has become neutralized by the alkaline
fermentation, but it is only characteristic of ammoniacal urines. This
sediment consists of yellow or brownish rounded spheres which are often
covered with thorny -shaped prisms and, because of this, are rather large
and resemble the thorn-apple. It reacts to the murexid test. It is dis-
solved by alkalies with the development of ammonia, and crj-stals of uric
acid separate on the addition of hydrochloric acid to this solution.
Calcium oxalate occurs in the sediment generally as small, shining,
strongly refractive quadratic octahedra, which on microscopical examina-
tion remind one of a letter-envelope. The cr}-stals can only be mistaken
for small, not fully developed cr}'stals of ammonium-magnesium phos-
phate. The}'^ differ from these by their insolubility in acetic acid. The
oxalate may also occur as flat. oval, or nearly circular disks \\-ith central
ca\nties which from the side apj^ear like an hour-glass. Calcium oxalate
mav occur as a sediment in an acid as well as in a neutral or alkaline urine.
NON-ORGANIZED SEDLMENTS. 679
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 ]ye
the diacid alkah phosphate, and the greater the quantity of this salt in the
urine the greater the quantity of oxalate in solution, ^^^len. as pre\'iously
mentioned (page 677) , the simple-acid phosphate is formed from the diacid
phosphate, on allowing the urine to stand, a corresponding part of the oxa-
late 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 i;i alkaline urines. It either has almost the
same appearance as amorphous calcium oxalate or it occurs as somewhat
larger spheres with concentric bands. It dissolves in acetic acid with the
generation of gas, which differentiates it from calcium oxalate. It is not
yellow or brown like ammonium urate, and does not give the murexid test.
Calcium Phosphate. The c.\lcium triphosphate, Ca3(P04)2^ which
occurs only in alkaUne urines, is always amorphous and occurs partly as a
colorless, ver}- fine powder and partly as a membrane coiLsisting of ven,'
fine granules. It differs from the amorphous urates in that it is colorless,
dissolves in acetic acid, but remains undissolved on warming the urine.
Calcium diphosphate. CaHP04-L2H20. occurs in neutral or only in vers-
faintly acid urine. It is found sometimes as a thin film covering the urine
and sometimes as a Sediment. In crj'stallizing. the crj-stals may be single,
or they may cross one another, or they may be arranged in groups of color-
less, wedge-shaped cr^-stals whose wide end is sharply defined. These cr^-s-
tals differ from crj^stalline alkali urates in that they dissolve without a
residue in dilute acids and do not give the murexid test
Calcium sulphate occurs very rarely as a sediment in strongly acid urine. It
appears as long, thin, colorless needles, or generally as plates grouped together.
Ammonium-magnesium phosphate, triple phosphate, mav separate
from an amphoteric urine in the presence of a sufficient quantity of am-
monium salts, but it is generally characteristic of a urine wliich is ammo-
niacal through alkaline fermentation. The ciystals 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 cr^-stals of the rhombic svstem
(coffin-shaped) which are easily soluble in acetic acid. Amorphous magne-
sium, triphosphate, ^Ig3(P04)2, occurs with calcium triphosphate in urines
rendered alkaline by a fixed alkali. CiystalUne magnesium phosphate,
]\Ig3(P04)2+22H20, has been ob.served in a few cases in human urine (also
in horse's urine) as strongly refracti\-e. long rhombic plates.
68C URINE.
Kyestein is the film which appears after a little while on the surface of the urine.
This coating, which was foruierly 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, hoema-
toidine. In alkaline urine blue crystals 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. Ebstein ^ considers the essential difference between an amorphous
or crystalUne 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 con-
trary, 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 23roduces catarrh accompanied 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 into the bladder. The calculus may have more than one nu-
cleus. In a tabulation made l)y Ultzmann of 545 cases of vesicular calcuh,
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 originall}^ of a simple stone may be converted 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,
' Die Natur und Behandlung der Harnsteine. Wiesbaden, 1884.
URINARY CALCULI. 681
the primary constituents of an originally primary calculus may be partly
dissolved and be replaced by phosphates. .Metamorphosed 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 primarily. Layers of uric
acid sometimes alternate with otlier layers of primary formation, most
frequently with layers of calcium oxalate. The simple uric-acid calculus
leaves very little residue when burnt on a platinum foil. It gives the
murexid test, but there is no material development of ammonia when acted
on by caustic soda.
Ammonium urate calculi occur as primary calculi in new-born or nursing
infants, rarely in grown persons. They often occur as a secondary forma-
tion. 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. They 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 covered
with prongs (mulberry calculi). These calculi produce bleeding easily,
and therefore they often have a dark-brown surface due to decomposed
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
682 URINE.
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 with-
out effervescence, and the solution gives the reactions for phosphoric acid
and the alkaline earths. The triple-phosphate calculi generate ammonia
on the addition of an alkali.
Calduyn-carhonate calculi occur chiefly in herbivora. The}^ 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 entirel}^ on the platinum foil burning with a bluish
flame. They give the above-mentioned reactions for cystine.
Xanthine calculi are very rarely found. They are also of primary formation.
They vary from the size of a pea to that of a hen's egg. They are whitish, yel-
lowish-brown or cinnamon-brown in color, of medium hardness, with amorphous
fracture, and on rubbing appear like wax. They burn up completely when
heated on a platinum foil. They give the xanthine reaction with nitric acid and
alkali but this must not be mistaken for the murexid test.
Urostealith calculi have been ob.served 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, inve.stigated by Krl'kenberg,^ consisted of
paraffine derived from a paraffine 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.
HoRB.\czEWSKi ^ has also analyzed a bladder stone which contained 958.7 p. m.
choledcrin.
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
' Chem. Untersuch. z. wissensch. Med., 2. Cited from Maly's Jahresber., 19, 422.
^ Zeitschr. f. physiol. Chem., 18.
URIXARY CALCULI. 683
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 imburnt 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 suspected
bases. The residue is then tested according to the following schema 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.
684
uraxE.
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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-membranes
of many horn-formations are nearly insoluble in caustic alkalies. Keratin
occurs in the horn structure mixed with other bodies, from which it is
isolated with difficulty. Among these bodies the mineral constituents in
many cases occupy a prominent place because of their quantity. Hair
leaves on burning 5-70 p. m. ash, which may contain in 1000 parts 230
parts alkali sulphates, 140 parts calcium sulphate, 100 parts iron oxide,
and even 400 parts silicic acid. Dark hair on burning seems generally,
although not always, to yield more iron oxide than blond. The nails are
rich in calcium phosphate, and the feathers rich in silicic acid, which
Drechsel/ claims exists in part in organic combination as an ester.
According to Gautier and Bertrand ^ arsenic also occurs in the epider-
mal formations. The arsenic is, according to Gautier, of importance in
the formation and growth of the same, and on the other hand these struc-
tures, hair, nails, and epidermis-cells, are of great importance for the
excretion of arsenic.
The skin of invertebrates has been the subject, in a few cases, of chemi-
cal investigation, and in these animals various substances have been
found, of which a few, though little studied, are worth discussing. Among
these bodies tunicin, which is found especially in the mantle of the tuni-
> Centralbl. f. Physiol., 11, 361.
''Gautier, Compt. rend., 129, 130, 131; Bertrand, ibid., 134.
685
686 THE SKIN AND ITS SECRETIONS.
cata, and the widely diffused chitin, found in the cuticle-formation of
invertebrates, are of interest.
Tunicin. Celluiose seems, according to the investigations of Ambronn, to
occur rather extensively in the animal kingdom in the arthropoda and the mol-
lusks. 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 investiga-
tions 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 ' and later confirmed by Win-
TERSTEIN.
Chitin is not found in vertebrates. In invertebrates chitin is alleged
to occur in several classes of animals; but it can be positively asserted
that true, typical chitin is found only in articulated animals, in which it
forms the chief organic constituent of the shell, etc. According to Kraw-
Kow^ chitin of the shell, etc., does not seem to occur free, but in com-
bination with another substance, probably a proteid-like body. Chitin
also occurs, according to Gilsox and Wintersteix,^ in certain fungi.
According to Suxdvik the formula of chitin is probably C5oHjooN8038 +
nCHfi), where n may vary between 1 and 4. According to Araki it has
on the contrary the composition CigHgoNjOj,. According to Krawkow the
chit ins of different origin show different behavior with iodine, and he
therefore concludes that there must exist quite a group of chitins, w^hich
seem to be amine derivatives of different carbohydrates, such as dextrose,
glycogen, dextrins, etc. According to Zaxder ^ 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. Schmiedeberg,
therefore, considers chitin as a probable acetyl acetic-acid combination
of glucosamine. Fraxkel and Kelly,^ on the contrarj^, consider chitin
as of a more complicated composition. The most characteristic cleavage
product obtained by them was a chitosamine acetylized at the nitrogen
atom, CgHjLsOsN.COCHg, and a second product, acetyldichitosamine,
Cj4H2eOioN2, which, according to Araki, has the same composition as
chitosan (see below), but is essentially different in many regards.
^ Ambronn, Maly's Jahresber., 20; Berthelot, Annal. de Chim, et Phys., o6, Compt.
rend., 47; AVinterstein, Zeitschr. f. physiol. Chem., 18; Franchimont, Ber. d. deutsch
chem. Gesellsch., 12.
2 Zeitschr. f. Biologie, 29.
' Gilson, Compt. rend., 120; Winterstein, Ber. d. deutsch. chem. GeseUsch., 27 and
28.
^Sundvik, Zeitschr. f. physiol. Chem., 5; Araki. ibid. 20; Zander, Pfliiger's Arch., 66.
^ Ledderhose, Zeitschr. f. physiol. Chem., 2 and 4; Schmiedeberg, Arch. f. exp. Path,
u. Pharm., 28; Friinkel and Kelly, Monatshefte f. Chem., 23.
CHITIX AND HYALIX. 687
According to Hoppe-Seyler and Araki/ on heating chitin with
alkali and a little water to 180° C. a cleavage takes place with the splitting
off of acetic acid, and the formation of a new substance, chitosan, whose
formula according to Araki is C\4H.gN20io, but according to v. Furth
and Russo^ more Ukeiy a multiple of Ci3H2„X2Qij. On iieating with acetic
anhydride chitosan is converted into a chitin-hke substance which is not
identical with chitin. Chitosan is insoluble in water and alkali, but dis-
solves in dilute acids. It splits into acetic acid and glucosamine by the
action of hydrochloric acid. According to v. Furth and Russo on acid
cleavage it yields 25 per cent acetic acid and 60 per cent glucosamine.
One nitrogen atom corresponds closely to 1 molecule acetic acid and f
molecule glucosamine. All the glucosamine complexes present in the
chitosan molecule seem to be acetylized. According to Krawkow the
various chitins behave differently with iodine or with sulphuric acid and
iodine, in that some are colored reddish brown, blue, or violet, while
others are not colored at all.
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 concen-
trated acids. It is dissolved without decomposing in cold concentrated
hydrochloric acid, but is decomposed by boiling hydrochloric acid. When
chitin is dissolved in concentrated sulphuric acid and the solution dropped
into boiling water and then boiled, a substance is obtained (glucosamine,
chitosamine) which reduces copper suboxide in alkaline solutions.
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, and lastly extracted with alcohol
and ether. If chitin so prepared is dissolved in cold, concentrated sul-
phuric acid and diluted with cold water, then pure chitin separates out,
having been set free from the combination withthe other bodies (Kraw^kow).
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 cjuantity (16 per cent) of lime salts (carbonate,
phosphate, and sulphate).
According to Lt'CKE ^ 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
'Araki. 1. c; v. Furth and Russo, Hofmeister; Beitrage 8.
* Virchow's Arch., 19.
688 THE SKIX AXD ITS SECRETIONS.
It differs from keratin on the one hand and from proteids on tlie 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 tlie 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-jormations are of different
kinds, but have not been much studied. Those occurring in the stratum
Malpighii of the skin, especially of the negro, and the black or brown pig-
ment 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 the
melanin of the eye, 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 matter of certain pathological swellings in man, the
phynuttorhusin (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 pigment,
which are free from sulphur (Laxdolt and others); others, on the con-
trary, 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 w'hether 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-
ati-^^e of haemoglobin. K. Morner and later also Br.^ndl and L. Pfeiffer
found, on the contrary, that this pigment did contain iron, and they
consider it as a derivative of the blood-pigments. The sarcomelanin
(from a sarcomatous liver) analyzed by Schmiedeberg contained 2.7 per
cent iron, which was in organic combination in part and could not be com-
pletely removed by dilute hydrochloric acid. The sarcomelanic acid pre-
pared by Schmiedeberg by the action of alkali on this melanin contained
1.07 per cent iron. The sarcomelanin investigated by Zdarek and v.
MELANINS. 689
Zeynek also contained 0.4 per cent iron. Recently Wolff' has pre-
pared two pigments from a melanotic liver of which one was no doubt
modified. The other, which was soluble in a soda solution, contained 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 obtained on the
contrary a melanin free from iron 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).^
The difficulties which attend the isolation and purification of the mela-
nins have not been overcome in certain cases, while in others it is ques-
tionable whether the final product obtained has not another composition
from the original coloring matter, owing to the energetic chemical processes
resorted to in its purification. 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 melanin prepa-
rations can only be of secondary importance.
The one or more pigments of the human hair have a low percentage
of nitrogen, 8.5 per cent (Sieber), and a variable but considerable amount
of sulphur, 2.71-4.10 per cent. The great quantity of iron oxide which
remains on incinerating hair does not seem to belong to the pigments-
The pigment of the negro's skin and hair was found entirely free from iron
by Abel and Davis.^ The pigment prepared by Spiegler from the
hair of animals also contained no iron.
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. Most
melanins — and this is also true for the melanoids produced from proteins
on cleavage with acids (Samuely) — yield indol or skatol and a pyrrol
substance, and we must therefore admit with Samuely* that the dif-
ferent chromogen groups contained in the protein molecule, which readily
yield aromatic and specially heterocyclic nuclei, which condense with the
withdrawal of water and absorption of oxygen, produce dark colored pro-
ducts the mixture of which forms the melanoids.
' Zdarek and v. Zeynek, Zeitschr. f. physiol. Chem., 36; Wolff, Hofmeister Beitrage
5. The literature on the melanins may be found in Schmiedeberg, "Elementarformeln
einiger Eiweisskorper, etc." Arch. f. exp. Path. u. Pliarm., 39; also in Robert, Wiener
Klinik, 27 (1901), and Spiegler, Hofmeister's Beitrage, 4.
^ The summary of the extensive literature on melanotic pigments may be found in
O. V. Fiirth, Centralbl. f. allgem. Path. u. Pathol. Anat. lo, 1904.
^ Journ. of Expt. Med. 1, 361.
* Hofmeister's Beitrage, 2.
690 THE SKIN AND ITS SECRETIONS.
It has also been found that by the action of tyrosinases upon tyrosine
dark products similar to melanin are formed, and these, like the animal
melanins, yield substances smelling like skatol on fusion with alkali.
Such a direct pigment formation caused by the presence of tyrosinase has
also been observed by Gessard in the maceration of the skin of frogs and
toads, and certain investigators, such as Gessard, v. Fijrth and Schneider,^
are therefore of the opinion that tyrosine is the mother-substance of the
melanins.
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 for mation 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 ver}' similar to
that of oxyhaemoglobin. It must be remarked that according to Laidlaw ^ turacin
or at least a pigment with the same properties can be obtained on boUing hsemato-
porphyi-in in dilute ammonia with ammoniacal copper solution. KRrKENBERG *
found a large number of coloring matters in birds' feathers, namely, zooerythrin,
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 verj^ 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. Hmnatoporphyrin,
according to Mac^Iunn,* also occurs in the integuments of certain of the lower
animals.
In certain butterflies (the pieridinpe) 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,® of an entirely different kind. In this case we are dealing
with a compound between proteid and a pigment which is allied to bilirubin or
urobilin, i.e., a compound similar to hsemoglobin.
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.
Caxminic acid, or the red pigment of the cochineal, gives on oxidation, accord-
ing to LiEBERMANN and VoswiNCKEL,' cochenillic acid, CioHgOy, and coccinic acid.
* Gessard, Compt. rend. 136, and Compt. rend., see. bid. 57; v. Furth and Schneider,
Hofmeister's Beitrage, 1.
^ Joum. of Physiol. 31.
3 Vergleichende physiol. Studien, Abth. 5, and (2. Reihe) Abth. 1, 151, Abth. 2, 1,
and Abth. 3, 128.
*Wunn, cited from Maly's Jahresber., 1; Halliburton, Joum. of Physiol., 6; Merej-
kow.ski, Compt. rend., 93; MacMunn, Proc. Roy. See, 1883, and Joum. of Physiol., 7.
"> Phil. Trans., 186.
"Pflijger's Arch., 98.
'Ber. d. deutsch. chem. Gesellsch., 30.
SEBUM. 691
CsHgOfi, the first being the tri-carboxyhe acid, and the other the di-carboxylic
acid of m-cresol. The beautiful purple solution of ammonium carminate has two
absorption-bands between D and E which are similar to those of oxyhremoglobin.
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, from 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 him stentorin, actiniochrom, honellin, poly per ythri?i, pentacrinin, antedonin,
crustaceorubin, janthinin, and chlorophyll.
Sebum when freshly secreted is an oily semi-fluid mass which solidifies
on the upper surface of the skin, forming a greasy coating. Sebum is
according to Rohmann and Linser a mixture of the secretion of the
sebaceous glands and of the constituents of the epidermis. Hoppe-
Seyler has found in the sebum a body similar to casein besides albumin
and fat. According to Rohmann and Linser true fat occurs only to a
very slight extent. On saponification the sebum gives an oil, dcrmolein,
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
cetylalcohol by v. Zeynek. The amount of cholesterin in this secretion
is small and originates essentially from the epidermoidal formation.
Cholesterin is found in especially large quantities in the vernix caseosa.
The solids of the sebum consist chiefly of fat, epithelium-cells, and protein
bodies; the vernix caseosa is made up chiefly of fat. Rltppel ^ found on
an average in the vernix caseosa 348.52 p. m. water and 138.72 p. m.
ether extractives. Besides cholesterin he found also isocholesterin.
On account of the generally diffused view that the wax of the plant
epidermis serves as protection for the inner parts of the fruit and plant,
Liebreich ^ 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 glycerine 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.
' 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.
' Virchow's Arch., 121.
692 THE SKIX AXD ITS SECRETIONS.
In the fatt}'' protective substance secreted by the Psylla alni Sundvik ' has
found psylla-alcohol, CajHggO, which exists there as an ester in combination with
psyllic acid, CgaHesCOOH.
Cenunen is a mixture of the secretion of the sebaceous and sweat glands
of the cartilaginous part of the outer passages of the ear. It contains
chiefly soaps and fat, fatt}^ acids, cholesterin and proteid, and besides
these a red substance easily soluble in alcohol and ^yith a bitter-sweet
taste. -
The preputial secretion, smegma pneputii, contains chiefly fat, also
cholesterin and ammonium soaps, which probably are produced from
decomposed urine. The hippuric acid, benzoic acid, and calcium oxalate
found in the smegma of the horse have probably 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, fat, resins, traces of phenol (volatile oil), and a non-nitrog-
enous body, castorin, crystallizing in four-sided needles from alcohol, insoluble
in cold water, but somewhat soluble in boiling water, and whose comj^osition is
little known.
In the secretion from the anal glands of the skunk butyl mercaptan and alkyl
sulphide have been found (Aldrich, E. Beckmaxn ^).
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 LiFSCHxJTZ have found other alcohols in wool-fat besides mjTistic acid, also
two oxyfatty acids, lanoceric acid, C3oH6g04, and lanopalmitic acid, C.eHgjOa.
According to Rohmaxn * wool-fat contains a body lanocerin, which is the internal
anhydride of the above-mentioned lanoceric acid. Lanocerin is obtained without
saponification by repeatedly boiling lanolin with method alcohol, dissolving the
insoluble residue in ether and precipitating with alcohol.
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, Ci,H3,0, which represents 40-45 per cent
of the ethereal extract (Rohmaxx). The fatty acids are oleic acid, small amounts
of caprylic acid, palmitic acid, and stearic acid, and optical i-somers of lauric and
myristic acid. The fatty acids are in great part combined with the octadecylic
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 bufidia
(Jorxara and Casali), hufotalin and the disputed bodies hujonin and Imjotenin
* Zeitschr. f. physiol. Chem., 17, 25, and 32.
^ See Lamois and Martz, Maly's .Jahresber., 27, 40.
'Aldrich, Joum. of Expt. Med., 1; Beckmann, Maly's Jahresber., 26, 566.
* Darmstadter and Lifschtitz, Ber. d. d. Chem., Gesellsch, 29 and 31; Rohmann,
Hofmeisters Beitrage 5 and Centrabll. f. Physiol. 19, 317.
PERSPIRATION. 693
(Faust, Bertrand and Phisalix '). Thalassin is the crystalline body discovered
by RiCHET ' which is the poisonous constituent of the feelers of the sea nettle.
The Perspiration. Of the secretions of the skin, whose quantity
amounts to about g^j of the weight of the body, a disproportionally 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
very numerous, and the quantity of sweat secreted must consequently
vary considerably. The secretion differs for 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 seba-
ceous glands. Filtered perspiration is a clear, colorless fluid with a salty
taste and of different odors from different parts of the body. The physio-
logical reaction is acid, according to most statements. Under certain
conditions also an alkaline sweat may be secreted (Trumpy and Luch-
sixGER, Heuss). An alkaline reaction may also depend on a decompo-
sition with the formation of ammonia. According to a few investigators
the physiological reaction is alkaline, and an acid reaction depends,
according to them, upon an admixture of fatt}' acids from the sebum.
Camerer found that the reaction of human perspiration in certain cases
was acid and in others alkaline. ^^Ioriggia found that the sweat from
herbivora was ordinarily alkaline, while that from carnivora was gener-
ally acid. According to Smith ^ horse's sweat is strongly alkaline.
' De Jonge, Zeitschr. f. physiol. Chem., 3; Roliniann 1. c; Zaleski, Hoppe-Seyler's
Med.-chem. Untersuch., 85; Faust, Arch. f. exp. Path. u. Phami., -tl; Jomara and Casali,
Maly's Jahresber., 3; Faust, Arch. f. exp. Path. u. Pharm., 47 and 49; Bertrand, Compt.
rend., 135; Bertrand and Phisalix, ibid.
2 Pfliiger's Arch., 108.
^ Trumpy and Luchsinger, Pfliiger's Arch., 18; Ileuss, Maly's Jahresber., 22; Camerer,
Zeitschr. f. Biologic, 41; Moriggia, Moleschott's Untersuch. zur Xaturlehre, 11; Smith,
Journ. of Physiol., 11. In regard to the older literature on perspiration, see Hermann's
Handbuch, 5, Thl. 1, 421 and 543.
694 THE SKIN AND ITS SECRETIONS.
The specific gravity of human perspiration varies between 1.001 and
1.010. It contains 977.4r-995.6 p. m., average about 982 p. m. water.
The soUds are 4.4-22.6 p. m. The molecular concentration is also very
variable and the freezing-point depression depends essentially upon the
content of NaCl. ARDix-DELTEiLfound A= -0.08-0.46°, average -0.237°.
Brieger and Disselhorst ^ found with perspiration containing 2.9, 7.07
and 13.5 p. m. NaCl, that the A was equal to - 0.322°, - 0.608° and - 1.002°,
respectively. The organic bodies are neutral fats, cholesterin, volatile fatty
acids, traces of jprotein (according to Leclerc and Smith always in horses,
and according to Gaube regularly in man, while Leube ^ claims only
sometimes after hot baths, in Bright's disease, and after the use of pilo-
carpin), also creatinine (Capraxica), aromatic oxyacids, ethereal- sulphuric
acids of -phenol and skatoxyl (Kast^), sometimes also of indoxyl, and
lastly urea. The quantity of urea has been determined by Argutinsky.
In two steam-bath experiments, in which in the course of h and | hour
respectively he obtained 225 and 330 c. c. of perspiration, he found 1.61
and 1.24 p. m. urea. Of the total nitrogen of the perspiration in these
two experiments 68.5 per cent and 74.9 per cent respectively belong to
the urea. From Argutinsky's experiments, and also from those of
Cramer,^ it follows that of the total nitrogen a portion not to be disre-
garded is eliminated by the perspiration. This portion was indeed 12
per cent in an experiment of Cramer at high temperature and powerful
muscular activity. Cramer has also found ammonia in the perspiration.
In uraemia, and in anuria in cholera, urea may be secreted in such quan-
tities by the sweat-glands that crystals deposit upon the skin. The mineral
bodies consist chiefiy of sodium chloride w^ith some potassium chloride,
alkali sulphate, and phosphate. The relative quantities of these in per-
spiration differ materially from the quantities in the urine (Favre,^ Kast).
The relationship, according to Kast, is as follows:
Chlorine
In perspiration 1
In urine 1
Phosphate : Sulphate
0.0015 : 0.009
0.1320 : 0.397
Kast found that the proportion of ethereal-sulphuric acid to the sul-
phate-sulphuric acid in perspiration was 1:12. After the administration
^ Ardin-Delteil, Maly's Jahresber., 30; Brieger and Disselhorst, Deutsch. med. Woch-
encshr., 29.
^Leclerc, Compt. rend., 107; Gaube, Maly's Jahresber., 22; Leube, Virchow's Arch.,
48 and 50, and Arch. f. klin. Med., 7.
^ Capranica, Maly's Jahresber., 12; Kast, Zeitschr. f. physiol. Chem., 11.
^Argutinsky, Pfliiger's Arch., 46; Cramer, Arch. f. Hygiene, 10.
* Compt. rend., 35. and Arch, g^ner. de Meu. (5), 2.
EXCHANGE OF GAS THROUGH THE SKIN. 695
of aromatic substances the ethereal-sulphuric acid does not increase to
the same extent in the perspiration as in the urine (see Chapter XV).
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 acid,
succinic acid, tartaric acid, iodine, arsenic, mercuric chloride, and quinine pass
into the perspiration. Uric acid has also been found in the perspiration in gout
and cystine in cystinuria.
Chromhidrosis is the name given to the secretion of colored perspiration.
Sometimes perspiration has been observed to be colored blue by indigo (Bizio), by
pyocyanin, or by ferro-pho.sphate (Kollmann'). 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 in man is of very little importance
compared with the exchange of gas by the lungs. The absorption of
oxygen by the skin, which was first shown by Regxault and Reiset
is very small, and according to Zuelzer amounts under the most favor-
able circumstances to j-Jo of the oxygen absorbed by the lungs. The
quantity of carbon dioxide eliminated by the skin increases with the rise
of temperature (Aubert, Rohrig, Fubixi and Ronchi, Barratt^), It
is also greater in light than in darkness. It is greater during digestion
than when fasting, and greater after a vegetable than after an animal
diet (FuBiNi and Ronchi). The quantity calculated by various inves-
tigators for the entire skin surface in twenty-four hours varies between
2.23 and 32.8 grams.^ In a horse, Zuntz with Lehmanx and Hagemann,*
found for twenty-four hours an elimination of carbon dioxide by the skin
and intestine which amounted to nearly 3 per cent of the total respiration.
Less than four-fifths of this carbon dioxide came from the skin respira-
tion. According to the same investigators the skin respiration equals
2^ per cent of the simultaneous lung respiration.
' Bizio, Wien. Sitzungsber., 39; KoUmann, cited from v. Gorup-Besanez's Lehrbuch,
4. Aufl., 5.55.
^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,
Joum. of Physiol., 21.
^See Hoppe-Seyler, Physiol. Chem., 580.
* 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
comm.unication 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. 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, hydrogen, hydrocarbons
and carbon monoxide. The nitrogen is found only in very small quan-
tities, 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 seems to be simply absorbed by the blood, at least in great part.
696
GASES OF THE BLOOD. 697
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 conditions, such as a difference in the
rapidity of circulation, a different temperature, rest and activity, cause a
change. In regard to the gases they contain the greatest difference is
observable between the blood of the arteries and that of the veins.
The quantity of oxygen in the arterial blood of dogs is on an average
22 vols, per cent (Pfluger). In human blood Setschenow found about
the same quantity, nameh% 21.6 vols, per cent. Lower figures have been
found for rabbit's and bird's blood, respectively 13.2 per cent and 10-15
per cent (Walter Jolyet). Venous blood in different vascular regions
has very variable quantities of oxygen. By summarizing a great num-
ber of analyses by different experimenters Zuntz has calculated that the
venous blood of the right side of the heart contains 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, PflIjger, P. Bert, Bohr and
Henriques and others), or a little above. Setschenow found 40.3 vols,
per cent in human arterial blood. The quantity of carbon dioxide in
venous blood varies still more (Ludwig, PFLiJGER, and their pupils,
P. Bert, Mathieu and Urbain, and others). According to the
calculations 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.^
Oxygen is absorbed only to a small extent by the plasma, which only
absorbs 0.65 per cent oxygen. 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.
' Afl the figures given above may be found in Zuntz's "Die Gase des Blutes" in
Hermann's Handbuch d. Pfiysiol., 4, Thl. 2, 33-43, which also contains detailed state-
ments and the pertinent literature, and Bohr in Nagel's Handbuch der Physiologie des
Menschen, Bd. 1. Hefte 1, 1905.
698 CHEMISTRY OF RESPIRATION.
The carbon dioxide of the blood occurs in part, and indeed, according
to the investigations of Alex. Schmidt,^ Zuntz,^ and L. Fredericq,^ 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. According to Bohr * a
pressure of about 30 mm. may be considered as the average pressure of
the carbon dioxide in the organism, and with such a pressure the quantity
of physically dissolved CO2 in 100 c.c. of the blood amounts to 2.01 c.c.
As the blood with this tension takes up about 40 vols, per cent CO,, hence
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 c.c. exists with the
corpuscles and 1.42 c.c. with the plasma.
As the blood corpuscles in 100 c.c. blood as above stated take up at
the above pressure about 14 c.c. COj only a small part of its COj is physi-
cally dissolved. The chief mass of the COj is loosely combined and the
constituent of these cells which unites with the CO, seems to be the alkali
combined with phosphoric acid, oxyhsemoglobin 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 comes again into play, so
that, with the carbon dioxide becoming free, a re-formation of alkali
diphosphate takes place. It is generally admitted that the blood-coloring
matters, especially the oxyhaemoglobin which can expel carbon dioxide
from sodium carbonate in vacuo, acts like acids; and as the globulins also
act similarly (see below), these bodies may also occur in the blood-
corpuscles as an alkali combination. The alkali of the blood-corpuscles
must therefore, according to the law of mass action, be divided between
the carbon dioxide, phosphoric acid, and the other constituents of the
blood-corpuscles which possess acidic properties, and among these espe-
cially 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 combinations, while
at a diminished partial pressure of the same gas, with the escape of carbon
* Ber. d. k. siichs. Gesellsch. d. Wissensch., math.-phys. Klasse, 1867.
'Centralbl. f. d. med. Wissensch., 1867, 529.
' Recherches sur la constitution du Plasma sanguin, 1878, 50, 51.
* In regard to the work of Bohr we will refer here and in future to Nagel's Handbuch
der Physiologie des Menschen, Bd. 1, Hefte 1.
THE CARBON DIOXIDE OF THE BLOOD. 699
•dioxide, the alkali diphosphate and the other alkali combinations must
be re-formed at the cost of the bicarbonate.
Haemoglobin must nevertheless, as the investigations of Setschenow *
and ZuNTz, and especially those of Bohr and Torup,^ have shown, be
able to hold the carbon dioxide loosely combined even in the absence of
alkali. Bohr has also found that the dissociation curve of the carbon-
dioxide haemoglobin corresponds essentially to the curve of the absorp-
tion of carbon dioxide, on which ground he and Torup consider the haemo-
globin itself as of importance in the binding of the carbon dioxide of the
blood, and not its alkali combinations. According to Bohr the haemo-
globin takes up the two gases, oxygen and carbon dioxide, simultane-
ously by the oxygen uniting with the pigment nucleus and the carbon
dioxide with the protein component. But as according to the researches
of ZuNTz ^ the combination of haemoglobin with the alkali is first split
to any great extent with a carbon dioxide tension of more than 70 mm., it
must be admitted that with the ordinary CO2 pressure in the organism, the
combination of the carbon dioxide in the blood corpuscles does not essen-
tially take place through the agency of the alkali but chiefly by means
of the haemoglobin.
The chief part of the carbon dioxide of the blood is found in the
blood-plasma or the blood-serum, which follows from the fact that the
serum is richer in carbon dioxide than the corresponding blood itself.
By experiments with the air-pump on blood-serum it has been found
that the chief part of the carbon dioxide contained in the serum is given
off in a vacuum, while a smaller part can be removed only after the
addition of an acid. The red blood-corpuscles also act as an acid,
and therefore in blood all the carbon dioxide is expelled in vacuo.
Hence a part of the carbon dioxide is in firm chemical combination
in the serum.
Absorption experiments with blood-serum have shown us further that
the carbon dioxide which can be pumped out is in great part looselv chem-
ically combined, and from this loose combination of the carbon ^dioxide
it necessarily follows that the serum must also contain simplv absorbed
carbon dioxide. For the form of binding of the carbon dioxide contained
m the serum or. the plasma there are the three following possibilities:
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 com-
bination.
> Centralbl. f. d. med. Wissensch., 1877. See also Zuntz in Hermann's Handbuch
76. '
2 Zuntz, 1. c, 76; Bohr, Maly's Jahresber., 17; Torup, ibid.
'Centralbl. f. d. med. Wissensch., 1867.
700 CHEMISTRY OF RESPIRATION.
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 c.c. of blood is given above as 1.42 c.c.
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 calcu-
lated 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.^
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 different from a solution of bicar-
bonate, or carbonate of a corresponding concentration; and the behavior
of the loosely combined carbon dioxide in the serum can be explained
only by the occurrence of bicarbonate in the serum. By means of
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 the bicarbonate
in the serum from which the carbon dioxide can be set free by simple
dissociation in vacuo, it must be assumed that the serum contains other
weak acids, in addition to the carbon dioxide, which contend with it for
the alkalies, and which expel the carbon dioxide from simple carbonates
in vacuo. The carbon dioxide which is expelled by means of the pump,
and which, without regard to the quantity merely absorbed, is generally
designated as " carbon dioxide in loose chemical combination," is thus
only obtained in part in dissociable loose combinations; in part it origi-
nates from the simple carbonates, from which it is expelled in vacuo by
other weak acids.
» E. Pfluger, Ueber die Kohlensaure des Blutes, Bonn, 1864, 11. Cited from Zuntz
in Hermann's Handbuch, 65.
COMBINATIONS OF THE CARBON DIOXIDE. 701
These weak acids are thought to be in part phosphoric acid and in
part globuHns. The importance of the alkaU phosphates for the carbon-
dioxide combination has been shown by the investigations of Ferxet;
but the quantity of these salts in the semm 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 Setschexow 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,^ whose \'iews have foimd 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-
senmi which, according to the law of mass action, contends with the
carbon dioxide for the alkalies. By a greater partial pressure of the
carbon dioxide the latter deprives the globulin alkali of a part of its alkali
and bicarbonate is formed; by low partial pressure carbon dioxide is set
free and it is abstracted from the bicarbonate by the globulin alkali.
The assumption that the proteins of the blood are bodies active in
combining with the carbon dioxide has received some support by the
investigations of Siegfried - on the combination of carbon dioxide by
amphoteric amino bodies. Siegfried has found that amino acids com-
bine ■v^ith carbon dioxide, therebv being converted into carbamino-
H
acids (glycocoll) for example, into carbamino acetic acid CH.^ — X — COOH
I
COOH
and that the carbon dioxide oan be readily split off from these compounds.
The peptones and serum proteids in the presence of calcium hydroxide
may also act in the same manner as amino acids. Proteid 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-seriun, as well as of the blood
m 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 condition
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 h^'drochloric acid had been introduced. In the coma-
tose state of diabetes mellitus the alkali of the blood seems to be in great
* Hoppe-Seyler. Med. chem. Untersuch.
* Zeitschr. f. physiol. Chem., 44 and 46,
702 CHEMISTRY OF RESPIRATION.
part saturated with acid combinations, /3-oxybutyric acid (Stadelmann,
Minkowski), and Minkowski^ 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 Hexsen ^ on the gases of human lymph
are at hand, but it still remains a question whether the lymph investi-
gated was quite normal. The gases of normal dog-lymph were first inves-
tigated by Hammarsten.' These gases contained traces of oxygen and
consisted of 37.4-53.1 per cent CO. and 1.6 per cent N at 0° C. and 760
mm. Hg pressure. About one half of the carbon dioxide w^as 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 Buchxer that the lymph collected
after asphyxiation is poorer in carbon dioxide than that of the breathing
animal is explained by Zuxtz * b}^ the formation of acid in the tissues,
and especially in the lymphatic glands, immediately after death, and this
acid decomposes the alkali carbonates of the lymph in part.
The secretions with the exception of the saliva, in which Pfltjger and
KuLZ found respectively 0.6 per cent and 1 per cent oxygen, are nearly
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 quantity 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 Pflu-
ger 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 chemically com-
bined carbon dioxide, making a total of 74.9 per cent. KiJLz^ found a
maximum of 65.78 per cent carbon dioxide for the parotid saliva, of
1 Walter, Arch. f. exp. Path. u. Pharm., 7; Stadelmann, ibid., 17; ^linkowski, Jlittheil
a. d. med. Klink in Konigsberg, 1888.
2 Virchow's Arch., 37.
^ Ber. d. k. sachs. Gesellsch. d. Wissensch., math.-phys. Kl.as.se, 23.
* Buchner, Arbeiten aus der pliysiol. An.stalt zii Leipzig, 1876: Zuntz, 1. c, 85.
^Pfluger, Pfliiger's Arch., 1 and 2: Kiilz. 2^itschr. f. Biologie, 23. It seems as if
KiJlz's results were not calcidated at 760 millimeters Hg, but rather at 1 meter.
EXCHANGE OF GAS. 703
which 3.31 per cent was removable by the air-pump and 62.47 per cent
was firmly combined. From these and other statements on 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 proteid 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 nearly
all removable by the air-pump, and a part seems to be loosely combined
with the sodium phosphate. The figures found by Pfluger for the
total quantity of carbon dioxide in milk and urine are 10 and IS. 1-19.7
per cent respectively.
EwALD ^ has made investigations on the quantity of gas in pathological
transudates. He found only traces, or at least only very insignificant
quantities of oxgyen 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 diox-
ide, especially that firmly combined, increases with the age of the fluid
while, on the contrary, the total quantity of carbon dioxide, and espe-
cially 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 tissues. It
is 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 occur exclusively 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.
' C. A. Ewald, Arch. f. (Anat. u.) Physiol., 1873 and 1876.
704 CHEMISTRY OF RESPIRATIOX.
This does not exclude the fact that in the lungs, as in every other tissue, an
internal respiration takes place, namely, a combustion with a consump-
tion of oxygen and formation of carbon dioxide. According to Bohr and
Henriques ^ the lungs take a very variable but ahvaj's 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
according to these experimenters upon the fact that the intermediary meta-
bolic products formed in the tissue 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-
hsemoglobin, and the law of the dissociation of oxyhsemoglobin is of funda-
mental 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 haemoglobin and HtJFXER ^ has made very careful and
important determinations on such solutions. Recent investigations of
Bohr ^ and his pupils, as well as of Loewy and Zuxtz,'* 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 hgemoglobin brought about
in its preparation. A haemoglobin solution combines firmer with oxygen
than the blood, and the dissociation tension of the oxygen is greater in
blood than in a haemoglobin solution. If we graphically represent the
influence of the oxygen pressure upon the power of the blood to take up
oxygen by representing the oxygen tension as abscissa and the quantity
of oxygen taken up as ordinate then the haemoglobin solution shows a
somewhat flatter oxygen tension curve than the blood.
The oxygen tension may be variable, as Loewy ^ has shown, with
different individuals and, as Bohr, Hasselbalch, and Krogh « have found,
that besides this the CO2 present also influences the oxygen taken up, in
that as the carbon dioxide tension (also within physiological limits)
» Centralbl. f. Physiol. 6 and Maly's Jahresber, 27.
=> Arch. f. (Anat.'u.) Physiol., 1890 and 1894.
3 See Nagel's Handbuch and Krogh, Skand., Arch. f. Physiol., 16.
*Arch. f. (Anat. u.) Physiol., 1904.
^Ibid.
•Centralbl. f. Physiol. 17 and Skand., Arch. f. Physiol, 16.
OXYGEN TENSION IN THE BLOOD. 705
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 ^ upon horse's blood at 38° and a constant
carbon dioxide tension will be given below. In calculating the results in
column 5 the quantity of oxygen chemically combined at 150 mm. oxygen
pressure is equal to 100.
In 100 cc. Blood Oxygen taken up
n,T.«^., Chemically Oxygen Per cent Tlic<;nlve.i in
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 0.4.55
From the above table we see that even with an oxygen tension
which only amounts to one half of the oxygen pressure in the air that
haemoglobin in greatest part is saturated with oxygen. The dissociation
is hence at 70-80 mm. pressure only slightly more than with a pressure of
150 mm. and indeed even with as low a pressure as 40-30 mm. still 90
-80 per cent of the entire quantity of oxygen taken up chemically at 150
mm. is combined with the haemoglobin.
From these and other observations it follows that the oxygen partial
pressure may sink to one half of that existing in the atmospheric air without
markedly influencing the oxygen content of the blood. This coincides
also with the experience of Frankel and Geppert - 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 mention-
able decrease observed. A. Loewy ^ has found that the lowest oxvgen
pressure of the alveolar air when the exchange of material can go on
normally both qualitatively and quantitatively, is equal to 30 mm. Hg.
It may be concluded from the large quantity of oxygen or oxvhaemo-
globin in the arterial blood that the tension of the oxygen in the arterial
blood must be relatively higher. From the investigations of several
1 Skand. Arch, f. Physiol., 16.
^ Uber die Wirknugen der verdriinnten Luft auf. den. Orsanismus. Berlin, 1883.
*A. Loewy, L^ntersuch., iiber die Respiration und Zirculation etc., Berlin 1895; also
Centralbl. f. Physiol., 13, 449 and Arch. f. (.Inat. u.) Physiol, 1900.
706 CHEMISTRY OF RESPIRATION.
experimenters, such as P. Bert, Herter, and Hufxer,^ who experi-
mented partly on living animals and partly with hsemoglobin solutions,
we may assume the tension of the oxygen in arterial blood at the tem-
perature of the body to be equal to a partial oxygen pressure of 75-80
mm. Hg.
According to Bohr - the facts are otherwise, and he has obtained
remarkably higher results 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 haemataerometer. 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 enclosed 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 ten.sion 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.
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 con-
taining 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.
HiJFXER and Fredericq^ 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, has presented strong objections to the acceptance of
Bohr's findings, while on the other* hand Bohr not only defends his experi-
ments but also finds errors in the experiments of his opponents. On the
other hand Hald.vxe and Smith's * experiments making use of an entirel}^
different principle speak for the high results found by Bohr.
' Bert, La pre.s.sion barometrique, Paris, 1878; Herter, Zeitschr. f. physiol. Chem.,
3: Hiifner, 1. c.
= Skand. Arch. f. Physiol. 2 and Xagel's Handbuch. der Physiologie.
3 Hiifner, Arch. f. (.\nat. u.) Physiol. 1S90; FreJericq, Centralbl. f. Physiol. 7 and
Traveanx du laboratoire de I'lnstitiite de physiologie de Li^ge 5. 1S96.
*Haldane, Journ. of Phj'siol., 18; Haldane and Smith, ibid., 20.
COMPOSITION OF THE ALVEOLAR AIR. 707
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 haemoglobin in the arterial blood with carbon
monoxide has become constant, as shown by a special titration method. This
percentage saturation is dependent upon the relation between the tension of the
oxygen in the blood and the tension of the carbon monoxide, as known from the
composition of the inspired air. When this last and the percentage saturation
with carbon monoxide and oxygen are known the oxygen tension in the blood can
be easily calculated.
According to this method Haldane and Smith found still higher
figures than Bohr for the oxygen tension in the blood, and they calculated
the average tension of the oxygen in human arterial blood as 38.5 per cent
of an atmosphere i.e., equal to about 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 fol-
lowing average composition in volume per cent:
Oxygen.
Atmospheric air 20.96
Expired air 16.03
The partial pressure of the oxygen of the atmospheric air corresponds
at a normal barometric pressure of 760 mm. to a pressure of 160 mm. Hg.
The loss of oxygen which the inspired air suffers in respiration amounts to
about 4.93 per cent, while the expired air contains about one hundred
times as much carbon dioxide as the inspired air.
The expired air is therefore a mixture of alveolar air with the residue
of inspired air remaining in the air-passages; hence in the study of the
gaseous exchange in the lungs the alveolar air must first be considered.
There does not exist any 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 ^ 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 nitro-
gen in the alveolar air does not essentially differ from the expired air, and
admit that the quantity of oxygen in the alveolar air is 6 per cent less
than the inspired air, it will be seen that the alveolar air contains 15 per
cent oxygen. As the total pressure of the air of the lungs after deducting
Nitrogen
(and argon).
Carbon
Dioxide.
79.02
0.03
79.59
4.38
' See Zuntz I. c. Hennann's Handbuch 105 and 106.
708 CHEMISTRY OF RESPIRATION.
the aqueous tension of about 50 mm. can be calculated 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.
There are several direct determinations of the alveolar air of dogs by
Pfluger and his pupils Wolffberg and Nussbaum.^ These determina-
tions which show that the alveolar air is not much richer in carbon dioxide
than the expired air have been performed by means of the so-called 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.
In the air thus obtained from the lungs Wolffberg and Nussbaum
found an average of 3.6 per cent COj. Nussbaum has also determined the
carbon-dioxide tension in the blood from the right heart in a case simul-
taneous with the catheterization of the lungs. He found nearly 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 enclosed parts of the lungs had
taken place. From these investigations it can be calculated that the
quantity of oxygen in the alveolar air of dogs is about 16 per cent, which
corresponds to an oxygen partial pressure of about 115 mm. Hg.
If the oxygen partial pressure in the alveoUi, is put at only 106-115 mm.
Hg, and compare this with about 80 mm. as found by certain investigators
for the oxygen tension of the arterial blood, we find that a considerable
excess remains in favor of the alveolii, and the taking up of oxygen in the
lungs can simply, according to physical laws, be explained 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 Haldane and Smith, as average for various races of animals,
indeed always higher than the tension in the lungs. In these cases the
passage of oxygen from the lungs to the blood cannot be simply explained
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 the views on the taking up of oxygen are disputed so also are the
views on the giving up of carbon dioxide.
' Wolffberg, Pfliiger's Arch. 6; Nussbaum, ibul. 7.
CARBON DIOXIDE TENSION. 709
The tension of the carbon dioxide in the blood has been determined in
different ways by Pflijger and his pupils, Wolffberg, Strassburg, and
NUSSBAUM.^
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 cjuantity of carbon
dioxide of the gas mixture corresponds as nearly as possible in the beginning to
the probable tension of this gas in the blood, we may learn the tension of the
carbon dioxide in the blood.
According to this method the carbon-dioxide tension of the arterial
blood is on an average 2.8 per cent of an atmosphere, corresponding to a
pressure of 21 mm. mercury (Strassburg). In the blood from the pul-
monary alveoli Nussbaum found a carbon-dioxide tension of 3.81 per cent
of an atmosphere, corresponding to a pressure of 28.95 mm. mercury.
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 41.01 mm. mercury.
Another method is the catheterization of a lobe of the lungs (see page
708). In the air thus obtained from the lungs Nussbaum and Wolffberg
found an average of 3.6 per cent CO2. Nussbaum, as previously mentioned,
has also determined the carbon-dioxide tension in the blood of the pul-
monary alveoli in a case simultaneously with the catheterization of the
lungs. He found nearly identical results, namely, a carbon-dioxide
tension of 3.84 per cent and 3.81 per cent.
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 706), 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 containing carbon
dioxide, from 0.9 to 57.8 mm. Hg. A comparison of the carbon-dioxide
tension in the blood with the bifurcated air gave in several cases a greater
carbon-dioxide pressure in the air of the lungs than in the blood, and as
maximum this difference amounted to 17.2 mm. in favor of the air of the
lungs in the experiments with inhalation of atmospheric air. As the
alveolar air is richer in carbon dioxide than the bifurcated nir this experi-
* Wolffberg, Pfliiger's Arch., 6; Strassburg, ibid.; Nussbaum, ibid., 7.
710 CHEMISTRY OF RESPIRATION.
ment unquestionably proves, according to Bohr, that the carbon dioxide
has migrated against the high pressure.
In opposition to these investigations, Fredericq,^ in his above-men-
tioned experiments, obtained the same figures for the carbon-dioxide ten-
sion in arterial peptone blood as Pflltger and his pupils found for normal
blood. Weisgerber,^ in Fredericq's laboratory, has made experiments
with animals which respired air rich in carbon dioxide, and these experi-
ments confirm Pfltjger'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 PFLiJGER's pupils. In opposition to these investiga-
tions Bohr has presented strong objections; he has demonstrated the
principles for the construction of the tonometer and according to him the
older experiments with the tonometer are not conclusive as he claims that
a complete equilibrium of the gas tension was not sufficiently accomplished.
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
statement, first made by Holmgren, has recently found an advocate
in Werigo. Still Zuntz has presented very important objections to
Werigo's experiments, and Bohr^ has later also shown that we have no
positive basis for the above assumption.
The conditions as to the elimination of carbon dioxide in the lungs is
also not quite clear, and from the above we see that in regard to the gas
exchange in the lungs w^e have two opposed views. According to the
older view suggested by the Pfluger school the exchange of gas follows
the simple physical laws and is on the whole a diffusion process. Accord-
ing to Bohr's view a diffusion does take place; but according to him the
lung is a gland which has the power of secreting gases, and the gas exchange
in the lungs is essentially a secretory process. According to Hammarsten
we cannot dispute the fact that the investigations made thus far speak
very much in favor of Bohr's view, and this latter also receives support
in the detectable secretion of gases 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 oxvgen 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-
' See footnote 3 page 706.
'Centralbl. f. Physiol. 10, 482; Falloise, see Maly's Jahresber, 32.
^Holmgren. Wien Sitzungsber., 48 Werigo, Pfluger's Arch., 51 and 52; Zuntz, ibul.,
52; Bohn, see Nagel's Handbuch der Physiologie.
INTERNAL RESPIRATION. 711
ing to BiOT and others, contain considerably more oxygen and even above 80 per
cent. MoREAU has also found that after emptying the swimming-bladder by
means of a trocar new air collected after a time, and this air was richer in oxygen
than the atmospheric air and contained even 85 per cent oxygen. Bohr, who
has proved and confirmed these statements, also found that this collection is under
the influence of the nervous system, because on the section of certain branches
of the pneumogastric nerve it is discontinued. It is beyond dispute that there is
here a secretion and not a diffusion of oxygen. Recently Jaeger '■ has given a
further explanation as to the secretory activity of the swimming-bladder.
From what has been said above (page 703) in regard to the internal
respiration, one can conclude that it consists chiefly in that in the capil-
laries the oxygen passes from the blood into the tissues, while the carbon
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 Peluger,^ and the oxygen tension in the
blood on entering the capillaries must be higher. The oxygen tension of
the plasma is of importance for the giving up of oxygen to the tissues as
the blood corpuscles only contain a supply of oxygen, which, as the tissue
removes oxygen from the plasma, replaces this again. 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 nearly or entirely free from oxygen a considerable difference in regard
to- the oxygen pressure must exist between the blood and the tissues.
The possibility that this difference in pressure is sufficient to supply the
tissues with the necessary quantity of oxygen is hardly to be doubted.
The animal body it seems also has the command over means of regu-
lating and varying the oxygen tension, and such a means is the carbon
dioxide produced in the tissue which, according to Bohr, Hasselbach,
and Krogh,^ raises the oxygen tension. Another regulating moment is,
according to Bohr, the specific oxygen capacity of the blood which means
the relationship of the maximum oxygen combination to the quantity of
iron of the blood or the haemoglobin solution.
As the haemoglobin obtained 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.* This c;[uantity, he claims, may be different not only
' 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.
^ Estor and Saint Pierre with Pfliiger in Pfliiger's Arch. 1.
» L. c.
*Centralbl. f. Physiol. 4 and Nagel's Handbuch.
712 CHEMISTRY OF RESPIRATION.
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 ecjual, 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
ox3rgen, and the animal body must, according to him, have means of varying the
tension of the oxygen in the tissues in a short time without changing the quantity
of oxygen contained in the blood. The great importance of such a property of
the tissues for respiration is evident; but it is perhaps too early to give a positive
opinion on Bohr's statements 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 ^ found in the urine of dogs and in the bile a carbon-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 atmos-
sphere. 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 laws of diffusion.
Several methods have been suggested f )r the study of the quantitative
relationship of the respiratory exchange of gas. The reader must be
referred to other text-books for more details as to these methods, and we
will here only mention the chief features of the most important methods.
Regnault and Reiset's Method. According to this method the animal or
person experimented upon is allowed to respire in an enclosed 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 oxygen used as well as the carbon dioxide produced, has since
been modified by other investigators, such as Pfluger and his pupils, Seegen
and XowAK, and Hoppe-Sevler, Rosenthal, and Zuntz.^
Pettenkofer's Method. According to this method the individual to be
experimented upon breathes in a room through which a current of atmospheric
air is passed. The quantity of air passed through is carefully measured. As it
is impossible to analyze all the air made to pass through the chamber, a small
fraction of this air is diverted into a branch line during the entire experiment,
carefully measured, and the quantity of carbon dioxide and water determined.
From the composition of this air the quantity of water and carbon doxide 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
' Pfliiger's Arch. 6.
^ See Zuntz in Hermann's Handbuch, 4, Thl. 2, and Hoppe-Seyler, Zeitschr. f .
physiol. Chem., 19; Rosenthal, Arch. f. (Anat. u.) Phj^siol., 1902; Zuntz, Verhandl. d.
Berl. physiol. Gesellsch., 1901.
LUNGS AND THEIR EXPECTORATIONS. 713
method. The large respiration apparatus of Sonden and Tigepstedt as well as
of AtwatePv and Rosa ^ are based upon this principle.
Speck's Method.- For briefer experiments on maa .'Speck has used the follow-
ing: He breathes into two spirometer-reeeivers, on which the gas- volume can be
read off very accurately, through a mouthpiece with two valves, closing the nose
with a clamp. 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 fiuantity 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 Zuxtz and his pupils).
IIaxriot and Richet's Method * 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 dio.xide 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 E.xpectorations.
Besides proteid bodies and the albuminoids of the connective-substance
group, lecithin, taurine (especially in ox-lungs), i^ric acid, and inosite have
been found in the lungs. Poulet ^ claims to have found a special acid,
which he has called pulniotartaric acid, in the lung-tissue. Glycogen
occurs abundantly in the embryonic lung, but is absent in the adult organ.
The proteolytic enzymes also belong to the ph3'siological constituents of
the lungs. They are active in the autolysis of the lungs (Jacoby) as well
as in the solution of pneumonic infiltrations (Fr. Muller^).
^ Pettenkofer's method; see Zuntz, 1. c; Sonden and Tigerstedt, Skand. Arch. f.
Physiol., 6; Atwater and Rosa, Bull, of Dept. of Agriculture, 63. Washington.
^ Speck, Physiologie des menschlichen Atmens. Leipzig, 1892.
^ Pfliiger's Arch., 42. See also Magnus-Levyin Pfliiger's Arch., 55, 10, in which the
work of Zuntz and his pupils is cited.
* Compt. rend., 104.
5 Cited from Maly's Jahresber., 18, 248.
'Jacoby, Zeitschr. f. physiol. Chem., 33; ^liiller, Verhandl. d. Kongress. f. inn.
Medizin, 1902.
714 CHEMISTRY OF RESPIRATION.
The black or dark-brown pigment in the lungs of human beings and domestic
animals consists chiefly of carbon, which originates from the soot in the air. The
pigment may in part also consist of melanin. Besides carbon, other bodies, such
as iron oxide, silicic acid, and clay, may be deposited in the lungs, being inhaled
as dust.
Among the bodies found in the lungs under pathological conditions
must be specially mentioned proteoses (and peptones?) in pneumonia and
suppuration, glycogen, a slightly dextrorotatory carbohydrate differing
from glycogen found by Pouchet in consumptives, and finally also cellu-
lose, which, according to Freund,^ 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 lu.ig the following: NaCl 130, K^O 13, Na^O 195, CaO 19, MgO 19,
Fe203 32, P.O., 485, SOg 8, and sand 134 grams. According to Oidtmann ^
the lungs of a 14-day old child contained 793.05 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
very variable, especially under pathological conditions when various pro-
ducts 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 peptone (?), which are prob-
ably produced by bacterial action or by autolysis (Wanner, Simon ^),
volatile fatty acids, glycogen, Charcot's crystals, and also crystals of
cholesterin, hsematoidin, tyrosine, fat and fatty acids, triple phosphates,
etc., have been found.
The form constituents are, under physiological circumstances, e ■>ithe-
lium-cells of various kinds, leucocytes, sometimes also red blood-corpuscles
and various kinds of fungi. In pathological conditions elastic fibres,
spiral formations consisting of a mucin-like substance, fibrin coagulum,
pus, pathogenic microbes of various kinds, and the above-mentioned
crystals occur.
1 Pouchet, Compt. rend., 96; Freund, cited from Maly's Jahresber, 16, 471.
^Schmidt, cited from v. Gorup-Besanez, Lehrbuch, 4. Aufl., 727; Oidtmann, ifeid,,
732.
^Wanner, Deutsch. Arch. f. klin. Med.. 75; Simon, Arch. f. exp. Path. u. Pharm., 49.
CHAPTER XVIII.
METABOLISM WITH VARIOUS FOODS, AND THEIR NECESSITY
TO MAX.
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,
etc. — which leave the organism, and which, moreover, being very poor in
energy, are for this reason of little or no value for 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 which man and animals
take with the food all cannot be equally necessary or have the same value.
Some perhaps are unnecessary, while others may be indispensable. We
have learned Ijy 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, carbo-
hydrates, and fats.
It is also apparent that the variovis 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 knov.l-
edge 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 con-
ditions, and also in deciding many other questions — for instance, the
proper nutrition for an individual in health and in disease.
Such knowledge can only be attained by a series of systematic and
thorough observations, in which the quantity of nutritive material, relative
715
716 METABOLISM.
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 several investi-
gators, but above all should be mentioned those made by Bischoff and
VoiT, by Pettenkofer and Voit, and by Voit and his pupils, by Rubner
and by Atwater.
It is absolutely necessary in researches on the exchange of material to
be able to collect, analyze, and quantitatively estimate the excreta of the
organism, so that they may be compared with the quantity and composition
of the nutritive bodies ingested. In the first place, one must know what
the habitual excreta of the body are and in what way these bodies leave the
organism. One must also have trustworthy methods for the quantitative
estimation of the same.
The organism may, under physiological conditions, be exposed to
accidental or periodic losses of valuable material — such losses as
only occur in certain individuals, or in the same individual only at a
certain period; for instance, the secretion of milk, the production of
eggs, the ejection of semen or menstrual blood. It is therefore apparent
that these losses can be the subject of investigation and estimation only
in special cases.
The regular and constant excreta of the organism are of the very
greatest importance in the study of metabolism. To these belong, in the
first place, the true final metabolic products — carbon dioxide, urea (uric
acid, hippuric acid, creatinine, and other urinary constituents), and a part
of the water. The remainder of the water, the mineral bodies, and those
secretions or tissue constituents — mucus, digestive fluids, sebum, perspira-
tion, and epidermal formations — which are either poured into the intestinal
tract, or secreted from the surface of the body, or broken off and thereby
lost to the body, also belong to the constant excreta.
The remains of food, sometimes indigestible, sometimes digestible but not acted
upon, which are contained in the fseces, 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 neces-
sary 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 faeces, cannot be sei)arated 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.
EXCRETA OF THE ORGANISM. 717
is very small as compared to the variation which is caused by different individu-
alities, different modes of living, different foods, etc. Xo general but only apj^roxi-
mate values can therefore be given for the constant excreta of the human body.
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 2.500-3500
Salts (with the urine) 20-30
Carbon dioxide 750-900
Urea 20-40
Other nitrogenous urinary constituents 2-5
Solids in the excrements 20-50
These total excreta are approximately divided among the various
excretions in the following way; but still it must not be forgotten tliat
this division may vary to a great extent under various 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 excrements
5-9 per cent. The eUmination by the skin and lungs, which is sometimes
differentiated by the name " perspiratio insensibilis^' 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 different 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 multiplying
the quantity of nitrogen in the urine by the coefficient 6.25 Oj^ -=6.2b),
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 dis-
charges from the intestine always contain some nitrogen, which as stated
in Chapter IX consists in part of non-absorbed remnants of the food, but
in chief part and sometimes entirely of constituents of the epithelium 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 excrements which originates from the digestive tract and
from the digestive fluids. It may not only vary in different individuals,
but also in the same individual after more or less active secretion and
718 METABOLISM.
absorption. In the attempts made to determine this part of the nitrogen
of the excrements it has been found that in man, on non-nitrogenous or
nearly nitrogen-free food, it amounts in round numbers to somewhat less
than 1 gram per twenty-four hours (Rieder, Rubner). Even with such
food the absolute quantity of nitrogen eliminated by the faeces increases
with the quantity of food because of the accelerated digestion (Tsuboi ',)
and is greater than in starvation. Muller ^ found in his observations on
the faster Cetti that only 0.2 gram nitrogen was derived from the intes-
tinal canal.
The quantity of nitrogen which leaves the body under normal circum-
stances by means of the hair and nails, with the scaling off of the skin, and
with the perspiration cannot be accurately determined. It is 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
faeces, a nitrogen deficit occurred in the visible elimination.
This question has been the subject of much discussion and of numerous
investigations.' These investigations have shown that the above assump-
tion is unfounded, and moreover several investigators, especially Petten-
kofer and Voit, and Gruber,* 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 faeces is equal or nearly equal to the quantity
contained in the food. Undoubtedly we must admit with Voit that a
deficit of nitrogen does not exist, or it is so insignificant that in
experiments upon metabolism it need not be considered. Ordinarily,
in investigations on the catabolism of proteins in the body, it is
only necessary to consider the nitrogen of the urine and faeces, 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 faeces (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.
^ Rieder, Zeitschr. f. Biologie, 20; Rubner, ibvL, 15; Tsuboi, ibid., 35.
2 Berlin, klin. Wochenschr., 1887.
^See Regnault and Reiset, Annal. d. chim. et phys. (3), 26, and Annal. d. Chem.
u. Pharm., 73: Seegen and Nowak, Wien. Sitzungsber., 71, and Pfliiger's Arch., 25;
Pettenkofer and Voit, Zeitschr. f. Biologie, 16; Leo, Pfliiger's Arch., 26.
* Pettenkofer and Voit, in Hermann's Handbuch, 6, Thl. 1 ; Griiber, Zeitschr. f.
Biologie, 16 and 19.
EXCRETION OF SULPHUR AND PHOSPHORUS. 719
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 hj the urine, which in man is only to a small extent derived
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, H2SO4, produced by their combustion is in the ratio 5.2:1, or about
the same as in the urine (see page 622) . 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 control
is especially important in cases in which it is expected to study the action
of certain nitrogenous non-albuminous bodies on the metabolism of pro-
teins. 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 sub-
stances 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 faeces must be determined. The sulphur of the catabolized proteins
is quicker eliminated, according to v. Wexdt, than the nitrogen, and this
behavior of sulphur gives a more positive picture of the temporal cata-
bolism of protein than the nitrogen. This is all the more important as
according to Falta ^ not only does the nitrogen corresponding to a certain
amount of protein require several days for elimination but also the chief
quantity of this nitrogen in man after taking different kinds of proteins
is eliminated with varying rapidity.
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 (Gumlich,
Saxdmeyer, ^Iarcuse, Rohmaxx, and Steixitz, Loewi,- and others).
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-
' V. Wendt, Skand. Arch. f. Physiol., 17; Falta, Deutsch. Arch. f. kliii. Med. 86.
^ 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, 1900: Loewi, Arch. f. exp. Path. u. Pharm.,
45. On the absorption of ca.sein, see Poda. Prausnitz, Micko, and P. ^liiller, 2^itschr. f.
Biologic, 39. The literature on the phosphorus metabolism can be found in Albu and
Neuberg, Physiol, u. Pathol, des Mineralstoffwechsels, Berlin, 1906.
720 METABOLISM.
phonis (see page 619). 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 faeces, that there is an excess of the first, this means that the
body has increased its stock of nitrogenous substances — proteins. If, on
the contrary, the urine and fsBces 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, a part of its own proteins has been decomposed. We
can, from the quantity of nitrogen, as above stated, calculate the corres-
ponding quantity of proteins by multiplying by 6.25.^ Usually, according
to Voit's proposition, the nitrogen of the urine is not calculated as decom-
posed proteins, but as decomposed muscle-substance or flesh. Lean meat
contains on an average about 3.4 per cent nitrogen; hence each gram of
nitrogen of the urine corresponds in round numbers to about 30 grams of
flesh. The assumption that lean meat contains 3.4 per cent nitrogen is
arbitrary, and the relationship of N: C in the proteins of dried meat, which
is of great importance in certain experiments on metabolism, is given
differently by various experimenters, namely, 1 : 3.22 — 1 : 3.68. Argutin-
SKY found in beef, after complete removal of fat and subtraction of glycogen,
that the relationship 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 faeces in the form of organic compounds, in which the quan-
tity of carbon can be determined by elementary analysis. It used to be
considered sufficient to calculate the quantity of carbon in the urine from
the quantity of nitrogen according to the relationship N: C= 1: 0.67. This,
does not seem to be trustworthy, as this relationship varies and depends,
according to Tangl and PFLtJGER, Lang.stein, and Steinitz," upon the
kind of food. Tangl has shown that the richer the food is in carbohydrates
the more carbon and 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 fat 0.747 gram C and 9.22 Calories; for carbo-
hydrate-rich diet he found 0.963 gram C and 11.67 Calories.
The quantity of gaseous carbon dioxide eliminated may be determined
by means of Pettenkofer's respiration apparatus or by other methods.
By multiplying the quantity of carbon dioxide found by 0.273 one obtains
the quantity of carbon eliminated as CO^. If the total quantity of carbon
eliminated in various ways is compared with the carbon contained in the
1 In calculating the protein catabolism from the nitrogen of the urine it must not
be forgotten that the food often contains nitrogenous extractions whose nitrogen cannot
be calculated as protein and for which a special correction must be made, if necessary.
2 Tangl, Arch. f. (Anat. u.) Physiol., 1899, Supplbd.; Pfluger in Pfiuger's Arch., 79;
Langstein and Steinitz, Centralbl. f. Physiol., 19.
CALCULATIOX OF THE EXTENT OF CATABOLISM. 721
food some idea can be obtained as to the transformation of the carbon
compounds. If the quantity of carbon in the food is greater than in the
excreta, then the excess is deposited; while if the reverse be the case it
shows a corresponding loss of body substance.
The nature of the substances here deposited or lost, whether they con-
sist of proteins, fats, or carbohydrates, 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 ma}- 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 sub-
stance has been consumed besides the proteins. If the quantity of carbon
in the proteins is considered in round numbers as 53 per cent,' then the
relation between carbon (53) and nitrogen (16) is as 3.3 : 1. If the total
quantity of nitrogen eliminated is multiplied by 3.3, the excess of carbon
in the eliminations ovef 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 24 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 com-
pounds. If we start from the simplest case, starvation, where the body
lives at the expense of its own substance, then, since the quantity of
carbohydrates as compared with the fats of the body is extremely small,
in such cases in order to avoid mistakes the assumption must be made
that the person experimented upon has used only fat and proteins. As
animal fat contains on an average 76.5 per cent carbon, the quantity of
transformed fat may be calculated by multiplying the carbon bv :rT^ = 1-3.
76.5
In the case of the above example, the person experimented upon would
have used 62.5 grams of proteins and 167X1.3 = 217 grams of fat of his
own body in the course of the twenty-four hours.
Starting from the nitrogen balance, it can be calculated in the same
way whether an excess of carbon in the food as compared with the quantity
of carbon in the excreta is retained by the body as proteins or fat or as
both. On the other hand, with an excess of carbon in the excreta one can
determine how much of the loss of the substance of the body is due to a
consumption of the proteins or of fat or of both.
The quantity of water and mineral bodies voided with the urine and
faeces can easily be determined. The quantity of water eliminated by the
* This figure is perhaps a little too high.
722 METABOLISM.
skin and lungs may be directly estimated by means of Pettenkofer 's
apparatus. The quantity of oxygen taken up is calculated as the difference
between the weight of the individual before the experiment plus all the
directly determined substances ingested, and the final weight of the indi-
vidual plus all his excreta.
The oxygen may also be determined directly, according to RECiNAULT-
Reiset's method, or in other ways, and such a determination with the
simultaneous estimation of the carbon dioxide eliminated is of great
importance in the study of metabolism.^
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 car-
bon dioxide, because it is not only used in the oxidation of carbon, but
also in part in the formation of water, sulphuric acid, and other bodies.
The volume of expired carbon dioxide is regularlv less than the volume of
CO
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 quo-
tient is therefore equal to 1. The same is true in the burning of carbo-
hydrates, 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 decompo-
sition 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 therefore gives important data on the
quality of the material decomposed in the body, naturally Avith the suppo-
sition that the elimination of carbon dioxide, independent of the formation
of carbon dioxide, is not influenced by special conditions, such as the
alteration of the respiratory mechanism.
It is also possible in systematized experimentation to carry on the
metabolism experiments so that the decomposable material of the body,
as shown by the respiratory quotient, remains qualitatively the same, at
' In regard to the methods for estimating the carbon-dioxide excretion and the oxy-
gen consumption, see Zuntz, Hermann's Handbuch d. Physiol., 4, Tl. 2; Hoppe-Seyler,
Zeitschr. f. physiol. Chem., 19; Sonden and Tigerstedt, Skand. Arch. f. Physiol., 6;
Speck, Physiol, des menschl. Atmens. Leipzig, 1892; Zuntz and Geppert, Pfiuger's
Arch., 42: Magnus-Levy, ibid., 55, 10, where the works of Zuntz and his pupils are
cited; Hanriot et Richet, Compt. rend., 104, and Atwater, Bull, of Dept. of Agric,
Washington, Nos. 44, 63, 69, and 109.
CALORIFIC VALUES FROM OXYGEN CONSUMPTION. 723
least for a short time. In such experiments it has been shown, especially
by ZuNTz and his pupils/ that the extent of oxygen consumption may
be taken as a measure for the action of different influences on the extent of
metabolism. This possibility is based on the fact proved by Pfluger and
his pupils, and by Voit,- that the consumption of oxygen within wide
limits is independent of the supply of oxygen, and is exclusively dependent
upon the oxygen demand of the tissues. For certain reasons the consump-
tion of oxygen gives indeed a better conclusion than the elimination of
carbon dioxide as to the extent of exchange of material and energy; but
as the same quantity of oxygen (100 grams) consumes different quantities
of fat, carbohydrates, and proteins in the body — namely, 35, 84.4, and
74.4 grams respectively — the respiratory quotient must also be deter-
mined, in order to ascertain the nature of the substance burnt in the body,
simultaneously with the determination of the carbon dioxide.
As the different foods require different amounts of oxygen in the com-
bustion of each gram of substance and yield different amounts of CO,,
each gram of oxygen taken up and each gram of carbon in the expired
!iir as carbon dioxide must correspond to different heat values. This
follows from the following table:
Calories Calories
per grm. C Relative pergrm. Relative
ill the CO2 of Value. Consumed Value.
the Expired Air. Oxygen.
In the combustion of cane-sugar .. . 9.5 100 3.56 118.6
" " " "meat 10.2 107 3.00 100.0
" " " "fat 12.3 129 3.27 109.0
Pfluger has found the following figures for the calorific value of 1
gram oxygen:
For muscle tissue free from fat 3 . 30 Cal.
Fat 3.29 "
Starch •. 3.53 "
The figures for the oxygen differ, as seen above, le.ss than those for the car-
bon, and this is the reason why, as above stated, the oxygen consumption gives a
much more correct conclusion as to the exchange of force than the elimination of
carbon dioxide.^
Kaufmann * encloses 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 of 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
.' See footnote, page 722.
^Pfluger, Pfiiiger's Arch., 6, 10, and 14; Finkler, ibid., 10; Finkler and Oertmann,
ibiii, 14; Voit, Zeitschr, f. Biologie, 11 and 14.
' See Ad. Magnus-Levj', Pfiiiger's Arch., 55, 7, and Pfluger, ihiil., 77, 78, and 79.
*Arch. d. Physiologic (5), 8.
724 METABOLISM.
formulae for the various possible transformations of the proteins, fats, and
carbohydrates in the body, it is clear that other values must be obtained
for the heat, carbon dioxide, oxygen, and nitrogen of the urine, when one,
for example, admits of a complete combustion of proteins to urea, carbon
dioxide, and water, or of a partial splitting off of fat. Another relation-
ship 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,
Kaufmaxx attempts to explain the various decomposition processes in
the body under different nutritive conditions.
I. The Energy and the Relative Nutritive Value of Various
Organic Foodstuffs.
With the organic foods the organism receives a suppl}' of chemical
energy which is converted into heat and mechanical work in the body.
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 following pages
large calories are to be understood. There are numerous investigations by
different experimenters, such as Fraxklaxd, Daxilewski, Rubxer,
Berthelot, Stohmaxx, 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 products, are taken from Stohmaxx 's ^ 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
^ See Rubner, Zeitschr. f. Biologie, 21, which also cites the works of Frankland and
Danilewski; see also Berthelot, Compt. rend., 102, 104, and 110; Stohmana. Zeitschr.
f. Biologie, 31.
CALORIFIC VALUE OF THE FOODSTUFFS. 725
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 physio-
logical 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 pro-
ducts, which, leaving the body with the excreta, still represent a certain
quantity of energy which is lost to the body. The heat of combustion of
the proteins is smaller within the organism than outside of it, and they
must therefore be specially determined. For this purpose Rubxer ^ 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 faeces, which corresponded
to the food taken plus the quantity of heat necessary for the swelling up of
the proteins and the solution of the urea. Rubxer has also tried to deter-
mine the heat of combustion of the proteins (muscle-proteins) decomposed
in the body of rabbits in starvation. According to these investigations,
the physiological heat of combustion in calories for each gram of substance
is as follows:
1 grm. 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 Rubxer 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 " fat 9.3
1 " carbohydrate 4.1
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
' Zeitschr. f. Biologie, 21.
726 METABOLISM.
ratio of replacement. The investigations made by Rubner have taught
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 pro-,
tein and carbohydrate are equal to or isodynamic with 100 grams of fat in
regard to source of energy, because each yields 930 calories on combustion
in the body.
By means of recent very important calorimetric investigations Rubner^
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 ^ 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 deter-
mined exactly 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 nearly an
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. By this law it is possible to consider the processes of
metabolism as more uniform transformations of energy. 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.
1 Zeitschr. f. Biologic, 30.
^ Bull, of Dept. of Agric, Washington, 44, 63, 69, and 109 and Ergebnisse des
Physiologic 3.
Total loss
Availability
in per cent.
in per cent.
10.20
89.8
9.60
90.4
10.70
89.3
7.60
92.4
17.90
82.1
26.50
73.5
23.20
76.8
HEAT VALUES OF THE FOOD. 727
The heat value of a foodstuff can be directly determined in a calori-
meter but may also be calculated from its composition. If one subtracts
from the gross heat value of the food obtained in one way or another, the
combustion heat of the fseces 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 'physiological availahility
by RuBNER.* In order to elucidate this we will give a few of Rubner's
values. The loss in calories, as well as the physiological availability, are
calculated in percentages of the total energy content of the food.
Loss in per cent.
Food. In urine. In the freces.
Cow's milk 5.13 5.07
Mixed diet (rich in fat) 3 . 87 5 . 73
" " (poor in fat) 4.70 6.00
Potatoes 2.00 5.60
Graham bread 2.40 15.50
Rye bread 2.20 24.30
Meat 16.30 6.90
In order to simplify the calculation of the energy exchange there exist,
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 Cal. Kohler - found 5.678 Cal. for 1 gram of ash
and fat-free dried-meat substance of the ox and 5.599 Cal. for the horse.
According to Frentzel and Schreuer ^ 45.4 Cal. is calculated for 1 gram
of nitrogen in fat and ash-free dried-meat fseces (dog), while 6.97 to 7.45
Cal. is calculated for 1 gram of nitrogen in meat-urine. The calorific
Cal
urine quotient ■ seems still, as above given, not to be constant for
human beings at least, 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 according to the following principle as
suggested by E. Voit.* If we designate the heat of combustion for 1 gram of the
substance by Cal. 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 =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
' Zeitschr. f. Biologie, 42.
^ Zeitschr. f. physiol. Chem., 31.
^ The works of Frentzel and Schreuer may be found in Arch. f. (Anat. u.) Physiol.,
1901, 1902, and 1903.
* Zeitschr. f. Biologie, 44. See also Krummacher, ibid.
728 METABOLISM.
can be readily determined. The value K is nearly constant for substances of the
same groups; but also different groups show among themselves only slight devia-
tion for this value. Voit obtained the following values for a few of the foodstuffs:
K. (in kg. Cal.) 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.
II. Metabolism in Starvation.
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 only continue for a limited time. 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 tw^o 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,^ 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 more than half
a year, and frogs more than a 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.
* Cited from Voit in Hermann's Hnndbuch, 6, Thl. 1, 100.
^ See Luciani, Das Hungern. Hamburg u. Leipzig, 1890.
STARVATION. 729
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 nearly
unchanged during starvation. The investigations of Zuntz, Lehmann,
and others^ on the professional faster Cetti showed on the third
and sixth days of starvation an average consumption of 4.65 c.c.
oxygen per kilo in one minute, and on the ninth to eleventh day an
average of 4.73 c.c. The calories, as a measure of the metabohsm, fell
on the first to fifth day of starvation from 1850 to 1600 calories, or
from 32.4 to 30 per kilo, and it remained nearly unchanged, if referred to
the unit of body weight.^
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 experiments
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, according
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 VoiT ^ on the same dog. This dog received 2500 grams of meat daily
before the first series of experiments, 1500 grams of meat daily before
the second series, and a mixed diet relatively poor in nitrogen before the
third series.
Day of Starvation. Grams of Urea Eliminated in Twenty-four Hours.
Ser. I. Ser. II. Ser III
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,
1 Berlin, klin. Wochenschr., 1887.
^ See also Tigerstedt and collaborators in Skand. Arch. f. Physiol., 7.
'See Hermann's Handbuch, 6, Thl. 1, 89.
730 METABOLISM.
TiGERSTEDT, Landergren,^ as follows: At the commencement of star-
vation the protein metaboUsm is reduced by the glycogen still present
in the body. After the consumption of the glycogen, which takes place in
great part during the first days of starvation, the destruction of proteins
increases as the glycogen action decreases, and then decreases again when
the body has become poorer in available proteins.
Other conditions, such as varying quantities of fat in the body, have
an influence on the rapidity with which the nitrogen is eliminated during
the first days of starvation. After the first few days of starvation the
ehmination 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 relationship between the protein and fat content
of the body.
Like the destruction of proteins during starvation, the decomposition
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
Hmit, then in order to supply the calories necessary a larger quantity of
protein is destroyed and the premortal increase now occurs (E. Voit ^) ,
Since the fat has a diminishing influence on the destruction of proteins
corresponding to what was said above, the elimination of nitrogen in star-
vation is less in fat than in lean individuals. For instance, only 9 grams
of urea were voided in twenty-four hours during the later stages of starva-
tion 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,^
who had been poorly nourished.
The investigations on the exchange of gas in starvation have shown, as
previously mentioned, that the absolute extent of the same is diminished,
but that when the consumption of oxygen and elimination of carbon
dioxide are calculated on the unit of weight of the body, 1 kilo, this quantity
' Prausnitz, Zeitschr. f. Biologie, 29; Tigerstedt and collaborators, 1. c; Landergren,
" Undersokningar ofver menniskans agghviteomsattning, Inaug.-Diss. Stockholm,
1902.
^Zeitschr. f. Biologie, 41, 167 and 502. See also Kaufmann, ibid., and N. Schulz,
ibid., and Pfliiger's Arch., 76.
3 Berl. klin. Wochenschr., 1887.
STARVATION. 731
quickly sinks to a minimum and then remains unchanged, or, on the con-
tinuation of the starvation, may actually rise. It is a well known fact
that the body temperature of starving animals remains nearly 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, and death 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 occasionally be
lower, 0.65-0.50, as observed in the cases of Cetti and Succi. As explana-
tion for this unexpected behavior one must admit of a storage of incom-
pletely oxidized substances in the body during starvation.
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 the same
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
natuially 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,! 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 per-
ceptible 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. INIunk also
observed in Cetti 's ^ case a relative increase in the phosphoric acid and
calcium in the urine during starvation, which was due to an increased
exchange of bone-substance.
Contrary to the above Bohtlingk with starving white mice, and Katsuyama^
with starving rabbits found a greater excretion of sodium than potassium.
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-
1 Arch, des sciences biol. de St. Petersbourg, 5.
2 L, c.
^ Bohtlingk, 1. c; Katsuyama, Zeitschr. f. physiol. Cheni., 26.
732 METABOLISM.
tion of this point we give the following results of Chossat's experiments
on pigeons, and those of Voit ^ on a male cat. The results are percentages
of weight lost from the original weight of the organ.
Pigeon (Chossat). Male Cat (Voir).
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 " "
Ner\'OUs system 2 " " 3 " "
The total quantity of blood, as well as the quantity of solids contained
therein, decreases, as Panum and others ^ have shown, in the same propor-
tion as the weight of the body. The statements in regard to the loss
of water by different organs are somewhat contradictory; according to
LuKJANOW ^ it seems that the various organs act somewhat differently in
this respect.
The above-tabulated results cannot serve as a measure of the metabo-
lism in the various organs during starvation. For instance, the nervous
system shows only a small loss of weight as compared with the other
organs, but from this it must not be concluded that the exchange of
material in this system of organs is least active. The condition may be
quite different; for one organ may derive its nutriment during starvation
from some other organ and exist at its expense. A positive conclusion
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 likely upon the disturbance in the nutrition of a few less vitally
important organs (E. Voit^).
In calculating or determining the loss of weight of the organs in star-
vation the original fat content of the organs must also 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. Voit ^ has found the following loss of weight in the supposed fat-free
' Cited from Voit in Hermann's Handbuch, 6, Part 1, 96 and 97.
^ Panum, Virchow's Arch., 29: London, Arch. d. scienc. biol. de St. P^tersbourg, 4.
^ Zeitschr. f. physiol. Chem., 13.
* Zeitschr. f. Biologie, 41.
^Ibid., 46.
STAR\'ATIOX. 733
organs in starvation, namely, muscles 41 per cent, viscera 42 per cent,
skin 28 per cent, and skeleton 5 per cent.
The knowledge of metabolism during starvation is of the greatest
importance in the study of nutrition, and it forms to a certain extent the
starting-point for the study of metabolism under different physiological
and pathological conditions. To answer the question whether the
metabolism of a person in a special case is abnormally increased
or diminished it is naturally xery important to know the average
extent of metabolism of a healthy person under the same circumstances,
for comparison. This quantity can be called the starvation requirement,
that is, the extent of metabolism used in absolute bodily rest
and inactivity of the intestinal tract. As a measure of this quantity
we determine, according to Geppert-Zuxtz, the extent of gaseoiLs
exchange, and especially the consumption of oxygen, of a person
lying down, best sleeping, in the early morning and at least twelve
hours after a light meal not rich in carbohydrates. The gas volume
reduced to 0° C. and 760 mm. Hg pressure is calculated on 1 kilo of body
weight and for one minute. The results varv' between 3 and 4. .5 cc. for
the consumption of oxygen, and between 2.5 and 3.5 cc. for the carbon
dioxide. As average 3.81 cc. oxygen and 3.08 cc carbon dioxide are
usually given. ^
The extent of protein destruction cannot be detennined in transient
experiments, and for the.se reasons only the values found after several
days of starvation are useful. In the star\^ation experiments on Cetti
and .Succi the elimination of nitrogen per kilo on the fifth to the tenth star-
vation day was 0.150-0.202 gram X. In a recent starvation experiment
made by E. and O. Freuxd - upon Succi the nitrogen excretion on the
twenty-first day sank to 2.82 grams X. The portion of the urea nitrogen
of the total nitrogen sank from 85-89 per cent on the first days
of starvation to 73 per cent on the fifteenth day and 56-54 per
cent on the day before the last day of starvation. Xone of the
other nitrogenous constituents examined increased to the samo
extent as the urea decreased. The amount of neutral sulphur rose
from 10 to 40 per cent of the total sulphur. In a recent series
of inve.stigations upon the faster Succi. Brugsch^ found on the
twenty-first to the thirtieth day that the urea only amounted to 54-69.4
per cent of the total nitrogen while the quantity of ammonia, because
of a high acidosis, rose to 15.4-35.3 per cent. The amino-acid fraction
was also above normal.
' See V. Xoorden. Lehrbuch der Pathlogie des Stoffwechsel, Berlin, 1906.
^Wien. klin. Rundschau. 1901. Xos. .5 and 6.
^ Zeitschr. f. exp. Path. u. Therap. 1.
734 METABOLISM.
III. Metabolism with Inadequate Nutrition.
The food may be quantitatively insufficient, and the final result is
absolute inanition. The food may also be qualitatively insufficient or, as
we say, inadequate. This occurs when any of the necessary nutritive
bodies are absent in the food, while the others occur in sufficient or perhaps
even in excessive amounts.
Lack of Water in the Food. The quantity of water in the organism is
greatest during foetal life, and then decreases with increasing age. Natu-
rally, the quantity differs in various organs. The tissue in the body being
poorest in water is the enamel, which is almost free, containing only 2 p. m.
water, the teeth about 100 p. m., the fatty tissues 60-120 p. m. 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.* If it is borne in mind that two
thirds of the animal organism consists of water; that water is of the very
greatest importance in the normal, physical composition of the tissues;
moreover that all flow of juices, all exchange of substance, all supply of
nutrition, all increase or destruction, and all discharge of the products of
destruction, are dependent upon the presence of water; and, in addition,
that by its evaporation it is an important regulator of the temperature of
the body, we perceive that water must be necessary for life. If the loss
of water be not replaced by fresh supplies sooner or later, the organism
succumbs and death may occur earlier with lack of water than with com-
plete inanition (Landauer, Nothwang).
If the water is withdrawn for a certain time, as Landauer and espe-
cially W. 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 withheld (by means
of the increased metabolism). The deprivation of water for a short time
may, according to Spiegler,^ 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 a previous chapter atten-
tion was called in several instances 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 con-
' See Voit in Hennann's Handbuch, 6, Tl. I, 345.
^Landauer, Maly's Jahresber., 24; Nothwang, Arch. f. Hygiene, 1892; Straub,
Zeitschr. f. Biologic, 37 and 38; Spiegler, ibiil., 40.
LACK OF MINERAL SUBSTANCES IN THE FOOD. 735
tains about 220 p. m. mineral bodies (Volkmann '), 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 min-
eral 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. (Volk-
mann) .
The mineral bodies seem to be partly dissolved in the fluids and partly
combined with organic substances. In accordance with this the organism
persistently retains, with food poor in salts, a part of the mineral sub-
stances, 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 combustion, and in part with organic nutritive
bodies poor in salts or nearly salt-free, which are absorbed from the intes-
tinal canal and are thus retained (Voit, Forster^).
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 substances.
Experiments to determine the action of an insufficient supply of min-
eral substances with the food in animals have been made by several inves-
tigators, 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, particularly
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 ^ found on partaking less than 0. 1 gram
salts per diem that the chief disturbance occurred in the muscular system.
BuNGE in opposition to these observations of Forster's has suggested
that the early death in these cases was not caused by the lack of mineral
salts, but more likely by the lack of bases necessary to neutralize the
sulphuric acid formed in the combustion of the proteins in the organism;
these bases must then be taken from the tissues. In accordance with
^ See Voit in Hermann's Handbuch, 6, Part 1, 353.
^ 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 and Pathologie des
Mineralstoffwechsel, Berlin, 190G.
^ University of Cahfornia Publications, Pathol. 1.
736 METABOLISM.
this view,. Bunge and Lunin ^ also found, in experimenting with mice,
that animals which received nearly ash-free food with the addition of
sodium carbonate were kept alive twice as long as those which had the
same food without the sodium carbonate. Special experiments also show
that the carbonate cannot be replaced by an equivalent amount of sodium
chloride, and that to all appearances it acts by combining with the acids
formed in the body. The addition of alkali carbonate to the otherwise
nearly ash-free food may indeed delay death, but cannot prevent it, and
even in the presence of the necessary amount of bases death results for
lack of mineral substances in the food.
In the above series of experiments made by Bunge the food of the
animal consisted of casein, milk-fat, and cane-sugar. While milk alone
was an adequate and sufficient food for the animal, Bunge found that the
animal could not be kept alive longer by food consisting of the above con-
stituents of milk and cane-sugar with the addition of all the mineral sub-
stances of milk than with the food mentioned in the above experiments
with the addition of alkali carbonate. The question whether this result
is to be explained by the fact that the mineral bodies of milk are chem-
ically combined with the organic constituents of the same and can be
assimilated only in such combinations, or whether it depends on other
conditions, Bunge leaves undecided. These observations, however, show
how difficult it is to draw positive conclusions from experiments made
thus far with food poor in salts. Further investigations on this subject
seem to be necessary.
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 KCl, 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 in NaCl, which therefore must be taken in greater amounts
from the outside (Bunge). This occurs habitually in herbivora, and in
man with vegetable food rich in potash. For human beings, and especially
for the poorer classes of people who live chiefly on potatoes and foods
rich in potash, common salt is, under these circumstances, not only a
condiment, but a necessary addition to the food (Bunge 2). On the be-
havior of chlorides, especially sodium chloride, in the animal body as well
' Bunge, Lehrbuch der phybiol. Chem., 4. Aufl., 97; Lunin, Zeitschr. f. physiol.
Chem., 5.
^ Zeitschr. f. Biologic 9.
LACK OF MINERAL SUBSTANCES IN THE FOOD. 737
as the elimination or the retention of NaCl in diseases we have an abundance
of investigations, 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 towards litmus
and neutral towards phenolphthalein, is chiefly due to the presence of
alkali carbonates and carbon dioxide. The alkali carbonates are also of
great importance not only as a solvent for certain protein bodies and as
constituents of certain secretions, such as the pancreatic and intestinal
juices, but they are also a means of transportation of the carbon dioxide
in the blood. It is therefore easy to understand that a decrease below
a certain point in the quantity of alkali carbonate must endanger life.
Such a decrease not only occurs with lack of bases in the food which brings
about various disturbances and death by a relatively great production of
acids through the burning of the proteins, but it also occurs when an animal
is given dilute mineral acids for a period. The importance of ammonia as
a means of neutralizing the acids produced or introduced into the body
as well as the different resistance of man and other animals towards this
action of acids has already been discussed in Chapter XV.
Lack of Phosphates and Earths. With the exception of the importance
of the alkaline earths as carbonates and more especially as phosphates in
the physical composition of certain structures, such as the bones and teeth,
their physiological importance is nearly unknown. The importance of
calcium for certain enzymotic processes and also the great importance of
calcium ions for the functions of the muscles and especially for cell life
give an indication of the great importance of the alkaline earths for the
animal organism. Very little is known in regard to the need of these
earths in adults, and no average results can be given for this. The same
is true for the need of phosphates or phosphoric acid, whose great impor-
tance is recognized not only for the construction of the bones but also
for 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 phos-
phorus are introduced, to retain more than is necessary. The need of
phosphates is relatively smaller in adults than in young, developing ani-
mals, and in these latter the question of the action of insufficient supply
of earthy phosphates and alkaline earths upon the bone tissue is of special
interest. In regard to this question we refer to Chapter 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
738 METABOLISM.
what extent are they necessary? The experiments of Rohmann and his
pupils ' with phosphorized (casein, vitelhn) and non-phosphorized pro-
teins (edestin) and phosphates show that with the introduction of casein
and vitelhn 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 phos-
phorized cell constituents necessary for cell life from non-phosphorized
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 development of genera-
tive organs of the salmon which are very rich in nuclein substances and
phosphatides from the muscles which are relatively poor in organic-com-
bined phosphorus seem to indicate a synthesis of phosphorized organic
substance from the phosphates. Other investigators, such as v. Wendt,^
also admit of a synthesis of phosphorized protein substances by the aid
of inorganic phosphates.
Lack of Iron. As iron is an integral constituent of haemoglobin, abso-
lutely necessary for the introduction of oxygen, just so is it an indispen-
sable constituent of food. Iron is a never-failing constituent of the
nucleins and nucleoproteids, and herein lies also another reason for the
necessity of the introduction of iron. Iron is also of great importance for
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 statements on this ques-
tion 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,^ 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, I;Andergren,^ and others, the protein metabolism may
' See Marcuse, Pfliiger's Arch., 67, and footnote 2, page 719.
^Skand. Arch., f. Physiol. 17.
5 Zeitschr. f. Biologic, 32.
* Hirschfeld, Virchow's Arch., Ill; Kumagawa, ifnd., 116; Klemperer, Zeitschr.
f. khn. Med., 16; Sivon, Skand. Arch. f. Physiol., 10 and 11; Landergren, 1. c. 11; footnote
1, page 730. See also iMaly's Jahresber., 32. .
ARTIFICIAL MIXTURES. 739
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 1 kilo as carbohydrates and fat) on the fourth star-
vation 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 absence of fats and carbohydrates in the food affect 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 and car-
bohydrates.^ 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 (Pflucjer^). Human beings and herbivora, on
the contrary, cannot live for any length of time on such food. On one
side 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, arrived at somewhat better results;
and RoHMANN ^ has arrived at still more conclusive results. He used
mice in his experiments, and fed them with a mixture of casein, white of
egg, vitellin, potato-starch, wheat-starch, margarine, and salts. With
this diet the animals maintained their body weight and brought forth
young. These latter could not be raised on artificial food. A better
result was obtained by adding some malt to the food. It was also possible
to further raise with artificial food to maturity' mice which had been
formed and born with artificial food. These mice remained somewhat
smaller than the normal, and no living young could be obtained from
them. If we exclude the fact that the foodstuffs fed were not all simple
(white of egg, malt), pure foods it seems as if artificial mixtures of food
are sufficient to maintain at least an adult animal for a long time, while
it is not quite sufficient for the development of a young animal.
' See Horbaczewski, Maly's Jahresber., 31, 715.
' Pfliiger's Arch., 50.
^ Hall, Arch. f. (Anat. u.) Physiol., 1896; Steinitz, Uber Versuche mit kiinstlicher
Ernahrung, Inaug.-Diss., Breslau, 1900; Falta and Noeggerath, Hofmeister; Beitrage,
7; Rohmann, Klin, therap. Wochenschr., 1902, No. 40.
'40 METABOLISM.
IV. Metabolism with Various Foods.
For the 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-
sivel}' 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 their catabolism and the elimina-
tion of nitrogen is increased, and this in proportion to the suppl}- of pro-
teins.
If a certain quantity of meat has been given to carnivora as food daily
and the quantity is suddenly increased, an augmented catabolism of pro-
teins, or an increase in the quantity of nitrogen eliminated, is the result.
If the animal is fed daily for a certain time with larger quantities of the
same meat, a part of the proteins accumulates in the body, but this part
decreases from day to day. while there is a corresponding daily increase
in the elimination of nitrogen. In this way a nitrogenous equilibrium is
established; that is, the total quantity of nitrogen eliminated is equal to
the quantity of nitrogen in the absorbed proteins or meat. If, on the
contrary, an animal which is in nitrogenous equilibrium, having been fed
on large quantities of meat, is suddenly given a small quantity of meat per
clay, then the animal 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 nitrogenous equilibrium, or nearly into this condition. These rela-
tions are illustrated by the following table {Yoit ^) :
Grams of Meat in the Food per Day.
1
Before the Test.
500
During the Test.
1500
2
1500
1000
Grams of Flesh Metabolized in Body per Day.
1
1222
1153
1310 1390 1410 1440
1086 1088 1080 1027
6
1450
7
1500
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 meta-
» Hermann's Handbuch, 6, Part I, 110.
METABOLISM WITH FOOD RICH IX PROTEINS. 741
bolism of flesh on the first day of the experiment, with only 1000 grams
meat, decreased considerably, and on the fifth day nearly a nitrogenous
equilibrium was obtained. During this time the animal gave up daily
some of its own proteins. Between that point below which the animal
loses from its own weight and the maximum, which seems to be depend-
ent upon the digestive and assimilative capacity of the intestinal canal, a
carnivora may be kept in nitrogenous equilibrium with varying quantities
of proteins in the food.
The supply of proteins, as well as the protein condition of the body^
affects the extent of the protein metabolism. A body which has become
rich in proteins by a previous abundant meat diet must, to prevent a lo.ss
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 * has found in man but not, or at least not to the same extent, in
dogs, that quite great differences exist between the different proteins.
Thus on feeding pure proteins the chief amount of the nitrogen is much
quicker eliminated after feeding casein than after genuine ovalbumin.
This latter is much easier demolished after a previous denaturization by
coagulation than in the native state, which indicates that an unequal resist-
ance of the different proteins towards the digestive juices plays a part.
Even on feeding with easily decomposable proteins it takes always 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 rapidity with which the different proteins are decom-
posed it follows that in the passage from a diet poor in protein to one rich
in protein the time within which nitrogenous equilibrium occurs depends
chiefly upon the kind of protein contained in the food.
Pettenkofer and Voit have made investigations on the metabolism
of fat with an exclusively protein diet. These investigations have shown
that by increasing the quantity of proteins in the food the daily meta-
bolism of fat decreases, and they have drawn the conclusion from these
experiments, as detailed in Chapter X, that even a formation of fat may
take place under these circumstances. The objections presented by
PFLtJGER 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 Pfll-ger'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-nitrogenous bodies and hence the fat and fat-forming carbo-
hydrates are spared. If sufficient protein is introduced into the food to
satisfy the total nutritive requirements, then the decomposition of fat
1 Deutsch. Arch. f. klin. Med. 86.
742 METABOLISM.
stops; and if non-nitrogenous food is taken at the same time, this is not
consumed, but is stored up in the animal body, the fats as such, and the
carbohydrates at least in great part as fat.
Pfluger defines the " nutritive requirement " as the smallest quantity
of lean meat which produces nitrogenous equilibrium without causing any
decomposition of fat or carbohydrates. At rest and at an average tem-
perature it is found for dogs to be 2.073 to 2.099 grams of nitrogen ^ (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, PFLiJGER has found that the protein meta-
bolism 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 Pflxjoer's all of the excess of protein
introduced was not 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 supph', and that the animal body has the property,
within wide limits, of accommodating the protein catabolism to the pro-
tein supply.
These and certain other peculiarities of protein catabolism have led
YoiT to the view that all proteins in the body are not decomposed with
the same ease. "S'oit differentiates the protein fixed in the tissue-elements,
so-called organized proteins, tissue-protei^is, 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 are destro^'ed. These circulating 'proteins are, according to Voit,
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 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 have the power of taking proteins from the fluids washing
the tissues and appropriating them, while their own proteins, the tissue-
* See SchondorfF, Pfliiger's Arch., 71.
TISSUE AND CIRCULATIXG PROTEINS. 743
pr>;teins, 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 and their ability to decompose nutritive proteins is also increased
to a certain degree. When nitrogenous equilibrium is obtained after an
increased supply of proteins, it denotes 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 proteids may have time to
become fixed and organized by the tissues, and in this way the mass of
the flesh of the body increases. During starvation or with a lack of pro-
teins in the food the reverse takes place, for a part of the tissue proteins
is converted into circulating proteins which are metabolized, and in this
case the flesh of the body decreases.
Voit's theory has been criticised by several investigators and espe-
cially by PFLtJGER. Pfluger's statement, based on an investigation made
by one of his pupils, Schoxdorff,^ 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 is not
verv contradictory of ^'oit if the author does not misunderstand Pflij-
GER. YoiT ^ has, as is known, stated that the conditions for the destruc-
tion of substances in the body exist in the cells, and also that the circu-
lating protein, likewise according to Voit, is first catabolized after having
been taken up by the cells from the fluids washing them. The point of
Yoit's theory is that all proteins are not destroyed in the body with the
same degree of readiness. The organized protein, which is fixed by the
cells and has become a part of the same, is destroyed less readily, accord-
ing to Voit, than the protein taken up by the cells from the nutritive fluid,
which serves as material for the chemical construction of the very much
more complicated organized proteins. This nutritive protein, which cir-
culates 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 supply 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 asser-
tion can hardly, for the present, be considered as refuted or exactly
proved.
1 Pfliiger, Pfliiger's Arch., 54: Schondorff, ibid., 51.
^ Zeitschr. f. Biologic, 11.
744 METABOLISM.
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
nearly independent upon the quantity of portein taken as food, while the
quantity of urea is determined by the protein partaken of, speaks without
any doubt in favor of Voit 's view that we must differentiate between the
real cell protein and the food protein. This has also led Folin to differen-
tiate between endogenous and exogenous protein metabolism. The ex-
perience on protein feeding and the endeavor of the body, as observed
by ScHREUER,^ on going to an ordinary diet after abundant protein feeding,
to remain at the old state previous to the over feeding of protein, speak
also for 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 and Falck and others ^ on the transitory prog-
ress of the elimination of urea after a meal rich in proteins throws light
on this question. From experiments upon a dog it was found that the
elimination of urea increases almost immediately after a meal rich in pro-
teins, and that it reaches its maximum in about six hours, when about one
half of the quantity of nitrogen corresponding to the administered proteins
is eliminated. If we also recollect that, according to an experiment of
Schmidt-^Iltlheim ^ upon a dog, about 37 per cent of the given proteins
are absorbed in the first two hours after the meal and about 59 per cent
in the course of the first six hours, it may then be inferred that the in-
creased elimination of nitrogen after a meal is due to a catabolization of
the digested and assimilated proteins of the food not previously organized.
If it is admitted that the catabolized protein must have been organized^
then the greatly increased elimination of nitrogen after a meal rich in
proteins supposes a far more rapid and 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-
tein is in great part due to a progressive demolition of the food protein in
* Folin, Amer. Journ. of Physiol., 13; Schreuer, Pfliiger's Arch., 110.
^ Panum, Nord. Med. Arkiv., 6; Falck, see Hermann's Handbuch, 6, Part I, 107.
For further statements in regard to the curve of nitrogen elimination in man, see Tschen-
loff, Korrespond. Blatt Schweiz. Aerzte, 1896; Rosemann, Pfliiger's Arch., 65, and
Veraguth, Journ. of Physiol., 21; Schlos.se, Maly's Jahre.sber., 31.
2 Arch. f. (Anat. u.) Phy.siol., 1879.
NUTRITIVE VALUE OF GELATINE. 745
digestion whereby certain atomic complexes are more readily split than
others. The abundant elimination of nitrogen by the urine after par-
taking considerable protein may also depend in great part upon these
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
after food rich in proteins, as observed by Nencki and Zaleski * seem to
speak in favor of this view.
In this connection it must be recalled that, according to the investi-
gations of RiAZANTSEFF, Substantiated by Schepski, after partaking of
food an increased nitrogen elimination depends in part upon the increased
work of the digestive glands. The observations of Riazantseff ^ that
after so-called "apparent feeding" an increased elimination of nitrogen
occurs has not been confirmed by the recent observations of Cohxheim
and therefore cannot be considered as conclusive.
It has been stated above that other foods may decrease the catabolism
of proteins. Gelatine is such a food. Gelatine and the gelatine- formers do
not seem to be converted into protein in the body, and this last cannot
be entirely replaced by gelatine in the food. For example, if a dog is fed
on gelatine and fat, its body sustains a loss of proteins even when the
quantity of gelatine is so large that the animal, with an amount of fat
and meat containing just the same quantity of nitrogen as the gelatine in
question, may remain in nitrogenous equilibrium. On the other hand,
gelatine, as Voit, Panum, and Oerum^ have shown, has a 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 Yoit's experiments upon a dog:
Food per Day. Flesh.
Meat. Gelatine. Fat. Sugar. Catabolized. Ou the Body.
400 0 200 0 450 -50
400 0 0 250 439 -39
400 200 0 0 356 +44
I. MuNK * has later arrived at similar results by means of more deci-
sive experiments. He found in dogs that on a mixed diet which con-
tained 3.7 grams protein per kilo of body, of which hardly 3.6 grams was
catabolized, nearly f could be replaced by gelatine. The same dog cata-
* Arch, des scienc. biol. de St. Petersbourg 4; Salaskin, Zeitschr. f. physiol. Chem.
25; Nencki and Zaleski, Arch. f. exp. Path. u. Pharm. 37.
^Arch. des scienc. biol. de St. Petersbourg, 4, 393; Schepski, Maly's Jahresber., 30;
Cohnheim, Zeitschr. f. physiol., Chem. 46.
' Voit, 1. c, 123; Panum and Oerum, Nord. Med. Arkiv., 11.
*Pfluger's Arch., 58.
746 METABOLISM.
bolized on the second day of starvation three times as much protein as
with the gelatine feeding. Munk states also that gelatine has a much
greater sparing action on proteins than the fats or the carbohydrates.
This ability of gelatine to spare the proteins is explained by Voit by
the fact that the gelatine 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 gelatine upon proteins. The extent of
protein destruction during gelatine 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 gelatine. The physiological availability of gelatine 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 gelatine. Kaufmann,^
who experimented upon dogs, found that ^ of the protein nitrogen could
be readily replaced by gelatine nitrogen, while in an experiment upon
himself with 93 per cent gelatine 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 gelatine replaced by amino acids had nearly the same
physiological value as the proteins.
Gelatine may also decrease somewhat the consumption of fat, although
it is of less value in this respect than the carbohydrates.
The question of the nutritive value of proteoses (and peptones) stands
in close relationship to the nutritive. value of the proteins and gelatine.
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 nitrogenous 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. According to Pollitzer this is true for differ-
ent proteoses as well as for true peptone; but this does not correspond
with the experience of Ellinger,- who finds that the true antipeptone
(gland peptone) is not able to entirely replace proteins or to prevent the
loss of protein in the animal body. On the contrary, according to him, it
' Knimmacher, Zeitschr. f. Biologic, 42; Kirchmann, ibid., 40; Kaufmann, Pfliiger's
Arch., 109.
2 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. Biologic, 33 (literature).
NUTRITIVE VALUE OF PROTEOSES AND PEPTOXES. 747
has, like gelatine, the property of sparing proteins. Voix long ago ex-
pressed a similar view. According to him the proteoses and peptone may
indeed replace the proteins for a short time, but not permanently; 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 re-
place the proteins of the food, while casein protoproteose had this property.
The question as to the nutritive value of proteoses and peptones has
turned in a new direction, due to the more recent views, as mentioned in
Chapter IX. on the absorption of proteins where the proteins are not ab-
sorbed chiefly as proteoses and peptones, but as simpler cleavage products.
From these simple products as mentioned in a previous chapter (IX on
absorption) a synthesis of protein can take place in the body. Even if
such a synthesis takes place and if it were possible to nourish the body for
a long time with a mixture of digestion products still it does not follow
that proteoses and peptones can completely replace the proteins of the food.
The proteoses and peptones are formed by cleagages. and perhaps certain
atomic complexes are absent which occur in the mixture of cleavage pro-
ducts and which are necessary for a regeneration of special protein bodies.
We have a number of investigations- upon the value of asparagin. and
the results are still not conclusive so that quite positive deductions can be
drawn from them. The experiments upon herbivora seem to indicate that
the asparagin has hardly any action upon the deposition of protein while
it can have an indirect protein sparing action and may serve in producing
temperature. The protein sparing action seems, at least in part, to be
explained by its excelerating action upon digestion. In carnivora (I.
Muxk) and in mice (Voix and Politis) it was found that asparagin has
onh" a very slight, if any. sparing action on the proteins. It is not known
how it acts in man.
Metabolisin on a Diet consisting of Protein, with Fat or Carbohydrates.
Fat cannot arrest or prevent the catabolism of proteifis: 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.
, « » ' .
Meat. Fat. Metabolized. On the Boay.
A 1500 0 1512 -12
B 1500 150 1474 -^26
» Zeitschr. f. physiol. Chem., 30: Voit. I. c. 394.
- Weiske, Zeitschr. f. Biologie. 15 and 17. and Centralbl. f. d. med. Wissensch., 1890,
945; Munk, Virchow's Arch.. 94 and 9S: Politis, Zeitschr. f. Biologie. 2S. See also
Mauthner. ibid., 2S: Gabriel, ibid.. 29: and Voit. ibid.. 29. 125: Kellner. Maly's Jahresber,
27. and Zeitschr. f. Biologie. 39: Kellner and Kohler, Chem. Centralbl. 1, 1906. Vsltz
Pfliiger's Arch. 107: v. Strusiewicz, Zeitschr. f. Biologie 47.
^ Voit in Hermann's Handbuch 6, 130.
748 METABOLISM.
According to Voir 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 equi-
librium, but may even add to the store of body proteins, ^Yhile 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 equi-
librium.
Like the fats the carbohydrates have a sparing action on the proteins.
By the addition of carbohydrates to the food the carnivora not only re-
mains 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: ^
Fooil. Flesh.
Meat.
Fat.
Sugar.
starch.
Metabolized.
On the Body.
500
250
558
- 58
500
300
466
+ 34
500
200
505
- 5
800
250
745
+ 55
800
200
773
+ 27
2000
200-300
1792
+ 208
2000
250
1883
+ 117
The sparing of protein by carbohydrates is greater, as shown by the
table, than by fats. According to Voit the first is on an average 0 per
cent and the other 7 per cent of the administered protein without a previ-
ous addition of non-nitrogenous bodies. Increasing quantities of carbo-
hydrates in the food decrease the protein metabolism more regularly and
constantly than increasing quantities of fat. Atwater and Benedict ^
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 to
that of the fats occurs, as shown by Laxdergren,^ to a still higher degree
with food poor in nitrogen or in nitrogen starvation, in which cases the
' Voit, ibid., page 143.
' See Ergebni.sse der Phy.siologie 3.
3 L. c, Inaug.-Diss., and Skand. Arcli. f. Physiol., 14.
SPARING ACTION OF CARBOHYDRATES AND FATS. 749
carbohydrates have double the protein-sparing action as compared to an
isodynamic quantity of fat.
The protein-sparing action of the carbohydrates and fats has generally
been studied by the one-sided feeding with one or the other of these two
groups of foodstuffs. The question may be raised whether the difference
observed between the fats and carbohydrates could not be brought about
also by the simultaneous supply of carbohydrates and fat in varying pro-
portions. Tallquist ^ has made a series of experiments on this subject.
In one of the periods 16.27 grams N, 44 grams fat, and 466 grams carbo-
hydrate were given; in a second. 16.08 grams X, 140 grams fat, and 250
grams carbohydrate, containing nearly the same number of calories,
namely, 2867 and 2873 calories. In both cases nearly a complete nitro-
genous 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 carbohydrate
when the quantity of carbohydrates does not sink below a certain mini-
mum, which is not known for the present.
This condition as well as the extent of protein-sparing action of the
carbohydrates stands, according to Laxdergrex,^ 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 proteins being
used in the sugar formation. This part can be spared by carbohydrates
but not by fats, from which, according to Laxdergrex, the carbohydrates
cannot be formed. In this lies also the probable reason why the fats, on
being fed exclusively but not with a sufficient supply of carbohydrates,
have a much lower protein-sparing action than the carbohydrates. The
fats cannot prevent the protein catabolism necessary for the formation of
sugar on a diet lacking in carbohydrates.
The law as to the increased protein catabolism with increased protein
supply applies also to food consisting of protein with fat and carbohydrates.
In these cases the body tries to adapt its protein catabolism to the supply;
and when the daily calorie-supply is completely covered by the food, the
organism can. within wide limits, be in nitrogenous equilibrium with dif-
ferent quantities of protein.
The upper limit to the possible protein catabolism per kilo and per day
has only been determined for herbivora. For human beings it is not
known, and its determination is from a practical standpoint of secondary
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
^ Finska Lakaresallskapets handl., 1901. See also Arch. f. Hygiene, 41.
*L. c, Inaug.-Diss. See also Skand. Arch. f. Physiol., 14.
750 METABOLISM.
dogs by HiRSCHFELD, KuMAGAWA, Klemperer, Munk, Rosenheim,* and
others. It follows from these experiments that the lower limit of protein
needed for human beings for a week or less is about 30-40 grams or 0.4-
0.6 gram per kilo with a body of average weight, v. Noorden ^ considers
0.6 gram protein (absorbed protein) per kilo and per day as the lower
limit. The above-mentioned figures are only valid for short series of ex-
periments; still there exist the observations of E. Voit and Constantinidi ^
on the diet of a vegetarian when the protein condition was kept nearly
normal but not completely for a long time with about 0.6 gram of protein
per kilo. Caspari * has also made observations upon a vegetarian and for
a period of 14 days with an average of 0.1 gram nitrogen (recalculated as
equal to 0.62 gram protein) per kilo where a nearly complete 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 working man 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 protein
the demand for calories was considerably greater; as, for instance, in cer-
tain cases it was 51 (Kumagawa) or even 78.5 calories (Klemperer). It
therefore seems as if the above very low supply of protein was only possible
with great waste of non-nitrogenous food; but in opposition to this it must
be recalled that in Voit and Constantinidi 's experiments upon the veg-
etarian, who for years was accustomed to a food very poor in protein and
rich in carbohydrate, the calories amounted to only 43.7 per kilo. In the case
studied by Caspari a supply of 41 calories per kilo was entirely sufficient.
SivEN has shown by experiments upon himself that the adult human
organism, at least for a short time, can be maintained in nitrogenous equi-
librium with a specially low supply of nitrogen without increasing the calo-
ries 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 only holds
for a short time, has no general validity follows from other observations.
Thus Caspari ^ also, in an experiment on himself, could not attain com-
1 See footnote 4, psge 738; also Munk, Arch. f. (Anat. u.) Physiol., 1891 and 1S96;
Rosenheim, ibul., 1891; Pfliiger's .\rch., 54.
' Grundriss einer Methodik der Stoffwechseluntersuchungen. Berlin. 1892.
^ Zeitschr. f. Biologie, 25.
'' Physiologische Studien liber Vegetarismus, Bonn, 1905.
^Siven, Skand. Arch. f. Physiol., 10 and 11; Caspari, Arch. f. (Anat. u.) PhysioU
1901.
DKPOSITIOX OF FLESH.
751
plete nitrogenous equilibrium on a much greater nitrogen supply. The
protein minimum seems also to be different for various individuals.
The very important question as to the conditions favoring the depo-
sition of fat and flesh in the body is closely associated with \Yhat has just
been said in regard to foods consisting of protein and non-nitrogenous
foodstuffs. In this connection it must be remembered in the first place
that all fattening presupposes an overfeeding, i.e., a supply of foodstuffs
which is greater than that catabolized in the same time.
In carnivora a flesh deposition may take place on the exclusive feeding
with meat. This is not generally large in proportion to the quantity of
protein catabolized. As shown by an experiment upon a male cat by
Pfluger ^ this may be so great that the body doubles in weight under
favorable conditions. In man and herbivora, on the contrary, the demand
for calories may not be covered by protein alone, 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 relationship 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 consider-
able addition of fat. then nitrogenous equilibrium is only 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 b}' day, and nitrogenous equi-
librium 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 ^'oIT may serve as example:
Number of
Days of Ex-
perimentation.
Food.
Total
Deposit of
Flesh.
Daily
Deposit of
Flesh.
Nitrogenous
Meat, Grams.
Fat, Grams.
Equilibrium.
32
7
500
1800
250
250
1792
854
56
122
Not attained
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
* PfliigeV's Arc-h., 7".
752 METABOLISM.
established after seven days; and though the deposition of fiesh per day
was greater, still the absolute deposit was not one half as great as in the
former case.
The experiments of Krug upon himself, under the directio.n of v.
NooRDEN, give us information as to the practicability of flesh deposition
in man. With abundant food (2590 cal. =44 cal. per kilo) Krug was
close to nitrogenous equilibrium for six days. He then increased the
nutritive supply to 4300 cal. =71 cal. per kilo for fifteen days by the addi-
tion of fat and carbohydrate, and in this time 309 grams of protein, corre-
sponding to 1455 grams of muscle, was spared. Of the excess of admin-
istered calories in this case only 5 per cent was used for flesh deposit and
95 per cent for fat deposit. On the other hand Bornstein,' also experi-
menting upon himself, without any considerable increase in calories, could
produce, an increase in his protein condition by about 100 grams of
protein, corresponding to 500 grams of flesh, in the course of fourteen days
simply by increasing the supply of protein (50 grams of nutrose = sodium
casein with 7 grams N per day).
BoRNSTEiN arrived at still better results in regard to protein retention
by simultaneous muscle work, as in these cases the nitrogen retention
corresponded to a flesh deposit of 800 grams. The importance of work
for the so-called protein deposition follows also from many other obser-
vations, and it is in agreement with daily experience that a man cannot
be made muscle-strong by over-feeding alone. A work-hypertrophy
must also be introduced.
BoRNSTEiN and Schreuer ^ have given further proof for the possi-
bility of a protein deposition in man and animals (dogs) and there is no
doubt that the body becomes richer in active cell masses after abundant
supply of protein. This increase seems still, according to Schreuer, not
to be continuous, and the question to what extent the nitrogen retention
in so-called protein overfeeding in full-grown animals and man is to be
considered as a true flesh enrichment i.e., a new formation of living tissue,
seems to require further proof.
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. The growing body of the nursling
also uses according to Rubner and Heubner,^ the protein of the food
' Krug, cited from v. Noorden, Lehrbuch der Pathologie des Stoffwechsel., 2 Aufl.
557; Bomstein Berl. klin. Wochenschr., 1898, and Pfluger's Arch,. 83 and 106.
'Pfluger's Arch., 110.
^ Zeitschr. f. exp. Path. u. Therap. 1.
ACTION OF WATER, SALTS, ETC., UPOX METABOLISM. 753
essentially to replace the quantity of protein catabolized and for deposi-
tion.
It is difficult to produce a permanent flesh deposit in man by overfeed-
ing alone. Flesh deposition is, according to v. Xoordex, a function of
the specific energy of the developing cells and the cell-work to a much
higher extent than the excess of food. Therefore there is observed,
according to v. Xoordex, 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 the same. The deposition of flesh is in this case
an expression of the regenerative energy of the cells. ^
The experiences of graziers show that in food-animals a flesh deposit
does not occur, or at least is only inconsiderable, on overfeeding. The
individualit}' and the race of the animal are of importance for flesh depo-
sition.
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 deter-
mined by the excess of calories administered over those actually needed.
If a large part of the calorie-demand is covered by protein, then a greater
part of the non-nitrogenous foodstuffs simultaneously ingested is spared, i.e.,
used for fat deposition. But as protein and fat are expensive nutritive
bodies as compared with carbohydrates, the supply of greater quan-
tities of carbohydrates is important for fat deposition. The body decom-
poses less substance 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 quantity
in excess of that which is necessary is introduced into the organism, the
excess is quickly and principally eliminated with the urine. This in-
creased elimination of urine causes in fasting animals (Voit, Forster),
but not to any appreciable degree in animals taking food (Seegex, Sal-
KOW'SKi and Muxk, ^Iayer, Dubelir ^), an increased elimination of nitro-
gen. 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,
' See also Svenson, Zeitschr. f. kiln. Med., 43.
^ Voit. Untersuch, fiber den Einfluss des Koch.salzes, etc. (Munchen, 1860); Forster,
cited from Voit in Hennann's Handbuch, 6, 153; Seegen, Wien. Sitzung.sber., 63; Sal,
ko-n-ski and Munk, 'S'irchow's Arch., 71-; Mayer, Zeitschr. f. klin. Med., 2; Dubelir-
Zeitschr. f. Biologie, 28.
75 1 METABOLISM.
is that because of the more active current of fluids, after taking large
quantities of water an increased metabolism of proteins takes place. Yoit
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. The views on this question are
still somewhat contradictory.^
When the body has lost a certain amount of water, then the abstinence
from water (in animals) is accompanied by a rise in the protein metabo-
lism (Landauer, Straub-). In regard to the action of water on the
fomiation of fat and its metabolism, the view 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.
Salts. The statements are somewhat contradictory 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. Recent investigations of Straub and Rost ^ have shown
that the action of salts stands in close relationship to their power of
abstracting water. Small amounts of salt which do not produce diuresis
have no action on metabolism. On the contrary, larger amounts which
bring about a diuresis which is not compensated by the ingestion of water,
produce a rise in the 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 somewhat increased by the ingestion of
water and thus increasing the diuresis, and the action of salts seems to
bear a close relationship to the demand and supply of water.
Alcohol. The question as to how far the alcohol absorbed in the intes-
tinal 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,'Bexe-
DicENTi *). As the alcohol has a high calorific value (1 gram = 7 calories),
then the question arises whether it acts sparingl}- on other bodies, and
whether it is to be considered as a nutritive substance. The older inves-
tigations made to decide this question have led to no decisive result. The
thorough investigations of Atwater and Benedict, Zuntz and Geppert,
•See R. Neumann, Arch. f. Hygiene, 36; Heilner, Zeitschr. f. Biologie, -1:7; Hawk,
University of Pennsylvania Med. Bull., xviii.
^ Landauer, Maly's Jahresber., 24; Straub, Zeitschr. f. Biologie, 37,
^W. Straub, Zeitschr. f. Biologie, 37 and 38; Rost, Arbeiten aus d. Kaiserliche
Gesundheitsamte, 18 (literature). See also Griiber, Maly's Jahresber., 30, 612.
*Arch. f. (Anat. u.) Physiol., 1896, which contains the literature.
INFLUENCE OF WEIGHT OF BODY AND AGE. 755
Bjerre, Clopatt, Neumann, Offer, Rosemann,^ and others, seem to
show positively that in man alcohol can diminish the consumption not
only of fat and carbohydrates, but also the proteins, although at first;,
due to its poisonous properties, it may increase the protein metabolismi
for a short time. The nutritive value of alcohol can only be of speciaP.
importance in certain cases, as large amounts of alcohol taken at one time,,
or the continued use of smaller quantities, has an injurious action on the-
organism. Alcohol may therefore be regarded as a. foodstuff only in
exceptional cases, and in other respects must be considered as an article^
of luxury.
Coffee and tea have no action on the exchange of material which can bfr
positively proved, 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.
V. The Dependence of Metabolism on Other Conditions.
The so-called starvation requirement which was previously mentioned,,
i.e., the extent of metabolism with absolute rest of body and in activity 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-,
starvation requirement belongs to the circulation and respiration work.
The magnitude of the starvation 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 relationship 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 metabolizes
absolutely less, but relatively more as compared with the unit of the
weight of the body. It must be remarked that the relation between flesh
and fat in the body exerts an important influence. The extent of th&
metabolism is dependent upon the quantity of active cells, and a very fat
* 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 hterature upon alcohol can be formed in Abderhalden, "Bibliographie der ges-
amten wissenschaftlichen Literature iiber den Alcohol und den Alcoholismus," Berlio
and Wien, 1904.
' Cited from v. Noorden's Handbuch. 2 Aufl.
756 METABOLISM.
individual therefore decomposes less substance per kilo than a lean person
of the same weight. According to Rubner ' the importance of the size
of the flesh or cell-mass in the body is overestimated. In his investiga-
tions 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. B}' approximately esti-
mating the quantity of fat in the body Rubner was also able, from the
protein condition, to compare the calculated exchange of energy with
that actually found. The exchange per kilo amounted to 52 calories in
the lean and 43.6 cal. in the fat boy, while, if the protein condition was a
measure, one would expect an exchange of calories of only 35 cal. for the
fat person. We cannot 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 metabolism in general is smaller, and the latter is ordi-
narily about four fifths that of men.
The question as to what extent gender 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 essential reason wh}' 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 meta-
bolism than older ones. If the heat production and carbon-dioxide
elimination is calculated on the unit of surface of the body, we find, on
the contrary, as the experiments of Rubner, Richet,^ and others show,
' Beitrage zur Ernahrung im Knabenalter, etc. Berlin, 1902.
2 Skand. Arch. f. Physiol, 6.
^ In regard to metabolism and its relationship to the phases of sexual life and esi>e-
cially under the influence of menstruation and pregnancy, see the investigations of A.
Ver Eecke (Bull. acad. roy. de m6d. de Belgique, 1897 and 1901, and Maly's Jahresber.,
SO and 31).
* Rubner, Zeitschr. f. Biologic, 19 and 21; Richet, Arch, de Physiol, 5. (2).
INFLUENCE OF AGE. 757
that they vary only sHghtly from a certain average in individuals of differ-
ent 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 not only applies to adult human beings
but also to children and growing individuals (Rubner, Oppenheimkr).
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,' 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 ^ found by many investigators,
which is sometimes not very small, speaks for 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 for
metabolism.
The more active metabolism in young individuals is apparent when
we measure the gaseous exchange as well as the excretion of nitrogen.
As example of the elimination of urea in children the following results of
Camerer ^ are of value:
Age. Weight of Body in Kilos, p^,. ]^lf '" ^'^'f^; Kiio.
l^years 10.80 12.10 1.35
3 " 13.30 11.10 0.90
5 " 16.20 12.37 0.76
7 " 18.80 14.05 0.75
9 " 25.10 17.27 0.69
12h " 32.60 17.79 0.54
15 " 35.70 17.78 0.50
In adults weighing about 70 kilos, from 30 to 35 grams of urea per day
are eliminated, or 0.5 gram per kilo. At about fifteen years of age the
destruction of proteins per kilo is about the same as in adults. The rela-
tively greater metabolism of proteins in young individuals is explained
partly by the fact that the metabolism of material in general is more active
* Rubner, Ernahrung im Knabenalter, page 45; E. Voit, Zeitschr. f. Biologie, 41 ;
Oppenheimer, ibid., 42.
^ See Magnus-Levy, Pfliiger's Arch., 55; Slowtzoff (u. Zuntz), ibid., 95.
^ Zeitschr. f. Biologie, 16 and 20.
758 METABOLISM.
In young animals, and partly by the fact that young animals are, as a rule,
.poorer in fat than those full grown.
According to Tigerstedt and Sonden the greater metabolism in young
animals depends nevertheless also in part on the fact that in these indi-
viduals 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
H^i 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 devia-
ttion from the above rule; still these differences may, according to Rubner,
^e dependent upon the work performed, the food, and the nutritive condi-
tion. Magnus-Levy and Falk ^ have reported observations which sup-
port the views of Sonden and Tigerstedt.
In old age the metabolism is very much reduced; and even when calcu-
lated upon the square meter of surface of body it is lower than in an indi-
vidual of medium age.
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 metabo-
lism.
Rest and Work. During work a greater quantity of chemical energy is
^converted into kinetic energy, i.e., the metabolism is increased more or
Hess on account of work.
As explained in a previous chapter (XI), work, according to the gener-
ially accepted view, has no material influence on the excretion of nitrogen.
It is nevertheless true that several investigators have observed in certain
''Cases an increased elimination of nitrogen; but these observations have
been explained in other ways. For instance, work may, when it is con-
nected with violent movements of the body, easily cause dyspnoea, and
"this last, as Frankel^ has shown, may occasion an increase in the elimi-
nation of nitrogen, since diminution of the oxygen supply increases the
protein metabolism. In other series of experiments the quantity of car-
■bohydrates and fats in the food was not sufficient; the supply of fat in the
body Avas decreased thereby, and the destruction of proteins was corre-
spondingly increased. Other conditions, such as the external temperature
and the weather,^ thirst, and drinking of water, can also influence the
excretion of nitrogen. According to the generally accepted views muscu-
lar activity has hardly any influence on the metabolism of proteins.
' Tigerstedt and Sonden, 1. c. ; Rubner, 1. c. ; Magnus-Levy, Arch. f. (Anat. u.) Physiol.,
1899, Suppl.
* Virchow's Arch., 67 and 7L
■*See Zuntz and Schumburg, Arch. f. (Anat. u.) Physiol., 1895.
li\FLUEXCE OF REST AND WORK. 759^
On the contrary, work has a very considerable influence on the elimina-
tion of carbon dioxide and the consumption of ox3^gen. This action,
which was first observed by Lavoisier, has later been confirmed by
many investigators. Pettenkofer and Voit ^ have made investigations
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:
Consamption of
Proteins. Fat. Carbohydrates. COj Eliminated. O Consumed.
^ ,. (Rest 79 209 ... 716 761
tasting ... •j^Qj.j. .,5 3gQ jjg^ jQ_^
,,. , .. , J Rest 1.37 72 352 912 8-31
Mixed diet ^ ^Qj.1. ^37 ^73 3-0 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 pupils ^ have made very 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 important
influence of muscular work on the catabolism of material, but they
also indicate in a very instructive way the relationship between the
extent of metabolism of material and useful work of various kinds.
We can only refer to these important investigations 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 ^ has also made experiments upon himself, and
finds that on the production of as complete a muscular inactivity as pos-
sible 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.
* Zeitschr. f. Biologie, 2.
^See the works of Zuntz and Lehmann, Maly's Jahresber., 19; Katzenstein, Pfliiger's
Arch., 49; Loewy, ibid.; Zuntz, ibid., 68, and especially the large work "Untersuch
uber den Stoffwechsel des Pferdes bei Ruhe und Arbeit," Zuntz and Hagemann, Berlin
1898, which also contains a bibliography. Zuntz and Slowtzoff, Pfliiger's Arch., 95 ;
Zuntz, ibid.
^ Nord. Med. Arkiv. Festband, 1897; also Maly's Jahresber., 27; Speck, "Physiol,
des menschl. Atmens," Leipzig, 1892. ■
760 METABOLISM.
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 Loewy ^ the
combustion processes in the animal body go on in the same way in work
as in rest, and a raising of the respiratory quotient (irrespective of the
transient change in the respiratory mechanism) takes place only with
insufficient supply of ox^-gen 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 Kat-
zenstein by the statement that during work two kinds of chemical pro-
cesses act side by side. The one depends upon the work which is con-
nected with the production of carbon dioxide also in the absence of free
oxygen, while the other brings about the regeneration which takes place
by the taking up of oxygen. When these two chief kinds of chemical
processes make the same progress the respiratory quotient remains un-
changed 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 per-
formed in a way so that irregularities and occasional changes in the circu-
lation 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 thereby in the first place determined by the nutri-
tive 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^' He has observed the reverse, namely, a diminution in the respira-
tory quotient during continuous excessive work, and this is not reconcilable with
the above statements. According to Laulanik, who considers sugar as the source
of muscular energy, the rise in the respiratory quotient is due to an increased
combustion of sugar. The diminution of the same he explains by a re-formation
of sugar from fat which takes place at the same time and is accompanied by an
increased consumption of oxygen.
' See footnote 2, page 7.59.
^ Pfliiger's Arch., 49.
3 Arch, do Physiol. (.-)), 8, 572.
INFLUENCE OF EXTERNAL TEMPERATURE. 761
In sleep metabolism decreases as compared with that during waking,
and the most essential reason for this is the muscular inactivity during
sleep. The investigations of Rubner upon a dog, and of Johansson ^
upon human beings, teach us that if the muscular work is eliminated the
metabolism during waking 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 proved that metabolism
is increased under the influence of light. Most investigators, such as
Speck, Loeb, and Ewald,- consider that this increase is due to the move-
ments caused by the light or an increased muscle tonus. Fubini and
Benidicenti ^ assume that the increase in metabolism due to light is inde-
pendent of the movements. They base this assumption on experiments
made on hibernating animals.
Mental activity does not seem to have any influence on metabolism
according to the means at hand for studying this influence.
Action of the External Temperature. In cold-blooded animals the pro-
duction of carbon dioxide increases and decreases with the rise and fall of
the surrounding temperature. In warm-blooded animals this condition is
different. By the investigations of Ludwig and Sanders-Ezn, Pflijger
and his pupils, and Duke Charles Theodore of Bavaria and others^ it
has been demonstrated that in warm-blooded animals the change in the
external temperature has different results according as the animal's own
heat remains the same or changes. If the temperature of the animal sinks,
the elimination of carbon dioxide decreases; if the temperature rises, the
elimination of COj increases. If, on the contrary, the temperature of the
body remains unchanged, then the elimination of carbon dioxide increases
with a lower and decreases with a higher external temperature. The
statements on this subject are somewhat disputed and cases have been,
observed ^^•here in warm-blooded animals the merabolism rises on cooling
and lowering the body temperature, while warming and raising the body
temperature produces a diminution (Krarup ^).
The increase in metabolism produced by a lowering of the external
temperature is explained, according to Pfluger and Zuntz, by the state-
1 Rubner, Ludwig-Festschr., 1887; Loewy, Berl. klin. Wochenschr. , 1891, 434;
Johansson, Skand, Arch. f. Pliysiol., 8.
* Speck, 1. c; Loeb, Pfliiger's Arch, 42; Evvald, Journ. of Physiol., 13.
' Cited from Maly's Jahresber., 22, 395.
* The pertinent Hterature may be found cited by Voit in Hermann's Handbueh, 6,
and also by Speck, 1. c.
' J. C. Krarup, Den omgifvende temperaturs indflydeke, etc., Inaug.-Diss. Kjoben-
havn, 1902. See also Falloise, Maly's Jahresber., 31; Predteschcnsky, ibid; Rubner,
Arch. f. Hygiene, 38.
762 METABOLISM.
nient that the low temperature, by exciting a reflex action on the sensitive
nerves of the skin, causes an increased metabolism in the muscles with an
increased production of heat, affecting the temperature of the body, while
Avith a higher external temperature the reverse takes place. The experi-
ments made upon animals are somewhat uncertain for several reasons, but
the determinations of the oxygen absorption, as well as the elimination of
CO,, made by Speck, Loewy, and Johansson ^ in human beings, have
shown that cold does not produce any essential increase in the metabolism
of man. The irritation caused by cold may reflexly cause a forced respi-
ration with an action on the gas oxchange, and weak reflex muscular
movements, such as shivering, trembling, etc., may cause an insignificant
increase in the elimination of carbon dioxide; in complete muscularinac-
tivity cold seems to cause no increased absorption of oxygen or increased
metabolism. Eykman's^ experiments upon inhabitants of the tropics
also show the same result, namely, that in human beings no appreciable
heat regulation occurs.
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. ZuxTz, A. Loewy, F. Muller and W. Caspari.^
^Metabolism is increased by the ingestion of food, and Zuntz has calcu-
lated that in man the consumption of oxygen is raised on an average 15
per cent above the amount during rest for about six hours after taking
a moderately hearty meal. This increase in the metabolism is caused,
according to the generally accepted view, probably only by the increased
work of the digestive apparatus on the partaking of food. Rjasantseff
has shown that the extent of nitrogen elimination is proportioned to the
intensity of the digestive work. It also follows from the works of i\L\GNUS-
Levy, Koraen and Johansson * that the proteins and to a lesser extent
the carbohydrates even by themselves produce a rise in metabolism which
does not seem to be true for the fats.
• Speck, 1. c; Loewy, Pfluger's Arch., 46; Johansson, Skand. Arch, f. Physiol., 7.
' Virchow's Arch., 133, and Pfluger's Arch., 64.
5 "Hohenklima und Bei^wanderungen in ihrer Wirkung auf den Menschen," Berlin,
1906.
* Zuntz and Levy, "Beitrag zur Kenntniss d. Verdaulichkeit, etc. ,des Erodes,"
Pfluger's Arch., 49; Magnus-Levy, ibid., 55; Koraen, Skand. Arch. f. Physiol., 11,
Johansson and Koraen, ibid., 13.
FOOD REQUIREMENT BY MAX. 763
VI. The Necessity of Food by Alan 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 indi-
viduals— soldiers, sailors, laborers, etc. — the average quantity of food-
stuffs required per head. Others have calculated the daily demand of
food from the quantity of carbon and nitrogen in the excreta or calculated
it from the exchange of force of the person 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 during a period of
several daj's the organic foodstuffs consumed daily by persons of various
occupations who chose their own food, b}^ 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 num-
ber 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 faeces. The gross results of calo-
ries calculated from the food taken must therefore, according to Rubxer,
be diminished by at least 8 per cent. This figure is true at least when the
human being partakes of a mixed diet of about 60 per cent of the proteins
as animal and about 40 per cent of the proteins as vegetable foodstuffs.
With more one-sided vegetable food, especially when this is rich in undi-
gestible 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 liv-
ing force which corresponds to the quantity of food in question, calculated
as calories, with the above-stated correction. The calories are therefore
net results, while the figures for the nutritive bodies are gross results.
764
METABOLISM.
Proteins. Fat. ^|rat*S^' Calories. Authority.
Soldier during peace ... . 119 40 529 2784 Playfair.*
" light service 117 35 447 2424 Hildesheim.
" in field 146 46 504 2852
Laborer 130 40 550 2903 Moleschott.
Laborer at rest 137 72 352 2458 Pettenkofer and VoiT.
Cabinetmaker (40 years). 131 68 494 2835 Forster.^
Young physician 127 89 362 2602
134 102 292 2476
Laborer (36 years) 133 95 422 2902
English smith 176 71 666 3780 Playfair.
pugihst 288 88 93 2189
Bavarian wood-chopper . 135 208 876 5589 Liebig.
Laborer in Silesia 80 16 552 2518 Meinert.^
Seamstress in London .. . 54 29 292 1688 Playfair.
Swedish laborer 134 79 485 3019 Hultgren and Landergren.*
Japanese student 83 14 622 2779 Eijkman.^
Japanese shopman 55 .6 394 1744 Tawara.^
We have a very large number of complete investigations upon the diet
of people of different vocations in America but they are too extensive to
enter into, hence we must refer to the original publications of Atwater.*^
It is evident that persons of essentially different weight of body who
live under unequal external conditions must need essentially different
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. Fat. Carbohydrates. Calories.
For men 118 grams 56 grams 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.
The quantity of food required by a woman engaged in moderate work
' In regard to the older researches cited in this table we refer the reader to Voit in
Hermann's Handbuch, 6, 519.
^ Ibid., and Zeitschr. f. Biologie, 9.
^ Armee-und Volkserniihrung, Berlin, 1880.
* Untensuchung liber die Ernahrung schwedischer Arbeiter bei frei gewahlter Kost
Stockholm, 1891. Maly's Jahresber., 21.
^ Cited from Kellner and Mori in Zeitschr. f. Biologie, 25.
» Report of the Storrs Agrio. expt. Station, Conn. 1891-1895 and 1896 and U. S.
Report of .\griculture, liull. 53, 1898.
FOOD REQUIREMENT BY MAX. 7(35
is about four-fifths that of a laboring man, and we may consider the f(jl-
lowing as a daily diet with moderate work:
Proteins. Fat. Carbohydrates. Calories.
For women C4 grams 45 grams 400 grams 2240
The proportion of fat to carbohydrates is here as 1: 8-9. Such a pro-
portion occurs often in the food of the poorer classes which live chiefly
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 was increased at the expense of the carbohydrates, but
unfortunately on account of the high price of fat such a modification can-
not always be made.
In examining the a]:)Ove numbers for the daily rations it must not be
forgotten that the figures 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, there-
fore, VoiT, as above stated, calculates the daily quantity of proteins
needed by a laborer as IIS grams, he starts M-ith 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 Pflijger
and his pupils Borland and Bleibtreu ^ 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 the same. 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
ma}' exist on considerably smaller quantities of proteins than Yoit sug-
gests. It follows from the investigations of Hirschfeld, Kumagawa and
Klemperer, Siven, and others (see page 750) that a nearly complete or
indeed a complete nitrogenous equilibrium may be attained by the suffi-
cient administration of non-nitrogenous nutritive bodies with relatively
very small quantities of jDroteins.
If we bear in mind that the food of people of different countries varies
greatl}^ and that the individual also takes essentially different nourish-
ment according to the external conditions of living and the influence of
climate, it is not remarkable that a person accustomed to a mixed diet
1 Bohland, Pfluger's Arch., 36; Bleibtreu, ibid., 38.
766 METABOLISM.
can exist for some time on a strictly vegetable diet deficient in proteins.
No one doubts the ability of man to adapt himself to a heterogeneously
composed diet when this is not too difficult of digestion and is sufficient
in quantity; also we cannot den}' that it is possible for a man to exist
also for a long time with smaller amounts of protein than Voit suggests,
namely 118 grams. Thus 0. Xeumaxx ^ experimented on himself during
764 days in three series of experiments, and his diet consisted of 74.2 grams
protein, 117 grams fat, and 213 grams carbohydrates = 2367 gross calo-
ries, with a weight of 70 kilos and with ordinary laboratory work. These
figures cannot be compared with those obtained by Voit's worker, weigh-
ing 70 kilos, whose work was harder than a tailor's and easier than a black-
smith's; for example, the work of a mason, carpenter, or cabinet-maker.
The very extensive investigations recently performed by Chittexdex ^
on the estimation 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 twent}' months and consisted of careful investigations and
observations upon the manner of living, food taken, nitrogen elimination,
and the ability of performing work. The different 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 corp of the United States army) which
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 Yoit's value or were somewhat higher,
was gradualh' reduced more or less. The total calories supplied v.ere
not increased above the original value but rather diminished to a reason-
able extent. The bodily as well as the mental abilit}' was repeate<-lly
tested. As it is not possible to enter into the details of the investigation
the following will be sufficient to show the results. With a diet corre-
sponding to Voit's values the amount of urine nitrogen per day was 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 aliove
three groups may be found in the following table where for comparison
Hammarstex includes also the figures for Voit's diet.
Urine Nitrogen. Catabolized Protein. Protein per Kilo.
Min. Max. Min. Max. Min. Max.
Group 1 5.69 8.99 35.6 56.19 0.61 0.S6
Group 2 7.03 8.39 43.9 52.44 0.74 0.87
Group 3 7.47 11.06 46.7 69.10 0.75 0.92
Voit's figuro« 16 100 1 43
' Arch. f. Hygiene. 45.
- R. H. Chittenden, Physiological Economy in Nutrition, New York, 1904.
FOOD REQUIREMENT BY MAN. 767
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 per-
sons 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 exist for a long time 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 nearly 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
At WATER and others ^) 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 ^
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 764 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, there-
fore, 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. It is
'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 Byrant, ibid., Bull. 75;
Jaffa, ibid., 83; Grindley, Sammis, and others, ibid., 91.
' Hultgreu and Landergren, 1. c. ; Lapicque, Arch, de Physiol. (5), 6.
768 METABOLISM.
certainly tnie that the amount of nutriment required by the body is not
directly proportional to the body weight, for a small body consumes rela-
tively 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 quantity of substance than a
small one, and in estimating the nutritive need one must also always con-
sider 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 ^
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 38.5 calories per kilo. In the ordinary sense
for a resting man the general food requirement is calculated in round num-
bers as 30 calories for every kilo. The minimum figure for metabolism
during sleep and in as complete rest as possible has been found by
SoxDEN, TiGERSTEDT and JoHANSsoN ^ 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 nearly the same power of serving as a source
for muscular work, and that the muscles, it seems, select that foodstuff
Avhich is supplied to them in the greatest quantity. As a natural sequence
it is to be expected that muscular activity requires indeed an increased
suppl}' of foodstuffs, but no essential change in the relation of the same, 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 con-
tradiction 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 confirmed in general. As example of this
'Skand. Arch. f. Physiol, 11.
'Sond(5n and Tigerstedt, Skand. Arch. f. Physiol., 6; Johansson, ibid., 7; Tigerstedt,
Nord. Med. Arkiv. Festband, 1897.
REST AXD WORK. 769
the following tables gives the rations of soldiers in peace and in the field
and the average figures from the detailed data of various countries.^
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 manoeuvre A
(hard work) and B (strenuous work) in war.
Proteids. 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 weli 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 carbohydrates.
The importance of the food-demand for working individuals is shown
by the figures given on page 764 for a wood-chopper in Bavaria. A need
of more than 4000 calories occurs only seldom, and with very hard work
the demand may rise even to 7000 calories (Atwater and Bryant,
Jaffa ^).
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.
^ 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.
'See footnote 1, page 767,
770 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 ex-
ample 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.^
Woman in poorhouse 80 49 266 1725
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 1733
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 con-
dition 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 insuf-
ficient nutrition. This is especially the case in obesity, and all the diet-
aries proposed for this purpose are chiefiy starvation cures which will be
shown below from those selected, namely, Harvey, Ebstein and Oertel's
cure.
The oldest and most gene'rally known diet cure for corpulency is that of
Harvey, which is ordinarily called the Banting method. The principle
of this cure consists in increasing, as far as possible, the consumption of
the accumulated fat of the body by as limited a supply of fat and carbo-
hydrates as practicable and a simultaneously increased supply of proteins.
A second, called Ebstein 's cure, based on the assumption (not correct)
that the fat of the food is not accumulated in a body rich in fat, but is
completely burnt. In this cure large quantities of fat are therefore
allowed in the food, while the quantity of carbohydrates is diminished
very materially. The third cure, called Oertel's^ cure, is based on the
correct view that a certain quantity of carbohydrates has no greater influ-
ence in the accumulation of fat than the isodynamic quantities of fat. In
this cure, therefore, carbohydrates as well as fat are allowed, provided the
* See Voit, Untersuchung der Kost. Miinchen, 1877, 142. See also Hirschfeld
Maly's Jahresber.. 30.
"^ See Voit, Intersuchung, der Kost, page 186.
^ Banting, Letter on Corpulence. London, 1864. Ebstein, Die Fettleibigkeit und
ihre Behandlung. 1882. Oertel, Handbuch der allg. Therapie der Kreislaufstorungen.
1884.
OBESITY CURES. 771
total quantity of the same is not so great as to hinder the decrease in
the fatty condition. A greatly diminished supply of water is also one of
the features of Oertel's cure, especially in certain cases. The average
quantity of the various nutritive substances supplied to the body in these
three cures is as follows, and we give also for comparison in the same table
VoiT 's diet necessary for a laborer.
Proteins. Fat. Carboliydrates. ^gJogg^f
Harvey- Banting's cure 171 8 75 1083
Ebstein's cure 102 85 47 1396
Oertel's " 156 22 72 1140
"(Max) 170 44 114 1573
Laborer, according to Voit 118 56 500 3055
If the fat in all cases is recalculated in starch, then the proportion of
the proteins to the carbohydrates is;
Harvey-Banting's cure 100 : 54
Ebstein's cure 100 : 240
Oertel's " 100 : 80
" (Max) 100 : 129
Laborer 100 : 530
In all these cures for corpulence the quantity of non-nitrogenous bodies
is diminished as compared with the proteins; but also the total quantity
of food, as is shown by the number of calories, is considerably diminished.
Harvey-Banting's cure differs from the others in a relatively very
much greater quantity of proteins, while the total number of calories in it
is the smallest. On this account this cure acts very quickly; but it is
therefore also more dangerous and more difficult to accomplish. In this
regard Ebstein's and Oertel's cures (especially Oertel's), having a
greater variation in the selection of food, are better. As the adipose
tissue has a protein-sparing action, we have to consider in using these
cures, especially Banting's, that the destruction of proteins in the body-
is not increased in the adipose tissue, and one must therefore carefully
watch the elimination of nitrogen by the urine. All diet cures for obesity
are moreover, as above stated, starvation cures; and if the daily quantity
of food required by an adult man, represented as calories, is in round
numbers 2500 calories (according to the average figures found by Forster
in the case of a physician), then one immediately sees what a considerable
part of its own mass the body must daily give up in the above cures. This
reminds us of the great care necessary in employing them; each special
case should be conducted with regard to the individuality, the weight of
the body, the elimination of nitrogen in the urine, etc., etc., and always
under strong control, and only by a physician, never by a layman. A
more detailed discussion of the many conditions which must be considered
in these cases does not enter the plan and scope of this work.
AXIMAL FOODS.
TABLE I.— FOODS. 1
773
I. Animal Foodstuffs.
1000 Parts contain
>, ■
•P"3
Relationship of
1:2:3.
Meat without Boxes.
Fat beef ^
Beef (average fat ^)
Beef 2
Corned beef (average fat) . .
Veal
Horse, salted and smoked. .
Smoked ham
Pork, salted and smoked ^.
Meat from hare
" " chicken ,
" " partridge
" " wUd duck
h. Meat with Bones.
Fat beef ^
Beef (average fat ^)
Beef, slightly corned. . . .
Beef, thoroughly corned.
Mutton, ver}' fat
" average fat
Pork, fresh, fat
" corned, fat
Smoked ham
c. Fishes.
River eel, fresh, entire
Salmon, " "
Anchovy, " "
Flounder, " "
River perch, fresh, entire. . . .
Torsk, " " . . . .
Pike, " ,"•■■■
Herring, salted, entire
Ancho\-y, " "
Salmon (side\ salted
Kabeljau fsalted haddock). . .
Codfish (dried ling")
" (dried torsk)
Fish-meal from variety of Gadus
183
196
190
218
190
318
255
100
233
195
253
246
156
167
175
190
135
160
100
120
200
89
121
128
145
100
86
82
140
116
200
246
532
665
736
166
98
120
115
80
65
365
660
11
93
14
31
141
83
93
100
332
160
460
540
300
220
67
39
14
2
1
1
140
43
108
4
5
10
7
11
18
18
117
13
125
100
40
12
11
14
12
9
15
85
100
8
10
5
60
70
6
10
11
11
6
100
107
132
178
106
59
640
688
672
550
717
492
280
130
744
701
719
711
544
585
480
430
437
520
365
200
340
352
469
489
580
440
455
461
280
334
460
472
257
116
170
150
150
167
180
88
150
70
80
90
333
333
333
250
450
450
4.50
340
400
100
100
100
150
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
ino
90
50
63
53
42
20
143
660
5
48
6
13
90
49
53
53
246
100
460
450
150
246
56
31
9
2
1
1
100
37
54
1
1
1
1
1 The resiilts in the following tableis are chiefly compiled from the simimary of Alm^n' and of
Koxic. "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.
774
FOOD TABLES.
TABLE I.— YOOT)^— {Continued).
I. Animal Foodstuffs.
d. Inner Organs (Fresh).
Brain
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 (grains)
Wheat-flour (fine)
" " (very fine)
W^heat-bran
Wheat-l)read (fresh)
Macaroni
Rye (grains)
Rye-flour
E,ye-I)read (dry)
" " (fresh, coarse)
" (fresh, fine)
Barley (grains)
Scotch liarley
Oat (grains)
" (peeled)
Corn
Rice (peeled for boiling) . . .
French beans
Peas (yellow or green, dry).
Flour from peas
1000 Parts contain
5 g
cs.S
"c >'-
116
196
184
1G3
221
150
182
190
220
7
3
304
35
35
41
37
230
334
89
106
122
160
103
103
56
92
106
38
170
150
160
850
990
35
7
9
257
270
66
70
93
107
307
7
11
123
17
110
10
92
11
150
39
88
10
90
3
115
17
115
15
114
20
77
10
80
14
111
21
110
10
117
60
140
60
101
58
70
7
232
21
220
15
270
15
50
50
38
35
40
50
456
4
5
676
740
768
439
550
768
688
720
725
480
514
654
720
563
660
656
770
537
530
520
4
5
C
0)
11
770
17
720
10
714
10
721
13
728
10
670
9
807
50
610
55
565
15
119
7
175
217
7
873
7
901
7
905
6
665
60
400
50
500
56
329
8
654
10
756
13
520
8
875
18
1^0
8
120
3
120
50
130
17
330
8
131
18
140
20
110
15
110
16
400
11
370
26
140
7
146
30
130
20
100
17
140
2
146
36
137
25
150
25
125
Relationship of
1:2:.3.
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
KJO
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
192
VEGETABLE FOODS AND LIQUORS.
TABLE L—FOOBS^iContinued).
1000 Parts contain
Relationship of
2. Vegetable Foodstuffs.
1
l|
2
3
"5 **
5 -a
4
<
5
c
a
"S
6
o
'S.
'A
1
:2
•3
Potatoes
20
14
10
25
19
27
31
14
10
12
32
219
4
5
242
140
2
2
2
4
2
1
5
3
1
1
4
25
537
480
200
74
90
50
49
66
33
22
23
38
60
412
130
90
72
180
10
7
10
8
12
0
19
10
4
I
61
3
6
29
50
760
893
873
904
900
888
908
944
956
934
877
160
832
849
54
55
8
10
15
9
18
12
8
i
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
Turnips
529
Carrot (vellow)
900
Cauliflower
200
Cabbage
Beans
258
244
Spinach
106
Lettuce
157
Cucumbers
230
Radishes
317
Edil)le mushrooms (average). . .
Same dried in the air (average)..
Apples and pears
188
188
3250
Various berries (average)
Almonds
1800
30
Cocoa
129
TABLE IL— MALT LIQUORS.
1000 Parts by Weight contain
c
<a
"S
o o
o
A
O
o
<
-a
'S
o
3
c
"S
o
P
.2
"S
<
o
o
>,
O
J3
<
Porter
871
887
885
911
903
881
916
945
2
2
2
2
3
54
28
32
35
40
47
25
22
76
55
58
72
59
7
15
7
8
4
6
5
7
13
3.0
2.0
1.5
1.7
4.0
2
2
4
Beer (Swedish)
65
73
5
" (Swedish export)
3
Draught-beer
10
7
13
31
47
?,
Lager-beer
?,
Bock-beer. ;
3
"Weiss-beer
9,
Swedish "Svagdricka"
23
3
776
FOOD TABLES.
TABLE III —WINES AND OTHER ALCOHOLIC LIQUORS.
1000 Parts by Weight contain
u
Is
Alcohol, Vol.
Per Cent.
1
M
3
g 6
111
1^5
(0
a
o
<
'cTo .
Bordeaux wine
883
803
776
801
808
795
774
791
790
479
94
115
90
94
120
170
164
1.56
164
263
460
550
442-590
23
23
134
105
72
35
62
53
46
6
4
115
87
51
15
40
33
35
332
260-475
5.9
5.0
6.0
6.0
7.0
5.0
4.0
5.0
5.0
1.0
1.0
9.0
6.0
2.0
3.0
4.0
2.0
2.0
1.0
2.0
3.0
5.0
3.0
3.0
4.0
White wine (Rheingau)
Champagne
,
Rhine wine (sparkling)
Tokay
(■ 60-70
Sherry
Port wine
Madeira
Marsala
Swedish punch
Brandy
French cognac
778 INDEX TO 6PECTRUM PLATE.
SPECTRUM PLATE.
1. Absorption spectrum of a solution of oxyhcemogldbin.
2. Absorption spectrum of a solution of hcemoglobin, obtained by the action of an
ammoniacal ferro- tartrate solution on an oxyhaemoglobin solution.
3. Absorption spectrum of a faintly alkaline solution of methcemoglohin.
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).
B C
E b
\s \s 7 e i| IP *i is 1^ U M *e /tr /a /o go
ji:!ri.ii;'iLimvi^''':i:::'iJil.,:|i.:toMtonitUL^h^^^^^^
?/ ?» S3 ?* ?5
! I
[]
ct X?
8.
■H
/?
- , a
/?
T"¥
■
I
u
m
w
^^ET?'"
J.
»
'iiint;[;i|iii]:t!1ini|iiiMiiii|ih!|iu|iii|ini|iiiipi|iiii|'^!|Mi,[iiiijii:!|iiiijiii|iL-i^ilili!i:l|:;^
't U E 7 a a ^ ^^ ^? ^» « /o /ff Tr la n 7o ?/ ?3 ?3 2V 25
BCD E b F G
GENERAL INDEX.
Abietinic acid, 336
Abiuret products, 54, 394, 417
Absorption, 412-427
, action of putrefactive pro-
cesses in the intestine on,
405-407
Absorption ratio, 218
of the blood pigments,
218
Acceptor, 6
Acetanilid, behavior in animal body, 633
Acethsemin, 212
Acetic acid in intestinal contents, 400
in gastric contents, 373, 376
, passage of, into urine, 608, 629
Aceto-acetic acid, 625, 671
in urine, 625, 668
Acetone, 32, 669,
in urine, 668
Acetonuria, 668, 669
Acetophenone, behavior in body, 367
Acetylene, compound with haemoglobin,
267
Acetyldichitosamine, 686
Acetyl equivalent, 137
Acetyl acid equivalent, 137
Acetylamino benzoic acid, 636
Acetylparaminophenol, 633
Achilles tendon, composition of, 429
Acholia, pigmentary, 329
Achromatin, 149
Achroo-dextrin, 128
Acid albuminates, 36
, properties, 48, 49
, formation in peptic di-
gestion, 55, 359
, absorption of, 413
Acid amines, behavior in the animal body,
630
Acid equivalent, 137
Acid fermentation of urine, 677
Acid haemoglobin, 205
Acid rigor, 466
Acids, organic, behavior in the animal
body, 624, 629-631
Acidity of urine, 544, 545
of the gastric contents, 374
of the muscles, 448, 466, 467, 469,
Acrite, 114
Acrolein, 132
Acrolein test, 132, 136
Acroses, 114
Acrylic acid diureid. See Uric acid.
Actiniochrom, 691
Adamkiewicz-Hopkins reaction, 42, 103
Adelomorphic cells, 349, 364,
Adenase, 16, 271, 273
Adenine, 157, 162, 271, 571, 578
, properties, reaction, 162
, in urine, 162, 578
Adhesion, importance in blood coagula-
tion, 227
Adipocere, 442
Adrenalin, 278, 279
, relation to glycosuria, 298
Adrenalin-like bodies, 278
iEgagropila, 411
^rotonometric method, 709
Age, influence on metabolism, 755-758
Agglutination, 195
Agglutines, 26, 195
Alanine. 26, 33, 54, 84, 305, 462
Alanylalanine, 396
Alanylglycine, 55, 396
Alanylleucine, 396
Albumin, 36, 45
, detection of, in urine, 640, 643
, quantitative estimation of, 645
See also Proteids.
Albumins, 36
, general properties, 39, 45, 60
. See also the various albumins.
Albuminates, 36
, properties, 49, 48
, ferruginous albuminate in
the spleen, 272
Albuminoids, 37, 72-82, 430, 433, 492
Albumoids. See Albuminoids.
Albuminose, in spermatozoa, 498
Albuminous bodies. See Proteids.
Albuminuria, 640
, alimentary, 413
Albumoses. See Proteoses.
Alcapton and alcaptonuria, 591, 597, 599
Alcohol. See Ethyl alcohol.
Alcoholase, 21
Alcoholic fermentation. See Ethyl alco-
hol.
Aldehydases of the liver, 18
Aldehydes, 105
, behavior in the animal body,
631, 636
Aleuron grains, 503
Alexines, 186
Aldoses, 105
Alimentary glycosuria, 299, 420
779
780
GENERAL INDEX.
Alizarin, in the urine, 639
, administration of, 438
Alkali albuminates, 36, 48, 49
, absorption of, 413
Alkali albumose, 49
Alkali carbonates, physiological impor-
tance for gaseous
exchange, 698-702
, action on secretion of
gastric juice, 350
, action on secretion of
pancreatic juice, 384
. See various tissues
and fluids.
Alkalies, relation to gaseous exchange,
223, 224
, diffusible and non-diffusible in
blood, 224
, division of, in blood corpuscles
and plasma, 224, 238
. See also the various fluids and
Alkali phosphates in urine, 619-622, 720
, occurrence. See the
various fluids and
organs.
Alkali proteose, 49
Alkali urates, 542, 575
in calculi, 680
in sediments, 542, 575, 677,
678
(Vlkali earths, elimination by the intestine,
619, 626
in urine, 619, 620, 626
in bones, 436
insufficient supply of, 438,
439, 727, 738
Alkaline fermentation of urine, 677
Alkalinity, determination of, in blood, 191
Alkaloids, action on muscles, 466
, passage of, into urine, 639
, retention by the liver, 280
Alkyl sulphide of the skunk, 692
Allantoin, properties and occurrence, 568,
573, 583, 584
in transudates, 259, 513
, formation from uric acid, 568,
574
Alloxan, 568
Alloxuric bases, 156, 578
Alloxuric bodies, 156
AUoxyproteic acid, 611, 613
Almen-Bottger-Nylander's sugar test, 116
655
Ambergris, 412
Ambrain, 412
Amide nitrogen, 26, 27
Amino acids, 88-103
, relation to formation of uric
acid, 572
, relation to formation of urea,
5.'')0-553, 630
, relation to carbohydrate
metabolism, 305
Amino acids, formation from protein sub-
stances, 30, 33, 54, 380,
394, 401, 416
, deamidation of, 305, 462, 550,
571
, passage of, in the urine, 614,
675
, conjugation of, 34
Amino-acetic acid. See Glycocoll.
Amino-benzoic acids, behavior in the ani-
mal body, 636
Amino-caproic acid. "See Leucine.
Amino-cerebrinic acid chloride, 486
Amino-cerebrinic acid glucoside, 486
Amino-cinnamic acid, 633, 635
Amino-glutaric acid. See Glutamic acid.
Amino-ethyl sulphonic acid. S°e Taurine.
Amino-phenyl-acetic acid, behavior in ani-
mal body, 634
Amino-phenyl-propionic acid, 30, 585
Amino-phenyl-propionic acid, behavior in
the animal body, 5S5, 633
Amino-propionic acid, 84. See also Al-
anine.
Amino-pyrotartaric acid. See Glutamic
acid.
Amino sugar, 107. See also Glucosamine.
Amino-succinic acid. See Aspartic acid.
Amino-thiolactic acid. See Cysteine.
Amino-valerianic acid, 84
Amidulin, 126
Ammonia, formation in autolysis, 380
, formation in protein putrefac-
tion, 401
, formation from protein sub-
stances, 26, 29, 380, 394, 401
544
, formation in tryptic disection,
394
, occurrence in blood, 241, 551
, occurrence in urine, 544, 549,
623
, elimination after administra-
tion of mineral acids, 544,
624, 625
, elimination in disease, 549
, elimination in diseases of the
liver, 554
, after extirpation or atrophy of
the liver, 554
Ammonia, estimation of, in urine, 623, 624
Ammonium salts, relation to formation of
glycogen, 291
, relation to formation of
urea, 572
, relation to formation of
uric acid, 551
Ammonium-magnesium phosphate in uri-
nary calculi, 678, 682
Ammonium-magnesium phosphate in in-
testinal calculi, 411
Ammonium-magnesium phosphate in uri-
nary sediment, 679
GENERAL INDEX.
781
Aimnoniura sulphate, method of separat
ing proteoses, 38.
51, 60
, method of separat-
ing carbohydrates,
289
Ammonium urate in urinary sediments,
681
in urinary calculi, 677,
678
Amniotic fluid, 512
Amphicreatine, 457
Amphopeptone, 51, 57
Amygdalin, 17
Amylose, 126
Amylodextrin, 126, 128
Amyloid, 36, 69, 431
, vegetable, 129
Amyloid degeneration, bile in, 329
, chondroitin -sul-
phuric acid in .
liver in, 431
Amylolytic enzymes, 16, 386, 387. See
also various tissues and secretions.
Amylopectin, 126
Amylopsin, 387, 388
Amylum. See Starch.
Ansemia, pernicious, 246
Anaerobic metabolism, 21, 461
Aniline, behavior in the animal body,
633
Anisotropous substance, 447
Antedonin, 691
Antialbumate, 359
Antialbumid, 56, 359
Antialbumose, 51
Antienzymes, 210. See also various en-
zymes.
Antifebrine, relation to elimination of uro-
bilin, 604
Antimony, passage of, into milk, 539
, action on the elimination of
nitrogen, 548
Antipeptone, 51, 54, 57
Antipyriiie, relation to formation of gly-
cogen, 291
, action on the urine, 604, 638,
639
Antitoxines, 25
Antoxyproteic acid, 611, 612
Anuria, in cholera, 694
Aorta elastin, 75, 76
Apatite in bone-earths, 436
Aqueous humor, 264
Arabinose, 108, 112
, relation to formation of gly-
cogen, 290
Arabinosimine, 108
Arabite, 106
Arachidic acid, 131, 518
Arachoidal fluid, 257
Arbacin, 62
Arbutin, relation to formation of glyco-
gen, 291, 592
, behavior in the animal body, 592
Arginase, 16, 21, 36, 271, 284, 550
Aj'ginine, 16, 59, 63, 96, 550
Argon in blood, 697
Arnold's aceto-acetic acid reaction, 672
Aromatic compounds, behavior in animal
body, 632-f339
Aromatic substances in the urine, 586
Arsenic, in the animal body, 187, 239, 685,
695
action on the elimination of
nitrogen, 548
Arsenious acid, action on peptic digestion,
359
Arseniuretted hvdrogen, poisoning with,
331-333
Arterin, 196
Ascitic fluids, 259, 262, 263
Asparagine, 88
, relation to formation of gly-
cogen, 291
, nutritive value, 747
Asparaginic acid. See Aspartic acid.
Aspartic acid, 87
, relation to formation of
uric acid, 572
, relation to formation of
urea, 550
, formation from protein, 33,
54, 88
, behavior in the organism,
550, 572, 630
Asparagus, odoriferous bodies of, in the
urine, 639
Assimilation limit, 259, 260
Ass's milk, 529
Atmidalbumin, 52
Atmidalbumose, 52
Atmidkeratin, 75
Atmidkerato.se, 75
Atropine, action of, elimination of uric
acid, 569
, on the secretion of saliva, 347
Auto-digestion, 372. See Autolysis.
Auto-intoxication, 25
Autolysis, 22, 23
, substances retarding coagula-
tion produced in, 234
See also the various organs
and tissues.
Auto-oxidation, 3-8
Bacterial proteins, 28
Bacterium urea?, 677
Banting cure, 770
Beer- vinegar bacteria, enzyme of, 11
Beeswax, 138
Bela's acetone reaction. 671
Bence-.Tones proteid, 645
Benzaldehyde, oxidation of, 5, 6
, substituted aldehydes, be-
havior in the animal
body, 636
Benzoic acid, formation from protein sub-
stances. 31. 32, 585
, passage of, into sweat, 695
782
GENERAL INDEX.
Benzoic acid, behavior in the organism, 3,
585, 635
, occurrence in the urine, 586
, substituted benzoic acids,
action in body, 635
Benzene, 31, 76
, behavior in the animal body,
632, 633
Benzoyl-amino-acetic acid. See Hippuric
acid.
Benzoyl-cystine, 93
Benzoar-stones, 411
Bial's reagent, 667
Bifurcated air, 706
Bile, 307-334
, analysis of, 326, 327
, antiseptic action, 405-407
, enzymes of, 325
, in disease, 329
, influence on protein digestion, 358,
393, 394, 398, 399
, on the secretion of bile, 309
, on the absorption of fat, 398, 405,
406, 422-425
, on tryptic digestion, 393, 394, 399
, molecular concentration of, 326
, passage of foreign bodies, 329
, occurrence of, in urine, 426, 652
, occurrence of, in gastric contents,
373, 398, 399
in meconium, 410
, decomposition in the intestine, 403
, chemical formation of, 329-333
Bile-concretions, 333, 334
Bile-pigments, 320-325
, origin and formation, 329-
333
, reactions, 321,322, 652, 653
, passage of, into urine, 652
Biliary fistulae, 307, 406
, influence of, on intestinal
putrefaction, 406
, influence on the food re-
quirement, 406, 407
Bile-salts, 310
Bile-acids, 311, 319
in urine 426, 652
, detection of, 319, 652
, absorption of, 426
, origin of, 330, 331
, Pettenkofer's test lor, 311
Bile-mucus, 310, 329
Bilianic acid, 315
Bilicyanin, 319, 322, 324
Bilifulvin, 320
BUifuscin, 319, 324
Bilihumin, 319, 324
Biliphffiin, 320
Biliprasin, 319, 324
Bilipurpurin, 324
BUirubin, 319, 320
, relationship to blood-pigments,
210, 215, 319, 331, 333
, relationship to hsematoidin,
216. 320, 321
Bilirubin, putrefaction of, 400
, occurrence of, 319
Biliverdin, 323
in fffices, 409
Biogen molecule, 4
Biogens, 4
Biological protein reaction, 186, 413
Bismuth, passage of, into milk, 539
Birotation, 88, 109
Bitch's milk, 529, 535
Biuret, 34, 555
Biuret base, 35; cleavage of, 395
Biuret reaction, 43, 44, 50, 555
Bladder. See Urinary calculi.
Bleeding, 248, 298, 697
Blister fluid, 265
Blonds, milk of, 534
Blood, 170-249
, general behavior, 170, 222, 225
, analyses, quantitative, 235-241
, analyses, physico-chemical, 191,
236
, arterial and venous, 196, 197. 241,
697
, defibrinated, 172
, asphyxiation, 197, 697
, quantity of, in the body, 248
, detection, chemico-legal, 216
, distribution of, in the organs, 249
, behavior in starvation, 244
, comiX)sition under varioxis condi-
tions, 241-247
in gastic contents, 373
in urine, 648, 650
Blood-casts, 648
Blood-clot, 172, 225
Blood coagulation, 170, 171, 176-178, 225-
235
Blood-corpuscles, white, 220, 221, 226, 227
, number of, 220, 225,
226, 246, 247
, relation to coagulation,
220
, red, 192, 193
, number of, 192, 244,
246
, relation to high alti-
tudes, 245
, passage of, into urine,
648
, permeability of, 195
, composition of, 218, 219
Blood gases, 696, 702
Blood-pigments, 195-221
in bile, 329
in urine, 648, 649
, estimation, 217, 218
, regeneration, 216
Blood-plasma, 172-183
, composition of, 187, 188,
238-240
Blood-plates, 220-222, 227, 228
, relation to coagulation of
blood, 227, 231-233
Blood-sarum, 172, 183-192
GENERAL INDEX.
rss
Blood-serum, composition of, 188-192
Blood-spots, 217
Blood transfusion, 245, 247, 248
Blueberries, coloring matter of, in urine,
639
Blue stentorin, 691
Boar spermatozoa, 497
Boas' reaction for HCl, 374
for lactic acid, 374
Bones and bone tissues, 434, 440
in starvation, 620
Bone-earths, 436, 437
Bone marrow, 172, 437
Bonellin, 691
Borneol, 609, 638
Bottcher's spermine crystals, 496
Bottger-Almen-Nylander's sugar test, 94,
116, 655
Bowman's disks, 488
Brain, 479-489
Bromadenine, 159
Bromanil, 32
Bromhypoxanthine, 159
Bromides, beliavior to secretion of gastric
juice, 364
Bromine, passage of, into saliva, 346, 347
Bromoform, from protein, 32
, behavior in the animal body,
631
Bromtoluene, behavior in the animal body,
636
Brunettes, milk of, 534
Brunner's glands, 377
Buccal mucus, 341
Buffy coat, 225
Bufidin, 692
Bufotalin, 692
Bufotenin, 692
Bufotin, 692
Bull, spermatozoa, 497
Burbot, spermatozoa, 62
Bursfe mucosae, contents of, 266
Butalanine, 81, 382
Butter-fat, 517, 518
, absorption of, 424
Butterfly, pigment of wings, 569, 690
Buttermilk, 528
Butyl alcohol, behavior in the animal body,
632
Butyric acid, in urine, 607
in gastric contents, 376
in milk fat, 517, 518
Butyric-acid fermentation, 5, 110, 516
in intestine,
397, 403
Butjdmercaptan, 692
Butyrinase, in blood, 185
Byssus, 37, 81
Cadaver alkaloids, 24
Cadaverine, 24, 98, 457
in intestine, 676
in urine, 615, 676
Csecum, solution of cellulose in the, 397,
398
Caffeine, 157
, action on the muscles, 466
, behavior in the animal body, 579
Calcium, lack of, in food, 438, 439, 737
, occurrence, 625. See also various
tissues and fluids.
Calcium carbonate in urine, 679
in urinary calculi, 682
in urinary sediments,
678
in bones, 436, 439
in tartar, 348
in otoliths, 494
Calcium casein, 441
Calcium oxalate in urine, 582
m urinary sediments, 678
In urinary calculi, 681,
682
Calcium phosphate, relation to the coagula-
tion of fibrinogen,
229
, relation to the coag-
ulation of casein,
520
, occurrence in the in-
testinal concretions,
410, 411
in the urine, 543, 619,
620, 625
in urinary sediments,
679, 680
in urinary calculi, 681
682
Calcimn salts, elimination, 619, 626
, importance to coagulation
of the blood, 171, 177,
229
, importance to coagulation
of milk, 520
. See various calcium salts.
Calcium sulphate, in urinary sediments, 679
, ion action, 168
Calculi, salivary, 348
, intestinal, 410-412
, urinary. 680-683
Calories of foodstuffs, 723-727
of different rations, 763-771
Campho-glucuronic acid, 122, 610, 638
Camphor, behavior in the animal body,
610, 638
Cane-sugar. See Saccharose.
Capranica's reaction for guanine, 161
Capric acid, 131, 518, 530
Caproic acid, 131, 518, 530
Caprylic acid, 131, 518
Caramel, 115, 124
Carbamino acetic acid, 701
Carbamic acid, 563
in blood, 186, 5,52
in urine, 552, 553, 563
poisonous action, 552
Carbamic-acid ethylester, 563
Carbazol, behavior in body, 634
Carboglobulinic acid, 701
Carbohaemoglobins, 207
r84
GENERAL INDEX.
Carbohydrates, 104-130
, importance in fat forma-
tion, 444, 445, 753
, importance in glycogen
formation, 290, 292, 293
, importance for muscular
activity, 459, 468, 469,
473, 474
, action on protein metabo-
lism, 738, 747, 748, 749,
751
, action on intestinal putre-
faction, 415, 406, 589
, absorption of, 419-421
, inadequate supply of, 739
See also the various car-
bohydrates.
Carbolic acid, action on peptic digestion,
359
See also Phenol.
Carbolic urine, 591
Carbon, relation to nitrogen in the urine,
628, 720
, calorific value, 723
Carbon dioxide, assimilation, 1
in the blood, 696-701, 708-
710, 712
in the blood in diabetes,
701, 702,
in the blood in poisoning
with mineral acids, 701
in the intestine, 401, 403
in the lymph, 251, 702
in the stomach, 372
in the muscles during rest
and activity, 468, 472
in the muscles in rigor
mortis, 466
in the secretions, 702, 703
in transudations, 703
action on the secretion of
gastric juice, 353
elimination, dependence of
external temperature
upon, 761
elimination in rest and ac-
tivity, 468, 472, 759,
760
elimination by the skin,
695
elimination in the in-
cubations of the egg,
510
elimmation in various
ages, 756-758
Carbon-dioxide haemoglobin, 207, 699
Carbon-monoxide poisoning, 205, 299, 460,
548
action on the formation of lactic acid,
460
action on the elimination of nitrogen,
548
action on the elimination of sugar,
299. 460
Carbon monoxide hajmochromogen, 209
Carbon-monoxide methsemoglobin, 207
Carbon-monoxide blood test, Hoppe-Sey-
ler's, 206
Carminic acid, 690
Carnic acid, 457
Carniferrine 457
Carnine, 158, 456, 457
, in urine, 578
Carnitine, 457
Carnomuscarine, 457
Carnosme, 454
Carp, s{)erma of, 63, 504
, eggs of, 71
Cartilage, 69, 431-434
, quantity of ash, 434
, behavior to gastic juice, 360
, behavior to pancreatic juice, 395
Cartilage gelatine, 433
Caseanic acid, 30, 33, 101
Caseid, 520
Casein, 36, 47, 100
, origin of, 537
, from woman's milk, 530
, from cow's milk, 518
, quantitative estimation of, 526
, absorption of, 413
, behavior towards rennin, 362, 520
, behavior to gastic juice, 47, 521,
530, 531
, heat of combustion, 724
Caseinokyrin, 58, 59
Caseinic acid, 30, 33, 101
Caseinogen, 521
Caseoses, 52, 522
, relation to the coagulation of
blood, 171
Castor bean, 25
Castoreum, 692
Castorin, 692
Catalases, 7, 20. See also the fluids and
Catalyzers, 7, 14, 15, 20, 201
Catheterization of the lungs, 708, 709
Cat's milk, 527
CeH, animal, 139-169
Cell constituents, primary and secondary,
140
Cell fibrinogen, 271
Cell globulins, 141, 194
Cell membrane, 142, 360
Cell nucleus, 149
Cellulose, 129
, fermentation of, 129, 130, 397,
404
, solution of, in the caecum, 398
Cement, (in tooth structure), 440
Cephalin, 485, 488
Cephalic acid, 485
Cephalopods, flesh of, 76, 455, 478
Cerebrin, 268, 480, 481 483, 484,
, in pus, 268
Cerebrinin phosphoric acid, 486
Cerebrinic acid, 486
Cerebron, 480, 483, 485
Cerebrosides, 194, 481, 482, 483
GENERAL INDEX.
785
Cerebrospinal fluid, 264, 489
Cerolein. 138
Cerotic acid, 138
Cerumen, 692
Cervical ligament, 75, 76, 429
Cetaceans, bones of, 438
Cetin, 138
Cetyl alcohol, 138
ChakLza, 506
Charcot's crj'stals, 247, 496
' 'Charge theory,' ' 364
Cheno-taurocholic acid, 315
Chief (adelomorphic) cells, 349, 364
Children's urine, 543, 549, 583
Chitaminic acid, 121
Chitaric acid, 121
Chitin, 81, 120, 686
, behavior in tryptic digestion, 395
Chitosamine, 120, 686. See also Gluco-
samine.
Chitosan, 687
Chitose, 121
Chloral hvdrate, behavior in the animal
body. 609, 632
, action upon the secre-
tion of bile, 309
Chloral secretin, 309
Chlorates, poisoning with, 203, 648
Chlorazol, 32
Chlorbenzene, beha^^or in the animal
body, 639
Chlorides, elimination by the urine, 616-
619, 694, 695
, elimination by the sweat, 695
, action on protein metabolism,
754
, insufficient supply of, 736
See also various fluids and
tissues.
Chlorochrome, 282
Chlorocruorin, 219
Chloroform, action on the elimination of
chlorides, 615
, action on the muscles, 466
, action upon proteins, 40
, behaA-ior in the animal body,
631
Chlorometer, 618
Clilorophan. 491
Chlorophyll, 2
, relation to blood-pigments,
197, 214
Clilorosis, 246
CMorphenylmercapturic acid, 639
Clilorrhodinic acid, 269
ClJortoluene, behavior in the animal bodv,
636
Cholagogues, 308
Cholamine, 315
Cholalic acid, 310, 315-318. See also
Cholic acid.
Cholanic acid, 317
Cholecyanin, 323, 324
Choleic acid, 312, 317, 318
Choleprasin, 319, 324
Cholepyrrliin, 320
Cholera, blood in, 240
, sweat in, 694
, ptomaines in, 25
Cholera bacilli, behavior with gastric
juice, 371
Cholestanol, 335
Cholestenone, 335
Cholesterilene, 334
Cholesterin, 334, 335
in blood-serum, 183, 194, 220
in the bUe, 310, 326, 327, 328
in gaU-stones, 333. 334
in the brain, 480, 487, 488
in the urine, 675, 682
, importance in thecell, 140, 143
, beha\ior toward saponin, 337
Cholest'erin calculi, 334
Cholesterin ester in blood-serum, 183
Cholesterin fat, as protective fat, 691
Cholesterin-propionic ester, 335
Cholesterinic acid, 315
Cholesterone, 334
Choletelin, 322, .323
, relation to urobilin, 603
Cholic acids, 315, 316, 335
Cholic acid azide, 312, 315
Cholic acid hydrazide, 312
Cholic acid uretliane, 315
Choline, 25, 145, 147, 264, 325, 394, 482
Cholohsematin, 324
Choloidic acid, 319
Cholylic acid, 316
Chondrigen, 77, 430
Chondrin, 81, 269
Chondrin balls, 433
Chondro-albumoid, 433
Chondroitic acid, 431
Choudroitin, 431
Chondroi tin-sulphuric acid, 42, 65, 66, 69,
70, 431
, in urine, 611,
647
, in kidneys,
542
Chondromucoid, 69, 430, 433
Chondroproteids, 65, 66. 69
in the urine. 647
Chondrosin from chondroitin - sulphuric
acid, 431
from sponges, 69
Chorda saliva, 340
Choroid coat, 493
, pigment of, 688
Clu-omatin, 149
Chromhidrosis, 695
Chromogens in urine, 601
in suprarenal capsule. 277
Chrvsophanic acid, action on urine, 639
Chyle. 250-252
Chyluria, 675
Chyme, 366
, investigation of, 373-376
7S6
GENERAL INDEX.
Chymosin, 13, 16, 186, 361, 520
, detection in gastric contents,
373
, occurrence in the pancreas,
386, 395
. See also Rennin.
Cilianic acid, 315
Cinnamic acid, behavior in the animal
body, 585
Citric acid in milk, 518, 532
Clupeine, 63, 64
Coagulated proteids, 36, 61
Coagulation of the blood, 170, 171, 176-
178, 225-235
, intravascular, 234
of milk, 362, 373, 396, 515,
519, 530
of muscle-plasma, 448, 452,
453, 467
Coagulins, 232, 233
Coaguloses, 56
Cobra poison, 234
Coccinic acid, 690
Coccygeal glands, 692
Cochineal, 690
Cochinillic acid, 690
Codfish, eggs, 504
, spermatozoa, 62
Coefficient, Haser's, 627
, respiratory, 445, 472, 721, 731
, dissociation, 190
, extinction, 218
, urotoxic, 615
CofTee, action on metabolism, 755
Collagen, 37, 76-78, 395, 428, 433, 435
Colloid, 68, 275, 499
Colloids, 39, 40
Colloid corpuscles, 499
Colloid cysts, 498
Colon, exclusion of, 427
Coloring-matters. See various pigments.
Colostrum, 528, 533
Colostrum corpuscles, 528
Comma bacillus, behavior with gastric
juice, 371
Compound proteids, 36, 65-72
. See also the different groups
of protein substances.
Conalbumin, 507
Conchiolin, 37, 81, 82
Concentration, molecular. See various
fluids.
Concrements. See various calculi.
Cones of the retina, pigm-jnt of, 491
Conglutin, calorific value of, 724
Connective tissues, 428-430
Copaiva balsam, actio.n on the urine, 639
Copper in blood, 187, 239
in bile, 310
in biliary calculi, 334
in hsemocyanin, 219
in protein substances, 26
in turacin, 690
Cornea, 434, 493
Cornein, 37, 81, 82
Cornicrystalline, 82
Corpora lutea, 216, 498
Corpse wax, 442
Corpulence, d et cures for, 779, 771
Corpus callosum., 488
Corpuscula amylacea, 486
Cow's milk, 515-529
, general behavior, 515, 516
, analysis of, 524-527
, anti-putrefactive action of,
405, 588, 589
, coagulation with rennin, 363,
373, 516, 530
, beliavior in the stomach, 530,
531
, composition of, 526-528
Cream, 530
Creatine, relation to formation of urea,
455, 550
, relation to muscular activity,
470
, properties and occurrence, 455
Creatinine, relation to muscular activity,
470, 472, 563
, properties and occurrences,
563, 564
zinc chloride, 564
Cresol, 30, 401, 588, 589
Cresol-sulphuric acid, 588, 589
Crotonic acid, 674
Crude fibre, digestion of, 427
Cruor, 172
Crusocreatinine, 457
Crustaceorubin, 691
Crusta inflammatoria or phlogistica, 225
Crystalbumin, 493
Crystalfibrin, 493
Crystallins, 491, 492
Crystalline lens, 492, 493
Crystalline seralbumin, 181
Crystalloids, 39
Cimiic acid, 634, 635
Cuminuric acid, 635
Curd, 442, 520
Cuorin, 144
Cyanhaemoglobin, 205
Cyanhydrines, 106
Cyanmethtemoglobin, 205
Cyanocrystalline, 509, 690
Cyanuric acid, 555, 568
Cyanurin, 601
Cyclopterine, 63
Cymene, 634
Cyprinine, 63
Cysteine, 28, 29, 33, 93, 94
, conjugation in animal body, 639
Cysteinic acid, 93
Cystine, 28, 29, 34, 92, 93, 94, 331, 675
, occurrence in urine, 611, 675, 676
, occurrence in urinary sediments,
680
, occurrence in urinary calculi, 682
, occurrence in sweat. 695
. behavior in animal body, 331,675,
676
GENERAL INDEX.
787
Cystinuria, 24, 92, 615, 675
Cvsts, tapeworm, 265
, ovarial, 498, 502
, thyroid, 275
, mucoid substances of, 498-501
Cvtin, 271
Cytoglobin, 36, 142, 228, 271
Cytosine, 152, 156, 165, 166
Cytotoxines, 186
Cytozym, 232
Damaluric acid, 616
Damolic acid, 616
Defibrinated blood, 172
Dehydrochloride hsemin, 212
Deliydrocholan, 311
Dehydrocholic acid, 315
Dehydrocholeic acid, 317
Dehydrocholesterin, 335
Delomorphoic or parietal cells, 349, 364
Denige's reaction for uric acid, 576
Denige's-Morner's tyrosine test, 91
Dentin, 437, 440
Dermoid cyst, 502
Dermocerin, 691
DeiTno olein, 691
Desaminoalbuminic acid, 49
Desaminoproteic acids, 31
Descemet's membrane, 69, 492
Desoxycholic acid, 316
Deuterocaseoses, 52
Deuteroelastose, 76
Deuteroproteose, 52, 60, 644
Deuterogelatose, 76
Deuteromyosinose, 79
Deuterovitellose, 52
Dextrins, 128
, formation from starch, 128, 344,
388
, loading the stomach with, 364
, occurrence in the gastric con-
tents, 367
, occurrence in muscles, 459
, occurrence in portal blood, 242,
419
Dextrin-like substance in the urine, 608
Dextrose, 114-118
in blood, 184, 237, 242, 297-304
in urine, 297, 608, 654-665
in the lymph, 250
in muscles, 459
in the vitreous humor, 491
, preparation of, 118
, fermentation of, 20, 115, 657
, detection of, 118, 655-660
, reactions of, 115-117
, absorption of, 419
, quantitative estimation, 600-
665
Diabetes mellitus, 297-306, 654
, elimination of ammo-
nia by the urine in,
625
, relationship of the liv-
er to, 300
Diabetes mellitus, relationship of the pan-
creas to, 301-303
, blood in, 240, 297
, amount of sugar in
blood in, 297
, urine in, 543, 628, 654,
670
, CO, in the blood in, 701
, oxybutyric acid in the
blood in, 702
, oxybutyric acid in the
urine in, 625, 673
Diacetic acid. See aceto-acetic acid.
Dialuric acid, relationship to formation
of uric acid, 573
Diamide, poisoning with, 584
Diamino acids, 33, 96-101
Diamines, 24, 97, 98
in the urine, 615, 676
in the intestinal contents, 24,
676
Diaminoacetic acid, 97
Diamino-caproic acid. See Lysine.
Diaminopropionic acid-dipeptide, 35
Diaminotrioxydodecanoic acid, 101
Diamino-valerianic acid, 97. See Ornith-
ine.
Diastatic enzymes, 16, 185, 295, 343, 388
. See also other enzymes.
Diastase in the blood, 185
Diazo reaction, Ehrlich's, 612, 613
Diazobenzol-sulphonic acid, reaction with
sugar, 117
Dibenzoylornithin, 97
Dicalcium casein, 519
Dichlorpurme, 157
Diet cures, 770, 771
Diet for various classes of people, 763-765
Digestion, 339-427
Digestibility of food stuffs, 367-370, 417,
418, 421, 423, 425
Diglycyl-glycine, 35
Dileucyl-glycyl-glycine, 35
Dileucylcystine, 35
Dimethylaminobenzaldehyde, 43, 638,
675
Dimethylaminobenzoic acids, 638
Dimethyltoluidine, 638
Dimethylketone. See Acetone.
Dioxyacetone, 114
Dioxybenzenes, 591, 592, 633
Dioxydiaminosuberic acid, 33, 101
Dioxynaphthalene, 633
Dipalmitylolem, 132
Dipeptides, 35-37
, behavior with trypsin, 395,
396
Diphtheria toxins, action of the gastric
juice upon, 371
Disaccharides, 123
in urine, 420, 665, 666
, inversion of, 123, 293, 360,
378, 419
as glycogen formers, 293,
294
788
GENERAL INDEX
Dissociation degree, 190
Dissociation coefficient, 190
Distearyllecithin, 143
Distearyipalmitin, 132
Doeglic acid, 135
Dog's milk, 529, 535
Dolphin's milk, 529
Donne's pus test, 651
Dotterplatchen, 37, 503, 509
Dropsical fluid, 262
Dulcite, 106
, relation to formation of glycogen,
291
Dysproteose, 51
Dyslysins, 319
Dyspeptone, 359
Dyspnoea, action on protein catabolism,
548, 758
Ear, fluids of, 494
Earthy phosphates, elimination by the
urine, 618, 619,
625, 626,
, absorption of, 426
, solubility in fluids
rich in protein, 439
, occurrence in bone-
earths, 436-439 _
, occurrence in calculi,
333, 411, 681, 682
, occurrence in sedi-
ments, 678, 679
See also different
earthy phosphates.
Ebstein's diet cure, 770, 771
Echinochrom, 219
Echinococcus cysts, cyst wall, 687
, cyst contents, 265
Eck's fistula, 552
Edestan, 62
Edestin, 33, 46, 62, 100
, absorption of, 413
Edible bird's nests, 69
Eel, flesh of, 475
Eel-serum, 171, 234
Egg, 502
, hen's, 502-512
, absorption in the intestine, 417
, incubation of, 510-512
Egg albumin. See Ovalbumin.
Egg-shell, 73, 323, 509
Egg yolk, 502
Egg-white, 506
, albumin of the, 507
Ehrlich's diazo reaction. 612, 613
test for bile-pigments, 654
glucosamine test, 121
urine test, 674
Elaic acid, 134
Elaidic acid, 135
Elaidin, 135
Elastin proteoses, 76, 77
Elastin, 37, 75, 76, 100
, behavior with gastric juice, 360
, behavior with tiypsin, 395
Elephant bones, 436
milk, 529
teeth, 440
Ellagic acid, 412
Embryo of the hen, development of, 511,
512
Emulsin, 13, 609
Emvdin, 509
Enamel (of the teeth), 440
Encephalin, 482, 484
Endoenzymes, 22
Endolymph, 494
Energy, potential, of foodstuffs, 723-728
Enterokinase, 379, 383, 384, 386
Enzymes in general, 9-23
, zymogens, 16
. See various enzymes in the
tissues, organs and fluids.
Epidermis, 73, 685
Epiguanine, 157, 578, 580
Epinephrin, 278
Episarkine, 157, 578, 580
Erepsin, 153
, importance for absorption, 380,
391, 416
Erucic acid, 131
, absorption of, 423
Erythyrite, relation to glycogen forma-
tion, 291
Erythrocytes, 191-196. See also red
blood-corpuscles.
Erythrodextrin, 128, 344
Erythropsin. See Visual purple.
Esbach's method for estimating proteid
in urine, 645.
Esters, cleavage of, 284, 389
, synthesis of, 390
Ethal, 138
Ether, action on the blood, 193, 195
, action upon proteins, 40
, action on the secretion of gastric
juice, 352
, action on the muscles, 466
Ethereal sulphuric acids in the bile, 310,
325, 327
Ethereal sulphuric acids in the perspira-
tion, 694
Ethereal sulphuric acids in the urine, 588-
595, 622, 633, 637
Ethereal sulphuric acids, synthesis of, in
the liver, 280
Ethyl alcohol, production by fermentation,
10, 11, 21, 302, 396, 461
, production in the intestine,
400
, passage of, into milk, 539
, behavior in the animal
body, 632, 754
, action on the secretion of
gastT'ic juice, 351, 352,
365, 369
, action on the pancreatic
juice, 385
, action on the muscles, 465,
466
GENERAL INDEX.
789
Ethyl alcohol, action on metabolism, 754
, action on digestion, 369
, action on proteins, 40, 41
Ethyl benzene, behavior in the animal
body, 634
Ethylene glycol, relation to formation of
glycogen, 291
Ethylenimine. See Spermine.
Ethylidene-lactic acid, 460. See also
other lactic acids.
Ethyl mercaptan, behavior in the animal
body, 632
Ethyl-sulphuric acid, behavior in the
animal body, 631
Ethyl sulphide, formation from protein,
28, 30, 33
, behavior in the animal
body, 612
Euglobulin, 179, ISO
Euxanthic acid, 122, 638
Euxanthon, 638
Euxanthon glucuronic acids, 610
Excrementsr408, 410, 717, 718
in dogs with biliary fistula, 407
Excreta, of the animal organism, 715-720
, division by the various channels,
717
Excretin, 410
Excretolic acid, 410
Exostosis, 438
Expectorations, 713, 714
Extinction coefficient, 218
Extracellular action of enzymes, 22
Exudates, 256-265
Eye, 489-494
Fseces. See Excrements.
Fat, origin in the body, 442-446
, general properties, detection and oc-
currence, 131-138
, relation to work, 471-474
to the formation of glycogen,
291, 292
, calorific value of, 723-728
, nutritive value of, 723-728, 747, 748,
751, 752
, rancidity of, 133,
, absorption of, 421-427
, behavior with gastric juice, 363, 364
, behavior with pancreatic juice, 388-
390
, saponification of, 133, 389, 423
, action of. on the secretion of bile, 308
, action of, on the secretion of gastric
juice, 349-351
, action of, on the secretion of pan-
creatic juice, 384, 385
, iodized, behavior of, in the animal
body, 442, 538
, estimation of, 136, 137, 526, 527
, metabolism of, in activity and at rest,
471
, metabolism of, in starvation, 729
, metabolism of, with various foods,
740, 741, 770, 771
Fat, sugar formation from, 304-306
Fat formation, from proteins, 442-446
, from carbohydrates, 442-
446
Fat-sweat, 692
Fatty acids, general properties, detection
and occurrence, 131-138,
441
, solubility in bile, 422, 423-
, absorption of, 422
, sjTithesis, 444, 445
to neutral fats, 421
Fatty deseneration, 443
Fatty infiltration, 282, 283, 443
Fatty series, behavior of members in the
animal bodv, 629
Fatty tissue, 441, 446
, behavior with gastric juice,
360
Feathers, 73
, pigments of, 692
Fehling's solution, 116, 660
Fellic acid, 318
Fermentation, 10, 11, 20,21, 109, 111, 11.5
in the intestine, 397, 399
403, 404
in the urine, 677, 678
in the gastric contents, 371,
373
. See also various fermen-
tations, alcoholic, etc.
Fermentation lactic acid, properties, occur-
rence, etc., 460-
462
in the gastric
contents, 353
in the souring
of milk, 460,
515, 516
, detection in the
gastric con-
tents, 374,375
Fermentation saccharometer. 664
Fermentation saccharomanometer, 664
Fermentation test in the urine, 657, 663
Ferments in general, 9-23
inorganic, 14
. See various enzymes.
Ferratin, 282
Ferrine, 282
Fevers, elimination of ammonia in, 625
, elimination of uric acid in, 569
, elimination of urea in. 54S
, elimination of potassium salts in,
623
, metabolism of proteins in, 543
Fibre, crude, utilization of, 427
Fibres, elastic, in sputum, 714
, reticulate, 428
Fibrin, 36, 171. 174. 183, 225, 228, 230
, occurrence in transudates, 260
, Henle's. 495
Fibrin coagulation, 175-178, 225-235
Fibrin calculi, 411, 682
Fibrin digestion, 356, 373, 392-394
790
GENERAL INDEX.
Fibrin ferment, 16. 175, 176, 177, 183, 228-
234
Fibrin formation, 175-178, 225-235
Fibrin globulin, 173, 175, 177, 183
Fibrm soluble. See Serglobulin.
Fibrinogen, 36, 172-177, 183, 228-230, 250
Fibrinolysis, 173, 175
Fibrinoplastic substance. See Serglobulin.
Fibroin, 37, 81, 82
Fischer-Weidel's reaction, 160
Fish-bones, 'f38
Fish-eggs, 37, 504, 509
Fish-scales, 81, 160
Fish, bile of, 310, 328
, spermatozoa of, 63, 151, 497
, swimming-bladder of, 160, 710
, visual purple of, 490
Fish-glue, 77
Flesh, metabolism, in starvation, 729
, metabolism, with various foods,
739-753
Flesh quotient, 476
Florence's sperma reaction, 49.6
Fluoride plasma, 231
Fluorine in bones, 436
in enamel, 440
Fly-maggots, formation of fat in, 443
Foods, influence on the secretion of intes-
tinal juice, 377
, influence on the secretion of bUe,
308
, influence on the secretion of gastric
juice, 350, 351
, influence on the secretion of pan-
creatic juice, 382, 383
, influence on the secretion of mUk,
536
, influence on the elimination of vu-ic
acid, 569
, influence on the elimination of
urea, 548
, influence on the elimination of
purine bases, 578
, influence on fseces, 408, 409, 417,
418,421,719,
, influence on metabolism, 762
, requirements, 763-771
, various, 739-753
, insufficient supply of, 734-739
Foodstuffs, necessity of, 715
combustion heat of, 723-728
Formaldehyde, formation in plants, 1, 114
, action upon proteins, 50
, combination with urea,556
, relation to sugar forma-
tion, 113, 114
Formic acid in gastric contents, 376
, passage of, into urine, 607,
629
Frog's eggs, 509
membrane of, 66
Fructose. See Levulose.
Fruit sugar. See Levulose.
Fundus glands, 349, 363, 364
Fungi, glycogen in, 288
Fungi, tyrosinase in, 18
Fumaric acid, 32;
Furfuracrylic acid, 637
Furfuracryluric acid, 637
Fiu"furol, from pentoses. 111
, relation to Pettenkofer's bile-
acid tests, 312
, reagent for urea, 555
, behavior in the animal body, 637
Fuscin, 491
Galactonic acid, 120
Galactose, 106, 120, 524
, from cerebrins, 484, 485
, relation to formation of glyco-
gen, 293
, passage of, in the urine, 665
Galactosamine, 71, 121
Galactosides, 108
Gallacetophenon, behavior in the animal
body, 038
Gallic acid, behavior in the animal bodv,
597, 637
Gallois's inosite test, 459
Galtose, 108
Gas, exchange of, in various ages, 755-757
, exchange of, through the skin, 695
, exchange of, in starvation, 730, 731
, exchange of, in various conditions of
the body, 445, 446, 472, 720, 721,
730, 733, 757, 759, 761
, exchange of, in the muscles, 468, 472
, exchange of, starvation requirement
of, 733
Gases of the blood, 696-702
of the intestine, 403, 404
of the bile, 329, 702
of the urine, 626, 702, 703, 712
of the hen's egg, 509, 510
of the lymph, 251, 702
of the muscles, 465, 468
of the transudates, 259, 712
of the stomach, 372
Gastric contents. See Chyme.
Gastric fistula, 350, 352
Gastric juice, 349
, composition of, 353, 354
, secretion of, 349-352
, estimation of acidity of, 373,
374, 376
, relation to intestinal putre-
faction, 371, 406-408
, action of, 356-363, 366-371
Gastric lipase, 363
Gastric mucosa, 348
Gelatine, 28, 77-80, 100, 582
, relation to glycogen formation
291 *
, relation to coagulation of blood,
233
, putrefaction of, 401
, nutritive value of, 745
, behavior with gastric juice, 360
, behavior with pancreatic juice,
391, 395
GENERAL INDEX.
791
Gelatina in the egg, 511
Gelatine and the (leteetion of trypsin, 290
Gelatine-forming substances. See Colla-
gen.
Gelatine peptones, 57, 58, 79
Gelatine sugar. See GlycocoU.
Gelatinous tissues, 429
Gelatoses, 79
, relation to blood coagulation,
171
Generation, organs of, 495-513
Gentisic acid, 599
, behavior in the animal
body, 637
Gentisic aldehyde, 599
Gerhardt's diacetic acid reaction, 672
Globan, 46
Globin, 62, 99, 197, 208
Globulins, 36
, general characteristics, 45
, in starvation, 187
, in urine, 643
, in protoplasm, 140
. See also the different globu-
lins.
Globuloses, 52
Glucalanine, 81
Glucase, 21, 185, 345, 346
Glucocyanhydrin, 106
Glucoheptose, 106
Gluconic, acid, 105, 114, 300
Glucosamine, 108, 120, 121
from chit in, 120
from proteins, 32, 65, 67,
500, 506-508
in diabetes, 300
relation to formation of
glycogen, 294
Gluconucleoproteids, 71, 72
Glucoproteids, 36, 65-72, 141, 428, 430,
500, 506
, relation to formation of
glycogen, 292, 293, 295
Glucoproteose, 55
Glucosaminic acid, 121
Glucosan, 115
Glucose, 105, 106, 114. See also Dex-
trose.
Glucosides, 13, 108, 110
Glucosoxirae, 106
Glucorone, 122
Glucothionic acid, 222, 272, 285, 431, 514,
542
Glucuronic acid, 121, 122
, relation to formation of
glycogen, 291
, conjugated, 122, 609,
632, 638, 666
in diabetes, 300
in blood, 184
in bile, 310, 325
in urine, 609, 632, 638,
667
Glutamic acid, 88
Gluteines, 77
Gluten casein, 100
Gluten proteins, 100
Glutin peptones, 58, 79
Glutokyrin, 58
Glutose, 108
Glutinase, 391, 392
Glutinic acid, 78
Glyceric acid, 630
Glycerine aldehyde, 80
Glycerine relation to formation of glyco-
gen, 291
relation to formation of sugar,
305
Glycerophosphoric acid, 144, 247, 272, 277,
325
Glycerophosphoric acid in urine, 608, 614
Glyceroses, 114
Glycine. See GlycocoU.
Glycocholeic acid, 312
Glycochohc acid, 310, 312
, occurrence in excre-
ments, 404
, occurrence in bile from
various animals, 328
, absorption of, 426
, behavior to intestinal
pvitrefaction, 406
Glycocholates from rodents, 313
GlycocoU, 83
GlycocoU, relation to formation of uric
acid, 56S, 572
, relation to formation of urea,
550, 551, 630
, synthesis with, 3, 585, 586, 635
Glycogen, 140, 287-307
, origin of, 289-307
, relation to muscular activity,
468, 472
, relation to muscle rigor, 466
, relation to pepsin secretion, 365
, occurrence in sputmn, 714
, occurrence in leucocytes, 221
, occurrence in the lungs, 713
, occurrence in the lymph, 251
, occurrence in protoplasm, 140.
148, 221, 268
Glycol aldehyde, 2
Glycolysis, 185, 302, 303, 396
Glycolytic enz^^ne, 20, 185, 302, 303
Glycosuria, 29'7-307
, alimentary, 298, 420
Glycosuric acid, 597
Glycuronic acid, 121. See Glucuronic acid.
Glvcvkilanine, 35, 395
Glycylglycine, 35, 396, 630
GlycyI-1-leucine. 35
Glycyl-1-tyrosine, 35
Glyoxji diureide. See Allantoin.
Glyoxylic acid, as reagent, 42
Gmelin's test for bile-pigments, 321
test for bile-pigments in urine,
652
Goat's milk, 528, 529
Gold equivalent of the proteins, 41
Goose-fat, absorption of, 423
792
GENERAL INDEX.
Gorgonin, 82
Gout, elimination of uric acid in, 570
Graafian follicles, 498
Grape-moles, 512
Grape-sugar. See Dextrose.
Guaiaconic acid ozonide, 649
Guaiacum blood test, 648
Guanase, 16, 271, 273, 571
Guanidine, 32, 33, 501, 550
Guanine, 158, 160
in urine, 578
Guanine gout, 160
Guano, 160, 569
Guano-bile acids, 313
Guanovulit, 510
Guanylic acid, 151, 153, 155, 156, 381
Gulonic acid lactone, 121
Gulose, 113, 118
Gums, various, 129
• , animal, 67
, animal, in urine, 608
Gunning-Lieben's acetone reaction, 670
Giinzberg's reagent for free HCl, 374
Hsemagglutination, 195
Hsemase, 20
Hsemataerometer, 706
Hsematin, 197, 209
, relation to bilirubin, 332
, relation to urobilin, 603
, neutral hsematin, 208
Hsematinogen, 216
Hsematino meter, 217
Hsematinic acids, 210, 211, 325
Hi3ematinic acid imide, 211, 320
Hsematinic acid ester, 211
Hsematocrit, 236
Haematogen, 503, 510
Haematoglobulin. See Oxyhseriioglobin.
Haematoidin, 216
, relation to bilirubin, 216,
320, 331
, occurrence in sputum, 714
, occurrence in corpora lutea,
498
, occurrence in excrements,
409
, occurrence in sediments, 680
Haematoporphyrin, 213
, relation to chlorophyl,
197, 214
, relation to bilirubin,
215, 320, 332
, relation to urobilin,
214, 332, 603
, occurrence in urine,
601, 649, 650
, occurrence in lower
animals, 690
Hsematoscope, 219
Hsematuria, 648
Hsemerythrin, 219
H^min, 211, 649
Haemin crystals, 211, 649
Haemochrom, 196, 199
Hsemochromogen, 197, 208, 209
, occurrence in muscles,
451
Hsemocvanin, 219
Haemoglobin, 36, 65, 197
, composition of, 198
, properties and behavior, 202
, quantity in blood, 197, 242-
247
, quantitative estimation,
217-219
. See also Oxyhaimoglobin
and the combinations of
haemoglobin with other
gases.
Haemoglobinuria, 648
Haemolysis, 193, 337
Hajmolysins, 186, 193
Hsemometer, 219
Haemopyrrol, 197, 214
Haemorrhodin, 207
Hsemoverdin, 208
Haser's coefficient,
Hair, 73, 685
, pigments of, 689
Hair-balls, 411
Half-rotation, 109
Hammarsten's reaction for bile-pigments,
322, 653
Haptogen-membrane, 517
Heat, action of, on metabolism, 761
of combustion of various foodstuffs,
723-727
Helicoproteid, 71
Heller's albumin test, 41
albumin test applied to urine, 640
Heller-Teichmann's blood test, 649
Hemicelluloses, 130 ,
Hemicollin, 79
Hemielastin, 76
Hemiindigotin, 594
Heminucleic acid, 154
Hemipeptone, 51
Hemp-seed calculi, 681
Hen, development of the embryo of, 511,
512
Hen's egg, 502-512
, incubation of, 510
white of the, 506-510
yoke of the, 502, 506
Heptoses, 105
Herring, spermatozoa of, 63, 498
Heterolysis, 23
Heteroproteoses, 51, 55, 56, 57
Heterosyntonose, 100
Heteroxanthine, 157
in urine, 579
Hexone bases, 63, 96-100
Hexoses, 113-120
See also the various hexoses.
High altitude, action on the blood, 245
Hippokoprosterin, 337
Hippomelanin, 688
Hippuric acid, 585
,properties and reactions^ 587
GENERAL INDEX.
793
Hipp uric acid, estimation of, 578
, formation in the body, 3,
585, 635
, cleavage of, 4, 584, 587, 588
, occurrence of, 585
as sediment, 679, 680
Hirudin, 231, 233
HLstidine, 63, 99, 100, 108
Histidylhistidine, 35
Histone, 27, 36, 61, 100, 208, 229, 270
in urine, 647
Histozyme, 588
Hofmann's tyrosine test, 91
Hog-fat, 423
, absorption of
Hog-flesh, 475
Holothuria, mucin of, 69
Holozyme, 232
Homocerebrin, 482^84
Homogentistic acid, 68, 90, 597-599
Hoppe-Seyler's CO blood test, 206
xanthine test, 160
Horn, 73, 685
Horn substance. See Keratins.
Horse's milk, 529
Human milk, 529-535
, behavior in the stomach,
530, 531
Humin substances in urine, 600
Himior, aqueous, 264
, vitreous, 491
Huppert's reaction for bile-pigments, 322
reaction for bile-pigments in
urine, 653
Hyalines, 68
of the walls of hydatid cysts, 687
of Rovida's substance, 141, 221,
267
Hyalogens, 65, 68
Hyalomucoid, 490
Hydatid cysts, 687
Hydrtemia, 246
Hydramnion, 513
Hydrazones, 107
Hydrobilirubin, 320
, relation to urobilin, 603
Hydrocele fluids, 259, 264
Hydrocephalus fluid, 264
Hydroquinone, 591. 639
Hydroquinone sulphuric acid, 588, 592
Hydrochloric acid, secretion in stomach,
353, 364, 365, 373
, anti-fermentive action
of, 371
, action of, on secretion
of pancreatic juice,
384
, action of, on secretion
of bile, 308, 309
, 'action of, on pylorus,
367
, material of, 371
, quantitative estima-
tion in gastric con-
tents, 375, 376
Hydrochloric acid, reagents for free HCl in
gastric contents, 374
Hydrocinnamic acid, behavior in the ani-
mal body, 585
Hydrocyanic acid, action on peptic diges-
tion, 359
, action on tryptic di-
gestion, 393
Hydrogen in putrefactive and fermentive
processes, 5, 401, 403
Hydrogen peroxide, decomposition of,
• by catalases, 7, 20
Hydrogenases, 19
Hydrolytic cleavages, 9, 16
. See also the various
cleavages.
Hydronephrosis fluid, 542
Hydroparacoumaric acid, 597
, in intestinal
putrefaction,
401
Hydroxylamine, poisoning with, 584
Hyoglycocholic acid, 313
HjTiergluciemia, 298, 299
Hyperisotonic solutions, 193
Hypisotonic solutions, 193
Hypnotics, relation to formation of gly-
cogen, 291
Hypogaeic acid, 138
Hyjjosulphites in the urine, 612
Hyposulphurous acid in urine, 612, 631
Hypoxanthine, 158
, properties, 161, 162
, passage of, into urine, 578
Ichthidin, 504, 509
Ichthin, 509
Ichthulin. 71, 504, 509
Ichthylepidin, 81
Ignotin, 457
Icterus, 307, 333, 652
, urine in.
Immunity, 25, 186
Incubation of the egg, 510
Indican test, Jaffe's, 594
, Obermeyer's, 594
Indican, urine, 592-594
, elimination in starvation, 404,
592, 593
, elimination in disease, 592, 593
Indigo, 402, 592, .594
, in sweat, 695
, in urinary sediments, 680
Indigo blue. See Indigo.
Indigo red, 594
Indigo sulphonic acid, 595
Indol, properties, 402
, formation from protein, 30, 34, 102
, formation in putrefaction, 401, 402,
588, 592
, formation from melanins, 689
, in the blood, 186
Indolacetic acid, 596
Indolaminopropionic acid, 30 102
Indophenol reaction, 18
794
GENERAL INDEX.
Indoxj'^1. See Indol.
Indoxyl-glucuronic acid, 592, 595
Indoxyl red, 594
Indoxyl-sulphuric acid, 592, 595
Inosinic acid, 155, 454, 457
Inosite, properties and occurrence, 458,
459
in urine, 668
, relation to formation of glycogen,
291
Integral factor, 574
Intestinal calculi, 410-412
Intestinal fistula, 377, 380, 399
Intestinal gases, 403, 404
Intestinal juice, 377-379
Intestinal mucosa, 377
Intestine, putrefactive processes in, 399-
408, 586-590
, reaction in, 399, 400, 406-408
, absorption in, 406, 411-427
, digestive processes in, 396-405
Intestine nucleic acid, 152
Intracellular enzymes, 22. See also the
various organs.
Inulin, 127
, relation to formation of glycogen,
290
, relation to the secretion of pepsin,
365
Inversion, 123, 361, 397, 419
Invertases, 16, 185, 361, 378, 419
Invert-sugar, 123
Iodides and secretion of gastric juice, 364
Iodine equivalent, 137
Iodine, passage of, into milk, 539
, passage of, into sweat, 695
. , passage of, into saliva, 347
, action upon protein, 30
, in the blood, 187, 243
, in glands, 27, 271, 275, 276
Iodized proteins, 30, 81, 275, 598
Iodized fats, 442, 538
Iodoform, behavior in the animal body,
631
test. Gunning's, 670
test, Lieben's, 670
lodogorgonic acid, 82
lodohgematin, 213
lodospongin, 81
lodothyreoglobulin, 275
lodothyrin, 275, 276
Ion action, 15, 39, 168, 169, 359, 393, 464
Iron in blood, 238, 239
in urine, 626
in new-born, 286, 535
, elimination of, 325, 332, 347, 626
and blood formation, 244, 245, 503
and bile formation, 332
, absorption of, 244, 245
See also various tissues and fluids
Iron starvation, 738
Isobilianic acid, 315
Isocholanic acid, 317
Isocasein, 520
Isocholesterin, 335, 337, 691, 692
Isocreatinine, 454
Isocysteine, 93
Isodynamic law, 726
Isoglucosamine, 108
Isoiactose, 16
Isoleucine, 87
Isomaltose, 125, 184, 344, 388
in urine, 608
Tsosaccharin, relation to formation of
glycogen, 291
Isoaerine, 96
Isotonic solutions, 193
Isotropovis substance, 447
Ivory, 440
Jaffe's indican test, 594
creatinine test, 566
Janthinin, 691
Japanese, nutrition of, 764
Jaune indien, 122
Jecorin, 147, 273, 283
, in blood, 184
Jequirity bean, 25
Jolles's reaction for bile-pigments, 653
Kathsemoglobin, 208
Kephir, 524, 528
, anti-putrefactive action, 405
Kephir lactase, 524, 610
Kerasin, 482, 283, 484, 488
Keratose, 73
Keratins, 37, 73-75_, 685
, behavior in the stomach, 360
, behavior with pancreatic juice,
396
Ketones, behavior in the animal body, 632,
637, 638
Ketoses, 105, 113, 119
Kidneys, 541
, relation to formation of urea, 553
, relation to formation of hippuric
acid, 586, 587
Kinases, 16,176,231,232,233,379,383,386
Kjeldahl's method of determining nitro-
gen, 556
Knapp's titration method, 662, 663
Knee-joint cartilage, 364-434
Rnop-Hiifner's method for determining
urea, 562
Koprosterin, 335, 337, 408
Krinosin, 486
Kumyss, 524, 528
Kyestein, 680
Kynurenic acid, 597, 600
Kyrins, 58, 59
Kyroprotic acids, 31
Laborer, diet of, 763-770
Laccase, 18
Lactacidase, 21 *
Lactalbumin, 36, 522, 523, 526
Ijactase, 524
in the intestine, 379, 419
in the pancreas, 386
Lactates. See I^actic acids, also 461-463
GENERAN INDEX.
795
Lactic-acid fermentation, 110, 116, 371,
373, 389, 400,
460, 516, 524
in intestine, 397
399
in stomach, 371,
373
inniilk,515,516,
523, 524
Lactic acids, 460
, in intestine, 397, 400
, in urine, 460, 572, 608
, in bones, 439
, in stomach, 353, 374
, relation to formation of uric
, acid, 572, 573
. See also Paralactic and Fer-
mentation lactic acids.
Lacto-caramel, 524
Lacto-globulin, 522
Lactolase, 21
Lactone of saccharic acid, 121
Lactones of varieties of sugars, 105, 106
Lactophosphocarnic acid, 523
Lactoprotein, 523
Lactose. See Milk-sugar.
Laiose, 665
Lakey color of blood, 225
Lamb, intestinal fluid in the, 377
Lanoceric acid, 692
Lanocerin, 692
Lanolin, 337, 642
Lanopalmitic acid, 692
Lanugo hair, 512
Lard, absorption of, 424
Large intestine, extirpation of, 426, 427
, secretion of, 380
Latebra, 502
Laurie acid, 131, 138, 51S
Lead in the blood, 239
in the hver, 286
passage of, into milk, 539
Lecithalbumins, 47, 349, 541
, relation to secretion of
gastric juice, 349
, relation to secretion of
urine, 541
Lecithans, 488
Lecithins, 143, 144
, in egg-yolk, 502, 503
, in the brain, 480, 488
, in the muscles, 463
, in milk, 518, 532
, in the liver, 283
, importance for cells, 143, 193
, putrefaction of, 146, 177, 404
Legal's acetone reaction, 671
Lens, (see Crystalline lens), 492
, capsule of, 69, 492
, fibres of, 492
Leo's sugar, 665
Lepidoporphyrin, 690
Lepidotic acid, 690
Lethal, 138
Leucaemia, blood, 158, 247
, uric acid, elimination in, 274,
569, 570
, purine bases in, 158, 247,
578
Leucin, 85-87
, relation to formation of uric acid,
572
, relation to formation of urea, 550,
551, 630
, passage of, into urine, 614, 675
, behavior in the animal body, 550,
551, 630
Leucin ester, 86, 87
Leucin ethylester, 87
Leucinic acids, 86
Leucinimide, 87
Leucocytes, relation to absorption, 416
, relation to formation of uric
acid, 570
, in thymus gland, 272
Leucomaines, 24
, in urine, 615
, in muscles, 457
Leuconuclein, 229, 270
Leucylalanyl-glycine, 35
Leucyl-1-tyrosine, 35
Levolactic acid, 460
Levuline, 127
Levulinic acid, 67, 113, 152, 524
Levulose, 106, 108, 118, 119
, in urine, 664
, in blood, 184
, relation to glycogen formation,
293
, absorption of, 419, 420
, behavior in diabetics, 300
, in transudates, 260, 512
Lichenin, 127
Lieben's acetone reaction, 670
Lieberkiilm's, alkali-albuminate, 48
, glands, 377
Liebermann's reaction for proteins, 43
liiebermann-Burchard's reaction for cho-
lesterin, 336
Liebig's titration method for estimating
urea, 557-560
Lienases, 273
Ligamentum nuchce, 75, 76, 429
Lignin, 129
Linoleic acid; 131, 135
Linolic acid, 504
Linseed-oil, feeding with, 442, 538
Lion's urine, 567
Lipanin, absorption of, 423
Lipase, 16
in blood, 185
in stomach, 363
in the intestine, 379, 380
in the liver, 284
in pancreatic juice, 384, 388
in milk, 523
Lipiawsky's acetoacetic acid reaction,
672
796
GENERAL INDEX.
Lipochromes, 186, 505
Lipoids, 193
Lipuria, 675
Lithium, in blood, 166, 239
Lithium lactate, 463
Lithium urate, 575
Lithobilic acid, 411
Lithofellic acid, 318, 411
Lithuric acid, 616
Liver, 280-287
, relation to coagulation of blood,
172, 234, 235
, relation to formation of uric acid,
571, 572, 573
, relation to formation of urea, 550,
551, 552, 554
, blood of, 242, 296, 297
, proteids of, 286, 287
, fat of, 282, 283
, quantity of sugar in, 295, 296, 297
Liver atrophy, acute yellow, 23, 284
, elimination of amino acids
in, 284, 285, 675
, elimination of ammonia in,
554
, elimination of urea in, 554
, elimination of lactic acid
in, 461, 608, 609
, autolysis, 23, 284
Liver cirrhosis, ascitic fluid in, 262, 263
, action of, on the elimina-
tion of ammonia and
urea, 554
Liver extirpation, elimination of ammonia
with, 554, 572
, elimination of uric acid
with, 572, 573
, elimination of urea with,
554
, elimination of lactic
acids with, 460, 572,
608
, action on formation of
bile, 330
Lotahistone, 62
Lung catheter, 708
Lungs, 703, 704, 713, 714
Luteins, 505
in corjjora lutea, 216, 498
, egg-yolk, 505
in blood-serum, 180
, relation to hsematoidin, 216, 505
Lymph, 250-256
Lymphagogues, 255
Lymphatic glands, 269
Lymph-cells, quantitative composition of,
272
. See also Leucocytes.
Lymph-fibrinogen. See Tisaue-fibrinogen.
Lysalbinic acid, 49
Lysatine and lysatinine, 99
Lvsinp. 33, 64. 98, 99, 186
Lysines, 25, 186, 193
Lysuric acid, 98
Lysyllysine, 35
Mackerel, flesh of, 475
, sperm of, 62, 63
Madder, feeding with, 438
Magnesium in urine, 625, 628
in bones, 436, 440
m muscles, 464, 475, 477
. See also various tissues and
fluids.
Magnesium phosphate in intestinal calculi,
411
in urine, 619, 625,
628
in urinary calculi,
680, 681, 684
in urinary sedi-
ments, 677, 679
in bones, 436, 440
Magnesium soaps in excrements, 408
Malic acid, behavior in the animal body,
544
Maltase, 16, 125, 345, 346, 388
Maltodextrin, 128
Maltoglucase, 21, 185, 344, 346
Maltose, 123, 124
, formation from starch, 124, 127,
344, 388
, absorption of, 419
, relation to glycogen formation,
293
in intestine, 397, 419
, occurrence in urine, 667
Mammary glands, 514, 537
Mandelic acid, 634
Man in poorhouse, diet of, 770
Mannite, 106
, relation to formation of glycogen,
291
Mannonic acid, 114
Mannose, 108, 113, 114, 118
Mare's milk, 530
Margarine and margaric acid, 134
Marsh-gas, formation in putrefaction, 30,
401, 404
Martamic acid, 154. See also Methane.
Maschke's creatinine reaction, 565
Meat extracts, action on secretion of gas-
tric juice, 365
, constituents of, 454, 456
Meat, utilization in intestinal tract, 417
, calorific value of, 724-726
, digestibility of, 368
, composition of, 443, 444, 474-476
. See also muscles.
Meconium, 410
Medulla oblongata, 486
Melanins, 154, 688-690
in the eye, 491
in the urine, 651
Melanogen in the urine, 651
Melanoidic acid, 688
Melanoidins, 27, -30, 154
Melanotic sarcoma, pigment of, 688
Melissyl alcohol, 138
Membranins, 69, 434, 492
Menstrual blood, 187, 242
GENERAL INDEX.
'9'i
Menthol, behavior in the anim:.! body, 638
Mercaptan, from proteins, 30, 33, 73, 401
Mercapturic acids. 63S
Mercury salts, passage of, into milk, 539
, passage of, into sweat, 695
, action on ptyalin, 346
, action on trj-psin, 393
Mesitylene, behavior in the animal body,
635
Mesitylenic acid, 635
Mesitylenuric acid. 635
MesoporphjTin, 214
Metaca.sein reaction, 521
Metabolism, dependence of external tem-
perature upon, 761, 762
in various ages, 755-758
in work and rest, 467-474,
758-761
in different sexes, 756
in starvation, 728-733 '
with different foodstuffs, 739-
754
in sleep and waking, 761
calculation of extent of, 720,
723
Metalbumin, 499, 500
Metallic sols, 14
Metaphosphoric acid, as reagent for pro-
teins, 41, 642
Metazym, 232
Methffimoglobin, 203, 218
in urine, 648
Methal, 138
Methane, formation in putrefaction, 30
401, 404
Methose, 114
Methylenitan, 114
Methvlethvlmaleic acid anhvdride, 211
Methylfurfurol. 336
Methyl glycocoU. Ree Sarcosin.
Methylguanidine, 455, 457, 565
Methylguanidin-acetic acid. See Crea-
tine.
Methylhydantoic acid, 631
Methyl iudol. See Skatol.
Methylimidazol. lOS
Methyl mercaptan in proteins, 30, 401,
404
Methyl pentose. See Rhamnose.
Methyl p\Tidjne, behavior in the animal
body. 634, 637
Met hvl-i ) vridvl-amraoniiun hydroxide,
639
Methylthiophene, 336
Methyl ura mine, 455, 457, 565
Methyl xanthine. 157, 579,
Micrococcus restituens, 415
Micrococcus urea, 677
Micro-organisms in intestinal tract, 24,
371, 398, 399, 405, 408
Milk, 515-542
, secretion of, 536, 537
, consumption of, in intestine, 417,
425. 530, 531
, blue, 540
Milk, anti-putrefactive action in intestine,
405, 589
in disease, 5.39
, passage of foreign bodies into, 538
, behavior in the stomach, 367, 371,
530, 531
. See also various kinds of milk.
Milk-fat, 517, 5.30
, formation of, 537, 538
Milk-globules from cow's milk, 516, 517
from human milk, 530
Milk-plasma, 518
Milk-sugar, 135, 524
, relation to formation of gly-
cogen, 293
, properties of, 524,
, fermentation of, 524
, calorific value of, 724
, quantitative estimation of,
527
, absorption of, 419
, passage of, into unne, 293,
420, 524, 665
, origin of, 538
Millon's reagent, 42, 43
Mineral acids, alkali-removing action of,
544, 624
, action on the elimination
of ammonia, 544, 624
Mineral bodies, elimination in starvation,
620, 623, 731
, insufficient supply of, 734-
738.
See also the various
fluids, tissues, and
j uices.
Modified proteins, 40
Molisch's sugar test, 117
Monaniino acids, 83-96
behavior in animal body,
305, 462, 550, 572,
614, 630
Monosaccharides, 105-123
Moore's sugar test, 115
Morner-Sjoqvist's method of estimating
urea, 561
method of estimating
acidity, 376
Momer's tjTOsins test, 89. See also
Denige's.
Morphine, passage of, into urine, 608,
639
, passage of, into milk, 539
Mucic acid, 120, 128, 524
, relation to formation of gly-
cogen. 291
Mucilages, vegetable, 129
Mucin, 36, 65-68
in sputum, 714
in cysts. 501
in urine, 615, 647
in .salivarj- glands, 339, 340
Mucin-like substances in bile, 309, 310,
329
in urine, 615, 646
798
GENERAL INDEX.
Mucin-like substances in kidneys, 541,542
Mucinogen, 66, 340, 509
Mucinoids. See Mucoids.
Mucin peptone, 67, 360
Mucoids, 36, 65, 68
in ascitic fluids, 262
in the vitreous humor, 491
in the cornea, 434
in connective tissue, 428-430,
435
in the hen's egg, 506, 508
in cysts, 498-501
Mucoproteoses, 360
Mucous glands, 66, 339, 348, 380
Mucous membranes of the stomach, 348
Mucous tissue, 430
Mucus of the bile, 310, 327
of the urine, 542, 615, 647
of synovial fluid, 265
Mulberrj^ calculi, 681
Murexide test, 575
Muscle, coagulation of. See Muscle-
plasma.
, chemical tones of, 467
, permeability of, 465
Muscle-fibres, 447, 465
, permeability, 465
Muscle-pigments, 453
Muscle-plasma, 448, 449, 452, 453
, coagulation of, 448, 452,
453, 466, 477
Muscle rigor, 465
Muscle-serum, 448
Muscle-stroma, 451
Muscle-sugar, 459
Muscle-syntonin, 451
Muscles, Bowman's disks, 448
, non-striated, 477
, striated, 447-477
, blood of, 242, 468
, chemical processes in work and
rest, 467-474, 759
, chemical processes in rigor, 465
, proteins of, 447-454, 466, 470
, extractives of, 453-464
, enzymes of, 453
, pigments of, 453
, fat of, 463. 472-474
, gases of, 465, 468
, calorific value of, 725-727
, mineral bodies of, 464, 477
, amount of water in, 476
, composition of, 474-477
Muscular energy, origin of, 472-474
Musculamine, 457
Muscular force, chemical processes in mus-
cles, 467-474
, action of, on urine, 544,
564, 567, 608, 616
, action of, on metabolism,
470-474, 758-761
Musculin, 450, 452, 478
Mussels, glycogen of, 287
, muscles of, 477
Mutton-fat, feeding with, 442
Mutton-fat. absorption of, 423, 424
Myeline forms. 481
Myelines, 481, 488
Myoalbumin, 449, 451
Myogen, 453
Myogen fibrin, 449, 453, 466
Myoglobulin, 449, 451
Myohaematin, 454
Myoproteid, 452, 453
Myosin, 36, 221, 449, 450, 452, 466
, absorption of, 413
Myosin ferment, 452, 453
Myosin fibrin, 449, 452
Myosinogen, 452
Myosinoses, 52
Myricin, 138
Myricyl alcohol, 138
Myristic acid in animal fat, 131
in butter, 518
in bile, 325
in wool-fat, 692
Mytolin, 451
Myxfjedema, 276
Myxoid cysts, 498
Nails, 73, 685
Naphthalene, action on urine, 639
, behavior in the animal body,
633
Naphthalene sulphocliloride as reagent,
675
Napthalene sulpho derivatives of amino
acids, 92, 675
Napthol-glucuronic acid, 639
Napthol, reagent for sugar, 117, 659
, behavior in the animal body,
609, 639
Napthyl isocyanate compound of the
amino-acids, 92
Narcotics, relation to glycogen formation,
291
Native proteins, 40
Navel cord, mucin of, 66, 67, 429
Negative phase, 234
Neosine, 457
Neossin, 69
Neozym, 232
Nerves, 479, 480, 488
Neuridine, 481, 486. 502
Neurine, 145, 277, 485, 615
Neurochitin, 489
Neuroglia, 480
Neurokeratin, 73, 480, 488, 489
Neutral fats. See Fats.
Nicotine action upon the gases of the
stomach, 372
Nitrates in the urine, 623
Nitric-oxide htemoglobin, 207
Nitriles, behavior in the animal body, 631
Nitro-benzaldehvde, behavior in the ani-
mal body, 636
Nitro-benzene, 635
Nitro-benzoic acid, 636
Nitro-benzyl alcohol, 638
Nitro-celluiose, 130
GEXEPwlL INDEX.
799
Nitro-hippiiric acid, 636
Nitro-phenol, 635
Nitro-phenyl-propiolic acid, reagent for
sugar, 117,
6.59
, behavior in
the animal
body, 592,
595
Nitro-toluene sulpho-compounds of amino
acids, 92
Nitro-tolulene, behavior in the animal
body, 638
Nitrogen, combined, quantity of, in intes-
tinal evacuations,
717, 718
, in meat, 443, 476
, in protein bodies, 27
, m urine, 548, 549
, estimation of, in
urine, 5.56-561
Nitrogen deficit, 718
Nitrogen elimination in work and rest,
470-473, 7.58, 7.59
in starvation, 728-
730
with various foods,
739-752
through the intes-
tine, 417, 418,
717, 718
through the urine,
548, 549, 611,
612, 620, 622,
717-719
through the epider-
mis, 718
through the sweat,
694; 718
, relation to the elim-
ination of phos-
phoric acid, 620
, relation to the elim-
ination of sul-
phuric acid, 622,
719
, relation to digestive
activity, 624,
717, 718, 762
Nitrogen, free, in blood, 696
, in intestine, 403
, in .stomach, 372
, in secretions. 702
, in transudates, 702
, in urine, 626
Nitrogen, residual, 183
Nitrogenous equilibrium, 718. See also
Chap. XVIIl
Nitroso-indol nitrate, 402
Non-striated muscles, 477
Non-biuret giving products, 54, 394, 417
Norisosaccharic acid, 121, 504
Nova in. 457
Nubecula, .542, 615
Nucleases, 153, 271, 391
Nucleic acids, 71, 1.50-151, 1.56, 195
in the urine, 647
Xuclein bases, 156-164
in blood, 1.58, 186
in the m-ine, 578
Nucleins, 72, 149
, relation to elimination of allox-
uric bases, 578
, relation to formation of uric
acid. 570, 571
, relation to elimination of P.Or,
619, 620
, behavior with gastric juice, 72,
149, 150, 3.50
, behavior with pancreatic juice,
394, .395
Nuclein plates, 222
Xucleoalbvunins, .36, 46, 141, 1.50
in the bile, 310, 329
in the liver, 281
in the urine, 646
in the kidneys, .541, 542
in protopla.sm, 141
in transudates, 2.58, 261
, behavior in pepsin diges-
tion, 46, 150, 522, 532
Nucleoglucoproteids, 71, 72
Nucleohistone, 62, 221, 269
, relation to coagulation of
blood, 229
in urine, 647
Nucleoproteids, 36, 71, 72, 142, 149
in the liver, 282
in gastric juice, 354, 355
in blood. 172, 178
in bile, 329
in mammarj' glands, 514
in muscles, 451, 477
in the kidneys, 541
in the pancreas, 149, 150,
381
in protopla.sm, 142
in cell nucleus, 142, 149
in thyroid gland, 275
in thymus, 270
, behavior in pepsin diges-
tion, 71, 1.50
, behavior with pancreatic
juice, 395
Nucleotin, 154
Nucleotin phosphoric acid, 154
Nucleon, 457, 477
in milk, 523, 532
Nucleosin, 165
Nutrition requirements, 763-771
, of man, 7.39-7.53
Nylander's reagent. See Almen-Bottger's
sugar test.
Obermeyer's indican test, 594
Obermuller's chole-sterin reaction, 337
Oblitin, 457
Odoriferous bodies in the urine, 586
CEdema, subcutaneous, fluid from, 265
Oertel's diet cure, for corpulency, 770, 771
800
GENERAL INDEX.
Oesophageal fistula, 350
Oleic acid, 135
Olein, 134
Oleodistearin, 132
01iga?mia, 246
Oligocythseniia, 246
Oliguria, 628
Olive oil, absorption of, 423
, action on the secretion of bile,
308, 309
Onuphin, 69
Oocyanin, 509
Oorodein, 509
Opalisin, 523, 531
Opium, passage of, into the milk, 539
Optograms, 491
Orcin test, 111, 666
Organic acids, behavior in theanimal body,
624, 629-631
Organized proteids, 742, 743
Organs, distribution of the blood in, 249
loss of weight in starvation, 732
Organs of generation, 495-513
Ornithine, 97, 635
Ornithuric acid, 97, 635
Orotic acid, 523
Orthonitrophenylpropiolic acid. See Nit-
rophenylpropiolic acid.
Orylic acid, 523
Osaminic acid, 107
Osamines of varieties of sugar, 107
Osazones, 107
Osmosis, relation to absorption, 427
, relation to lymph formation,
255, 256
Osmotic pressure of blood, 189, 159
of urine, 546
Osones, 107
Ossein, 76, 435
Osseoalbvmioid, 435
Osseomucoid, 66, 435
Osteomalacia, 438, 439
Osteoporosis. See Osteosclerosis.
Osteosclerosis, 438
Otoliths, 494
Ovalbumin, 32, 507
, relation to glycogen formation,
290
Ovarian cysts, 498-502
Ovaries, 498
Ovoglobulin, 33, 506
Ovomucoid, 68, 508
Ovomucin, 506
Ovovitellin, 36, 503
Ovum, 502-512
Oxalate calculi, 680, 681
Oxalate of lim.e. See Calcium oxalate.
Oxalates, action on blood coagulation, 171,
225
Oxalic acid, in the urine, 582,583,678,680
behavior in the animal body
582, 783, 629
Oxaluric acid, 568, 582
Oxaluric-acid amide, 32
Oxamide, 29, 32
Oxaminic acid, 32
Oxidases, 8, 16-19, 185
. See also the tissues and fluids.
Oxidation ferment. See Oxidases.
Oxidations, 3-9, 17-19, 629-631, 633, 703
in diabetes, 300
Oximes, 106
Oxonic acid, 568
Oxvacids, formation in putrefaction, 30,
401
, detection of, 597
, passage of, in \n-ine, 401, 596
, in the sweat, 694
Oxybenzoic acid, behavior in the animal
body, 635, 636
Oxybenzenes, 633
Oxbutyric acid, 668, 072, 673
, detection and estimation,
673-675
in the blood, 702
, passage of, into the urine,
625, 668, 669, 672, 673
Oxydiaminosuberic acid, 1.9, 33
Oxydiaminosebacic acid, 282
Oxyethylsulphonic acid, behavior m the
animal body, 632
Oxyfatty acids in animal fat, 131
Oxygen, consumption, 199, 704, 705
in work and rest,
468, 472
in starvation, 729,
730
through the skin,
695
Oxygen, activity of, 3-8
in the blood, 697, 704-709
in the intestine, 403
in the lymph, 251, 702 '
in the stomach, 372
in the swimming-bladder of
fishes, 710
in secretions, 702, 703
in transudates, 703
, tension of, in blood, 203, 698
, lack of action on protein destruc-
tion, 461, 569, 611
, lack of, action on elimination of
lactic acid. 461, 469, 608
, lack of, action on elimination of
sugar, 461
Oxygen capacity, specific, 711, 727
Oxygen-carriers, 7, 648, 649
Oxygen, calorific value in the combustion
of different foo s, 623-624, 723
Oxygen, consumption in the blood,
Oxygenases, 17
Oxyhfematin, 210
Oxyhspmocyanin, 219
Oxyhaemoglobin, 198
dissociation of, 198-200,
704, 705
, properties and reactions,
198-202
, quantity of, in the blood,
196, 197, 239, 241-246
GENERAL INDEX.
801
Oxyhsemoglobin, quantity in the muscles,
45 -i
, passage of, into the urine,
64S
, behavior with gastric
juice, 360
, behavior with trypsin,
Oxj'^hydroparacoumaric acid, 597
Oxymandelic acid, 597, 600
Oxymonamino acids, 29, 33, 95
Oxymonaminosuberic acid, 29, 96
Oxymonaminosuccinic acid, 29, 96
Oxynaphthalene, 632
Cxyphenyl-acetic acid, 90, 401,596,597,
637
C xjTjhenylaminopropionic acid. See Ty-
rosine.
Oxyphenylethjdamine, 30, 54
Oxyphenylpropionic acid, 401, 597, 637
Oxyproteic acid in urine, 612, 613
Oxj'^proteins, 31
Oxyprotosulphonic acid, 31
Oxji^yrrolidincarboxyUc acid, 34, 102
OxyquinoHne, 638
OxyquinoUnecarboxylic acid, 600
Ozone, 3
Ozone traasmitter, 201
Palmitic acid, 134
Palmitin, 134
Pancreas, 381
, relation to glycolysis, 185, 302,
396
, extirpation of, action on absorp-
tion,
, extirpation of, elimination of
sugar, 418, 421, 425
, pepsin, 385
, change during secretion, 381
Pancreatic calculi, 396
Pancreatic diabetes, 301, 302
Pancreatic diastase, 388
Pancreatic protein, 150, 381
Pancreatic rennin, 396
Pancreatic casein, 396
Pancreatic juice, 382-388
, secretion of, 382-385
, enzymes of, 386
, action on foodstuffs, 387-
395
, action upon peptides, 35,
396
Parabamic acid, 568
Paracasein, 522
Parachyrao.sin, 361, 362
Paracresol, formation in putrefaction, 401,
588
Paraglobulin. See Serglobulin.
Paraglycocholic acid, 313
Parahaemoglobin, 201
Parahistone, 63
Paralactic acid, 460
, relation to formation of
uric acid, 572, 573
Paralactic acid, properties and occurrence,
460-463
, formation from glycogen,
461, 466, 467
, formation in osteomalacia
bones, 439
, formation in muscle dur-
ing work, 468-470, 472,
473
, formation in rigor mortis,
467
, formation in lack of oxy-
gen, 460, 461, 469
, formation in animals with
extirpated livers, 460,
461, 572
, passage of, into the urine,
460-462, 572, 608
Paralbumin, 275, 500
Paralytic saliva, 340
Paraminophenol, 633
Paramucin, 500
Paramyosinogen, 450, 452
Paranuclein. See Pseudonuclein.
Paranuclsic acid, 522
Paraox^T^henylacetic acid, 90, 401, 5C6,
597, 637
Paraoxvphenvlaminopropionic acid, 89,
401, o96, 597, 637
Paraoxj^aropiophenone, behavior in ani-
mal body, 638
Parapeptone, 359
Paraxanthine, 157, 580
in urine, 578, 580
Parenterally introduced protein, 412
Parietal or delomorphic cells, 349, 364
Parotid, 339
Parotid saliva, 341
Parovarial cysts, 501
Partition of the nitrogen in the urine, 548,
549, 568, 569, 611
Peas, utilization in the intestine, 421
Pemphigus chronicus, 265
Penicillium glaucum, 86
Pennacerin, 692
Pentacrinin. 691
Pentamethylendiamine. See Cadaverin.
Pentosanes, 110
digestion of, 427
Pentoses, 110
, relation to glycogen formation,
111, 290
in blood, 184
in urine, 110, 666
in pancreas, 110
in nucleic acids, 152, 155
in nucleoproteids, 72, 110, 285,
514
Penzoldt, acetone reaction, 671
Pepsin, 354-361
, detection in gastric contents, 373
, quantitative estimation, 357
, occurrence in the urine, 426, 615
Pepsin charge, in the stomach, 364
, in the pancreas, 385
802
GENERAL LXDEX.
Pepsin cells, 349
Pepsin digestion, 356-362
, products of, 52, 53, 359
Pepsin glands, 348
Pepsiu-glutin peptone, 57
Pepsin-hydrochloric acid, 360, 361
Pepsin-like enzyme, 354
Pepsinogen, 364
Pepsin peptones, 51, 57
Pepsin test, 356
Peptides, 35, 54, 394, 395, 416, 614
, relation to trypsin, 35, 395
Peptochondrin, 433
Peptones, 29, 30, 36, 49-61, 359
, assimilation of, 413-417
, absorption of, 414, 415
, passage of into m'ine, 414, 643
Pepto'.e blood, 235
Peptone-plasma, 171
Peptozym, 234
Percaglobulin, 510
Perch eggs, 66, 504, 509, 510
Pericardial fluid, 257, 260
Perilymph. 494
Peritoneal fluid, 257, 261
Permeability, of the blood-corpuscles, 195,
196
of the vascular walls, 257,
258
of the muscles, 465
Peroxidases, 17, 18
See also the tissues and
fluids.
Peroxyproteic acid, 31
Perspiratio insensibilis, 717
Perspiration, 692-695
Pettenkofer's test for bile-acids, 135, 312,
652
respiration apparatus, 712,
Phacozymase, 493
Phaseomannite, 458
Phenaceturic acid, 588, 634, 635
Phenol-glucuronic acid, 590, 610, 638
Phenol-svilphuric acid in the urine, 589,
592, 637
in sweat, 594
Phenols, elimination by the urine, 401, 588
-591, 633, 637, 638
in starvation, 404
, estimation in urine, 590, 591
, formation in putrefaction, 30,
401, 588
behavior in the animal body,
401, 402, 588, 589, 637, 638
Phenylacetic acid, formation in putrefac-
tion, 30, 401
, behavior in the ani-
mal bodv, 588, 634,
635
Phenylalanine, 33, 91
, behavior in the animal
body, 5S5, 633
, in alcaptonuria, 597-599
Phenylaminoacetic acid, behavior in the
animal body, 634
Phenylaminopropionic acid, 91
Phenylbutyric acid, 634
Phenylglucosazone, 107, 117
Phenylhydrazine test, 107, 117
in the urine, 657
Phenylketopropionic acid, 633, 634
Phenyllactic acid, 598, 633, 635
Phenyllactosazone, 524
Phenylpropionic acid, behavior in the
animal body, 585, 634
Phenvlpropionic acid, formation in pu-
trefaction, 30, 401, 585
Phenylvalerianic acid, 634
Plilebin, 196
Plilorhizin, poisoning with, 282, 297, 610,
670
Phlorhizin diabetes, 297, 610
Phloroglucin as reagent. 111, 374, 666
Phosphate calculi, 681
Phosphates in urine, 543, 619-622, 640,
677-682
. See also the different phos-
phates 143 144
Phosphatides, 146, 325, 328^ 480, 481, 488
Phosphaturia, 620
Phosphocarnic acid, 454, 457
in the milk, 523,
532
in blood, 186
in brain, 480, 481
in the urine, 614
in relation to the
elimination of CO,
and lactic acid,
462
in relation to muscu-
lar activity, 470,
474
Phosphoglucoproteid, 71, 509
Phosphoric acid, elimination by the urine,
617-622, 625, 628
, formation in muscular
activity, 470
, quantitative estimation
of, 620-622
Phosphorized combinations in the urine,
614
Phosphorus poisoning, action on the elim-
ination of am-
monia, 554
, action on the elim-
ination of urea,
548, 554
, action on the elim-
ination of lactic
acid, 460, 462,
608
, action upon the
blood, 172, 175
, fatty degeneration
caused by, 282,
283, 443
, liver autolysis in,
23, 283, 284, 285
GENERAL INDEX.
803
Pnosphorus poisoning, change in the urine.
285, 460, 54S
554, 613
Photomethaemoglobin, 205
Plirenin, 486
Phrenosin, 483, 485, 488
Phthalic acid, behavior in the body, fi33
from choHc acid, 315
PhthaUmide malonic ester, 102
PhyUocyanin, 214
Phylloporjihyrin, 197, 214
Phylloerythrin, 324
Phymatorusin, 688
in the urine, 651
Physetoleic acid, 138
Pliysiological availability, 727
Phytosterines, 335
a-Picoline, behavior in the animal body,
637
Picric acid, reagent for protein, 42, 646
, reagent for creatinine, 565
567
, reagent for sugar, 117, 56G
Pigment calculi, 334
Pigments of the eye, 489-491
of the blood, 196-219
of the blood-sermn, 186, J8f-
505
of the corpora lutea, 216, 49S
of the egg-shell, 509
of feathers, 690
of the fat-cells, 441
of the bile, 319-325, 328, 331
of the urine, 600- 607
of the skin, 688-691
of the lobster, 509, 690
of the liver, 282
of the muscles, 453, 454
of lower animals, 218, 219
, medicinal pigments in the urine,
639, 654
Pigmentary acholia, 329
Pig's milk, 529
Pike, flesh of, 476
Pilocarpine, action on the secretion of in-
testinal juice, 377
, action on the elimination of
CO^ in the stomach, 372
, action on the secretion of
saliva, 347
, action on the elimination of
uric acid, 569
Piqure, 299
Piria's tyrosine test, 90
Placenta, 512
Plant gums, 128, 129
Plant nucleic acids, 156
Plants, chemical processes in, 1, 2
Plasma. See Blood-plasma.
Plasminic acid, 156
Plasmoschisis, 227
Plasmozym, 232
Plastein, 56, 363, 396
Plasteinogen, 57
Plastin, 149
Plattner's cr^'stallized bile, 311
Plethora polycythaimia, 245
Pleural iiuid, 257, 261
Plums, action on the elimination of hip-
puric acid, 585
Pneumonic infiltration, solution of, 23, 268,
713
Poikilocytosis, 247
Polarization test, 664
Polycythicmia, 245, 248
Polypeptides. See Peptides.
Polyperythrm, 691
Polysaccharides, 126
Polyuria, 628
Pons varolii, 486
Poorhouses, diet of inmates of, 770
Pork, 475
Pork-fat, absorption of, 423
Portal vein, blood of, 241, 295, 296, 419
Positive phase, 234
Potassium combinations, division of, in
the form-ele-
ments and
fluids, 166,
167, 464
, elimination of,
in fevers, 623
, elimination of
in starvation,
623, 731
, elimination by
the saliva, 847
in the urine, 623
Potassiiim chlorate, poisoning with, 203
Potassimn phosphate in yolk of eggs, 505
in muscles, 464, 465,
477
in cells, 166, 167,
168
in spermatozoa, 496
Potassium sulphocyanide in the urine, 611
in saliva, 341,
343
in gastric con-
tents, 354
Potatoes, absorption of, in the intestine,
421
Potential energy of various foods, 724-728
Precipitins, 186, 413
Preglobulin, 141, 228, 271
Preputial secretion, 692
Primary proteoses, 52
Prisoners, food-ration for, 770
Proliferous cvsts, 498
ft-Proline, 34", 74, 101
Prolineglycyl anhydride, 35
Propepsin, 364
Prope}>tones, 50
Propylalanine. 35
Propyl benzene, behavior in the animal
body, 634
Propylene glj'^col, relation to formation of
glycogen, 291
Prosecretin, 379, 384
Prostatic calculi, 498
804
GENERAL INDEX.
Prostatic secretion, 496
Prosthethic group, 71
Protagon, 148, 271, 480, 481, 482
Protalbinic acid, 49
Protojiroteoses, 51
Protamines, 36, 58, 62, 63, 149, 497, 498
.Proteids, 36, 37-65.
, See also tlie various protein
groups.
Protein, separation from fluids, 44
, approximate estimation in the
urine, 646
, circulating and tissue protein,
741-745
, action on the formation of gly-
cogen, 292, 294, 303-305
, active, 4
, living and non-living, 4
, detection and quantitative esti-
mation of, 43-45, 639-646
, regeneration of, 415, 416, 417
, absorption of, 412-418
, passage of, into the urine, 640
, heat of combustion of, 723-726
, digestibility in gastric juice, 356,
368
, digestibility in pancreatic juice,
393
, formation of sugar from, 303-305
Protein bodies in general, 26-83
, summary of the various,
36, 37
. See also the various pro-
tein bodies of the tissues
and fluids.
Protein hydrogele, 39, 40
Protein hydrosole, 39, 40
Protein content affected by inoculation,
188
Protein metabolism in work and rest, 471-
475, 758 _
in starvation, 728,
729
in various ages, 757,
758
with different foods,
739-745
ante-mortem increase,
730
after feeding with
thyroid extracts,
276 _
Protein overfeeding, 751, 752
Protein putrefaction, 30, 401-408, 585,
588, 589
Protein, relation to the albuminates, 49
Proteincystine, 92
Proteinochromogen, 30, 102
Protein substances, 26-82
, synthesis of, 34, 35
, action upon coagula-
tion of the blood,
233
See also individual
protein bodies.
Proteoses, 36, 52
, general properties and prepar-
ation, 50-61
in blood, 183, 247, 415
, formation in protein putrefac-
tion, 50, 401, 415, 416
, relationship to the coagiilation
of the blood, 171, 226, 234,
235
, nutritive value, 745, 746
, absorption of, 414-417
, transformation into protein, 415
, occurrence in urine, 643
Prothrombin, 176, 231, 232, 233
Protic acid, 454
Proteolytic enzymes, 16, 263
Protocatechuic acid, behavior in the body,
591
Protoelastose, 76
Protogelatose, 79
Protogen, 49
Protokyrin, 58
Protones, 63
Protoplasm, 4, 140, 141
and protein decomposition
of, 548
Protosyntonose, 100
Pseudocerebrin, 485
Pseudochvlous fluid, 262
Pseudoglobulin, 179, 643
Pseudoglycogen formers,
Pseudohsemoglobin, 203 293
Pseudolevulose, 108
Pseudomucin, 68, 500
in ascitic fluids, 262
in cysts, 500
in the gall-bladder, 329
Pseudonucleins, 47, 151, 359,
from casein, 521, 531
from vitellin
Pseudopepsin, 354
Pseudotagatose, 108
Pseudoxanthine, 457
Psittacofulvin, 690
Psylla-alcohol, 692
Psyllic acid, 692
Psychical period of secretion, 350
Ptomaines, 24
in the urine, 615, 676
Ptyalin, 343, 344
, behavior with acid, 345, 366
, action on starch, 344-346
, tests, 345, 346
Pulmotartaric acid, 713
Purine, 157
Purine bases, 156, 578
, See also Nuclein bases.
Purple, 691
Purple cruorin, 202
Pus, 266-269
, blue, 269
cells, 267
in urine, 651
corpuscles, 267
serum, 266
GENERAL INDEX.
805
Putrefactive processes, 2.5, 30
in intestine, 400-
40S, 585, 588-
596
Putrescine, 24, 97
in intestine, 24, 676
in the urine, 615, 676
Pyin, 261, 266, 269
Pyinic acid, 269
Pyloric gland, 348
Pyloric secretion, 365
Pyocyanin, 269
in sweat, 695
Pyogenin, 268, 483
Pyosin, 268, 483
Pyoxanthose, 269
Pyridine, beliavior in the body, 639
a-Pyridine-carboxylic acid, 632, 636
Pyrocatechin, 591
, occurrence in urine, 591
, occurrence in transudates,
259, 265
Pyrocatechin-sulphuric acid, 591
Pyromucic acid, 637
Pyromucinornithuric acid, 637
Pyrrol derivatives, 210, 689
a-Pyrrolidinecarboxyhc acid, 101-103, 197.
See «-Proline.
Pyrrolidonecarboxylic acid, 74
Quercite, relation to glycogen formation,
291
Quinic acid, behavior in the animal body,
586
Quinine, passage of, into urine, 639
, passage of, into sweat, 695
, action of, on the elimina.tion of
uric acid, 569
, action on the spleen, 274
Quotient, respiratory, 306, 446, 472, 722
731, 760
Quotient, urea to nitrogen, 628, 720
, nitrogen to sugar, 304, 306
Racemic acid, behavior in the animal body,
539
Rachitis, bones in, 438, 440
Rape-seed oil, feeding with, 442
Reductases, 19
Reduction processes, 2, 5, 18, 19. See also
the various chapters.
Reichert-Meissl's equivalent, 137
Reindeer, milk of, 529
Rennin, 25, 56. See also Chymosin.
Rennin cells, 349
Rennin glands. 349
Rennin zymogen, 349, 361. 362
Reproductive organs, 419-513
Resacetophenon, 637
Residual nitrogen, 183
Resin acids, transition into urine, 639, 641
Respiration, anaerobic, 21, 461
, external, 696, 703
, internal, 696, 703, 712"
of the hen's egg, 511, 512
Respiration of plants, 2
See also Chemistry of res-
piration, 696-714, and
Exchange of gas under
various conditions.
Respiratory quotient, 306, 446, 472, 722,
731, 760
Rest, metabolism during, 467-472, 758-
761
Reticulin, 37, 80, 428
Retene, 335, 336
Retina, 489
Reversion, 124
Revertose, 16
Reynolds' acetone reaction, 671
Rhamnose, relation to glycogen formal ion,
290, 336
Rheometer, 706
Rhodizonic acid, 458
Rhodophan, 491
Rhodopsin, 489
Rhubarb, action on the urine, 639
Rib-cartilage, 434
Rigor mortis of the muscles, 465
Roberts' method of estimating sugar, 663
Roch's reaction for protein, 642
Rodents, bile-acids of, 318
Rods of the retina, pigments of, 490
Rosenbach's bile-pigment test, 852
urine test, 675
Rotation, specific, 109
Rosin's levulose reaction, 119, 665
Rovida's hyaline substance, 141, 221, 267
Rubner's sugar test, 117, 665
Rye bread, utilization, 417, 421, 727
Saccharic acid, 106, 107, 121
, lactone of, 121
, relation to glycogen for-
mation, 291
, behavior in diabetes, 300
Saccharose, 123, 124
calorific value, 724
absorption of, 419
Salicylase or aldehydase, 18
Salicylic acid, action on pepsin digestion,
359
, action on trypsin digestion,
393
, behavior in the animal
body, 635
Salicylic-acid amyl ester, 284
Salicylsulphonic acid as protein reagent,
42
Saliva, 339-348
, secretion of, 346, 347
, mixe4, 342
, physiological importance, 348
, behavior in the stomach, 348,
366, 367
, action of, 345, 348, 367
, gases of, 340, 702
, composition of, 346, 347
Salivary calculi, 348
Salivary diastase. See Ptyalin.
806
GENERAL INDEX.
Salivary glands. 339
Salkowski's cholesterin reaction, 336
Salkowski-Ludwig's method of estimating
uric acid, 336
Salmine, 63, 64
Salmon, flesh of, 454
, sperma of, 63, 155, 497
Salmonucleic acid, 154
*Salts, action of, upon metabolism, 754
, antagonistic action of, 377, 375
. See also the various salts.
Salt-plasma, 171
Salts of vegetable acids, behavior in the
organism, 544
Samandarin, 692
Santonine, action on the urine, 639, 654
Sapokrinin, 385
Saponification equivalent, 136
Saponification. 133, 388, 398, 422, 423
Saponin, 193, 337
Sarcolactic acid. See Paralactic acid.
Sarcolemma, 447
Sarcomelanin, 688
Sarcomelanic acid, 688
Sarcosine, 455
, behavior in the animal body,
630
Sarkine. See Hypoxanthine.
Scherer's inosite test, 459
Schiff's reaction for cholesterin, 336
reaction for uric acid, 576
reaction for urea, 555
Schreiner's base, 496
Schiitz-Borissow's law, 390, 392
Schweitzer's reagent, 107
Sclerotic, 493
Scombrine. 59, 63, 64
Scombron, 62
Scyllite, 272
Scymnol, 310
Scymnol-sulphuric acid, 310
Seal-fat, 138
Sea-urchin, sperm of, 62
Sebacic acid, 135
Sebum, 691
Secondary proteoses, 52
Secretin, 309, 377, 379, 384
Secretin enzymes, 22
Sediments. See Urinary sediments.
Sedimentum lateritium, 543, 575, 607,
677
Seliwanoff's reaction for levulose, 119,
665
Semen, 495-497
Semicarbazide, poisoning with, 584
Semiglutin, 79
Seminose. See Mannose.
Senna, action on the urine, 639, 654
Sepsine, 24
Seralbumin, 36, 181
detection of, in the urme, 640,
643
, quantitative estimation of,
183, 645
, absorption of, 413
Serglobulin, 36, 178
, detection of, in the urine, 640,
643
, quantitative estimation of,
180, 645
Sericin, 37, 82
Serine, 33, 95
Serolin, 183
Serosamucin, 258
Serous fluids, 256-265
Serum. See Blood-serum.
Serum casein. See Serglobulin.
Sex. influence on metabolism, 756
Sharks, bile of, 310, 325
, urea in. 240, 325, 547
Sheep's milk, 529
Shell-membrane of the hen's egg, 73,
509
Silicic acid in feathers, 685
in hair, 685
in urine, 626
in hen's egg, 505, 509, 510
in connective tissue, 429
Silicic acid ester in feathers, 685
Silk gelatine, 82
Sinistrin, animal, 71
Silver, in blood, 239
Skatol, 29, 34, 102, 401, 402
, formation in putrefaction, 29, 401,
588
, behavior in the animal body, 400,
401, 588, 595, 634
Skatolacetic acid, 30, 102
Skatolaminoacetic acid, 30, 102
Skatolcarboxylic acid, 102, 596
Skatol-pigment, 595, 596, 607
Skatosine, 103
Skatoxyl, 401, 588, 634
Skatoxylglucuronic acid, 595
Skatoxylsulphuric acid, 588, 595
in sweat, 694
Skeletins, 81
Skeleton at various ages, 438
Skin, 685-695
, excretion through, 690, 692-695,
717
Sleep, metabolism, 761
Small intestine, 378, 380
extirpation of, 427
Smegma prseputii, 692
Smith's reaction for bile-pigments, 654
Smooth muscles, 477
Snail mucin, 66
Snake poison, action upon blood, 193,
226, 234
Soaps in blood-serum, 183
in chyle, 251, 423
in pus, 268
in excrements, 408, 426
in bile, 310, 325
in milk, 532
, imi^ortance of, in the emulsification
of fats, 389, 390, 398, 423
Sodium alcoholate as a saponification
agent, 136, 659
GENERAL INDEX.
807
Sodium cliloride, elimination by the urine,
616, 617, 694, 695
, elimination by the sweat,
694, 695
, physiological i m p o r t-
ance, 736
, quantitative estimation,
616-619
, influence on the quan-
tity of urine, 754
, influence on the elimina-
tion of urea, 754
, influence on the secre-
tion of gastric juice,
364, 736
, behavior with food rich
in potassium, 736
, insufficient supply of,
364, 736
, action on the secretion
of intestinal juice, 377,
378
, action on pepsin diges-
tion, 358, 359
, action on trypsin diges-
tion, 393, 394
Sodium compounds, elimination by the
urine, 623
, division among the
form-elements and
fluids, 166
. See also the vari-
ous tissues and
fluids.
Sodium phosphate in the urine, 619,
620
Sodium salicylate, action on the secretion
of bile, 309
Sodium tartrate, relation to glycogen
formation, 291
Solanin, 193
Soldiers, diet of, 769
Sorbin, 119
Sorbinose, 113, 119
Sorbite, 106
Source of mviscular energy, 472-474
Spawn of tlae frog, 71
Specific rotation, 109
Spectrophotometry, 218
Sperma, 63, 495-498 '
Spermaceti, 138
Spermaceti oil, 138
Spermatin, 498
Spermatocele fluids, 263
Spermatozoa, 497
Spermine, 496
Spermine crystals, 496
Spherules, 37, 503, 509
Sphingosin, 485
Sphygmogenin, 278
Spider excrement, guanin therein, 160
Spiegler's reagent, 642
Spirographin, 69
Spirogyra, 114, 167
Spleen, 272-275
, relation to formation of blood, 274
, relation to formation of uric acid,
274, 570, 573
, relation to digestion, 385
, blood of the, 242
Spleen pulp, 272
Splitting processes in general, 1, 2, 9. See
also the various enzymes.
Spongin, 37, 81, 82
Sputum, 714
Sputum mucin. See Mucin from mucous
membrane, 66
Starch, 126
, hydrolytic cleavage by diastase,
128, 387, 388
, hydrolytic cleavage by pancreatic
diastase, 388
, hydrolytic cleavage by saliva, 344
, calorific value, 724
, absorption, 419, 421
Starches, digestion of, 367, 388
Starch, cellulose, 126
Starch granulose, 126
Starvation, action on the blood, 244, 731,
732
, action on the urine, 404, 548,
585, 592
, action on the elimination of
indican, 404, 592
, action on the elimination of
oxalic acid, 582
, action on the secretion of bile,
307, 308
, action on the secretion of
pancreatic juice, 382
, action on the elimination of
phenol, 404
, action on metabolism, 722,
728-733
, death from, 728
Starvation cures, 770, 771
Starvation requirement, 733, 755
Steapsin, 388
Stearic acid, 133
Stearin, 133
, absorption of, 423
Stentorin, blue, 691
Stercobilin, 320, 409, 603
Stercorin, 337
Stethal, 138
Stokes's reduction fluid, 203
Stokvis' reaction for bile-pigments, 653
Stojnach, gases in the, 372
, importance in digestion, 369
, pepsin charge in, 364
, relation to intestinal putrefac-
tion, 371, 406, 407
, auto-digestion of, 372
, digestion in the, 365-372
Stomachic glands, 350
Stone-cystine, 92
Streptococcus, behavior with gastric juice,
371
808
GENERAL INDEX.
Stroma fibrin, 195
Stroma of the blood-corpuscles, 194
of the muscles, 451
of the ovaries, 498
Strontium salts and blood coagulation,
171
Struma cystica, 275
Strychnine, passage of, into the urine, 639
and sugar elimination, 299
Sturgeon, sperma of, 63
Sturine, 63, 100
Sublingual glands, 339
Sublingual saliva, 341
Submaxillary glands, 339
Submaxillary mucin, 66, 67
Submaxillary saliva, 340, 341
Succinic acid in putrefaction, 32
in the fermentation of milk,
516
in the intestine, 400
in the spleen, 272
in transudates, 259, 264
in the thyroid glands, 275
from phosphocarnic acid,
457
, passage of, into the urine,
630
, passage of, into the sweat,
695
Sugar, relation to work, 469, 473
, formation from fats, 306, 473
, formation from protein, 303-305
Sugar formation, in the liver, 295-301,
306
after pancreas extirpa-
tion, 301-305
Sugar, behavior on subcutaneous in-
jection, 293
, behavior to blood-corpuscles, 195
, quantitative determination, 659-
665
. See also various kinds of sugar.
Sugar tests in the urme 655-659
Sulphsemoglobin, 207
Sulphocyanides in the urine, 611, 631
in gastric iuice, 354
in the saliva, 341, 343
Sulphonal intoxication, urine in, 213, 650
Sulphonic acids, behavior in the animal
body, 540
Sulphur, of proteins, 27. See also various
proteins.
, in the urine, 471, 611, 612
, elimination of, in activity, 471
, elimination of, with lack of oxy-
gen, 611
, neutral and acid sulphur in urine,
611
, behavior in the organism, 611,
631
Sulphur methjemoglobin, 207
Sulphuretted hydrogen in putrefaction in
the intestine,
401, 404
in the urine, 612
Sulphuric acid, ethereal and sulphate, in
the urine, 588, 589, 611,
622, 623
, elimination of, in activity,
471
, elimination of, by the
urine, 543, 622, 623, 628
, elimination of, by the
sweat, 694
, estimation of, 622
, relation to elimination of
nitrogen, 471, 611, 622
, action on pepsin digestion,
358
Suprarenal capsule, 277
Suprarenin, 278. See also Adrenalin.
Swallow's-nests, edible, 69
Sweat, 692-695
Swimming-bladders of fishes, gases of, 7 10
, guanine in,
160
Sympathetic saliva, 340
Synproteose, 55
Synovia, 265
Synovial fluid, 265
Synovial mucin, 258
Synoviamucin, 265
Synovin, 266
Synthesis, 1, 2,
of ethereal sulphuric acids, 280,
401, _ 588, 590, 593, 594, 637
of conjugated glucuronic acids,
122, 589, 593, 609, 610, 632,
638
of uric acid, 567, 568, 572, 573
of urea, 547, 550, 551, 552
of hipinu'ic acid, 3, 585, 635
of varieties of sugars, 106, 114
of polypeptides, 36,
Syntonin, 48, 100
, calorific value of, 726
Tagatose, 108
Talonic acid, 120
Talose. 108, 113, 120
Tapeworm cysts, 265
Tannic acid, behavior in the animal body,
637
Tartar, 348
Tartaric acid, relation to glycogen forma-
tion, 291
, passage of, into sweat, 695
, behavior in the animal
body, 630
Tartronic acid, 573
Tatalbumin, 506
Taurine, 94, 95, 310, 313, 331
, behavior in the animal body, 629
Taurocarbamic acid, 631
Taurocholeic acid, 314
Taurochohc acid, 310, 313, 328
, occurrence in meconium.
410
, decomposition in
intestine, 404
the
GENERAL INDEX.
809
Taurocholic acid, protein-precipitating ac-
tion, 42, 647
Tea, action on metabolism, 755
Tears, 418
Teeth, 440
Teichmann'i crystals, 211, 649
Tendon mucin, 66
Tendon mucoid, 428
Tendon svnovia. 265
Tension of the CO. in the blood, 708-710
in the tissues, 712
in the Ivmph, 251
O in the blood, 703-708
Terpen-glucuronic acid, 667
Terpentine, action of, on the secretion of
bile, 308
, action of, on the urine. 639
, behavior in the animal bodv,
609, 638
Tetraglycylglycine, 35, 396
Tetraoxj^aminocaproic acid, 96
Tetrapeptides, 35, 395
Tetronerj'thrin, 219, 690
Testes, 495
Tetroses, 105
Theobromine, 157
, behavior in the animal
body, 579
Theophylline, 157
, behavior in the animal
body, 579
Thioalcohols, behavior in the animal
body, 632
Thioglycolic acid, 74
, behavior in the animal
bodv, 632
Thiolactic acid, 28, 33, 94
Thiophene, behavior in the animal body,
637
Thiophenic acid, 637
Thiophenuric acid, 637
Thiotolene, 637
Thrombin, 13, 16, 175, 228, 233
Thrombogen, 231-233
Thrombokinase, 231
Thrombosin, 229
Thymine, 152, 154, 165
Thymic acid, 154
Thymonucleic acid, 151, 152, 153, 154,
155
Thymus, 270
Thyreoglobulin, 276, 277
Thyreoidea, 275-277
Thyreoproteid, 276
ThjTeotoxalbumin. 277
Thyroid gland, 275, 276
Thyroiodin. See lodothyrin.
Tissue-fibrinogen, 141, 271
Tissue proteids.
ToUens' reaction for pentoses, 111, 112
Toluene, behavior in the animal bodv, 585,
634 _
Toluric acid, 635
Toluylenediamine, poisoning with, 333 '
Toluic acid, 635
Tonas, chemical of the muscle, 467
Tooth structure, 440
Tortoise, bones of, 436
Tortoise-shell, 73, 691
Toxalbumins, behavior with gastrin juice,
371
Toxines, 24, 25, 186, 2S0
Tracheal cartilage, 420, 433
Transudates. 256-266
Tribromacetic acid.
Tricalcium casein. 520
Tricliloracetic acid as reagent, 42, 45
Trichlorethyl-glucuronic acid. See Uro-
cliloralic acid.
Triglycylglycine, 35, 396
Triolein, 34
Trioses, 105
Tripalmitin, 1.34
Tripeptides. 35, .395
Triple phosphate in urinary sediments,
678, 679
in urinars' calculi, 68J,
681, 682
Tristearin, 133
Triticonucleic acid, 152, 156
Tronmier's test for sugar, 116, 655
, behavior with
uric acid, 575
, behavior with
creatinine,
565
Tropics, metabolism in inhabitants of, 762
Trypsin, 186, 390-395
, action on proteins, 54. .55, 392, 416
, action on peptides, 35, .395. 396
, importance in absorption, 416
Tr>T)sin digestion, 392-396
, products of, 394
Trj-psinogen, 383-386
Trvqjsin peptone, 51, 54, 55, 57, 58
Tryptophane, 30, 102, 355
Tubo-ovarial cysts, 501
Tunicin, 685
Turacin, 690
Turacoverdin, 690
Trj-osine, 18, 33, 54, 89-91
, in urine. 675
, in sediments, 680
, detection of, 91, 675
, behavior in putrefaction, 400,
585, 588
, behavior in the animal body,
598, 599, 633, 670
Trj'osinases, 18, 90, 690
Tyroslne-sulphuric acid, 90
UfTelmann's reaction for lac ic acid, 374
Umikoff's reaction. 532, 533
Uracil, 152, 156. 164
Uraemia, bile in, 329
, gastric contents in, 373
, sweat in, 694
Uraminobenzoic acid, 636
Urates, 542
, in sediments, 575, 678
810
GENERAL INDEX.
Urea, 547
, elimination in starvation, 548
, elimination in children, 549, 665
, elimination in disease, 547, 548, 554
, properties and reactions, 554
, formation and origin, 550-554, 630
, quantitative estimation, 558-562
, synthesis, 547, 551-553
, occurrence in the blood, 186, 240,
242, 551
, occurrence in the bile, 325, 329, 547
, occurrence in the liver, 547, 550,
551
,,occurrence in the muscles, 455, 547
, occurrence in transudates, 259
Urea glucuronic acid, 609
Urea nitrate, 555
Urea oxalate, 555
Ureides, 30, 568, 583
Urein, 562
Urethane, 563
Urease, 16, 677
Ureido-gluciu-onic acid, 609, 611
Uric acid, 157, 550, 567, 568
, elimination in disease, 570
, elimination after feeding with
nuclein, 569, 571
, relation to urea, 567-573
, properties and reactions, 573-
576
, formation in the animal body,
570-574
, quantitative estimation, 576-
. 578
, syntheses of, 568, 572, 573
, behavior in the animal body,
573, 574
, occurrence of, 568, 569
, occurrence of, in sweat, 547, 694
, occurrence of, in sediments, 543,
575
Uric-acid calculi, 681
Urinary calculi, 680-683
Urinary pigments, 601-607
, medicinal, 639, 654
Urinary sand, 680
Urinary sediments, 542, 543, 677-680
Urine, 541-684
, excretion of, 626, 627
, inorganic constituents of, 616-626
, poisonous constituents of, 615
, organic pathological constituents
of, 639-676
, physiological constituents of, 546-
616
, enzj^nes of, 615
, casual constituents of, 629-639
, color of, 542, 601, 628, 639, 648-654
, solids, calculation of, 626, 627
, quantity of solids, 626, 627
, alkaline fermentation of, 677
, acid fermentation of, 677
, gases of, 626, 703, 712
, quantity of, 626, 627
, physical properties of, 542-547
Urine, osmotic pressure of, 546
, physico-chemical analysis of, 628
, reaction of, 543-546
, acidity of, 543-545
, estimation of acidity, 545
, specific gravity of, 546, 627, 628
, passage of foreign bodies into, 629-
639
, composition of, 628
, reducing power of, 608
Urine indican, 592
Urine indigo, 592, 601
Urine poison, 615
Urine purines, endogenous and exogenous,
_ 578-581
Urine sugar. See Dextrose.
Urinometer, 546
Urobilin, 601, 602-607 _
, relation to bilirubin, 319, 332,
404, 603
, relation to choletelin, 603
, relation to hsematin, 332, 603
, relation to hsematoporphyrin,
214, 603
, relation to hydrobilirubin, 332,
603
Urobilin icterus, 604
Urobilinogen, 601, 605
Urobilinoid bodies, 603
Urocamic acid, 526, 615
Urocliloralic acid, 122, 632
Urochrome, 601, 602
Urocyanin, 601
Uroerythrin, 601, 607
Uroferric acid, 611, 613
UrofvLscohsematin, 650, 651
Uroglaucin, 601
Urohfematin, 601
Urohodin, 601
Uroleucic acid, 597, 60
Uromelanins, 601
Uronitrotoluolic acid, 638
Urophsein, 601
Uroproteic acid, 613
Urorubin, 601
Urorubrohsematin, 650
Urorosein, 596, 601, 651
Urospectrin, 650
Urostealith calculi, 682
Urotheobromine, 580
Urotoxic coefficient, 615
Uroxanthine, 592
Uroxonic acid, 568
Ursocholeic acid, 319
Uterine milk, 512
Uterus colloid, 502
Utilization of the various foodstviffs, 417,
421, 425, 530, 531
Valerianic acid, 29
Vegetable acids, behavior of the alkali
salts of, in body, 544
Vegetable gums, 120, 129
Vegetable mucilages, 128, 129
GENERAL INDEX.
811
Vegetarians, food of, 750, 767
, excrement, 408
Vernix caseosa, 336, 691
Vesicatory blisters, 265
Vesicle calculi,
Vesiculase, 496
Virtual sugar, 184
Visual purple, 489-491
Visual red, 489
Vitali's pus-blood test, 649
Vitellin, 36
in yolk of egg, 503
in protoplasm, 141
Vitellolutein, 505
Vitellorubin, 505
Vitelloses, 52
Vitreous humor, 491
"Water, drinking of, action in the elimina-
tion of chlorides,
616
, action on the elimina-
tion of uric acid,
569
, action on the elimina-
tion of urea, 753
, action on the deposi-
tion of fat, 754
, action on the excre-
tion of urine, 626
, elimination of, through the urine,
626-628, 717
, elimination of, through the skm,
693, 717
, elimination of, in starvation, 731
, elimination of, importance for the
animal body, 734
, elimination of, quantity of, in the
various organs, 734
, elimination, lack of, in the food,
734
Wax, 138
in plants, 691
Weidel's xanthine reaction, 160
Weyl's reaction for creatinine, 565
Wheat bread, absorption of, 418
Whey, 516, 528
Whey proteid, 521
White of egg, 506-510
, calorific value of, 724
, absorption of, 413, 414
Witch's milk, 534
Woman's milk. See Human milk.
Wool-fat, 337, 692
Work, action on the elimination of
clilorine, 616
, action on the elimination of sul-
phur, 471
, action on the excretion of nitrogen,
470, 471
, action of the necessity for food,
768, 709
, action on metabolism, 468-474, 758
-761
Worm-Miiller's sugar test, 655
Wound secretion, 265
Xanthine, 157, 159
in the urine, 578
in urinary calculi, 682
in urinary sediments, 680
, detection and quantitative esti-
mation, 159, 160, 163, 164,
580, 581
Xanthine bodies. See Nuclein bases.
Xanthine calculi, 682
Xanthine oxidase, 18, 73, 571
Xantho-creatinine, 457, 470, 567
Xantho-melanin, 32
Xanthophan, 491
Xanthophyll groups, 505
Xanthoproteic reaction, 42
Xylene, behavior in the animal body, 634
Xyliton, 689
Xyloses, 113, 282
, relation to the formation of
glycogen. 111, 290
Yeast-cells, relation to fermentation, 10,
11
Yeast maltase, 17
Yeast nucleic acid, 152, 156
Yeast nuclein, 150
Yolk of the hen's egg, 502-506
Yolk-spherules, 37, 503, 509
Zein, 27, 98
Zinc in the bile, 325
in the liver, 286
, passage of, into milk, 539
Zooervthrin, 690
Zoofulvin, 690
Zoorubin, 690
Zymase, 10, 11, 20, 21, 396
Zymogens, 15. See various enzymes.
Zymoplastic substances, 176, 228, 231
INDEX TO AUTHORS.
Abderhalden, E., protein hydrolysis, 29,
54, 55 \ carbohydrate group in the pro-
teins, 33, 182; digestion products. 54,
55; histone, 62; elastin, 75; monanimo
acids, 83, 84, 85, 88. 89. 91, 101; in tlie
urine, 614, 675; cystine and cvstinuria,
92, 631, 675; histidine, 99, 208; dia-
mino trioxydodecanic acid. 101; pro-
teoses in the blood, 183; blood serum,
188; globin, 208; blood corpuscles. 219;
blood analyses, 238, 239; quantity of
haemoglobin, 239. 243: iron prepara-
tions, 245; blood and air dilution. 246;
adrenalin, 278; sugar formation, 296;
cholesterin, 334, 335; duodenal secre-
tion, 377; tniJsin, 394, 395, 417; pro-
tein absorption, 400. 413, 416, 417;
ovalbumin, 507; milk, 529, 536; poly-
peptides in urine, 551, 614; glycylglycin,
behavior in animal body, 630; Bence-
Jones protein, 645; alcohol, 755; dipep-
tides from protein bodies, 35; spongin,
81
Abel, J., epinephrin, 278; spermin, 496;
carbamic acid, 552; melanines, 689
Abeles, M., sugar, 118; uric acid, 186;
liver sugar, 295; carbohydrates in the
urine. 608
Abelmann, M., protein digestion, 418; fat
absorption. 425
Abelsdorf. G.. 490
Abelous, J., enzymes, 8, 17-20
Ach, L., 568
Achard, Ch., 185
Ackermann, D., 195
Adamkiewicz, A., protein reaction, 42;
proteoses, nutritive value of, 746
Aducco, v., acidity of urine, 544; urine
poison, 615
Aders, R. H., hydrolysis of gelatine, 78,
83, 84
Adler, O., pentose, 112; levulose, 119;
blood, 217, 649
Adler, R., pentose, 112; levulose, 119;
blood, 217, 649
Adrian, C, 594
Adriance, J., human milk, 531 ; colostrum,
531
Adriance, V., human milk, 531; colos-
trum, 531
Afanassiew, M., 333
Albanese, M., 579
Albert. R., 10
Albertoni, P.. bile, 308; trypsinogen, 390;
sugar, absorption of, 419
Albrecht, E., 193
Albro, A., 393
Albu, A., urine poisons, 615; mineral
metabolism. 616, 625, 719, 735, 737
v. Aldor, L., 644
Aldrich. J. B., adrenalin, 278; skunk,
secretion of, 692
Alexander. F., proteoses, 55, 522
Alexandroff. D., 102
V. Alfthan, K., carbohydrates of lu-ine,
609; glucuronic acid in the urine, 667
Allard, E., 672
Allen, S., 359
Allihn, F., sugar determination, 290,
663
Almagia, M., amino acids and carbohy-
drate formation, 305; uric acid demoli-
tion, 574
Almen, A., sugar test, 116. 655; xanthine,
159; meat, 476; foodstuffs, 773
Aloy, J., enzymes, S, 19
Alsberg, C, pseudonucleic acid, 46; nu-
cleic acid. 154
Altmann, 147
Altmann, R., nucleic acids, 152, 156
Ambronn. H., 686
Amermann, G., pepsin digestion, 358;
stomachic digestion, 370
Amiradzibi. S., 457
Amthor. K, fats, 135, 442
Andersson, J., 277
Andrlik. K, 89
V. Anrep. B.. carbon monoxide haemoglo-
bin. 207; benzoic acid, 588
Anselm. R.. 326
Ansiaux, G., protein coagulation, 40;
fibrinogen and phosphorus poisoning,
172
Anthon. 115
Araki. T.. met haemoglobin. 204; sulphur-
met haemoglobin. 207; glycosuria, 299;
nucleic acids. 395; lactic acid, 460, 461-
463; in urine. 608; acetoacetic acid,
672; chitin, 686; chitosan, 687
Ardin-Delteil, P., 694
813
814
INDEX TO AUTHORS.
Argutinsky, P., meat, 476, 720; perspir-
ation, 694
Armstrong, E. F., enzymes, 13, 16;
osones, 107
Arnheim, J., 303
Arnold, J., 277
Arnold, V., neutral hsematin, 208; aceto-
acetic acid, 672
Arnschink, L., 423
Arnstein, R., 581
Aron, H., colloids, 41; bone eartlas, 436,
438
Arrhenius, S., 190
Arronet, H., 238
Arteaga, T. F., 298
Arthus, M., blood coagulation, 170, 171,
226, 227, 229, 231; fibrinolysis, 174;
glycolysis, 185; blood lipase, 185; peri-
toneal fluid, 231; sugar formation, 295;
casein, 519; rennin coagulation, 520
Ascherson, 517
Ascoli, A., pyrimidine bases, 152, 164;
nucleic acids, 152; plasminic acid, 156;
protein absorption, 414; placenta, 512;
urea formation, 550; uric acid demoli-
tion, 574
Asher, L., blood-sugar, 184; lymph, 250,
254-256; saliva, 340; protein absorp-
tion, 414; lactic-acid formation, 461
Aso, K., 19
Astaschewsky, 469
Athanasiu, J., blood coagulation, 234;
fatty liver, 283; fat formation, 443
Atwater, W. O., metabolism, 471, 474, 716,
726, 748; respiration apparatus, 713,
722; alcohol in metabolism, 754; diets,
764, 767, 769
Aubert, H., 695
Austin, A. E., 290
Austrian, C. R., 571
Autenrieth, W., oxalic acid, 583; potas-
sium, 623
Azemar, L., 670
Ayres, W. C, 489
Akermann, J., 365
Baas, H., cleavage of esters, 389; intestinal
putrefaction, 586; demolition of tyro-
sine, 633
Babkin, B., oil soaps and pancreatic
secretion, 385; urea formation, 551
Babcock, 523
Bach, A., autoxidation, 7; peroxides and
oxidases, 7, 8, 17; enzymes, 8, 19
Baer, J., 544
Baeyer, A., assimilation, 1, 114; oxi-
dations, 6; indol, 402
Baginsky, A., bile, 327; bones, 439
Baglioni, S., 240
Bainbridge, F. A., lymph formation, 255;
lactase, 386
Baisch, C, carbohydrates in urine, 608;
Vienzoylation, 659
Baker, J. L., 125
Balch, A., 307
Baldi, D., jecorin, 283, 481
Baldoni, A., 396
Balean, H., 216
Balke, P., purine bases, 159, 163; phos-
phocarnic acid, 457; episarkine, 580
Bang, B., 539
Bang, I., histones, 62, 269, 271; nucleic
acids, 151, 155, 156, 270; leucocytes,
221; lymph glands, 269, 272: thymus,
270-272; parachymosin, 361, 362;
proteoses in urine, 644
Banting, W., 770
Barbera, A. G., lymph, 250, 254, 255; bile,
308; protein absorption, 414
Barbieri, I., phenylalanine, 91; isochole-
sterin, 337
Barker, L. F., 675
Barral, 185
Barratt, W., 695
Barth, H., 583
Bartoschewitsch, S. T., 589
Basch, K., casein formation, 537; milk,
538
Baserin, O., 332
Bashford, E., 586
Bassow, 349
Bastianeili, G., 378
Battelli, F., enzymes, 20, 21
Bauer, 605
Bauer, J., protein absorption, 412; fatty
degeneration, 443
Bauer, M., 100
Bauer, R., 73
Baumann, E., diamines, 24; in urine and
faeces, 615, 676; cystine and cystinuria,
93, 675; thiolactic acid, 94; carbohy-
drates, benzoylation of, 117, 659;
iodothyrin, 276; diamidation, 305; pu-
trefaction and products produced, 401,
586, 588; hippuric acid, 586; ethereal
sulphuric acid, 588, 592, 637; phenol in
urine, 590; pyrocatechin, 591; hydro-
quinone, 591; urine indican, 592, 593;
bile acids, 597; oxyacids, 597; homo-
gentisic acid, 597-600; carbohydrates in
the urine, 608; sulphuric acid of the
urine, estimation of, 622; sarcosin, de-
struction of, 631; demolition of com-
povmds containing sulphur, 632; be-
havior of aromatic bodies, 633, 636,
639 ; mercapturic acids, 639 ; benzoyl cys-
tine, 676
Baumann, K., 60
Baumgarten, O., 300
Baumstark, F., brain, 487; protagon, 481;
cholesterin, 480; urinary pigments, 650
Bayer, H., 57
Bayliss, W. M., intestinal enzymes, 379;
erepsin, 380, 391; enterokinase, 379,
383; trypsinogen and trypsin, 383, 386,
390, 392; secretin, 384
Beatty, W. A., 35
Beaumont, W., gastric fistula, 349; gas-
tric digestion, 368
Beccari, L., 326
INDEX TO AUTHORS.
815
Bechamp, A., crj'stalline lens, 493; oval-
bumin, 507; milk dextrin, 525
Bechhold, H., 656
Beck, A., urobilin, 603; urine poisons,
615
Beck, 0 , 471
Beck, R. F., 538
Beckmann, E., 692
Beckmann, W., 624
Becquerel, A., blood, 243; milk, 534
Beebe, S. P., 166
Begar, C, 536
Behrend, R., 568
Beier,. K.. 330
Beitler, C, 102
Bellamy, H., 385
Belloni, E., 523
Bence, J., 224
Bence-Jones, H , 645
Bendrix, E., pentoses, 72, 110, 111; glyco-
gen formation, 292
Benedicenti, A., formalin protein com-
bination, 49; alcohol in metabolism,
754; light and metabolism, 761
Benedict, F. G., metabolism in work, 471,
474; protein sparing, 748; alcohol in
metabolisin, 754
Benedict, H., elimination of sulphur, 471,
'H611
Benedikt, R., fats, 134, 137
Benrath, A., 364
Berard, E., protein coagulation, 40;
ovglobulin, .506; ovalbumin, 507
Berdez, J., melanins, 688
Berenstein, M., 409
Bergell, P., carbohydrate groups in pro
teias, 33, 182; protein hydrolysis. 35,
395; amino acids, 92; lecithm, 147;
adrenalin, 278; placenta. 512; glycyl-
glycine. behavior in the body, 630;
.5-oxybutvric acid, 674
Berger, W. M.. 344 ^
Bergh, E., elastin, 75
v. der Bergh, 615
Bergin, T. .1., 407
V. Bergmann, G., residual nitrogen, 183;
taurine from cystine, 331
Bergmann. P., thyroidea, 276; pseudo-
pepsin, 355; CEPCum, 427
Bergmann, W.. 620
Berlatzki, G. B., 401
Berlioz, A., 616
Berlinerblau. M., 241
Bernard, Claude, glycogen 148, 287
glycolysis, 184; sugar of the blood, 240
sugar formation, 295-298; diabetes, 299
pancreas, 382, 389; fat splitting. 389
fat absorption, 424; muscles, glycogen
consumption in, 468
V. Bernek. M., 14
Bernert, R., oxyprotic acids, 31; mon-
oxvstearic acid, 131; pseudochylous,
262
Bernheim, A., transudates, 261, 263
Bernheim, R., 623
Bernstein, J., 477
Bernstein, N. O., 382
Bert, Paul, mammary gland, 514, 539;
blood gases, 697, 706
Bertagnini, C, 636
Berthelot, M. P., fat splitting, 389; calori-
metric determinations, .511, 724
Bertin-Sans, H., blood pigments, 204,
207
Bertrand, G., oxidation enzymes, 8, 18,
19; arsenic, 166, 275, 685; adrenalin,
278; epidermis formations, 685; poi-
sons, 693
Bertz. F., 440
Berzelius, J. J., 347
Besbokaia, M., .385
■Bethe, Alb., 486
Bial, M., pentoses, 110, 112; reage t,667;
conjugated glycuronates, 122, 667;
diastase in the blood, 185, 295, 297;
lymph, 251; glycogen formation, 294
sugar formation, 295
Bialobrzeski, M., 212
Bialocour, F., 371
Biarnes, G., oxidation enzymes, 8, 18
V. Bibra, E., 286
Bidder, F., buccal mucus, 341; saliva,
347: gastric juice, 353; pancreatic juice,
387; biliary fistula, 406; fat absorption,
424
Biedermann, W., 18
Biedert, Ph., 530
Biedl. A., 241
Biel, J., mare's milk, 529; human milk,
530
Bielfeld, P., 285
Binet. P., 426
Bienstock, B., 408
Biernacki, E., alkalinity of blood, 223;
blood analysis, 236; pepsin, 355; trj'p-
sin, 390; putrefaction processes, 405,
407, 588
Bierrj-, A., lactase formation, 386; mal-
tase, 388
Biffi, X., 522
Bins;. H. J., jecorin and blood sugar, 184,
283. 296
Binz, C, 631
Biot, J. B.. 711
Biscaro, G., 523
Bischoff, Th., 716
V. Bitto, Bela, 671
Bizio, G., 695
Bizio, J., 287
Bizzozero, J., blood plates, 222, 227
Bjerre, P., 755
Blachstein, A., 459
Blankenhorn, E., protagon, 481, 482
Bleibtreu, L., blood analyses, 235; quan-
tity of plasma. 2.38; urea estimation
561; protein requirement. 765
Bleibtreu. M.. blood analyses. 235; quan-
tity of plasma, 238; respiratory quo-
tient, 446
Bleile, A. M., 240
816
INDEX TO AUTHORS.
Blendermann, H., tyrosine, behavior in
the body, 305, 633; oxyhydropara-
coumaric acid, 597
Bliss, C. L. 49
Bhx, M. G., 236
Bloch, C, 587
Blondlot, N., 406
Blum, F., halogen proteins, 30; Millon's
reaction, 4.;; protogen, 49; thyroid
gland, 277; adrenalin glycosuria, 279
Bliun, L., autolysis, 23; protein nitrogen,
27; cystine, demohtion of, 631; pro-
teoses, nutritive value of, 747
Bhunenthal, F., acetone, 32, 668; nucleo-
proteids, 72, 282; pentoses, 110, 666;
glycogen, 292; assimilation limit, 420;
hippuric acid, 587; vu'ine indican, 593
Boas, J., chymosin, 361; reagent for lactic
acid, 374; for hydrocliloric acid, 374
Bocarius, N., 496
Bock, C, blood sugar, 297; diabetes, 299
Bock, J., blood pigments, 206, 207; haemo-
globin and gases, 207
Badlander, G., 754
Bodon, K, 260
Bodong, A., blood coagulation, 231; hiru-
din. 233
Boedeker, C, 591
Boedtker, E., urine nitrogen, 548; clilor-
ine estimation, 617
Boehm, R., glycogen, 459, 466
BoehtHngk. R., 731
Boekelmann, W. A., 674
Boemer, A., 60
Boeri, G., oxalic acid, 582; sulphur of
urine, 611
Boettcher, 496
Boettger, 116
Bogdanow-Beresowski, 343
Bogomoloff, Th., 602
Bohland, G. W., 269
Bohland, K., urine nitrogen, 548; esti-^
niation of urea, 557, 561, 613; of
ammonia. 625; uric acid elimination,
569; protein requirement. 765
Bohr, Chr., blood pigments, 196-200,206;
oxygen absorption, 199, 200, 704;
oxygen tension, 706-708, 711; blood-
gases, 696-698, 704; carbon dioxide
tension, 710; swimming-bladder, 711;
oxygen capacity, 711, 712; lungs,
metabolism of, 704, 714; secretion of
gases, 708, 710; egg, incubation of, 510,
512
DuBois-Reymond, E., muscle work, 469;
smooth muscles, 477
DuBois-Reymond, R., 225
Bokorny, T,, reserve protein, 5; carbohy-
drate formation, 114
Boldireff, W. N.. intestinal enzymes, 378,
379: gastric digestion, 398
Boll. F., 489
Bonanni, A., bile, 326; conjugated glu-
curonates, 638
Bondi, J., 512
Bondi, S., sericin, 82; glycocholic acid,
312; taurocholic acid, 313; cholic acid,
316. 318; acetoacetic acid, 672
Bondzynski, St., oxyprotic acid, 31; kop-
rosterin, 337; ovalbmnin, 507; \irinary
purines, 578; oxyproteic acid in the
urine, 612, 613; uroferric acid, 614
Bonnema, A., 517
Borchardt, L., diastatic enzyme, 295;
acetone estimation, 673
Bordet, J., precipitins, 186; blood coagu-
lation, 227, 231
Borissow, law of pepsin action, 358
Bori.ssow, P., bitter principles ,350; allan-
toin, 584
Borkel, C, 57
Bornstein, K., work and protein metabo-
lism, 471; protein feeding, 752
Boruttau, H., 459
Bosshard, E., 85 _
Bottazzi, Ph., sea animals, osmotic press-
ure, 189; erythrocytes, 220; intestinal
protein, 379; heart muscles, 451;
smooth muscles, 478; placenta, 512;
tunicin, 686
Bouchard, Ch., autointoxication, 25, 280;
glycogen formation, 291; poison of
\irine, 615
Boudet. 183
Boulud, conjugated glucoronates, 122, 184;
pentoses, 184; virtual sugar, 184;
maltose in vu'ine, 667
Bouma, J., urinary indican, 595; reaction
for bile pigments, 653; /?-oxybutyric
acid, 674
Bourcet, P., iodine, 187, 243; arsenic, 187,
243; menstrual blood, 243
Bourquelot, E., oxidation enzymes, 8;
sugar elimination, 293
Bourquet, F., 191
Bo vet, v., 30
Brahm, C, 638
Brand. J., bile, 307, 326, 327
Brandberg, J., 646
Brandenberg, K., alkalinity of the blood,
223
Brandl, J., 688
Brat, H., 112
Brauer, L., 329
Braunstein, A., glycolysis, 303; estima-
tion of urea, 561
Bredig. G., enzymes, 11, 12; inorganic
ferments, 14
Brenziger, K., 675
Brieger, L., ptomaines, 24; putrefaction
products, 401; skatol, 409, 596; neuri-
dine, 481, 486, 502; urinarj' indican,
592, 593; diamines in urine, 615;
cystinuria, 676; perspiration, 694
Brinck, Julia, 415
Brion, A., 630
Brocard, M., 386
Brodie, T. G., fibrin ferment, 176; dia-
betes, 298; pancreatic casein, 396
Brook, F. W., 30
INDEX TO AITHORS.
817
Brown, E. W., cholesterin esters, 183;
allantoin, 574, 584
Brown, H., enzyme action, 14; isomaltose,
125; hydrolysis of starch, 128, 346;
invertase, 379
Browne, C. A., (Jr.), 518
Brubacher, H., bones, 438, 439
V. Brucke, E., enzymes, 11; peptone, 52;
coagulation of blood, 226; fibrin. 229;
glj'cogen, 289, 290; pepsin, 355-357,
in urine, 615; emulsification of fat, 398;
absorption of proteins, 412; carbohy-
drates in the urine, 608
Brugsch H., 733
Bruhns, G., 164
Brunner, Th., 534
Bruno, G., action of steapsin, 389; action
of bile and trj-psin, 393, 397
Bruno, B., 616
Brunton-Blakie, 455
de Bruyn-Lobry', osamines, 107; varieties
of sugar, 108
Bryant, A. P., diets, 767, 769
Buchanan, A., 175
Buchner, E., fermentation. 10, 11; zy-
mases, 11,21; gases of the lymph, 702
Buchner, H., fermentation, 10, 21
Budde, v., 664
Bulow, K.. proteid, 38; starch, 126, 128
Bunz. R., 487
Burger, L., 429
Burker, K., 227
Buffalini, 244
Bugarsky, St., acid combinations of pro-
teins, 38; blood serum, molecular con-
centration of, 189; degree of dissocia-
tion, 191; conductivity blood analyses,
236; urine, 546
Bulnheim, G., 316
V. Bvmge. G., blood servmi, 188; blood
corpuscles, 220; blood plasma, 238;
blood analyses, 237; blood and air
dilution, 246; iron, of the liver, 285,
534; absorption, 245; secretion of
hydrocliloric acid, 364; gastric juice,
371; cartilage, 434; haematogen. 503,
510; milk. 527. 532. 5.35, 537; and
growth, 535 536; hippuric acid, 586,
587; mineral requirements, 736; arti-
ficial feeding, 739
Bunsen, R., 561
Buntzen, J., blood in starvation, 244;
blood and digestion, 244
Buraczewski, J., 215
Burchard, H., 336
Burckhardt, A. E. 188
Burian, R., xanthine oxidase. 18. 158. 273,
571; purine bases, 15S. 163. 454, 470;
in the urine, 579; formation of uric acid.
275, 569, 570, 573; destruction of uric
acid, 574; histones in the urine, 647
Burow, R., 532
Busch, P. W., lymph formation, 254. 255
Busch. W., 368
Butlerow, A., 113
Cade, A., 352
Cahn, A., secretion of hydrochloric acid,
364; digestion products, 370; retina,
489
Camerer, W., milk, 526, 527, 531, 533-535;
urinary nitrogen, 548; elimination of
uric acid, 569; purine bases, 581; meta-
bolism, 756, 577
Camerer, W., Jr., milk, 533, 535; elimina-
tion of ammonia, 624; perspiration, 693
Cammidge, P. J., 676
Campani, A., 621
Campbell, G., ovovitellin, 503; ovomucin,
506; ovalbiunins, 507
Campbell, J. F., bilicyanin, 322, 324
Camps, R., 600
Camus, L., enterokinase, 383; secretin,
484; vesiculase, 496
Cannon, W. B.. gastric movements, 366;
food, digestibility, 369
Cappelli, J., erythrocytes, 220; smooth
muscles, 278
Capranica, St., guanine, 161; perspira-
tion, 694
Carlier, E. W., blood coagulation, 226;
chyle. 251
Carl Theodore (Duke of Bavaria). 761
Carnot. Ad., bone earth. 436; dentin, 440
Carvallo. J., blood coagulation, 234; ex-
tirpation of the stomach, 369, 371
Casali, A., 692
Caspari, W., high altitudes, 246, 761; pro-
tein metabolism, 471, 750; milk fat,
538; vegetable diet, 750
Castaro, X., arginine, 97; hemicellulose,
130
Cathcart, E. P., glycogen formation, 294;
absorption, 415, 417
Cavazzani, E., cerebrospinal fluid. 264;
destruction of glycogen, 296; carbohy-
drate absorption, 421; phosphocarnic
acid, 457; muscle work, 468; semen, 495
Chabbas, J., 491
Chabrie. C, 440
Chandelon, Th., 468
Chaniewski. 445
Chanoz. M.. 284
Charcot, J. M., 496
Chassevant, A., 574
Chauveau, A., sugar formation, 306; fat
formation, 444-446; muscle work, 468,
473
Chigin. P., 350
Chittenden, R. H., proteoses and peptones,
51, 52, 56, 60; keratin, 73; elastin, 75,
76; gelatine. 77, 79; saliva. 343-346;
peptic digestion, 358, 359; gastric diges-
tion, 369: trj^Jtic digestion. 393;
tendon mucoid. 428. 435; mvosin, 450;
neurokeratin, 73, 480. 488, 489; nutri-
tive requirements, 766
Chodat, R.. peroxides and peroxidases,
7,8, 17, 19; enzymes. 8, 19
Chossat, Th., metabolism in starvation,
828, 732
818
INDEX TO AUTHORS.
Christenn, G., 531
Cliristensen, A., 646
Ciamician, G., 403
Cingolani, M., 568
Citron, H., 623
Clar, C, 569
Claus, R., 303
ClayiDton, J. L., 23
Clemens, Paul, conjugation of glucuronic
acid, 638; Ehrlich's urine test, 675
Clemm, C. G., 533
Clerc, A., 185
Cleve, P. T., 316
Cloetta, M., hsematin, 210, 212
Cloez, 330
Clopatt, A., alcohol and metabolism, 754
Closson, O. E., 8
Cohn, Felix, 371
Cohn, M., saliva, 343; pancreas secretion,
387
Cohn, R., protein hydrolysis, 29; leucine,
85; leucinimide, 87; tyrosine, 89, 633;
amino acids and carbohydrate forma-
tion, 306; amino benzoic acids, 636;
nitrobenzaldehyde, 636; furfurol, 637;
pyridine, 639
Cohn, Th., blood, 189; sperm crystals, 496;
allantoin, 584
Cohnheim, J., ptyalin, 344; urine dias-
tase, 616
Colmheim, O., alcoholic fermentation, 21;
proteins, 26, 38; glycolysis, 302, 303;
erepsin, 378, 380, 416; pancreatic juice,
384; apparent feeding, 745
Cohnstein, J., 243
Cohnstein, W., alkalinity of the blood,
223; lymph formation, 256
Colasanti, G., metabolism of muscles, 467;
lactic acid, 469, 608; xanthocreatinine,
567
Cole, S. W., protein reaction, 42, 43; tryp-
tophane, 102, 103
Colenbrander, M., glycolysis, 185
Collmann, 695
Colls, P. C, 61
Comaille, A., lactoprotein, 523; milk, 537
Connstein, W., 185
Conradi, H., autolysis, 23, 281; anti-
thrombin, 234; intestinal bacteria, 408
Constantinidi, A., foodstuffs, 421; vege-
table diet, 750
Contejean, Ch., blood coagulation, 234;
phlorhizin diabetes, 298; gastric juice,
353; acid secretion, 364; pyloric secre-
tion, 365
Copemann, M., 649
Coranda, G., urea formation, 551; am-
monia elimination, 624
Cordua, H., 216
Corin, G., protein coagulation, 40; fibrin-
ogen, 172; ovoglobulin, 506; ovalbu-
min, 507
Coronedi, G., 442
Corvisart, L., 390
Le Count, E. R., 101
Courant, G., milk, 515, 520, 529; casein
salts, 519
Cousin, H., 504
Couvreur, E., 291
Cramer, C. D., fibrinogen, 173; blood co-
agulation, 229
Cramer, E., fibroin, 82; sericin, 82; per-
spiration, 694
Cramer, Tr., 418
Cramer, W., protagon, 481 ; hippuric acid,
586;
Cremer, M., pentoses. 111, 290; glycoly-
sis, 185; glycogen, 288-291, 293;
phlorhizin diabetes, 298; sugar from
glycerine, 308; fat formation, 445
Croftan, A., bile acids in blood, 238;
suprerenal capsule, 279
Croft-Hill, A., 16
Croner, W., 375
Cronheim, W., 375
Croockewitt, J. H., 82
Cummins, G. W., trypsin digestion, 393;
myosin, 450
Cunningham, R. H., 425
Curtius, Th., peptide syntheses, 34; cholic
acid, 315 .
Cutter, W. D., tendon-mucin, 66, 428;
saliva, 340
CybuLski, N., 278
Cerny, T., lactic acid fermentation, 461
Czerny, A., 221
Czerny, F., alcoholic fermentation, 21
Czerny, V., extirpation of stomach, 369;
absorption, 413
Czerny, Zd., 644
Daddi, L., fat in blood, 244; milk fat, 538
Daenhardt, C, 702
Dakin, H. D., arginase, 36, 97, 550; pro-
tamines, 63, 64, 84, 95, 97, 101; hexone
bases, 98, 100; adrenalin substances,
278; ester cleavage, 284
Daland, J., 237
Danilewsky, A., protein sulphur, 28;
plasteines, 56, 363; anti enzymes, 372,
379; muscle protein, 448, 451, 475;
milk globules, 517
Danilewsky, B., 724
Danilewsky, W., 146
Dareste, C, testicles, 495; yolk of egg, 502
Darmstadter, E., 674
Darmstadter, J., 692
Darmstadter, L., 337
Dastre, A., fibrinogen, 172; fibrinolysis,
174, 175; blood coagulation, 227, 234;
glycogen, 222, 251, 295, 296; liver, 282,
286; sugar elimination, 293; bile, 307,
324,325; in stomach, 398; trypsinogen,
386; fat absorption, 424
Dautzenberg, P. J. W., 636
Dauwe, F., 12
Davis. W. S., 689
Day, H., 367
Dean, A. L., inulin, 127; protein regener-
ation, 417
INDEX TO AUTHORS.
. 819
Decaisne, E., 536
Dehn, W. M., 618
Delezenne, C, blood coagixlation, 170, 226,
231, 234, 255; intestinal juice, 378;
enterokinase and pancreatic juice, 379,
383, 386; secretin, 384
Delfino, A., 512
Demant, B., 378
Deniges, G., tyrosine, 91; uric acid, 576;
determination of purine bases, 582;
homogentisic acid, 600
Denis, P. S., 177
Derrien, E., methaemoiglobin, 202, 205
Desgrez, A., 291
DeucherJ P., pancreas, 418, 425
Devoto, L., determination of proteoses,
60, 61, 644
Devillard, P., 264
Diaconow, C, lecithin, 143, 147
Diamare, V., pancreas, 302, 381
Diels, O., cliolesterin, 334, 335
Diesselhorst, G., 693
Dietschy, R., 644
Dietze, A., 393
Dillner, H., 506
• Disque, L., urobilin and urobilinogen, 601,
603, 604
Ditthorn, Fr., galactosamine, 65, 71, 121
Dittrich, P., 205
Dock, F. W., 299
Dorpinghaus, Th., carbohydrate groups in
proteins, 33, 182; products of protein
hydrolysis, 74, 84, 85, 88
Dombrowski, St., oxy|Droteic acids in
urine, 613; viroferric acid, 614; urinary
ptomaines, 615
de Dominicis, N., 300
Donath, J., 264
Donne, A., 651
Dormeyer, C, 137
Doyon, M., fibrinogen, 172; lipases, 185,
284; bile, 308, 309, 329; biliverdin,
324
Dragendorff, D., 652
Drechsel, E., oxidation, 8; protein sub-
stances, 26-29, 36, 38; gorgonin, 82;
diaminoacetic acid, 97; hexone bases,
78, 98, 100; lysuric acid, 9S; purine
bases, 159; thyroidea, 276; jecorin,
283; urea formation, 550, 552; car-
bamates, 552; silicic acid ester, 685
Droop-Richmond, IL, 517
Dubelier, D., 753
Dvicceschi, V., blood coagulation, 235;
heart-muscle, 451
Duclaux, E., protein coagulation, 40;
milk fat, 518
Dull, G., 128, 129
During, Fr., 437
Dufourt, L., bile, 308, 309, 329; work,
carbohydrate consumption in^ 468
Duggan, C. W., 40
Dumas, J. A., 553
Dunlop, J. C, work and protein meta-
bolism, 471; oxalic acid, 582
Ebstein, E., pentoses, 72, 110, 111
Ebstein, W., saliva, 345; pyrocatechin,
591; urinary calculi, 680; diet cures,
770, 771
Eckhard, C, 340
Edkins, J. S., pepsinogen, 364; pancreatic
rennin, 396
ver Eecke, A., 756
Ehreiifeld, R., protein hydrolysis, 29;
oxyglutaric acid, 32; halogen proteins,
32; leucine, 86; tyrosine, 90
Ehrenreich, M., 391
Ehrenthal, W., 409
Ehrlich, E., 719
Ehrlich, F., 87
Ehrlich, Paul, aldehyde reaction, 121, 675;
precipitines, 186; bilirubin reaction,
321, 654; urine tests, 674
Ehrstrom, R., histone, 62; elimination of
phosphorus, 620; peptonuria, 643
Eichholz, A., globulins, 180; glycoproteids,
506; urobilins, 602
Eichhorst, XL, 413
Einhorn, M., 403
Eiselt, Th., 651
Ekbom, A., 317
Ekehorn, G., 619
van Ekenstein, Alberda, 108
Ekholm, K, 768
Ekunina, M., 461
Embden, G., cysteine and cystine, 28, 92,
94; proteoses in the blood, 183, 415;
ethereal sulphuric acids, 280; conjugated
glucuronic acids, 280; glycolysis, 303;
amino acids, carbohydrate formation,
305; passage of, into urine, 614; pro-
tein regeneration, 416; lactic-acid for-
mation, 416; acetone formation, 262
Embden, H., 598
Emerson, R. L., 394
Emich, Fr., 406
Emmerling, A., 74
Emmerling, O., enzymes, 12, 16, 17; re-
version of sugar; 125
Ellenberger, W., gastric digestion, 370;
protein absorption, 413; milk, 529;
appendix, 397
Ellinger, A., isoserine, 96; diamines, 97,
98; diamine acids, 97, 98; indol-acetic
acid and indol-propionic acid, 102; co-
agulation of bood, 234; lymph forma-
tion, 255,256; pancreatic secretion, 387;
urinary indican, 592-594; kynurenic
acid,600; proteoses, nutritive valueof,746
Elvove, E., 8
Ely, J., saliva, 343, 345
Engel, H., lipases, 363, 389
Engelmann, G. J., 471
Engler, C, 6, 7
Eppinger, 258
Erb, W., 38
Erben, Fr., oxystearic acid, 131; blood
in disease, 246, 247; fat of chyle, 251;
nitrogen partition in the urine, 549;
urein, 563
■820
INDEX TO AUTHORS.
Erlandsen, A. W. E., 143, 144
Erlanger, J., 427
Erlenhieyer, E., leucine, 85; tyrosine,
89, 90
Erlenmeyer, E. jr., tyrosine, 89; cystine,
93; serine, 96
Esbach, G., 645
Estor, A., 711
Etard, A., 457
Etti, C, 512
Eulenburg, A., 295
V. Euler, H., catalysis, 14, 15, 20
Eves, F., 345
Ewald, Aug., keratin, 73; gelatine, 78;
hsematoidin, 216; digestion of gelatine,
395; of cartilage, 395; visual purple,
489; corpora lutea, 498
Ewald, C. A., protein absorption, 413;
gases of transudates, 703; metabolism
and light, 761
Ewan, Th., 6
Eykman, C, isotony, 193; blood-cor-
puscles, 195; blood analysis, 236; inhab-
itants of tropics, 762
Eymonnet, 614
Fabian, E., 294
Falck, F. A., 744
Falck, C. Ph., 744
Falk, Ernst, 758
Falta, W., alcaptonuria, 598; protein
metabolism, 719, 741; artificial nutri-
tion, 738
Falloise, A., secretion of bile, 309; intes-
tinal enzymes, 379; carbon dioxide ten-
sion, 710; metabolism, 761; lipase, 363
Fano, G., 171
Farkas, G., 191
Farkas, K., 512
Farnsteiner, K., 136
Farwick, B., 475
Faust, E., sepsine, 24; gelatine, 77;
samandarin, 692
Favre, P. A., 694
Fawitzki, A., 554
Fedeli. C, 590
Feder, L., 551
Fehling, H., reagent, 116; sugar estima-
tion, 660
Fehrsen, A., 244
Feinschmidt, J., 303
V. Fenyvessv, Bela, conjugated glucuronic
acids, 610, 638
Fermi, CI., fibrin, 175; foodstuffs, digesti-
bility of, 368; autodigestion of the
stomach, 372; trypsin test, 392
Fernet, E., 701
Filehne, W., 32.9
Filhol, 539
Fingerling, G.,^36
Finkler, D., oxidations, 3, 723
Fischer, Emil, enzyme's, 13, 16; protein
hydrolyses, 29, 35. 54, 55, 74, 78, 82;
peptides, 35, 55, 395; amino acids, 83-
92, 101; ornithine, 97; diamhiotri-
oxydodecanoic acid, 101; oxypyrroli-
dincarboxylic acid , 102; carbohy-
drates, 105-109, 113, 114, 120; glucosi-
aes, 108; sugar syntheses, 114; glu-
cosamine, 107 108, 120; glucuronic
acid, 121; isomaltose., 125; purine
bases, 156, 157, 160-162; pyrimidine
bases, 164, 16;5; trypsm, 394, 395, 416;
lactose fern'^entaition, 524; uric acid,
568; urinary purines, 579; conjugated
glucuronic acids, 610
Fischer, Chr., 83
Fischler. M., 324
Flatow, R., 578
Flaum, M., 3o8
Fleckseder, R., 343
Fleig, C, bile, 309; pancreatic juice, 384;
sapokrinm, 385
Fleischl, E., hsemometer, 219; formation
of bile, 330
Fleitmann, 28
Fleroff, A., 63
Fletcher, H, M., 347
Flint, A., stercorin, 337; work and pro-
tein catabolism, 471
Florence, 496
Floresco, N., blood coagulation, 234;
liver, 282, 286; bXliprasiai, 324
Flueckiger, M., urine, 609
Foa, C, saliva, 343; milk, 515, 530
Folin, O., animal gum, 67; alkalinity of
blocd, 223; muscle rigor, 466; acidity
of urine, 545-; urea, 561, 562; urein,
563; creatinine, 563, 565, 567, 744;
uric acid, 569, 571, 578, 744; sulphur
of urine, 611, 744; sulphuric acids of
urine, 623; ammonia, 625; protein
metabolism, 744
Fordos, M., 269
Forrest, J. R., 437
Forssner, G., 614
Forster, J., transfusion of blood, 248;
mineral metabolism, 735; water and
metabolism, 753; dietaries, 764, 770,
771
Fraenckel, P., blood, determination of
alkalinity, 192; methods oi blood
analysis, 236; gastric juice, 354
Fraenkel, A., urine in phosphorus poison-
ing, 554; actioin of diluted air, 705;
dyspnoea metabolism, 758
Fraenkel, Sig., proteins, 28, 33, 45; pep-
tones, 53, 57, 60; thiolactic acid, 28, 94;
histidine, 99; thyreoidea, 276; forma-
tion of glycogen, 294; gastric juice,
363; homogentisic acid, 599; chitin,
686
Framm, F., glutin, 79; sugar and alkali,
115; glycolysis, 185
Franchimont. A. P., 686
Franks O., estimation of fat, 137; fat
absorption, 421-423; cui'are poisoning,
467
Frankenstein, W., 6
Frankland, E., 724
Franz, Fr., 171
Fredericq, L., protein coagulation, 40;
INDEX TO AUTHORS.
82 1
serglobiilin, ISO; osmotic pressure, 190;
hsemocyanin, 219; blood coagulation,
226; blood gases, 698, 706, 710
Fremy, E., muscles of cephalopods, 478;
yolk-splierules, 509
Frentzel, J., glycogen, 288, 290; work
and fat catabolism, 472, 474; nitrogen
in meat and meat extracts, 477; calories
and nitrogen, 727
Frericlis. F. Th., synovia, 266; human
bile, 326; saliva, 347; uric acid, de-
struction of, 573
Freudberg, A., 223
Freund, E., serglobulin, 179; h;T'matin-
ogen, 216; coagulation of blood, 226;
determination of chlorine, 617; star-
vation metalx)lism, 733; cellulose in
consumptives, 714
Freund, O., 733
Frevtag. Fr., cerebrosides, 268, 482, 483,
484; protagon, 481-483
Friedenthal, H., oxidases, 15; determi-
nation of alkalinity, 192; pepsin, 355;
digestion oi starches, 367; protein
assimilation, 412
Friedjung, J. K.,533
Friedlander, G., 413
Friedmann, E., sulphur of proteins, 28;
proteoses, 56; thiolactic acid, 74, 94;
thioglycolic acid, 74; cystin, 93; taur-
ine, 95, 321 ; adrenalin, 278; mercap-
turic acids. 639
Friend, W. M., erythrocytes, 194; peri-
cardial fluid, 261
Fromm, E., 638
Fromme-, A., 363
Frommer, V.^ 671
Frouin, A., gastric juice, 351; intestinal
juice, 378; enterokinase, 383; pan-
creatic juice, 383
Fubini, S., respiration by skin, 695;
metabolism and light, 761
Furbringer, P., oxalic acid, 582; protein
reagent, 643
V. Fiirth, O., tyrosinase, 18; peroxyprotic
acid, 31; xanthomelanin, 32; supra-
renin, 278; bile and fat cleavage, 398;
muscle proteins, 448, 451-453, 477, 478;
muscle rigor, 466; smooth muscle'^, 477,
478; chitosan, 687; melanins, 689,
690
Fuld, E., fibrin formation, 176; blood
coagulation, 231, 232, 234; chjinosin,
361, 362; remiet coagulation, 520, 521
V. Fvinke, 471
Gabriel, S, cystine, 93; bones, 438;
teeth, 440; ovalbumin, 507; asparagin,
nutritive value of, 747
Gachet, J., 3S'>
Gad. J., emulsification of fat, 398; mve-
line, 481
Gartner, G., 219
Gaglio, G., lactic acid, 241, 460; oxalic
acid, 629
Galdi, F., 258
Galeotti, G., 39
Galimard, J., 74
Gallois, 459
Gamgee, A., nueleoproteids, 72; nucleic
acids, 153; oxy haemoglobin, 200, 202;
int-estinal juice, 377; protagon, 481,
482; pseudocerebrin, 485
Ganassini, D., 343
Gamier, L., 598
Garrod, A. E., htematoporphyrin, 213-
215, 650; homogentisic acid, 598-600;
vu-ocln-ome, 601 ; lu-obilin, 603, 605, 606;
uroerj-tlirin, 607; cystinuria, 676; di-
amines, 676
Gatin-Gruzewska, Z., seralbumin, 181;
glvcogen. 287, 288
Gauhe. T., 694
Gaud. F., 115
Gaule, J., 245
tlnnnus, 271; glycogen, 290; fat forma-
tion, 444, 445; muscles, 457; proteid of
egg white, 507, 508; xanthocreatine,
457, 567; epidermis formations, 685
V. Gebhard, F., 467
Geelmuyden, H. C, acetone bodies, 669,
670; estimation of, 674
Geissler, 643
Gengou, O., blood coagulation, 227, 261
V. Genser, 534
Gentzen, M., trj-ptopliane, 103; indol, 593
Geoghegan, E. G., cerebrin, 483, 484;
brain, mineral constituents of. 489
Geppert, J., alkahnity of the blood, 223;
oxj'gen of blood and air pressure, 705;
respiration investigations, 713, 722,
733; alcohol, nutritive. value of, 754
Gerard, E., reductases, 19; cholesterin,
335; uric acid fermentation, 568
Gerber, N., 531
Gerhardt, C, 672
Gerhardt, D., 604
Gerngross, O., 165
Gertner, W., 309
Gessard, C, 690
Geyer, J., 658
Gej'ger, A., 598
Giacosa, P., mucin substances, 66. 509;
estimation of blood ])igments, 217;
frog's eggs, 509; urinary iron, 626;
demolition of aromatic substances, 634
Giertz. H., 47
Gies, W. J., mucin substances. 66. 67. 68,
360, 428. 429, 43^; elastin. 75. 76, 100;
gelatine, 77, 100; liexone bases. 100;
action of ions. 168. 169; hnnnh forma-
tion. 2.i4, 255; pancreatic juice. 387;
Achilles tendon, 429; ligamentura
nucha\ 428, 429; bones, 435; protagon,
4S2; ureine, 563
Gilbert. 455
Gilson. E., lecithin, 143, 147; chitln, 686
Ginsberg, S., 421
Githens, Th. St., 188
822
INDEX TO AUTHORS.
Gittelmacher-Wilenko, G., urine purines,
582; urine reducing power of, 609
Giunti, L., 630
Gizelt, A., 385
Glaessner, K., ethereal sulphiuic acids,
280; gastric enzymes, 355, 362; Brun-
ner's glands, 377; pancreatic juice, 387;
protein absorption, 413, 416; kvnurenic
acid, 600
Glassmann, B., 561
Gleiss, W., 469
Gley, E., iodine, 187; coagulation of
blood, 234, 235; lymphogogues, 255;
enterokinase, 383, secretin, 484; vesic-
ulase, 496
Glikin, W., 137
Gluzinski, A., 369
Gmelin, L., test for bile pigments, 321,
322, 652; saliva, 347
Gmelin. W., 353
Gogitidse, S., 538
Goldmann, E., cystine and cystinuria, 93,
675, 676; iodothyrine, 276; demolition
of sulphurized compounds, 632; dia-
mines, 675
Goldmann, H., 215
Goldschmidt, C, 556
Goldsclunidt, F., 55
Goldscluuidt, H., 344
Gonnermann, M., tyrosinases, 18, 90;
givcocoll, 83; action of trypsin, 395
Goodbody, W., 401
Goodwin, R., 52
Gorodecki, H., 332
V. Gorup-Besanez, E. F., pericardial fluid,
260; human bile, 326
Gossip, B., 460
Gossmami, H., pancreas, 382
Goto, M., protamines, 63, 64; uric acid,
576
Gottleib, R., urea in blood, 240; iron in
the bile, 326; in the urine, 626; urinary
purines, 578; oxyproteic acid, 612
Gourlay, F., 275
de Graaff, C. J. H., 645
Graebe. C, 412
Graffenberg-er, L., 438
Graham, Th., 39
Grawitz, E., 246
Green, E. H., 81
Green, J. R., fibrinolysis, 174; blood
coagulation, 229
Gregor, A., iJ67
Gregor, G., 623
Grehant, N., urea in blood, 240, 242;
urea formation, 553; fatty acids in the
urine, 629
Griffiths, A. B., neurokeratin, 489; urine
poison, 615
Grimaux, E., protein synthesis, 34, 234
Grimbert, L., 606
Grimm, F., 604
Grimmer, W., 367
Grindley, H. S.. 767
Griswold, W., 345
Grober, J., 355
Groeber, A., 595
Grohe, B., 870
Groll, S., 244
Grosjean, A., 234
Gross, A., pseudochylous effusions, 262;
ovovitellin^ 503, 504
Gross, O., 625
Grosser, P., 596
Grouven, H., 475
Gruber, D., dextrins, 128, 129
Gruber, I\I., nitrogen deficit, 718; water
and metabolism, 754
Griibler, G., 38
Griinbaum, D., 512
Griindler, J., 631
Griinhagen, A., 264
Griiixs, G., 195
Griitzner, B., 642
Griitzner, P., estimation of pepsin, 357;
gastric contents, 366; Brunner's glands,
377; pancreatic diastase, 388
Grund, G., pentoses, 72, 110, 111
Grutterink, 645
Gscheidlen, R., sulphocyanides, 343, 611;
lactic acid, 460; urea formation, 553
Gubler, A., lymph, 252; witch's milk, 534
Gumlxjl, Th., 27
Giirber, A., .seralbumin, 181, 182; serum,
mineral constituents of, 187; alkalinity of
blood, 224; bile, 329; amniotic fluid, 512
Guerin, G., 615
Giinzburg, A., 374
Guillemonat, A., spleen, 274; liver, 285
Guinochet, E., 262
Gulewitsch, W., arginine, 96; choline, 145;
nucleic acid, 154; thymin, 165; tryp-
sin, 395; extractives of the muscles, 457
Gullbring, A., 314
Gumilewsky, 378
Gumlich, E., protein absorption, 413;
urinary nitrogen, 548, 554; phosphorus
metabolism, 619
Gunning, J. W., 670
Gusserow, A., 583
Guth, F., 132
Gyergyai, A., proteoses, 414, 746
Haas, K, 214
Habermann, J., proteins, 29, 32; amino-
acids, 87, 88, 91
Handel, M., 434
Hansel, E. W., 372
Haser, 627
Hiuisermann, E., 245
Hafner, A., 132
Hagermann, O., cutaneous respiration of
the horse, 695; work and metabolism,
760
Hagen, W., 164
Hahn, M., fermentation, 10; digestion of
casein, 521; Eck's fistula, 552; urea
formation, 552, 553
Haig, A., 569
Haiser, F., 155
INDEX TO AUTHORS.
823
Haldane, J., blood pigments, 204-206;
estimation of, 218; quantity of blood,
248; oxygen tension, 707, 708
Haliff, E., 20
Hall, W., pmine bases, 163, 408, 454, 5S2;
absorption of iron, 245; artificial nutri-
tion, 739
Hallauer. B., 329
V. Hallervorden, E., urea formation, 551,
554; elimination of ammonia, 625
Hallibiu-ton, W. D., protein coagulation,
40, 61; nucleoproteids, 72; dextrins,
129; cell globulins. 141, 194; fibrin
ferment, 176; seralbimiin, 181; blood
serum, 188; blood corpuscles, 194;
blood coagulation, 234; tetroneiAthrin,
219. 690; pericardial fluid. 261; cere-
brjspinal fluid, 264, 488: liver, 281;
glycogen, 289; pancreatic rennin, 396;
myxcedema, 429; bone marrow, 437;
muscles, 448-453; proteins of the brain,
479; diseases of the nervous svstem,
488; kidnevs, 542
Halsey, J. T.^ 90
Ham, C. E., 216
Hamburger, C, amyolysis, 185, 345
Hambm-ger. H. J., theorv of ions, 41;
colloid^s. 169; blood sefmn, 187, 189,
190; stroma of blood-corpuscles, 192,
220; isotony, 193; permeability of the
blood corpviscles, 195, 196; of the
muscles, 465; leucocytes, 220; alka-
linity of blood, 187, 224; hanph for-
mation, 255; ascitic fluid, 263; intes-
tinal juice, 377, 378; enterokinase, 383;
absorption, 427
Hamburger, E. W., 326
Hammarsten, O., chymosin, 13; globulins
46.179-181; nucleoalbumin. 47; mucin
substances, 66, 67, 68, 509; helico-
proteid, 71; nucleoproteids, 110. 282
381; protoplasm, 141; fibrinogen,
fibrin and blood coagulation, 173, 176,
177, 183, 230; fibrin globulin, 177, 183;
seralbimiin, 181, 183: blood plasma and
serum, composition of, 187; hsema-
topoq)hyrin, 215, 650; casein, 179, 520;
coagulation with rennet, 520; l\Tnph
gases, 251, 702; transudates, 258, 260-
263: synovial fluid, 265; storage of
carbohydrate, 294; bile mucin, 310;
bile acids, 312, 314, 319; scj^nol
sulphuric acids, 310; dehvdrocholic
acid. 315; bile of various anmiais, 307,
312. 315, 319, 327; reaction for bile pig-
ments, 232, 653; cholohsematin, 324;
hmnan bile, 326, 327; phosphatides,
325, 328: urea in the bile, 325, 547;
saliva, 345; antichjinosin, 361; pan-
creas infusion. 391: pseudomucin. 500;
perch-eggs, 504, 509; lactoprotein. 523;
urinar\' protein, 645; phenylhydrazine
test, 658
Hammerbacher, F., 347
Hammerl, H., 409
Hammerschlag, A., 222
Hanriot, M., enzymes, 13; lipases, 17,
185; diabetes, 300; respiratory quo-
tient, 446; respiration, 713, 722
Hansen, C, lecithin, 143; protein regener-
ation, 417; fatty tissues, 441; yolk-fat,
504; milk-fat, 538
Hansen, W., 132
Hardv, W. B., 169
Harley, G., 600
Harley, v., hver and blood sugar, 297;
intestinal putrefaction, 401; pancreas,
418, 425; large intestine, 426; urobilin,
604
Harms. H., 436
Harnack, E., ash-free protein, 38; iodo-
spongin, 81; blood-pigments, 205, 207;
hydramnion, 513; oxalic acid poisoning,
593; m-inary sulphur. 611; demolition
of chlorine substituted methanes, 631;
gallic and tamiic acid, 637
Harris, J. F., protein nitrogen. 27;
nucleic acids, 152, 156; pyrimidine
bases, 152, 156
Hart, E., protein nitrogen, 27; proteoses,
56; histidine, 99; hexone bases, 100
Hart, A. S., elastin, 75
Hartogh, 306
Hartung, C, 509
Hart well, J. A.. 52
Harvey. W., diet cures, 770, 771
Hasebroek, K., lecithin, 146, 404; peri-
cardial fluid, 261; digestion products,
360
Haskins, H. D., vireine, 563; carbamic
acid, 563
Haslam, H. C, salting out of proteins, 39,
179, 181; proteoses, 55, 5(3, 60
Hasselbalch, K. A., incubation of the egg,
510; absorptionof oxygen, 704; oxygen
tension, 711
Hauff . 534
Hausmann, W., nitrogen in protein sub-
stances, 27, 78- koprosterin, 338;
cholesterin, 337
Hawk. P. B., bones, 435; water and
metabolism, 754
Hay. M.. 262
Haycraft. J. B., protein coagulation. 40;
blood coagulation, 171, 226; diabetes,
300; biliverdin. 323; test for bile-acids,
652
Hayem, G., hsematoblasts, 226: per-
nicious ansemia. 246: estimation of
hydrochloric acid. 376
Hedenius, J,, gizzard. 74; bile pigment,
325
Hedin, S. G., elastin, 75: protein hvdrolv-
sis. 78: hexone bases, 96. 98-100;
blood-coriniscles, 193-195; blood analy-
sis. 236: haematocrit. 237; lienases, 273;
action of trypsin, 392; enzjnnes of the
muscles, 454
Hedon, E., pancreatic diabetes, 301; ab-
sorption of sugar, 419; of fat, 425
824
INDEX TO AUTHORS.
Heffter, Aj, liver. 283; muscle reaction of,
447, 466; lactic acid, 463, 469; hypo-
sulphites in the urine, 612; foreign
bodies in the urine, 629; synthesis of
hippuric acid, 635
Heger, P., 280
Heidenhain, M., 44
Heidenhain, R., IjTnph, 250, 253-255;
traiisudates, 257; bile secretion, 307,
308; saliva, 341, 437; stomach, 349,
350, 363, 365; pyloric secretion, 365;
pancreas and its secretion, 381, 382;
trypsin, 386, 391; protein regeneration,
416; sugar absorption, 420; smooth
muscles, 477; milk, 537
Heilner, E., 754
Heinemann, H. N., 474
Heintz, W., fat, 133; lactic acid, 460;
estimation of uric acid, 569, 576
Heiss, E., 439
Heitzmaim, C, 439
Hekma, E., intestinal juice, 377-379;
enterokinase, 383; trypsinogen, 386
Hele. T. Sh., 599
Helier, H., 609
Heller, Fl., protein test, 41, 641; uro-
xanthine, 592; urinary pigments, 601;
blood test, 649; urinary calculi, 683,
684
Helm, 349
Helmholtz, H., 470
Helhvig, smooth muscles, 477
Hemmeter, J. C, 407
Henderson, Y., protein nitrogen, 27;
protein regeneration, 416, 417
Henkeh Th., 518
Henneberg, W., 397
Henninger, A., 60
Henocque, A., 219
Henri, V., enzymes, 13, 14; oxyhsemo-
globin, 199
Henriques, R., 138
Henriques, V., lecithin, 143; lecithin-
sugar, 184; blood, reducing substance,
240; protein regeneration, 417; adi-
pose tissue, 441 ; fat of yolk, 504; milk-
fat, 538; blood gases, 697; lungs, oxi-
dation, 704
Hensen, V., lymph, 253; lymph-gases,
702
Henze, M., gorgonin, 82; iodogorgonic
acid, 82; aspartic acid, 88; hsemocy-
anin, 219; copper in cephalopods, 286;
spongosterin, 337; extractives of mus-
cle, 455; muscles of octopods, 478
Heptner. F. K., 327
THeritier, 534
Herlant, L., nucleic acids, 154-156
Hermann, L., muscle work, 9, 466;
haemoglobin in starvation, 244; forma-
tion ot fa?ces, 409; muscle rigor, 466;
allantom, 583
eron, J., amylolysis, 128, 344; invertin
Bin the intestine, 378
Herrmann, Aug., trypsin, 394; uric acid,
569
Herrmann, E.. 166
Herter, E., saliva, 347; ethereal sulphuric
acids, 589, 591, 637; oxybenzoic acids,
636; oxygen tension, 706
Herth, R., proteosis and peptones, 52, 60
Hervieux, Ch., indol and indican, 186,
592-594; skatol red and urorosein, 596;
uroerythrin, 607
Herzen, A., spleen and digestion, 274;
secretion of gastric juice, 350; charging
theory, 365-385; pancreatic juice, 384,
385
Herzfeld, A., 107
Herzog, R. O., zymases, 11; histidine, 100
Hesse, A., 296
Hetper, J., htemin, 212; ha'mopyrol, 215
Heubner, O., casein, utilization, 530;
nurslings metabolism, 752
Heubner, W., fibrinogen, 177; mytolin,
451
Heiiss, E., 693
Hewlett, A. W., 427
Hewlett, R. T., 40
Hewson, W., 226
Heymann, F., pseudomucin, 500
Heymans, J. F., 481
Heynsius, A., salting out of proteins, 51;
bilicyanin, 322, 324
Hiestand, O., 144
Hilbert, P., 634
Hildebrandt, H., oxalic acid, 583; amino-
benzoic acid, 636; conjugations with
glucuronic acid, 638
Hildebrandt, P., mammary glands, 514,
537; conjugations with glucuronic acid,
610
Hildesheim, 764
Hilger, 69
Hilger, A., 458
Hiller, E., 438
Hirsch, R., glycolysis, 303; amino-acids in
the urine, 614
Hirschberg, A., 222
Hirschfeld, E., 371
Hirschfeld, F., work and nitrogen excre-
tion, 471; uric acid, 569; acetone bodies,
669; metabolism, protein, 738, 750, 765;
nutritive requirements, 770
Hirschfeld, H., 643
Hirschl, J. A., 658
Hirschler, A., putrefaction processes in the
intestine, 405, 407, 589; ethereal
sulphuric acids, 589
His, W., 434
His, W., Jr., pvu-ine bases, 164; uric acid,
575; pyridine, 639
Hlasiwetz, H., protein bodies, 29; amino-
acids, 87, 88, 91
Hochhans, H., 245
Hoeber, R., colloids, 169; osmotic pres-
sure, 189; determination of alkalinity,
191; permeability of the blood-cor-
INDEX TO AUTHORS.
825
puscles, 195; of the muscles, 465;
absorption, 427; acidity of the urine,
545
Hone, J., 652
Hofbauer, L., 343
van't Hoff, J., 6
Hoffmann, A., 586
HofTmann, F. A., transudates, 259, 261;
oedematoas fluid, 265; blood-sugar,
297; diabetes, 299
Hoffmann, J., 544
Hoffmann, P., 626
Hofmann, tyrosine test, 91
Hofmann, Fr., fat and fat formation, 441-
443
Hofmann, K. B., muscles, 474; brain, 486
Hofmeister, F., cellular enzymes, 23; pro-
tein nitrogen, 27; halogen proteins, 30;
glucosamine in proteins, 33; amino-
acids, 3; bindings, 34; removal of pro-
teins, 44; synproteose, 55; collagen,
77; gelatine, 79; serglobulin, 178, 180;
pus, 267; gastric movements, 366;
protein absorption, 414, 416; assimi-
lation limit, 420; ovalbumin, 507;
crystallization of proteins, 507; urea
formation, 553; creatinine, 564; glo-
bulin in urine, 646; lactose, 666
Hofmeister, V., gastric digestion, 370;
cellulose, 397; protein absorption, 413
Holde, D., 132
Holmgren, E. S., submaxillary glands,
340; phenylhydrazine test, 658
Holmgren, Fr., blood-gases, 697; elimi-
nation of carbon dioxide, 710
Holmgren, J., muscle stroma, 448, 451
V. Hoist, G., mucins, 66, 265; transudates,
258
V. Hoogenhuyze, C. J., creatinine, 563,
567
Hooker, D., 255
Hopkins, F. G., halogen proteins, 30;
Adamkiewicz's reaction, 42; trypto-
phane, 102, 103; protein crystallization,
182, 507, 508; uric acid, 569, 577; uro-
bilin, 603, 605, 606; butterflies. 690
Hoppe-Seyler, F., oxidations, 3; cleavage
processes, 5; proteins, 36, 47; compound
proteids, 65; collagen, 76; protoplasm,
141, 145, 148; lecithins, 143, 145, 147;
glycogen, 148, 287; cholesterin, 148;
nuclein, 149; xanthine, 160; blood-
plasma, 187; blood-corpuscles, 194,
219; blood-pigments, 190, 197, 198,202,
204-210,213,214, 217; urobilinoids, 214,
603; blood analysis. 237; chyle, 251;
pericardic fluid, 260; pus, 267-269;
cerebrin, 268; struma, 275; bile, 326,
329; excretin, 410; cartilage, 434: bones
and teeth, 437, 440; lactic acids. 461,
463, 469, 608; retina, 489; ovovitellins,
47, 503; casein, 518; milk, 526; bile
acids in urine, 652; inosite, 668;
chitosan, 687; sebum, 691; respiration
apparatus, 712, 722
Hoppe-Seyler, G., determination of blood
pigments, 217; elimination of phenol,
590; of indoxyl, 592-595; urobilm,
604, 606
Horbaczewski, J., keratin, 73; elastin,
75, 76; glutamic acid, 88; purine bases,
159, 160; uric acid, 568; formation of
uric acid, 274, 570; urostealith, 682;
metabolism, 739
Hombcrg, A. J., gastric juice, 352, 353
Home, R. M., 171
Horodynski, W., 241
Hoagardy, A., 181
Howell, W. H., 464
Huber, A., blood coagulation, 171; fibrin-
olysis, 174; liver diastase, 295
Huebner, R., 403
Hiifner, G., leucine, 85; blood-pigments,
197, 198, 199, 203-200, 209, 218;
spectrophotometry, 218; dissociation
of oxy haemoglobin, 199, 704; glyco-
cholic acid, 313; bile, 313; bird's eggs,
gases of, 509; urea determination, 562;
oxygen tension, 706; gases of the
swimming-bladder, 711
Hiirtlile, K., 183
Hugounenq, L., biliverdin, 324; haema-
togen, 503; ash of child and milk, 535
Huiskamp, W., fibrinogen and fibrin for-
mation, 173, 174, 175, 176, 177; nucleo-
proteids, 178,270, 271; thymus histones,
270, 271
Hultgren, E. O., foodstuffs, 417, 421;
appendix, 427; diets, 764, 767
Humnicki, V., koprosterin, 337
Hundeshagen, F., sponges, 81; lecithin, 143
Hupfer, Fr., 586
Huppert, H., digestion products, 55;
glycogen, 221, 288; reaction for bile
pigments, 322, 653; estimation of
pepsin, 357; meat, 476; urea, 555; uro-
leucic acid, 600; urine, estimation of
proteins, 646; laiose, 665; determin-
ation of acetone, 673
Hurtley, W. H., 600
Husson, 539
Hybbinette, S., 608
Ide, 457
Ignatowski, A., 675
Ikeda, K, 14
Inagaki, C., 181
Inoko, Y., 198
Inonye, K., intestine nucleic acid, 151,
154; cytosine, 165; lacticacidinurine,608
Inonye, Z., 364
Irisawa, T., lactic acid, 241, 608
Ito, M., 643
Iversen, A., secretion of prostate, 496;
concretions, 498
Iwanoff, 621
Iwanoff, L., nucleases, 153, 394
Jaarsveld, G. J., 588
Jablonsky, J. 387
826
INDEX TO AUTHORS.
Jackson, H. C, lactic-acid formation, 461;
kynurenic acid, 600
Jacobsen, A., 184
Jacobsen, O., human bile, 326, 327; be-
havior of aromatic substances in the
body, 635
Jacobson, J., 19
Jacoby, M., oxidation enzymes, 8, 18;
autolysis, 22, 281, 713; phosphorus
poisoning, blood in, 172, 175; demoli-
tion of uric acid, 574
Jacubowitsch, 347
Jaeckle, H., human fat, 132, 134, 441; fat
determinations, 136; lipomia, 442
Jiiderholm, A., blood pigments, 204,
209
Jaeger, A., 711
Jaffa, M. E.,. diets, 767, 769
Jaffe, M., ornithin and ornithuric acid, 97,
635; bile-pigments, 322; creatine, 456;
phenylsemicarbazide, 556; urethane,
563; creatinine, 565, 567; uric acid,
572, 575; urinary indican, 592-594;
kynurenic acid, 600; urobilin, 409, 601,
602, 604, 605; behavior of aromatic
substances, 633, 635-638; furfurol, 637;
thriophenic acid, 637
V. Jaksch, R., alkalinity of blood, 223;
urea in the blood, 240; brain, 480;
urine, partition of nitrogen in, 549;
volatile fatty acids, 608; melanin, 651;
phenylhydrazine test, 657; pentosuria,
666; acetone, 668
J.akowsky, M., 400
Jalowetz, E., 125
Jamieson, G. S., 82
Jantzen, F., 537
Jaquet, A., oxidation enzymes, 8; hsemo-
globin, 198; estimation of blood pig-
ments, 219
Jastrowitz, M., pentoses, 110, 666
Jelinek, J., fermentation, 21, 461
Jensen, P., 459
Jerome, W. Smith, 612
Jesner, S., 491
Joachim, J,, serglobulin, 179; transuda-
tes, 2^8, 261, 263
Jodlbauer, 436
Johansson, J. E., serglobulin, 182; ex-
change of material and gas, 474, 759-
762, 768
Johnson, E. G., chymosin, 361
Johnson, St., creatinine, 563-566
Johnson, T. B., triticonucleic acid, 150;
cytosine, 165
John, S., pig-bile, 313; elimination of
ammonia, 624
JoUes, A., blood-catalases, 20; oxidation
of protein, 32; determination of iron,
217; bile-pigments, 321, 653; milk, 533;
urinometer, 547; uric acid, 578; uro-
bilins, 602; urine, iron of, 626; protein
of, 642; nucleohistone, 647
Joly, 5.39
Jolyet, 697
Jones, W., autolysis, 22; nucleoproteids,
72; nucleic acids, 153; enzymes of
nucleic acids, 158, 273, 571; thymine,
165; nucleases, 271; thymus, 271;
spleen, and uric acid, 273, 275
de Jonge, D., 693
Jorissen, W. P., 6
Jornara, D., 693
Josephsohn, A., 600
Jowett, H. A. D., 278
Junger, E., 318
JungHeisch, E., 463
Justus, J., 166
Juvalta, N., 633
Kalberlah, F., 670
Kanitz, A., steapsin, 389; trypsin, 393
Kareff, N., 173
Kast, A., intestinal putrefaction, 407;
virine sulphur, 611; elimination of
chlorine, 616; methane substituted by
halogens, 631; demolition of sulphur-
ized substances, 632; sweat, 694
Kastle, J. H.„enzymes, 7, 8, 16
Katsuyama, K., lactic acid, 241, 461;
inanition, 731
Katz, A., 604
Katz, J., 477
Katzenstein, A.,630
Katzenstein, G., 760
Kauder, G., globulin, ISO; seralbumin,
181
Kaufmann, M., glycogen, 295; sugar of
the blood, 297; svigar formation from
fat, 306; fat formation, 444-446;
respiratory quotient, 446; muscles,
urea of, 455j sugar consumption of,
468; virea formation, 5'54; methods of
metabolism investigations, 723
KaufmanUj M., urine purines, 579
Kaufmann, Martin, hunger, protein cata-
bolism in, 730; gelatine, nutritive value
of, 746
Kaup, J., 471
Kausch, W., 293
Keller, A., 614
Keller, W., 635
Kellner, O., toodstuffs, utilization of, 418;
work and protein, 471; asparagin, 747;
diets, 764
Kelly, A., extractives of muscles, 455;
chitin, 686
Kermauner, F., 409
Kerner, G., 564
Kiliani, H., 105
Kirschmami, J., 746
Kirk, R., 600
Kisch, F., 459
Kishi, K., 277
Kistermann, 658
Kitagana, F., 485
Kjildahl, nitrogen determination, 556,
557
Hages, A., 318
V. Klaveren, H. K. L.. 208
INDEX TO AUTHORS.
827
Ivlecki, K, 409
Kleine, F., urinary sulphur, 611; formani-
lide, 63'4
Klemensiewicz, R., stomach, formation of
acid, 363; pylorus secretion, 365
Klemperer, G., chymosin, 361; uro-
chrome, 601 ; oxalic acid, behavior of,
630; metabolism of protein, 73S, 750, 765
af ICIercker, O., 563
Klingemann, F., 539
Klingenberg, K., 634
Klug, F., tryptophane, 102; pepsin and
pseudopepsin, 354; products of di-
gestion, 360; trypsin, 390; elimination
of phosphoric acid, 620
V. Knalli-Lenz, E., 286
Knapp, K., sugar test, 116; determination
of sugar, 660, 662
V. Knieriem, W., digestion of cellulose,
397; formation of urea, 550, 551; for-
mation of vu'ic acid, 572
Knopfelmacher, W., human fat, 132, 441
Knoop, F., histidine, 99; methyl imi-
dazole, 108; proteoses in the blood, 183,
415; protein regeneration, 416; demoli-
tion of aromatic substances, 633, 634
Knop, W., 562
Kobert, H. U., 196
Kobert, R., haemolysis, 193; cyanmet-
hiemoglobin, 205; urinary iron, 626;
melaiiines, 6S9
Kobrak, E., 531
Koch, W., lecithans and lecithins, 146
148, 487, 488; neurokeratin, 480
protagon, 482; cephalin, 485, 486, 488
cholesterin, 480; brain analysis, 487
milk, 532
Kocher, Th., 276
Kochs, W., ethereal sulphuric acids, 280;
hippuric acid, 586
Koefoed, E., 518
Kohler, A., meat 476; calorific value,
727; asparagin, 447
Konig, J., muscle, 475; milk, 527-529,
638; foods, 773
Koppe, H., blood corpuscles, 192-196,
220; volume determination, 237; iso-
tomy, 192, 193; haemoglobin, 193, 194;
secretion of hydrochloric acid, 364
Koster, H., 521
Koettgen, E., 490
Kohlrausch, Fr., 191
Kolisch, R., blood, 240; creatinine, 567;
urinary histone, 647
Koraen, G., muscular activity, 474; food
and metabolism, 762
v. Koranyi, A., . blood, 224; osmotic
pressure, 256
Kossel, A., nitrogen of the proteins, 27;
protein hydrolysis, 29, 78; arginase,
36, 97, 550; protamines, 59, 61-64, 84,
96, 98, 498; histone. 62, 63, 96, 97;
nucleoproteins, 71, 72; aminovaler-
ianic acid, 84; serine, 95; hexone bases,
96, 98-100; a-proline, 101; saponifica-
tion, 136; the cell, 140, 141, 142; nucleo-
histone, 141, 270: nucleic acitls, 152-
156, 498; pyrimidine group, 152, 164-
166; pi isminic arid, l.)6; purine bases,
152, 156, 158, 161-164, 271, 284, 382,
454; haemoglobin, 197; blood plates,
222; carebrosides, 268, 482-485; pro-
tagon, 431, 4S2; ichtlmlin, 504
Kossler, A., phenol determination, 589,
590
Kostin, S., 206
Kostytschew, S., nucleic acid, 153, 155
Kotake, Y., nucleic acid of the intes-
tine, 151, 154; cytosine, 165; vanillin,
635
Kotliar, E., 280
Kowalewsky, K., products of digestion,
53; uric acid, 572
Kowarski, A., 455
Kramm, W., 565
Kranenburg, W. R. H., 364
Krarup, J. C, 761
Kratter, Jul., 442
Kraus, Fr., fatty liver, 283; amino acids
and formation of carbohydrates, 305
Krauss, E., 594, 595
Krawkow, N. P., amyloid, 69, 70, 431;
chitin, 686, 687
Krehl, L., 647
Kreis, H., 132
Kresteff, S., 365
Kreusler, W., protein substances, 29
Kreuzhage, C, 471
Krieger, H., protein substances, 38; seral-
bumin, 181, 182; phcsphaturia, 621
Krimberg, R., 457
Krober, E., Ill
Kronig, B., 168
Krogh, A., oxygen in the blood, 704, 711
Kronecker, F., 588
Kriiger, A., denaturization of proteids,
40; haemoglobin, 201; leucocytes, 221;
blood, 221,241,242; spleen, 274; liver,
286; saliva, sulphocyanide, 343; intes-
tinal juice, 377
Kriiger, M., saponification, 136; alloxuric
bases, 156, 159, 163; in fieces, 408; in
urine, 578-581; ammonia, 625
Kriiger, Th. R., peptones, 58, 80; phospho-
carnic acid, 457, 474
Krug, B., 752
Krukenberg, F. C. W., hyalogens, 68;
keratin proteoses, 73; skeletins, 81, 82
lipochromes, 186; haemerythrin, 219;
extractive bodies of muscle, 455, 456;
bird's eggs, 509; urostealith, 682; bird's
feathers".' 690
Krummacher, O., determination of fat,
137; activity, work, metabolism, 471;
heat of combustion, 728; gelatine, its
nutritive viTue, 746
Krumbholz, C. J., 422
Kiibel. F. , saliva, 345, 346
Kiihling, O., behavior of aromatic sub-
stances, 637, 638
828
INDEX TO AUTHORS.
Kiihne, W., enzymes, 10; proteose and
peptones, 51-60, 359; gelatine, 78; ty-
rosine, 89; paraglobiilin, 178; hsema-
toidin and lutein, 216; glycogen, 287;
gastric digestion, 367, 371 ; the pancreas
and its enzymes, 387-391, 395, 396;
emulsion of fat, 422; proteins of the
muscles, 448, 449; smooth muscles,
477, 478; neurokeratin, 73, 480, 489;
pigments of the eye, 490, 491; lens, 493;
corpora lutea, 498
Kiilz, C, 259
Kiilz, E., cystine, 92; pentoses, 110, 666;
amylolysis, 125, 344, 389; glycogen,
287, 288, 291, 295; diabetes, 298, 300;
gastric juice, 364; muscle activity, 468;
gases in milk, 533; conjugated glu-
curonic acids, 632, 638; oxybutyric
acid, 673
Kiilz, R., methffimoglobin, 204; deter-
mination of glycogen, 290; gases of the
saliva, 342, 702
Kueny, L., 117
Kiister, W., blood-pigments, 209-212;
hsematinic acids, 211, 320; haemo-
pyrrol, 214; bile-pigments, 210, 320,
321, 323, 324
Kuhn, 491
Kuliabko, A., pancreas, 302; ureine, 563
Kumagawa, ]\I., formation of fat, 444;
determination of sugar, 663; protein
metabolism, 738, 750, 765
Kunkel, A. J., arsenic, 166; carbon-
monoxide blood-test, 206; iron prepar-
ations, 245; iron and bile, 326, 332;
urinary iron, 626
Kuprianow, J., 460
KurajefT, D., halogenic proteins, 30; plas-
teine, 56; protamines, 63; tryptophane,
102
Kurbatoff, D., 135
Kurpjuweit, O., 407
Kusmine, K., 255
Kutscher, Fr., nitrogen of the proteins,
27; products of digestion, 54, 57; his-
tones, 61, 63, 88, 89; protein hydrolysis,
78, 88, 89; hexone bases, 96-100; nu-
cleic acids, 154; martamic acid, 154;
cytosine, 166; thymine, 271; erepsin,
380; guanidine, 394; choline, 394; intes-
tinal contents, 400; absorption of pro-
tein, 413; bases of meat extracts, 457;
gelatine, oxidation, 582; neurine, 615
Kuwschinski, P., 387
Kyes, P., lecithin, 146; in hsemolysis, 193
Laache, S., 246
V. Laar, J., 73
Ladenburg, A., 496
Laidlaw, P. P., blood-pigments, 216;
turacin, 690
Lanceraux, 301
Landauer, A., bile and putrefaction, 406;
water and metabolism, 734, 754
Landergren, E., foodstuffs, 417, 421;
metabolism, 730, 738, 748; sugar forma-
tion, 749; diets, 764, 767
Landois, L., stroma fibrin, 195; trans-
fusion, 248
Landolt, H., 688
I^andwehr, H., animal gum, 67, 525, 608
Lang, G., 646
Lang, J., taurine, 95; lymph, 253
Lang, S., deamidation, 305; lactic acid
formation, 572; nitriles, 631
de Lange, C, milk, 533, 535
Langendorff, O., 464
Langer, L., 134
Langgaard, A., 530
Langhaus, Th., 216
Langley, J. N., saliva, 345, 347; propep-
sin, 364; cells of the gastric glands, 365
Langstein, L., carbohydrate from protein
substances, 33, 65, 180, 181, 506; diges-
tion products, 53, 57; scatosine, 103;
proteoses in the blood, 183, 415; resid-
ual nitrogen, 183; serum, 188; animo
acids, deamidation, 305; ovoglobulin,
506; ovalbumin, 507; ovomucoid, 508;
alcaptonuria, 598, 600; kynurenic acid,
600; urinary quotient, 628, 720; lactic
acid in the urine, 665
Lankester, E, R., 219
Lannois, E., absorption of sugar, 419;
cerumen, 592
Lapicque, L., spleen, 274; liver, 285, 286;
diets, 767
Lappe, J., 379
Laptschinsky, M., 493
Laqueur, E., casein and rennet coagula-
tion, 518-521; paracasein, 521; lipase,
363
Larin, A. M., 358
Lassaigne, J. L., 583
Lassar, O , 223
Lassar-Cohn, bile acids, 315, 317; human
bile, 318; myristic acid in the bile, 325
Latarjet, A., 352
Latham, 4
Latschenberger, J., blood, 170; bile-pig-
ments, 330, 332; iron of liver, 332;
protein absorption, 413
Latschinoff, P., bile-acids, 317
Laulanie, F., respiratory quotient, 446;
work and sugar consumption, 473, 760
de Laval, G., 526
Laves, E., diabetes, 300; milk-fat, 530
Laves, M., 295
Lavoisier, A. L., 759
Lawrow, D., digestion products, 53;
histones, 61; histidine, 99; blood-pig-
ments, 208; conjugated glucuronic
acids, 638
Lawrow, Maria, 56
Lawes, 445
Laxa, O., casein and rennet coagulation,
519,521
Lazarus-Barlow, W. S., 255
Lea, Sh., 346
INDEX TO AUTHORS.
829
Leathes, J. B., autolysis of spleen, 273:
fat formation, 297; proteosis, absorp-
tion of, 415, 417; ovarial fluid, 500
Lebedeff, A., liver fat, 282; fat of food, 442
Leclerc, A., 694
Leconte, P., 351
Ledderhose, G., glucosamine, 120; chitin,
686
Ledoux, A., 234
van Leersum, E. C, pentoses, 112; con-
jugated glucuronic acids, 122
Legal, E., indol, 402; acetone, 671
Lehmann, C, fat formation, 445; meta-
bolism in starvation, 729; in work, 760
Lehmann, C. G., saliva, 347; protein of
egg-white, 506
Lehinann, Fr., 695
Lelimann, K. B., hgemorrhodin, 207;
adipocere, 442; sugar determination,
663
Leichtenstern, O., blood, 243, 244
Leick, 441
Leipziger, R., 719
Lelli, G., 480
Lemaire, F., isomaltose, 608; and lactose
in the urine, 665
Lemus, W., 518
Leo, H., fatty liver, 282; diabetes, 300;
acids of gastric juice, 375, 376; fatty
degeneration, 443; laiose, 665; nitrogen
deficit, 718
Lepage, L., 384
Lepine, R., conjugated glucuronic acids,
122; pentoses, 184; virtual sugar, 184;
glycolysis, 185, 251, 302; glycogen, 291;
absorption, 419; sulphur of urine, 611,
612; phosphorus compounds in the
urine, 614; urine poison, 615; maltose
in the urine, 667
Lepinois, E., 616
Lerch, 493
Lesem, W., 482
Lesnik, M., 633
Lesser. 1^. J., 417
Lesser, K. A., 254
Leube, W., urine, 640; sweat, 694
Leuchs, H., serin, 95; oxypyrrolidine car-
boxvlic acid, 102; glucosamine, 107,
108,' 120
Levene, P. A., autolysis, 22, 495; pseudo-
nucleic acid, 46; proteoses, 56; tendon-
mucin, 66, 428; hydrolysis of gelatine,
78, 79; amino acids, 83; nucleic acids,
151-156, 514; pjTimidine bases, 152,
154, 156, 394; nucleoproteids, 272, 479;
glucothionic acids, 222, 273, 285, 431,
514; phlorhizin diabetes, 298; trj-psin,
390; ichthulin, 504; dipeptides, 35
Levites, S., 27
Le^T, A. G., 223
Lev)% H., 637
Le\j, L., 454
Le^T. ^I- ^^9
Lewandowski, M., 412
Lewin, K , uric acid and hippuric acid,
586; indican, 593
Lewin, L., ha?moverdin, 208; hydro-
quinone sulphuric acid, 591
Lewinskv, J., 187
Lewis, fh., 478
Lewy, B., 496
Lewy. Benno, 227
V. Leyden, E., 496
V. d. Leyen, E., 593
Lieben, A., hsematoidin and lutein, 216;
iodoform test, 670
Liebermann, C, cholesterin, 336; shell of
bird's eggs, 509; carminic acid, 690
Liebermann, L., catalases, 20; protein
and acid compounds, 38; protein re-
action, 43; pseudonucleins, 47, 151;
lecithalbumins, 47, 349, 542; fat
determination, 137; nucleins, 150, 151;
mucosa of stomach, 349; volk of egg,
502, 504-506, 510, 511; kidneys, 542;
guaiac test, 648
V. Liebig, J., inosinic acid, 155; mineral
bodies, 166; fat formation, 445; work
and metabolism, 471, 472; m'ea, 557;
diets, 764
Lieblein, V., wound-secretion, 265; liver
and urea formation, 553
Liebrecht, A., 30
Liebreich, O., neurine, 145; protagon,
481; cholesterin fat, 691
Liepmann, W., 512
Lifschutz, J., isocholesterin, 337; wool-fat,
692
Likhatscheff, A., 637
Lilienfeld, L., histone, 62, 270; nucleo-
histone, 141, 270; cell nucleus, 142;
fibrin ferment and blood coagulation,
176,227; blood-plates, 222, 227: thymus,
271.272; leuconuclein, 229, 270
V. Limbeck, R., 223
Limpricht, H., 455
Lindberger, W., tryptic digestion, 393;
bile and putrefaction, 406
Lindemann, L., Ill
V. Linden, M., 690
Lindvall, V., 73
Ling, A. R., 125
Lingle, D. J., 464
Linossier, G., 543
Linser, P., sebum, 691
Lintner. C. J.. 128, 129
Lintwarew, S. J., pyloric reflex,' 368;
pancreatic juice, 383
Lipliawskv, A., 672
Lipp, A., 89
Lippich, Fr., 563
V. Lippmann, E. O., enzjTnes, 21;
tyrosine, 90; carbohydrates, 105
Lister, J., 226
Lloyd- Jones. E., 222
Loeb, J., ion action, 166, 168, 169; mus-
cles, 464: metabolism, 761
Loeb, L., blood coagulation, 232, 235
830
INDEX TO AUTHORS.
Loeb, W., carbon-dioxide assimilation, 1;
muscle work, 474
Lobisch, W. F., bilipurpurin, 324; con-
nective tissue, 428
Lobisch, W., mammary glands, 514; case-
in formation, 537
LoUein, W., pepsin estimation, 357; tryp-
sin estimation, 392
Lonnberg, J., cartilage, 433; kidneys, 542
Lonnquist, B., 351
Lore her, G., 361
Loeschcke, K., 289
Loevenhart, A. R., enzymes, 7, 17, 446
Loew, O., active proteins, 4; catalases,
7, 20; protein nitrogen, 27; proteins,
30, 34, 49; sugar syntheses, 114; cell
nucleus, 167
Loewenthal, W., 398
Loewi, O., phlorhizin diabetes, 298; sugar
formation, 306; protein legeneration,
416; urea formation, 550; uric-acid
formation, 571; allantoin, 584; con-
jvigated glucvironic acids, 610; phos-
phorus metabolism, 619, 719
L6\Yit, M., 227
Loewy, A., diamines, 97, 98; alkalinity
of the blood, 223; high altitudes, 246,
614, 762; workandmetabolism with, 471,
760; amino acids in the urine, 614,
oxygen absorption and pressure, 704,
705; metabolism, 762
Lohmann, lysine, 99; choline, 394; neurine,
615
Lohnstein, Th., urometer, 547; saccharo-
meter, 657, 664; sugar estimation, 664
Lohrisch, H., 397
Lombroso, 421
London. E. S., digestion, 370, 400, 401;
blood in starvation, 732
Long, J. H., casein, 518; urine coefficient,
627
Lorrain Smith, J., chlorose, 246; quantity
of blood, 248
Lossen, F., 32
Lubarsky, E., 138
Luchsinger, B., glycogen, 291, 293; sweat,
693
Luciani, L., starvation, 244, 728
Ludwig, C., pseudohaemoglobin, 203;
formation of bile, 330; gastric diges-
tion, 369; pancreatic juice, 382; ab-
sorption of protein, 414; of sugar, 420;
blood-gases, 696, 697; temperature and
metabolism, 761
Ludwig, E., fat of demoid cysts, 138, 502;
uric acid estimation, 576
Lucke, A., hyalin, 69, 687; pus, 269;
benzoic acid reaction, 587
Liidecke, K., glycerophosphoric acid, 144
Liithje, H., sugar formation, 303-305;
oxalic acid, 582
Liittke, J., 376
Lukjanow, S., bile, 307, 308; starvation,
732
Lummert, W., fat, 283; fat formation, 445
Lunin, N., requirement for mineral bodies,
736; artificial feeding, 739
Lusk, Graham, phlorhizen diabetes, 298,
462: sugar formation, 306; lactose in
the intestine, 419; lactic-acid forma-
tion, 462; fermentation of sugar, 665
Lussana, F., 295
Luther, E., 659
Luzzatto, A., 583
Maas, O., 49
MacuUum, A. B., potassium, occurre/ce
of, 167, 464; iron preparations, 245
Maccadam, J., 471
Mac Galium, J. B., 378
Macfayden, A., fermentation, 10; intes-
tinal contents, 399
V. Mach, W., 572
Mackay, J. C. H., 652
Macleod, J., phosphocarnic acid, 457, 470;
carbamic acid, 563
Mac Munn, Ch. A., hfematoporphyrin,
213, 649, 690; urobilinoids, 214, 603;
echinochron, 219; cholohje matin, 324;
myoha?matin, 454; tetronerythyrin, 690
Madsen, Th., 337
Maetzke, G., 400
Magnanini, G., 403
Magnier, 626
Magnus, G., 696
Magnus, R., 284
Magnus-Levy, A., thyreoidea, 277; liver,
281; diabetes, 306; fat formation, 445;
hippuric acid, 586; volatile fatty acids
in the urine, 608; Bence-Jones's pro-
teid, 645; acetone bodies, 669, 674;
respiration experiments, 713, 722, 723;
metabolism, 757, 758, 762
Maillard, L. C, 594
Maillard, M. L., 168
Majert, W., 496
Makris, C., 530
Malcolm, J., 619
Malengreau, F., thymus and nucleohis-
tone, 270, 271
Malfatti, H., tryptophan, 54; purine
bases, 581
Mall, F., 80
Mallevre, A., 397
Maly, R., oxyprotic acids, 31: peptones,
60, 746; bile-pigments, 320-323; hy-
drobilirubin, 320, 603; saliva, 341;
secretion of hydrochloric acid, 364;
pancreatic juice, 387; bile and putrefac-
tion, 406; luteines, 505; creatinine, 566
Manasse, A. 92
Manasse, P., 283
Manche, M., 648
Manchot, W., oxidations, 6, 7, 19
Mandel, A. R., lactic acid formation, 462
]\Iandel, J. A., nucleic acids, 152, 153, .154,
514; spleen nucleoproteid, 272; glu-
cothionic acids, 222, 272, 273, 285, 514;
mammary gland, 514; organic phos-
phorus in the urine, 614
INDEX TO AUTHORS
831
Mandelstaram, E., 308
Manicardi, C, 457
Manning, T. D., 378
Mansfield, G., 264
Maqueiine, L., starch, 126; inosite, 458
Murcet, xanthine, 159; excretin, 410;
excretolic acid, 410
Marchetti, G., 442
Marclilewski, L., pigments of leaves and
blood, 197, 214; h»min, 212; hsemo-
pyrrol, 214; phylloerythrin and cholo-
liBematin, 324
Marcus, E., serglobulins, 178
Marcuse, G., phosphorus metabolism, 719
Marcuse, W., glycogen, 468; lactic cid
formation, 469
Mares, Fr., 569
Marfori, P., 630
Margulies, 658
Mark, H., 283
Marquardsen, E., 407
Marshall, J., 597
Martin, C. J., fibrin formation, 176; blood
coagulation, 234
Martin, S. H., 397
Martins, F., 376
Martz, 692
Maschke, O., protein crystals, 38; crea-
tinine, 565
Masius, J. B., stercobilin, 320, 409
Masloff, A., 377
Massen, V., 552
Masuvama, M., 503
Mathieu, E., 697
Mathews, A., arbacine, 62; protamines,
63, 498; lysine, 98; nucleic acids, 154,
498; ion action, 169; fibrinogen, 172
Matthes, M., intestinal contents, 407;
urinary pepsin, 615; urinary histone,
647
Maurenbrecher, A. D., 113
Mauthner, J., cholesterin. 334; asparagin,
1 ; nutritive value of, 747
Maximowitsch, S., seralbumin, 181, 182
May, R., Ill
Mayer, A., 626
Maver, J., 753
Mayer, L., 418
Mayer, M., 188
Mayer, P., cystine, 92-94; mannoses, 108,
109; conjugated glucuronic acids, 122,
123, 184, 609, 610, 667: oxalic acid, 583;
indican, 593; skatoxyl glucuronic acid,
595
Mayo-Robson, A. W., 307
Mays, K., trj^psin; 390; fuscin, 491
Maze, P., 21
Meara, F. S., 52
Medwedew, A., oxidations, 818; gly-
cocholic acid, 312
V. Mehring, J., uiochloralic acid, 122, 632;
blood-sugar, 184; portal blood. 242,
419; glvcogen formation, 291; phlor-
hizin diabetes, 297, 298; pancreas
diabetes, 300; amylolysis, 344, 388;
assimilation of proteins, 412; absorp-
tion of sugar, 420; sarcosin, 631;
acetonuria, 670
Mehu, C, pleural fluid, 261; urobilin, 604,
605
Meillcre, G., 616
Meinert, C. A., utilization of foodstuffs,
418; diets, 764
Meinert z, J., 283
Meisenheimer, J., fermentation, 11,21
Meissl, F]., 445
Meissl, Th., 513
Meissner, G., products of digestion, 359;
egg-white, 506; urea formation, 553;
allantoin, 583; hippuric acid, 585
Meister, V., 553
Mendel, L. B., lymph formation, 255;
alcohol, 369; trypsinogcn, 385; ab-
sorption of proteid, 412, 413, 414;
muscle extractives, 455; uric acid, 574;
allantoin, 584; kynurenic acid, 600
Mendes de Leon, M. A., 531
Menzies, J. A., 205
De Merejkowski, C, 690
Mesernitzki, 502
Messinger, J., 673
Mester, Br., intestinal putrefaction, 407;
indol and skatol, 596; urinary sulphur,
611
Mett, S., estimation of pepsin, 357; esti-
mation of trypsin, 392
Meyer, C, 286
Meyer, E.. blood-pigment and bile, 329;
alcaptonuria, 598, 600; nitrobenzene,
635
Meyer, G., 417
Meyer, H., camphor glucuronic acid, 122;
adrenaline substances, 278; uric-acid
formation, 572
de Mever, J. . 302
Michaelis, H., 185
Michaelis, L.. 186
Michel, A., seralbumin, 181-183
Micko. K. , excrements, 409; absorption
of casein, 719
V. Middendorff, M., 242
Miescher, F., protamines, 63, 64, 497;
nucleins, 149, 152; pus, 267, 269;
sperm, 497; salmon, metabolism of,
738
Milchner, R., 67
Millon, M. E., protein reaction, 30, 43;
lactoprotein, 523
Mills. W., 582
Milroy, J. A.. 209
Milroy. T. H., nucleins, 150; phosphorus
metabolism, 619
Minkowski, O., blood alkalinity. 223;
ascites, 262; glycogen. 295; blood-
sugar and liver, 297; phlorhizin dia-
betes, 297, 298; pancreas diabete.-=. 300,
301; formation of bile pigments. 331-
333; fat absorption. 421; pancreas and
absorption, 418, 425; lactic acid, 460,
572, 573; uric acid 572, 573; allantoin,
832
INDEX TO AUTHORS.
584; histozyme, 588; acetone bodies,
670, 673, 674; blood in diabetes, 702
Mitjukoff, K., 500
JVIittelbach, F., fibrinogen, 173; homo-
gentisic acid, 600; estimation of pro-
tein, 646
Miura, K., blood-sugar, 184; glycogen
formation, 290; intestinal invertase,
379
Miyamota, S., 355
Mochizuki, J., 394
Modrzejewski, E., 70
MoUenberg, R., 243
Moller, J., 409
Morner, C. Th., membranins, 69, 434;
albu inoid, 74; gelatine, 77, 79, 80.
433, 434; ichthylepidin, 81; tyrosin
test, 91; vitreous humor, 429, 491;
cartilagenous tissue, 430-433, 435;
cornea, 434, 492; bones, 436; crystal-
line lens, 492, 493; ovomucoid, 508;
perkaglobulin, 510; homogentisic acid,
600; estimation of chlorine, 619; gallic
and tannic acids, 637
Morner, K. A. H., sulphur of the proteins,
28; cysteine and cystine, 28, 74, 92;
thiolactic acid, 28, 74, 94; hydrolysis
of protein, 29; albuminates, 49, 45;
tyrosin, 89; proteids of the serum, 170-
182; ha^matin and ha?min, 212, 213;
blister fluid, 265; estimation of hydro-
chloric acid, 576; chondroitin sul-
phuric acid, 431, 542, 647; pigments of
muscles, 454; urinary nitrogen, 548,
554; estimation of urea, 561; fatty
acids in the urine, 608; nubecula, 615;
acetanilide, 634; proteid in the urine,
640; nucleoalbumin in the urine, 646,
647; melanins, 651, 688; bile-acids in
the urine, 652.
Mohr, Fr., reagent for hydrochloric acid,
374; chlorine titration, 617
Mohr, L., sugar formation, 306; urinary
purines, 579; oxalic acid, 583
Mohr, P., 73
Moitessier, J., blood catalases, 20; carbon
monoxide metha!moglobin, 207; chlor-
ine compounds in the urine, 616;
Bence-Jones proteid, 645
Moleschott, J., 764
Molisch, H., 117
Moll, L., 46
Monari, A., creatinine, 455, 470; muscle-
work, 468, 470; xanthocreatinine, 567
Monery, A., 431
Montuori, A., 297
Moor, Ovid, 562
Moore, B., bile and fatty acids, 398, 422;
reaction of intestine, 407; synthesis of
fat, 421; fat emulsion, 422
Moore, J., 115
Moraczewski, W., formation of fjeces, 409;
digestion of casein, 521 ; diabetes, 593
Morat, J., work and carbohydrates, 468
Morawitz, P., blood coagulation, 227, 231,
232,234; cadaver-blood, 235; detection
of proteoses, 644
Morax, V., 589
Moreau, A., 711
Moreau, J., 129
Morel, A., fibrinogen, 172, 173; blood
lipase, 185; hsematogen, 503
Morgen, A., 536
Morgenroth, J., antichymosin, 25, 361
Mori, Y., foodstuffs, utilization of, 418;
diets, 764
Moriggia, A., 693
Morishima, K., liver, 281; lactic acid, 461
Moritz, 366
Moritz, F., transudates, 258; phlorhizin
diabetes, 298; alimentary glycosuria,
298
Moriya, G., lactic acid, 460
Morkowin, N.,63
Morochowetz, L., 430
Morris, G. H., fermentation, 10; isomal-
tose, 125; amylolysis, 128
Moscatelli, R., ascites, 259, 263; lactic
acid, 469, 608
Mosen, R., 222
Mosse, M., pseudochylose fluid, 262; blood
sugar, 296; acid formation in the
stomach, 364; ethereal sulphuric acids,
589
Mott, F. W., cerebrospinal fluid, 264, 488;
diseases of the nervous system, 488
Mouneyrat, A., 102
Mlihle, P., 57
Muhsam, J., 241
Miiller, Erich, digestion of cellulose, 397
Miiller, Ernst, bile-acids, 312, 313, 316, 318
Miiller, Franz, high altitudes, 246, 762
Midler, Friedrich, autolysis of pneumonic
infiltrations, 23, 268, 713; glucosamine
from protein substances, 32, 66-68, 500;
mucous-membrane mucin, 66; star-
vation (indican), 404; fat absorption,
424, 425; ethereal sulphuric acids, 589;
urobilin, 603, 607; urinary sulphur, 611,
612; demolition of aniline, 633; acetone
bodies, 668; faeces nitrogen, 718
Miiller, Johannes, work and sugar con-
sumption, 469; enzymes of the egg-
yolk, 502
Miiller, Julius, 591
Miiller, M., 457
Miiller, Paul, proteoses, 60; koprosterin,
337; excrements, 409; utilization of
casein, 530, 719
Miifler, Paul Th., fibrinogen, 172, 188;
bone-marrow, 172, 437
Miiller, W., cerebrin, 483, 485
Miintz, A., 539
Miinzer, E., formation of urea, 551; liver
and urinary nitrogen, 554
Miither, A., 113
Muirhead, A., 552
Mulder, G. J., 73
Munk, H., 276
Munk, I., chyle and lymph, 251-253;
IXDEX TO AUTHORS.
833
sulphocyanides, 343, 611; contents of
the intestine, 407; absorption of pro-
tein, 414: of sugar, 420; of fat, 423, 424,
427; fat synthesis and fat formation,
421, 442, 445; smooth muscles, 47S;
milk, 52.5; formation of urea, 551;
elimination of phenol, 5S9: work and
catabolism. 471, 620: reaction for bile-
pigments, 633; titration of sugar. 661;
starvation, 730, 731; gelatine, nutritive
value of, 745; nutritive value of pro-
teoses, 746; of asparagin. 747: protein
requirement, 75u; water and metabolism
7. J 3
Murray, Fr. W., 3S7
MuscuJus, F., amylolysis, 12S, 344, 388;
urochloralic acid, 632; urease, 677
Mygge, J., 646
Mylius, F., iodide of starch, 127: iodo-
cholalic acid, 316; bile acids, 311, 312,
315-317
V. Naegeh, C, 126
Naegeli, O., 545
Nagano, J., intestinal juice, 378; absorp-
tion, 419, 420
Nagel, W.. 697
Nakaseko, R., 290
Nakayama, ^M.. erepsin, 153, 380; reaction
for bile-pigments, 653
V. Name, W. G.. gelatine, 77, 80
Nasse, H., blood, 243: hinph, 254; spleen,
273
Nasse, O., activation of oxygen, 5; pro-
tein bodies, 27, 42, 79; dextrines, 129;
glycolysis, 185: glycogen, 287, 289, 467,
46S: saliva, 344. 346: musculin, 450,
452; smooth muscles, 477
Naunyn, B., formation of glycogen, 291;
liver and bile, 331-333: demolition of
aromatic substances, 633
Nawratzki, 264
Nebelthau, E., glycogen, 291 ; hcematopor-
phvrin, 650
Neilson, H., 342
Keimarm, W., conjugated glucuronic acids,
122, 123, 609. efO; carbohydrats-like
substance of the liver, 285, 296
Nencki. L., 635
V. Nencki, M., oxidation, 8; protein sul-
phur, 28; putrefaction of proteins, 30;
trvtophane, 102: blood-pigments, 197,
201, 211-213: phyUocyanin, 214; uro-
bilinoids, 603: haematoporphvrin, 213,
214, 332; ammonia, 241. 550,' 624, 625,
745; diabetes. 300; gastric enzjmes,
354, 355; gastric juice, 354, 364;
splitting of esters, 389: intestinal di-
gestion, 398, 399, 401: indol. 402: re-
action of the intestine, 407; butyric
acid fermentation, 445; urea, 455, 549,
551, 553; carbamic acid, 552; uro-
rosein, 601, 651; acid amides, 630;
demolition of aromatic substances, 634,
635, 637; urine, odor of, 639; melanins,
688
Nerking, J., determination of fat, 137;
glycogen, 289
Nerast, W., permeability of a membrane,
142; fluid chains, 191
Nessler, 374
Neubauer, C, creatine, 456; creatinine,
563, 586, 567; ammonia, 624
Neubaner, O., protein reaction, 43; alcap-
tonuria, 598; urobilinogen, 605; con-
jugation of glucuronic acid, 632;
Ehrlich"s reaction, 675
Neuberg, C, acetone, 32, 668; glucosa-
mine, 67, 121, 500, 504; amyloid, 69,
70; amino-acids, isolation of,' 92; cys-
tine, 92-94; isoserine, 96; oxj-amino-
succinic acid, 96; tetraoxyaminocap-
roic acid, 96, 431; diamines, 97, 98
mannoses, 108; pentoses, 109, 111-113
osazones, 117; levulose. 118, 119, 259.
glucuronic acid, 121-123; conjugated
glucuronic acids, 122, 123, 609, 610; l^'sine
in the blood, 186: liver, 284; glycogen,
290; deamidation, 305; cholesterin,
335, 336: chondrosin. 431; galactosa-
zone, 524; heteroxanthine, o79; esti-
mation of phenol, 591; skatox>i glu-
curonic acid, 595; mineral metabolism.
616, 626, 719. 737; phenylhydrazine
test, 658; detection of glucuronic acids.
667
Neumann, -\lb.. orcin test. 112; nucleic
acids, 152-155; pyrimidine bases, 152,
165: urinarj- iron, 626: phenylhy-
drazine test. 658
Neumann. E., 332
Neumann, O., nutritive value of alcohol.
755; protein requirement, 766
Neumann. R.. 754
Neumann. Walt., 57
Neumeister. R.. proteoses and peptones.
51. 52. keratins, 73: tryptopliane, 102;
dextrins, 129: glycogen. 289: absorp-
tion of proteins, 412, 413; ovomucoid,
508
Neusser. E., 649
Nickles, J., 509
Niemilowicz, L., purine bases, 582; re-
ducing power of the urine, 609
Nierenstein, E., 357
Nilson, G., 127
Nilson, L. F., milk-fat, 527, 528
Le Nobel, C, urobilinoids, 214, 603;
hiematoporphyrin, 649; acetone test,
67
Noeggerath, C. T., 739
Noet-Paton, D., h-mph, 253; liver, 282-.
formation of fa't, 294, 296; bile, 307,
328: work and protein, 471
Noguchi, H., 337
Nolf, P., fibrinogen, 172, 175; fibrinolysis,
175: proteoses in the blood, ?.83, 415;
blood coagulation, 234; saliva, 340;
carbamic acid, 563
Noll, A., nucleic acid, 154; protagon, 488
V. Noorden, C, spectrophotometry', 218;
831
INDEX TO AUTHORS.
alimentary glycosuria, 298; diabetes,
301; formation of sugar, 306; liver and
urinary nitrogen, 554; ethereal sul-
phuric acids, 5S9; proteid in the urine,
040; metabolism, 733, 7 JO, 752, 753
Notkin, J., 276
Nothwang, Fr., 734
Novi, J., pseudoha?moglobin, 203; iron of
the liver, 285; of the bile, 325; saliva,
347
Novy, F., 49
Nowak, J., meat, 476; respiration ap-
paratus, 712; nitrogen deficeti, 718
Nussbaum, M., alveolar air, 70S; carbon-
dioxide tension, 709
Nuttal, G., 405
Nylander, E., sugar test, 116, 655
Nylen, S., 345
Obermayer, Fr., precipitation of protein,
45; indican test, 594
ObermiiUer, K., saponification, 136; chol-
esterin, 334, 335, 336
Oddi, R., bile in the stomach, 398; amy-
loid, 431 ; acidity of the urine, 544
Odenius, R., 514
Oertel. diet cures, 770, 771
Oertel, Horst, phosphorus compounds in
the urine, 614; phosphorus metabolism,
719
Oertmann, E., oxidations, 3, 723
Oerum, H. P. jr., blood-pigments, 217;
human bile, 327; estimation of indican,
595
Oerum, H. P., cystic fluids, 502; nutritive
value of gelatine, 745
Offer, Th. R., glycogen, 294; nutritive
value of alcohol, 755
Ofner, R. 119
Ogata, M., 369
Ogden, H., 598
Oidtmann, H., lymph-glands, 269; thy-
mus, 272; spleen, 274; thyroid, 275;
salivary gland, 339; pancreas, 382;
kidneys, 542; lungs, 714
Oker-Blom, M., dissociation, 190; supra-
renal capsule, 277
Okunew, W., 56
Oliver, G., 278
Ollendorff, G., 107
Olsavszky, V., 620
Omeliansky, 23, 397
van Oondt, 298
Oppenheim, M.,20
Oppenheimer, C., ferments, 12, 13. 390;
seralbumin, 181 ; proteoses in the blood,
183; parental protein assimilation, 412;
law of surface, 757
Orban, R., 379
Orgler, A., acetone, 32, 668; tetraoxy-
aminocaproic acid, 96, 431; uric acid
and nutrition, 569
Orglmeister, G., 97
Orndorff, W. R.. bilirubin, 320, 321
Orton, K. J. P., 599, 600
Osborne, T. B., proteins, 27, 28, 46, 62;
nucleic acids, 152, 156; pyrimidine
bases, 152, 156; ovovitellin, 503;
ovomucin, 506; ovalbumins, 507
Osborne, W. A., 459
Ost, H., 125
Ostwald, W., catalysis, 14; action of ions,
168
Oswald, A., halogen proteins, 31; thyroid
gland, 275-277; urinary globulins, 643
Otori, J., mucin, 67; transudates, 259;
autodigestion of the pancreas, 394;
pseudomucin, 501
V. Ott, 415
Ott, A., transudates, 259; phosphates,
619
Otto, J. G., blood-sugar, 184, 237; blood,
242, 243, 419; blood-plasma, quantity,
238; blood-pigments, 197, 204, 205,
218; skatoxyl-glucuronic acid, 595;
estimation of sugar, 660, 662
Overton, E., boundary layer of the pro-
toplasm, 142, 143,148; permeability of
the blood corpuscles, 195; of the
muscles, 465; mineral bodies of the
muscles, 464
Owen-Rees, 251
Paal, C., proteins, 27, 49, 51, 53, 59; gela-
tine peptone, 79, SO; animo-acids, 102
Pachon, V., coagulation of the blood, 234,
235; extirpation of the stomach, 369,
371; trypsinogen, 385
Paderi, 185
Pages, C, coagulation of the blood, 229,
230; rennet coagulation, 520; milk, 536
Paijkull, L., exudates, 257, 258, 261, 262;
bile-mucus, 310
Painter, H. M., proteoses, 52; saliva, 346
Panek, K., oxyproteic acids in the mine,
612, 613; uroferric acid, 614; phos-
phaturia, 621
Panella, A., phosphoric acid, l86, 457, 477
Panormoff, A., muscle-sugar, 459; oval-
bumin, 507; egg-white, 508
Pantanelli, E., 87
Panum, P. L., serum casein, 178; blood in
starvation, 244, 732; transfusion, 245,
248; elimination of urea, 744; nutritive
value of gelatine, 745
Panzer, Th., halogen protein, 32; chyle,
251; cerebrospinal fluid, 264; colloid,
499, 500
Paraschtschuk, S., 538
Parastschuk, S. W., 362
Parens, E., cerebrin, 483; homocerebrin,
483
Parke, J. L., 505, 506
Parker, W. H., 422
Parmentier, E., 530
Partridge, C. L., purine-base enzymes,
158, 274, 571
Pascheles, W., 631
Paschutin, V., lymph, 254; intestinal
juice, 378
Pascucci, O., blood-corpuscles, 193, 194
Pa.squalis, G., 608
INDEX TO AUTHORS.
835
Pasteur, L., fermentation, 10, 11; micro-
organisms and digestion, 40o
Patten, A. J., cysteine, 28, 94; histidine,
lOU; hexone bases, 100
Paul, Th., action of poison, 168 uric acid,
575
Pauli, W., protein, 39, 40; gelatine
solutions, 78
Paiily, H., histidine, 99; adrenaline, 278
Pautz, W., aqueous humor, 265; lactase,
379; vitreous humor, 491
Pavy, F. W., carbohydrate in proteins, 32,
293; isomaltose, 184; glycogen, 290,
295; blood sugar and diabetes, 295-298;
autodigestion of stomach, 372; work
and metabolism, 471; estim^ation of
sugar, 660, 663
Pawlow, J. P., biliary fistula, 307; saliva,
342, 348; stomach and gastric juice,
349, 351, 353, 356; gastric enzymes,
354, 362; pylorus-reflex, 368; pan-
creatic juice, 382-384, 386; entero-
kinase, 383; pancreas enzymes, 386,
389, 392, 397; ammonia in the blood,
551; Fck's fistula, 552; urea formation,
553
Paver, A., 243
Peiper, G., 223
Peju, P., 173
Pekelharing, C. A., trj'ptophane, 54;
fibrin ferment and blood coagulation,
175, 176, 229, 230; nucleoproteids,
178. 451 ; gastric enzymes, 355, 356, 362
Pemsel, W., 38
Penny, E., 589
Penzoldt, Fr., acetone, 671, 673
Pernossi, L., 12
Pernou, M., 274
Peskind, S., 193
Petersen, P., 475
Petit, A., 616
Petrone, A., 227
Petrowski, D., 486
Petry, E., blood-corpuscles, 196; rennet
coagulation, 521
V. Pettenkofer, M., test for bile acids, 311;
formation of fat, 442-444; work and
metabolism, 471-473, 759; respiration
apparatus, 712, 722; metabolism ex-
periments, 716, 718, 741, 759; diets, 764
Pfaff, F., 307
Pfannenstiel, J.,499
Pfaundler, M., digestion products, 53, 55;
nitrogen in the urine, 549
Pfeiffer, E., woman's milk, 531, 533
Pfeiffer, L., 688
Pfeiffer, Th., 587
Pfeiffer, W., 574
Pfleiderer, R., 359
Pfliiger, E., oxidations, 3. 4, 723; ethereal
sulphuric acids, 280: glvrogen. 288-292,
434; diabetes, 301-303. 306: gases of
the saliva, 340; bile and fattv acids.
397, 423; fat absorption, 422. 423: fat
formation, 443, 445, 741; nitrogen in
meat, 444; muscle metabolism, 467,
469, 471, 473, 474; ovaries, 498; gases
of milk. 528; urinarj' nitrogen, 548;
estimation of, 560, 561; estimation of
urea, 557-561, 613; sugar tests, 657,
663, 664; blood-gases and respiration,
251, 696, 697, 700. 702, 708-711; res-
piration apparatus, 712; N : C quotient
in the urine, 720; calorific value of
oxygen, 723; protein metabolism, 739,
741, 742, 751; nutritive requirements,
742; external temperature ami meta-
bolism, 761 ; protein requirements, 765
Phisalix, C, 693
Picard. J., 491
Piccard, 63
Piccolo, G., hsematoidin and lutein, 216,
498
Pick, A., 358
Pick, E. P., proteoses and peptones, 57,
55-57, 60; serglobuUn, 178; peptozyme.
234
Pick, Friedel, 295, 296
Pickardt, M., transudates, 259; cartilage
434
Pickering, J. W., products similar to the
proteins, 34, 234
Piegand, J., 259
Pierallini, G., 630
Piettre, M., blood-pigments, 202, 212
Piloty, O., glucosamine, 121; conjugated
glucuronic acids. 610
Pilzecker, A., 329
Pinkus, S. N., proteins, 30, 39; crystalli-
zation, 182. 507, .508
Piontkowski, L. F., 351
Piria, 90
Planer, J., 372
Plattner, E., 310
Plant. M., 614
Playfair, 764
Plimmer, R. H., lactase, 386
Plosz, P., blood corpuscles, 194; liver, 281,
282: elimination of proteo.ses. 414;
nutritive value of proteoses, 746: uri-
nary pigments, 601; urinary protein,
640
Poda. H., excrements, 409; absorption of
casein. 719
Podiischka, P.. 574
Poelil. A., intestinal putrefaction, 405;
spermine, 496
Pohl, J., oxidations. 8; dextrin, 129;
estimation of globulin, 180, 646; liver,
281; urea formation, 551; allantoin,
574. 584; oxalic acid. 630; demoliiion
of fatty bodies. 630
Poleck. egg. 505, 509
Polimanti. O., estimation of fat, 137;
fattv degeneration, 443
Politis. G., 747
Pollak. L., glutinase, 391, 392; sulphocy-
anide, 631
Pollitzer, S., 746
Pommerehne, H., 563
836
INDEX TO AUTHORS.
Ponfick, E., 248
Ponomarew, Brunner's glands, 377
Popel,W.,244
Popielski, L., enterokinase, 383; pancrea-
tic juice, 383, 384
Popoff, X., 415
Porcher, Ch., lactose, 539; urinary indi-
can, 592-595 ; skatol red and urorosein,
596; uroerythyrin, 607
Porges, O., 179
Porteret, E., 291
Portier, P., fermentation, 21; lactose in
the intestine, 419
Tosner, C, semen, 495, 496; urinary pro-
tein, 640
Posner, E. R., mucin, 67, 68; mucoids,
360; protagon, 482
Posselt, L., 82
Posternak, S., musculamine, 457; oxj''-
methylphosphoric acid, 458
Pottevin, H., isomaltose, 125; syntheses
of esters, 390
Pouchet, A. G., carmine, 456; urinary
purines, 578; urinary poison, 615;
lungs, 714
Poulet, v., 713
Poulsson, E., 455
Pozerski, E., kinases, 383; secretin, 384
Pozzi-Escot, E., 19
Prausnitz, W., phlorhizin diabetes, 298;
excrements, 409; absorption of casein,
719; metabolism in starvation, 729, 730
Predteschensky, E., 761
Pregl, Fr., protein hydrolysis, 88, 101;
carbon monoxide ha'mochromogen, 209;
dehydrocholon, 311, 316; bile-acids,
316-318; intestinal juice, 377, 378;
ovalbumin, 507; oxj^^roteic acid, 613;
peptides in the urine-, 614; C. : N.
quotient, 628
Presch, W., urinary sulphur, 611; hypo-
sulphrites, 612
Preusse, C, phenols in the urine, 591;
behavior of aromatic substances, 633,
639
Prevost, J. L., bile, 426; formation of
urea, 553
Preyer, W., globin, 208; blood-crystals,
208; placenta-pigment, 512
Preysz, K., 620
Pribram, E., 633
Pribram R., 623
Pribram, H., 453
Prochownick, L., 513
Proscher, F., bilirubin, 321; milk, 529,
536; Erhlich's urine test, 675
Prutz, W.. 592
Prynn, O., spleen and digestion, 274, 385;
trypsin, 383
Pugiie.se, A., 232
Puis, J., 525
Pupkin, Z., 223
Quevenne, Th., lymph, 252; witch's milk,
534
Quincke, G., 517
Quincke, H., hsematoidin, 216; iron
preparations, 245; poikilozytosis, 247
Quinquaud, Ch., urea, 240, 242; muscle-
work, 468; fatty acids in the urine, 629
Raaschou, C. A., guanylic acid, 155, 156
Rachford, B. K., pancreatic diastase, 388;
trypsin digestion, 393; bile and fat, 398
Radenhausen, P., 517
Radziejewski, S., 442
Radzikowski, C, 350
Raehlmann, E., 491
Raikow, P. N., 28
Raineri, G., 512
Ramsden, W., 61
Ranke, H., 569
Ranke, J., division of blood, 249; bile,
307
Ransom, H., haemolysis, 193, 337
Rapp, R., 10
Rauchwerger, D., cholesterin, 335, 336
Raudnitz, R. W., milk and casein, 515,
519, 521
Reach, F., tyrosine, 89; pseudopepsin,355;
digestion, 370; protein absorption, 413;
muscular activity, 474
Reale, E., oxalic acid, 582; urinary
sulphur, 611
Reese, H., 614
Regnault, H. V., cutaneous respiration,
695; method of respiration experiment,
712; nitrogen deficit, 722
Reh, A., 269
Reich, O., 625
Reichel, H., 521
Reich-Herzberge, F., 395
Reinbold, R., Molisch's reaction, 117;
met haemoglobin, 204; trypsin digestion,
394; benzoylation of carbohydrates, 659
Reinders, W., 14
Reinecke, 441
Reiset, J., cutaneous respiration, 695;
method of respiration experiment, 712,
722; nitrogen deficit, 718
Reiss, E., 180
Reissner, O., 376
Reitzenstein, A., 578
Rekowski, L., 637
Rennie, J., pancreas, 302, 381
Renwall, G., 625
Rettger, L. F., 385
Reuss, A., transudates, 259, 261
Reye, W., 174
Reynolds, J. E., 671
de Rey-Pailhade, J., enzymes, 8, 19
V. Rohrer. L., acid compounds of proteins,
38; acidity of the urine, 545
Riazantseff, N. V., elimination of nitrogen,
745, 762
Ribaut, H., enzyme actions, 17, 19
Richards, A. N., albuminoids, 75, 76, 77;
hexone bases, 100; saliva, 343
Richet, Ch., gastric juice, 349, 353;
digestion, 368; fat formation, 446,
INDEX TO AUTHORS.
837
urea, 550; uric acid, 574; thalassin,
693; method of respiration experiment,
713, 722; law of surface, 756
Richter, Max, 496
Richter, P. F., liver, 186, 284, 554
Riecke, R.. 587
Rieder, H., 718
Riegel. M., 518
Riesel, secretion of gastric juice, 353
Riegler, R., 210
Riess, L., oxymandelic acid, 597; lactic
acid, 608
Ringer, L., 229
Ringstedt, O. T., 544
Ritter, A., plilorhizen diabetes, 298; fat
absorption, 42; fermentation of urine,
677
Ritter, E., bile, 328, 329: gall-stones, 334
Ritter, E., cholesterin, 338
Ritthausen, H., proteins, 29, 38; leucin-
imide, 87; milk, 525
Riva, A., urinary pigments, 601, 607, 650
Rivalta, F., 258
Roberts, W., pancreas rennet, 396; esti-
mation of protein, 646; estimation of
sugar, 663
Roch. G., 642
Rockwood, C. W., 614
Rockwood, D., bile and fatty acids, 398,
422; fat absorption, 422; reaction of
the intestine, 407
Rockwood, E. W., protein assimilation
and absorption, 413; uric acid, 570
Rodier, A., 243
Roden, H., 361
Roeder, G., pyrimidine bases, 164
Rolunann, F., oxidation en2}Tnes, 8, 18;
leucin ethylester, 86; detection of glu-
cose, 118; amylolysis, 125, 345; gly-
colysis 185; fat in the blood, 241;
diastase, 185, 251, 295, 297; glycogen
formation, 291; intestinal juice, 378;
bile and putrefaction, 406: absorption,
419, 421, 424; muscles, 447, 466; casein
salts, 519; phosphorus metabolism, 619,
719, 738; sebum, 691; anal-gland
secretion, 692; artificial nutrition, 739
Rohrig, A., metabolism of the muscles,
467; cutaneous respiration, 695
Rose, B., 521
Rosing, E., 6
Roger, G. H., 280
Rohde, E., 43
Rokitausky, P., 608
Rona, P., histone, 62; gelatine, 78; <X-
proline, 101; sugar formation, 296;
duodenal secretion, 377; protein re-
generation, 417
Ronchi, J., cutaneous respiration, 695
Roos, E., iodothyrine, 276; phosphorus
metabolism, 619; sugar tests, 658,
659
Roosen, O., 568
Rosa, E., 713
Rosemann, Rud, milk, 539; eliminatio
of nitrogen, 744; nutritive value of
alcohol, 755
Rosenbach, (), urine tests, 652, 675
Rosenbaum, A., blood catalases, 20;
glycolys s, 303
Rosenberg, Br., 298
Rosenberg, S., secretion of bile, 308;
pancreatic juice, 383; bile and putre-
faction, 406; pancreas and absorption,
418, 421, 425
Rosenfeld, Fritz, indican, 593; volatile
fatty acids, 608
Rosenfeld, G., fat and fat formation, 282,
283, 442, 443; glycogen and fat, 283;
uric acid, 569; phenylhydrazine test,
658; acetone bodies, 669
Rosenfeld, M., 210
Rosenfeld, R., 184
Rosenheim, Th., 750
Rosenqvist, E., 306
Rosenstein, A., chvle and Ivmph absorp-
tion, 252, 253, 414, 420. 423
Rosenstein, W., 299
Rosenthal, J., 712
Rosin, H., levulose, 119, 665; indican,
594; skatol pigments, 596: reducting
power of the urine, 609; Rosenbach's
urine test, 675
Rossi, O., 264
Rost, E., gallic and tannic acids, 637;
salts and metabolism, 754
Rostoski, O., 645
Roth, W., 256
Rothberger, C. J., liver, 280; carbamic
acid, 552
Rothera, C. H., protein nitrogen, 27;
cystine, 92
Rotmann, F., 259
Rotschv, A., 214
Roux. E., 126
Rovida, C. L., hyaline substance, 141, 194,
267
Rovighi, A., putrefaction, 405, 589
Rowland, S., fermentation, 10; leinases,
273; muscle enzjTnes, 454
Rubbrecht,R.,lS8
Rubner, M., protein sulphur, 28; sugar
reaction, 117, 658, 665; utilization of
foodstuffs, 417, 421, 424, 530, 727, 763;
nitrogen in meat, 444; fat formation,
445; milk, 532; nitrogen of fsces, 718;
heat of combustion of foodstuffs, 724-
727; metabolism experiments, 716, 726,
730, 752, 756-758, 761; law of surface,
757, 758
Rubow, W., 464
Riidel. G., 575
Ruff, O., 107
Ruge, E., 404
Rulot, H., fibrinolysis, 174, 175
Rumpf, Th., sugar formation, 306; esti-
mation of phenol, 589; ammonia, 625
Runneberg, J. W., 261
Ruppel, W. G., protagon. 481, 482; milk-
fat, 530; vernix caseosa, 691
■838
INDEX TO AUTHORS.
Russel, 523
Russo, M., 687
Rywosch, D., glycolysis, 185; blood-
corpuscles, 193
iSaarbach, L., 204
Sabanejew, A., 60
Sabbatani, S., 229
Sacharjin, 238
Sacharow, N., 57
Sachs, Fritz, nucleases, 153, 380, 395;
elimination of salts, 364
Sachsse, R., carbohydrate, 116, 127
Sackur, O., casein, 519, 520
Sadikoff, W., 77
Sahli, H., hsemometer, 219; titration of
sugar, 663
Saiki, T., 608
Saillet, urobilin and urobilinogen, 601-
606; hfpmatoporphyrin, 213, 650
de Saint Martin, L., 205
Saint Pierre, C, 711
Saito, S., lactic acid, 241, 461
Salaskin, S., digestion products, 53;
plastein, 56; leucinimide, 87; alkalinity
of the blood, 223; anunonia, 241, 551,
624, 745; erepsin, 380; urea, 550, 553,
561 ; liver and acid formation, 554, 572;
uric acid formation. 572
Salkowski, E. , oxidation enzymes, 8 ; auto-
digestion, 22; putrefactive products,
30,401; protein, 40, 43; pseudonucleic
acid, 46, 521, 522; pseudonuclein, 47;
proteoses, 52, 644; skatolcarbonic acid,
102, 596; pentoses, 110-112, 290, 666;
glucuronic acid, 121; cerebrospinal
fluid, 264; synovin, 265; liver proteid
282; glycogen, 290; cholesterin, 336;
saliva, 347; pancreas, 391; indol, 403;
adipocere, 443; nitrogen of meat, 476!
ovomucoid, 508; casein, 521, 522;
xirea, 550, 551, 553; creatinine, 565,
566; uric acid, 573, 576; purine bases,
581; oxalic acid, 582, 583, 630;
allantoin, 584; hippuric acid, 585;
phenaceturic acid, 588; ethereal sul-
phuric acid, 589; indican, 595; urobilin,
605, 644; urine, fatty acids of, 608, 677;
carbohydrates of, 609; sulphur com-
pounds of, 611, 612; alkalies of, 623;
estimation of the sulphur acids of the
urine, 623; destruction of various sub-
stances, 631, 632, 634, 636; h^mato-
porphyrin, 649, 650; fermentation test,
657; estimation of acetone, 673; water
and metabolism, 753
Salkowski, H., putrefaction, 401, 585;
demolition of aromatic substances, 634
Salomon, George, glycogen, 148; purine
bases, 158; lactic acid, 241; urme
purines, 578-580
Salomon, H., dieamidation, 305; oxalic
acid, 583; acetone, 670
Salomon, W., 551
Salvioli, G., 414, 415
Sammis, J. L., 767
Samuely, F., melanoidins, 30, 689; amino-
acids in the urine, 614; cystine, demoli-
tion of, 631
Sanders-Ezn, H., 761
Sandmeyer, W., pancreas diabetes, 301;
absorption, 418, 425, 426; phosphorus
metabolism, 719
Satta, G., urinary nitrogen, 549; acetone
bodies, 669
Sauerbeck, E., pancreas, 302, 381
Sawitsch, W., 385
Sawjalow, W., plastein, 56; chymosin, 362
Schafer, E., coagulation of blood, 229;
suprarenal capsule, 287
Schaffer, Ph., uric acid, 578; ammonia, 625
Schaffer, F., 28
Schalfejeff, M.. hsemin, 211, 212
Schardinger, F., 460
Scheermesser, \V., peptons, 57, 58, 80
Scheibe, A., 532
Scheibler, C, 124
Schemiakine, A. J., 365
Schenck, Fr., detection of glucose, 118;
blood-sugar, 240, 297, 469
Schenck, M., protein substances, oxida-
tion of, 32, 582; martamic acid, 154
Schepowainikow, N. P., 383
Schepski, N. W., 745
Scherer, J., lymph, 252; inosite, 458, 459,
meta- and paralbumin, 500
Scheuer, M., 353
Scheunert, A., 367
Schierbeck, N. P., saliva, 345; gases of
the stomach, 372; tryj^sin digestion,
393; faeces, 408
Schiff, A., 357
Schiff, H., protein, 27; biuret reaction, 43;
cholesterin, 336; urea, 555; uric acid,
576
Schiff, M., spleen, 274; liver, 280; liver
sugar, 295; bile, 309, 426; charging
theory, 364, 385
Schindler, S., 271
Schittenhelm, A., xanthine oxidases, 18,
158, 273, 571, 574; elastin, 75; amino-
acids, 83-85, 91 , 614, 630; a-proline, 101 ;
nucleic acids, 153; purine bases, 158,
163, 408; coagulation of the blood, 231,
233; spleen, 273; uric acid, 275, 571,
574; estimation of ammonia, 625;
cystinuria, 675
Schlatter, K., digestion, 370, 371
Schlesinger, A., 345
Schlesinger, W., 606
Schlosing, Th.,625
Schlossberger, J. E., milk, 534, 539
Schlossmann, A., milk, 526, 529, 532
Schmey, M., liver, 285; muscle, 464, 477
Schmid, Jul., 581
Schmidt, Ad., intestinal putrefaction, 400;
excrements, 410
Schmidt, Albr., 497
Schmidt, Alex., catalysis, 10; cell protein,
141, 228, 271; coagulation of the blood.
INDEX TO AUTHORS.
S39
176, 177, 261, 227-232; fibrinoplastic
substance, 178, 221, 228; blood cor-
puscles of the frog, 195; leucocytes,
221. 227-229; blood-gases, 698.
Schmidt, C, serum, 188; blood, 238, 239;
lymph, 252^ transudates, 257; buccal
mucus, 341; saliva, 346; gastric juice,
353, 354; pancreatic juice, 387; biliary
fistula, 406; absorption of fat, 424;
osteomalacia, 439
Schmidt, C. H. L., 31
Schmidt, C. W., 714
Schmidt, E., 455
Schmidt, Fr. , 670
Schmidt, P., 579
Schmidt-Mulheim, A., coagulation of the
blood, 171; protein absorption, 413,
414,744
Schmidt-Nielsen, S., enzymes, 12; chy-
mosin, 362
Scluniedeberg, O., oxidations, 7; protein
crystals, 38; desamino-albuminic acid,
49; salmine, 63; onuphin, 69; campho-
glucuronic acid, 122, 638; nucleic acids,
152, 154, 155; nucleosin, 165; fen at in,
282; chondroitin sulphuric acid, 431,
432; urea, 551; hippuric acid, 586, 587;
histozym, 588; phenolgl neuronic acid,
590; indoxylglucuronic acid, 595; chi-
tin, 686; melanin substances, 688
Schmitz, K., putrefaction, 405, 407, 589
Schneider, A., 238
Schneider, E., saliva, 343; kynurenic
acid, 600
Schneider, H., tyrosinases, 18; melanins,
690
Schoffer, A., 85
Schonbein, C. F., 623
Schondorff, B., urea, 240, 455, 532; esti-
mation of, 562; thyroidea, 277; glyco-
gen, 288, 292, 459; uric acid, 569; pro-
tein metabolism, 742, 743
Schone, A., Ill
Scholz, H., indican, 593
Shore, L. E., absorption, 415, 416
Schotteliug, M., 405
Schotten, C, fellic acid, 318; intestinal
putrefaction, 586; fatty acids in the
urine, 608, 629; damaturic and damolic
acids, 616; behavior of aromatic sub-
stances, 633, 634
Schoubenko, G., 28
Schoumow-Simanowski, E. O., pepsin,
355; gastric juice, 364, 371
Schreiber, E., 571
Schreiner, Ph., 496
Schreuer, M., nitrogen of meat, 477;
calorific value of nitrogen, 727; protein-
overfeeding, 744, 752
Schrodt, M., 437
V. Schroeder, W., uric acid, 240, 241, 547,
572; urea, 551, 553
Schroter, F., 153
Schrotter, H , proteoses and peptone, 52,
53, 60
Schryver, S. B., 23
Schiile, 353
SchiUe, A., 343
Schutz., E., digestion products, 55; esti-
mation of pepsin, 357, 358; stomach,
366; lactic acid, 608
Schiitz, J., pepsin, estimation of, 357;
action of, 359; bile and fat, 398
Schutze, .\lb., 186
Schiitzenberger, P., proteins, 29, 34, 51, 59
Schultze, B., 445
Schultze, E., 548
Schultzen, O., diabetes, 300; urea, 550,
551; oxymandelic acid, 597; lactic
acid, 608; acid amides, 631; sarcosin,
631; behavior of aromatic substances,
633, 635
Schulz, .\rth., 215
Sclmlz, Fr. N., protein, 28, 31, 33, 38, 41,
59; oxyjirotein, 31; hi.stones, 62, 208;
galactosamine, 65, 71, 121; estimation
of fat, 137; seralbumin, 181; starva-
tion, 244, 730
Schulz, H., 429
Schulze, C, 345
Schulze, E., hydrolytic products of pro-
teins, 29, 84, 85, 87, 90, 91; hexone
bases, 96, 100; hemicelluloses, 130;
isocholesterin, 337; lecithans, 144
Schulze, E., 522
Schulze, F. E., 166
Schumburg, W., chymosin, 361; metabo-
lism, 758
Schumm, O., blood, 217, 247; spleen. 273;
sugar formation, 306; pancreatic cysts,
387
Schunck, C. A., pigments, 197, 505
Schunck. E., 601
Schur, H., uric acid, 569, 570, 574; urinary
purines, 579
Schurig, 285
Schuster, A., utilization of foodstuffs, 418;
diets, 770
Schuurmanns-Stekhoven, 202
Schwalbe, E., blood plates, 227; fat, 283,
443
Schwann, Th., biliary fistula, 307, 406
Schwarz, H., 117
Schwarz, Hugo, elastin, 75, 76, 89
Schwarz, L., protein compounds, 49;
secretion of hydrochloric acid, 364;
acetone bodies, 669
Scliwarz. O., antipepsin, 373; aceto-acetic
acid, 672
Schwarzschild, M., biuret bases, 35;
trypsin, 390, 395
Schweissinger, O., 642
Schwinse, W., 243
Scofield, H., 323
Sebelein, J., peptones, 52; digestion of
casein, 521; milk, 515, 521, 522, 525,
528
Seegen. J., blood-sugar, 118. 184. 295, 296,
468, 473; carbohydrate substance in the
liver, 285, 296, 297; amylolysis, 344;
840
INDEX TO AUTHORS.
respiration, 712; nitrogen deficit, 718;
metabolism and water, 753
Seelig, P., 4U5
Seemann, J., proteins, oxidation of, 32, 78;
carboiiydrate in the proteins, 33, 508;
nucleic acid, 154; erepsin, 380; in-
testinal contents, 400; absorption, 413;
ovomucoid, 508
Segale, M., 166
Seitz, W., 287
Selitrenny, L., 78
Seliwanoff, Th., levulose, 119, 665
Selmi, 24
Semmer, G., 195
Senator, H., 593
Senkowski, M., bile-acids, 316
Senter, G., 20
Serdjukow, A., 368
Sertoli, E., 701
Sestini, L., 568
Setschenow, J., blood-gases, 697, 699, 701
Shepard, C. U., 585
Shore, L. E., protein absorption, 415, 416
Siau, R. L., isomaltose, 184; phlorhizin
diabetes, 298
Sieber, N., protein sulphur, 28; glycolysis
185; blood-pigments, 201, 211, 213
haematoporphyrin, 213, 214, 215, 332
urobilinoids, 214, 603; diabetes, 300
gastric juice, 354, 355, 362, 371; in.
testinal digestion, 399; Umikoff's re-
action, 532; urorosein, 601, 651; nitro-
benzaldehyde, 636; melanins, 688, 689
Siegert^ F., 441
Siegfried, M., hydrolysis of proteins, 29;
peptone substances and kyrines, 54, 58,
59, 60, 80; reticulin, 80, 81, 428; glu-
tamic acid, 88; nitrotoluene sulpho-
compounds, 92; lysine, 99; pseudo-
haemoglobin, 203; jecorin, 283; phos-
phocarnic acid, 457, 458, 462, 470, 474;
orylic acid, 523; milk nucleon, 532;
carbanimo-acids, 701
Silbermaim, M., isoserine, 96; oxyanimo-
succinic acid, 96
Silbermann^ O., 333
Simacek, E., fermentation, 21, 396; lactic
acid, 461
Simon, G., milk, 526, 528
Simon, O., autolysis, 23, 268, 714; car-
bohydrate substance of the liver, 296
de Sinety, L., 665
Siven, V. O., uric acid, 569, 570; protein
metabolism, 738, 750, 765
Siwertzow, D., 145
Sjoqvist, J., proteins, 38, 60; estimation of
hydrochloric acid, 375, 376; urinary
nitrogen, 546, 554; urea estimations,
561
Skita, A., fibroin hydrolysis, 82-84, 89
Skraup, Zd., protein hydrolysis, 29, 78;
oxyanimo acids, 96, ioi; caseanic and
caseinic acids, 101; benzoylation of
carbohydrates, 117; kyrines, 58
Slosse, A., urinary nitrogen, 553, 744
Slowtzoff, B., pentosanes, 111; liver, 286;
semen, 495; metabolism, 57, 759
Smals, Fr., 575
Smirnow, A., 636
Smith, F., 693
Smith, Herbert, saliva, 345, 346; bones,
435
Smith, Lowain, chlorosis, 246; quantity
of blood, 248; oxygen tension, 707, 708
Smith, W. G., 654
Smith, W. J., urinary sulphur, 612;
demolition of sulphur compounds, 631
Smits, H., 46
Socin, C. A., 293
Socoloff, N. 326
Soldner, F., milk, 518-520, 525-527, 531,
533-535
Sorensen, S. P. S., amino-acids, 83;
ornithin, 97; a-proline, 102
Soetber, F., 621
Solera, L., 343
Solley, Fr., gelatine, 77, 79
Sollmann, T., gall-bladder, 329; muscles,
449, 453; fibroma of uterus, 502
Sommer, A., 283
Sommerfeld, 327
Sonden, K., respiration aparatus, 713,
722; metabolism, 756, 758, 768
Sorby, H. C., 509
Sourdat, 534
Southgate, 393
Soxhlet, F., glucose, 117; maltose, 125;
fat formation, 445; milk, 518, 520, 526,
536, 538; titration of sugar, 661
Spampani, G., 538
Spangaro, S., 170
Speck, C, gas exchange, 713, 759-762
Spiegler, A., 734
Spiegler, E., protein reagent, 642; mel-
anins, 689
Spiro, K., proteins, 38, 45; phenylalanine,
78; glycocoll, 83; serglobulin, 179;
blood coagulation, 231, 232, 234; pep-
tozym, 234; lactic acid, 469; rennet
coagulation, 520, 521
Spitzer, W., oxidation enzymes, 8, 18, 20;
xanthin oxidases, 18; glycolysis, 20,
185, 302; liver protein, 282; uric acid
formation, 571
Spriggs, E. J., 357
Ssubotin, M., 536
Staal, J. Ph., 596
Stade, W., 363
Stadelmann, E., tryptophane, 102; ict-
erus, 216, 332, 333; suprarenal capsule,
277, 330; bile, 307-309, 328, 331-333,
426: intestinal putrefaction, 407; elin i-
nation of nitrogen, 554; elimination of
ammonia, 624; peptonuria, 643; /^-oxy-
butyric acid, 673; blood in diabetes, 702
Stadthagen, M., diamines, 24, 615;
adenine, 162; xanthocreatinine, 567;
urinary sulphur, (111; poison of urine,
615; cystine, 676
Staedeler, G., 324
INDEX TO AUTHORS.
841
Staehelin, R., 258
Stanek, V., 145
Stange, M., 132
Starke, J., proteins, 40, 182; globvilin, 46
Starke, K, 181, 182
Starling, E. H., lymph formation, 255,
256; intestinal enzymes, 379; entero-
kinase and trypsinogen, 379, 383-386;
secretin, 384; pancreatic erepsin, 391
Stassano, H., 386
Stavenhagen, A., 10
Steensma, F. A., 402
Steilf, R., 589
Steigef, E., 96
Steil, H., muscle-fat, 474, 475
Stein, G., 334
Steinitz, phosphorus metabolism, 619,
719; C. : N. quotient, 628, 720; lactose
in the urine, 665; artificial nutrition,
739
V. Stejskal, R., 240
Sternberg, S., 526
Stepanek, J. O., 502
Stern, H., 331
Stern, Heinrich, 496
Stern, L., 20
Stern, R., bile 329; ethereal siilphuric
acids, 589
Steudel, H., mucin, 67; arginine, 97;
glucosamine, 121; nucleic acids, 151;
pyrimidine, bases, 152, 164-166
Stewart, C. W., 359
Stewart, G. N., blood, 236; muscle, 449,
453
Steyrer, A., muscle, 453, 472; urine, 546
Sticker, G., saliva, 343, 346
Stiles, P., 298
Stintzing, R., 467
Stockmann, R., 471
StofTregen, A., 643
Stohmann, F., digestion of cellulose, 397;
calorific determinations, 724
Stoklasa, J., fermentations, 21, 396, 523;
lecithin, 146; glycolysis, 302; lactic-
acid formation, 461
Stokvis, B. J., bile-pigments, 322, 323,
603, 653; benzoic acid, 588; urobilin,
604, 644; haematoporphyrin, 649
Stolnikow, J., 646
Stolte, K., 550
Stoltz, Fr., 278
Stone, W. E., Ill
Stookey, L. B., gelatine, 79; glycogen,
292
Stoop, F., cystine, 93; serine, 93, 96
Storch, v., milk, 517, 539
Stradomsky, N., oxalic acid, 583, 630
Strashesko, N. D., 350
Strassburg, G., gases of the lymph, 251;
carbon-dioxide tension, 709, 712
Strassburger, J., 410
Straub, W., glj'^cosuria, 299; metabolism,
734, 754
Straus, J., 262
Strauss, Edw., spongin, 81, 82
Strauss, H., le\nilose, 118, 119, 184, 259;
blood, 123; transudates, 259, 262; bile,
326; lactic-acid fermentation, 371;
urine, 546
Strecker, A., lecithin, 143; bile-acids, 315
Strickler, E., 528
Strohmer, F., 445
Stusiewicz, B., 747
Struve, H., 649
Strzyzowski, C, 213
Subbotin, V., blood, 244; alcohol, 754
Sugg, E., 523
Suida, W., 334
Sulima, A. Th., digestion, 370, 400
O'Sullivan, C, 13
Sundl?erg, C, 356
Sundvik, E., glucosamine, 121; xanthine
bodies, 157; uric acid, 568; conjugated
glucuronic acids, 610, 632; chitin, 686;
psylla alcohol, 692
Suter, F., protein sulphor, 28; thiolactic
acid, 28, 74, 94
Suto, K., 663
Suzuki, W., 92
Svenson, N., 753
Swain, R. E., 103
Symmers, D., 614
Syniewski, V., starch, 126; dextrin, 128
Szekely, S., 137
V. Szontagh, F., milk enzymes, 523; case-
ins, 529, 531
Szydlowski, Z., 361
Szymonowicz, L., 278
Takamine, J., 278
Tallquist, T. W., 749
Tammann, G., enzymes, 12, 14
Tangl, Fr., blood-serum, 189, 190; blood-
analyses, 191, 236; blood-sugar, 297;
297; development of the egg, 511, 512,
C : N = quotient, 628, 720
Tanret, Ch., 524
V. Tappeiner, H., celliilose, 397, 404; bile-
acids, 426
V. Tarchanoff, J., bile pigments, 331; tata-
proteid, 506
Tarulli, L., 544
Taveau, R., 278
Tawara, 764
Taylor, A. E., fatty liver, 283; fat for-
mation, 443; lack of mineral bodies, 735
Tebb, Chr., reticulin, 81; glycogen, 289;
amylolysis, 345, 388; intestinal inver-
tase, 379; cholesterin, 487
Teeple, J., bilirubin, 320, 321
Teichmann, L., htemin crj'stals, 211, 649
Tensstrom, B. St., bile, 311, 314
Terfat, P., 616
V. Terray, P., bile and putrefaction, 406,
407; oxalic acid, 582; lactic acid in the
urine, 608
Terroine, E. F.,388
Terry, O. P., 342
Teruuchi, Y. , 551
Tetzner, E., saliva, 343
842
INDEX TO AUTHORS.
Thelen, M., 563
Thesen, J., isocreatinine, 455; indican, 593
Thiele, O., 613
Thiemich, M., 283
Thierfelder, H., galactose, 120, 484;
bariiini, 166; digestion and micro-
organisms, 405; protagon, 482; cere-
bron and cerebrosides, 484, 485; sphin-
gosin, 4S5; mammary gland, 514, 539
Thiroloix, J., 301
Thiry, L., intestinal fistula and intestinal
juice, 377, 378
Thorner, W., 515
Thompson, W. H., 550
Thorns, H., 556
Thudichum, L. W., lecithins, 143; bile-
pigments, 321; brain phosphatides
480-482, 485; cerebrosides, 483-486
luteins, 505; urotheobromin, 580
urinary pigments, 601; alcohol in the
animal body, 754
Tiedemann, F., 347
Tiemann, H., milk, 522, 528
Tigerstedt, K., 620
Tigerstedt, R., respiration apparatus, 713,
722; metabolism, 729, 730, 756, 758,
768
Tissot, J., 468
Tobler, L., digestion, 370; phosphaturia,
621
Toepfer, G., 617
Tollens, B., carbohydrates, 105, 111, 113,
117, 118; glucuronic acid, 123; urea, 556
Tolmatscheff, milk, 526, 531, 534
Tompson, F. W., 13
Toppelius, M., 563
Torup, S., carbohsemoglobin, 206, 207;
globulins, 701
Tower, 81
Toyonaga, M., 286
Traube, M., oxidations, 3, 6, 8
Traube, W., 157
Treupel, G., urinary carbohydrates, 609,
659
Trifanowski, D., 326
Trillat, A., 19
Tritschler, F.,630
Troller, J., 353
Trommer, C, sugar test, 116, 655
Trumpy, D., 693
Tschenloff, B., 744
Tscherwinskv, N., 445
Tschirjew, S', 248
Tsuboi, J., 244
Tuczek. F., 346
Tullner, H., 568
Turby, H., 378
V. Udrdnsky, L., diamines, 24, 615, 676;
bile-acids, 312, 652; urinary pigments
and humin substances, 601; sugar test,
659; cystine, 676
TJffelmann, J., 374
Uhlenhuth, 186
Uhlik, M., blood-crystals, 201, 203
Ulpiani, C, lecithins, 146, 147; brain, 480;
uric acid, 568
Ulrich, Chr., 125
Ultzmann, R., 680
LTzer, F., 137
Umber, F., proteoses, 55; nucleoproteids,
150, 381; transudates, 258; gastric
juice, 352, 353; levulose, 665
Umikoff, N., 532
Underbill, F. P., 234
Urbain, V., 697
Ure, A., 635
Ussow, 393
Ustjanzew, W., 427
Vahlen, E., bile-acids, 316, 317
Valenciennes, A., muscles of cephalopods,
478; yolk-spherules, 509
Valenti, A., 532
de Vamossv, Z., 280
Vandegrift' G. W., 429
Vandevelde, A. J. J., 523
Vanlair, C, stercobilin, 320, 409
Vaubel, W., halogen protein, 30; Millon's
reagent, 42; estimation of acetone, 673
Vauquelin, L. N., 583
\sLy, Fr., liver proteid, 282; glycogen, 468
V. d. Velde, A., 588
V. d. Velden, R., 588
Velichi, J., 477
Vella, L., 377
Veraguth, O., 744
Verhaegen, A., gastric secretion, 353, 365
Verneuil, 349
Vernois, M., 534
Vernon, H. M., erepsin, 379, 380, 391;
pancreas enzymes, 386, 388, 390, 391,
396; tryptic digestion, 394
Verploegh, H., creatinine, 563, 567
Verworm, M., 4
Viault, P., 245
Vierordt, K., spectrophotometry, 218;
expiration air, 707
Vigni, L., 413
Vignon, L., 82
Vila, A., blood- igments, 202, 205; mus-
culamine, 457
Ville, J., blood-catalases, 20; blood-pig-
ments, 202, 205, 212; fat absorption,
425; estimation of chlorine in urine, 616
Villiers, A., 615
Villiger, V., 6
Vincent, Sw., 478
Vines, S. H., plant-enzymes, 380, 390
Viola, E., 191
Virchow, R., amyloid, 69; hsematoidin
216; suprarenal capsule, 330 »
Vitali, A., 649
Vitali, D., 616
Vitek, E., 21
Voltz, W., milk-corpuscles, 517; nutritive
value of asparagin, 747
Vogel. J., pentoses, 110, 666; amylysis,
125, 344, 3S9; lactase, 379
Vogel, R., 467
INDEX TO AUTHORS.
843
Vogelius, 288
Voges. O., 548
Vohl, H., 458
Voirin. G,, 597
Voit. C, glycogen formation, 291, 293.
420; bile and putrefact'on, 406; fajces,
408; absorption. 413. 419, 424; fa^
formation. 442-445, 742; work anJ
metabolism, 471-473, 759; nitrogen in
meat, 444, 476, 720; urea formation,
553; elimination of phosphoric acid,
620; detection of milk sugar, 665;
st ndard figures for dietaries, 728, 750,
764, 765, 767; nitrogen deficit, 718; oxy-
gen requirements, 723; water content of
the body, 734; starvation metabolism,
728, 729, 732, 738; mineral metabolism,
735; protein catabolism, 740-744, 751;
organized and circulating protein, 742-
744; nutritive value of gelatine, 745;
proteoses, 746; asparagin, 747; water
and metabolism, 753; salts, 753;
dietaries, 764, 770. 771
Voit, E., f^t determination, 137; glycogen,
293; bones, 438, 439; fat formation,
4-i4, 445; heat of combustion, 727;
starvation metabolism, 730, 732, 738;
vegetable diets, 750; law of surface, 757
Voit, Fr., fermentation of sugar, 120; sugnr
elimination, 293, 420; thyroidea, 277;
?;lycogen formation, 293; formation of
aeces, 409; curare poisoning, 467;
acetone bodies, 669, 670
Volhard, F., estimation of pepsin, 357;
lipase, 363; estimation of trypsin, 392
Volhard, J., titration methods, 290, 581,
617
Volkmann, A. W., 735
van Voornveld, J. A., 246
Vossius, A., 331
Voswinckel, H., 690
Vozarik, A., acidity of urine, 544, 545
Vulpian, A., suprarenal capsule, 277
Wachsmann, M., 388
Wachsmuth, L., 260
Walchli, G., 76
de Waele, H., 523
Wagner, B., 664
Wagner, H., extractives of the mucle, 455.
456
Wahlgren, V., biliary mucus, 310, 329;
glycocholeic acid, 312
Wait, Ch., 471
Wakeman, A. J., 284
Waldvogel, R., uric acid, 571; acetone
bodies, 669
Walter, Fr., \irea formation, 551; blood-
ga.ses, 701
Walter, G., ichthulin, 71, 504
Walther, A.. 382
V. Walther, P., fat absorption, 421, 423
Wanach, R., 220
Wang, E., 594
Wanklyn, J. A., 518
Wanner, Fr., 714
Warren, J., 469
Wasbutzki, M., 407
Wassermann. A., 186
Wassiliew, W., 387
Wawrnsky, R., 368
Waymouth Reid E., 419
Weber, 509
Weber, O., 439
Wedenski, N., carbohydrate of the urine,
608
Weidel, H., xanthin reaction, 160; camine
456
Weigert, Fr., 98
AVeigert, R., 185
Weinland, E., glycogen, 288, 293; anti-
enzymes, 355, 372, 380; lactase, 379,
386; lactose in the intestine, 420
Weintraud, W., diabetes, 300; elimination
of nitrogen, 554; phosphorus meta-
bolism, 619
Weisbach, 486
Weisgerber, G., 710
Weiske, H., cellulose digestion, 397;
bones, 438, 439; nutritive value of as-
paragin, 747
Weiss, J., alkalinity of the blood, 223;
formation of sugar, 297; uric acid, 586
Weiss, H. R., trypsin, 392, 393
Weiss, Sigm., glycogen, 291, 293, 468
Weissberg, J., 6
Wells, H. G., 101
V. Wendt, G., metabolism, 719, 738
Wenz, J., 51
Werenskiold, F., 529
Werigo, B., proteins, 38; respiration, 710
Wertheimer, E., bile, 329; pancreatic
juice, 384
Werther, M., saliva, 347; glycogen, 467;
lactic acid, 469
Westphal, C, 217
Wetzel, G., conchiolin, 81; histidine, 99
Weydemann, H., 32
Weyl, Th., protein crystals, 38; fibroin,
82; alanine, 84; carbon monoxide
methsemoglobin, 207; cholesterin, 334;
muscle work, 470; amniotic fluid, 512;
creatinine, 565; benzoic acid, 588;
nitrates, 623
Wheeler, H. L., iodogorgonic acid, 82;
nucleic acids, 156; cytosine, 165
White, B., allantoin, 574, 584
Wichmann, A., proteins, 33, 181, 522
Widdicombe, J. H., 379
Wiechowski, W., hippuric acid, 586, 587
Wiener, H., autolysis, 23; uric acid, 18,
569, 571, 572, 573, 586; oxalic acid, 583
Wild. W., 7
Willdcnow, C, 98
Williams, D., 397
Willstatter, R., 1 4
Wiman. A., 47
Windaus, A., histidine. 99; methylimi-
dazole, 108; cholesterin, 334
Winkler, 441
844
INDEX TO AUTHORS.
Winteler, L., 309
Winter, J., estimation of hydrochloric
acid, 376; milk, 530
Winterberg, H., ammonia, 241, 544;
liver, 280; carbamic-acid intoxication,
552
Winternitz, Hugo, estimation of blood
pigments, 217; quantity of haemoglobin,
243; contents of the gall-bladder, 329;
putrefaction, 405, 588; iodized fat, 442,
537, 538
Wi ternitz, M. C, purine-base enzymes,
15S, 273, 571
Winterstein, E., amino-acids, 84, 87, 90;
hexone bases, 97, 98, 100; inosite, 458;
colostrum, 528; tunicin, 686; leci-
thans, 144
Wislicenus, J., 473
Wittmdack, K, 532
Wohler, Fr., synthesis of hippuric acid,
635; ^of urea, 547; decomposition of uric
acid, 573; allantoin, 583
Worner, E., protagon, 482; cerebron, 485;
creatinine, 563; uric acid, 578
Wohl, A., 106
Wohlgemuth, J., liver proteid, 96, 282;
oxyaminosuberic acid, 96; oxydiamino-
sebacic acid, 101; pentoses, 109, 111,
112, 290; glycogen formation, 290, 292;
sulphurized products of metabolism,
331; enzymes of the egg-yolk, 503;
amino-acids, demolition of, 636
Wolff, E., 471
Wolff, H., glucosamine, 121; transudates,
262; melanins, 689
Wolff, L. K., 46
Wolff berg, S., glycogen formation, 293;
respiration, 708, 709
Wolkow, M., 597
Woll, F. W., 516
Woltering, H., iron preparations, 245
liver-proteid, 282
Woods, H. S., 487
Wooldridge, L. C, tissue-fib rinogen, 141
stroma of blood-corpuscles, 194; coagvi-
lation of the blood, 228, 233, 234
Worm-Muller; J. ,blood, 244, 245, 248
sugar test, 655; estimation of sugar,
660, 662, 663
Wright, A., fibrin ferment and coagu-
lation, 175, 226, 233; alkalinity of the
blood, 223; phlorhizin diabetes, 298
Wroblewsky, A., fermentation, 10;
pseudonuclein, 47; starch, 126; pepsin,
354, 358; gastric digestion, 370, 371;
action of enzymes, 397; milk, 531
Wulff, C, purine bases, 161, 164, 580
Wurm, W. A., 690
Wurster, C, 625
Wurtz, A., lymph, 251; colloid, 499
Young, P. A., 325
Young, R. A., dextrin, 129; glycogen,
289
Yvon, P., 611
Zadik, H., 719
Zangerle, M., 500
Zahor, H., 646
Zaitschek, A., enzymes of milk, 523;
casein, 521, 529, 531
Zak, E., 258
Zaleski, J., plant- and blood-pigments,
197, 214; blood-pigments, 211, 2^3, 214j
216; hsemopyrrol, 214; ammonia in
the blood, 241, 551; in the glands, 624,
774; estimation of, 625; formation of
urea, 553, 554; estimation of urea, 561;
liver and acid formation, 554, 572
Zaleski, St., iron of liver, 282, 286: iron
in sucklings, 535; reaction of the
intestine, 407; milk, 536
Zalesky, N., bones, 436; samandarin,
692
Zalocostas, P., 81
Zander, E., 686
Zanetti, C. U., glucoproteid, 180; bile
310; ovomucoid, 508
Zangemeister, W., estimation of blood-
pigments, 217; amniotic fluid, 513
Zaudy, 569
Zdarek, E., 688
v. Zebrowski, E., 342
Zeehuizen, H., 631
Zeidlitz, P. V., 656
Zsitler, X., 470
Zeller, A., elimination of chlorine, 616;
destruction of cWorine compounds,
631
V. Zeynek, R., fat of dermoid cysts, 138,
502, 691; blood-pigments, 204, 205,
209; liver, 286; bile, 327; sarcomela-
nin, 689
Zickgraf, G., oxidation of proteins, 32,
78
Ziegler, 583
Ziegler, E., 635
Zillesen, H., 469
de Zilva, L., 387
Zimnitzki, S., 405
Zink, J., fats, 135, 437, 442
Zinoffsky, O., 197
Zinsser, A., 363
Zobel, S., 459
Zoja, L., oxyproteic acids, 31; elastin, 75,
76; ovalbumin, 507: urobilin, 604;
uroerythyrin, 607; hsematoporphyrin,
649, 650
Zsigmondy, R., 41
Zuelzer, G., lecithin, 147; myelin, 486;
cutaneous respiration, 695
V. Zumbusch, L., 324
Zunz, E., digestion jDroducts, 53, 55;
digestion, 370, 418; trypsinogen, 386;
absorption, 413; extractives of meat,
457
Zuntz, L., 223
Zuntz, N., blood, 224, 243, 246; glycogen,
288; blood-sugar. 296. 469; phlorhizin
diabetes, 298: digestion, 393, 427;
protein assimilation, 412; muscle- fat.
INDEX TO AUTHORS.
845
463 472; muscle metabolism, 467, 471,
473- high altitudes, 246, 694, 761;
cutaneous respiration, 695; blood-gases,
697-699; alveolar air, 707: respiration,
710 712 713, 722, 723, 733; metabo-
lism, 723, 729, 755, 757, 758, 761 ; nutri-
tive value of proteoses, 746; of alcohol,
754; digestibility of bread, 762
Zweifel, P., ptyalin, 344; pancreatic
diastase. 388; eclampsia, 608
Zwerger, R., 58, 59
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