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PHYSIOLOGICAL CHEMISTRY

LONG

BY THE SAME AUTHOR

Elements of General Chemistry

Fourth Edition. 33 Illustrations, x-j-443
pages. Cloth. $1.50 net.

A Text-Book of
Elementary Analytical Chemistry

Third Edition. 10 Illustrations, x-f 297
pages. Cloth. $1.25 net.

P. BLAKISTON'S SON & CO.

PHILADELPHIA

A TEXT-BOOK

OF

Physiological Chemistry

FOR

STUDENTS OF MEDICINE

JOHN H. LONG, M.S., Sc.D.

PROFESSOR OF CHEMISTRY IN NORTHWESTERN UNIVERSITY MEDICAL SCHOOL, CHICAGO

SECOND EDITION, REVISED
WITH 42 ILL US TEA TIONS

PHILADELPHIA

P. BLAKISTON'S SON & CO.

1012 Walnut Street

1909

Copyright, 1909, by P. Blakiston's Son & Co.

\^bs

Press of

The new era Printing Company

Lancaster, pa.

PREFACE TO THE SECOND EDITION.

In the preparation of this revision a number of important changes
have been made. The new protein classification of the American
societies has been added, while a few points based on older notions
of protein relations have been dropped. Additions have been made
in several other chapters, also, and most frequently in the general
text.

A much fuller discussion has been given to the subject of the urine,
and a new chapter has been added on the methods of urine analysis.
These methods embrace not only the usual clinical tests, but the most
important quantitative processes, in certain directions, as well, and
are given in sufficient detail for practical metabolism work.

I have endeavored to keep the book within reasonable limits as to
size and to make it conform to the needs of the classes of students
for whom it is written. I believe it covers the ground which should
be required in the chemical courses in medical schools, or in scientific
schools where preparation for medicine is given. Some points of
minor importance for beginners are printed in smaller type, but in
general the details of interest to specialists only are omitted, as there
is danger in presenting more to the student than he has time to
properly master. This danger is as apparent in the teaching of
physiological chemistry as it is in certain other lines of work.

In the preparation of the index and in the reading of the proof I
have been greatly aided by my wife, Catherine Stoneman Long, and
by my son, Esmond R. Long, to both of whom my thanks are due.

J. H. Long.

Chicago, July, 1909.

FROM THE PREFACE TO THE FIRST EDITION.

" In the following pages I have attempted to present a brief ac-
count of the important principles of physiological chemistry in a form
suitable for the use of medical students who may be assumed to have
completed courses in the elements of general inorganic and organic
chemistry. From the very necessities of the case a work of this
character, dealing with many topics in an elementary way, must be
largely a compilation; in the selection of material, besides consulting
the standard hand books, I have made free use of the recent mono-
graphs by Cohnheim, Effront and Oppenheimer, as well as of numer-
ous articles in the Zeitschrift fur physiologische Chemie, the Beitrage
zur chemischen Physiologie und Pathologie and other journals. As
the book is intended for beginners I have not thought it necessary to
make any special quotations of literature references."

" A considerable number of illustrative experiments are given in
the text, but distinguished by being printed in smaller type. These
experiments are sufficiently numerous and comprehensive to serve the
purpose of a laboratory course parallel with the general course."

vi

Chapter

Chapter
Chapter
Chapter
Chapter

TABLE OF CONTENTS.

INTRODUCTION
I. Scope and Methods

SECTION I
THE NUTRIENTS

II. Inorganic Elements. Water. Air. Salts 7

III. The Carbohydrates and Related Bodies 17

IV. The Fats and Substances Related to Them 40

V. The Protein Substances 51

SECTION II

FERMENTS AND DIGESTIVE PROCESSES

Chapter VI. Enzymes and Other Ferments. Digestion 96

Chapter VII. Saliva and Salivary Digestion 121

Chapter VIII. The Gastric Juice and Changes in the Stomach. . . 126

Chapter IX. The Products of Pancreatic Digestion 144

Chapter X. Changes in the Intestines. Feces 158

SECTION III

THE CHEMISTRY OF THE BLOOD, THE TISSUES AND
SECRETIONS OF THE BODY

Chapter XL The Blood 175

Chapter XII. The Optical Properties of Blood. The Use of the

Spectroscope and Other Instruments 193

Chapter XIII. Further Physical Methods in Blood Examination.
Freezing Point and Electrical Conductivity.

The Hematocrit 204

Chapter XIV. Some Special Properties of Blood Serum. Bac-
tericidal Action. Precipitins, Agglutinins, Bac-

teriolysins, Hemolysins 216

ptee XV. Transudations Related to the Blood 230

"i EB XVI. Milk 236

vii

viii CONTENTS.

Chapter XVII. The Chemistry of the Liver. Bile. Cells in

General 249

Chapter XVIII. Chemistry of the Pancreas and Other Glands.

Muscle, Bone, the Hair and Other Tissues 272

SECTION IV

THE END PRODUCTS OF METABOLISM. EXCRETIONS.
ENERGY BALANCE

Chapter XIX. The Excretion of Nitrogen, Sulphur and Phos-
phorus. The Urine 289

Chapter XX. Some Practical Urine Tests 313

Chapter XXI. The Gaseous Excretion. Respiration 356

Chapter XXII. The Energy Equation 366

Index 380

PHYSIOLOGICAL CHEMISTRY.

INTRODUCTION.

CHAPTER I.

Scope and Methods. In our study of the organized world the most
fundamental problems which present themselves are essentially chem-
ical. Beginning with the mysterious transformations wrought through
the energy of the sun's rays in such simple substances as the carbon
dioxide and aqueous vapor of the atmosphere, when these bodies come
in contact within certain vegetable cells, and following the history of
the products thus formed through their many changes in the plant
organism and later through the highly complex animal structures,
for whose formation the plant cell must prepare the raw material, and
finally as we note the gradual breaking down of these same elaborate
combinations, with liberation of energy and ultimate restoration of
carbon dioxide and water and nitrogen to the air and soil which once
had held them, we see that, step by step, the various transformations
which occur are such as may be represented by the equations of organic
chemistry. It may not always be possible to express these equations in
simple or exact form, because of the lack of knowledge in details,
but the theoretical feasibility of writing such expressions we every-
where recognize.

In following the migrations of atoms of carbon, hydrogen, oxygen
and nitrogen through the vegetable and animal worlds, our inquiry
naturally widens beyond the field legitimately claimed by chemistry.
We find ourselves at the very outset confronted by the question of the
final forces inaugurating the changes, the chemical expression of
which appears often so extremely simple. It may be the part of wis-
dom to admit at once that this question is one beyond our power to
answer. Then, again, we find ourselves attracted by questions of
form, function and general conditions of the existence of organisms
upon the earth, in addition to those of composition and mode of forma-
tion. In touching these we enter upon the field of General Biology

2 PHYSIOLOGICAL CHEMISTRY.

and soon recognize that in this vast and independent science there is
much contained which has no possible bearing on our problems of
chemistry. But some knowledge of biological science is certainly
essential to a proper understanding of the chemistry of living beings.

As proper subjects of inquiry in Physiological Chemistry we recog-
nize mainly the following: (a) The nutrition of plants and animals
and the composition and properties of the nutrient substances, (b)
The changes which the nutrients undergo before and during the
processes of assimilation, (c) The agents of preparation for assimi-
lation and the general conditions of their activity, (d) The fate of
the assimilated nutrients and the nature of the products of degrada-
tion. (<?) The absorption or liberation of energy.

In the broader sense the discussion is extended to the conditions
appearing in the life history of plants, and the lower animals as well
as of man, but ordinarily the narrower field of the life of man and
the higher animals alone is considered, and in the present work the
latter limits will be observed. But a brief discussion of the general
relations of plants to animals will not be out of place.

At one time it was very generally held that the cell activities in the
plant are essentially different from those in the animal, the work in
the one case being looked upon as wholly synthetic, while in the other
it was assumed to be disintegration or analysis. But this is not quite
correct. The reactions in the two classes of structures are qualitatively
much the same, although the quantitative differences are so great
that we almost lose sight of qualitative similarities. The reactions in
the plant world are largely endothermal and require for their com-
pletion the constant expenditure of external energy. This energy is
derived from sunlight and through its agency chlorophyll-bearing
plants are able to effect a remarkable condensation, viz. : that of carbon
dioxide with water accompanied by liberation of oxygen. In its
simplest terms this condensation may be represented as

C02 + H20 = H2CO + 02;

that is, formaldehyde and oxygen result.

Formaldehyde is the first member of the series containing the mono-
saccharoses and may be the actual starting point in their elaboration
by the vegetable cell. Of the mechanism of further transformations
by the plant we know but little ; it is likely that many of the following
changes are brought about by the action of soluble ferments or en-
zymes, which will be referred to in a subsequent chapter. With the
completion of this synthesis a large amount of kinetic energy of the

INTRODUCTION. 3

solar rays is transformed into the potential energy of protein, fat or
carbohydrate. In the oxidation of the plant as fuel or food the op-
posite change is accomplished, and the stored up energy in complex
organic molecules is liberated as heat, electricity or muscular motion.
These reactions are so characteristic for plants and animals that we
are apt to lose sight of others which also take place. In plants there
is a respiration process as in animals, in which oxygen is absorbed and
carbon dioxide liberated, and in the dark this may be readily observed,
since then it is not obscured by the much more prominent reduction
process. Indeed, this respiration may be followed in the light in the
case of those plants which are free from chlorophyll. Further than
this there are parasitic plants which, free from chlorophyll, must de-
pend on other plants for their nourishment; they consume organic
and not inorganic materials, and in this behavior resemble animals
completely.

Then it must be remembered that the activity in the animal is not
wholly oxidation or degradation. It is well known that some syn-
theses are constantly taking place which are commonly overlooked
because of the much greater importance of the oxidation reactions.
In recent years low forms of animal life have been found which
contain chlorophyll grains and which are able to produce oxygen in
presence of sunlight. In some of these cases the chlorophyll may be
present in a symbiont organism, but in others it appears to be diffuse,
and therefore brings the animal structure containing it into close rela-
tion with vegetable cells.

In the essential phenomena of life plants and animals have, then,
much in common ; it is only when we follow them into details that the
characteristic differences appear.

From the nature of the materials entering into the structure of
plants and animals it follows that the discussions of physiological
chemistry are, in the main, but special cases of the general field of
organic chemistry. The important nutrients, the carbohydrates, the
fats and the protein substances, are all organic and the products ap-
pearing as stages in their metabolism are also organic. Therefore
much which the student has met with in his study of organic chemistry
may be found repeated in his work in physiological chemistry. Inas-
much as many of the relations to be now traced out are quantitative,
it is highly important, also, that the student should bring to the work
before him a good knowledge of the principles of volumetric analysis,
as these will be applied frequently in what is to follow.

4 PHYSIOLOGICAL CHEMISTRY.

Historical. To trace the beginnings of Physiological Chemistry
we are not obliged to go far back in the development of science.
With the old medical chemistry of the so-called iatro school it has
nothing in common, and in fact is in no sense a development of that
science of the sixteenth and seventeenth centuries. Before the days of
Lavoisier, it is true, some little advance had been made in the study
of bodies of animal or vegetable origin, but without a rational theory
of chemical combination the isolated facts established led to little of
real value. With the nature of respiration explained, however, and
the identification of its phenomena with other phenomena of oxidation,
the way was opened for true scientific progress. At the same time
accurate methods of ultimate organic analysis were suggested and
soon developed by the followers of Lavoisier. In the hands of Ber-
zelius, Gmelin and others these soon began to furnish results. This
brings us to the end of the first quarter of the nineteenth century, a
point which marks the real beginning of our science. A peculiar dis-
tinction between inorganic and organic bodies had gradually arisen,
and had come to be commonly accepted. This was founded on the
notion that while the former might be produced by laboratory proc-
esses synthetically, for the latter group nothing similar was possible.
By this arbitrary limitation research was naturally greatly curtailed.
However, in 1828, Wohler made the important discovery that urea
could be easily formed by warming a solution of ammonium cyanate,
and this was in time followed by others of equal value, pointing to
the same conclusion, that the production of organic compounds is in
no wise dependent on the aid of a so-called vital force. It was soon
demonstrated that the chemist's laboratory, no less than nature's
laboratory, could take part in the formation of these substances, and
in the next ten years, to about 1840, many products of physiological
interest were made. The great Wohler made many of the most fruit-
ful discoveries of this epoch, but it is to Liebig that we owe the most.
For many years he was busily engaged in perfecting methods of
analysis, and with these developed he turned his attention largely to the
chemical phenomena of vegetable and animal life. This led to the
publication, in 1840, of his epoch-marking work, "Organic Chemistry
in Its Relations to Agriculture and Physiology." This was followed
in 1842 by a work giving evidence of his broadened and strengthened
views, " Organic Chemistry in Its Relations to Physiology and Path-
ology." These works passed through many editions and were trans-
lated into several languages. In them we find much that is now
considered fundamental in physiology and physiological chemistry,
and they suggested or called out the active efforts of many succeeding

INTRODUCTION. 5

investigators. Not a few of these men are still living, so young is
our science, and it will be sufficient to merely call attention to the names
of the more prominent workers who followed the active pioneers. In
Germany C. G. Lehmann made many important contributions to the
chemistry of the blood and published a text-book of Physiological
Chemistry which reached a third edition in 1853. In 1858 F. Hoppe-
Seyler published the first edition of his Physiological and Pathological
Chemical Analysis, and from 1877-81 his Physiological Chemistry
in four parts or volumes. This work contributed greatly to our sys-
tematic knowledge. C. Voit, since 1863 professor of physiology in
Munich, began his valuable studies in nutrition and metabolism about
1856, and continued them nearly forty years. W. Kiihne, of Heidel-
berg, did much to develop the chemistry of the protein substances,
his studies dating from 1859. The pupils o-f these German scholars
are to-day among the most active investigators in all fields of physi-
ological chemistry.

In France, Pasteur must be mentioned in this connection on account
of his pioneer investigations on fermentation and ferments, a subject
of far-reaching importance. CI. Bernard investigated the chemistry
of the digestive secretions and especially the behavior of sugar in the
organism. He published valuable works in 1853 and 1855, which
went through later editions. Somewhat later P. Schuetzenberger, in
Paris, made important additions to our knowledge of the chemistry
of the protein bodies and published a work on fermentation which
for many years ranked as our only systematic treatise on the subject.

At the present time physiological chemistry has become a recog-
nized department of study in the United States, England and other
continental countries as well as in Germany and France, and journals
are now published devoted solely to its interests. The rapidly increas-
ing number of investigations published in these journals and elsewhere
attests the growing importance of the science from the theoretical
standpoint as well as in its practical relations to medicine.

It remains to briefly mention the development of another field of
scientific study because of its bearing on certain problems of physi-
ological chemistry. In the last quarter of the eighteenth century
Lavoisier clearly showed the nature of combustion and the relation of
animal heat to respiration and the oxidation of the tissues. Lavoisier
and Laplace carried out the first quantitative experiments in which a
c-ilorimeter was employed to measure the evolution of body heat.
These were repeated by Desprctz in T824 and later by Dulong. Since
then by greatly improved methods many similar investigations have
been made.

6 PHYSIOLOGICAL CHEMISTRY.

A little later than the date on which Lavoisier and Laplace an-
nounced their important researches on the relation of animal heat to
oxidation of foodstuffs, Benjamin Thompson, Count Rumford, an-
nounced a discovery of equally far-reaching consequences. He made
the observation that the heat of friction between two pieces of metal
may be absorbed by water and so measured, and that there is a relation
between the mechanical work lost in the friction and the heat gen-
erated. He made also the curious observation that the work per-
formed by the horse in one of his experiments in which friction was
produced depended in turn on the combustion or oxidation of the food
of the horse, from which it followed that indirectly the heating of the
water was due to the combustion of a certain amount of food. But
all the consequences of his experiments Rumford did not see. He was
mainly interested in showing the absurdity of the notion of the ma-
terial nature of heat, then commonly held, which he did completely.
It remained for Joule of England and Mayer of Germany to point out,
nearly fifty years later, the true relation between heat and work. In
fine, by establishing the work equivalent of heat they made it possible
to calculate the food equivalent of work, since the food equivalent of
heat had been already proven. These relations are all of the highest
value in the study of metabolism, to be considered in the sequel.

The discussions of the earlier part of the eighteenth century placed
in clear light finally the full meaning of the doctrine of the In-
destructibility of Matter. These later discussions developed a new
doctrine, that of the Conservation of Energy, the recognition of which
played no small part in the gradual advance of physiological as well
as physical science.

It is the intention of the following chapters to present the funda-
mental facts and theories of physiological chemistry in the simplest
possible manner. Much matter found in the larger hand-books must
necessarily, therefore, be omitted from a work of this elementary
character. But enough will be given to furnish the student, it is
hoped, a satisfactory view of that which is most important in the
science at the present time. It will be found convenient to make four
general divisions of the subject, as follows :

Section I. The Nutrients and Related Substances.

Section II. Ferments and Digestive Processes.

Section III. The Chemistry of the Tissues and Secretions of
the Body.

Section IV. The End Products of Metabolism. Excretions.
Energy Balance.

SECTION I.

CHAPTER II.

THE NUTRIENTS.

INORGANIC ELEMENTS. WATER. AIR. SALTS.

Composition of the Body. The living animal body is composed
in the mean of about 35 to 40 per cent of solids and 60 to 65 per cent
of water. In adults the solids are somewhat in excess of this amount,
while in infants they are lower, perhaps not over 30 per cent. The
elements most abundantly present are carbon, oxygen, hydrogen, nitro-
gen, phosphorus, sulphur, chlorine, potassium, sodium, calcium, mag-
nesium and iron. In traces only, or in particular tissues, we find
iodine, fluorine, bromine, silicon, manganese, copper and lithium.
The presence of these in minute amount seems to be necessary for
the existence of certain animals. These elements are not present in
the free state, but exist combined in more or less complex compounds,
the degree of complexity varying between that illustrated in such
simple bodies as water or common salt, and that found in the large
protein molecules with possibly thousands of atoms present.

In point of abundance these elements are found in the body in about
the order given on the following page.

The solids of the body are both organic and inorganic, and ap-
proximately the composition of the whole may be thus represented :

Per Cent.

Water 65

Protein substances 15

Fats 14

Other organic extractives 1

.Mineral matters 5

It must be remembered, however, that the fat may vary widely from
the above number and therefore change the ratio, fat : protein. Among
the mineral matters calcium phosphate holds the first place, as it
makes up the larger part of bone ash ; carbonates and chlorides of the
alkali metals make up the remainder largely.

In view of this composition of the body, it is important to learn
how its waste is replenished, and what substances must be or may be

7

PHYSIOLOGICAL CHEMISTRY.

Table of the Elements in the Body.

Name of Element.

Oxygen
Carbon
Hydrogen
Nitrogen

Calcium

Phosphorus

Potassium

Sodium

Chlorine
Sulphur

Magnesium
Iron

Iodine, Fluorine,
Silicon

Per Cent.
Amount.

66.0
17-5

10.2
2.4

1.6
0.9

0.4

0.3

0.3
0.2

0.05
0.004

traces.

Occurrence.

In the water of the body, in the fats, the protein sub-
stances and in nearly all the tissues and salts.

In the fats, protein substances and in most of the im-
portant compounds produced in the body.

In water, the fats, protein substances and in the im-
portant products of metabolism.

Found mainly in the protein substances of the body.
Also in many of the metabolic products derived from
these.

This element occurs mainly in the bones, but is found
in the blood also and in several secretions in small
amount.

Is found principally with calcium in the bones, but
occurs also in several complex compounds in organic
combination.

Found as chloride, carbonate or phosphate in many of
the body tissues and secretions. Exists also in
organic combination.

Occurs combined, as does potassium; the chloride and
carbonate are the most important salts and are found
in several body fluids.

Found in combination with sodium and potassium, also
as hydrochloric acid in the gastric juice.

This element is important as occurring in the protein
compounds of the body, and is found also in minute
amount in other combinations.

Is found mainly as phosphate and carbonate in the bones.

Iron occurs in the important hemoglobin of the blood,
as an integral part of the complex molecule. It is
found also in inorganic compounds in traces.

Fluorine is found in the teeth, iodine in the thyroid
gland, silicon in the hair. Besides these, other ele-
ments have been found occasionally, but do not appear
to be necessary.

consumed to repair the constant losses and enable the body to do its
proper work. This leads to the question of foods or nutrients in the
broad sense. Beginning with the inorganic materials used by the body,
we have first :

• Water. As it appears on the surface of the earth water is classed
conveniently as hard and soft. The descending rain, after the dust is
washed from the air, consists of nearly chemically pure water. It
holds no mineral matters dissolved, and is contaminated mainly with
a small amount of dissolved carbon dioxide. Such water on reaching
the earth is soft and can replace distilled water for most purposes.
The changes which follow after contact with the soil depend on the
composition of the latter. If the strata over which the water flows
or through which it percolates consist of sand, quartz or silicate rocks
or other insoluble materials the water is left in practically pure con-
dition, and is the water usually spoken of as soft water. But, on the
other hand, if the rain water comes in contact with limestone, gypsum,

THE NUTRIENTS. 9

or other slightly soluble substances, something goes into solution and
the product is now known as hard water, the degree of " hardness "
depending on the amount of dissolved solids. Waters containing the
carbonates of calcium and magnesium are described as temporarily hard,
since the carbonic acid which holds these carbonates in solution may
be removed by boiling, which causes precipitation. Calcium sulphate
or chloride in water can not be precipitated by boiling and the presence
of these and a few other substances produces permanent hardness.

Moderate amounts of these mineral matters in water are not ob-
jectionable; in fact waters with some lime and magnesia are preferable
to absolutely soft water for drinking purposes. But along with the
inorganic substances the water may take other things from the soils
with which it comes in contact that are not so desirable. These are
various partly decomposed organic matters of animal or vegetable
origin and, what is more important, minute living vegetable cells,
mostly bacteria, which are capable of causing much mischief when
taken into the stomach of man. It is very generally believed that
several diseases in man have their origin in the consumption of water
contaminated in this way.

Natural Purification of Water. But it must not be supposed that
these bacteria are always harmful. On the contrary some of them
are the common agents which effect the natural purification of waters
containing organic matter, in which they incite destructive fermenta-
tion or putrefactive changes and finally oxidation. As a result of
these changes harmless inert substances such as nitrogen or nitrates,
carbon dioxide, methane and water are produced from the relatively
complex waste or excreta of the higher organisms. When we speak
of the spontaneous or self-purification of water we refer to a series
of changes in which these bacteria play a leading part.

Artificial Purification of Water. On a smaller scale water may
be rendered safe and suitable for household use by several methods.
By distillation all objectionable matters may be rejected and a whole-
some drinking water obtained. It is possible, also, to separate prac-
tically all bacteria and other solid matters by filtration through beds
of fine sand. In this way the supplies of many cities are obtained at
the present time. Frequently the filtration is preceded by coagulation
or precipitation of the organic substances by means of some suitable
agent such as alum or salts of iron, or lime.

Tests of Drinking Water. In the sanitary examination of water
it is not necessary to make very full analyses to determine its value
for household use. A few tests usually suffice to discover the pres-

IO PHYSIOLOGICAL CHEMISTRY.

ence or absence of objectionable substances. For example, in un-
contaminated waters from ordinary springs, lakes, rivers or wells,
chlorine is present in small amount only. Any excess of chlorine
suggests contact with sewage or household waste somewhere, and a
quantitative test is of prime importance to settle this point. Such a test
and a few others will be illustrated below.

Experiment. The Test for Chlorides. A test is often made in this way:
Measure out 200 cc. of the water, add to it a few drops of a solution of pure
neutral potassium chromate, and then from a burette run in, with constant stirring,
solution of tenth normal silver nitrate until a faint reddish precipitate of silver
chromate appears. Each cubic centimeter of the silver solution precipitates 3.54 mg.
of chlorine from common salt or other chloride, and when the last trace of chlorine
is combined, the silver begins to precipitate the chromate with production of red
color. The chromate acts here as an " indicator," as it shows just when the chlorine
is all combined by beginning to precipitate itself.

In making this test it is well to take two similar beakers, place them side by side
on white paper, pour equal amounts of water in each, add to each the same number
of drops of the indicator, and then with one make the actual test by adding the
silver solution. Note the amount used to give a light shade and then discharge it by
adding a drop of salt solution. Now, with this opalescent or turbid liquid for com-
parison add silver nitrate to the second beaker until the light yellowish red shade
just appears. This reading is usually somewhat more accurate than the first.

As a result of the decomposition of various nitrogenous matters
ammonia is frequently found in natural waters. Its amount is there-
fore a measure of contamination to some extent, and tests for its
presence are always made in sanitary examinations. In practice the
test is usually made on a distillate from the water in question, but
the following experiment will illustrate the behavior of the reagent
employed.

Experiment. Test for Ammonia. Solutions of ammonia or ammonium salts
possess the peculiar property of giving a yellowish brown color with what is known
as Nessler's reagent (a solution of mercuric-potassium iodide, made strongly alka-
line with sodium or potassium hydroxide). With more than traces of ammonia a
precipitate is formed.

To make the test measure out 50 cc. of the water in a large test-tube, or tall
narrow beaker, and add to it 2 cc. of the Nessler solution. By placing the beaker
on a sheet of white paper and looking down through it, the depth of color can
be observed. A few parts of ammonia in one hundred million parts of water can be
readily seen and measured.

The Oxidation Tests. Pure water absorbs free oxygen from
the atmosphere but has no tendency to decompose compounds to
secure it. On the other hand, waters containing organic matters or
certain inorganic contaminations have the power of decomposing oxy-
gen salts to secure the oxygen they desire, and the amount of oxygen so
taken up becomes a measure of the impurity of the water. Potassium

THE NUTRIENTS. I I

permanganate is a salt, which, under certain conditions, gives up its
oxygen to waters containing organic bodies in solution and is fre-
quently employed in water analysis for this purpose. An experiment
will show one way in which it is used.

Experiment. Measure out about ioo cc. of pure, carefully distilled water, pour
it into a clean beaker in which water has just been boiled and add 5 cc. of pure
dilute sulphuric acid (1 to 3). Place the beaker on wire gauze and heat to boiling.
Now add 5 drops of a dilute permanganate solution (300 milligrams to the liter)
from a burette or dropping tube and boil five minutes. The pink color persists.

Repeat the experiment, using 100 cc. of common hydrant water to which a trace
of egg albumin or urea has been added, and after running in the permanganate boil
again. The color fades out and more may be added. Finally, after sufficient has
been added the pink color remains. The number of drops or cubic centimeters used
is a measure of the contamination of the water, although often, as in this experi-
ment, a very rough one.

The Tests for Nitrites and Nitrates. Nitrogenous matters
undergoing oxidation in water and soil usually give rise, in time, to
nitrites and finally to nitrates. These compounds are therefore looked
for in water as evidence of past contamination. In most instances
nitrites, as a less advanced stage of oxidation than nitrates, suggest
comparatively recent contamination. The tests are especially inter-
esting in the examination of well and spring water.

Chemists are acquainted with a number of methods for the detec-
tion of traces of nitrogen in the form of nitrites and nitrates, but at
the present time certain color reactions are, because of their simplicity,
mainly in favor. These are illustrated by the following tests :

A reagent for nitrites is prepared by dissolving 0.5 gm. of sul-
phanilic acid in 150 cc. of acetic acid of 25tper cent strength, and
mixing this with a solution of 0.1 gm. of pure naphthylamine in 200
cc. of dilute acetic acid. This mixture keeps very well for a time in
the dark.

Experiment. To about 50 cc. of water in a clean beaker add 2 cc. of the above
solution. If the water is quite free from nitrites the reagent imparts no color to
it. One hundredth of a milligram of nitrogen as nitrite in the water gives a faint
pink color at the end of five minutes ; with large quantities the color may become
deep rose red.

Experiment. A nitrate test may be illustrated in this manner : To the residue
obtained by evaporating 50 cc. of an ordinary river or lake water to dryness in a
porcelain dish add 1 cc. of phenolsulphonic acid. Rub the acid over the bottom of
the dish, and add a few drops of dilute sulphuric acid. Warm the dish a few
minutes and add 25 cc. of water. This should show now a faint yellow color. By
supersaturating with ammonia the color becomes deeper. In this experiment picric
acid is at first formed if a nitrate is present and the addition of ammonia yields
ammonium picrate, the color of which is more marked.

For the interpretation of all these tests works on sanitary analysis must be
consulted.

12 PHYSIOLOGICAL CHEMISTRY.

Physiological Importance of Water. This is suggested by the
large proportion in which it is present in the animal body, as shown
above. It serves primarily as the general solvent for all the solid
foodstuffs taken into the system and assists in the removal of the
solid waste products or excreta. To accomplish these ends it must be
drunk in sufficient quantity. It is a well recognized fact that most
people in the United States drink too little water, from which various
ills result. Important chemical changes within the body are dependent
on the so-called hydrolytic action of water. These appear mainly in
the phenomena of digestion, in which starches, sugars, protein bodies
and fats are altered before absorption, and will be discussed in detail
in sections to follow.

It must be remembered further that water plays a very important
part in the removal of heat from the body. For each gram of water
evaporated as perspiration or in the breath nearly 600 units of heat
are absorbed, and in this way over 20 per cent of the heat expenditure
may be accounted for.

The average amounts of water found in the important tissues is
shown in the following table :

Per Cent. Per Cent.

Dentine 10. Pancreas 78

Fatty tissues 20 Blood 79

Bones 50 Kidney 83

Elastic tissue 50 Brain (gray matter) .... 86

Liver • • 70 Milk

Skin 72 Vitreous humor 98.5

Muscles 75 Cerebro-spinal fluid .... 99.0

Spleen 76 Saliva 99.5

Air. Besides its content of oxygen, nitrogen and argon the at-
mosphere contains several other gases in small amount. The most
abundant of these is water vapor, with smaller traces of carbon di-
oxide, helium, neon, etc. As the amount of aqueous vapor present is
extremely variable it is customary to give the analysis of the dry air
only, which in volume per cent is about this, the rarer gases being
included with the nitrogen and the argon :

Per Cent.

Nitrogen 78.40

Oxygen 20.94

Argon 0.63

Carbon dioxide 03

The water vapor present varies with the temperature and other
physical conditions and may sometimes make up one per cent, or even
more, by weight of the whole mass. A cubic meter of fully saturated
air contains 30.1 grams of aqueous vapor at 300 C, and 75 per cent of

THE NUTRIENTS. I 3

this is frequently present in the hot, " close " weather of our summers.
It is this high proportion of moisture which renders further evapora-
tion from the skin so difficult, and which therefore contributes greatly
to our bodily discomfort.

The normal carbon dioxide content is given above as 0.03 per cent
or three cubic centimeters in ten liters. This amount is greatly ex-
ceeded in the air of poorly ventilated houses, but is not in itself the
cause of the unpleasant sensations experienced in going into such an
atmosphere, although this was long believed. It has been found by
experiment that one can breathe, although not comfortably, in a pure
atmosphere containing as much as 3 per cent of carbon dioxide, while
an atmosphere contaminated to the extent of 1 per cent by human
respiration would be practically unbearable. This condition is doubt-
less due to the traces of organic products thrown off in the breath and
perspiration, and especially to the decomposition of organic matter
on the unclean skin. The carbon dioxide is often made the approxi-
mate measure of the contamination of inhabited rooms, because of
the practical difficulty of measuring anything else.

In respiration the air is modified about as shown by these figures:

Inspired Air Expired Air

Per Cent. Per Cent

Nitrogen, argon, etc 79.0 80.0

Oxygen 21.0 16.0

Carbon dioxide 03 4.0

The amount of oxygen inhaled each day by a full-grown man is
not far from 500 liters, while the volume of carbon dioxide exhaled
is somewhat less, about 450 liters in the mean. Later something will
be said about the numerical relation existing between the volume of
carbon dioxide eliminated and the volume of oxygen absorbed.

The most accurate method of finding the amounts of aqueous vapor
and carbon dioxide in the air is to aspirate a measured volume through
a series of weighed absorption tubes. The first of these contain dry
granular calcium chloride or some other good water absorbent, while
the following tubes contain soda-lime or a strong potassium hydroxide
solution to absorb the carbon dioxide. The increase in weight of
the tubes shows the amount of vapor and gas absorbed from the given
volume of air. For quick determinations somewhat less exact methods
are often used in practice.

The atmosphere often contains traces of other gases, as ammonia,
sulphurous oxide, oxides of nitrogen and ozone, which are of little
physiological importance and need not be here considered. Of greater
importance are the minute organized forms everywhere present to

14 PHYSIOLOGICAL CHEMISTRY.

some extent, at least, and which include bacteria and many other
agents of putrefaction and fermentation. Most of these are prac-
tically harmless in respiration, but the presence of others is an element
of the greatest danger, because of the disturbances they occasion when
taken into the body.

MINERAL SUBSTANCES REQUIRED.

Salts. The table some pages back gives the percentage amount of
the different elements which make up the human body, some being
united in organic and the others in inorganic compounds. Aside from
water the most abundant and important of the inorganic materials
are the phosphates and carbonates of the alkali-earth metals found in
the bones, the alkali chlorides and the alkali carbonates. The solid
mineral matter or ash of the adult body amounts in the mean to about
5 per cent; not far from four-fifths of this content comes from the
skeleton, while about one-tenth of it is derived from the muscles.
The proportion of ash in the different tissues, taken in the moist
condition, is approximately as follows :

Per Cent. Per Cent.

Bones 33 Pancreas, brain 1.0

Cartilage 2 Lung, heart 0.95

Liver and spleen 1.5 Blood 0.93

Muscles 1.3 Skin 0.75

Kidney 1.2 Milk 0.70

Leaving traces out of consideration, it appears that the body con-
tains four metallic elements, calcium, sodium, potassium and mag-
nesium, which exist in combination with four acids, viz., phosphoric,
hydrochloric, carbonic and sulphuric. Of all these compounds the
calcium phosphate of the bones is the most abundant.

Phosphates. The phosphates of the body are salts of the common
or orthophosphoric acid, H3P04. The three kinds of salts possible
here are :

Primary phosphates, MH2P04,

Secondary phosphates, M2HP04,

Tertiary phosphates, MsP04,

The alkali salts of the three classes are readily soluble in water.
The secondary and tertiary phosphates are mostly insoluble, those
of the alkali metals excepted. Secondary phosphates are converted
into pyrophosphates by heat and the primary phosphates into meta-
phosphates. To most indicators the primary phosphates show acid
behavior, while the secondary phosphates are feebly alkaline. The
soluble tertiary phosphates are strongly alkaline. The action on
different indicators must be remembered in attempting to estimate
the acidity or alkalinity of urine.

THE NUTRIENTS. I 5

The phosphates furnished us in various animal and vegetable foods
are mainly those of calcium and potassium, but it is likely that the
larger part of the phosphorus utilized in the body is combined in
relatively complex organic compounds, the lecithins and nucleins, for
example, which yield phosphoric acid and phosphate in the final oxi-
dation. We find therefore the tertiary calcium and magnesium phos-
phates, Ca3(P04)2 and Mg3(P04)2 in bones. Acid calcium phos-
phate of the formula Ca(H2P04)2 occurs in some of the body fluids,
and is an important urinary excretion. The phosphate CaHP04
may sometimes be deposited from the urine. Secondary potassium
phosphate, K2HP04, is a constituent of all animal cell structures,
possibly in soluble form, but possibly, also, in organic combination.
The muscular juice is rich in alkali phosphates.

Chlorides. Chlorine is found in the body as sodium chloride and
potassium chloride, also as free hydrochloric acid in the gastric juice.
In our foodstuffs it comes to us mainly as sodium chloride, but this
by double decomposition may give rise to the potassium chloride later :

K2COs + 2NaCl = 2KCI + Na2C03

In the gastric and pancreatic juices and in the blood sodium chloride
is more abundant than potassium chloride, but the latter is in excess
in the cell structures. Chlorine is utilized in the animal body only as
it is found in the metallic compounds or chlorides. The various
organic combinations of chlorine can not replace the salts. It has
been pointed out by Bunge that sodium chloride is much more neces-
sary in the food of man or animals consuming a vegetable diet than
it is when the diet is mainly flesh.

Carbonates. The carbonates found in the human body are pro-
duced there from the carbonic acid of oxidation. Hard waters contain
the carbonates of calcium and magnesium, but these must suffer de-
composition when taken into the stomach, and the traces of acid gas so
expelled. Besides the carbon dioxide of tissue oxidation we must
consider also that formed by several ferment processes in the intestines.
A large amount of the gas is produced in this way and part of this
is absorbed into the circulation. Under certain conditions the " weak "
carbonic acid is able to decompose sodium chloride and produce
sodium carbonate and free hydrochloric acid. The origin of the latter
in the gastric juice is now accounted for in this way, while the sodium
carbonate formed at the same time is carried into the blood and
through this to other parts of the body, where other carbonates may
be made by double decompositions. The soluble alkali carbonates are

1 6 PHYSIOLOGICAL CHEMISTRY.

the most abundant, but in the bones and in the teeth calcium carbonate
forms an important part. Some carbonates are always excreted by
the urine, and the alkali-earth carbonates may occasionally appear in
the sediment.

Sulphates. Sulphur may enter the body in a variety of combina-
tions but nearly all of it is finally excreted in the completely oxidized
form, that is, as sulphates, by the urine. Sulphur in organic combina-
tion is found in all protein substances and in the final oxidation of
these compounds in the body sulphuric acid is produced. The manner
in which this is combined before excretion will be discussed later.
The amount of sulphur normally present in the body is but a small
fraction of one per cent of the weight of the latter and practically all
of it is found in the protein or protein-like substances. Among these
keratin is characterized by its relatively high sulphur content. Of the
exact manner in which the sulphur is combined in most of these bodies
but little is known.

Bases. The several acid radicals referred to occur in combination
with metals and four of these only are present in the body in appre-
ciable quantity. These are calcium, magnesium, sodium and potas-
sium, and they exist in the form of salts with the acid radicals. To
these four the iron of the blood must be added, but it is present in
organic combination. In a following chapter it will be shown to what
extent these bases are present in ordinary foodstuffs. Of some it is
important that they enter the body in certain forms only, otherwise
their utilization — assimilation — is imperfect or even impossible. This
is especially true of the iron, the chief use of which is in the building up
of hemoglobin. For this purpose the iron of the mineral salts is prob-
ably not available, but it may be taken from certain peculiar organic
combinations. Many mineral matters are as essential for the growth
of the body as are the organic foods to be described in the next
chapters and care must be observed to provide them in sufficient
quantity, especially in the feeding of the young. There is some reason
for believing that most of the basic material assimilated in the body
is in the form of complex salts or organo-metallic combinations of
some kind. The sulphur, phosphorus and carbon are so found and
possibly the metals also. It has been pointed out that in the oxidation
of such organo-metallic compounds carbonates or basic salts must
result, and some portion at least of these salts must be available to
combine with the sulphuric acid arising from the oxidation of the
protein substances. According to Bunge common salt is the only
mineral substance, in excess of that furnished by the usual organic
foods, which the body actually demands in large amount.

CHAPTER III.

THE CARBOHYDRATES AND RELATED BODIES.

Under the term carbohydrate it has long been customary to include
a number of bodies with closely related properties and similar com-
position, which may be expressed by such simple formulas as C6H10O5,
C0H12O6, or multiples of these. The term carbohydrate came into
use long before the structure of the bodies in question was known.
It is now possible to describe these substances in their relations to the
fundamental hydrocarbons or alcohols and this classification will be
therefore briefly explained.

NATURE OF THE CARBOHYDRATES.

In their chemical behavior these bodies resemble aldehydes or ke-
tones in certain important characteristics. Like the latter they are
often strong reducing substances and most of them form combinations
with phenyl hydrazine. These and other properties suggest that they
may be considered as aldehyde or ketone derivatives of the poly-
hydric alcohols, which relationship is shown by the table on pages
1 8 and 19, which contains also some acid derivatives for further
illustration.

Some of the bodies in the table are naturally occurring substances
and are highly important, but most of them are artificial. The aldo-
hexoses and the ketohexoses are closely related to two groups of more
complex bodies, in which cane sugar and starch are the best illustra-
tions, and with them form the important class of carbohydrates in
the more restricted sense.

CARBOHYDRATES PROPER.

Following the usual classification we have then :

Monoses, or monosaccharides,
Saccharodioses, or disaccharides,
Saccharotrioses, or trisaccharides,
Polysaccharides.

These bodies are mostly of vegetable origin, but some, such as
sugar of milk, are found in the animal kingdom. The synthetic
preparation of some of these sugars has been accomplished, starting
from either formaldehyde or the mixture called above glycerose.
When formaldehyde, CH20, is treated with lime or other weak
3 17

PHYSIOLOGICAL CHEMISTRY.

bases it polymerizes or condenses to a mixture of sugars, one of which
has been isolated in pure condition and is known as a-acrose. By the
condensation of the mixture of glyceraldehyde and dioxy-acetone

Relations of the Carbohydrates.

Polyhydric
Alcohols.

Aldehyde Deriva-
tives.

Ketone Deriva-
tives.

Acid Derivatives.

Acid Derivatives.

CH2OH

CH2OH
Glycol.

CH.OH

1
CHO

Glycol aldehyde,
diose.

CH2OH

! "

COOH

Glycollic acid

COOH

1
COOH

Oxalic acid.

CH2OH

CHOH

1

CH2OH
Glycerol.

CH.OH

1 "
CHOH

1
CHO

Glyceraldehyde.

CH,OH

1
CO

1

CH2OH
Dioxyacetone.

CH.OH

I
CHOH

1
COOH

Glyceric acid.

COOH

1
CHOH

1

COOH
Tartronic acid.

V

Glycerose or triose.

CH2OH

1
CHOH

1
CHOH

1

CH2OH
Erythrol.

CH2OH

CHOH

1
CHOH

1
CHO

Aldotetrose.

CH.OH
I "
CHOH

1
CO

1

CH2OH
Ketotetrose.

CH.OH

1
CHOH

1
CHOH

1

COOH
Erythritic acid.

COOH

CHOH

1
CHOH

1
COOH

Tartaric acids.

Erythrose.

CH.OH

1
CHOH

1
CHOH

1
CHOH

CH2OH

Pentitols,

arabitols, etc.

CH.OH
1
CHOH

CHOH

1

CHOH
1

CHO
Aldopentoses,
arabinose, etc.

CH.OH
1
CHOH

CHOH

1
CO

1

CH2OH

Ketopentoses.

CH2OH

1
CHOH

1
CHOH

1
CHOH

COOH

Tetrahydroxy-

monocarboxylic

acids, arabonic

acid, etc.

COOH

1

CHOH
1
CHOH

1
CHOH

1
COOH

Trihydroxydi-

carboxylic

acids, trioxyglu-

taric acids.

V

Pentoses.

CH2OH

CHOH

1
CHOH

1
CHOH

1
CHOH

1

CH2OH

Hexitols,

mannitol, etc.

CH.OH

1
CHOH

1
CHOH ■

1
CHOH

CHOH

CHO
Aldohexoses,
glucoses, etc.

CH.OH

1 "
CHOH

1
CHOH

1
CHOH

1
CO

CH2OH

Ketohexoses,
fructose.

CH.OH

1
CHOH

1
CHOH

1
CHOH

CHOH

1

COOH

Pentahydroxy-

carboxylic acids,

mannonic acids,

dextronic acid.

COOH

1
CHOH

1
CHOH

1
CHOH

1
CHOH

1
COOH

Tetrahydroxy-

dicarboxylic

acids, saccharic

acid, etc.

Y

Hexoses.

THE CARBOHYDRATES AND RELATED BODIES.
Relations of the Carbohydrates. — Continued.

19

Polyhydric Aldehyde Deriva- Ketone Deriva-

Alcohols. tives. tives.

C7H160T

CSH1S0S

CoHonOo

C7H140T.

Acid Derivatives.

^s-n-i6^'s

^d-^-is^'9

CH-OH

(CHOH),

COOH

CHoOH

(CHOH),

COOH

CH.OH

(CHOH),

COOH

Acid Derivatives.

COOH

(CHOH)5

COOH

COOH

(CHOH)8

COOH

COOH

(CHOH)r
COOH

(glycerose or triose) mentioned in the table above the same acrose
has been obtained. This acrose is identical with the sugar mixture
known as (d -f- /) -fructose.

A number of sugars have also been obtained by a general method of synthesis
which depends on the fact that as aldehydes and ketones they have the power to
unite with hydrocyanic acid and produce nitriles of acids which may be reduced
to new aldehydes with a larger number of carbon atoms than the original substance
contained. This may be illustrated by starting with arabinose, C5Hi0O5, as figured
above. This with hydrocyanic acid yields a cyanide as follows :

CH2OH.(CHOH)3CHO + HCN = CH2OH.(CHOH)3.CHOHCN, .

and this by the usual reaction gives arabinose carboxylic acid:

CH2OH.(CHOH)3CHOHCN + 2H20 = CH2OH.(CHOH)3.CHOH.COOH + NH,.

By loss of water from this acid the corresponding lactone is formed:

CH2OH.(CHOH)4COOH — H20 =

CH,OH.CHOH.CH.CHOH.CHOHCO = C0H10Oc.

-0-

By reduction with sodium amalgam this lactone becomes a sugar, identical with
that obtained by the other condensation :

CH.OH.CHOH.CH.CHOH.CHOH.CO + H, =

o !

CH2OH.(CHOH)4.CHO = QH]2O0.

By an extension of the principle, sugars with 7, 8 and 9 carbon atoms have been
obtained. In what is to follow a brief discussion of the more important natural
substances will be given.

THE MONOSES OR MONOSACCHARIDES.

A number of pentose and hexose bodies must be considered here.

Pentoses. Small amounts of these sugar-like compounds exist in
nature, but they are mostly derived from simple antecedent substances
called pentosans. The pentoses bear the same relation to the pentosans

20 PHYSIOLOGICAL CHEMISTRY.

that dextrose bears to starch; by hydration the latter compounds are
converted into the former, thus :

C5Hs04 + H20 = C5H10O5.

C6H10O5 + H2Ot=C6H12O6.

Among the pentoses two, known as /-arabinose and /-xylose, are the
most important.

Arabinose, or pectin sugar, is made by warming cherry gum,
wheat bran, gum arabic, quince or gedda mucilage, exhausted brewers'
grains and various other substances with dilute acids. It has a specific
rotation [a]D= -f- 104.50. When boiled with dilute hydrochloric
acid it yields furfuraldehyde.

Xylose, or wood sugar, is obtained by boiling wood gum with
dilute sulphuric acid. Its specific rotation is -f- 19.40. Like the pre-
ceding body it is a reducing sugar, but non-fermentable. The nutri-
tive value of these substances for man is low, but for the herbivora
these and the antecedent pentosans are more important, as they appear
to be rather easily digested in the alimentary tract of many animals.
Traces of pentoses have occasionally been found in human urine.

All these bodies are distinguished from the sugars proper by yield-
ing relatively large quantities of furfuraldehyde when distilled with
hydrochloric acid or sulphuric acid, which reaction may be illustrated
by the following test :

Experiment. In a small retort or flask fitted with a delivery tube mix 5 gm. of
bran and 100 cc. of ten per cent hydrochloric acid. Connect the retort or flask with
a condenser, apply heat and distil over about half the liquid. With a few drops of
this make the Schiff furfuraldehyde test. Moisten a small strip of paper with
aniline acetate obtained by mixing equal volumes of glacial acetic acid and aniline.
Touch this test-paper with a glass rod holding a drop of the bran distillate. A
bright red color appears, due to the formation of furoaniline, C4H3O.CH. (C6H4NH2)2.
The reaction is extremely delicate and serves for the detection of traces of the
products yielding furfuraldehyde, C4H3O.CHO.

The Hexoses. These are important substances represented by the
general formula C6H1206. Several occur widely distributed in nature,
being found in ripe fruits and elsewhere. A few are artificial products
formed by laboratory operations. Complex combinations of these
bodies known as glucosides are also common and are essentially
ethereal salts of the hexoses. These hexose bodies are all sweet,
soluble in water, nearly insoluble in alcohol and are all reducing sub-
stances with oxidizing agents like Fehling's solution. They undergo
fermentation readily, and all the important forms yield alcohol and
carbon dioxide under the influence of the yeast organism. Some of
them yield lactic acid when acted upon by the proper ferment. By

THE CARBOHYDRATES AND RELATED BODIES. 21

partial oxidation they yield monocarboxylic acids, such as mannonic
acid or dextronic acid, and by the more pronounced oxidation they
are converted into dicarboxylic acids, as saccharic or mucic acid.

A reaction of great importance in connection with the hexoses is
that which they exhibit when acted upon by phenyl hydrazine. While
this is a general aldehyde or ketone reaction, the behavior of the
hexoses is so characteristic as to require mention. With one molecule
of phenyl hydrazine, C6H5NH — NH2, the hexoses yield hydrazones,
C6H1205 — N — NH.C6H5, the ketone or aldehyde oxygen being re-
placed by the hydrazine group. The hydrazones are mostly soluble
in water. An excess of phenyl hydrazine, enough to give two mole-
cules of that substance to one of the hexose, yields bodies called osa-
zones, which are mostly yellow, insoluble crystalline compounds
of great importance for the separation and identification of sev-
eral of the sugars. They are represented by the formula C6H10O4
(N — NH.C6H5)2, and by warming with strong hydrochloric acid
they yield peculiar compounds called osones, which are mixed ketone
and aldehyde structures. These reactions will all be illustrated below.

Glucose (tf-glucose), known also as dextrose, grape sugar, or
diabetic sugar, is the best known representative of the group. It is
found in honey and many fruit juices, often associated with levulose
or fruit sugar. It may be produced by the action of weak acids on
cellulose or starch and on the large scale is so made from the latter
substance. Weak sulphuric acid was at first commonly employed and
the hydration or conversion was effected under pressure. Hydro-
chloric acid is now generally employed in making a commercial
glucose, the acid being neutralized with sodium carbonate at the end
of the reaction. With other acids the action is much slower or less
complete. In the section below on the behavior of starch the nature
of the reaction will be explained. The following experiment will
illustrate the production of the sugar by the aid of sulphuric acid :

Experiment. Make a paste by boiling about a gram of starch with ioo cc. of
water in a glass flask. Add 10 drops of dilute sulphuric acid (1:5) and boil five
to ten minutes. Now allow the liquid to cool, remove 5 cc. with a pipette, dilute
this to 25 cc. with water and add a few drops of iodine solution; a blue violet color
results, showing that starch or a starch-like substance is still present. The re-
mainder of the acid liquid in the flask is next boiled steadily for one hour, a little
water being added from time to time to replace that lost by evaporation. At the
end of an hour remove 5 cc, dilute and test with iodine solution as before. The
characteristic starch reaction is now absent, while the liquid has become thin and
transparent.

Neutralize the free sulphuric acid by addition of a slight excess of chalk or fine
marble dust, heat gently to complete the reaction. Then filter and evaporate the

22 PHYSIOLOGICAL CHEMISTRY.

filtrate nearly to dryness on a water-bath. Allow to cool and notice that the
residue has a sweet taste. It is, in fact, glucose and the experiment illustrates the old
method of manufacturing glucose on the large scale. Test it by dissolving a little
in water and adding a few drops of Fehling's solution, described below. On boiling,
a yellowish precipitate appears which becomes bright yellow and finally red.

Under certain conditions it is possible to obtain a pure crystalline
product from the syrup made as just illustrated. This is known as
crystallized grape sugar or pure anhydrous dextrose. The common
commercial product, sold as glucose syrup, often contains much un-
converted dextrin from the incomplete hydrolysis of the starch. At
the present time large quantities of glucose, both solid and liquid, are
made and used in the fermentation industries, by bakers and confec-
tioners and in the household. Glucose is sweet, but not as sweet as
cane sugar, and, because of the fact that it readily undergoes fermen-
tation, it can not replace cane sugar for certain purposes, such as the
preparation of the syrups of the pharmacopoeia or the canning or
preserving of fruits.

The typical aldose reactions are well shown with a solution of glu-
cose. For the first of these we require a reagent, referred to above
as Fehling's solution, which is made as follows :

Fehling's Solution. Ex. Dissolve 69.28 gm. of pure crystallized copper sulphate
in distilled water and dilute to make a liter of solution. In a second portion of dis-
tilled water dissolve 100 gm. of pure solid sodium hydroxide and 350 gm. of pure re-
crystallized Rochelle salt by aid of heat. Cool and dilute to make one liter. These
solutions when mixed yield the Fehling's solution proper. It is best to mix equal
volumes, quite accurately measured, just before the reagent is needed for use.

This Fehling's solution is commonly employed as a qualitative test
for reducing sugars in general, but the reaction may be first illus-
trated by a simpler method:

Experiment. Trommer's Test. Add to a dilute solution of glucose a consid-
erable excess of strong potassium hydroxide solution and then a very few drops
of a dilute copper sulphate solution. This produces no precipitation but imparts a
deep blue color. On warming the solution a yellowish precipitate forms, which
grows bright red by boiling. This is cuprous oxide, and the test is known as
" Trommer's" test. It is frequently employed to detect the presence of sugar in
liquids, especially in urine, but on the whole is not as satisfactory as the next one.

Experiment. Fehling's Test. To a very weak glucose solution add an equal
volume of diluted Fehling's solution. The mixture remains deep blue in the cold,
but on heating a yellowish precipitate turning to red is soon produced. This pre-
cipitate of cuprous oxide comes from the reduction of the cupric compound held
in solution in the test reagent. In the first or Trommer test the first indication of
the presence of a sugar is the formation of a deep blue clear solution rather than
a greenish blue precipitate of cupric hydroxide which would result from the action
of the copper sulphate and alkali alone. But cupric hydroxide dissolves in solu-
tions of sugars and other polyhydric alcohols, the solution being deep blue, and
stable in the cold. In the case of glucose and other reducing (aldehyde or ketone)

THE CARBOHYDRATES AND RELATED BODIES. 23

sugars this stability is only temporary, since reduction follows on boiling. If the
polyhydric alcohol employed to produce the deep blue solution is not a reducing
substance the liquid remains clear and stable, even on boiling. This is the case with
the Fehling's solution, in which the cupric hydroxide is held dissolved through the
alcoholic behavior of the Rochelle salt. Similar solutions are made by the aid
of glycerol (trihydric) and mannitol (hexahydric). The Fehling test has this ad-
vantage over the Trommer test, that in the latter if too much copper sulphate is
used, and little sugar is present the precipitate on boiling may be mainly black
cupric oxide instead of the red cuprous oxide. With the Fehling liquid no black
precipitate can form.

Experiment. Bismuth Reduction Test. Add to a glucose solution some strong
potassium hydroxide solution and then a very small amount of bismuth subnitrate.
For an ordinary test a few milligrams will be enough. On boiling, a black pre-
cipitate appears, which frequently forms a bright mirror on the walls of the test-
tube. This precipitate seems to be a mixture of metallic bismuth with some oxide,
and shows the strong reducing power of the sugar. Similar reductions may be
obtained from alkaline solutions of several heavy metals, but these tests illustrate
the general principle.

Another test which serves for the recognition of even minute traces
of glucose and other sugars, is the following, proposed by Molisch:

Experiment. To a small amount of a dilute sugar solution add two drops of a
solution of a-naphthol, containing about 20 gm. in 100 cc. On shaking the liquid
becomes turbid. Now add to it an equal, or slightly greater, volume of pure strong
sulphuric acid and shake. A deep violet color appears, which gives place to a
violet precipitate on addition of water. This reaction has been shown to be due
to the combination of the a-naphthol with furfuraldehyde produced by the action
of sulphuric acid on the sugar present.

Experiment. Phenyl Hydrazine Test. A characteristic reaction of great prac-
tical value, referred to above, is given on the addition of phenyl hydrazine to a
solution of glucose under certain definite conditions.

To 20 cc. of a dilute glucose solution add about a gram of phenyl hydrazine
hydrochloride, and two grams of sodium acetate. Heat on the water-bath half an
hour, and then allow the liquid to cool. There will now be found a beautiful
yellow crystalline precipitate of phenyl glucosazone, the nature of which is best-
seen under the microscope. This test is one of great delicacy, and has been applied
to the detection of traces of sugar in urine. But care must be taken to keep the
reagent in considerable excess, since otherwise the soluble hydrazone may be formed.
The melting point of the pure osazone is 205° C. The following reactions illustrate
the combination of the phenyl hydrazine :

C,HuO. + C6HB.NH.NH2 = C.HuO^N.NH.C.H. + H20,

C.HuO. + 2C,H8NH.NH2 = C6H]0O4.(N.NH.QHB)2 + 2H20 + H2,

Phenyl glucosazone

C.H..NH.NH, + H2 = CeH8NH2 + NH3.

By treatment with strong hydrochloric acid the osazone decomposes to yield an
osone and phenyl hydrazine hydrochloride :

CH1.04.(N.NH.C,H,)I + 2HCI + 2H20 =

2C.H5.NH.NH2.HC1 + CH20H.(CH0H)8.C0.CH0.

Glucosone

24 PHYSIOLOGICAL CHEMISTRY.

This osone on reduction with nascent hydrogen yields a sugar, not glucose, but
d-fructose, or levulose:

CH2OH.(CHOH)3.CO.CHO + H2 = CH2OH(CHOH)3.CO.CH2OH.

Glucose and levulose (cf-fructose) yield the same glucosazone; we are therefore
able by this reaction to pass from one sugar to the other.

The ready fermentation of glucose will be shown later in the dis-
cussion of fermentation reactions in general. The production of glu-
cose from cane sugar will also be explained. The specific rotation
of glucose in 20 per cent solution is given by the formula [a]z> = 53°,
and increases slightly with the concentration.

gJ-Fructose, fruit sugar, levulose, is a ketohexose similar to d-
glucose in some respects but very different in others. It occurs in
honey and sweet fruits, but is not easily separated in a pure state
because it is very soluble and does not readily crystallize. The prepa-
ration of glucose from ordinary starch has been referred to above.
In like manner fructose may be obtained from certain less common
starches, especially from inulin by hydrolysis with very weak acid.
In pure condition this sugar has no technical importance.

The various reduction and fermentation reactions are shown by
fructose as well as by glucose, but the quantitative relation between
copper hydroxide and fructose is not quite the same as with glucose.
As they yield the same osazone the phenyl hydrazine test can not be
employed to distinguish between them. The most characteristic prop-
erty of fructose is found in its optical behavior. While the specific
rotation of c?-glucose is about 53 ° to the right, that of d-fructose is,
at 200 C, and for a strength of 20 per cent, about 93 ° to the left.
Because of this behavior the sugar is commonly called levulose. An-
other reaction which may be applied is this :

Experiment. Dissolve resorcin in 20 per cent hydrochloric acid and heat a little
of this solution with the levulose solution to be tested. A red color results. At
the same time a precipitate forms which may be dissolved in alcohol with a red
color. One-tenth gram of resorcin to 5 cc. of the acid is sufficient.

^-Galactose. This is the third hexose of importance, but it is not
a natural substance. In the inversion of milk sugar by weak acids
galactose is formed along with glucose, and it results also from the
action of acids on several gums. The sugar is readily soluble in
water, fermentable and dextro-rotatory like glucose. It forms a char-
acteristic osazone. It reduces Fehling's solution but not in the same
proportion as glucose. On reduction it yields dulcitol, which shows
its chemical relations most characteristically. On oxidation it yields
galactonic and mucic acids.

J-Talose is an unimportant aldose of artificial origin.

THE CARBOHYDRATES AND RELATED BODIES. 25

Sorbinose is a ketone sugar obtained from the juice of the moun-
tain ash berry. It is levorotatory and non- fermentable with yeast.

Invert Sugar. This name is given technically to the mixture of
glucose and fructose, equal molecules, produced by the action of weak
acids on cane sugar, as described below.

THE CANE SUGAR GROUP.

The Saccharobioses or Disaccharides. These are sugars of the
formula C12H22011 and are important substances. The best known
representatives of the class are cane sugar, milk sugar and malt sugar,
all of which are natural products, with cane sugar the most abundant.
These bodies are closely related to the hexoses, two molecules of the
latter being in some manner condensed or united to produce one of the
former. The disaccharides, on the other hand, by treatment with
weak acids or certain ferments break up easily into two molecules of
a hexose, water being added in the reaction :

CJSJOa + H20 = CHuO, + QH120,.

The hexose molecules formed may be alike or different, and the
process of converting the disaccharides into monosaccharides in this
manner is called " inversion." By this inversion the following changes
should be noted :

Saccharose or cane sugar yields glucose and fructose.

Lactose or milk sugar yields glucose and galactose.

Maltose or malt sugar yields glucose and glucose.

Saccharose. This sugar has been known from earliest times to
some peoples, but did not become an article of commerce until after
the discovery of the Americas. It is found in the juices of various
canes, several kinds of beets, the saps of many trees and in many seeds
and nuts. On the commercial scale it is produced from the beet and
canes, and in smaller amount from maple sap.

Cane sugar does not undergo fermentation directly with pure yeast,
but by prolonged action of common yeast on a dilute solution of the
sugar fermentation appears. This is due to the fact that the crude
yeast contains an inverting enzyme known as invertase which produces
glucose and fructose which then yield to the true fermentation. Cane
sugar gives no combination with phenyl hydrazine, and is not a re-
ducing sugar. These facts point to the absence of aldehyde or ketone
groups in the large molecule. An experiment illustrates this:

Experiment. Prepare a dilute solution of pure cane sugar and boil it with
Fehling solution in the usual manner. Observe that no reduction of the copper
compound takes place. Next boil a similar cane sugar solution with a few drops

26 PHYSIOLOGICAL CHEMISTRY.

of dilute hydrochloric or sulphuric acid several minutes, neutralize with sodium
carbonate, and then apply the Fehling test. The characteristic red precipitate now
appears. In this reaction the cane sugar is broken up by the acid into a molecule
of glucose, and a molecule of fructose, both reducing sugars as explained above.

The behavior of cane sugar solutions with polarized light is charac-
teristic and affords the simplest and most accurate means for quanti-
tative determination. The specific rotation is practically independent
of the concentration and is represented by the formula [a] D =+66.5°.

Strong solutions of cane sugar, " syrups," are used in the house-
hold and in pharmacy to prevent fermentation. Hence the use of this
sugar in the canning or preserving of fruit.

Lactose. This is the characteristic sugar of all kinds of milk,
with possibly one or two exceptions. It may be separated from the
"whey" which is the product remaining after skimming and precipi-
tating the casein. It is made commercially in large quantities as a
by-product in the cheese industry, and in pure crystallized form has
the formula C^H^On-H^O.

Milk sugar resembles cane sugar in respect to the conditions under
which it may be fermented, but it is a reducing sugar directly, acting
strongly on copper or bismuth solutions. In its behavior with polar-
ized light it resembles glucose closely, having a specific rotation,
[a]D= -f- 52. 50. Inverted milk sugar ferments readily, and products
known as kumyss, from mare's milk, and kephir, from cow's milk,
are made in this way. In digestion lactose splits up into glucose and
galactose readily, while cane sugar yields glucose and fructose, but
less readily. With phenyl hydrazine a yellow lactosazone is formed.

Milk sugar is much less soluble in water than is cane sugar and has
but a slightly sweet taste. It is used mainly in the production of
infant and invalid foods and in manufacturing pharmacy in tablets,
pills, etc.

Maltose, or the sugar of malt, is produced by the action of malt
diastase on starch. It therefore occurs in germinating seeds and
grains, and is present wherever a diastase acts on starch. In the
action of weak acids on starch paste malt sugar is produced as a
transition stage, glucose finally resulting by inversion. In this country
malt sugar is not a common article of commerce, but in several Euro-
pean countries it has been produced in considerable quantities to be
used as an article of food in the place of glucose or cane sugar. The
manufacture of a relatively pure sugar by the use of malt diastase
and a starchy material, such as corn, seems to be attended, however,
with great practical difficulties.

THE CARBOHYDRATES AND RELATED BODIES. 27

Maltose is readily soluble in water, sweet but not to the same
degree as cane sugar, and is not directly fermentable. But an invert-
ing enzyme in common yeast changes it so quickly that it was long
classed among the true fermenting sugars. The view is now gener-
ally held that the disaccharides must first be converted into mono-
saccharides before real fermentation can take place. In the industries
malt sugar is thus fermented on the large scale. Toward oxidizing
solutions its behavior is like that of glucose, although its reducing
power is not quite as great. With phenyl hydrazine it forms a malt-
osazone, and on polarized light its rotating power is very great, the
specific rotation being at 200 C. [a]0 = + 1370. In the body maltose
is changed into glucose by action of an inverting enzyme occurring
both in the pancreas and in the true intestinal juice. This inversion
seems to take place much more readily than in the case of cane sugar,
which is a fact of considerable physiological importance.

Isomaltose. This is a sugar which has been made by the action of
fuming hydrochloric acid on glucose. It also accompanies the true
maltose in the products formed by the action of diastase on starch.
It differs from maltose in rotating power, which is much less, in the
character of its phenyl osazone, and in water solubility. It reduces
copper and bismuth solutions but undergoes fermentation with yeast
very slowly.

Other disaccharides known have but little importance. Mycose or trehalose is
found in certain fungi, agarose is obtained from the juice of the agave plant.
Melibiose and turanose are formed in the hydrolysis of certain polysaccharides.

The Saccharotrioses or Trisaccharides. This group contains a
few sugars and but one of these is important at the present time.

Melitose or raffinose. This sugar, having the formula C1SH32016
-f- 5H20, is found in certain kinds of manna and also in sugar beets
in small amount. It is characterized by having a strong rotation,
la]D= I04-5°- Being more soluble than saccharose it is found in the
last crystallizations from beet juice, and thus sometimes contaminates
the beet sugar. Its high rotation may cause an error in the estima-
tion of sugar by the polarimeter. When inverted with acids it yields
fructose, and the disaccharide melibiose.

THE DETERMINATION OF SUGARS.
This is carried out in several ways. In one method the reactions
depend on the reducing power of sugars on alkaline copper or other
metallic solutions. The Fehling reagent is usually employed. Methods
with the polariscope will be described later.

28 PHYSIOLOGICAL CHEMISTRY.

Method with Fehling's Solution. Fehling's solution, as described
above, is made arbitrarily of such a strength that one cubic centimeter
is reduced by 5 milligrams of glucose, on the supposition that the
sugar and copper salt react on each other in the proportion of one
molecule of glucose to five molecules of crystallized copper sulphate.

It was formerly held that the reaction was a perfectly definite and
simple one, and could be expressed in this manner, but it is now
known that the dilution of the solutions is a very important factor in
determining the amount of copper reduced. The best conditions to be
employed in practice have been determined by Soxhlet, who found the
reducing power of several sugars to vary as follows, when they were
tested in solutions of 1 per cent strength :

0.5 gm. of invert sugar in 1 per cent solution reduces 101.2 cc. of Fehling's
solution, undiluted.

0.5 gm. of invert sugar in 1 per cent solution reduces 97.0 cc. of Fehling's
solution, diluted with 4 volumes of water.

0.5 gm. of glucose in 1 per cent solution reduces 105.2 cc. of Fehling's solution,
undiluted.

0.5 gm. of glucose in 1 per cent solution reduces 101.1 cc. of Fehling's solution,
diluted with 4 volumes of water.

0.5 gm. of milk sugar in 1 per cent solution reduces 74 cc. of Fehling's solution,
undiluted. The reducing power in diluted solution is the same.

0.5 gm. of maltose in 1 per cent solution reduces 64.2 cc. of Fehling's solution,
undiluted.

0.5 gm. of maltose in 1 per cent solution reduces 67.5 cc. of Fehling's solution,
diluted with 4 volumes of water.

The oxidizing power of 1 cc. of Fehling's solution with each kind of sugar may
be tabulated as follows, assuming the sugars to be in solutions of approximately
1 per cent strength when acted upon.

One cubic centimeter of Fehling's solution oxidizes :

When When Diluted with

Undiluted. 4 Vols, of Water.

Glucose 4.75 mg. 4.94 mg.

Invert Sugar 4.94 " 5.15 "

Milk Sugar 6.76 " 6.76 "

Maltose 7.78 " 7.40 "

The practical application of the test is best shown by an experiment.

Experiment. Measure out accurately into a flask holding about 250 cc, 25 cc.
of the copper solution and the same volume of the alkaline tartrate. Heat the
mixture, or Fehling's solution, on a wire gauze and note that it remains clear. Fill
a 50 cc. burette with a dilute glucose solution and run 10 cc. into the hot liquid.
Boil one minute, shaking the flask continuously, and allow the mixture to settle. If
the supernatant liquid appears yellow this indicates that the sugar solution is much
too strong and must be diluted with at least an equal volume of water before begin-
ning another test. If, on the other hand, the liquid is still blue, add 2 cc. more of
the sugar solution, boil again for a minute and allow to settle. If the color is now
yellow an approximate value for the amount of sugar in the solution becomes
known, but if still blue, the operation of adding solution and boiling must be con-

THE CARBOHYDRATES AND RELATED BODIES. 29

tinued until, after settling, a yellow color appears. Approximately 250 mg. of
glucose is required to reduce the Fehling solution taken, and this must be con-
tained in the sugar solution added. From this preliminary experiment calculate the
amount of sugar present in each cubic centimeter.

Experiment. With the data obtained in the above experiment as a basis, make
now a new sugar solution, having a strength of about 1 per cent. Measure out
50 cc. of the Fehling's solution, heat to boiling and run the new sugar solution from
the burette as before, the first addition being about 20 cc. Boil and note the color
after settling and then cautiously continue the addition of sugar solution, a few
tenths of a cc. at a time, boiling after each addition, until the blue color gives place
to a yellowish green and then, by the addition of a drop or two, to a pale yellow.

Sometimes the final disappearance of the copper from the solution is determined
by filtering a few drops through a very small filter and adding a drop of acetic
acid and a drop of ferrocyanide solution to the filtrate, when the characteristic
reddish color is given if a trace of copper is present.

To determine cane sugar by the Fehling's solution it must first be converted or
" inverted " into a mixture of glucose and fructose. If the sugar is in the dry
condition the inversion can be accomplished as follows : Weigh out 9.5 gm., dis-
solve in 700 cc. of water, add 20 cc. of normal hydrochloric acid and heat for 30
minutes on the water-bath. Then neutralize with 20 cc. of normal sodium hydroxide
solution and make up to 1000 cc. on cooling. This gives now a 1 per cent solution,
which is employed as given for glucose, using the factor 4.94 instead of 4.74 as
the amount of sugar oxidized by each cc. of the copper solution. On completion
of the experiment calculate 95 parts of cane sugar for each 100 parts of invert
sugar found.

The attention of the student is directed to the fact that malt sugar and milk
sugar may be determined by the aid of Fehling's solution without previous inver-
sion. This should be verified by experiment.

Method by Use of Ammoniacal Copper Solutions. The deter-
mination of glucose in pure aqueous solution by the above method is
simple and accurate, but in liquids containing other organic matters
the precipitate sometimes fails to settle readily, so that the recognition
of the end point is difficult. This is often the case with urine and
other physiologically important liquids. Advantage may be taken of
the fact that cuprous oxide dissolves in ammonia without color to pre-
pare a quantitative solution with which this difficulty may be largely
overcome. Pavy was the first to employ such a reagent practically
and his solution was made by diluting the ordinary Fehling's solu-
tion with ammonia in certain proportions. His suggestion has re-
ceived several modifications. In all of these the weak sugar solution
is added to the boiling ammoniacal copper solution until the color of
the latter is just discharged, at which point the reduction of the copper
hydroxide by the sugar is complete. In place of using' the Fehling's
solution it is well to make the Loewe solution with glycerol the basis
of the dilution. A solution of this kind may be made by the formula
below, in which the proportions have been found by the present writer
to give the best result in practical work. One cubic centimeter of the
solution oxidize-, one milligram of glucose in 0.2 per cent solution.

30

PHYSIOLOGICAL CHEMISTRY.

It is made with the following amounts per liter :

Copper sulphate, cryst 8.166 gm.

Sodium hydroxide (100 per cent) 15.000

Glycerol 25.000 cc.

Ammonia water, 0.9 sp. gr 350.000

Water to make 1,000.000 "

Experiment. Of this solution, measure 50 cc. into a flask and dilute with water
to 100 cc. To prevent too rapid an escape of ammonia and avoid reoxidation to
some extent, add to the mixture, while warming, enough pure white solid paraffin
to make a layer 3 or 4 millimeters in thickness when melted. The burette tip for
discharging the sugar solution is made long enough to pass down the neck of the
flask and below this paraffin. By boiling gently and adding the weak saccharine
liquid slowly, very close and constant results may be obtained. At the end of the
titration the paraffin is solidified by inclining the flask and immersing it in cold
water, or by flowing cold water over it. The reduced liquid is then poured out
and the cake of paraffin is thoroughly washed for the next test. A flask so pre-
pared may be used for a hundred titrations. To prevent bumping and facilitate
easy and uniform boiling, it is well to add a few very small fragments of pumice-
stone.

A solution made as above is not too strong in copper for accurate work, but the
volume of ammonia necessary to hold a much larger amount of the reduced oxide
in solution would render the process very inconvenient. Some practice is neces-
sary to show just how fast the saccharine solution may be safely added. If added
too rapidly the end point may be overlooked and the sugar content made to
appear too low.

Polarization Tests and the Use of the Polariscope. This is the proper place
to show the applications of the polariscope in the examination of sugars and other
substances. For a description of the various forms of polariscopes and discussion
of the optical principles involved in their construction the reader is referred to the

Fig. 1. A common form of Laurent polariscope. The polarizing prism is situated in the
tube below H, the analyzer at E, B is the reading microscope and C a vernier.

author's translation of Landolt's work, "The Optical Rotation of Organic Sub-
stances and its Practical Applications," but a few words of elementary explanation
may be in order here. A simple form of polarimeter in common use is shown in
the illustrations.

In the polariscopes in common use for general scientific studies homogeneous

THE CARBOHYDRATES AND RELATED BODIES. 3 I

yellow light is employed and this is first polarized by passing through a specially
designed prism in the front part of the instrument. This prism is usually some
form of a Nicol prism and is so constructed that only one of the polarized rays
produced at the start is allowed to emerge and pass through the instrument. The
plane in which this ray vibrates is called the plane of polarization. Such plane
polarized light passes through air, water, alcohol, ether, glass and many other trans-
parent substances without change ; that is the direction in which the light vibrates
remains unaltered. But many organic substances, liquids or solids dissolved, have
the remarkable property of causing this plane of polarization to change direction;
in other words the plane of vibration of the light suffers a twist or rotation in
passing through a column of the liquids. Substances which have the power of
changing the direction of the plane of vibration of polarized light passing through
them are called " active " substances and the extent of the rotation is dependent on

GO

■* ^

Fig. 2. This represents the course of the light through the Laurent polariscope, the
direction being reversed, however, from that of the last figure. a is a bichromate plate
to purify the light, b the polarizing Nicol, c a thin quartz plate covering half the field
and essential in producing a second polarized plane, d the tube to contain the liquid
under examination, e the analyzing Nicol and / and g the ocular lenses.

the number of molecules which the light passes. In the case of homogeneous
liquids like oil of turpentine the rotation varies with the length of the column
through which the light must pass, while in the case of dissolved solids, sugar
solutions for example, the amount of the twist or rotation varies with the length
of the column of solution, and also with its concentration or number of molecules
in a given volume. An instrument which has some device which enables the
observer to read off this rotation in degrees is called a polarimeter, and the number
of degrees read constitutes a measure of the strength or concentration of the
substance.

In order to compare the rotation of substances the term " specific rotation "
has been introduced. This, as applied to liquids, may be defined as the rotation
which a substance would exhibit if examined in a column ioo millimeters in length
having a concentration of i gram of active substance to each cubic centimeter.
This rotation must therefore be a calculated one, and is found as illustrated by this
concrete case. Consider a solution made by dissolving 25 gm. of pure cane sugar
in distilled water and diluted to make exactly 100 cc. This is then examined in a
polarization tube, which is a long tube of glass or metal having ends of plane
polished glass perfectly parallel to each other. The sugar solution forms then a
clear transparent column of definite length, which, assume in this case, is 200
millimeters. By examination in the polarimeter it is found now that this solution
rotates the plane of polarized sodium light through 33. 250. For a solution with 100
grams to 100 cc. the rotation by calculation should be four times this, or 1330, in the
200 mm. tube or 66.50 in the 100 mm. or standard tube. This is then the specific
rotation, and we express it by the formula:

[a] D = 66.5°,

in which [a] is the usual symbol for the specific rotation, and the D the indication

32 PHYSIOLOGICAL CHEMISTRY.

that the observation is made with sodium light, a without the brackets is the
angle of rotation as read off. Remembering the definition of specific rotation we
have this general formula as applied to solutions :

, ioo X iooa io4a

[a]= ixc -rr

in which / expresses the length of the observation tube in millimeters and c the
concentration or strength of the solution in grams per ioo cubic centimeters.

For many substances this rotation is so characteristic and so easily observed that
it constitutes a good test of purity or identity. With the specific rotation known
the following relation enables us to find the amount of active substance in solution :

io4q

C~[a]-l

The following are some specific rotations which have importance from the stand-
point of physiological chemistry, the temperature being 200 C. in each case :

Cane sugar, [a]x) = -|- 66.50 c = 10 to 30

Milk sugar (+H20), [a]D = + 52.5° c= 3 to 40

Malt sugar (+H,0), [a]D = + 137.0° c— 2 to 20

Glucose, [oi]d = -\- 53-0° c = 20

Levulose, [a]o = — 93.00 c= 10 to 20

Invert sugar, [a]x> = — ■ 20.20 c = 15

The protein substances, dextrin, glycogen and a number of other compounds to be
referred to later have also a high rotating power, which finds application in
investigations.

THE POLYSACCHARIDES.

We have here a very important group of bodies, some of which
appear to have an extremely complex structure. Formerly these com-
pounds were assumed to be simpler than the sugars and were repre-
sented by the general formula C6H10O5. The action of water in
producing glucose was assumed to consist merely in the addition of
one molecule as shown by the formula :

CJJttO, + H,0 = CiHfflO»

But this view is no longer held; the starches, cellulose bodies and
certain gums belonging to the group have been shown to exist in the
form of large and probably very complex molecular aggregations, and
the formula (C6H10O5)n is now usually employed to indicate this fact.

These polysaccharides are related to the real sugars by several
reactions. By certain treatment most of them may be converted more
or less readily into maltose, glucose or fructose, and besides this they
yield the ester derivatives characteristic of polyhydric alcohols. In
their natural condition they are mostly insoluble in water and other
solvents. It is customary to make three classes of these compounds, of
which the starches or amyloses, as the most important, will be treated
first.

THE CARBOHYDRATES AND RELATED BODIES. 33

The Amyloses. In the vegetable kingdom starch is a common
and widely distributed reserve material, a sugar, probably saccharose,
being first formed as a synthetic product. Starch is found in many
seeds, grains and tubers in the form of minute granules which are
often characteristic in shape or size. They may be extremely small,
pepper starch for example, or relatively large, as in the case of arrow-
root starch. Under the microscope these granules often appear to be
built up of concentric layers, and furthermore they are not homo-

Fig. 3. Wheat starch magnified about 350 diameters.

geneous in composition. The outer part of the granule consists of a
covering or sheath of starch cellulose within which is the large mass of
starch granulose. The cellulose sheath is insoluble in water at the
ordinary temperature, but with elevation of temperature in presence
of an excess of water the protective layer breaks and allows the
granulose to form a more or less perfect solution of so-called soluble
starch.

On the technical scale starch may be obtained from a variety of sub-
stances. The common sources are potatoes, corn, rice and arrowroot.
The manufacture is largely a mechanical operation, which may be
illustrated as follows :

Experiment. Grate a potato to a pulp by means of an ordinary tin grater, mix
the pulp with water and squeeze through a piece of coarse unbleached muslin,
collecting the strained liquids in a large beaker. Allow the mixture to settle a
half hour or longer and pour off the water, which contains some soluble albuminous

4

34 PHYSIOLOGICAL CHEMISTRY.

substances, some cellular floating matter, but very little starch. Most of this will
be found in the bottom of the beaker. Add some fresh water, stir up and allow
to settle. Now pour the water off again and repeat these operations until the
starch appears perfectly clean and white. Transfer this starch to a clean shallow
dish and allow what is not intended for immediate use to dry spontaneously in an
atmosphere free from dust. The dried product will consist of minute glistening
particles resembling small crystals. Save this starch for tests given below.

Experiment. Examine starch from several sources under the microscope, em-
ploying a power of about 300 diameters. Clean a glass slide thoroughly, place in
the center of it a small drop of water, and stir into this by means of a needle, or
glass rod, a minute quantity of starch. Now drop on a clean cover glass in such
a manner as to exclude air bubbles, and place under the microscope for observation.

Experiment. Repeat the last experiment, using an aqueous solution of iodine
instead of water. The starch granules will now appear blue. For the detection
of starch in mixtures the use of iodine is often indispensable.

Some idea of the size of the starch cells may be obtained from this
table which gives the mean diameter in fractions of a millimeter.
For starch granules which are oval instead of circular, the averages
of the longer and shorter diameters is given:

Potato 0.06 -0.10

Common arrowroot 0.01 -0.07

Corn 0.007-0.02

Wheat 0.002-0.05

Rice 0.005-0.008

Pea 0.016-0.028

Bean 0.035

Barley 0.013-0.040

Rye 0.002-0.038

Starch may be recognized by a number of chemical tests, the best of which are the
following :

Experiment. Boil a small amount of starch with water so as to make a thin
paste. Allow this to cool, and add a few drops of an aqueous, or alcoholic solu-
tion, of iodine. A deep blue color is formed, which disappears on boiling the
mixture. This test is exceedingly delicate and characteristic, and serves for the
detection of minute traces of iodine as well as starch. The blue color is destroyed
by alkalies or much alcohol as well as by heat.

Experiment. That starch is insoluble in cold water may be shown by stirring
some with water in a beaker, allowing to settle, and pouring the liquid through a
paper filter. The filtrate tested with the iodine solution does not give a blue color.
Use the potato starch of the experiment for this test.

When boiled with dilute acids starch is converted into soluble com-
pounds. The nature of these compounds depends on the acid used
and on the duration of the heating. By prolonged heating glucose is
the main product of the reaction, as already illustrated, but various
intermediate steps may be recognized, maltose and forms of dextrin
being readily demonstrated. With strong acids the results are quite
different. With sulphuric acid the reaction is completely destructive,
water, carbon dioxide and sulphurous acid from reduction being

THE CARBOHYDRATES AND RELATED BODIES. 35

formed. Strong nitric acid acts as an oxidizing agent and by proper
manipulation oxalic acid may be obtained in quantity as a product of
the oxidation.

Experiment. Add 15 cc. of strong sulphuric acid to a gram of starch in a flask
holding about 200 cc. Heat to the boiling point and observe that a black mass is
soon produced. By prolonged heating this is further decomposed, while fumes of
sulphurous oxide escape, leaving finally a colorless liquid.

Experiment. Add 15 cc. of strong nitric acid to one gram of starch in a flask
holding 200 to 300 cc, place this on a sand-bath in a fume chamber and apply
heat. After a time copious red fumes are given off. Remove the lamp and allow
the reaction to continue until the fumes cease to be evolved. Finally, transfer the
liquid to a porcelain dish and evaporate to a small volume. On cooling, a crystalline
residue remains which consists mainly of oxalic acid.

When carefully heated starch may be largely converted into a form
of dextrin, which, as will be fully explained later, is one of the impor-
tant stages in the common transformations of starch. The reaction
is employed on the large scale in the manufacture of British gum
which is used in the preparation of size and paste for various purposes.

Experiment. Heat about 10 gm. of starch in a porcelain dish on a sand-bath to
a temperature short of the point where it begins to scorch. It is necessary to stir
well all the time, and continue the heat ten minutes after the starch has become
uniformly yellowish brown. Then allow the dish to cool, add water and boil
thoroughly, which brings part of the product into solution. When sufficiently
diluted this solution can be filtered. The filtrate is precipitated by alcohol. The
addition of a few drops of iodine solution to the aqueous liquid gives rise to a
reddish color characteristic of dextrin.

The chief uses of starch have been referred to in other connections.
Much is directly employed as food and large quantities are converted
into glucose as shown above. The production of various kinds of
dextrin and British gum is also extremely important and consumes
enormous quantities of starch. In the form in which it occurs in
nature, that is mixed with other substances in small amount, starch is
the most abundant of our foodstuffs, and the one consumed in largest
amount. Great interest therefore attaches to the reactions by which
this starch is made soluble or digested as a step in its assimilation.
The discussion of this fundamental point will be left, however, for a
following chapter, when the theory of digestive operations can be
explained as a whole.

In certain plants a variety of starch called inulin occurs. It is best
obtained from tubers of the dahlia, and is interesting from the fact
that by hydrolysis it yields fructose instead of glucose. It differs from
the ordinary starch in yielding a true solution with hot water, and in
giving a yellow instead of a blue color with iodine.

36 PHYSIOLOGICAL CHEMISTRY.

Glycogen, or animal starch. This product, which is formed in
the liver, is related in many ways, both chemically and physiologically,
to common starch. In some respects it resembles also the simple
sugars, from which it is indeed derived, and may be said to stand
between them and vegetable starch. It is readily soluble in water,
giving, however, an opalescent solution. This is especially character-
ized by a strong action on polarized light, [a]D = + 1960 to 2130,
according to different authorities. Like common starch glycogen is
a reserve material, being formed from the absorbed sugar of the
digestive process, and, in turn, being reconverted into sugar from the
liver as this is required for oxidation in the body. After death the
store of glycogen in the liver rapidly diminishes, glucose being pro-
duced. The amount of glycogen present in the liver varies greatly
with the diet and time after eating. It may make up 12 to 16 per
cent of the total weight of the organ, but is usually much below this,
perhaps in the mean 2 to 3 per cent. In addition to its occurrence in
the liver glycogen is found in variable amount in the muscles, and in
traces in other body tissues. It occurs also in the vegetable kingdom,
and has been recognized in certain fungi. The laboratory production
of glycogen and some of the simple reactions are illustrated by the
following experiments, while the physiological relations will be
reserved for further discussion in a later chapter.

Experiment. Kill a rat or a rabbit which has been well fed; remove the liver
as quickly as possible, and without delay cut it into small bits, which throw into a
vessel of boiling water. The weight of the water should be about five times that
of the minced liver. Boil five minutes, then remove from the water and rub up
in a mortar with fine clean quartz sand. In this way the fragments of liver be-
come thoroughly disintegrated. The contents of the mortar, sand as well as liver,
are thrown into boiling water again and kept at ioo° 15 minutes. At the end of this
time enough dilute acetic acid must be added to impart a faint acid reaction. This
coagulates and precipitates some albuminous matters, which are separated when the
hot mixture is filtered. In the opalescent filtrate, which must be collected in a
cold beaker, a further precipitation of albuminous matter is effected by adding a
few drops of hydrochloric acid and some potassium mercuric iodide as long as a
precipitate forms.

Filter again and use the dilute aqueous solution of glycogen resulting for tests
below.

Experiment. Evaporate about half of the liquid above to a small volume and
precipitate impure glycogen as an amorphous white powder by addition of strong
alcohol.

Experiment. Add a little tincture of iodine to a small portion of the solution,
and note the red color produced. This color is discharged by heat. Boil some of
the solution with dilute hydrochloric acid ten minutes; neutralize the acid nearly,
-cool and again test with iodine. No color is now produced, as glycogen has dis-
appeared under the treatment, having been converted into sugar.

It has been remarked above that after death the store of glycogen in the liver

THE CARBOHYDRATES AND RELATED BODIES. Z7

rapidly disappears, so that tests applied at the end of a day or two fail to show
its presence. This may be shown as follows :

Experiment. Cut some beef liver from the market into small bits and extract
with boiling water. Boil longer to coagulate protein bodies, after adding some
sodium sulphate. Apply the iodine test for glycogen, which is found absent, and
the Fehling test for sugar, which is found present in quantity. It is an excellent
exercise also to determine the amount of sugar which may be obtained from a
given weight of liver. In this test the extraction must be repeated with several
portions of water.

The Gums. Some of these occur in nature as products related to
the pentose group of sugars referred to some pages back. Others are
related to starch, and on transformation yield finally hexoses. The
group of gums includes further the dextrin bodies formed from starch
by several reactions, one of which has been already illustrated. The
transformation of starch by the action of weak acids or enzymes is
far from being a simple process and much uncertainty still exists as
to the number of intermediate products between the parent substance
and the final maltose or glucose. Some writers have attempted to
distinguish several well defined stages in the reaction and describe as
definite bodies erythrodextrin, achroodextrin, amylodextrin and
nialtodcxtrin. The first gives a violet-red color with iodine and is
easily precipitated by alcohol ; the second gives no color with iodine,
but is still precipitated by alcohol. It shows reducing power with
Fehling's solution, and may be looked upon as one of the end products
of the action of diastase on starch, maltose being the other. The
name amylodextrin is given to a product of diastatic action and also
to a dextrin formed by the treatment of starch with very dilute acids.
It is said to show a purple color with iodine, and to exhibit very strong
dextro rotation. The existence of maltodextrin is affirmed by several
writers, but the properties of the substance are not well established.
Some authors have gone so far as to recognize several modifications
of achroodextrin, which are described as a, /? and y forms, and which
differ in optical properties and reducing power. The more recent
extended investigations seem to disprove this notion, however, and the
most that can be safely said is that along with maltose an end product
is produced by diastatic conversion of starch which is probably a single
substance. What is called erythrodextrin is more likely to be a mix-
ture, possibly, of soluble starch and achroodextrin. Under the name
crythrogramdose a similar complex has been described. In a later
chapter on the action of ferments more will be said about the theory
of the transformation of starch into these products.

The true dextrins are not directly fermentable with yeast, but they

38 PHYSIOLOGICAL CHEMISTRY.

appear to be aldehyde bodies and as such have reducing power. They
react also with phenyl hydrazine and yield osazones, which, however,
are not easily purified, because of their solubility. The dextrins have
a slightly sweetish taste and all show a specific rotation about
[a]D = -f- 1960. Beyond the empirical formula C6H10O5 it is not
possible to go in describing the constitution of these bodies.

The natural vegetable gums are often mixtures of several sub-
stances, and but few of them have been studied. Gum arabic and gum
Senegal are the potassium and calcium salts of arabic acid to which
the formula (C6H10O5)2 + H20 is given. On treatment with weak
sulphuric acid both arabinose and galactose appear to be formed.
Agar-agar is said to yield lactose and then galactose, while cherry gum
yields arabinose.

Cellulose. The cell walls of vegetable substances consist of cellu-
lose mixed always with related compounds of which a body called
lignin is the most important. The cellulose resists the action of strong
oxidizing or other agents much more perfectly than do' the accom-
panying bodies, and may therefore be freed from them by various
treatments. In washed Swedish filter paper we have an illustration of
nearly pure cellulose, as all the other bodies in the original fibers have
been removed by the bleaching and washing processes to which the
raw material was subjected in the manufacture of the paper. A pure
cellulose paper may be made from wood also, but only by more com-
plicated operations.

The pure cellulose is characterized by insolubility in water, weak
acids, alkalies, alcohol or ether. It may be dissolved rather readily
in a solution known as Schweitzer's reagent and by prolonged di-
gestion with acids is converted into hexoses and pentoses.

The natural celluloses may be divided roughly into three groups:
(a) those which resist hydrolytic action very perfectly and are not
capable of serving as foodstuffs for any animals; in this group we
have linen and cotton fibers, hemp, China grass, etc. (b) Those
which are less resistant to hydrolytic action and which contain active
CO groups. These bodies may be called oxycelluloses ; they yield also
furfuraldehyde by distillation with hydrochloric acid. In this group
we have the mass of the material found in the fundamental tissues of
flowering plants, and a large part of ordinary woody tissue. This
lignified tissue is made up of compound celluloses or lignocelluloses
from which the cellulose proper may be isolated in a variety of ways.
Some of the bodies in this sroup are partly digestible and have some
value as foods for the herbivora. (c) In this third group we have

THE CARBOHYDRATES AND RELATED BODIES. 39

substances described as pseudocelluloses or hemicelluloses and which
offer little resistance to hydrolysis. They are easily attacked by weak
acids and alkalies and also suffer digestion by enzymes, so that they
may be classed among the foodstuffs of limited value for the herbivora.
These bodies resemble starch much more than they resemble the ligno-
celluloses. By action of weak acids fermentable sugars are formed
almost quantitatively from pseudocelluloses, while from the ligno-
celluloses not over about 20 per cent of fermentable sugars may be
obtained.

Experiment. Prepare Schweitzer's reagent by first precipitating copper hydrox-
ide from copper sulphate solution, in the presence of a little ammonium chloride,
by addition of sodium hydroxide in excess. Wash the precipitate thoroughly and
then dissolve it in the smallest possible quantity of strong ammonia water. This
yields a deep blue solution, the reagent in question. It dissolves cotton rather
easily. This solution may be filtered after dilution, and from the filtrate a pure
cellulose is thrown down by addition of acids.

By action of strong nitric acid, aided by sulphuric acid, cellulose is converted into
a series of nitrates or " nitro-celluloses." The number of N03 groups added de-
pends on the strength of the acid mixture and time of its action. The more
highly nitrated products constitute the well-known explosives. Products not so
highly nitrated are used in the preparation of collodion and celluloid. This latter
is essentially a mixture of camphor and nitrated cellulose from cotton or paper.
These bodies are esters and therefore may be decomposed by alkalies.

CHAPTER IV.

THE FATS AND SUBSTANCES RELATED TO THEM.

In nature we find a large number of esters composed of the fatty-
acids united to glyceryl. These are the ordinary fats and as foodstuffs
they are nearly as important as the carbohydrates. In structure they
are practically all of the type C3H5(C»H2re_102)3, but include bodies
of widely different physical properties. Some are liquids, while others at
the ordinary temperature are hard solids. Nearly all vegetable products
contain fats of some kind; often the amount is very small, but fre-
quently it constitutes fully 50 per cent by weight of the seed, nut or
fruit in question. In the animal kingdom fats are always present,
in some amount, in all organisms. The animal fats are often derived
from the vegetable fats consumed as food.

THE NATURAL FATS.

The important fatty acids combined with the radical of glycerol,
C3H5(OH)3, are given in the following table. The combinations are
essentially like that illustrated by this structural formula of stearin:

CH2— 0-QsH350

CH _0-ClsH350

CH2 — O — C]8H350

SATURATED ACIDS, CnH2re02.

Formic acid, HCHO, "|

Acetic acid, HC2H302 l-glycerides not natural substances.

Propionic acid, HC3H502 J

Butyric acid, HQH702, occurs in butter fat as glyceride.
Pentoic acid, HC5H902,. valeric acid occurs as a natural compound.
Caproic acid, HC6Hn02, in butter fat as glyceride.
(Enanthylic acid, HC7Hi302, does not occur as glyceride.
Caprylic acid, HCsH1502, as glyceride in butter and other fats.
Pelargonic acid, HC9H1702, in vegetable kingdom, but not as glyceride.
Capric acid, HQ„H]902, in butter and other fats as glyceride.
Undecylic acid, HC„H2102, not found as natural glyceride.
Laurie acid, HC12H2302, as glycerol ester in several fats.
Myristic acid, HC14H2702, in nutmeg butter and other fats as glyceride.
Palmitic acid, HC16H3102, as glyceride in many fats.
Margaric acid, HC17H3302 obtained as artificial glyceride.
Stearic acid, HCJSH3502, as glyceride in many fats.
Arachidic acid, HC20H39O2, as glyceride in peanut oil.
Behenic acid, HC22H4302„ as glyceride in certain oils.

40

THE FATS AND SUBSTANCES RELATED TO THEM. 4 1

A few other acids of this series are known in glycerol combina-
tions but they are unimportant.

NON-SATURATED ACIDS, CnH2n-202 AND CnH2n.402.

Not many of these acids occur as natural glycerides.

Hypogaeic acid, C16H30O2, as glyceride in peanut oil.
Oleic acid, CsH^Oo, as glyceride in many oils.
Linoleic acid, C1SH3202, as glyceride in drying oils.
Ricinoleic acid, dsH^Os, as glyceride in castor oil.

A large number of the acids in the first list and two in the second
occur in the edible fats, while some are found in products of little
importance, or in such as have technical uses only. The fats which
are most commonly used as foods are those consisting largely or
wholly of stearin, palmitin and olein. Butter fat, however, contains
relatively large amounts of other glycerides.

Reactions of Fats. Certain general reactions are common to
practically all fats and will be explained here in detail.

Saponification. This term is applied to the change which fol-
lows when fats are treated with alkali solutions, usually with applica-
tion of heat. The fats decompose more or less readily, and as products
we have soaps and glycerol, according to these equations :
C3H5(C1SH3502)3 + 3KOH = C3H5(OH)3 + 3KC1SH3302
2C3H5(CISH3302)3 + 3PbO + 3H20 = 2C3H5(OH)3 + 3Pb(C18H3302)2

In the first case potassium stearate is formed and in the second
lead oleate, which is the important constituent of lead plaster. Here
lead oxide and water are equivalent to lead hydroxide. Much of the
glycerol of commerce is produced by such decompositions. When
sodium hydroxide is used with the common fats ordinary hard soap
results.

An analogous change occurs when fats are subjected to the action
of water at a high temperature or superheated steam. We have here
hydrolysis purely, although the term saponification by steam is some-
times applied. The same reaction is brought about by certain enzymes
at the ordinary temperature, for example by the enzyme known as
lipase or steapsin in the pancreatic secretion. This will be discussed
fully later as it is important in the digestion of fats. Sometimes the
reaction is complete as shown by this equation :

C3Hr,rC,JI,,02)3 + 3H20 = C3Hr,(OH)3 + 3CISH30O2
But products of partial hydrolysis, monostearin and distcarin, for
example, may be left, as :

rC .n <>■ ("OH

C,llA C,JT,,r,02 + H20 = C,l I'M' ) .. I- HC«H„Oa

Ic.j Lc,j( .' >.

42 PHYSIOLOGICAL CHEMISTRY.

ILLUSTRATIVE TESTS.

Some of the saponifications are illustrated by these experiments:

Experiment. Boil about 25 gm. of tallow with a solution of 10 gm. of potas-
sium hydroxide in 100 cc. of water. Stir the mixture thoroughly until it becomes
homogeneous; that is, until no oil globules are seen floating on top of the aqueous
liquid, which may require half an hour. Add water from time to time, to make
up for that lost by evaporation. The resulting mass is a mixture of glycerol,
excess of alkali and soft soap. To this now add a solution of 15 gm. of common
salt in 75 cc. of water and heat again, which brings about a conversion of the
soft soap into the hard or sodium soap. On cooling, this separates and floats on
the excess of spent lye and salt solution.

Experiment. The presence of fatty acids in the above soap may be shown by
adding to a portion of it enough hydrochloric acid to decompose the soap. Use
about half the product of the experiment, dilute with water, and add the acid
in slight excess, about 10 cc. of the strong commercial acid. Warm on the water-
bath, which will cause the liberated fatty acids to collect on the surface as a liquid
layer as soon as the temperature becomes high enough. Add more water and
allow the whole to cool. A semi-solid layer of fatty acids can now be lifted from
the surface of the liquid. The hardness of the mixed acids depends on the fat
taken for experiment. Mutton and beef tallows yield very solid acids ; with lard
the mass is softer, while with some oils the acids do not solidify at all at the ordi-
nary temperature.

Experiment. Dissolve a small portion of the fatty acids in warm alcohol, nearly
to saturation. On cooling, the acids separate in crystalline scales.

Experiment. The presence of glycerol as one of the products formed by the
saponification of fats is best shown as follows :

Mix 50 cc. of cottonseed oil with 25 gm. of litharge and 100 cc. of water in a
porcelain dish. Place over a Bunsen burner on gauze and stir until all oil
globules have disappeared, adding a little water from time to time. The litharge
with water acts as lead hydroxide and saponifies the fat, forming an insoluble lead
soap, or plaster, and glycerol. When the saponification is complete add more water,
heat and stir well to dissolve glycerol. Allow to settle a short time and pour
the aqueous solution through a filter. To the residue add water again, heat, allow
to settle and pour through the same filter. Concentrate the mixed filtrates to a
small volume and after cooling observe the sweet taste of the thickish residue.

Experiment. Dissolve a portion of the sodium soap in water with aid of a little
alcohol. Then add some solution of calcium chloride or lead acetate. A white
precipitate is formed, as the calcium and lead salts of the fatty acids are not
soluble in water. Hard waters, which contain salts of calcium and magnesium,
decompose soap in the same manner.

Other Reactions. The common fats are insoluble in water and
when mixed with the latter tend to separate immediately. However,
it is possible to convert the fats and water into a peculiar mixture
called an emulsion which does not separate into two layers on standing.
In this condition the fat consists of extremely minute globules which
remain in suspension and which may be passed through the pores of
coarse filter paper. It does not seem possible to secure an emulsion
with perfectly neutral fats, and in most cases the phenomenon de-
pends on the presence of a trace of soap formed. In the processes of

THE FATS AND SUBSTANCES RELATED TO THEM.

43

digestion of fats in the animal body emulsification plays a very im-
portant part as will be shown later. It follows, probably, the partial
hydrolysis of the fats by lipase, referred to above.

As they exist in the animal or vegetable organism the fats are
doubtless all amorphous substances, but in the separated condition the
solid fats always become more or less crystalline. This may be

Fig. 4. Mutton tallow crystallized fror
chloroform. 300 diameters.

Fig. 5. Cat fat crystallized from chlo-
roform. 300 diameters.

Fig. 6. Beef tallow crystallized from
chloroform. 300 diameters.

Fig. 7. Beef tallow crystallized from
chloroform. 300 diameters.

readily shown by dissolving some fat in a proper solvent which is
then allowed to evaporate slowly.

The common fats can not be distilled under the ordinary pressure
without decomposition, and the distillation of the fatty acids is also
difficult. When the common fats are strongly heated they emit a pe-
culiar odor, due to the acrolein formed by the partial destruction of
glycerol. The following experiments illustrate some of the points
referred to :

44 PHYSIOLOGICAL CHEMISTRY.

Experiment. Note the solubility of small bits of tallow in ether, chloroform,
benzine and alcohol, using in each case the same volume of liquid, with equal
weights of fat. The solubility in alcohol will be found much less than in the
other menstrua.

Experiment. Dissolve some mutton or beef tallow in chloroform and with a
glass rod put two or three drops of the nearly saturated solution on the center of
a glass slide. As the chloroform evaporates a film begins to form on the top of
the drop. Now put on a perfectly clean cover glass and allow to stand until
crystallization is complete, which may require only a few minutes or some hours,
the time necessary depending on the temperature and on the concentration of the
solution. Examine the crystals with a microscope. Use a power of 200 to 300
diameters. By repeating the experiment with different fats considerable variation
in the form of the crystals may be noticed, which is shown in the annexed cuts.

Experiment. Add to 5 cc. of cottonseed oil half its volume of strong white of
egg solution and shake thoroughly. The liquids mix and form a white mass or
emulsion which, however, is not usually stable.

Experiment. To 5 cc. of cottonseed oil containing a little free fatty acid add 10
drops of strong sodium carbonate solution and shake. A good stable emulsion is
made in this way, as the sodium of the alkali solution forms a soap with the free
acid and this appears to form a film around the little fat globules which prevents
their flowing together again.

Origin of Fats in the Body. The question of the formation of
fats in the animal organism has been much discussed. It was once
assumed that, like protein substances, the fats are products of the
vegetable world only, and that the animal has not the power of build-
ing them up from compounds which are not fats. But this view is
not correct, as we have abundant proof that fats may be made in
other ways. Much has been learned from the results of cattle feeding
experiments carried out in agricultural experiment stations, where
the gain in fat is often much in excess of what could be accounted for
by the amount of fats in the food consumed. This gain must in some
way be due to the effect of the carbohydrate and protein substances
in the rations fed. The .fattening power of sugar has long been
recognized, but this has been in part accounted for on the theory that
the sugar acts to protect the fats of the body from oxidation, by being
readily oxidized itself to keep up the body energy. But much evi-
dence has been accumulated to show that carbohydrates take part
directly in the production of fats. How this is accomplished is not
known, but in the processes syntheses and oxidations both must be
concerned, since the fat molecules are more complex than those of the
carbohydrates.

It is further likely that protein compounds are important factors
in fat production. Many writers have indeed assumed that we have in
the breaking down of protein molecules the chief sources of fats, but
this view has been strongly combated. Indirectly the transformation

THE FATS AND SUBSTANCES RELATED TO THEM. 45

may follow in this way : It is known that sugars are formed as cleav-
age products of certain albumins in the ordinary katabolic processes of
the body and possibly a portion of the sugars thus formed may be
then built up to produce fats.

The existence of the substance known as adipocere has long been
held to furnish a pretty strong proof of the production of fatty acids
from protein. This adipocere or cadaver wax is often found in large
masses in old cemeteries and consists of fatty acids, calcium and am-
monium soaps in the main. It is held that this substance could not
possibly have come from the small amounts of fat ordinarily present
in cadavers but must have been produced from the muscular portions
of the body. This view has met with objections, however, and at-
tempts are made to account for the development of the adipocere in
other ways.

In the body fats constitute a reserve material in which potential
energy is conveniently stored up. In sickness or in wasting diseases
where there is a partial or complete failure in nutrition this fat is
called upon to supply the needs of the body. The fats are oxidized
while the muscular tissue is preserved.

PHYSIOLOGICALLY IMPORTANT FATS.

Stearin or Tristearin, C3H5(C18H3502)3. This is a simple fat
which does not occur in nature unmixed with other fats. When sepa-
rated in purest possible condition it shows a melting point of 550 to
580. It is the hardest of the common simple fats and apparently the
least soluble in alcohol or ether. It may be separated in the form of
rectangular plates by crystallizing from hot alcohol. Stearic acid
may be obtained in the form of pearly crystalline plates or scales. It
melts at 71 °.

Palmitin or Tripalmitin, C3H5(C16H3102)3. The perfect sepa-
ration of this fat from stearin, with which it is usually associated,
is a matter of considerable difficulty. The fats are much alike.
The melting point is variously stated by different observers, but
appears to be about 510. A mixture of stearin and palmitin was
formerly supposed to be a distinct fat and was called margarin,
C3H5('Ci7H3302)3. This fat has been produced artificially but it
does not appear to be a natural product. Palmitic acid resembles
stearic acid in appearance and solubility; both acids are slowly soluble
in strong hot alcohol and yield crystalline plates on cooling. The
melting point of palmitic acid is about 620.

Olein or Triolein, C3H5(C18H3302);!. This is a liquid fat at the

46 PHYSIOLOGICAL CHEMISTRY.

ordinary temperature and is a constituent of most of the natural fats
and oils. Some fatty oils are nearly pure olein and become solid at a
low temperature. The soft consistence of lard, human fat and sev-
eral other natural mixtures is due to the olein present. Olein is a
nonsaturated fat and will therefore show an absorption coefficient
as explained below. By the action of reagents yielding nitrous acid
it is converted into an isomeric substance known as elaidin.

Oleic acid in pure condition is not very stable, because of its un-
saturated structure. It is an oily liquid at the ordinary temperature,
but below 140 is converted into a crystalline solid. By treatment
with nitrous acid it yields the isomeric elaidic acid. Oleic acid is
characterized by forming a lead salt which is soluble in ether while
lead palmitate and stearate are practically insoluble.

Lard and Tallow are essentially mixtures of the three fats, pal-
mitin, stearin and olein. By heating lard to its melting point, cooling
slowly and subjecting the warm mass to pressure in a filter press the
softer portion, consisting mainly of olein, may be separated. This is
known as lard oil, while the harder residue is sometimes called lard
stearin. It has about the consistence of butter. By subjecting beef
suet to the same treatment a soft portion known as oleo oil is separated
from a solid residue called beef stearin. The oleo oil is the material
most often employed under the name of oleomargarin as a substitute
for butter. A mixture of somewhat similar consistence is made in
other ways ; for example by combining cottonseed oil with beef stearin.

Oleomargarin is the name given by law to these butter substitutes
in the United States. Sometimes the fats are churned with milk or
mixed with a certain amount of real butter to furnish a product with
flavor suggesting butter. The name butterine is usually given to
such mixtures and when properly made they are wholesome and in
every way as good as butter from the standpoint of nutritive value.

Butter. The fat of milk is a very complex mixture and an exact
separation has not yet been made by the methods of chemical analysis.
According to the older notion butter fat contains essentially olein,
stearin and palmitin, with a little butyrin, to which the flavor and odor
are largely due, but it has been shown that other glycerol esters are
certainly present. The results of some recent examinations may be
approximately expressed as follows :

Glyceryl butyrate 7.0

Glyceryl caproate, caprylate and caprate 2.0

Glyceryl oleate 36.0

Glyceryl myristate, palmitate and stearate 55.0

100.0

THE FATS AND SUBSTANCES RELATED TO THEM. 47

From various investigations it appears that these fats are not
present as simple esters, but may possibly exist in combinations repre-
sented by formulas like this :

rC18H3502
C3Ha< CieH3I02
I QH702

The melting point of butter fat is between 38 ° and 45 °. On melting
100 parts by weight of pure butter fat, saponifying, separating the
fatty acids and washing out everything soluble in hot water (butyric
acid, etc.) it is found that the insoluble residue left amounts in the
mean to 88 per cent, but may be more or less with different grades of
butter. Commercial butter contains in the mean about 85 per cent
of fat, 10 per cent of water and 5 per cent of salt.

Human Fat. This consists essentially of olein, palmitin and
stearin. In the fat of children the solid glycerides apparently are in
excess, while in later life the proportion of olein increases, so that the
separated fat may appear quite soft. In the human adult the olein
may amount to 75 per cent of the whole fat, but the proportion varies
with different parts of the body.

Glycerol. Since it is a constituent of all the true fats a few words
about this alcohol will be in order here. The substance was first recog-
nized in the aqueous liquid left in the preparation of lead plaster and
for many years all used in pharmacy and in the manufacture of cos-
metics was made by the same reactions. Since its importance in
technology was recognized it has been produced on the large scale by
other kinds of saponification or hydrolysis. In pure condition it is a
thick, sweetish liquid with a specific gravity of 1.266 at 150, referred
to water at the same temperature. It mixes with water and alcohol
in all proportions, but is not soluble in pure chloroform, benzene, car-
bon disulphide or petroleum ether. It is very slightly soluble in ether.
Like other alcohols it may be combined with acids to form esters.
With stearic acid mono-, di- and tri-stearin are produced, the last
being identical with the natural fat. When fed to animals glycerol
may be oxidized to a limited extent only. Any excess of it fails to be
assimilated and soon produces disorders in digestion and absorption.
Its food value, in free form, is therefore very slight.

Recognition and Determination of Fats. In physiological chemical investiga-
tions fats are separated from accompanying substances through their solubility in
warm ether, chloroform or petroleum ether. The carbohydrates, protein bodies
and salts are not soluble in these liquids. The saponification test is also of value in
identification. Many of the fats contain unsaturated acid groups and are there-
fore able to absorb certain amounts of halogens from specially prepared solutions.

48 PHYSIOLOGICAL CHEMISTRY.

Oleic acid, C^H^O^ absorbs iodine or bromine to form C^H^IsOu or dsH^BrjOj.
The fat to be examined is dissolved in chloroform and treated with the standard
solution. After a time the excess of iodine (or bromine) is determined, and that
absorbed by the fat is a measure of the non-saturated acid present. Linoleic acid
absorbs twice as much iodine or bromine as oleic acid, as the formula C1SH3202
becomes C1SH32I402.

The determination of the amount of insoluble fatty acids furnished by a given
weight of fat is also a valuable factor in the study of these bodies. In another
method the fat is saponified, the soap formed decomposed by dilute sulphuric acid
and the resultant product subjected to distillation. Fatty acids which are volatile
pass over and collect in the distillate, where their amount may be determined in
terms of KOH or NaOH, by titration. In this treatment stearin and palmitin
yield acids not volatile with steam. This is a valuable test and is applied in the
examination of butter supposed to be adulterated with other fats.

In certain lines of investigation and especially in the examination of tissues by
aid of the microscope, fat is recognized by its coloration through the coaltar product
known as Sudan III. This is one of the few colors which are soluble in fats
and fatty oils. A yellowish-red color is imparted.

Lecithin. This is a peculiar complex body which contains phos-
phoric acid and an organic basic group in place of one of the fatty
acid radicals in the common fats. It is found in the vegetable king-
dom, but commonly and most characteristically in many animal tis-
sues, in the brain and nerve tissue, blood, lymph, milk, pus, yolk of
egg, etc. It is most readily prepared from the last named substance.
The following formula represents the supposed structure of the body,

-_0-C18H350
C3)\,A -0-C18H350^

' OH

— O — PO-HO-0-C2H4 — N| c-

in which distearylglycero-phosphoric acid is combined with the base
choline :

(CH3)3 = N {QH4oH

It appears that several forms of lecithin exist, containing oleic and
palmitic acid as well as stearic. They undergo saponification, yielding
fatty acids, glycero-phosphoric acid and choline. They are soluble
in ether and alcohol and in other respects resemble the true fats. With
water lecithin swells to a gelatinous mass which under the microscope
presents a characteristic appearance. The function of lecithin in the
body is not understood, but from the fact of its wide distribution and
its occurrence in milk it is reasonable to assume that it performs some
important part.

The above formula represents the simple or typical lecithin. As recent investiga-
tions have shown that a number of related bodies exist containing other propor-
tions of nitrogen and phosphorus it has been proposed to give the name phos-
phatides to the whole group. The group name lecithan is also used. The chemistry

THE FATS AND SUBSTANCES RELATED TO THEM. 49

of these bodies is far from simple. Some of them appear to be associated with
sugars, and others with proteins in the animal and vegetable tissues, but as they
suffer decomposition very easily their separation in pure form for study is an
extremely difficult problem.

The Waxes. These bodies bear some resemblance to the fats and
will be briefly mentioned here. They consist largely of esters of
the higher monohydric alcohols of the saturated series, and in
most cases are complex mixtures of which the composition is not
exactly known. Spermaceti seems to consist largely of cetyl palmitate,
C16H33OC16H310. Beeswax contains some free acid, cerotic acid, in
addition to the esters. The most important constituent is apparently
myricin or myricyl palmitate, C30H61OC16H31O. The waxes are not
easily saponified and as a rule clear soap solutions are not obtained.

Cholesterol. This substance is an alcohol, but in appearance it
resembles some of the solid fats and is associated with them in several
natural products. Hence it is in place to describe it here. The for-
mula C27H45OH probably represents the composition of the body
which is found in the brain, yolk of egg, the liver, blood and other
tissues and is especially abundant in the fat of wool. It constitutes
also the main substance in certain gall-stones from which it may be
separated in nearly pure condition. In wool fat it exists partly in the
free state and partly in combination with fatty acids in the form of
esters.

It is readily soluble in hot alcohol, ether, benzene and chloroform,
but not in water or alkali solutions. It is therefore left as an insoluble
residue in the saponification of fatty mixtures containing it. Under
the microscope pure cholesterol appears as a mass of white plates with
sharp angles. The cholesterol esters combine with water to form
stable emulsions, and it is probably on account of the presence of these
esters that the substance known as lanolin is practically valuable. This
lanolin is made from purified wool fat and is largely used in the prepa-
ration of salves and ointments.

An isomeric substance known as isocholesterol is often found asso-
ciated with the true cholesterol, especially in wool fat. In the vege-
table kingdom other forms of cholesterol are widely distributed in
small quantities, being found in most oils and seeds. All forms of
cholesterol have a marked action on polarized light.

Experiment. If gall-stones are obtainable the following reactions may be car-
ried out in illustration of properties of cholesterol. Crush the stones to a powder
and boil with water to remove anything soluble. Extract the residue several times
with hot alcohol, filter, unite the solutions and allow the cholesterol to crystallize
on cooling. As some fat may be present this must be removed by saponifying with

5

50 PHYSIOLOGICAL CHEMISTRY.

a little alcoholic potassa solution. After saponification boil off the alcohol and
extract the dry residue with ether, in which soaps are insoluble. On evaporation
of the ether a nearly pure cholesterol is obtained. It may be further purified by
recrystallization from hot alcohol. With the substance these tests may be made:

Salkowski's Test. Dissolve about 10 milligrams of cholesterol in 2 cubic centi-
meters of chloroform and shake with an equal volume of strong sulphuric acid.
The chloroform becomes colored blood red, then cherry red and finally purple.
The acid shows a dark green fluorescence. If the chloroform is poured into a dish
the color changes to blue, then green and finally yellow.

Burchard-Liebermann Test. Dissolve about 10 milligrams of cholesterol in 2
cubic centimeters of chloroform, add 20 drops of acetic anhydride and 1 drop of
strong sulphuric acid. A violet pink color results.

The appearance of cholesterol plates should be studied under the microscope.

The presence of cholesterol in the ester form in lanolin and similar
preparations of wool fat may be shown by the above tests.

CHAPTER V.
THE PROTEIN SUBSTANCES.

These extremely important bodies, usually called albuminous bodies,
are found in vegetable and animal organisms of all kinds, and in some
form are essential elements in cell growth. Unlike the fats and carbo-
hydrates, they seem to be elaborated in the vegetable kingdom only;
or, at any rate, the fundamental structures in them appear to be formed
in vegetable growth only. The animal is able to modify and trans-
form to some extent, but apparently can not build them up from
simple materials. In composition the protein bodies are extremely
complex; qualitatively they contain carbon, hydrogen, oxygen, nitro-
gen and sulphur. In an important group of these bodies phosphorus
is also present, and a few contain iron. The quantitative composition
of some of the best known protein compounds is expressed approxi-
mately as follows :

Per Cent.

C 50.0-55.0

H 6.5- 7-3

O 19.0-23.0

N 15.0-17.0

S 0.3- 2.4

Attempts have been made to calculate formulas for certain protein
bodies from the results of analyses, but no great importance attaches
to the empirical formulas so reached. The best analyses made of the
compounds differ among themselves to an extent that makes a definite
result quite impossible. This is largely due to the fact that there are
great practical difficulties in the way of properly purifying the sub-
stances as a preliminary to analysis ; they all occur mixed with other
compounds, such as fats, carbohydrates and mineral matters, and to
remove these without in any way altering the composition of the
complex albuminous molecule is extremely difficult, if not impossible.
The formulas which have been published are interesting chiefly in
showing roughly how complex the structures certainly are. For serum
albumin TTofmeister has given this minimum formula:

V^4BoH J20-N 116^5»^-'n0!

while for t^ albumin he has given this:

51

52 PHYSIOLOGICAL CHEMISTRY.

Even more complex formulas are given, for example this :

C755.H.1215JN 195 "10*-' 235-

These formulas are in a measure based on an assumption as to the
number of sulphur atoms present, about which something will be said
later.

The usual methods of fixing organic formulas by aid of a molecular
weight determination can not be successfully applied in these cases.
In the cryoscopic method, for example, the traces of mineral im-
purities present have possibly as much influence on the result as the
whole weight of dry protein. Because of changes in composition at a
high temperature the boiling point method, even if otherwise reliable,
can not be applied, and methods based on osmotic pressure observations
lead to results of no value.

CLASSIFICATION OF THE PROTEIN BODIES.

The substances thus far studied have been divided into groups or
classes dependent on chemical composition or structure. With the
protein compounds this is only partially possible because of our lack
of full knowledge in this direction. Of the structural relations of the
protein molecules nothing whatever is known, while of composition
only a few general facts are clearly enough established to be available
in a scheme of classification. The first efforts at classification, which
we owe largely to the work of Hoppe-Seyler, were therefore essen-
tially empirical. Other schemes were later proposed as more facts
were brought to light, so that finally a grouping like the following
came to be gradually accepted by physiological chemists, with slight
differences in details only. The arrangement below is that of Ham-
marsten, in the form given by Cohnheim. He makes four principal
divisions as follows :

Protein Bodies

' True or Native Albumins.
Derived Albumins or Transformation

Products.
Proteids.
Albumoids.

THE PROTEIN SUBSTANCES.

53

These four great divisions may be further subdivided :

Albumins proper.

Serum albumin, egg albumin, lactalbumin.
Globulins.

Serum globulin, egg globulin, lactoglobulin, cell
globulin, vegetable globulin.
Coagulating albumins.

Fibrinogen, myosin, gluten protein.
Nucleoalbumins.

Casein, vitellins, mucin-like nucleoalbumins.
Histones.

Scomber-histone, salmo-histone.
Protamines.

Salmin, clupein, sturin, scombrin.

TRUE OR NATIVE
ALBUMINS.

DERIVED OR f Coagulated or Modified Albumin.
TRANSFORMATION J Acid and Alkali Albumins, Albuminates.
PRODUCTS. [ Albumoses, Peptones.

PROTEIDS.

Nucleoproteids.

Nucleic acid with histone, protamine, etc.
Hemoglobins.

Hematin with histone.
Glucoproteids.

Combination of a protein and carbohydrate group,
mucin, mucoids, phosphoglucoproteids.
Lecithoproteids.

Combination of a protein with a lecithin body.

ALBUMOIDS.

Collagen, forming gelatin, glue.
Keratin, in horn, hair, nails, etc.
Elastin, elastic tissue.
Amyloid, in pathological formations.
. Spongin, in sponge.

In recent years enormous additions have been made to the literature
of the proteins, and many new substances have been described. Our
knowledge of certain groups has been advanced largely through the
labors of the Yale school of chemists, and especially by Chittenden
and Osborne. Much of our systematic knowledge of vegetable pro-
teins must be credited to these investigators. In view of these im-
portant extensions of our acquaintance with the details of protein
chemistry a committee representing the American Society of Bio-
logical Chemists and the American Physiological Society has recom-
mended the following classification of the bodies in question. The
known substances are thrown into three main groups in place of four,
as above. The term proteid is abandoned.

54

PHYSIOLOGICAL CHEMISTRY.

SIMPLE PROTEINS

CONJUGATED PROTEINS.

' Albumins.
Globulins.
Glutelins.
- Alcohol-soluble Proteins.
Albumoids.
Histones.
Protamines.

Nucleoproteins.

Glycoproteins.

Phosphoproteins.

Hemoglobins.

Lecithoproteins.

DERIVED PROTEINS.

Primary Derivatives.

f Proteans.
-j Metaproteins.
[_ Coagulated proteins.

f Proteoses.
Secondary Derivatives. < Peptones.
(^ Peptides.

The differences in the two classifications are not great. The albu-
moids are here considered as simple proteins, which is probably an
advantage. The term phospho protein in the new classification covers
substances like the nude o albumins of the old.

GENERAL REACTIONS OF THE PROTEINS.

The various substances in the protein group respond to a number
of reactions which, taken together, are sufficient to characterize and
identify the bodies in question. They all contain nitrogen in a form
to be liberated as ammonia when the dry substance is heated with
soda-lime. A positive result with this test does not, of course, prove
the presence of a protein compound, since all ammonium salts and
amino compounds in general respond to it; but with a negative result
proteins as well as these other compounds are certainly excluded.
The reaction therefore serves as a preliminary test in the examina-
tion of unknown substances for proteins. The test may be easily
carried out and is delicate.

Experiment. Mix some dried albumin or some wheat flour with an equal bulk
of soda-lime in a dry test-tube. Apply heat and note the escape of ammoniacal
vapors as shown by the odor, or reaction on moist litmus paper. The fixed alkali
decomposes the protein matter very quickly, and ammonia always results.

COAGULATION TESTS.

Many of the protein substances undergo a peculiar change known
as coagulation when heated, or treated with certain reagents. The
test is characteristic of most of the bodies except some of the products

THE PROTEIN SUBSTANCES. 5 5

of transformation. This coagulation is usually accompanied by pre-
cipitation, that is, the body is thrown out of solution and as a rule
can not be restored to its original condition. But there are cases of
precipitation without coagulation; the terms must not, therefore, be
used as synonymous. In coagulation proper the protein body becomes
permanently altered, so that it can not be brought into its original
condition again by addition of reagents or by other means. On the
other hand, it is in many cases possible to throw a protein body out of
solution by simply adding an excess of some inorganic salt, without
at the same time producing any decided alteration in the character of
the protein precipitate. By largely diluting with water the precipitate
may be brought into the soluble condition again. This will be illus-
trated later by the use of ammonium sulphate which behaves in a
characteristic manner with different proteins. Many of these have
definite precipitation limits with the sulphate. That is, they begin
to separate when the amount of the salt reaches a certain value, and
precipitate completely with a greater concentration.

Experiment. The simplest coagulation may be shown by boiling a dilute white
of egg solution. As long as it is perfectly neutral coagulation follows at once,
but in presence of alkali acid must be added to the point of neutrality. This
behavior finds practical application in the ordinary test for serum albumin as it
occurs pathologically in urine. The precipitate may be redissolved only by some
digestion or chemical process which produces a new substance.

Coagulation by Reagents. By the addition of certain chemicals
many of the protein compounds are easily thrown into the coagulated
condition. Some of the most characteristic reactions in this direction
are shown by simple experiments with mineral acids, salts of heavy
metals and the so-called alkaloid reagents.

Experiment. Among the acids which bring about coagulation, nitric acid is the
most certain in its action and is commonly used in practical cases where it is
desired to recognize a small amount of serum or egg albumin in solution. The
test may be made by adding about a cubic centimeter of strong nitric acid to four
or five cubic centimeters of white of egg solution and warming. Coagulation fol-
lows at once. With a very dilute albumin solution the substance separates in
flakes, while with a strong solution a stiff, jelly-like mass may result. The test
is a common one in urine analysis, but must be conducted with certain precautions.

Experiment. Solutions of most protein substances are precipitated by addition
of alcohol in excess. This may be shown by mixing white of egg solution with
strong alcohol, the latter being added gradually until the maximum of precipitate
is obtained. With dilute alcohol precipitates are usually not secured, and besides
the alcohol precipitation is usually not a permanent coagulation as in the above
case with the acid.

Experiment. Precipitation by Salts. Some of the salts of heavy metals give
characteristic precipitates with protein solutions. The behavior may be illustrated
by adding to dilute egg albumin solution small amounts of solution of mercuric

56 PHYSIOLOGICAL CHEMISTRY.

chloride, lead acetate, copper sulphate or ferric chloride. The reagents must not
be added in excess, as in some cases this causes a resolution of the precipitate.
Similar reactions may be obtained with solutions of most of the heavy metals,
but the salts mentioned are often used in practice. The behavior of mercuric
chloride or corrosive sublimate as an active disinfecting agent depends on its
property of coagulating the protein matter of the pathogenic bacteria, to destroy
which it is used. The precipitates may be formed in neutral, acid or alkaline
solution as a rule, and chemically may be regarded as salts of the metals used as
precipitants.

Experiment. Precipitation by Alkaloid Reagents. In acid solution the pro-
tein bodies are very generally precipitated by addition of solutions of picric acid,
tannic acid, potassium-mercuric iodide, phosphomolybdic acid and other reagents
employed in the detection of alkaloids. The precipitates are voluminous, and in
most cases complete in presence of sufficient acid.

White of egg, much diluted, may be used in illustration.

The above reactions may be explained on the assumption that the
proteins here act as pseudo-acids or pseudo-bases. In perfectly pure
solution they are neutral and indifferent to some indicators, but the
addition of a mineral acid imparts to them the character of pseudo-
ammonium bases which yield precipitates as the alkaloids do under
like circumstances. On the other hand, with salt solutions they be-
come pseudo-acids and form now insoluble precipitates of complex
salts. But it has been shown that while some of the proteins may be
neutral to litmus they may at the same time be quite distinctly acid to
phenolphthalein, and require a decided amount of decinormal alkali
solution to give a reaction by displacing the acid in combination with
them. This is taken to indicate that they should be looked upon as
true bases, rather than as pseudo-bases. The amount of acid which
may unite with certain proteins has recently been found with con-
siderable accuracy, and becomes in some degree a measure of the
basicity. In other cases it may be shown that they have a true rather
than a pseudo-acid character and unite with alkali to form real salts.

Behavior with Millon's Reagent. In this we have one of the most
characteristic reactions of the protein bodies. Millon's reagent is made
by dissolving mercury in twice its weight of strong nitric acid, com-
pleting the reaction by heat. The solution obtained is diluted with
twice its volume of water. It contains a little nitrous acid. When
warmed with white of egg and other proteins it imparts a deep red
color to the coagulum produced and often to the containing solution.
The reaction is common to benzene derivatives which have a hydroxyl
group attached to the nucleus, and is given by phenol for example.
The reaction in the protein substances is due to the presence of the
tyrosine group in the complex molecule. This group seems to be
present in all protein bodies with the exception of gelatin, so that the

THE PROTEIN SUBSTANCES. 5 7

reaction is a nearly universal one. The protein derivatives which still
contain the tyrosine complex likewise show the reaction. Tyrosine is
represented by the formula C6H4OH.CH2.CHNH2.COOH, and will
be referred to later, as it is an important decomposition product of
proteins.

Experiment. Test the behavior of Millon's reagent by adding some to white of
egg solution, milk or flour, and applying heat. The characteristic color appears
almost immediately. Its depth depends somewhat on the concentration of the
protein substance used. Presence of much salt interferes with the test or may
even prevent the reaction.

Experiment. Apply the same test to weak solutions of phenol, salicylic acid and
thymol. Note the color and character of the reaction. These bodies all have a
benzene nucleus with hydroxyl combination. If pure tyrosine is available a very
dilute solution may be employed for tests. It is said that I part in iooo of water
gives a distinct reaction. Hydroquinol, resorcinol and a- and /3-naphthol give like-
wise decided reactions, but the colors are orange yellow.

The Biuret Reaction. This, like the above, is a protein test de-
pending on the presence of certain groups in the complex molecule.
When biuret and several substances of related composition are mixed
with an excess of alkali solution, either sodium or potassium hydrox-
ide, and then a few drops of a weak copper sulphate solution are
added, a blue-violet to reddish-violet color is produced. The shade
depends on the concentration of the solution, and on the composition
of the reacting group. It has been shown that the reaction seems to
follow with compounds which contain two groups — CONH2 directly
united or joined by a carbon or nitrogen atom, as for example :

CONH, yCONH, /CONH. CO • CONH2

| HN( H2C< " I

CONfL XCONH2 NCONH2 NH • CONH2

Oxamide Biuret Malondiamide Oxaluramide

The combination of copper and alkali with these bodies has been
recently studied and formulas determined. If, in place of using a
solution of one of these compounds, some white of egg, or other
protein solution, is employed the same color appears. The absorption
spectra from pure biuret and egg albumin solution, treated in this
manner, are the same, which shows that the albumin must split off this
group under the influence of the alkali used. The reaction is one of
extreme delicacy and may be employed for the recognition of traces
of protein compounds. It is used especially in the detection of peptone,
one of the derived protein substances. Derivatives of simpler nature,
that is, the products of the decomposition of proteins, do not give the
reaction. It is therefore of value in following the course of experi-
ments on the digestion or hydrolysis of proteins, as the reaction dis-
appears with the breaking flown of the last protein complex.

58 PHYSIOLOGICAL CHEMISTRY.

Nickel salts exhibit an analogous behavior, but show orange yellow
colors. Cobalt solutions give reddish colors, but not very strong.

Experiment. Prepare a dilute white of egg solution and add to 5 cc. of it some
solution of potassium or sodium hydroxide. Then add one or two drops of weak
copper sulphate solution, or enough to impart the characteristic color. An excess
of the copper yields a precipitate and must be avoided. The reaction is much
sharper with albumose and peptone derivatives than with the original native pro-
tein. Repeat the test with solution of nickel sulphate. The test is obscured by the
presence of ammonium salts, which is sometimes a matter of importance in prac-
tical work.

The a-Naphthol Test of Molisch. In the chapter on the sugars
it was shown that a very marked color reaction is given by mixing a
few drops of a weak alcoholic solution of a-naphthol with the sugar
solution and then adding some strong sulphuric acid. The same be-
havior is shown by solutions of some protein substances, which indi-
cates that they must contain a carbohydrate group of some kind. The
reaction depends on the formation of furfuraldehyde by the decompo-
sition of the sugar by the strong acid. This furfuraldehyde combines
then with the a-naphthol to produce a deep violet color, the reaction
being similar to that between furfuraldehyde and aniline acetate de-
scribed in the pentose test in a former chapter.

Experiment. To a few cubic centimeters of white of egg solution add five drops
of 10 per cent, solution of a-naphthol in alcohol. Then carefully add three or
four cubic centimeters of strong sulphuric acid, which sinks below the lighter solu-
tion. Note the color at the zone of contact and throughout the liquid on shaking.
Alkalies change the color to yellow. Thymol solution is sometimes used instead
of a-naphthol. This gives a deep red color. These furfuraldehyde reactions are
extremely delicate, and appear in a great variety of tests. Their general character
and importance should therefore be recognized.

The Xanthoproteic Reaction. This is a delicate test, depending
on the formation of yellow nitro derivatives of the phenol groups
in the protein complex. Similar reactions are given by many simpler
organic substances where nitric acid is mixed with them and heat
applied. The color produced by nitric acid in contact with the skin
is due to the same general reaction.

Experiment. In illustration, add some strong nitric acid to white of egg solu-
tion. On application of heat the yellow color appears. By neutralizing with
ammonia the color changes to orange yellow.

Make a similar test by warming some phenol solution with nitric acid. In this
case a nitro-phenol is formed. Pure phenol and strong nitric acid, it will be
recalled, yield trinitrophenol or picric acid, C6H2-OH- (N02)3 Add ammonia to
neutralize, as before.

The Lead Hydroxide Test. The protein bodies contain sulphur
which may be removed by action of an alkaline lead solution, or

THE PROTEIN SUBSTANCES. 59

alkaline bismuth solution or mixture. The second reaction has some
importance, as it is the source of a fallacy in the so-called bismuth test
for sugar in urine. In presence of albumin, sulphide of bismuth is
formed in place of the reduction product indicative of sugar. As all
protein bodies contain sulphur the test is a general one. It may be
made as follows :

Experiment. Produce first a soluble alkaline compound of lead by adding to a
few cubic centimeters of lead acetate solution enough strong alkali, sodium or
potassium hydroxide, to form a precipitate and redissolve it. Then add the protein
substance, white of egg for example, and boil. A brown or black color appears
and sometimes even a precipitate of black lead sulphide. Only a part of the sul-
phur, however, may be separated in this simple manner. Another portion seems
to be much more firmly combined in the protein molecule.

The reactions which have just been explained are the most impor-
tant and characteristic of all which have been suggested for the recog-
nition and identification of the proteins. Numerous other reactions
are known, however, which are easily observed. Several of these are
color tests, depending on the formation and combination of furfur-
aldehyde, but they need not be described as in principle they do not
differ much from the Molisch test.

QUANTITATIVE DETERMINATION OF PROTEINS.

The above tests serve for the recognition of proteins but not for
their determination, and for the latter purpose it may be said further
that no one method is perfectly suited to all cases. Many of the
simpler protein bodies are determined by complete coagulation, fol-
lowed by weighing of the precipitate formed. This involves several
operations, all of which must be very carefully performed. For
example, a pure native albumin in solution may be coagulated by
adding a few drops of acetic acid and boiling thoroughly. The coagu-
lum is collected on a weighed paper filter or in a Gooch funnel, thor-
oughly washed, dried and weighed. Instead of drying and weighing
the precipitate it may be decomposed according to the Kjeldahl process,
in which the nitrogen is converted into ammonia by digestion with
sulphuric acid. The ammonia is easily separated and measured. The
nitrogen is 14/17 of it. By multiplying the nitrogen found by the
factor 6.25 we obtain the original protein content on the assumption
that these bodies contain 16 per cent of nitrogen. This method is
now commonly followed in the determination of crude protein for
many technical and scientific purposes. But in many cases an error is
naturally introduced because of the uncertainty of the factor; 6.25
is the mean value for the native proteins and the closely related bodies.

60 PHYSIOLOGICAL CHEMISTRY.

COMPONENT GROUPS IN THE PROTEIN COMPLEX.

In the study of the protein molecule as a whole, a limit is soon
reached in any attempt to fix its composition, but much may be learned
by observing the various products formed in reactions by which the
molecule is broken down under the influence of different agents.
Some of these reactions are apparently largely hydrolytic in character,
and in a degree may be compared to the decomposition of a fat by
superheated steam. In this very simple case glycerol and fatty acids
are obtained and we conclude that they were not actually formed in
the process, but that they were present in combination in the original
fat. In treating protein bodies in a similar manner or in subjecting
them to the decomposing influences of acids or alkalies, a number of
products are formed. These must be either results of peculiar disinte-
gration and subsequent synthesis, or they must represent groups in
some way existent in the original complex. The latter view is
strengthened by the fact long observed that certain products result,
whatever the method of decomposition. Leucine, for example, is
found abundantly among the products liberated by subjecting protein
to the action of superheated steam, hot hydrochloric, nitric or dilute
sulphuric acid, concentrated alkali solutions, bromine water under
pressure, or to prolonged pancreatic digestion. The almost necessary
conclusion must be that in these varied reactions the leucine could not
have formed from smaller disintegration groups, but must have been
set free from something holding it in the protein complex.

The decomposition reactions are therefore considered very impor-
tant as suggesting probably the component groups in the large mole-
cule. In the following pages a few of the most important of these
reactions and their products will be described.

Decomposition by Steam under Pressure. By prolonged heating
of protein substances with water certain changes take place, even
below a temperature of ioo° C. Following the coagulation, which
appears in most cases, a gradual hydration and solution begins, and a
small portion of the substance is brought into the form of albumose or
possibly peptone. At a higher temperature, that is, by heating with
water or steam under pressure, more profound changes take place.
Ammonia and hydrogen sulphide are split off from the molecule and
relatively large amounts of albumose and peptone are formed. If the
temperature is high enough the reaction extends to the complete de-
struction of the molecule and such bodies as leucine and tyrosine are
produced in quantity.

THE PROTEIN SUBSTANCES. 6 1

Effect of Alkalies. Much more decided changes are noticed when
the protein body is heated with alkali solutions. Experiments of this
kind were long ago carried out by Schiitzenberger and have since been
frequently repeated. Numerous compounds have been identified
among the decomposition products, and of these the most important
are leucine in quantity, tyrosine, ammonia, carbonic acid, butyric
acid, formic acid, acetic acid, oxalic acid, aspartic acid, amino-valeric
and amino-butyric acid. Barium, potassium and sodium hydroxide
solutions have been used for the purpose. By melting the dry pro-
teins with alkali some of the same products are formed, especially
leucine and tyrosine.

Effect of Acids. Many experiments have been made on the de-
composition of protein bodies by boiling with acids, and particularly
with strong hydrochloric acid, the hydrolyzing power of which is
very great. The most important of the products isolated in this way
are the following:

The Hexone Bases. The term hexone bases has been given by
Kossel to a group of bodies which occur commonly in the decomposi-
tion products of practically all the protein substances. We have here
arginine, C6H14N402, lysine, C6H14N202, and histidine, C6H9N302.
The first appears to be a guanidine derivative of amino-valeric acid,
the second is diamino-caproic acid, while the third appears to be a
diamine acid of composition as yet unknown, or is, possibly, an imino-
azol derivative of amino-propionic acid. The isolation of these com-
pounds was a very important step in the direction of clearing up the
constitution of the proteins, inasmuch as some of the simplest of these
bodies, the protamines, seem to consist almost wholly of the hexones.
More will be said about this relation later. The hexones are soluble,
crystalline, optically active, compounds, and because of their wide
occurrence have been very thoroughly studied. They contain the
amino group in the a position, and in this respect resemble the other
common disintegration products. All the a amino acids appear to
have a sweetish taste, which is illustrated by the first of the products
to follow.

Glycocoll or Glycine, C2H5N02, amino-acetic acid. Obtained
abundantly from gelatin and also from a few other proteins. It is
very soluble in water to which it imparts a sweetish taste, and is
insoluble in alcohol or ether. From a theoretical standpoint the im-
portance of glycine is very great, as it is the starting point in various
syntheses, to be explained later. It also combines with benzoic acid

62

PHYSIOLOGICAL CHEMISTRY.

to form hippuric acid in the body metabolism. Hippuric acid is
benzoyl glycine.

Amino Propionic Acid, or Alanine, C3H7N02. This is in a
sense the nucleus substance corresponding to tyrosine and phenyl-
alanine, to be referred to. It is a soluble product rather widely dis-
tributed in protein bodies, and because of this marked solubility is
hard to isolate.

Amino Valeric Acid, C5H11N02. This substance is apparently
the a product. It is usually mixed with leucine and is separated only
with difficulty from this body. Combined with guanidine,
NHC(NH2)2, it yields the important arginine, referred to above.
Diamino valeric acid is known as ornithine.

Leucine, C6H13N02, a-amino-caproic acid, or a-amino-isobutyl-
acetic. acid. This has been already mentioned as found abundantly
among the products of protein decomposition by other agencies. In
some cases it appears to constitute 30 per cent of the reaction products,
and must therefore play a very important part in the original complex
molecule. Leucine is found in several different forms; the common
product obtained by acid hydrolysis is right rotating and shows in
hydrochloric acid solution [a]D = -f- 18. 90. In water solution it
rotates in the opposite direction.

Serine, C3H7NOs, amino-hydroxy-propionic acid, was first discov-
ered in silk, and hence the name. But it is present in many of the
protein bodies as well, although in not very large amount.

Aspartic Acid, C4H7N04, amino-succinic acid. Slightly soluble
in water, the solutions being apparently left rotating at the ordinary
temperature. But in hydrochloric acid it is strongly right rotating.
While the acid is found commonly in proteins the amounts are not
large.

Glutaminic Acid, C5H9N04, a-aminoglutaric acid. The acid is
found in several optical modifications. The common form is but
slightly soluble in water and is right rotating. In hydrochloric acid
the right rotation is marked. This acid is obtainable in considerable
quantities from many proteins, and is one of the extremely important
constituent groups. It is probably more abundant than leucine.

Proline, C5H9N02, (C4H7.NHCOOH), a-pyrrolidine carboxylic
acid. From the conditions under which it has been found this inter-
esting body is supposed to be a primary product. It is an imino not an
amino derivative. It was first obtained from casein and later from
other proteins. It has a very sweet taste and a high left rotation. The

THE PROTEIN SUBSTANCES. 63

closely related hydroxy-a-pyrrolidine carboxylic acid has been obtained
from gelatin.

Tyrosine, CgHuNOs, />-oxyphenyl amino-propionic acid. This
appears to be a component part of all the common proteins, with the
exception of gelatin, and from some of them has been obtained in
quantity. As already mentioned this is probably the substance which
reacts commonly with Millon's reagent. Tyrosine is but slightly
soluble in water, from which it crystallizes in bundles of fine needles.
Its solutions are optically active. In presence of hydrochloric acid
[a]D= — 8° to — 150, the rotation varying with the amount of acid.

Phenylalanine, CgHuNOg, phenylamino propionic acid. This
substance resembles tyrosine closely in structure and behavior and is
a common product of protein decomposition. It is slightly soluble
in water and has a sweetish taste. It appears to be present in many
cases when tyrosine is lacking, and must be considered as a very
important decomposition product.

Tryptophane, CnH12N202, indol-amino-propionic acid. This
complex product has been obtained from several of the proteins, and
probably occurs in most of them. Many of the peculiar color reactions
of proteins are due to the small amount of tryptophane present. The
most characteristic of these color reactions is given by the addition of
bromine water to a liquid containing the substance. A marked violet
color results. This is shown well in advanced stages of the tryptic
digestion of proteins. More will be said about the body later.

From the above list of decomposition products it will be seen that
the comparatively simple amino acids predominate; most of them,
possibly all, are the a compounds, and as will be shown below, they
make up a very large portion of the whole protein molecule.

Glucosamine, C6Hn05(NH2). This appears to be an important
constituent in some groups of protein bodies. It has been obtained in
quantity from the glucoproteids and is possibly present in small
amount in all. It is usually obtained as a salt, hydrochloride or hydro-
bromide, which is readily soluble in water and optically active. It is
regarded, usually, as a secondary product of dissociation.

Ammonia, XH,. This is always found in relatively large amount,
but in the main may be a secondary product.

Sulphur Compounds. Hydrogen sulphide, ethyl sulphide, thio-
lactic acid, C3H6S02, cystin, C0Hi2N2S2O4, and traces of other bodies
which contain sulphur have been identified in small amount among
the decomposition products. Of these sulphur compounds cystin is

64 PHYSIOLOGICAL CHEMISTRY.

the most important. It exists in two isomeric forms, one of which is
found in certain calculi.

Carbonic Acid is apparently a constant derivative, but may appear
as a result of some secondary reaction. Its significance, therefore, is
obscure.

With acids other than hydrochloric very similar reaction products
are secured. It will be shown below that the effects of prolonged
tryptic digestion are very nearly the same as observed with hydro-
chloric acid. This is a point of the highest practical importance, as it
gives us some insight into the complex physiological process. And
in peptic digestion also, where very weak hydrochloric acid and pep-
sin are employed, essentially the same products result provided the
time of the action be made sufficiently long.

RESULTANT CHARACTER OF THE PROTEIN MOLECULE.

While the various decompositions detailed above give us some in-
sight into the number and kind of groups combined in the protein com-
plex, they do not, unfortunately, show us much as to the manner in
which these groups are combined. We are not, as yet, able to picture
to ourselves a large molecule in which the leucine, tyrosine, aspartic
acid, glycocoll, and so on, are united to form a molecule with the
general properties and molecular weight as large as we assign to even
the simplest proteins, but a step has been made in that direction
through the synthesis of various polypeptides carried out by Fischer
and Curtius, who have succeeded in condensing several amino acids
into one molecule with certain properties suggesting those of the
peptones. These bodies will be referred to in a later chapter. Hof-
meister has suggested the possibility of the combination of amino
acids in large groups by the following general scheme :

— NHCHCO — NHCHCO — NHCHCO — NHCHCO —

I I I I

CH2 CH2 CH2 (CH2)3

CH3-CH-CH3 C,HtOH COOH CH2NH2

Leucine Tyrosine Aspartic Acid Lysine

The recognition of the various component groups suggests some
reasons why the proteins may exhibit acid and basic behavior at the
same time. Of most of these protein compounds the basic character
is the more pronounced and more readily observed ; that is, their acid
combining power. Some writers consider these bodies as so-called
pseud o bases and pseudo acids, because of the very peculiar manner
in which they unite with acids and bases. But several investigations

THE PROTEIN SUBSTANCES. 6$

of the last few years indicate that they are more properly true bases
and acids, but so weak in their combinations that hydrolysis follows
very readily. This hydrolysis obscures the reactions which must take
place in the formation of salts. In aqueous solutions the pure proteins
and the component amino acids are practically non-electrolytes, which
has been explained on the assumption that the basic part of one group
is linked to the acid part of another, with little or no dissociation.
Possibly, also, a ring-like structure is formed by a kind of internal
saturation. By various methods it may be shown that the simple pro-
teins and the amino acids combine in rather definite proportions with
the inorganic acids and bases, as will be pointed out in later chapters.
But the salts so formed suffer marked hydrolytic dissociation and
conduct the electric current essentially as the acid or base used. Gly-
cocoll hydrochloride, for example, CH2NH2.HC1 — COOH, hydro-
lyzes so as to leave free hydrochloric acid, while sodium glycocollate,
CH2NH2 — COONa, hydrolyzes to yield sodium hydroxide, and the
conductivity observed is due to this latter essentially. With casein,
which is a protein easily obtainable in pure condition from milk, the
phenomena of salt formation both with acids and bases may be very
easily observed. Something will be said about this later. One mole-
cule of casein combines, apparently, with four or five molecules of
sodium hydroxide to form a salt. In their basic capacity serum and
egg albumins combine with a large number of molecules of hydro-
chloric acid.

The presence of sulphur in the proteins was shown by a test referred to some
pages back. Investigations have shown that sulphur is present in at least two kinds
of combinations in the protein complex; there must be at least two sulphur atoms
in the molecule. Some of the sulphur is easily separated by hot alkali solutions,
while the rest of it is not. No part of this element appears to be combined in oxi-
dized form, that is, in the condition of a sulphite or sulphate. The sulphur com-
pounds which have been obtained in protein decomposition are such as may be
derived from a breaking down of the cystin group. It has been shown that cystin
gives up its sulphur very slowly to boiling alkali, and only in part as sulphide.

The general reactions and characteristics of the protein bodies hav-
ing been discussed, a brief description of the more important indi-
vidual substances will now follow.

TRUE OR NATIVE ALBUMINS.

In the scheme of classification given some pages back the true or
native albumins have the first place. The best known representatives
of the protein group are included here.
6

66 PHYSIOLOGICAL CHEMISTRY.

ALBUMINS PROPER.

These bodies are characterized by solubility in water and in weak
cold acid or alkali solutions. They are readily coagulated by heat
and by shaking with strong alcohol. Although usually considered as
amorphous, the albumins have been obtained in well crystallized form,

The characteristic color reactions previously referred to are all
given by the true albumins and they are precipitated by ammonium
sulphate or zinc sulphate added to saturation. With strong sodium
chloride precipitation follows only after addition of acid.

Serum Albumin. This is the important protein body of blood
serum, of which it constitutes three to four per cent by weight, the
related substance, serum globulin, making up nearly as much. While
closely resembling each other, it is not definitely known that the serum
albumins of different animals are identical. In fact, certain reactions
to be referred to later suggest peculiar points of difference. In blood,
the albumins are associated with globulins, fibrinogen, mucoids, salts
and other bodies, the perfect separation of which is practically im-
possible. The purification of serum albumin by crystallization is not
easily carried out with all blood serums; in some cases the formation
of crystals is slow and incomplete.

Serum albumin contains a relatively large amount of sulphur, about
two per cent in the mean, and is characterized further by a high
specific rotation. The values which have been given for this are not
constant, but in the mean are about [a]D= — 6o°.

Crude serum albumin is now an article of commerce, being made in
large quantities from blood collected at the slaughtering houses. It
is usually mixed with globulin, and besides is partly insoluble because
of the high temperature employed in drying it. For the following
experiments fresh blood must be used.

Experiment. Collect blood in a clean vessel and stir it thoroughly to separate
the fibrin and part of the corpuscles, as a clot. Some of the corpuscles, however,
remain with the serum and may be separated by allowing the latter to stand in a
tall, narrow jar, or better, by rotating the serum in a centrifugal machine. Most
of the corpuscles may be deposited in this way, leaving a yellowish liquid. A pure
white serum can not be obtained because a little of the hemoglobin dissolves from
the corpuscles and remains in solution. With this prepared serum make the fol-
lowing tests :

Experiment. To a little of the serum add finely powdered magnesium sulphate
to saturation ; this produces a precipitation of serum globulin, which separates on
standing. Pour off the clear liquid and add to it powdered ammonium sulphate,
which gives now a precipitate of albumin.

Experiment. Mix a little of the serum with two or three volumes of water in
a test-tube, and test the temperature of coagulation. It will be found near jo° C.

THE PROTEIN SUBSTANCES.

67

Lactalbumin. Milk contains two protein substances, the most
important of which is casein. The other is a true albumin which is
present to the extent of about one-half per cent in cow's milk. It
resembles serum albumin very closely but appears to have a much
lower specific rotation, [a]D — — 380.

Egg Albumin. White of egg contains this body as its character-
istic constituent along with some globulin and mucoid, and traces of
salts. Common albumin reactions are usually made with white of egg
solution. Although this substance is
always described as a true albumin,
some of its reactions seem to suggest
that it may belong to the group of
glucoproteids, or, at any rate, may
contain such a compound in rela-
tively large amount. On heating egg
albumin with weak acid glucosamine
is split off and in quantity sufficient
to indicate a rather large sugar con-
tent in the original substance. The
specific rotation is much lower than
that of serum albumin and may be
taken at [a]D = — 380, as for milk
albumin. Besides this difference, egg
albumin has a much lower coagulat-
ing temperature than has been given
for serum albumin, viz., 560. Egg
albumin is much more easily coagu-
lated by ether than is serum albumin.
Egg albumin becomes very quickly
insoluble when mixed with strong
alcohol. From serum albumin it differs, further, by this interesting
property. When its solution is injected into the blood circulation it
passes unchanged through the kidneys into the urine ; the same thing
happens when large quantities of white of egg are eaten. It seems to
escape digestion in this latter case and be absorbed in pure condition, to
be later discarded by the kidneys. These various points of behavior
indicate, then, a rather marked difference between the two kinds of
albumin.

Experiment. So-called pure egg albumin may be obtained in this way : The
white of egg is shaken in a bottle with some broken glass to thoroughly break up
the membranes. The foamy mass js filtered through fine, unsized muslin, and to

Fig. 8. Typical form of Graham
dialyzer frequently used in purifica-
tion of proteins. The substance to be
purified is placed in the cell a, which
has a parchment bottom and floats on
water in the large vessel b. The simple
parchment tube dialyzers now obtain-
able are more efficient.

68 PHYSIOLOGICAL CHEMISTRY.

the filtrate an equal volume of saturated ammonium sulphate solution is added.
This produces a precipitate of globulin which after 24 hours is filtered off. Am-
monium sulphate in this strength does not precipitate the true albumin. To this
filtrate a little more saturated ammonium sulphate is added and until a precipitate
or turbidity just begins to show. This is caused to disappear by the cautious
addition of water, a few drops at a time. Finally, acetic acid of ten per cent
strength saturated with ammonium sulphate is added until a turbidity again appears,
and then the mixture is allowed to stand 24 hours in a cool place. A part of the
albumin separates in the crystalline form. This is collected, redissolved in a very
little cold water and reprecipitated with ammonium sulphate and acetic acid as
before. The crystals are collected on a filter, then transferred to a dialyzer with
water for the separation of the sulphate by dialysis. In this way a nearly pure
albumin may be obtained in solution, but the crystallized substance has not been
secured free from salts.

White of egg contains in the mean about 86 per cent of water, 13
per cent of proteins, 0.6 per cent of mineral matters and a little fat.
The yellow of egg is a substance of very different composition. The
water present amounts to about 50 per cent, the proteins to 16 per
cent, the fat to 30 per cent, or more, while the ash is about 1 per cent.
The fat contains a notable quantity of lecithin.

GLOBULINS.

The proteins of this group differ from the albumins mainly with
respect to solubility in water. In pure water they are practically
insoluble, but they dissolve in moderately dilute salt solutions. On
diluting a globulin solution of this kind precipitation follows. Globu-
lin solutions coagulate by heat in much the same manner as observed
with albumins, but in general they become permanently insoluble
even more readily than do the albumins.

The preparation of pure globulin is even more difficult than the
preparation of pure albumin. The globulin must first be separated
by precipitation with some salt ; as the salt is later removed by dialysis
the globulin remaining becomes insoluble, which makes further treat-
ment difficult. Globulins are not well known in crystalline condition.

Serum Globulin. This substance makes up a large fraction of
the protein in blood serum, amounting to nearly as much as the serum
albumin. For a long time it was confounded with the latter, and it
was only after a lengthy series of investigations by different chemists
that its true nature was recognized. This globulin may be discovered
easily in the serum when the latter body is diluted with water, but the
separation is never quite complete by the water treatment alone, as a
portion always remains in solution. By salting out with ammonium
sulphate to half saturation, or with magnesium sulphate completely,
the desired end is reached.

THE PROTEIN SUBSTANCES. 69

The coagulation temperature of serum globulin is given as 75 ° and
the specific rotation as [a]fl = — 4§°> but these numbers are some-
what uncertain, especially the latter.

Bence-Jones Proteid. This is the name given to a substance oc-
casionally found in pathological urines, and which has usually been
considered an albumose. When purified, however, it has been found
to have the properties of a globulin. It may be held in solution in the
urine by the salts present.

Other Globulins. Several other bodies are described as globulins.
The most important of these is the so-called cell globulin, which is
possibly identical with serum globulin. This substance has been ob-
tained from different organs, from the liver, from the pancreas, from
muscle plasma, etc. Some of the globulins described as cell globulins
have a lower coagulating temperature than the true serum globulin.

In the crystalline lens of the eye a body has been long known which
is called crystallin. In coagulation temperature and specific rotation
this crystallin appears distinct from serum globulin, and further, it
seems to be made up of two related substances, a and /? crystallins.

Globulins have been described in milk and egg and also in the
vegetable kingdom under the name of phy 'to globulins or phytovitellins.
This last designation indicates that' they may be classed under the
head of the nucleo-albumins, with which bodies they have much in
common. Among the best known of these bodies we have the abun-
dant protein called edestin.

Edestin. According to Osborne this is a true globulin, and of
the vegetable products of this class has been among the most thor-
oughly studied. It has been obtained from many seeds and nuts, but
most readily from hemp seed. On analysis it shows, in the mean,
about 18.7 per cent of nitrogen, and 0.9 per cent of sulphur. Its
specific rotation is about — 440. Edestin can be secured in the crys-
talline condition, which has facilitated greatly its study. When hemp
seed meal is extracted with sodium chloride solution and this is fol-
lowed by dialysis or sharp cooling a portion of the edestin separates
in the crystalline form. The name edestan is given to a slightly hy-
drolyzed form of the original substance. The ending an is employed
in describing primary protein derivatives, formed by the action of
water or weak acids. These protcans are insoluble in salt solution, as
well as in water.

COAGULATING PROTEINS.

Several extremely important substances belong in this group, which,
like fibrinogen, have the property of spontaneous coagulation. In

70 PHYSIOLOGICAL CHEMISTRY.

nature they exist normally in the soluble and dissolved form, from
which, under certain influences, not always well understood, they pass
to the solidified condition. This coagulation is a different thing from
that produced by heating to a high temperature or by the addition
of reagents ; the changes in the latter case seem to be more profound.
We use in English the term coagulation to describe both classes of
alterations, which are really of a very different character, as will
appear from what follows.

Fibrinogen. Blood contains a peculiar protein body in small
amount, to which it owes its property of spontaneous coagulation.
This body is called fibrinogen and the product of coagulation is known
as fibrin. The nature of and important factors in this change have
been long subjects of investigation and discussion; it can not be said
that the matter has been fully explained in all its bearings. The essen-
tial points of what is known will be given in the chapter on the blood.

As a chemical substance fibrinogen is not known in perfectly pure
condition, since to hold it in soluble form various agents must be
added to the blood. But the fibrin formed, doubtless through ferment
action, is easily obtained and its properties are well established. As
usually prepared it is a white, elastic, stringy mass, insoluble in water,
but somewhat soluble in salt solutions. Like other proteins it under-
goes true coagulation through elevation of temperature or action of
various reagents. Fibrinogen, as prepared by salting out from plasma
at a low temperature, coagulates when warmed to 560. Its specific
rotation has been found only in presence of salt or alkali and varies
from [a]I) = — 360 to — 53 ° according to the nature of the admix-
ture or method of preparation. It undergoes digestion with the body
ferments very readily and has therefore often been used as a starting
point in digestion experiments.

Myosin and Myogen. The living muscle plasma contains a num-
ber of protein substances, one of which, at least, possesses the prop-
erty of spontaneous coagulation as observed in the solidification of the
muscle after death. At one time the term myosin was applied to this
body and it was supposed to be very simple in nature. Numerous
investigations, however, have shown that the chemistry of the muscle
proteins is comparatively complex and that the results of experiments
do not well agree. In the older sense this myosin was assumed to be
derived from a preexisting body, myosinogen, in the living muscle,
much as fibrin is considered as derived from fibrinogen. The solidi-
fied myosin behaves as a globulin, which may be illustrated by the
following experiment :

THE PROTEIN SUBSTANCES. 7 1

Experiment. Free muscle (round steak) as far as possible from traces of fat
and sinews, and then thoroughly disintegrate it by passing through a sausage mill.
Then wash it repeatedly with cold water until the latter is no longer reddened, and
the residue appears white. This is placed in a ten per cent solution of ammonium
chloride and allowed to remain about a day, with occasional shaking. Myosin
dissolves in the ammonium chloride and is found in the filtrate when the mixture
is filtered. Pour the filtrate into twenty times its volume of distilled water, which
causes a precipitation of the insoluble myosin. Allow to settle and wash three
times by decantation. Collect the precipitate and observe that portions of it dis-
solve readily in ten per cent solutions of sodium chloride and ammonium chloride,
or in a o.i per cent solution of hydrochloric acid. The solution in salt is pre-
cipitated by the addition of more to saturation.

By this treatment with the dilute ammonium chloride solution nearly
all of the protein of the muscle plasma may be removed, leaving the
stroma. It is now pretty generally recognized that this solution con-
tains two substances instead of one. The first of these is still called
myosin, and is said to make up about 20 per cent of the plasma protein,
while the name myogen is given to the other, constituting 80 per cent
of the soluble protein. Myosin is the part of the plasma which co-
agulates or solidifies the most readily and may be separated from the
plasma by adding ammonium sulphate to make 28 per cent of the
solution. On filtering, the myogen may be separated by adding am-
monium sulphate nearly to complete saturation. The coagulation
temperature of myosin is given as 470, while that of myogen is 560.
The former becomes quickly insoluble on addition of alcohol, while
myogen seems to be partly soluble in alcohol. Myosin-fibrin and
myogen-fibrin are the names given to the coagulated forms of these
bodies. More will be said of these relations when we come to con-
sider the muscular substance as a whole.

NUCLEO-ALBUMINS.

This group contains bodies which in the pure state are rather
markedly acid in character. They are called nucleo-albumins because
of the earlier fancied resemblance to the nucleo-proteids. The char-
acteristics of the latter group, such as the presence of nucleic acid and
the xanthine bases among the decomposition products, are wholly
wanting in the nucleo-albumins. Both groups contain phosphorus,
and in both cases the phosphorus is separated in complex combinations
on digestiorl with pepsin and hydrochloric acid; the character of the
phosphorus compound separated is very different in the one case,
however, from what it is in the other.

The free acids are but slightly soluble in water, but in the salt form
they are very soluble and these solutions do not coagulate on boiling,

72 PHYSIOLOGICAL CHEMISTRY.

as shown by the behavior of casein in milk. The addition of weak
acids to these salt solutions forms precipitates of the free nucleo-
albumin acids. From very weak solutions the precipitate may not
separate until after heating. A large number of bodies have been de-
scribed as nucleo-albumins, but only those will be mentioned here
which are well known. In the newer classification referred to above
all these compounds are described simply as phospho-proteins. No
assumption is made regarding the exact form in which the phosphorus
is held, but the combination may be in a general way that of an ester
of phosphoric acid.

Casein. Of all the nucleo-albumins this is the best known and
most important. It occurs in milk as a neutral calcium salt, and in
the case of cow's milk makes up nearly 4 per cent by weight. It may
be readily separated from milk by the addition of a little acetic acid.
In precipitating, the fat is usually carried down too, but may be re-
moved after drying by treatment with ether or petroleum spirit.
Rennin, a peculiar enzyme of the stomach, to be described later, causes
a kind of coagulation in casein solutions; if lime salts are present,
which is practically the case in milk, the coagulation extends to the
formation of a curd or cheesy mass which is very characteristic. The
first product formed by the rennin is known as paracasein and the
curd, or cheese, is the calcium combination of this.

Casein was formerly considered as an alkali albuminate because of
its behavior with acids and alkali solutions. Many of its alkali com-
binations are now produced in a technical way as by-products in the
butter and cream industries. Plasmon and nutrose are apparently
sodium-casein compounds. These are used as foods, but some of the
others find application in other directions. Casein forms two series of
salts with calcium hydroxide and other bases and the amount of metal
in several of these has been found with considerable accuracy. Most
of these salts form opalescent rather than perfectly clear solutions.
The addition of sodium chloride or magnesium sulphate to these solu-
tions in sufficient amount completely precipitates the casein. Like the
other nucleo-albumins, casein leaves a pseudo-nuclein residue on di-
gestion with pepsin and hydrochloric acid.

In combining casein with alkali 1 gram of the former may be dis-
solved in 4.5 cc. of N/10 sodium hydroxide or equivalent solution.
But this is still acid toward phenol-phthalein. To obtain a solution
neutral with phenol-phthalein just twice as much alkali must be used.
The second reaction corresponds to an equivalent weight of nil for
the casein. Casein shows also a basic behavior and unites readily with

THE PROTEIN SUBSTANCES. 73

many acids, i gram combines with 7 cc. almost exactly, of N/10
hydrochloric or equivalent strong acid, the reactions being completed
without the aid of heat. These reactions illustrate very beautifully
the chemical behavior of complex groups of amino acids. Something
will be said later about the method of preparing pure casein used in
such tests.

Vitellin. While white of egg contains essentially albumin proper
and globulin, the yellow part is extremely complex, containing many
substances. At least two of these compounds hold phosphorus in com-
bination; one of these is lecithin, referred to earlier, and the other is
the nucleo-albumin called vitellin. The separation of these substances
from each other is extremely difficult. Vitellin is not soluble in water,
but dissolves in weak alkali solutions; on digestion with pepsin and
hydrochloric acid it yields a pseudo-nuclein residue which contains
iron as well as phosphorus. The name hematogen has been given to
this, and it is considered as of great physiological importance because
of its iron content. It is possibly one of the parent substances of
hemoglobin.

Other Nucleo-albumins. In the eggs of fishes there is found a
peculiar vitellin called ichthulin, which has been obtained in crystalline
form. It is not soluble in water, but yields a clear solution with weak
alkalies.

In cell protoplasm several different nucleo-albumins are found.
These bodies contain iron, are insoluble in water in pure condition, but
with alkalies form salts which are readily soluble.

Vegetable Proteins. Most of the protein bodies thus far referred
to have belonged to the animal kingdom, but as plant constituents
fully as great a number occur. The exact nature of some of these is
obscure, but many valuable observations have been made by Osborne
and other chemists in the last few years which have cleared up some
of the points in dispute. Only brief mention can be made here.

In wheat flour, for example, four or five protein bodies appear to
be present. The most abundant of these is called by Osborne glutenin
and makes up over 4 per cent of the weight of the grain. Next in
abundance is another important compound known as gliadin, amount-
ing to about 4 per cent of the grain weight. These two proteins unite
in the formation of gluten which is essential in the production of an
elastic dough, which on leavening yields a porous and light bread.
Gliadin is soluble in dilute alcohol and forms an opalescent solution
with water. In some respects it resembles a globulin. In its behavior
with weak alkalies glutenin bears some resemblance to casein. Wheat
flour contains also a true globulin in small amount.

74 PHYSIOLOGICAL CHEMISTRY.

A peculiar protein body known as zein, or maize fibrin, is found
in corn meal. It is soluble in alcohol but not in water, and is not
soluble in dilute alkali solutions. Corn contains also three globulin-
like bodies and one or more substances to be classed with the albumins
proper.

Legumin is found in peas, beans and related seeds ; it was formerly-
placed in the group of nucleo-albumins, but in its solubility conditions
resembles the typical globulins and is now so included. The legumin
obtained from vetches does not coagulate on boiling. On boiling a
solution of pea legumin a jelly-like substance is formed.

Recently Osborne has proposed the nzmtprolamins for the seed proteins soluble
in alcohol. As the best representatives of this class we have the gliadin of wheat
and the zein of corn, just mentioned, and the hordein from barley. These proteins,
which are soluble in all proportions in alcohol of 70 to 80 per cent, are found in the
seeds of all cereals, apparently, and constitute a relatively large proportion of their
reserve material. They do not appear to occur in other parts of the plant. On
decomposition these prolamins yield relatively large amounts of glutaminic acid.

The glutelins, according to the same author, make up a large part of the protein
matter of cereals. They are said to be insoluble in all neutral solvents, but dis-
solve in weak acid or alkalies. The glutenin, mentioned above from wheat flour,
is the best known member of the group, because of its ready accessibility and ease
of preparation. It is difficult to separate the glutelins from other seeds in a form
pure enough for study, because they yield no coherent gluten, to begin with.

Seeds contain, also, compounds which appear to be true nucleo-proteins, that is
combinations of nucleic acid with a protein group. But the separation and identi-
fication of these bodies has not been, thus far, satisfactorily carried out.

THE HISTONES.

These are relatively simple proteins which, apparently, always occur
in combination with certain groups to form the nucleo proteids, or
conjugated proteins. They behave as rather strong bases and yield
basic groups on cleavage. In consequence of their basic character they
are precipitated from solution by addition of alkalies, especially by
ammonia. In presence of salts they are coagulated by boiling, and are
also precipitated in cold solution by nitric acid; this precipitate disap-
pears on warming, to return on cooling. They yield precipitates with
the alkaloid reagents in neutral as well as in acid solutions. The
nitrogen content of the histones is relatively high and the sulphur
content low. They contain no phosphorus. Histones are obtained
from several sources, and the best known are the following :

Globin. This makes up about 96 per cent of the hemoglobin of the
red blood corpuscle, existing in combination with the iron-containing
constituent, hematin. It is precipitated by a relatively small amount
of ammonia, and redissolved by a slight excess. On cleavage it yields

THE PROTEIN SUBSTANCES. 75

much histidine and leucine. Of all the histones this is the one most
readily obtained for experiment.

Salmo-histone, Scomber-histone, and Gadus-histone. These
bodies are obtained from the immature testicles of the salmon, the
mackerel and the codfish, and were first classed as albumoses. But
their precipitation reactions throw them into the group of histones.
Similar products have been obtained from the testicles of other
animals.

Nucleo-histone. This name was given to a product separated
from the thymus glands of the calf and was one of the first studied.
On cleavage it yields much arginine and tyrosine, and is characterized
by easy digestibility.

As strongly basic bodies the histones show the interesting property
of forming precipitates with many of the other simple albumins,
especially with casein, egg albumin and serum albumin. Their pre-
cipitates contain the component proteins in definite proportions.

PROTAMINES.

We come here to the simplest of all the naturally occurring proteins.
They do not exist free in nature but, like the histones, in combination
with nucleic acids, hematin or other simple "prosthetic group." The
protamines contain no sulphur but are very rich in nitrogen and low
in carbon as compared with the ordinary proteins. They are not
coagulated by heat and do not give the Millon's reagent reaction or
that of Adamkiewicz. The biuret reaction is marked and the alkaloid
reagents produce precipitates. Some of the groups in the common
proteins are therefore wanting in the protamines. Several of these
bodies have been isolated, particularly from the nucleo-proteids of
fish spermatozoa and the names given to them suggest their origin.
Thus, we have salmin, sturin, scombrin and clupein. In recent
analyses the following formulas have been found for the more im-
portant protamines :

Salmin C80H67N„O,

Clupein Ca„H02H14O9

Scombrin C32H7:N10O8

Sturin C34H7,N17O0

When warmed with weak acid, or when subjected to pancreatic

digestion, they yield at first proiones, corresponding to the peptones

of ordinary digestion and finally simpler products, among which the

■ne bases, arginine, lysine and histidine predominate. From

salmin, for example, over 80 per cent of arginine has been obtained.

76 PHYSIOLOGICAL CHEMISTRY.

In some cases of decomposition the cleavage into the hexone bases
has been nearly quantitative, which is an important step toward estab-
lishing the empirical formula of the parent protamine. The prota-
mines appear to have rather marked toxic properties.

The histones are more complex bodies than the protamines, and
possibly contain the latter as a component part; It is also possible that
the histones represent a stage in the development of the protamines,
since while the former are found in immature spermatozoa, the latter
are commonly obtained from the mature organisms. In basic prop-
erties the protamines are more marked than are the histones, and are
precipitated easily by alkalies. They do not seem to be altered by
peptic digestion, but by trypsin and erepsin they may be reduced to
crystalline products. The protones, referred to above, are stages in
this cleavage.

From a purely scientific standpoint these bodies possess great in-
terest and importance, since they represent, apparently, the beginnings
in the formation of protein molecules. On cleavage they yield groups
of amino acids which are quantitatively more readily measured than
are the products from the more complex proteins.

TRANSFORMATION PRODUCTS.

The protein bodies which have been described in the foregoing
pages are natural unmodified substances or primary products. We have
now to consider briefly a class of important protein compounds which
includes secondary or modified substances which in the main are de-
rived from the native albumins just discussed. These modified forms
may be obtained in various ways, but for convenience three groups of
transformation products may be made, as shown below.

COAGULATED OR MODIFIED ALBUMINS.

It has been shown already that white of egg dissolves easily in
water. The solution so made undergoes a change when heated or
when treated with certain reagents. This change is called coagulation
and the resultant product is so essentially altered that it may no longer
be brought into the original form, or a similar form, by any known
means. Some of the conditions of coagulation have been explained
above and illustrated by experiments. While the simple or native
egg albumin is soluble in water the modified product is insoluble. It
is, however, soluble in weak acids or alkalies, but is insoluble in solu-
tions of neutral salts. It follows, therefore, that while coagulation or
modification of a native albumin always follows on heating, pre dpi-

THE PROTEIN SUBSTANCES. 77

tat ion may not result. This depends on the reaction of the mixture.
Coagulation or modification on the one hand and precipitation on the
other are perfectly distinct phenomena. In the case of egg albumin
in solution, for example, a precipitate forms on heating as long as
the solution is nearly neutral. In presence of salts the precipitation
is more complete. But if the original solution is alkaline modification
of the albumin takes place but without precipitation, as soluble alkali
albuminate is now formed. In presence of acid in proper amount
soluble acid albumin is formed. Although often used synonymously
the terms coagulation and precipitation have here distinct meanings.

The exact nature of the change which takes place when native albu-
mins are heated is not known. Hence the terms used in describing
the phenomenon are somewhat indefinite. They are " modified," or, to
freely render a German expression, " denatured." To bring them
again into the original condition is not possible. White of egg may
sometimes be modified or altered without becoming opaque, and the
same is true of clear blood serum. In both cases we have coagulation
without precipitation.

Some of these changes in condition of the protein are termed re-
versible, and others irreversible. Many of the precipitation reactions
are reversible; that is, the protein may afterwards be returned to its
former condition. But the change produced in a protein by coagula-
tion, for example, is irreversible.

ACID AND ALKALI ALBUMINS.

These products represent the most important forms of the coagu-
lated modified albumin, and may be looked upon as forming salts of
the albumin nucleus acting as an acid or basic ion. They are most
readily secured by the action of acid or alkali in excess on some native
albumin, usually white of egg. These actions of alkali or acid are
but the beginning of the profound changes in which the protein mole-
cule finally breaks down into small groups. They may not be looked
upon, therefore, as absolutely sharp and definitely limited conditions,
which may always be exactly duplicated.

Alkali Albuminates. Strong alkali solutions act very energetically
on white of egg and the reaction is always accompanied by some de-
composition of the latter. There is a loss of nitrogen in the form
of ammonia, and of sulphur as hydrogen sulphide. The reaction with
lead solution, production of lead sulphide, disappears after the alkali
treatment. The most characteristic product of alkali action on native
albumin is a thick jelly-like mass and is known as " Lieberkuehn's
jelly." It may be obtained as follows :

78 PHYSIOLOGICAL CHEMISTRY.

Experiment. Add strong sodium hydroxide solution to white of egg, with con-
stant stirring, until a thick jelly is formed. Too much alkali must not be added
here, but just enough to make the maximum of jelly. This is next cut into small
pieces and washed in distilled water several times until the lumps are white through-
out. They are then heated with fresh pure water, but very gently, until they go
into solution. This is then filtered and the nitrate precipitated by acetic acid, avoid-
ing any excess. The precipitate is washed with pure water, and used for experi-
ments below.

This precipitate is the modified alkali albumin or alkali protein
proper. It is likewise insoluble in salt solutions. In the treatment
with the alkali a salt of the modified protein is formed, and this is
called an alkali albuminate. The salt is readily soluble, while the
alkali-protein itself is not.

Experiment. Use some of the alkali albumin of the last experiment to test other
properties. Dissolve a portion in weak hydrochloric or sulphuric acid and observe
that the solution does not coagulate on boiling. An acid solution is precipitated
by addition of sodium chloride to saturation, and it is also precipitated by adding
weak alkali to the point of neutrality. When this neutral point is reached more
alkali brings about solution again.

The formation of Lieberkuehn's jelly illustrates the production of
the alkali albumin at once in the cold. A similar result is obtained by
heating some white of egg solution for a time with very weak alkali.
A clear solution is finally obtained.

Experiment. Dilute white of egg with water and add a small amount of N/io
alkali solution. A few cubic centimeters will suffice. Keep the mixture at a tem-
perature of about 400 to 45° on the water-bath through an hour, and then test some
of it by boiling in a test-tube. It should not coagulate. To a portion of the
clear solution add a few drops of phenol-phthalein indicator and then run in dilute
sulphuric acid to neutralization. A precipitate forms as shown above.

Acid Albumin. According to the view held at one time the solu-
tion of the alkali albuminate in water yields an acid albumin on acid
treatment. But the weight of evidence now indicates that the group
in the albuminate having an acid function is different from the group
in the so-called acid albumin which certainly plays the part of a basic
radical. Although the albuminate and the acid albumin have certain
points in common, as will be shown, they are not identical. It appears,
however, that while the albuminate may not be converted into acid
albumin by action of weak acid, the opposite conversion is possible;
that is, weak alkali will change acid albumin into albuminate. Some
simple experiments may be made here :

Experiment. Dilute white of egg with four volumes of water, take 25 cc. of
the mixture, add 5 cc. of 0.2 per cent hydrochloric acid and warm it on the water-
bath for about two hours to a temperature of 450 C. Then carefully neutralize
the solution with dilute sodium hydroxide, using phenol-phthalein as indicator.

THE PROTEIN SUBSTANCES. 79

This precipitates insoluble acid albumin, which can be washed with water by decan-
tation. It is essential that just the right amount of alkali be added here; an
excess would redissolve the precipitated acid albumin with formation of alkali
albuminate. The washed acid albumin can be used for a number of tests.

Experiment. Dissolve a little of the washed acid albumin in water by the aid
of weak hydrochloric acid, and note that the solution does not coagulate on boil-
ing. Observe, however, that the addition of common salt to the acid solution
brings about precipitation. The same thing was found to be true with the solution
of alkali albuminate in acid.

In forming acid albumin from a native albumin the action of the
weak hydrochloric acid employed is much less destructive than is the
action of the alkali in producing albuminate. The actual modification
of the protein molecule is much less profound. Nothing is split off as
is the ammonia or hydrogen sulphide in the other case, and this may
account for the observed fact that the acid albumin may be changed
into albuminate by use of weak alkali. It must of course be remem-
bered that a stronger acid may not be used in making the acid albumin,
since here too the reaction may become destructive.

Syntonin. This appears to be an acid albumin, resulting from the
action of dilute acids on muscle, and is very readily formed in pres-
ence of the ferment pepsin. The name is often applied to all acid
albumins, but it is perhaps preferable to restrict its use to describe the
product from muscle.

Experiment. Free the muscle part of meat from fat as for as possible and run
it through a sausage mill several times to bring it to a fine state of subdivision.
Wash this chopped mass with distilled water until the washings remain clear. Now,
to about s gm. of the moist residue in a small flask, add 50 cc. of dilute hydro-
chloric acid, containing 0.1 per cent of the true acid. Warm the mixture slightly
(to 350 or 40° C), and keep at this temperature about three hours. Then filter
and test the filtrate. It contains the soluble syntonin, held by the excess of weak
acid used.

Experiment. To a small portion of the filtrate add weak caustic soda, which
produces a precipitate soluble in excess of the alkali. This latter solution contains
albuminate.

Boil another portion of the filtrate. It does not coagulate directly, but after
the addition of common salt precipitation follows.

It must be remembered that the action of both acids and alkalies on
the native albumins may easily extend beyond the formation of the
simple products here mentioned. These are merely limiting cases. It
has been already shown that by more prolonged action various prod-
ucts of disintegration are obtained and the substances just described
represent the first stages. With slightly stronger acids or alkalies or
by elevation of temperature the more easily separated of the amino
complexes begin to split off. The condition of stability is only relative.
With molecules as large as these it may even be possible to separate

80 PHYSIOLOGICAL CHEMISTRY.

some of the outlying groups without greatly impairing the integrity
of the whole.

It will be recalled that in the second classification of the protein
substances, given at the outset, a group of so-called metaproteins was
mentioned. This group includes the alkali albumins and the acid
albumins, but not the salts. That is, it does not include the so-called
albuminates or the opposite class of bodies, which consists of combina-
tions of acid proteins with acids. This distinction should be kept in
mind. It will be recalled, further, that the less highly modified pro-
tein, formed by the action of water alone, is called in this classification
a protean. The proteans are, like the acid and alkali albumins, in-
soluble in water.

ALBUMOSES AND PEPTONES.

By the simple treatment with weak acids or alkalies alone, the
changes in the native protein bodies are of the character described in
the last paragraph. But in presence of certain enzymes further modi-
fications are reached and these have received the names of albumoses
and peptones when they are produced by the ferments of the digestive
tract. It is indeed true that these substances may be produced in
fairly large amount by the simple chemical treatment or by heating
the protein substances with water under pressure. But the names, in
practice, are usually restricted to the products of enzymic formation.

Of the exact nature of the reactions by which these substances are
reached little is known. They represent the very last stages in the
process of breaking down complex native protein bodies which still
give the characteristic protein tests. Further disintegration leads to
bodies which are no longer proteins, but which, as amino acids, are
simply constituent groups of the complex protein molecule. The
peptone substances represent a more advanced stage of modification
than do the albumoses. In both groups of bodies we find the reactions
with the alkaloid reagents and with the precipitating metallic solutions
in most cases still marked; the biuret reaction is also still present.
But for the peptones we find lacking the property on which the salting
out processes depend. By adding plenty of ammonium sulphate or
zinc sulphate it is possible to throw the albumoses out of solution;
the peptones do not respond to this treatment and in other points also
they are further removed from the original proteins than are the
albumoses.

But it must not be understood that the distinction between the two
groups is perfectly simple and clear. Unfortunately much confusion

THE PROTEIN SUBSTANCES. ol

still prevails in the literature of the subject and an elementary pre-
sentation which is satisfactory and consistent is not yet possible. In
this chapter only a brief outline of the relations now generally accepted
among chemists and physiologists will be attempted, while in a follow-
ing chapter on digestion some of the more practical details will receive
consideration.

Basis of Classification. The general classification of these sub-
stances commonly recognized is that of Kiihne, which was elaborated
mainly in conjunction with Chittenden. The scheme has been en-
larged and modified somewhat by other workers but in its important
features the ideas of Kiihne still hold the first place. In the weak acid
as well as in the enzymic treatment it is easily seen that the common
proteins are not homogeneous or symmetrical bodies. On the con-
trary they seem to contain two great groups which respond very differ-
ently to the action of the digestive agent, whether acid or ferment.
A part of the original complex appears to break down rather quickly
and go into a soluble form ; while a second portion resists this breaking
down process pretty effectually as far as weak acid and pepsin fer-
mentation is concerned, at any rate, and in subsequent treatment with
the more active pancreatic ferment it yields products different from
those derived from the first group. To the first or less resistant frac-
tion, Kiihne gave the name hemi group, and to the second or more
resistant portion, the name anti group. It was later noticed that most
protein bodies seem to contain a third group which in the subsequent
breaking down yields a sugar of some kind. Hence a further or
carbohydrate group may be assumed to exist in the native protein
molecules, or in most of them, at least. But the latest researches seem
to show that the amount of this complex present is, in most cases,
not large.

Albumoses. In the first stage of the action of the acid and fer-
ment on the protein body a kind of acid albumin appears which passes
by continued digestion into the next or albumose stage. Different
albumoses seem to be derived from the several native proteins, and
these may be called, in general, proteoses. Names have also been
given to them corresponding to their origin. We have, accordingly,
fibrinoses, caseoses, myosinoses, globulinoses, and so on. Several de-
grees of albumose digestion are recognized; that is, bodies are pro-
duced which behave differently on treatment of the digesting mixture
with precipitating reagents, and we have, therefore, primary and
secondary albumoses. The secondary albumose stage represents a
more advanced condition of change on digestion than does the primary
7

82 PHYSIOLOGICAL CHEMISTRY.

albumose. Finally, the secondary albumose, by prolonged contact
with the digestive agents, passes into the peptone stage. Some idea
of the existence of these three stages of change may be obtained from
the following experiment in which commercial peptone is taken for
illustration. This is a substance made by the partial digestion of
fibrin, gelatin, serum and other bodies and is not uniform or homo-
geneous in structure. It contains representatives of the several classes
of derived digestion products.

Experiment. Dissolve about 5 gm. of commercial peptone in 50 c.c. of water
and use small portions of the solution for these tests : To one portion add some
strong nitric acid; this produces a precipitate. To a second portion add a little
copper sulphate solution, which gives a light greenish precipitate. To a third por-
tion add a few drops of acetic acid and then some potassium ferrocyanide. This
makes a turbidity or may even cause a precipitate. Now to the remaining and large
portion of the original solution add an equal volume of a saturated solution of am-
monium sulphate. A marked precipitate of primary albumose separates and may
be filtered off after a time. When the liquid has all passed through the filter note
that the precipitate may be easily dissolved by adding . fresh water, and further
that this new solution is not coagulated by boiling. Note also that the solution gives
a good biuret reaction.

Experiment. Use the filtrate from the primary albumose precipitate for a further
test. Add to it powdered ammonium sulphate to complete saturation, that is, as
long as the powder dissolves on thorough shaking. Then add five to ten drops of
a weakly acid solution of ammonium sulphate (which may be obtained by adding
to 10 cc. of saturated ammonium sulphate solution five drops of concentrated sul-
phuric acid). This last treatment with the acid ammonium sulphate gives a new
albumose precipitate which after a time may be separated by filtration. Save the
filtrate and test the precipitate as follows : Dissolve it in fresh water and test
portions with copper sulphate, nitric acid and the potassium ferrocyanide. These
reagents gave precipitates with the original peptone solution, but yield nothing with
the solution of the new albumose, which is called secondary albumose.

Experiment. The filtrate from the secondary albumose may finally be tested.
Add to it an excess of concentrated sodium hydroxide solution and then a drop
of dilute copper sulphate solution. This gives a purple red biuret color, showing
the presence of a soluble product not precipitated by ammonium sulphate in excess.
This soluble product is the peptone, representing the last stage of the true digestion.
This peptone gives no precipitation reactions with the reagent used above.

The first of these fractions, or the primary albumoses, may be con-
verted by further acid treatment or by digestion into secondary albu-
moses no longer precipitated by half saturated ammonium sulphate.
By solution in water and addition of alcohol it is possible to separate
this primary albumose into two sub- fractions which are pretty well
characterized. The first of these is known as heteroalbumose and is
insoluble in weak alcohol, while the second, or alcohol-soluble portion,
is called protalbumose. The heteroalbumose belongs to the above men-
tioned anti group and is further changed only with difficulty. The
protalbumose belongs to the hemi group. It is quite soluble in water,

THE PROTEIN SUBSTANCES. 83

and in dilute alcohol even more soluble. By prolonged peptic diges-
tion the protalbumose passes into the secondary albumose known as
dcuteralbumose A, and then into peptone B, so called.

An enormous amount of labor has been devoted to the study of the
various fractions obtained by digestion under different conditions, and
a complex nomenclature describing the products has grown up. But
the value of much of this is now doubted, as there is no great con-
stancy in the results secured by different observers. This much is true,
however, that in the earliest stages of digestion certain amino com-
plexes are very readily split off, while others are not. Tyrosine and
tryptophane, for example, separate relatively quickly, and would be
considered as belonging to the hemi group. On the other hand
glycine, phenylalanine and proline separate slowly and should
be referred to the anti group. But from the present point of view
the assumption of these two groups is arbitrary and without real justi-
fication. The classification based on it need not be further developed
in this book, which must be kept within elementary bounds.

Peptones. The amount of real peptone formed by the pepsin di-
gestion is always small; the large amount of peptone produced in the
body is a consequence of the action of the pancreas enzyme known "as
trypsin. The peptone of gastric digestion was assumed to be a mixture
of products from the hemi and anti groups and was called ampho-
peptone. The term antipeptone is generally applied to the final product
of the energetic pancreatic digestion. Amphopeptone has been ob-
tained as a yellow powder, very soluble in water and very hygroscopic.
It diffuses pretty well through parchment and has a sharp bitter taste.
It is not possible to salt out the peptone from solution, but the alkaloid
reagents give precipitates, which are soluble in excess. Precipitates
are formed by solutions of several of the heavy metallic salts also, but
not by copper salts.

The two forms of amphopeptone which have been described are
known as amphopeptone A and amphopeptone B. The first is insol-
uble in 96 per cent alcohol and is further characterized by giving a
strong reaction with the Molisch reagent which relates it to the carbo-
hydrate group. The second is soluble in 96 per cent alcohol and does
not give the Molisch reaction. Both forms give a strong biuret
reaction.

As to the exact nature of the antipeptone referred to above, there is
still much uncertainty. This was assumed by Kiihne to represent the
final product of pancreatic digestion, and it was supposed that even
prolonged digestion would not change it further. It was found later,

84 PHYSIOLOGICAL CHEMISTRY.

however, that various amino acids appear here in considerable quantity,
and that the digestion may be carried so far as to yield a product
which no longer gives the characteristic biuret reaction; that is, a
product from which everything of a really protein nature has disap-
peared. This matter will be more fully discussed in a following
chapter. The reactions described as characteristic of antipeptone are
similar to those for the amphopeptone in the main. A good biuret
reaction is obtained if the digestion is not too prolonged, and the alka-
loid reagents give precipitates which are soluble in excess. Some of
the metallic salts precipitate, but copper sulphate not.

The name kyrine has been given to certain kinds of peptones which
in a marked degree resist the action of hydrolysis through pepsin and
trypsin, and which are basic in character. In some cases the compo-
nent groups in these kyrines have been determined. For example, a
kyrine from casein is apparently made up of i molecule of arginine, I
molecule of glutaminic acid and 2 molecules of lysine.

In the formation of the albumoses and peptones from native protein
molecules a large amount of water is added; roughly the action may
be compared to the hydration of starch, producing malt sugar and
finally glucose. As the original molecule is very large the percentage
amount of water taken up in the hydration is much less than is the
case in the carbohydrate conversion. It is also very interesting to
note that with the progress of the hydration the amount of hydro-
chloric acid which may be held by the product increases; the smaller
molecules in the aggregate resulting from the hydration have a much
greater capacity for combining with free hydrochloric acid than the
parent substances have. This question assumes considerable practical
importance in connection with the subject of gastric digestion and
acidity of the stomach, as will be shown later.

It must be remembered that the various commercial products sold
as " peptones " may contain many other substances, and may be quite
unfit for use as a food or in medicine. While in some cases a con-
siderable portion of real peptone (with albumose) is present, in others
the main constituents are decomposition products formed by too long
digestion of the meat or fibrin with the acid and pepsin mixture.
Some of these commercial peptones appear to be formed by digesting
with weak acid under pressure, which results in the formation of bodies
of little nutritive value; indeed, it is likely that such products are dis-
tinctly harmful when taken into the stomach of man.

It should be mentioned further that many artificial products are
known which are related in properties to the peptones of advanced

THE PROTEIN SUBSTANCES. 05

hydrolysis. These may be called peptides, or polypeptides. Some-
thing will be said about them later.

THE PROTEIDS.

The term proteid, as already explained, is used to designate a certain
group of protein compounds. This use is a perfectly arbitrary one as
the word was once employed to describe all the bodies discussed in this
chapter. It would be perhaps well to drop the term proteid, and
describe the bodies included under it as conjugated or compound pro-
teins. According to the generally accepted modern classifications the
bodies now called proteids are compound substances in which a true
or native albumin is found in combination with some other group
which often may be separated as such. In the table given earlier in
the chapter three such combinations are mentioned : the nucleo-proteids,
the hemoglobins and the gluco-proteids. A brief description of each
group will here follow.

NUCLEO-PROTEIDS.

These proteins are important as making up a large part of the cell
nucleus. In treating tissues rich in cells with the pepsin-hydrochloric
acid digestive mixture it was long ago recognized that a certain portion
went easily into solution, while another portion was always left undis-
solved. This residue was called nuclein and was found to contain all
the phosphorus of the original protein. If in place of the pepsin mix-
ture some other hydrolyzing agent is used the general result is similar ;
a separation into two component parts takes place, and one of these
parts is a simple native protein substance and the other the nuclein or
further and final decomposition product, nucleic acid. The nucleo-
proteids are therefore described as combinations of native albumins
with nucleic acid.

In breaking down the complex nucleo-proteid it appears that several
stages must be distinguished, the body described as a " nuclein " con-
taining still some native albumin. Finally, however, the residue or
characteristic part, the nucleic acid, is left. Although many investi-
gations have been made there is still much uncertainty about the nature
of this acid. Indeed, from different parent substances acids of some-
what different properties have been obtained, so that it is customary to
speak of the nucleic acids. These will be considered below.

Like the native proteins already described the nucleo-proteids are
coagulated by heat and by acids. They are soluble in water, salt solu-
tions and also in alkali solutions. By means of large excess of salt

86 PHYSIOLOGICAL CHEMISTRY.

they suffer precipitation. In the last few years nucleo-proteids from
different sources have been studied, especially from yeast cells, the
thyroid gland, the pancreas and different kinds of spermatozoa. The
sperm and spermatozoa of sea urchins and fish have furnished a num-
ber of these substances because of their relative richness in cell struc-
tures. Thus, characteristic products have been obtained from the
spermatozoa of the salmon, the mackerel, the sturgeon and so on.
These appear to be distinct bodies, but more exact investigations may
show that the apparent differences depend on foreign proteins not
completely separated in their preparation.

As intimated above, the nucleo-proteids are found characteristically
in the organs rich in cells. The larger part of the solid portion of
certain glands and of the heads of spermatozoa consist of these conju-
gated bodies. The thymus of the calf has been frequently used in the
investigations of these bodies, as over 75 per cent of the dried cells of
these glands consist of a nucleo-histone. Fish spermatozoa are easily
obtainable from hatcheries and have, perhaps, furnished the main
material for investigation. The dry, fat-free portion of the heads,
which are easily separated, contains 95 per cent, or more, of protamine
or histone combinations of nucleic acids. The abundance of these
" nucleates " in cell structures, especially in young cells, shows their
great physiological importance. The nucleo-proteids of various organs
have been investigated in recent years, but the details can not be
explained in an elementary book.

Iron seems to be contained in the so-called " masked " or non-ionic
condition in the nucleo-proteids. It can not be recognized by the usual
qualitative tests, because of its peculiar organic combination. Iron in
this form has long been supposed to be important in the formation of
red blood corpuscles, which contain hematin. Special methods have
been devised for showing the organic iron.

The Nucleic Acids. The occurrence of these important compounds
in combination with protamines, histones and other proteins has been
referred to several times in the last few pages. They constitute, in
fact, the important part of the nucleo-proteids. By different processes
of separation a number of these acids have been obtained from various
cell structures, and especially from yeast and fish sperm or sperma-
tozoa. The results of analyses lead to formulas of about the follow-
ing character in nearly all cases : C40H52N14O25P4. These acids have
not been obtained in crystalline condition. They are but slightly sol-
uble in cold water, but soluble in weak alkali solutions when they form
salts. A number of salts of the heavy metals, which are insoluble in

THE PROTEIN SUBSTANCES. 87

water, have been made and studied. When boiled in aqueous or acid
solution the nucleic acids break up, yielding finally the characteristic
basic bodies, long known as the purine bases, the pyrimidine bases,
phosphoric acid and certain carbohydrates.

Attempts have been made to establish the structural formula of some
of the nucleic acids, but without much success, as they are evidently of
complex composition. Among the purines the following have been
separated :

Xanthine C5H4N402

Hypoxanthine C5H4N40

Adenine C6H5N5

Guanine C5H5N50

Three pyrimidine derivatives are known:

Uracil C4H4N202

Cytosine C4H5N30

Thymine C5H6N202

More will be said later about the relations of these bodies to each
other and to the uric acid of the urine. The first are important from
that standpoint, as in structure they are closely related to uric acid, and
may be forerunners of it.

From the amount of phosphorus and nitrogen found by analysis of
the nucleic acids and the amount of the bases secured on cleavage, it
has been suggested that they may be complex esters of 4 molecules
of phosphoric acid, in which different bases may be combined. This
would explain the existence of a large number of closely related acids.
It is not necessary to give here the numerous empirical formulas which
have been suggested for the acids from different sources. The typical
one given above is sufficient as an illustration of the general com-
plexity. Nucleic acids from yeast and other sources have found some
application in medicine. The acids from other vegetable sources have
been studied, especially by Osborne.

As acids these bodies have rather marked properties; they combine
not only with inorganic bases, but also with simple proteins and many
toxin bodies. The medicinal uses depend on these facts. The free
acids are rather easily hydrolyzed by water and mineral acids, but are
stable with alkalies. The salts with sodium and potassium form stiff
jellies when dissolved in water by aid of heat, and then cooled below
certain temperatures.

HEMOGLOBINS.

The discussion of the important subject of hemoglobins may prop-
erly be left to be taken up with the study of the blood in which ^hey

88 PHYSIOLOGICAL CHEMISTRY.

are contained. The term is used here in the plural since from different
kinds of blood bodies of somewhat different properties have been
obtained. Hemoglobin in general must be classed among the com-
pound bodies because it is distinctly- made up of two characteristic
parts, a histone, already referred to, and hematin.

GLUCO-PROTEIDS.

We have here a group of bodies containing a number of important
members about which our knowledge in most cases is not very ex-
tended or exact. As the name indicates the proteins here concerned
contain a carbohydrate constituent which may be recognized by its
reducing properties when the substance in question is warmed with a
weak acid and afterwards treated with Fehling's solution in the usual
way. The carbohydrate group separated appears to be, in most cases
at any rate, glucose amine. Familiar illustrations of these gluco-
proteids are found in the mucins and related bodies called mucoids.
As a class these substances are characterized by relatively low nitrogen
and high oxygen content, due to the presence of the carbohydrate
group. The amount of carbon present is also lower than in the com-
mon proteins. Of the exact nature of the albumin combined with the
carbohydrate little is known, because in separating the two groups by
acid or alkali treatment the protein constituent is so changed that no
safe conclusion can be drawn as to its original nature.

The gluco-proteids behave as acid bodies. They are not coagulated
by heat alone, but heating with acids or alkalies produces a complete
alteration. With weak acetic acid a precipitate is in most cases formed
which is not easily soluble in excess.

Mucins. These bodies are found in various secretions, especially in
the saliva, bile, vaginal fluid, tears, nasal mucus, etc. The amount
present, however, is always small and the separation, in pure condition,
very difficult. The mucin of the submaxillary gland is probably the
best known.

These bodies contain one of the complex protein groups, since they
give the reaction with Millon's reagent, the xanthoproteic and the
biuret reactions. They are only slightly soluble in water and in pres-
ence of alkali produce a viscous stringy liquid which is extremely
characteristic, even in great dilution. On warming with dilute alkali
the viscous condition disappears through formation of alkali albumi-
nate. On treatment with strong alkali or superheated steam a peculiar
body is formed which, from its discoverer, is known as Landwehr's
animal gum. This is now known to contain the protein and carbohy-

THE PROTEIN SUBSTANCES. 89

drate complexes; after diluting with weak acid and boiling, the sugar
reaction may be easily obtained.

The mucins are much more resistant than the nucleo-proteids against
the action of reagents or ferments, but they undergo both peptic and
pancreatic digestion slowly. In urine the identification of mucin is
often a matter of importance, as it is frequently mistaken for albumin.
The detection of mucin depends on the behavior with cold dilute acetic
acid and also on the solubility in hot water after precipitation with
strong alcohol. Albumin is permanently coagulated but mucin not.

Mucoid Bodies. These substances are found in the tendons, carti-
lage, the vitreous body of the eye, the cornea and elsewhere, and are
closely related to the mucins. They have the viscous properties of the
latter but in general, in concentrated condition, form stiffer jelly-like
masses. The cornea and sclerotic coat of the eye are made up largely
of mucoids and collagen dissolved in water.

The mucoids from tendon have been the most thoroughly studied.
An extract is made by prolonged treatment with weak lime-water.
The solution is precipitated by acetic acid and the precipitate taken up
with ammonia. These operations repeated several times give a nearly
constant product. The analyses show 48-49 per cent of carbon, 30 of
oxygen and below 12 of nitrogen. A small amount of sulphur is
present.

In cartilage, along with collagen and albuminoid bodies, a very im-
portant mucoid known as chondro-mucoid is found. This has a com-
position not very different from the tendon product just given, but
contains over 2 per cent of sulphur, part of which is in peculiar ethereal
combination. This ethereal product is separated by cleavage with
dilute acids or alkalies and is known as chondroitin sulphuric acid, and,
according to Schmiedeberg, has the composition Ci8H27N014.S03.
On hydrolysis this acid yields chondroitin, C18H27N014, and sul-
phuric acid ; the chondroitin furnishes acetic acid and chondrosin,
C12H21NOn; finally further hydrolysis breaks the chondrosin down
into glucoseamine and glucoronic acid, according to the same author,
but later researches seem to indicate that the cleavage is not as simple
as suggested. It has been shown also that this complex acid is not
peculiar to cartilage, but is found in many substances belonging to the
albuminoid group of proteins as well. Although widely distributed
the physiological importance of the body has not yet been determined.

In addition, mucoid substances have been recognized in urine, in
blood serum, in white of egg and several pathological transudates in
small amount.

go PHYSIOLOGICAL CHEMISTRY.

ALBUMOIDS OR ALBUMINOIDS.

These substances differ from the real proteins both physically and
chemically ; the physical differences are, however, the most pronounced
and characteristic. The second general classification of proteins places
these bodies in the group of simple proteins, that is, they are treated
as true proteins. The important bodies grouped here contain the dif-
ferent kinds of gelatin or glue-forming compounds, the horn sub-
stances, the spongin of the sponge, elastin of the so-called elastic
tissues of the body and other substances of less importance. They
are all firmer and harder than the common proteins and as a rule quite
insoluble in water, and in general resistant against the action of rea-
gents. While by prolonged treatment with superheated steam or acids
or alkalies they yield most of the cleavage products described as char-
acteristic of the albumins, some are, however, lacking. The tyrosine
group, for example, is absent from gelatin, or present in minute
amount at most.

In food value the albuminoids are quite distinct from the other
proteins. Most of these substances are so insoluble in the digestive
fluids that really no importance as foods could be ascribed to them.
Collagen, which yields gelatin, has a limited food value of a peculiar
kind which will be referred to below. All these substances serve as
supporting, connecting or protective tissues in the body, and they are
characterized necessarily by a kind of permanence, which depends on
insolubility in the first degree. With increasing age of the body the
albuminoid tissues become harder, firmer and less elastic.

COLLAGEN.

The best known of all these albuminoids is the collagen, or glue-
forming substance, found as ossein in bone, in cartilage, in the fibrils
of connective tissue, in tendons, in fish scales and elsewhere. This
substance, wherever found, is insoluble in cold water, but by pro-
longed heating with water it passes into the soluble form known as
gelatin, glutin or, in impure condition, as glue. The change seems to
depend on the taking up of a molecule, or more, of water. At the
present time it is made in enormous quantities from slaughter house
by-products and according to its purity is employed for different pur-
poses. When made by hot water extraction from clean bones or car-
tilage it is used as an adjunct to food and also in the preparation of
emulsions for photographic plates or gelatin paper. The product from
common material is used as joiner's glue.

Gelatin softens and dissolves in water at a temperature above 300.

THE PROTEIN SUBSTANCES. 9 1

But this solution point depends largely on the treatment to which it
has been previously subjected. By long heating with water, and espe-
cially under the action of superheated steam gelatin gradually breaks
down into the usual cleavage products of the proteins. As this cleav-
age progresses a point is finally reached where the mixture no longer
solidifies on cooling; a permanent liquid solution is obtained. By
hydrolysis with acids this condition is much sooner reached. Many
bacteria also have the power of " liquefying " gelatin, which depends
of course on their ability to decompose the complex into the more
easily soluble amino acids and other compounds.

Among the final cleavage products of gelatin easily recognizable
glycocoll and glutaminic acid are probably the most abundant. Leu-
cine, alanine and various other amino acids are found in smaller
amount. Like other proteins gelatin yields in peptic or tryptic diges-
tion bodies which have been called gelatoses, gelatin peptones and so
on. These resemble but are not identical with the true peptones, which
fact has some bearing on the long-discussed question of the food value
of gelatin. Gelatin is not converted into true protein in the animal
body and for this reason cannot wholly replace the albumins as food.
But to some extent it has the power of protecting the so-called circu-
lating albumin from katabolism, by undergoing destruction itself.
This sparing or protecting power is limited, however, and the gelatin
substances can not permanently replace the native proteins in this way.

Experiments to Illustrate Properties of Gelatin. Dissolve enough gelatin
in hot water to make a solution of about one-half per cent strength. Use portions
of this for tests :

To some of the solution add a solution of tannic acid; this gives a buff colored
precipitate. Gelatin solution is, conversely, employed as a test for tannic acid.

To some of the solution add an excess of strong alcohol; this causes precipitation.
This behavior is of importance in the estimation of gelatin.

Use some of the solution with the test reagents. Apply Millon's reagent, the
biuret test and the xanthoproteic test.

Prepare a strong solution of gelatin in hot water. To some of this add solution
of potassium dichromate and pour the mixture out to cool in a thin layer exposed
to sunlight. This treatment produces an insoluble mass which is not attacked by
hot water. This property finds application in photo-engraving processes.

To more of the strong gelatin solution add a trace of alkali to neutralize any
acidity and then some formaldehyde. On evaporating to dryness a hard mass is
obtained which is quite insoluble in water hot or cold and which has found many
applications in the arts.

Gelatin to be used in cooking should be nearly white and should dis-
solve in hot water to form a practically colorless, odorless solution.
Inferior gelatin gives off a bad odor when heated with water.

Isinglass is a kind of collagen made from the swimming bladder of

92 PHYSIOLOGICAL CHEMISTRY.

certain large fishes. On heating with water it yields a peculiar gelatin
which dissolves completely. Common isinglass is largely used in clari-
fying beer and wine, while the pure white varieties are employed in
thickening soups and jellies.

KERATIN.

This is the important insoluble substance in horn, the hoofs of cattle,
finger nails, hair and feathers. As can be inferred from the condi-
tions under which it exists, it is not easily attacked by water hot or
cold, by weak acids or alkalies, or by digestive fluids. By prolonged
action of hot hydrochloric acid, however, it undergoes gradual hydro-
lysis and cleavage with formation of the usual amino acids and other
products. Leucine is apparently the most abundant of these products,
as much as 18 per cent of the weight of the horn shavings taken having
been obtained by certain investigators. Other important cleavage
products found are tyrosine, a-aminoisovaleric acid, aspartic acid, glu-
taminic acid, phenylalanine, a-pyrrolidine-carboxylic acid, glycocoll, etc.

All keratin bodies contain large amounts of sulphur; some of this
is easily split off in the form of hydrogen sulphide. A large part
of the sulphur appears to be present in the complex body cystin,
C6H12N2S204. The xanthoproteic and Millon's reagent reactions are
very characteristic with horn substance. The behavior of a drop of
nitric acid on the finger nail is well known. The reactions with alka-
line lead solutions, yielding lead sulphide, are easily obtained. The
use of lead salts in hair dyes depends on this behavior.

ELASTIN.

Elastin differs from keratin mainly in its higher content of carbon
and low sulphur content. In their behavior toward reagents they are
much alike. Like keratin, elastin can be dissolved only by change in
composition. Leucine is produced in large amount by the hydro-
chloric acid cleavage, and glycocoll, tyrosine and other amino products
in smaller amount. Subjected to peptic and pancreatic digestion
elastin is slowly dissolved, yielding albumins and a kind of peptone.
Most of the protein reactions may be obtained from elastin after bring-
ing it into solution with alkali.

AMYLOID SUBSTANCE.

This is a body which is found in the so-called amyloid degeneration
of the liver and kidney. It is particularly characterized by the reddish
brown color it assumes when heated with a solution of iodine in potas-

THE PROTEIN SUBSTANCES.

93

sium iodide. The analysis of amyloid shows a large amount of carbon
and some sulphur. It is insoluble in cold water, but partly soluble by
long heating. It gives the usual protein reactions when brought into
alkaline solution, and contains also a complex group which yields
chondroitin sulphuric acid.

FOOD STUFFS.

In the preceding pages the individual substances used as foods or
occurring as essential principles of the animal body have been briefly
discussed. In nature these compounds do not occur in the pure free
condition, but are practically always mixed with other compounds.
Before passing to the subject of digestion it will be necessary to have
some idea of the general composition of the ordinary foods as used
by man. This information will be presented in tabular form, the
figures being average values from tables of Atwater. The fuel
values are given in so-called large calories.

ANIMAL FOODS.

Loin of beef, edible portion

Flank of beef, edible portion..

Ribs of beef, edible portion

Round of beef, edible portion..

Canned corned beef

Canned roast beef

Breast of veal, edible portion..
Leg of veal, edible portion....
Leg of lamb, edible portion....
Leg of mutton, edible portion..

Lean ham, edible portion

Fat ham, edible portion

Loin of pork, edible portion..

Chicken, edible portion

Turkey, edible portion

P.lack bass, edible portion

Catfish, edible portion

Salmon, edible portion

Trout, edible portion

Oysters

Hens' eggs, edible portion

Butter

Cheese, full, American

Lard, unrefined

Oleomargarin

Gelatin

Water

Protein

Fat

Ash

Per Cent.

Per Cent.

Per Cent.

Per Cent.

6l.3

19.0

19. 1

I.O

59-3

19.6

21. 1

0.9

57-0

17.8

24.6

0.9

67.8

20.9

10.6

I.I

51.8

26.3

18.7

4.0

58.9

25-9

14.8

1-3

68.5

20.4

10.5

1.1

71.7

20.7

6.7

i.l

58.6

18.6

22.6

1.0

55-0

17.3

27.1

0.9

60.0

25.0

14.4

1-3

38.7

12.4

50.0

0-7

50.7

16.4

32.0

0.9

74-8

21.5

2.5

I.l

55-5

21. 1

22.9

1.0

76.7

20.6

i-7

1.2

64.1

14.4

20.6

0.9

64.6

22.0

12.8

1.4

77-8

19.2

2.1

1.2

834

8.8

2.4

1-5

73-7

134

10.5

1.0

II.O

1.0

85.0

3-o

31.6

28.8

35-9

3-4

4.8

2.2

94-0

0.1

9-5

1.2

83.0

6.3

13.6

91.4

0.1

2.1

Fuel Value
in Calories
per Pound.

1 155
1255
1370

835
I28o
1 105

820

67O
1300
1465
1075

2345
1655

505
1360

455
1 135

950

445

335

720
3605
2055
4010
3525
1705

In the above table, it will be observed, the animal foods contain all a
large amount of water. The solids consist essentially of proteins and
fats. In the vegetable foods the water is much less; in most of them

94

PHYSIOLOGICAL CHEMISTRY.

carbohydrates are the characteristic principles present. The protein
is generally much lower than in the animal foods.

VEGETABLE FOODS.

Corn-, whole

Cornmeal

Popcorn

Oatmeal

Rice

Rye flour ,

Wheat flour, entire

Wheat flour, California

Wheat flour, general average..

White bread, wheat

Whole wheat bread ,

Crackers

Beans, dry

Beans, dry, Lima

Peas, dry

Peas, green, edible

Corn, green, edible

Cabbage, edible portion ,

Egg plant . _.

Potatoes, edible portion

Squash, edible portion

Apples, edible portion ,

Bananas, edible portion ,

Chestnuts, edible portion

Hickory nuts, edible portion . .
Peanuts, edible portion

15-0
12.5

4-3

7-3
12.3
12.9
11.4
13.8
12.0
35-6
38.4

7-1
12.6
10.4

9-5
74-6
75-4
91.5
92.9

78.3

88.3

84.6

75-3

5-9

3-7

9.2

•S g

8.2
9.2

10.7

16.1
8.0
6.8

13.8
7-9

1 1.4
9-3
9-7

10.2

22.5

18. 1

24.6

7.0

3-1

1.6

1.2

2.2

1.4

0.4

1.3

10.7

15-4

25.8

3-8

1.9
5-o
7.2

0.3
0.9
1.9
1.4
1.0
1.2
0.9
8.8
1.8
i-5
1.0
o.S
1.1

0.3
0.3
0.1
0.5
0.5
0.6
7.0
67.4
38.6

-2" Ph

68.7
75-4
78.7
67.5
79.0
78.7
71.9
76.4
75-i
52.7
49-7
72.4
59-6
65-9
62.0
16.9
19.7
5.6
5-1
18.4
9.0
14.2
22.0
74.2
1 1.4
24.4

fa u

1.9

1.0

1.4
0.9

0.2

0.4
O.9

0-3
0.5

1.2
0.4

4.4

4-5
1-7
0-5
I.I

0.8
0.4
0.8
1.2
1.0

2.7
2.5

1.4

1.0

i-3
1.9
0.4
0.7
1.0

0.5
0.5

1.2

i-3
1.5
3-5
4.1
2.9
1.0
0.7
1.0

0.5

1.0

0.8
0.3
0.8
2.2
2.1
2.0

>.2 o

1610

1655
1875
i860
1630
1630
1675
1625
1650
1205
1 140
1905
1605
1625
1655
465
470

145
130

385
215

290
460

1875

3345
2560

FLOUR AND MEAL.

As illustrating the composition of a common vegetable food the fol-
lowing tests may be made :

Experiment. Boil a small amount of wheat flour with Millon's reagent. The
red color produced shows presence of proteins.

Experiment. Moisten about 25 gm. of flour with water and work it into a dough.
Then hold this under a fine, slow stream of water and by kneading between the
fingers slowly work out a portion of the mass as a thin milky liquid. This is
largely starch. After some time an elastic residue is left insoluble in water. This
is " gluten " and is the chief nitrogenous element of the flour, which has been
already referred to. It may be separated into several constituents.

Experiment. To about 5 gm. of flour add 10 cc. of water, shake thoroughly and
allow to stand until a nearly clear liquid appears above a white sediment. Filter
the liquid and test for sugar by the Fehling solution. Boil some of the residue with
water and add iodine solution as a test for starch.

Experiment. To about 5 gm. of fine corn meal in a test-tube add 10 cc. of ether.
Close the tube with the thumb and shake thoroughly. Then cork and allow to stand
half an hour. Shake again and pour the mixture on a small filter, collect the
ethereal filtrate in a shallow dish and evaporate it by immersion in warm water.
A small amount of fat will remain.

THE PROTEIN SUBSTANCES. 95

Action of Yeast on Flour. The following experiment is intended to illustrate
the work done by yeast in leavening dough :

Experiment. Crumble two or three grams of compressed yeast into 15 cc. of
lukewarm water and shake or stir the mixture until the yeast is uniformly dis-
tributed. Then stir in enough flour to make a thick cream and allow to stand over
night at room temperature. In this time fermentation of the small amount of sugar
in the flour begins and the " sponge " swells up by the escape of bubbles of gas.
At this stage mix in uniformly and thoroughly enough flour to make a stiff dough,
using for the purpose perhaps 25 gm. Put the dough in an evaporating dish, keep
it for an hour or more at a temperature of 30°to 350 C. and observe that it increases
very greatly in size, from the continued action of the yeast in liberating bubbles of
carbon dioxide. If a good hot air oven is at hand the experiment is completed by
baking the leavened mass. The nature of the yeast fermentation will be explained
later.

Milk. In milk we have a substance in which all the essential food
elements are present. The average composition of cow's milk is given
in this table.

Water 87.4

Fat 3.5

Sugar 4.5

Proteins 3.9

Salts 07

100.0

Human milk contains more sugar and less protein than the milk of
the cow. Details of this will be given later.

SECTION II.

FERMENTS AND DIGESTIVE PROCESSES.

CHAPTER VI.

ENZYMES AND OTHER FERMENTS. DIGESTION.

In the course of time the conception of fermentation has undergone
many changes. The notion was first associated with those processes
in which a bubbling or boiling condition without application of heat
was observed, and later, as the most familiar kind of fermentation was
more closely studied, this phenomenon was found to be due to the
escape of gas. This escape of gas came finally to be recognized as the
essential feature of fermentation and many operations bearing no rela-
tion whatever to alcoholic fermentation were, through confusion of
ideas, frequently associated with it.

The real fermentation, which follows when saccharine juices are
exposed to the air, had been studied in a way from the remotest
antiquity, but no rational attempts at an explanation of the process
were made until after the middle of the seventeenth century, when the
relation of alcohol and the gas to the destruction of the sugar seems
to have been fully recognized. Several other reactions were asso-
ciated with the alcoholic fermentation; in the leavening of bread the
production of a gas was recognized, and it was noticed that in the
changes going on in the animal intestine gases were also liberated fol-
lowing the digestion of foods. Along with the alcoholic fermentation
there was included under the general name the peculiar change which
takes place when the wine formed from saccharine liquids was allowed
to stand exposed to the air. To be sure no gas was formed in this
action, as in the other, but something in common was recognized. In
both cases it was noticed that a scum formed over the liquid and that
a small amount of this substance was capable of quickly inciting sim-
ilar fermentation in more saccharine liquid or wine. The nature of
this scum became in time the subject of microscopic investigation (by
Leuwenhock) and we have here probably the beginning of our real
study of ferments. It was in 1680 that Leuwenhock recognized that
this scum in the case of beer yeast consisted of minute globules with
peculiar properties; the full value of this discovery, however, was not

96

ENZYMES AND OTHER FERMENTS. 97

generally admitted and more than a century passed before any great
advance was made by others. Lavoisier toward the end of the eight-
eenth century gave the first explanation of the chemistry of alcoholic
fermentation, as he was able to point out the relation of carbon dioxide
and alcohol to the parent sugar. But the cause of the action was not
much discussed and just what the function of the cells or globules of
Leuwenhock is remained obscure until the time of Pasteur.

Before taking up the important work of Pasteur something must
be said of discoveries in other directions. The older conception of
fermentation was widened by the addition of new facts. In 1780
Scheele isolated lactic acid from sour milk and later investigators
began to look for the agent responsible for the production of this acid.
About 1848 the probable nature of an organism which appeared to be
always associated with lactic fermentation was pointed out by Blon-
deau. Various formulas were given for the production of lactic acid
in quantity, but it often happened that the final product was an entirely
different substance, viz : butyric acid. Butyric fermentation was
therefore added to the list of these peculiar reactions, and various
speculations were advanced to connect the different phenomena. Mean-
while the situation became still further complicated by the gradual
recognition of a new group of reactions which exhibited many of the
essential features of the alcoholic and acetic fermentations, and which,
therefore, of necessity were classed as ferment reactions. Several
chemists had observed the peculiar behavior of a substance produced in
germinated barley; this substance possessed the power of converting
starch into a sugar which, from its origin, was called malt sugar.
Payen and Persoz, in 1833, succeeded in isolating the assumed ferment
from the sprouted barley, which they termed diastase. About the
same time it was recognized that saliva contains a similar starch-
converting agent which was later separated and called salivary diastase
or ptyalin. The seeds of the bitter almond were studied by several
scientific men and Liebig and Wohler isolated a ferment body which
they termed emulsin. This has the property of converting the gluco-
side called amygdalin into glucose, prussic acid and oil of bitter
almonds, or benzoic aldehyde. As the saliva was found to contain a
ferment acting on starches, so the gastric juice was recognized as
active through the presence of an analogous body called pepsin which
acts on proteins.

Most of these discoveries were made before 1840. The ferments
in the bitter almond, in sprouted barley, in the saliva and in the gastric
juice were all found to be soluble in water. They were therefore
8

98 PHYSIOLOGICAL CHEMISTRY.

called soluble ferments as distinguished from the yeasts and the fer-
ments of acetic, lactic and butyric acids, and from the conditions of
their action Liebig was led to formulate the first general theory of
fermentation, the molecular vibration theory.

THEORIES OF FERMENTATION.

Liebig's Theory. Liebig advanced and maintained for years this
view : A ferment is a chemical substance whose particles or molecules
exist in a peculiar state of vibration, and in contact with other bodies
this ferment is able to set up similar states of vibration which result
in the breaking down of the bodies mixed with the ferment. Fer-
ments were considered along with bodies undergoing putrefaction, and
many such substances were supposed to be able to bring about real
fermentations. According to a somewhat older view ferments were
said to act by their mere presence; that is, they exerted what was
described as a catalytic action. No real attempt, however, was made
to define more closely what was meant by this catalytic or contact
action.

The Theory of Pasteur. The real nature of yeast as a vegetable
growth had finally become established. With this admitted Pasteur
advanced the proposition that alcoholic fermentation is a consequence
of the life of the organism in contact with sugar and away from the
air. Alcohol and carbon dioxide are products of the yeast cell metabo-
lism under these conditions. The cell, according to the Pasteur view,
must be furnished with a proper supply of oxygen and this, under the
fermenting conditions, it takes from the sugar, giving off carbon
dioxide as an oxidation product and producing alcohol at the same
time, as a result of the breaking down of the sugar molecule. Fer-
mentation is then to be considered from a purely biological standpoint
with alcohol and carbon dioxide as excretory and respiratory products
respectively. This Pasteur theory soon found favor with the majority
of scientific men and gradually supplanted the mechanical notion of
Liebig which could not be brought into accord with experience in other
lines. Although the Pasteur view that the yeast produces alcohol only
in absence of free oxygen was shown to be incorrect the theory com-
mended itself as otherwise satisfactory and tangible.

Following this a similar explanation was offered for the action
taking place in the formation of acetic acid, lactic acid and butyric
acid. Here microorganisms are also concerned. These live on cer-
tain substances and produce others as metabolic excreta. As to the
mechanism of this metabolism we know, of course, nothing; to describe

ENZYMES AND OTHER FERMENTS. 99

the products formed as excreta is perhaps not really warranted by what
is actually known. It must be remembered then that the term is used
in a broad and general sense only to indicate some kind of a metabolic
product.

The work of Pasteur gave an enormous impetus to the study of the
common fermentations, but it was evident that this biological expla-
nation was of no value in accounting for the changes produced by the
active agents described as diastase, pepsin, emulsin, etc. These, it was
pointed out, are as truly " ferments " as are yeast and the mother of
vinegar. To avoid confusion it became customary to speak of the
organised and unorganised ferments, or the insoluble and soluble fer-
ments. The term ensyme was later applied to these soluble unorgan-
ized agents of change, but this new expression did nothing toward
explaining the difficulty or toward relating the two classes of ferments.

Certain scientists from the start, however, refused to admit any
fundamental difference between the work of the yeast ferment on the
one hand and that of bodies like diastase on the other. Even after
the biological theory of Pasteur had become current Berthelot, Hoppe-
Seyler and other chemists of prominence maintained that the living
cell ferments are active because they secrete soluble or enzymic bodies.
In the one case the actual " fermentation " takes place within the cell,
as appeared to be the fact with yeast; in other cases enzymes are pro-
duced by cells and thrown off to do their work elsewhere. This is
true, for example, in the stomach where certain groups of cells produce
the active ferment pepsin which, however, does its work of dissolving
coagulated protein, or digesting it, outside the cells themselves. In
germinating barley the living cells secrete diastase which may be
leached out and used to digest starch of other grains. If not leached
out the diastase gradually digests the starch of the barley kernel itself,
unless the action be checked by heat or other means.

The Work of Buchner. In principle, therefore, the two kinds of
ferment action were held to be alike, but although many attempts were
made no chemist succeeded in isolating the assumed enzyme from the
active cells. Repeated failure in this direction only served to strengthen
the belief of the advocates of the vital theory according to which alco-
holic and similar fermentations by fungi are processes which cannot
be thought of dissociated from the function of life itself. But finally
the problem was solved by the German chemist Buchner, who in 1897
succeeded in isolating the active enzyme from yeast cells and in quan-
tity too. This enzyme he called symase; it was found to be as active
as the yeast itself and to do all that could be expected of yeast. It has

IOO PHYSIOLOGICAL CHEMISTRY.

since been produced on the commercial scale in the endeavor to sup-
plant the use of yeast in practice. More recently it has been shown
that other ferment cells secrete enzymes and it is possible that all the
so-called organized ferments work in this way; but the isolation of the
soluble active principle seems to be very difficult in most cases.

All this, however, affords us no real insight into the nature of the
ferment process. We have as yet no satisfactory theory as to how
these active chemical principles behave in the breaking down of other
organic substances. It has not been found possible to prepare any
enzyme in a condition of even approximate purity and all analyses
made of such substances are doubtless wide of the truth. These
analyses appear to show that the enzymes are of protein character, but
the impurities in the products analyzed may be responsible for this
indication. With this lack of knowledge regarding the chemical com-
position of the ferments it is naturally impossible to offer a chemical
explanation of how they act. It is the effects only that we are familiar
with and all our classifications are practically based on what the fer-
ments can do rather than on what they are. It is known that all
ferments are destroyed by heat and by the action of even rather weak
acids and alkalies. In this they resemble the cells that produce them.
While the true ferments or enzymes are apparently complex chemical
substances their formation is due in every case, as least it so appears,
to cell action. They are organic, but not organized; yet they possess
many of the properties of organized bodies. On the other hand cer-
tain finely divided metals, especially colloidal platinum, bring about a
number of reactions which were long supposed to be characteristic of
the true ferments; these reactions are further modified or suspended
by the same substances which modify or suspend the ferment actions
in question. Based on this behavior it has been attempted to relate
the true ferment action to the " catalytic " action of the " inorganic "
ferments. All the ferments seem to have the power of decomposing
hydrogen peroxide in quantity or catalytically, and this property has
been considered as perfectly typical or characteristic. The addition of
a number of mineral substances interferes with this catalytic decom-
position ; in this respect the action of prussic acid is remarkably ener-
getic. It has been found that a minute trace of colloidal platinum in
dilute solution decomposes a greatly excessive amount of the peroxide,
and further that extremely dilute prussic acid or corrosive sublimate
checks the reaction here just as with the true ferment. These analogy
reactions are very suggestive, even if they do not explain.

Considering the enzymes as catalytic agents it is to be noted that their

ENZYMES AND OTHER FERMENTS. IOI

distinctive function is, therefore, to hasten certain changes which
would take place without them, but, in many cases, with extreme slow-
ness. They have not only the power of hastening or effecting decom-
positions, as in the alteration of sugars or starches, but also of effecting
many syntheses. For example, maltase has the power of bringing
about the conversion of glucose into isomaltose. In some cases the
enzymes act to retard, in place of hastening reactions, and in a great
number of instances they seem to act as aids to other catalyzers. In
this sense they are spoken of as co-enzymes, kinases or activators.

Enzymes consist of very large molecules which are usually unable
to pass through animal membranes or fine porcelain filters. In cases
where they can be so filtered the rate of passage is very slow. In addi-
tion to large size and probably complex structure the enzymes seem
to possess a certain sort of specificity. That is, their activity is exerted
in certain directions, or on certain compounds only. The enzyme
which aids the conversion of starch into sugar is inactive as far as the
digestion of albumin is concerned, although the reactions have much
in common. But the specific behavior does not end here. Certain
enzymes will effect a decomposition in one form of a glucoside, but
not in its optical isomer, and in a large number of reactions it has been
found that pancreatic extracts will hydrolyze certain artificial poly-
peptides, but not their isomers, or related bodies. Such behavior points
to a peculiar chemical structure on the part of the enzyme which must
bear some relation to the structure of the thing acted upon, or the
" substratum," as it is frequently called. Following out this idea there
has been no little speculation as to the manner in which the enzymes
hasten reactions. It appears that the enzyme, through its structural
configuration, unites with the substratum in such a way as to produce
a new compound which yields the same end products as the substratum
alone would yield, but much more rapidly. In the decomposition to
furnish the end products the enzyme group is liberated to combine with
a new portion of substratum, and so on, until the reaction is complete,
or has reached a condition of equilibrium. It has been suggested that
enzymes act as very weak acids or weak bases, or that they combine
both properties as do the amino acids, and through this behavior they
are able to unite with corresponding groups of the substratum. There
is evidence that in a number of such combinations studied the reaction
follows the mass action laws with a fair degree of closeness.

102 PHYSIOLOGICAL CHEMISTRY.

CLASSIFICATION OF THE FERMENTS.

With our present knowledge of ferments they are most satisfactorily
classified according to the character of the .decompositions they effect.
Two distinct kinds of action are easily recognized. Many ferment
changes are clearly hydrolytic ; that is, the reaction follows through the
addition of a molecule or more of water to one substance, causing it
to break up into smaller groups. In other cases the reaction appears
to be in the nature of an oxidation process in which the ferment causes
or brings about the addition of oxygen to convert one substance into
another. Some authors limit the true ferment reactions to changes
which may be referred to one or the other of these heads. But there
are a great many decompositions which, while they may not be so
clearly defined as those just mentioned, must still be looked upon as
of ferment origin. These are produced by bacteria, and in all proba-
bility by the enzymes secreted by bacteria. It will be well therefore
to add a third general group to make the classification complete.

KINDS OF FERMENT REACTIONS.

We may make three general divisions of the ferment changes, as
follows :

A. Hydrolytic Reactions.

B. Oxidation Reactions.

C. Bacterial Decompositions.

A brief discussion of the more important changes coming under
each one of these heads will now follow.

A. HYDROLYTIC REACTIONS.

The most important of our ferment reactions, with one exception,
perhaps, and at the same time the most thoroughly studied, the changes
involving hydrolysis have long claimed the attention of chemists. The
true nature of some of these reactions is easily recognized and the
earlier workers in this field were able to compare the behavior of the
enzymes in question with that of dilute hydrochloric or sulphuric
acid. In other very important cases this analogy is far less readily
pointed out and it remained for recent workers to satisfactorily estab-
lish the true relations. When malt digests starch or when certain
enzymes convert the malt sugar formed into glucose the general nature
of the changes, as requiring the addition of water, may be shown
without difficulty. But with the behavior of pepsin in digesting pro-
tein we have more difficulty. Here the reaction is not so easily fol-
lowed, and the quantitative relations between the original substances

ENZYMES AND OTHER FERMENTS. 103

and the products formed are more complicated than is the case with
the carbohydrate decompositions. However, these reactions likewise
have been shown to involve true cases of water addition and therefore
may be properly grouped with the carbohydrate reactions as hydrolytic.
This hydrolytic ferment activity is exhibited mainly in the following
directions :

1. In the modification of carbohydrates as illustrated by the sac-
charification of starch and further changes in the sugar thus formed,
and in other sugars.

2. In the breaking down of glucosides.

3. In the splitting of fats.

4. In the digestion of proteins.

5. In the so-called fermentation of urea.

Some of these reactions may be represented by definite equations.
In general they correspond to the changes produced in the same sub-
stances by weak acids with some variations in the details. The salient
points will be indicated here, leaving in most cases the fuller discussion
for following chapters which deal with the details of the digestion
phenomena.

CHANGES IN CARBOHYDRATES.
Amylase or Diastase. Certain enzymes convert starch paste into
malt sugar by a reaction which is indicated by the equation :

( CjjH^oO^) n + (HoO)ra= (C^H^OiOn

The enzymes here active are usually described as diastases or amylases,
the terms being employed in the plural, since the action is not confined
to a single substance. Of these two terms the word diastase is fre-
quently employed in the broad sense to include all the enzymes which
act on the starches and sugars formed from them, while the term
amylase is employed to describe the enzyme which changes starch into
malt sugar. In this sense it will be used here. In nature ferments
of this character are very widely distributed and serve very important
functions. They are active in the changes going on in the vegetable
kingdom during the growth of plants and the ripening of fruits, as
well as in the germination of seeds. On the commercial scale malt
represents the best known diastase-containing substance. In the ani-
mal body similar substances are found in the saliva and in the pan-
creatic secretion. The first of these is called salivary diastase or
ptyalin and the second pancreatic diastase or amylopsin.

These diastases have never been secured in anything like pure con-
dition. Very active solutions which digest starch quickly may be

104 PHYSIOLOGICAL CHEMISTRY.

obtained by extracting ground malt with water, which will be illus-
trated later. These solutions may be concentrated at a moderate tem-
perature, but the activity of the enzyme is destroyed by heat. A
stronger product may be secured by extracting with 20 per cent alcohol
and precipitating the solution so obtained by absolute alcohol. This
precipitate in turn may be redissolved in water and precipitated again
with strong alcohol or with ammonium sulphate, to secure a purer and
more active product.

Besides the well-known ferments in malt, in the saliva and in the
pancreatic secretion the following may be mentioned here. By many
authors the active substance in the liver which converts glycogen into
glucose is supposed to be a form of diastase. Others hold the conver-
sion of sugar into glycogen and the subsequent and gradual formation
of sugar from glycogen to be specific vital functions performed by the
liver cells. The name cellulase or cytase is given to a ferment body
which is found in many vegetable substances and which has the power
of converting cellulose into sugar. Inulase is the enzyme which acts
on the peculiar starch known as inulin found in many vegetable sub-
stances, converting it into fructose. Inulase does not appear to act on
ordinary starch and on the other hand, malt diastase is not able to
convert inulin into sugar. Pectinase is another little known vegetable
enzyme which converts the so-called pectin jelly substances into a
reducing sugar. The original pectose in the seed or fruit is first
changed into pectin by a kind of coagulating enzyme called pectase.
The true behavior of these bodies is not yet fully known.

Maltase or Glucase. The sugar formed by amylase from starch
is known as maltose. This is a primary product and may readily be
further converted into glucose by another enzyme occurring in malt
and properly known as maltase. The nomenclature of these enzymes
is unfortunately somewhat confused. An effort has been made to
name them systematically, using in each case the name of the carbo-
hydrate or other body on which the enzyme acts, as the first part of
the descriptive term, to be followed by the suffix ase. Thus amylase
refers to the enzyme acting on amy lose or starch and maltase to the
enzyme which acts on maltose or malt sugar. But many authors do
not follow this system consistently; hence we have as describing the
same ferment the term glucase in use, since glucose is the product
formed. It is preferable to employ the first designation or maltase.
This enzyme belongs to the class of so-called inverting ferments which
convert disaccharides into monosaccharides. In this special case malt
sugar yields glucose :

ENZYMES AND OTHER FERMENTS. 105

CuHaA, + H20 = 2C6H12Oc.

This maltase is found not only in malt extract, but in various yeasts
and elsewhere in the vegetable kingdom. It is also present in saliva,
but in small amount, in the pancreas, the liver and in the blood. The
formation of glucose in most cases is probably a secondary reaction,
maltose being formed first as the primary product. The general im-
portance of this reaction will be pointed out later, as it plays a very
essential part in the digestion of the carbohydrate foods.

Lactase. In analogy with the conversion of malt sugar into glucose
we have the conversion of its isomer, lactose or milk sugar, into two
monosaccharide groups. This is accomplished by the ferment called
lactase which is found in several kinds of yeast, and which appears to
be distinct from the maltase just described. The change of milk
sugar is represented by this reaction :

CaH^O* + H20 = CH^O, + CH^O..

Glucose Galactose

Lactose and glucose have nearly the same specific rotation, [oc]z)=52.5°
for the first and 53 ° for the second, while for the galactose it is about
83 °. The inversion may be readily followed by the polariscope,
therefore.

As to the distribution of this enzyme in nature there is still some
dispute. According to some authors lactase is not present in the gas-
tric juice or in the pancreatic secretion, but other investigators have
reported finding it in both secretions. It was formerly held that the
disappearance of milk sugar in the body is due largely to bacterial
actions, as some of these organisms are able to secrete an enzyme
which acts on the sugar.

Invertase or Sucrase. One of the most common and important of
these enzymic reactions is the inversion of cane sugar, forming glucose
and fructose.

C12H22On + H20 = C6H1208 + C.HuO..

Glucose Fructose

The name invertin or invertase has been given to the enzyme which
accomplishes this, but sucrase would be in better accord with the gen-
eral nomenclature. The presence of this inverting ferment in many
kinds of yeast has been long known. The yeast cell alone is not able
to convert cane sugar into alcohol and carbon dioxide; an inversion
must first be brought about in some manner. In old yeast or in yeast
in which the cell has been destroyed by heat or by mechanical means
the inverting enzyme seems to be present in greatest abundance.
Invertase is found in various animal secretions, especially in the

106 PHYSIOLOGICAL CHEMISTRY.

intestinal juice. The inverting power of this secretion is marked,
while with the pancreatic secretion the inverting power is much less
pronounced. In the gastric juice the inverting enzyme is said to be
present in some amount and is sufficient to change part of the cane
sugar of the food independently of the acid likewise present.

The blood does not appear to contain this invertase, since a solution
of cane sugar injected into the veins is eliminated later by the kidneys
unchanged. If injected into the portal vein, and thus made to pass
the liver, inversion takes place rapidly, as that organ possesses the
enzyme in quantity.

Many of the higher as well as the lower plant organisms contain
invertase, which accounts for the change of the cane sugar into invert
sugar in certain cases. In general this reaction may be easily fol-
lowed by the polariscope, as the strong dextro-rotation of cane sugar
gives place to the levo-rotation of invert sugar. It is possible to make
a fairly pure invertase solution from some kinds of yeast, and such a
solution has certain practical applications in analytical investigations.
By extracting yeast with thymolized water a solution is obtained which
rapidly inverts cane sugar, but which is practically without action on
malt sugar or milk sugar and which, at the same time, will not induce
alcoholic fermentation. This property of the yeast extract is made
use of in the determination of cane sugar in presence of the others just
mentioned.

GLUCOSIDE REACTIONS.

For our purpose it will not be necessary to go into many details here.
A few decompositions only need be mentioned by way of illustration.
The glucosides are peculiar compounds which may be looked upon as
more or less complex ethers of glucose. They are decomposed in
various cleavage processes, with the separation, usually, of glucose as
one of the constituent products.

Emulsin. The best known reaction in this group is that which
takes place spontaneously in the crushed bitter almond. Along with
other substances this kernel contains a characteristic nitrogenous gluco-
side known as amygdalin and the enzyme called emulsin. In presence
of water the amygdalin breaks up in this way :

CJH^NOa + 2H20 = aQH^Oe + HCN + CGHBCHO,

that is, glucose, hydrocyanic acid and benzoic aldehyde are formed.
In the uncrushed dry almond this reaction does not take place because
the enzyme and glucoside are not in direct contact, but are contained
in different cells. The same result is accomplished by distillation of
the bitter almond with dilute acids.

ENZYMES AND OTHER FERMENTS. 107

Similar reactions are observed with salicin,

C13H1S07 + H;0 = C.HuO, + C6H4.OH.CH2OH,

Saliginin

and with coniferin, arbutin and other glucosides. A related ferment,
known as myrosin, converts the potassium myronate found in black
mustard into allyl mustard oil, C3H5NCS, glucose, and potassium acid
sulphate.

THE SPLITTING OF FATS.
The general reactions of fats have been already referred to and it
has been shown that in general they may be broken up by the action
of water in the form of superheated steam :

C3H5(CnH2„.102)s + 3H20 = C3H503H3 + 3^CnU2n^02.

The action of the pancreas in the emulsification of fats was recognized
as early as 1834 and in seeking for the cause of this it was finally
found to depend on a ferment reaction, and subsequent soap formation.
Lipase or Steapsin. The active principle in the pancreas which
accomplishes this fat splitting was first called steapsin and afterwards
lipase. The details of its behavior in the digestion of fats will be
explained in a following chapter. Besides its constant occurrence in
the pancreas, it has been found in the blood, the liver and the kidney.
More recently the existence of lipase in many vegetable substances has
been observed and thoroughly studied. It has been found that it
hydrolyzes some of the simpler ethereal salts very readily and on this
behavior is based a method of recognition of convenient application.
Of these ethereal salts ethyl butyrate is possibly the best, as it suffers
but very slight change by the action of water alone at ordinary tem-
peratures. The fat-splitting power, or enzyme strength, of various
extracts may be compared by noting the amount of the ethyl butyrate
decomposed in a given time under the influence of the extract. The
extent of hydrolysis of the ethereal salt is determined by titrating the
butyric acid liberated with dilute alkali.

PROTEOLYTIC REACTIONS.

While it is not possible to write equations illustrating accurately the
absorption of water in the digestion of proteins, as may be done for
the carbohydrates and the fats, yet there is abundant evidence to show
that water addition is in most cases the characteristic preliminary
change here also. The action of superheated steam has been referred
to in a former chapter, but at the ordinary temperature certain pro-
teolytic changes take place which are the results of enzyme action. At

108 PHYSIOLOGICAL CHEMISTRY.

least three of these changes have been thoroughly studied, and are of
great importance in the digestion of foods.

Rennet or Rennin. It has long been known that a certain product
found in the stomachs of young animals and especially in the calf's
stomach, has the power of clotting milk rapidly, which property has
been applied in the manufacture of cheese. The same substance is
found also in the pancreas, and the same or a quite similar enzyme
in a number of plants. In fact, this curdling or clotting ferment, like
others already described, is quite widely distributed in nature. As
occurring in the stomach it is mixed with another ferment, which will
be described below, known as pepsin. The two substances are appar-
ently quite distinct from each other and may be more or less perfectly
separated. Some chemists are, however, inclined to consider them as
essentially similar.

Rennet acts on the protein substance casein, throwing it into a coag-
ulated or clotted form. The chemistry of the reaction is obscure and
not thoroughly worked out. The essentials of what is known about
it will be given later. It is possible to obtain an active extract from
the stomach of the calf or young pig which may be kept indefinitely
and used for cheese making or other purposes. Formerly in the cheese
industry small fragments of the dried calf's stomach, preserved for the
purpose, were mixed with the milk and stirred about to induce coagu-
lation. At the present time a liquid extract is made on the commercial
scale by the action of an appropriate solvent on the cleaned stomach.
Glycerol may be used, or water plus a small amount of salicylic acid
to prevent putrefaction. In some European countries certain plants
have been employed in the place of animal rennet in the cheese indus-
try. Rennet works well in an acid medium and is easily destroyed by
alkalies.

Pepsin. The best known and most thoroughly studied of the pro-
teolytic enzymes is pepsin which has the power of digesting coagulated
albumin in an acid medium. It may be obtained best from the mucous
membrane of the hog's stomach by extraction with acidulated water
or glycerol. In the stomach it appears to exist as a propepsin or
zymogen, in which condition it is known as pepsinogen. The action
of acid converts this into the true ferment. Pepsin is very sensitive
to the action of alkalies which even in weak solution destroy it or
materially lessen its power of dissolving protein. In presence of weak
acids, preferably hydrochloric acid of o.i to 0.2 per cent strength, it
forms from the native or coagulated proteins the derived products
known as albumoses and peptones. This change is unquestionably

ENZYMES AND OTHER FERMENTS. IO9

associated with the addition of a number of molecules of water to the
original protein group.

Commercial " pepsin " appears in commerce in the form of powder
or scales. The latter are obtained by drying the extract from the
glands of the stomach on glass plates. The product is far from pure,
as it contains a large excess of other extractives. Yet, as now made,
one part by weight of the scale or powder is capable of digesting or
rendering soluble two to four thousand parts of coagulated albumin in
the form of hard-boiled eggs. In an experimental way products of
enormously greater activity have been prepared; it is said that one
part of a dry pepsin may be made to dissolve three hundred thousand
parts of coagulated egg albumin. The relative strengths of pepsin
products are always compared by noting the amount of egg albumin
or washed fibrin which they will digest in an acid medium of definite
concentration.

Pepsin, like most of the enzymes, is precipitated by alcohol. In
aqueous solution with a little acid it is most active at about 400 C,
and loses its power at about 560. In the dry condition it withstands
perfectly a much higher temperature. While hydrochloric acid is
usually employed as an aid to pepsin digestion, other acids may be used
with equally good results. Oxalic acid, lactic acid and formic acid
work well, but the action with acetic and propionic acids is weak. In
presence of alkalies there is no activity and certain salts also interfere
with the digestive power.

Through fractional precipitations and by other means many attempts
have been made to obtain a " pure " pepsin. The strongest, that is, the
most active, products so secured have been analyzed. The results do
not differ greatly from those found on analyzing the proteins, yet with
some of these products it is not possible to obtain the ordinary protein
reactions. We have no clew to the real composition of the substance.
It is not at all diffusible through parchment and must have a high
molecular weight.

It is stated above that pepsin and rennin are possibly identical substances. The
view commonly held by the majority of chemists and physiologists has been that
they are distinct ferments produced possibly by different regions of the stomach, but
in late years a mass of evidence has been accumulating which appears to throw
doubt on this notion and suggest the perfect identity of the two proteolytic enzymes.
The chemists of the Pawlow school have been particularly active in advancing this
theory. According to them an extract which shows the digesting power will also
show the milk curdling action. If it is strong in the one case it will be found
strong in the other if the conditions are made right. This amounts to saying that
the same enzyme does the two kinds of work, but the conditions of reaction, con-
centration, salt content, etc., must be different in each case. A commercial rennet,

IIO PHYSIOLOGICAL CHEMISTRY.

for example, if largely diluted with 0.2 per cent hydrochloric acid, will show a strong
proteolytic reaction, while without such dilution it may appear quite inactive. It is
held further that the milk curdling ferment of the pancreatic extract is probably
identical with the trypsin to be now described.

Trypsin. One of the most active and important of the body fer-
ments is the substance which occurs in the pancreas and known as
trypsin. It may be extracted from the minced organ in a variety of
ways and in crude form is a commercial product. In its action it bears
some resemblance to pepsin, but works under different conditions.
While pepsin digests protein compounds in dilute acid medium trypsin
is most active in presence of weak alkali, preferably sodium carbonate.
Action may be observed, however, in neutral solution and even in pres-
ence of a trace of acid. In its hydrolysis of proteins trypsin goes
farther than pepsin. The action of the latter, under ordinary condi-
tions, ends with the production of albumoses and peptones, while the
pancreatic enzyme carries the splitting process to the extent of pro-
ducing a number of comparatively simple amino acids, and the hexone
bases. In this respect the behavior of the trypsin is comparable with
that of weak sulphuric acid when heated with the protein bodies. As
already pointed out in a former chapter this acid resolves the proteins
into complexes which may be considered as the constituent groups of
the large molecule. The trypsin digestion may be carried far enough
to leave products which fail to show more than a very faint biuret
reaction. This reaction, it will be remembered, persists as long as
anything having the characteristics of the original protein substance
remains. From all this it is evident that the trypsin is a hydrolyzing
agent of marked power.

Of the real nature of the enzyme nothing is known. It has never
been isolated in a condition of even approximate purity. The pan-
creatic extracts of the market contain the enzymes acting on fats and
carbohydrates as well, in addition to a very large amount of other
matter. The active ferment is very soluble in water and in dilute
glycerol or dilute alcohol, but in strong alcohol or glycerol it is insol-
uble. In presence of weak hydrochloric acid trypsin is quickly digested
or destroyed by pepsin, and at temperatures much above 500 C. it soon
becomes inactive. The temperature optimum is probably about 40 °
to 450, in weak alkaline solution, but the statements in the literature
on this point are somewhat discrepant.

Erepsin. In this connection another peculiar ferment, which in
some respects resembles trypsin, must be mentioned. Erepsin is found
in the walls of the small intestine and is concerned in the final splitting

ENZYMES AND OTHER FERMENTS. I I I

of protein derivatives. It acts on proteoses and peptones, essentially,
and carries the hydrolysis to the formation of comparatively small
amino acid groups, that is, to practically complete hydrolysis.

THE UREA FERMENTATION.
Urease and Other Ferments. Urine exposed to the air soon
becomes alkaline and the presence of ammonia is easily shown. This
behavior is due to the formation of ammonium carbonate from the
urea by a reaction which may be expressed in this way :

(NH2)2CO + 2H20 = (NHJ2C03.

The agency concerned in this addition of water molecules was for a
long time in doubt, but investigation finally showed it to be a case of
ferment action. In all urines undergoing this change numerous bac-
teria are present and by separating and making pure cultures of these,
several species have been found which are capable of decomposing pure
solutions of urea. The name micrococcus urecc has been given to one
of the most active of these bacterial organisms. It has been found,
however, that the action is certainly due to a soluble product or enzyme
secreted by the bacteria, since it may be brought into solution. This
solution, after the most careful filtration even, is very active when
properly made and will quickly induce the ammoniacal decomposition
in urea solutions.

The name urease has been given to this soluble ferment, which must
belong to the hydrolytic group. It is active up to about 500 C. and is
much more stable in presence of alkalies than with acids, as would be
supposed from the reaction it produces. The enzyme is not readily
extracted from the living bacterial cells ; these must first be destroyed
or allowed to die out in process of making strong cultures. The cells
holding this enzyme are very widely distributed in nature, being found
in the air, in most soils and in river water. This accounts for the
usually rapid fermentation of urine.

B. OXIDATION REACTIONS.

Under the head of oxidation reactions it is very easy to include some
in which the essential phenomenon is clearly one of addition of oxygen
to the decomposing substance. This is certainly the case in the pro-
duction of acetic acid from weak alcohol. In other cases, however, the
actual nature of the chemical change which occurs is more obscure and
the classification of such reactions as oxidation reactions is possibly
open to doubt, as will appear below. In some cases the classification

112 PHYSIOLOGICAL CHEMISTRY.

appears very arbitrary, as the grouping of the alcoholic fermentation
among the oxidation processes illustrates. But there is sufficient rea-
son for this to justify the place the reaction is given.

ALCOHOLIC FERMENTATION.

As mentioned at the outset the phenomena of alcoholic fermentation
were the first to claim attention and many of the fundamental condi-
tions were empirically established long before the part played by yeast
in the process was recognized. After the investigations of Pasteur
our knowledge in this field rapidly widened.

Yeast. The common agent of alcoholic fermentation is known as
yeast, but under this term are included a very large number of really
distinct species. In fact several different genera may be and actually
are employed in practice. The following table gives an idea of the
relations of the commoner organisms classed among the alcoholic fer-
ments. The yeasts with many other cells are classed in a group of the
budding fungi, or Eumycetes, as distinguished from the fission fungi
or Schizomycetes.

Family Saccharomycetes

Genus Monospora Saccharomyces Schizosaccharomyces

|" Cerevisise
Species J Ellipsoideus

] Pastorianus
Land others.

A few molds, also, bring about alcoholic fermentation. We have
included here Mucor mucedo, M tic or racemosus, Mucor Rouxii and
others which are not in any way technically useful.

Ordinarily, however, we take as the type of a yeast the common beer
yeast Saccharomyces cerevisice, which is a cultivated species employed
in fermentation by brewers and distillers. In the natural wine fer-
mentation other species seem to be the most active. These are found
on the skin of the grape and hence find their way into the juice after
crushing. .S. apiculatus and vS\ ellipsoideus are the names of two of
the most important of the species active in this way. The common
beer yeast appears in the form of nearly spherical cells having a diam-
eter of 8 to 9 /a. It is active through a comparatively wide range of
temperature. In practice the fermentation of malt wort to produce
beer is carried out at a very low temperature, while a grain mash to
produce common alcohol or whisky is fermented at a high temperature.

ENZYMES AND OTHER FERMENTS. I I 3

In the one case, however, weeks are required to complete the change,
in the other one or two days.

Like most similar reactions brought about by living cells a limit to
the quantity of product formed is soon reached. With ordinary glu-
cose the reaction follows approximately according to this equation :

CeH^Oe = 2C2H60 + 2C02.

The mechanism of the reaction is not known, but it may be considered
as one of internal oxidation, since the carbon of the liberated gas is in
the fully oxidized condition.

As the alcohol formed accumulates a point is reached where the
activity of the yeast cell is impeded and the fermentation finally stopped.
This occurs when about 20 per cent of alcohol has accumulated as a
reaction product. Very strong sugar solutions do not ferment at all.
Indeed some of the common uses of cane sugar in preserving fruits
depend on this fact. Some of the conditions of fermentation may be
readily illustrated by simple experiments.

Experiment. Make a strong cane sugar syrup, by boiling or heating together 10
gm. of sugar and 10 cc. of water. Allow to cool and add about a gram of crumbled
compressed yeast, and then set aside for several days. The solution should be
found free from any signs of fermentation.

Experiment. Prepare a 20 per cent solution of commercial glucose and pour 50
cc. of it into a small flask. Add some yeast and allow to stand two days in a
moderately warm place. At the end of this time it should be found in active
fermentation, as shown by the escape of gas bubbles and the odor of alcohol. If
allowed to stand several days longer in the ordinary atmosphere the liquid in the
flask usually becomes sour from acetic fermentation.

Experiment. Prepare a tube with sugar solution and yeast as in the last experi-
ment. Close it loosely with a plug of absorbent cotton and heat to boiling, allowing
steam to escape through the cotton. If the tube is now left to itself for several
days it will be found that fermentation has not taken place, showing that heat
destroys the characteristic property of the yeast cell.

Experiment. Prepare another tube with sugar and yeast and add 10 cc. of strong
alcohol. Shake the mixture and allow to stand. No fermentation appears, as the
activity of the yeast cell is destroyed by alcohol. We have good familiar illustra-
tions of this in the self-preservation of certain " heavy " wines, as ports, sherries and
malagas, while " light " wines, which contain 10 to 12 per cent of alcohol usually,
must be kept tightly bottled for preservation.

Experiment. Test for Alcohol. We have many tests by which the presence or
formation of alcohol may be shown. The fermentation of a saccharine liquid is
followed by a lowering of the specific gravity as may be easily found by experiment,
and a practical quantitative test is based on this fact. A simple chemical test for
the presence of alcohol, which in most cases is sufficient, is the following: Add to the
clear liquid to be examined a few small crystals of iodine, warm to about 60° C,
and then add enough sodium hydroxide or sodium carbonate to produce a colorless
solution. An excess of the alkali must not be used. In a short time bright yellow
crystals of iodoform precipitate, easily recognized by their color and odor. Certain
other liquids give the same test.

9

114 PHYSIOLOGICAL CHEMISTRY.

Ordinary yeast contains the soluble ferment called invertase, which
has been already referred to. This may be shown by experiment,
as follows :

Experiment. Crush some yeast, add water and wash by decantation or on a
filter thoroughly. Then rub up the washed yeast with some water in a mortar and
add the mixture to a solution of pure cane sugar which has previously been treated
with a few drops of a strong alcoholic solution of thymol. 50 cc. of a 5 per cent
sugar solution will answer. The thymol prevents the action of the yeast cell fermen-
tation, but does not prevent the action of the invertase. The mixture should be
kept about 24 hours at a temperature of 400 to 500 C. At the end of this time it
is filtered and the filtrate tested for invert sugar by means of the Fehling solution.
Ether and chloroform are sometimes employed in place of the thymol; the latter
must be removed by heating before making the Fehling test.

Zymase. It has been intimated already that the activity of yeast
as an alcoholic ferment is due to the presence of an enzyme. This
fact, long suspected and much debated, was finally demonstrated by E.
Buchner, as explained above. Buchner rubbed the yeast with fine,
sharp sand and water and then subjected the mixture to great pressure.
The liquid pressed out was carefully filtered and was found to be as
active as the original yeast. The enzyme in it he called zymase. It
clings tenaciously to the yeast cell, hence the necessity of destroying
the structure by grinding with sand, and employing great pressure.

Zymase is not a very stable ferment and in the solution obtained is
soon destroyed by other ferments present. The yeast extract may,
however, be concentrated at a low temperature and obtained in dry
form which is more stable. Extracts made from yeast by simple treat-
ment with water may contain invertase but no zymase. It seems prob-
able that the ferment is not confined to the yeast cell. It has long been
known that many overripe fruits produce a small amount of alcohol,
even when the possibility of the presence of yeast cells is entirely
absent. This formation of alcohol was finally ascribed to the cell
activity of the fruits themselves, but since the work of Buchner it
seems more rational to refer the appearance of alcohol to the presence
of an enzyme in the ripe fruit.

It should also be said that sugar may be made to yield alcohol by a
much simpler process. It has been found that a sugar solution mixed
with a little potassium hydroxide and placed in bright sunlight yields
some alcohol and carbon dioxide. This is of course a purely chemical
decomposition, and suggests the possibility of chemical reactions in
other cases. The old notion as to the necessity of the presence of
living cells to break down the sugar is thus completely disproved.

ENZYMES AND OTHER FERMENTS. I I 5

ACETIC FERMENTATION.

In this a true oxidation takes place, the oxygen of the air being
employed to convert weak alcohol into the acid according to this
reaction :

CHcO + 02 = C2H402 + H20.

The active agent concerned in the fermentation oxidation is the cell
found in "mother of vinegar."

Mother of Vinegar is an old name given to the slimy scum or sedi-
ment which forms in weak alcoholic liquids that turn sour, in wine or
cider, for example. Microscopic examination shows this substance to
consist of minute cells which have received the name of Micoderma
a-ceti; more recently the name Bacterium accti has been given to the
plant organism. Thus far it has not been found possible to isolate
a soluble enzyme from the cell ferment. One may be present, but
attempts to obtain it have failed.

Besides this Bacterium aceti several other vinegar ferments are
known. Most of them float in the air, and when lodged in a weak
alcohol containing certain mineral substances produce a fermentation
quickly. A dilute aqueous solution of pure alcohol will not ferment
in the same way; the presence of various salts and organic matters in
addition is necessary. An experiment may be made to illustrate vin-
egar or acetic acid fermentation.

Experiment. If available a fruit juice, freshly expressed and left in contact with
the skin, should be allowed to undergo alcoholic fermentation. Or, a sugar solution,
as described some pages back, may be allowed to ferment. The weak alcoholic
liquid obtained in the case of the fruit juice will next turn sour from the pro-
duction of acetic acid by the action of the germs on the skin. In the case of
the alcohol from the sugar it may be necessary to add a little " mother of vinegar "
from a vinegar factory to induce the fermentation. Presence of the air is neces-
sary to complete the change. The acid strength of the product may be finally tested
by means of a standard alkali solution and phenol-phthalein.

THE OXIDASE ENZYMES.
We come now to a very brief consideration of an obscure but inter-
esting subject about which our knowledge is of comparatively recent
origin. In certain vegetable substances reactions occur which are
ascribed to the presence of a class of oxidizing enzymes called oxidases.
These changes are illustrated by the blackening of an apple, potato or
beet which is cut and exposed to the air. The cut surfaces soon turn
dark. If the same substances are thoroughly heated before the cutting
the color change does not follow. Potato or apple pulp speedily
darkens in the air, but if previously cooked the natural light color per-

Il6 PHYSIOLOGICAL CHEMISTRY.

sists. To account for these and many similar reactions it has been
assumed by many chemists that the fruits or vegetables in question
contain an oxygen-carrying enzyme and at the same time some chem-
ical substance on which this can act with the production of color, the
oxygen necessary for the change being taken from the air. The
action of this enzyme or oxidase may be shown in other ways, espe-
cially by the use of hydroquinol and pyrogallol, which substances yield
very dark solutions when oxidized. It is simply necessary to make
an aqueous extract of certain vegetables and fruits and add this to
the aqueous solution of the hydroquinol to produce the dark color.
Here the enzyme appears to be active enough to carry oxygen to the
hydroquinol.

Laccase and Tyrosinase. These are the names which have been
given to two of these oxidases. The first was originally found in the
sap of the Japanese lac tree, which when expressed and exposed to the
air darkens and produces the well-known lacquer. The same laccase
is said to be one of the agents which brings about the darkening in
many other saps and juices. Tyrosinase acts on the phenol derivative
tyrosine which is found in traces in many vegetables and causes its
oxidation.

These two reactions may be taken to represent a wide range of
changes in which phenol bodies are concerned. In another group of
reactions aldehyde bodies are turned into acids, as happens to salicylal-
dehyde. It is possible that many of the obscure oxidative changes of
the animal body are brought about by enzymes of this type, but our
knowledge here is not very definite. It is known that extracts from
the liver and spleen have the power of changing hypoxanthine and
xanthine to uric acid, but of the more profound oxidations of the body
much less is known. Cohnheim has described a glycolytic ferment,
active in the combustion of sugar, when aided by a co-enzyme, or
activator, but the nature of the change is not one which can be clearly
explained.

Peroxidases. Recently the term peroxidase has been introduced to describe a
peculiar enzymic ferment which occurs in animal and vegetable cells, the striking
feature of which is to induce the oxidation of a great variety of substances through
hydrogen peroxide. Milk, for example, when fresh has the power of bringing
about the oxidation of phenol-phthalin to phenol-phthalein by hydrogen peroxide,
and the same behavior has been observed in other animal secretions. The intensity
of the oxidation is much increased by the presence of various other substances
which serve as accelerators. As hydrogen peroxide is very readily formed by a
wide range of reactions, it is possible that it is produced in living cells, to undergo
immediate destruction through the activity of the ferment bodies. This may have
some bearing on the explanation of animal oxidations, but as yet our knowledge
on this point is scarcely beyond the speculative stage.

ENZYMES AND OTHER FERMENTS. 117

C. BACTERIOLYTIC PROCESSES.

The term bacteriolytic is applied to such fermentation-splitting proc-
esses as may be carried out by bacteria without the addition of oxygen.
In the acetic acid fermentation, which is likewise a bacterial process,
the presence of oxygen is necessary, but there are several somewhat
analogous reactions in which oxygen is not required and these are
included in the present group. It must be admitted of course that the
division is a perfectly artificial one based on convenience rather than
on marked differences in agents or products. Some of the reactions
classed here have long been described as fermentations and have been
studied in connection with the other common ferment changes. These
will be taken up first. But we have, in addition, further changes which
are certainly of the same general character and call for like treatment.

LACTIC AND BUTYRIC FERMENTATIONS.

Why milk turns sour spontaneously in warm weather is an old
question, but it was not satisfactorily answered until after the time of
Pasteur's pioneer labors. Following his work on the yeasts Pasteur
took up other problems of fermentation and pointed out the general
nature of the reaction by which the sour substance present, lactic acid,
is formed. He found the production of lactic acid to depend on the
ferment activity of certain microorganisms, which have later been
more fully described by bacteriologists.

Lactic Acid Bacteria. It was found that lactic acid is formed
from the simple sugars by a splitting process which for a long time
was illustrated by an equation supposed to represent the facts quanti-
tatively :

C6H1=O0 = 2C3H0O3.

It was also recognized that not merely one, but many species of bac-
teria are capable of decomposing sugar solutions in this way. Of these
the form known as Bacillus acidi lactici has been perhaps the most
thoroughly studied ; it appears to be always present in milk which has
soured spontaneously, and can be found in the air, especially of pas-
tures or cowsheds. Many soils also contain the organism. In no
case, however, is the reaction a perfectly sharp one; along with the
lactic acid other products are formed, acetic acid, alcohol, formic acid,
carbon dioxide and hydrogen being the most common. In some cases
the proportion of lactic acid is relatively small. The formation of
lactic acid may be illustrated by a laboratory experiment.

Experiment. To ioo cc. of 20 per cent cane sugar solution add an equal volume
i/f aqueous malt extract and 10 to 15 grams of precipitated chalk. Inoculate this

Il8 PHYSIOLOGICAL CHEMISTRY.

mixture with a culture of lactic acid bacteria and keep at a temperature of about
400 C. for some days. The chalk is necessary to take up the acid as fast as
formed; without it the fermentation soon ceases, as the ferment is extremely sensi-
tive to the action of free acid. The mixture must be shaken from time to time.
As the fermentation progresses the slightly soluble calcium lactate begins to sepa-
rate. In a good fermentation enough of this forms to fill the fermenting vessel
with a mass of crystals. These crystals are redissolved in hot water, and the solu-
tion filtered. The filtrate on concentration deposits crystals of calcium lactate,
Ca(C3H503)2.sH20, which may be collected and dried between folds of filter paper.
The free lactic acid may be obtained by decomposing the calcium salt with sul-
phuric acid in the proper amount and shaking out with repeated small portions of
ether. The lactic acid dissolves in the ether and is left when this is evaporated.
Zinc oxide may be employed in place of calcium carbonate to neutralize during
the fermentation. In this case zinc lactate forms, from which the acid may be
separated by dissolving the crystals in hot water and decomposing the solution by
means of hydrogen sulphide.

Several pure cultures of lactic acid bacteria can now be obtained for technical use.
For the rapid production of the acid Lafar recommends Bacillus acidificans
longissimus.

Pure lactic acid as prepared by fermentation is a thickish liquid, with
marked acid taste and but slight odor. It is optically inactive, but may
be resolved into active components by treatment with strychnine, which
crystallizes with the levo modification. This common fermentation
acid is employed for several purposes in the industries and is now com-
paratively cheap since the introduction of methods of fermentation
with pure cultures.

Lactic acid fermentations are concerned in many common opera-
tions. In the leavening of bread along with yeast fermentation there
is usually a bacterial fermentation with production of acid. In some
kinds of bread this is extremely important. In the preparation of
sauerkraut and many pickles a lactic acid fermentation is the charac-
teristic feature. Several well-known beverages produced from milk
are fermented in such a manner that they contain lactic acid; kephir
and kumyss are illustrations. Yeasts and the lactic acid bacteria work
together in many instances and symbiotic products are the rule, per-
haps, rather than the exception in fermentations. In the milk indus-
tries these mixed fermentations are apparently essential in the ripen-
ing processes, and in certain distillery fermentations with yeast a. lactic
acid fermentation is encouraged to prevent the growth and action of
the bacteria. This fermentation lactic acid is found also in the stomach
and the intestine. In the stomach the formation of any large amount
is usually impossible because of the presence of hydrochloric acid.
About o.i per cent of free hydrochloric acid is sufficient to impede the
lactic fermentation. Free mineral acids are not present in the intes-
tine; the organic fermentation acids may therefore be formed in appre-

ENZYMES AND OTHER FERMENTS. 119

ciable quantities. Fermentation lactic acid must not be confounded
with the isomeric sarcolactic acid found in the muscles.

Butyric Acid Fermentations. Another very important kind of
acid fermentation is that which results in the formation of normal
butyric acid :

CeH3208 = 2H2 + 2C02 -f C4H802.

As in the case of lactic acid this butyric acid fermentation is not the
result of the action of one organism only, but it may be produced by
several, and furthermore several by-products are always produced in
quantity. The above reaction is then merely a limit reaction, which is
approached but never absolutely realized.

In the milk fermentation the lactic acid or calcium lactate formed
may be further changed to butyric acid, the necessary ferment entering
from the air. Most river waters contain butyric acid bacteria, which
bring about the characteristic fermentations when the water is mixed
with some sterilized milk, as in one of the common tests carried out in
the sanitary examination of water. Garden soils are also rich in some
of these butyric acid-producing organisms, and may be used in starting
a fermentation, as may be illustrated in this way:

Experiment. Mix 100 cc. of a 5 per cent glucose solution with four or five
grams of fibrin and heat to boiling. To the hot solution add a few grams of garden
loam and allow the liquid to cool rapidly. The bacterial spores resist the heat
while other forms succumb and are thus disposed of. Keep the mixture at a tem-
perature of about 27° to 400 C. Fermentation begins in about two days and is
assisted by neutralizing with a little sodium hydroxide from time to time. After
several days the presence of butyric acid may be shown by warming some of the
liquid with sulphuric acid, or with sulphuric acid and alcohol. In the latter case
the odor of ethyl butyrate formed is very characteristic.

Butyric acid in pure condition is a strongly acid liquid possessing a
rather disagreeable odor. It is frequently present in the stomach, but
its occurrence there is really abnormal. If the gastric juice contains
the proper amount of hydrochloric acid a butyric acid fermentation is
not possible. With diminished hydrochloric acid, however, bacterial
fermentations can take place. In the arts, while lactic fermentation
is desirable frequently, and encouraged, the butyric fermentation is
usually considered very objectionable and is prevented if possible.

Other Fermentations. It will not be necessary to explain at
length any other cases of bacterial fermentations, as these two just
given are sufficient for illustration. What is known as the mucous
fermentation sometimes takes place in saccharine liquids or in wines
which have not been completely fermented. A slimy mucilaginous
product is formed here which contains a kind of gum. Certain micro-

120 PHYSIOLOGICAL CHEMISTRY.

organisms have the power of decomposing cellulose and the operation
is called the cellulose fermentation. The products of this reaction
with certain bacteria are mainly gaseous, hydrogen and marsh gas pre-
dominating. Certain organisms are able to produce fatty acids also.
In the intestines of the herbivora changes of this character take place,
and the acids produced are doubtless of value in aiding in some of the
other digestive processes which take place there.

CHAPTER VII.

SALIVA AND SALIVARY DIGESTION.

It has already been said that the saliva contains an enzyme known
as ptyalin, the function of which is to begin the digestion of starchy
foods. It remains now to look into the nature of this process a little
more closely, and to study the conditions of this kind of fermentation.
The saliva as secreted by the three large pairs of glands of the mouth
is a thin liquid with slightly alkaline reaction. Because of the constant
presence of mucus and epithelial cells it is never clear but presents
always an opalescent appearance. The amount secreted daily varies
between i and 2 liters. In the last few years Pawlow has shown how
a normal saliva may be collected from animals.

In the older literature several complete analyses of saliva are given,
but less importance is now attached to these than formerly, since a
great degree of exactness is not possible in such tests and besides the
composition of the secretion cannot be a constant one. In the mean
the amount of water present is 99.5 per cent. In the 0.5 per cent of
solids about 0.2 per cent consists of inorganic salts and the remainder
of organic substances, including the ferment. Among the salts there
is a minute trace of potassium thiocyanate, KSCN, which may fre-
quently be recognized by the test with ferric chloride. It is not known
that this substance exerts any specific function, and in different indi-
viduals it is present in different amounts. Some of the important
properties of saliva may be illustrated by simple experiments.

Experiment. After washing out the mouth thoroughly with water chew a piece
of rubber or other neutral insoluble substance to stimulate the flow of saliva.
Collect 25 to 50 cc. in a clean beaker and after diluting with an equal volume of
distilled water allow to stand a short time to settle. Then filter through a small
filter paper into a clean vessel and use the filtrate for the following tests :

To a few cc. of the clear saliva in a test-tube add several drops of a dilute solu-
tion of ferric chloride. This gives a more or less marked red color from the
formation of ferric thiocyanate. A very strong reaction must not be expected.
Make a comparative test by adding a like amount of ferric chloride to dilute
solutions of potassium thiocyanate.

The addition of solution of mercuric chloride discharges the color. This test
is of value in distinguishing between a thiocyanate and a meconate, which sometimes
has value in medico-legal work.

Test the reaction of saliva with neutral litmus paper. It will be found slightly
alkaline. Now add two or thre< drops of dilute acetic acid and note that a stringy

121

122 PHYSIOLOGICAL CHEMISTRY.

precipitate of mucin separates. Filter off this precipitate and test the filtrate for
proteins by boiling with Millon's reagent or by the xanthoproteic reaction.

Make a thin starch paste, about a gram to 200 cc. of water, and observe that it
does not respond to the Fehling sugar test already described. Mix 10 cc. of this
paste with 5 cc. of the filtered saliva and warm to a temperature not above 400 C.
for about 15 minutes. At the end of this time apply the sugar test again. A
yellow or red precipitate will appear now, showing that the starch has been con-
verted, in part at least, into sugar.

The saliva alone fails to reduce the copper solution, as should be shown by trial.

Pour about 5 cc. of the clear saliva into a test-tube and boil a few minutes;
add the starch paste and allow to stand as in the above experiment. On testing
with the copper solution no sugar will be found, showing that heat destroys the
activity of the ferment.

The digesting power of the saliva is destroyed also by the addition of a small
amount of strong acid or alkali solution, which the student should prove by
experiment.

Saliva is practically without action on raw starch, as may be shown in this
way. Stir a small amount of uncooked potato starch into 5 cc. of saliva, and allow
to stand 15 minutes at 3S°-40°, and filter. Now apply the Fehling test, and note
the absence of precipitated copper suboxide.

THE CONVERSION OF STARCH.

The action of ptyalin on starch is a complicated one and in all details
cannot be satisfactorily described. In many respects the digestive
behavior of the enzymes of the saliva and of malt is similar to that of
weak acid. The complex insoluble starch molecule is in some manner
broken up and partly soluble bodies result. This change is at first
unaccompanied by hydration, but later the normal enzymic reaction of
water addition follows and the dextrin bodies first produced become
sugars. Malt sugar is formed first, and in the case of acids this gives
rise finally to glucose by further conversion. But with ptyalin the
main action seems to end with the production of maltose; at all events
no large amount of the hexose sugar is formed. A little maltase is
said to be present. Furthermore the whole of the starch is not brought
into the sugar condition; a portion remains in the form of a dextrin.
In an earlier chapter something was said about the character of these
dextrins.

In most respects the behavior of ptyalin is very similar to that of
malt diastase, which can be shown by a simple experiment with com-
mercial malt. This substance is usually made by germinating barley
and permitting the growth to continue some days, the barley in moist
condition being spread out on a so-called malting floor to encourage
the growth and prevent overheating. In the germination the enzyme
is developed, probably from a portion of the protein substance present.
When the action has gone far enough, which the malster recognizes

SALIVA AND SALIVARY DIGESTION. 1 23

by the appearance of the rootlet thrown out, the action is checked by
quick drying, leaving the diastase in permanent stable condition. This
malt is made in enormous quantities for use in breweries and distil-
leries. In the germinating seed in the ground the same enzyme is
formed which converts starch into soluble food for the young plant.

Experiment. Mix about 10 gm. of pale ground malt with 50 cc. of lukewarm
water, and allow the mixture to stand a short time, with frequent stirring and
shaking. Then filter and add the clear, bright filtrate to a thin starch paste made
of 10 grams of starch with 250 cc. of water. The starch paste must be cool when
the malt extract is added. Place the mixture on the water-bath and warm to 500-
6o° C, and maintain this temperature. Note that the liquid gradually becomes
thin and transparent. From time to time remove a few drops by means of a
pipette, and test with iodine solution. At first a deep blue color appears, but this
grows weaker, giving place to violet, then to yellowish brown and finally no color
is obtained, indicating completion of the reaction. The starch paste is first con-
verted into dextrin and finally into maltose. Evaporate the solution to a very
small volume and observe the taste and appearance of the residue. In the end
product there is usually about 80 per cent of maltose and 20 per cent of dextrin
when made at the temperature of this experiment.

It has been found in practice that the amount of malt sugar formed
depends on the temperature and duration of the digestion with diastase.
At a lower temperature with longer action the conversion of the dextrin
becomes more perfect. This corresponds with the behavior of the
pancreatic diastase which is active through a longer period usually than
is possible with the saliva.

BEHAVIOR OF THE DIASTASE.

The question of the identity of the malt diastase with that from
saliva is still a disputed one ; while some writers describe them as iden-
tical, others apparently find characteristic points of difference. The
behavior of saliva with various reagents has been pretty thoroughly
studied; stronger acids and alkalies have, of course, a destructive
action, but experiments seem to show that very weak acids favor rather
than retard the digestive power. When the acid strength is gradually
increased up to that of the gastric juice, the effect of the ptyalin on
starch paste grows weaker and finally becomes zero long before the
maximum acidity is reached. In the mouth the action of the saliva is
certainly largely mechanical, since the time for any other action is
entirely too short, but with the passage of the food into the stomach it
does not follow that all diastatic digestion ceases because of the acid
condition of that organ. After the beginning of a meal some time is
required for the commencement of hydrochloric acid secretion, and a
further time before enough has accumulated to seriously interfere with
the activity of the diastase. The effect of the acid is dependent on its

124 PHYSIOLOGICAL CHEMISTRY.

concentration, not on the gross amount present. Up to a concentration
of about o.oi per cent the acid seems to have but little inhibiting action.
Therefore while this amount of free acid is accumulating we may sup-
pose the salivary digestion to go on in the stomach. Later, with
increase in acid, the ptyalin disappears, possibly through gastric
digestion.

Many salts exert an influence on the rate of diastatic digestion.
Usually this is to retard the action, but sodium chloride and other
neutral salts in small amount have a beneficial effect. With other sub-
stances the action is generally unfavorable. Small amounts of protein
matter, or preferably the syntonin or acid albumins formed from the
proteins by combination with traces of hydrochloric acid, seem to
increase slightly the activity of the salivary diastase. This is a point
of considerable importance in explaining possibly the continuation of
the ptyalin reaction in the stomach. Acid combined with protein
behaves as free acid toward certain indicators, while with other indi-
cators it does not show. Starch digestion with saliva in a mixture
containing protein and hydrochloric acid, as indicated by dimethyla-
minoazobenzene,' cannot continue, but if the indication is merely by
phenol-phthalein the ptyalin action may still go on, since in this case
the acid shown may possibly be wholly or largely combined with pro-
tein substances. Recent investigations have shown that under such
conditions, which are probably duplicated in the stomach, the digestion
of starch may go on at practically the normal rate, the hydrochloric
acid being rapidly combined with protein, and therefore comparatively
inert with ptyalin. The alkalinity of human saliva is usually referred
to as due to the presence of sodium carbonate, but soluble phosphates
are present which may account for the reaction as shown by certain
indicators, especially by litmus. With phenol-phthalein the reaction
appears neutral ordinarily or even slightly acid. With the latter sub-
stance as indicator it is generally necessary to add a little alkali to
secure neutrality. With litmus as indicator the average alkalinity,
expressed in terms of Na2COs, is 0.15 per cent. This reaction seems
to vary with the time of day and is strongest before breakfast. Although
carbon dioxide is present in saliva, it probably occurs as bicarbonate
rather than as carbonate, which would account for the reactions
noticed.

Many soluble substances introduced into the blood in any way soon
appear in the saliva. This may be shown by an experiment which
illustrates also the rapidity of absorption.

SALIVA AND SALIVARY DIGESTION. 1 25

Experiment. Swallow about a gram of potassium iodide in a gelatin capsule.
In this manner the salt is gradually dissolved in the stomach without having come
in direct contact with the mouth. After a few minutes begin testing the saliva for
iodine. At first the tests are all negative, but in time a reaction appears on treat-
ing the saliva with something to liberate the iodine in presence of starch paste.
Solution of sodium hypochlorite may be used for this purpose. The time required
to exhibit this absorption and secretion with the saliva varies greatly in different
individuals.

CHAPTER VIII.

THE GASTRIC JUICE AND CHANGES IN THE STOMACH.

The gastric juice free from saliva and particles of food is a thin
liquid with specific gravity ranging from i.ooi to i.oio. It contains
besides certain enzymes some small amounts of protein matters, a little
sodium chloride and traces of other salts and free hydrochloric acid.
Lactic acid is also frequently present in traces. The older analyses of
human gastric juice, which have been frequently quoted, are mislead-
ing, as they were made with material containing saliva and food
products. By aid of a fistula it has been possible to obtain a fairly
normal secretion from certain animals, especially from the dog, and
much of our knowledge of the conditions of secretion and variations
in composition has been secured in this way. In this direction the
work of Pawlow has been of the greatest importance, and his experi-
ments have given us new ideas on the subject of the gastric secretion.
The physiologically important substances in the gastric juice are free
hydrochloric acid, pepsin, rennin, and a lipase.

The secretion is furnished by two kinds of glands known as the
pyloric glands and the fundus glands. Both groups of cells yield the
two enzymes, but the pyloric cells do not seem to furnish an acid secre-
tion. It is probable that certain of the fundus cells only are concerned
with the acid secretion. The gastric secretion is promoted by two
kinds of stimuli. Certain chemical substances when taken into the
stomach have the power of exciting a flow of the juice from the mucous
membrane, and are themselves not subject to gastric digestion. Small
amounts of alcohol, ether, spices and meat extracts act in this way.
But more important than this is the so-called "psychic" stimulus,
depending on the desire for food and the satisfaction derived from
partaking of it. The amount of the secretion varies with the nature
and kind of food.

THE DIGESTIVE AGENTS.

Origin of the Free Hydrochloric Acid. The material from which
the fundus cells produce the enzymes and the acid is the blood. But
this is always slightly alkaline and to account for the secretion of a
characteristic acid from such a source has long been a puzzle to physi-
ologists. Several hypotheses have been advanced, but these are all

126

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. 12J

more or less faulty. The difficulty is not with the liberation of hydro-
chloric acid, which is a purely chemical question, and one which may
now be explained, but with its secretion.

The blood contains always a small amount of sodium chloride and
an excess of carbonic acid. In a solution containing these two things
some double decomposition must take place with production of a little
free hydrochloric acid and sodium carbonate. According to the older
view hydrochloric acid is so much stronger than carbonic acid that the
liberation of the former from a chloride by the action of the latter is
impossible. But this view leaves out of consideration the effect of
a much greater mass of the weaker acid through which in fact a disso-
ciation of the chloride is to a certain extent accomplished. But,
granting this kind of a double decomposition, it is still beyond our
powers to explain how the free acid formed in the cells is able to pass
in one direction into the stomach, while the sodium carbonate produced
at the same time wanders in the other direction into the blood.

This acid is not liberated in constant amount at all times but its flow
is subject to the influence of the various stimuli referred to above.
The quantity present then in the stomach may vary from a mere trace,
or zero even, to a maximum. This maximum may be 0.5 or 0.6 per
cent of the liquid contents. It has usually been given as much lower.
How it is measured will be shown below. Just what is meant by the
term free hydrochloric acid will be presently explained.

The Enzymes. In an earlier chapter the general nature and beha-
vior of the three gastric ferments, the pepsin, rennin and the lipase was
pointed out. Whether the first two bodies are always secreted simul-
taneously and in corresponding amounts is not definitely known, but
that this is the case is often assumed; it will be recalled that the fol-
lowers of the Pawlow school consider the enzymes identical, as referred
to above. In fact, one of the clinical methods in use for the estimation
of "peptic" activity is based on the measurement of the rennet action
through milk coagulation. The process seems, however, of doubtful
value. In speaking of gastric digestion in adults we are concerned
mainly with what takes place through the action of pepsin, which will
now be discussed. A briefer discussion of the other ferments will

follow.

PEPTIC DIGESTION.

In presence of free acids of the so-called "stronger" type pepsin
has the power of effecting remarkable changes in protein substances,
which have been the subject of numerous investigations. In the
stomach hydrochloric acid only comes into play and it first gradually

128 PHYSIOLOGICAL CHEMISTRY.

converts the proteins present into acid-albumins or syntonin bodies.
This is the preliminary stage in the digestion of these food substances
and must be accomplished before the other steps in the stomach are
possible.

In this reaction the hydrochloric acid enters into a kind of combi-
nation with the protein. The product has just been spoken of as acid
albumin, but it is evidently through the basic character of the protein
complex that the combination can take place. The protein here is in
effect a very weak base. The amount of acid which may be so held
is considerable, and may in fact amount to 5 per cent or more of the
weight of the protein. With certain of the derived protein products
the weight of hydrochloric acid combined is even larger, at times as
much as 15 per cent of the protein weight being so held. These
derived products are hydrolysis products with smaller molecular weight
and evidently more available amino groups to hold the acid.

It is generally held, as just stated, that this acid fixation is the first
step in the gastric digestion, although some authors claim to have rec-
ognized the albumose stage as the primary one in some cases. While
this acid reaction may take place in pure aqueous-acid solution, it is
much more quickly reached in presence of pepsin, as is the case in the
stomach. Experiments with artificial mixtures show that the combi-
nation then is almost immediate, as is made evident by the loss of
" free " acidity, to be explained below. Then the hydrolysis goes on
and the various derived products mentioned in a former chapter are
produced. In the gastric digestion it is likely that the cleavage does
not usually extend beyond the production of the secondary albumoses ;
that is, not much real peptone is formed in the time naturally con-
sumed in normal digestion. In practice the larger part of the peptone
production is doubtless left for the trypsin conversion.

Hydrolytic cleavage beyond the acid albumin stage is favored by
abundance of free acid, but in absence of this it still goes on. In
actual digestion the whole of the hydrochloric acid may be combined
with albumin, leaving some of the latter in excess even, yet primary
and secondary albumoses will appear, leaving the remaining native
albumin to begin the reaction later. In other words, it is not neces-
sary that one stage of the digestive process must be complete before
the following may begin. All these reactions may be in progress
simultaneously, and if needed hydrochloric acid will be taken from the
advanced products to hasten the beginning hydrolysis of the protein
yet to be digested. It has in fact been shown that hydrochloric acid
in combination with leucine and other amino acids, which it will be

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. I 29

recalled are advanced products of proteolysis, will still digest fresh
albumin rather rapidly, but not as well, of course, as the equivalent
amount of free acid.

The amount of acid taken up by an original native protein substance
during gastric digestion has been referred to already. Starting with
a given weight of pure protein, hydrochloric acid may be added until
a distinct reaction is shown by dimethylaminoazobenzene. This indi-
cator behaves as a very weak base and will show no free acid until the
protein, considered as a basic body, is saturated. As digestion pro-
ceeds more and more acid must be added to complete the saturation.
The amount of acid which may be so added is to some extent a measure
of the advancing cleavage. With phenol-phthalein, which is a very
weak acid, the whole of the hydrochloric acid behaves as " free " acid.
The acid joined to the protein is "combined" acid as far as the
dimethylaminoazobenzene is concerned and this indicator may be used
to show the excess of free acid in examinations of stomach contents.
More will be said about this below.

As hydrolytic digestion goes on the amount of water combined
becomes appreciable, and finally may reach three or four per cent, as
has been determined by direct experiment. In a series of investiga-
tions carried out in the author's laboratory with casein the water addi-
tion amounted to 3.6 per cent, and the acid addition, at the same time,
to J.2 per cent. The water and acid are added in molecular propor-
tions, therefore. The analysis of the albumose and peptone products
shows practically the same thing; these substances are always lower in
carbon than are the original proteins since oxygen and hydrogen have
been taken up in the cleavage. These products of diminished molec-
ular weight pass from the stomach in the condition of hydrochloride
salts into the small intestine, where they undergo a new order of
changes.

THE ISOLATION OF PEPSIN.

It has been stated already that not one of the enzymes is known in
even approximately pure condition. Very strong active extracts of
the secretion of the gastric glands of animals may be made by the use
of various solvents. Such extracts naturally contain much besides the
pepsin, but they are suitable for experimental and other purposes. A
good process originally suggested by Wittich is illustrated by the fol-
lowing experiment.

Experiment. Separate the fresh mucous membrane of the hog's stomach from
the outer coatings and mince it fine in a meat chopping machine. To 10 gm.
of the minced membrane add 200 cc. of glycerol to which a little hydrochloric acid
10

130 PHYSIOLOGICAL CHEMISTRY.

has been added. The acid should amount to about o.i per cent of the weight of
the glycerol, and may be added in the form of the " normal " volumetric acid
of which 5 cc. will be sufficient. Allow the mixture to stand a week with frequent
shaking, then filter it by aid of the pump. This extract, bottled, will keep many
months. For use 5 cc. of it may be diluted with ioo cc. of water containing the
right amount of hydrochloric acid, generally o.i to 0.3 per cent.

For many laboratory experiments a fresh aqueous extract is prefer-
able which may be secured in this manner :

Experiment. The washed mucous membrane of the hog's stomach is chopped
fine and then rubbed up in a mortar with sharp sand or powdered glass. Water
is added (plus 0.1 per cent HC1) in amount ten times as great as the weight of the
minced membrane, the mixture is thoroughly stirred, and is allowed to stand over
night. It is then filtered and is ready for use. Such an extract is relatively strong.

A much purer product may be secured by the following process as
worked out by Kuehne and Chittenden :

Experiment. Remove the mucous membrane of a hog's stomach, wash it thor-
oughly with water and spread it out on a plate of glass. Scrape the membrane
with a knife or piece of glass and mix the scrapings with hydrochloric acid of 0.2
per cent strength. About half the membrane should be reduced to the form of
scrapings and for this mass 500 cc. of the acid may be used. Allow this to digest
at a temperature of 400 C. for about two weeks in order to convert as much as
possible of the protein present into peptone. The mixture is filtered, and to the
filtrate powdered ammonium sulphate is added to complete saturation. The object
of this is to throw down the pepsin and some albumose, the peptone formed in
the digestion being left in solution. This precipitate is collected on a filter, washed
with saturated ammonium sulphate solution and redissolved in a little 0.2 per cent
hydrochloric acid. The solution so obtained is placed in a tube dialyzer with a
little thymol water and dialyzed in running water until the sulphate is all removed.
The pepsin solution left is mixed with an equal volume of 0.4 per cent hydro-
chloric acid and kept at 400 C. 5 days to complete peptonization of albumose still
present. Then precipitation with ammonium sulphate to saturation is again effected,
the precipitate collected and washed as before and taken up with 0.2 per cent
hydrochloric acid. This solution is dialyzed in running water for the removal of
all sulphate. The liquid remaining in the dialyzer is a comparatively pure pepsin
solution. It may be concentrated in shallow dishes or on glass plafes at a tem-
perature not above 400 C, and leaves finally a scale residue. It may be evaporated
perhaps better in shallow dishes placed in a large vacuum desiccator with sulphuric
acid. The flakes or scales resulting may be kept in dry form almost indefinitely
and will be found extremely active.

Commercial Pepsin. What is commonly known as pepsin is a
product prepared on the large scale from the hog's stomach and pre-
served in dry form. Sometimes the mucous membrane is cut into
shreds, dried at a low temperature and ground to a powder, in which
condition it keeps very well. In presence of weak hydrochloric acid
this powder becomes active and is able to digest a large amount of
albumin. Usually, however, the mucous membrane is extracted in
some manner as illustrated by the first steps described in the Kuehne-
Chittenden process. In the commercial processes the following steps

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. I 3 I

are much simpler however. As carried out in the United States, they
aim to furnish a finished dry product, one part of which will digest
3,000 parts of egg albumin prepared in a certain way. Several dif-
ferent methods are in use by manufacturers for purifying and concen-
trating the extract from the stomach glands.

Some Reactions with Pepsin. The behavior of peptic extracts
may be easily shown by experiment. For this purpose an extract
made by the use of glycerol, as described above, is very convenient.
An aqueous extract will answer if freshly prepared.

Experiment. Boil an egg until it is hard, take out the white portion and rub
it through a clean wire sieve with fine meshes, by means of a spatula. Add about
five gm. of this egg to 100 cc. of 0.2 per cent hydrochloric acid in a flask, and then
add 2 cc. of the glycerol extract. Keep the flask at a temperature of 400 C, with
frequent shaking. In time the egg albumin will dissolve, forming an opalescent
liquid. Unless the flask is very frequently shaken the solution of the albumin will
be slow. Use the solution for experiment to be described.

Experiment. To 2 cc. of the glycerol extract in a test-tube add a little water
and boil a few minutes. Now add this boiled liquid to albumin and 0.2 per cent
hydrochloric acid, as in the last experiment, and note that under the same conditions
digestion does not take place, the heating having destroyed the active enzyme. In
like manner it may be shown that the enzyme is destroyed by alkalies or stronger
acids.

Experiment. Tests for proteoses and peptones. Some instructive experiments
may be made with the digesting mixture just described. Some hours after the
beginning of the digestion pour or filter off as much of the liquid as possible
and use it in this way. Divide the filtrate into several small portions. Boil one
portion in a test-tube and observe that it does not coagulate. On cooling the con-
tents of the tube the addition of alcohol produces a rather voluminous precipitate.
With other small portions, a few drops is enough, try the xantho-proteic and
the Millon's and other color tests. These all give good reactions. Then neutra-
lize the remainder of the liquid with ammonia, exactly, and add powdered am-
monium sulphate to saturation. In this way we secure a precipitate of the proteose
fraction. After a time filter off this flocculent precipitate and test the filtrate for
the more advanced digestion product, the peptone. The biuret reaction may be
employed, adding enough sodium hydroxide to cause a separation of sodium sul-
phate first. Because of the extreme solubility of the peptone bodies a real sepa-
ration is extremely difficult, but by concentration, and crystallization of the greater
part of the ammonium sulphate after cooling, followed by precipitation by alcohol,
it is possible to secure a solution in which the peptones may be more clearly
recognized.

In practice pepsin is always valued by the amount of protein matter
it will digest in a given time. Hard-boiled white of egg is generally
employed with hydrochloric acid of 0.3 per cent strength. Sometimes
well-washed fibrin is used, with a somewhat weaker acid. As an illus-
tration of practical pepsin testing the following may be given, which is
essentially the process of the U. S. Pharmacopoeia :

Pepsin Valuation. A. Prepare AV10 hydrochloric acid such as is employed in

volumetric analysis. B. Dissolve 66 milligrams of pepsin in ioo cc. of water. Mix

I32 PHYSIOLOGICAL CHEMISTRY.

175 cc. of A with 25 cc. of B, giving a solution of about 0.32 per cent acid strength.

Boil a fresh hen's egg fifteen minutes, then cool it by placing in cold water.
Separate the coagulated white part and rub it through a clean sieve having 40
meshes to the linear inch. Reject the first portions which pass through. Weigh
out exactly 10 gm. of the clean disintegrated substance, place it in a 100 cc. flask
and add 40 cc. of the acid-pepsin mixture last described. Put the flask in a large
water-bath or thermostat kept at 50° C. and let it remain three hours, shaking
gently every fifteen minutes. At the end of this time the albumin should have
practically disappeared, leaving at most only a few insoluble flakes. Much de-
pends on keeping the temperature constant, and shaking at regular intervals.

In the above test if the albumin is all digested it shows that the pepsin has a
converting power of 3,000 or over, which meets the practical requirement of the
Pharmacopoeia. The relative digesting power of stronger or weaker pepsin may be
ascertained by finding through repeated trials how much of a pepsin solution mixed
with the acid and made up to 40 cc. will be required to dissolve the 10 gm. of
disintegrated white of egg under the same conditions. The process, although not
thoroughly satisfactory, is a good one for practical purposes.

THE EXAMINATION OF STOMACH CONTENTS.

From the clinical standpoint the examination of the contents of the
stomach at any given time is a matter of considerable importance.
The examination may extend to the detection or recognition of the
nature of various solid products present, but ordinarily it is confined
to the detection or estimation of the acid and the enzymes on which
the functional activity of the organ depends. For such examinations
it is necessary to collect the liquid contents of the stomach by the aid
of some kind of stomach tube. Vomited matter may be used for the
same tests. In any event it is preferable to have as much of the solid
contents as possible along with the liquid.

Inasmuch as the secretion of the gastric juice does not take place all
the time, as was pointed out above, but depends largely on the action
of certain stimuli, of which the passage of food down into the stomach
is the common and most important one, it is customary to encourage
the flow of the secretion by giving what is called a " test-meal " some
time before introducing the stomach tube. Unless this is done it might
be possible to collect from the stomach a liquid practically free from
either acid or enzyme. The Ewald test-meal consists of wheat bread
and water or tea without sugar; 50 gm. of bread to 400 cc. of water
is an average meal. The content of protein in this would amount to
less than 5 gm. ordinarily, and in the normal stomach enough hydro-
chloric acid to more than combine with this would soon be secreted.
After about an hour therefore " free " acid should be detected by the
tests given below. With a meal richer in proteins more time would
be consumed in producing an excess of hydrochloric acid. In such a
case two or three hours might elapse before it would be possible to

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. I 33

detect the free acid. The Riegel test-meal consists of a plate of broth
or soup, 200 gm. of beefsteak, 50 gm. of wheat bread and 200 cc. of
water. The protein in this would amount to about 60 gm., which
would require 2 to 3 gm. of hydrochloric acid for preliminary satura-
tion. Some hours would therefore be consumed in producing this.
The detection of free acid, then, in such a case would be evidence of
relatively high secreting power.

The Detection of Free Acid. In the early digestion stages of a
meal rich in carbohydrates organic acids, especially lactic acid, may be
formed by bacterial fermentation. But the amount so produced is
usually very small if the normal secretion of hydrochloric acid begins
in the proper time. The organic acids produced are in amounts ordi-
narily below 0.1 per cent. Pathologically, when the bacterial fer-
mentation goes on unchecked by the production of hydrochloric acid,
the organic acid may accumulate far beyond this and may then be
readily detected by the processes given below. At present the detec-
tion of the free hydrochloric acid will be considered. Some of the
gastric secretion collected by a tube or otherwise is filtered, and the
filter (always a small one) is washed with a very little water. The
mixed filtrate and washings is used for the following tests :

Dimethylaminoazobenzene Test. To a few cc. of the gastric filtrate add a
drop or two of this reagent used in weak alcoholic solution (about 0.2 per cent).
Free hydrochloric acid present strikes a pink or even red color with the indicator.
Combined acid and the traces of organic acids which may be present have no such
action.

Congo Red Test. This substance in aqueous solution is turned blue by very
dilute hydrochloric acid. Organic acids do not give the test, except when present
in relatively much stronger solution.

The reaction is most conveniently carried out by means of test papers made by
dipping filter paper in a solution of the coloring matter and drying. These strips
are dipped in the gastric filtrate and allowed to dry spontaneously.

Methyl- Violet Test. A dilute violet-colored aqueous solution of this substance,
when mixed with weak hydrochloric acid, turns blue. The reaction with gastric
juice is faint, but when care is observed, characteristic. Organic acids, even when
present in quantity, do not give the test, which was first successfully used for the
detection of traces of mineral acids in vinegar. Use a few drops with 2 cc. of the
gastric filtrate.

Guenzberg's REAGENT. This is a well-known solution and is made as follows:

Phloroglucin 2 grams

Vanillin 1 gram

ihol 100 cubic centimeters

To make the tesl for free hydrochloric acid, mix 5 cc. of this solution with
5 cc. of the gastric filtrate and concentrate in a u'ass or porcelain vessel on the
water-bath. In presence of free hydrochloric acid the liquid gradually becomes
red as the concentration proo

134 PHYSIOLOGICAL CHEMISTRY.

Boas' Reagent for Free Hydrochloric Acid.

Resorcinol 5 grams

Cane sugar 3 grams

Alcohol, 50 per cent ioo grams

Add a few drops and evaporate as above. Color appears as in the other test.

Total Hydrochloric Acid. By the use of the above tests the excess
of hydrochloric acid beyond that which the proteins and bases will hold
is recognized. At one time this acid was supposed to be all that could
have any physiological value. The importance of that combined with
the proteins in the form of acid albumin was not considered. From
the explanations given above it is evident that in some stages of the
digestive process the hydrochloric acid may be largely or wholly in
combination and therefore not in a form to be recognized through the
aid of the tests just given. From experiments made under such con-
ditions it would be wrong to conclude that the stomach is secreting no
acids. It has been found that by making the test in a different way,
employing phenol-phthalein instead of the reagents mentioned above,
the combined acid may be readily recognized. To do this we must
make practically a quantitative analysis, and the method employed
depends on the proper use of certain indicators. This will be taken
up presently.

The Organic Acids. Under normal conditions, as already stated,
these are present in the stomach contents in very small amounts only.
As their formation depends on bacterial fermentation processes, they
appear only when hydrochloric acid is absent, or present in relatively
small proportion. Mineral acids arrest bacterial fermentation quickly,
from which it follows that in the healthy stomach there is never oppor-
tunity for the accumulation of much lactic or other acid of like origin.
These acids are never products of secretion as is hydrochloric acid;
they are not formed in the cells of the walls of the stomach, but in the
food contents. If from some pathological cause the fundus glands
fail to secrete hydrochloric acid or secrete it in traces only, then the
fermentation bacteria can work unhindered on the carbohydrates in
the stomach and produce relatively large amounts of acid. Lactic
acid is usually the most abundant of these fermentation products, but
butyric acid is occasionally formed and also acetic acid.

The recognition of these organic acids is not difficult if they are

alone or mixed with only a little mineral acid. These are of course

the cases of practical importance; much hydrochloric acid and much

lactic acid could not be found together. Among the simpler reactions

• employed the following with iron salts are the most useful.

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. 135

Test for Lactic Acid. Prepare a dilute solution of phenol by dissolving I gm.
of the pure crystallized product in 75 cc. of water. To this add 5 drops of a strong
solution of ferric chloride, which produces a deep blue color. Five cc. of this mix-
ture suffices for a test. Add to it a few drops of the liquid containing lactic acid,
and note the change from blue to yellow. (Uffelmann's test.)

A weak, colorless solution of ferric chloride serves also as a test substance, as its
color becomes much deeper by addition of a trace of lactic acid. (Kelling's test.)

This reaction is not influenced by the presence of small amounts of hydrochloric
acid, as can be readily shown by adding some to the liquid to be tested. The color
change depends on the peculiar behavior of ferric salts with organic acids in general.
These acids are relatively weak and with ferric iron tend to form " undissociated "
salts which all have a deeper color than have those with the stronger acids.

Both of these tests are much more delicate if applied to the product obtained by
shaking out the gastric juice or stomach content's with ether. About 10 cc. of the
filtered juice may be shaken with 100 cc. of ether in a separatory funnel through
half an hour. When the ether is drawn off and evaporated slowly the lactic acid,
if present, is left as a residue. This residue is taken up with a few cc. of water
and used for the tests.

The Amount of Acid. It has just been shown how we are able to
recognize the free acids existing in the gastric juice, and also, under
certain conditions, that in combination with the protein. An equally
important problem is the determination of the proportions in which
these fractions of the total acid exist. Several different schemes have
been proposed by which these degrees of acidity may be measured.
The total acid not combined in the form of inorganic salts may be most
accurately found by the methods of ordinary quantitative analysis.
The total chlorine is found by precipitation or by the Volhard titration.
The total bases are found by the usual gravimetric methods. On cal-
culating the amount of chlorine necessary to combine with these bases
an excess is left over which must be considered as existing in the form
of free acid. In very exact work the traces of phosphates and sul-
phates present must be also determined and these first combined with
bases. The method is one which requires great care in manipulation,
and besides does not distinguish between free acid and that held as
acid albumin, and this is a very important point.

The principle of another general method may be illustrated in this way. Three
portions of the gastric juice of 5 cc. each are measured off. The first is mixed
with a little pure sodium carbonate, evaporated and ignited. The total chlorine is
so retained and may be found by the Volhard titration. The second portion is
eyaporated slowly to dryness at a low temperature, mixed with sodium carbonate
and ignited. The chlorine is determined in the residue. This represents the
fractions combined to proteins and to inorganic bases, as the free hydrochloric acid
t in the original evaporation at low temperature. Finally, the third portion
is evaporated and ignited without any addition. The chlorine now found in the
residue is that which originally existed in inorganic combination. With these
three operations, as is at once apparent, it is possible to measure the element in
the three kinds of combination. The process has been modified and improved so
as to be fairly exact.

I36 PHYSIOLOGICAL CHEMISTRY.

Attempts are now made to determine the acid accurately volumet-
rically by the aid of indicators, and here, it may be said, if we can
neglect the lactic acid present, pretty good results are possible. But
if the lactic acid is present in amount more than traces, as suggested
by the qualitative tests above, the process becomes more difficult.
Before describing the details of a method something must be said
about the indicators themselves, as an understanding of their nature
and behavior is necessary for much that is to follow.

Theory of Indicators. The indicators employed in acidimetry and alkalimetry are
all weak acids or weak bases themselves, and in general much weaker than the
acids or bases in the determination of which they are employed. These indicators,
as acids or bases, form salts with the bases or acids to be titrated; it is on the
peculiar properties of these salts that the value of the indicators depends. As is
well known the change in " reaction " in employing an indicator is accompanied by
a change in color. This change in color is accounted for in two general ways.
According to one view, which is usually described as the " chromophoric theory "
substances which may be employed as indicators must be capable of existing in
fwo modifications, one of which, at least, must possess a so-called chromophoric
group. By change of reaction one of these modifications must pass over into the
other practically instantaneously, and by the addition of the smallest excess of
alkali or acid. Hundreds of substances show this phenomenon in a general way,
but to be of use as indicators the change must be both rapid and delicate.

Phenol-phthalein, for example, may be assumed to exist in two forms, one of
which is an extremely weak carboxylic acid, and weaker than the acids which are
to be titrated by its aid. The acid itself is not stable, but it forms more stable
red salts with alkalies. By addition of acids the phenol-phthalein passes over into
the other form, which is a lactone and colorless. The value of the indicator
depends on the fact that these changes are extremely sharp. The salt form has
a chromophoric complex which appears to be of quinoid structure.

In methyl orange, or its related substances, we have evidently two chromophoric
groups, one of which is found in the yellow salt form, given with alkalies, and the
other in the red isomer produced when combined with acids. The stable yellow
form is produced by even very weak alkalies, while the weakest acids are not able
to effect a transformation into the red isomer. This property has its advantages,
in the titration of mixtures containing both strong and weak acids.

The other, and perhaps more commonly accepted, view of indicators is based
on the ionization theory. Phenol-phthalein is assumed to possess a red ion which
does not appear in the acid form because of its slight dissociation, but when com-
bined with alkalies the salt dissociates and the red ion then shows itself. With
extremely weak alkalies this change does not follow, but the weakest acids are
able to suppress the ionization and with it the color. Hence the value of the
indicator in titrating weak organic acids. As weak bases do not form stable salts
with very weak acids, the whole of the hydrochloric acid combined with protein
may be titrated, as illustrated by this equation :

Prot. HC1 + NaOH = Prot. + NaCl + H20.

This reaction will be studied more fully later.

Methyl orange exhibits the opposite behavior, and is assumed to act as a weak
base which in the undissociated form is yellow. The ion of the salts, formed with
acids, is red. With weak acids it forms extremely unstable salts and therefore
cannot be used in the titration of such acids. Carbonic acid is practically inert

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. 137

with it. But bases, even very weak ones, are able to displace it from its combina-
tions with acids, just as weak acids displace phenol-phthalein. Weak ammonia,
for example, which combines imperfectly with phenol-phthalein, is strong enough
to react with the acid combinations of the dimethylaminoazobenzene or methyl
orange. Protein in the so-called acid albumin combination, in which the protein
is really basic in character, is stronger than the indicator and able to displace it
from it's salts. If we add a weak alkali to a solution of the red salt of methyl
orange the color changes immediately on the neutralization of any free acid which
may be present. The yellow color of the undissociated base takes the place of the
red of the salt or free ion. With the very weak solutions of the indicator used
the merest drop of alkali should be sufficient to bring about the change in the
indicator salt alone. Assuming in solution a mixture of free hydrochloric acid,
protein and hydrochloric acid, and the red methyl orange-hydrochloric acid salt,
addition of weak sodium hydroxide would produce a change in color immediately
after the neutralization of the last trace of free hydrochloric acid. Any excess of
alkali added would separate the protein-acid combination, but the protein would
behave itself as a base and furnish hydroxyl ions to decrease the dissociation of
the indicator and produce the characteristic yellow. Hence the " neutral " point
is reached with the disappearance of the actually uncombined HC1. With a weak
acid, like lactic acid, present in small amount the condition would be practically the
same. Such an acid is but slightly ionized and not able to form stable salts with
the indicator.

Illustration. Before taking up the actual titration of the stomach contents a
practical illustration of the steps may be found useful. To this end a mixture of
about 10 gm. of finely divided coagulated egg albumin with 10 milligrams of
powdered pepsin and ioo cc. of 0.4 per cent hydrochloric acid should be made
up to 200 cc. with water. This will give an acid strength at the very outset of
0.2 per cent.

Immediately after diluting measure out three portions of 25 cc. each of the
thoroughly shaken mixture. Filter one portion (A) at once and wash the residue
on the filter with water several times, adding the washings to the filtrate. Add a
few drops of phenol-phthalein solution and titrate this liquid with AVioNaOH,
preferably after warming. Warm the second portion (B) of 25 cc. and titrate with
the alkali and phenol-phthalein without filtering. In general the result here will be
higher than in the first case. It represents the total acidity and corresponds to
one-eighth of the acid taken. In the titration of A the result will be lower because
a part of the acid combined at once with the albumin and is left in a form not yet
soluble. To the third portion (C) of 25 cc. measured off add 2 drops of a weak
dimethylaminoazobenzene indicator and titrate directly with the N/10 alkali. The
result will be found distinctly lower than that with A or B, even in this beginning
stage of the process.

The remainder of the albumin and acid mixture in a loosely stoppered flask is
placed in a water-bath and kept as exactly as possible at a temperature of 400 C.
through five or six hours. The mixture is shaken frequently as in the pepsin test
described above. At the end of two or three hours measure out two portions of
25 cc. each ; titrate one with phenol-phthalein addition and the other with dimethyl-
aminoazobenzene. The result with phenol-phthalein present should be nearly the
same as before, while with the other indicator it will probably be a little less
than in the first case and not much more than half the acidity shown by the phenol-
phthahin. After the digestion lias continued six hours, or until practically com-
plete, te»1 two further portions of 25 cc. each in the same way. The total acidity
as measured by the aid of phenol-phthalein will be found but slightly changed,
while with the dimethylaminoazobenzene the "free" acid should be found still
further lowered probably, and not over half the total acid. The exact relation of

I38 PHYSIOLOGICAL CHEMISTRY.

the free to the total acidity depends on the strength of the pepsin and the amount
of albumin taken. In a long-continued artificial digestion or in presence of much
pepsin the acid is gradually combined more and more completely because the
basic digestion products formed have relatively lower molecular weights and com-
bine with the acid more or less perfectly, and as shown below the total acidity as
measured by phenol-phthalein will be increased. Exact numerical relations here
have not yet been established by sufficiently numerous or detailed experiments.

Titration of the Gastric Juice. The illustration given above shows
about how this should be carried out. In general as large a volume
as there used will not be available, but 5 or 10 cc. should be collected
by the tube or otherwise for each test. In testing for the total acidity
the mixture should not be filtered, unless the digestion is far advanced,
for the reason just pointed out. A part of the hydrochloric acid may
be held in the insoluble residue. In testing for the free acid, however,
the measured portion should be filtered and the residue on the filter
washed with a little water. The whole of the free acid will then be
found in the filtrate. As the color change with the indicator here used
is not as sharp as in the other case a clearer liquid is essential for
the test.

These two titrations give us the total acidity and the free hydro-
chloric acid, but do not measure the organic acid which may possibly
be present. Attempts have been made to estimate this by aid of
another indicator. Sodium alizarin sulphonate has been used for this
purpose, but the reaction is not as sharp as desirable. This substance
appears to behave as a weak acid, but one not as weak as phenol-
phthalein. Lactic acid may be titrated with it, but protein separated
from HC1 behaves as a base toward it. Theoretically the three indi-
cators are related in this way, as illustrated by diagrams, in which
H Pht represents phenol-phthalein, HA1 alizarin sodium sulphonate,
Or CI the hydrochloric acid salt of dimethylaminoazobenzene and HL
lactic acid :

H Pht 1 HA1 ■) Or CI ^

HClProt. L + Na0H. HClProt. L + Na0R HClProt. I + Na0H.

HC1 J HC1 J HC1 J

1 2 3

It has been shown above how phenol-phthalein and the methyl orange
bodies act. The alizarin sulphonate as standing midway between
them in properties is influenced by the protein which may be separated
from acid albumin in titration with NaOH. Therefore the difference
between the titrations in schemes numbers 2 and 3 must measure the
lactic or similarly acting organic acid.

Under some conditions this appears to be true. When there is rela-

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. 139

tively much lactic acid present and not much of the digestion products
a fairly good end reaction is obtained. This corresponds of course to
a practical case and the indicator then has some value. But as diges-
tion goes on the products formed are more or less basic. While not
strong enough to affect the phenol-phthalein they do appear to act on
the alizarin compound in such a manner as to diminish the alkali
required for titration ; the free hydrochloric acid is thus made to appear
low. It is plain that the indicator has but limited value.

The Amount of Pepsin. Thus far the detection and estimation of
the acid in the stomach contents has alone been considered, but the
question of the amount of pepsin present may be of equal importance.
We have no very satisfactory tests to determine this amount, but
approximate values may be obtained by observing the action of a
filtered portion of the gastric juice on some albumin solution to which
weak hydrochloric acid has been added. Comparative tests may be
made in this manner :

Prepare some egg albumin solution of about 2 per cent strength (2 per cent of
dry albumin) and mix this with 0.4 per cent hydrochloric acid in equal propor-
tions ; that is for every cubic centimeter of the albumin solution take one cubic
centimeter of the acid solution. The resultant mixture has an acid strength of
0.2 per cent and an albumin strength of 1.0 per cent. Measure out 20 cc. of the
acid-albumin mixture and add to it 5 cc. of the filtered gastric juice or stomach
contents. To another 20 cc. of the mixture add 5 cc. of a 0.2 per cent pepsin
solution. Incubate both mixtures through 24 hours at a temperature of 400 C,
with frequent shaking. At the end of the time examine both the incubated liquids
for digestion products. To this end neutralize a few cc. of each portion with
very weak alkali, using pbenol-phthalein, and observe whether a precipitate forms
or not, directly or on warming gently. If no precipitate forms in either fraction
the digestion has gone beyond the acid albumin stage, which should be the case
of course in the comparison sample with the pepsin. Next test 5 cc. portions of
each mixture in the Esbach albuminometer, adding the usual Esbach reagent (10
gm. picric acid and 20 gm. citric acid with water to 1 liter). This reagent precipi-
tates proteoses but not peptones, when used in excess, and from the extent of the
reaction in the tube some conclusion can be drawn as to degree of digestion. A
similar test should be made with potassium ferrocyanide and acetic acid in place
of the picric and citric acids. Ferrocyanhydric acid does not precipitate peptones.

In another general method the action on solid coagulated protein is observed.
White of egg is drawn up into narrow glass tubes having an internal diameter of
about 2 mm., and coagulated by heat. The tubes are then cut into lengths of 1
centimeter, thus exposing the ends of the coagulated columns of albumin. These
prepared tubes are then immersed in. the filtered gastric juice and in standard
pepsin solution to be compared and kept at a temperature of 400 some hours. The
change in length of the albumin column is taken as the measure of the peptic
activity. The filtered gastric juice must be largely diluted with water before making
the test, as salts and carbohydrate- present interfere with the normal solution of
the end of the coagulated mass. The amount of albumin dissolved under these
conditions is said to be proportional to the square root of the ferment strength,
but the rule is far from exact.

I4-0 PHYSIOLOGICAL CHEMISTRY.

It has been explained above that according to late researches pepsin
and rennin are believed by many chemists to be identical substances.
As the milk coagulating behavior seems to be much more easily fol-
lowed and measured than the proteolytic, the ferment strength is fre-
quently determined by observing the extent of the coagulating power.
The test may be made in a number of ways and has already found
clinical application, but the real value of the process remains to be
demonstrated.

PRODUCTS OF PEPTIC DIGESTION.

Frequent reference has already been made to the question of what
is produced from the food proteins during peptic digestion. In an-
swering this question it is necessary to distinguish between what may
be formed under the influence of pepsin and hydrochloric acid, with
sufficiently long time afforded for the action, and what actually is
formed in the few hours in which food, under normal conditions,
remains in the stomach. On this subject the views of physiological
chemists have undergone various and marked changes. The stomach
has long been considered, popularly, as the chief organ of digestion,
and this view appeared to be confirmed by the results of the earlier
experiments carried out with artificial digestive mixtures. The gradual
disappearance of coagulated egg albumin or of fibrin, with the simul-
taneous formation of soluble products, is a phenomenon easily observed.
Various precipitation reactions served to recover from the mixture
the products formed and these were early spoken of as " peptones."

At this time, however, the distinction between real peptones and
proteoses was not thought of. It remained to be shown that all this
abundant mass of material formed in the course of a few hours' diges-
tion consists actually in the main of products preliminary to peptones.
This later knowledge came somewhat slowly and led to a radical view
of just the opposite order from the early popular one of the function
and importance of the stomach. If the stomach is not the principal
organ of digestion, it was asked, what is its real value? If the opera-
tions carried out there may be accomplished as well later in the intes-
tine, if its work is wholly preliminary and if in turn these preliminary
stages are not really essential, what are the functions for which the
presence of the stomach appears to-be "practically" necessary? A
number of remarkable experiments made with animals threw some
light on the question. It was found that dogs were able to live and
thrive without the stomach, mixed foods of various kinds being almost,
if not quite, perfectly digested in the intestine. One of these dogs
was kept under observation several years after complete removal of

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. I4I

the stomach, and in other cases dogs have been fed through long
periods by direct injection of food into the small intestine, the connec-
tion with the stomach being meanwhile completely broken by ligature.
The feces of these animals were found to be practically normal in
most cases.

With such facts in view a school of chemists following Bunge have
come to the conclusion that the main use of the stomach is in the
destruction of bacteria taken in with the food. The acid usually
present in the gastric juice is assumed to be sufficiently strong to
destroy most of the ferment organisms, which if allowed to live and
pass into the alkaline intestine would certainly work great harm. It
must be granted that this view appears plausible; the protection of the
intestine through the sterilizing action of the acid is beyond question
of prime importance and that the stomach actually accomplishes this
to some extent must not be forgotten in any discussion of the relations
of the one organ to the other. It is well known what happens in the
human stomach when, from some cause, the hydrochloric acid is tem-
porarily absent or greatly diminished. A great development of organic
acid-producing bacteria follows, and the products of these are a source
of much discomfort without being at the same time strong enough to
cheek the growth of certain pathogenic bacteria.

But after all these facts have been given due weight we must still
admit that the peptic digestion if not actually " essential " is in practice
really important. Some peptone, although not a large amount, is
formed in the stomach and this is ready for immediate absorption, or
for the further conversion by erepsin. The proteoses are ready for
the final conversion into peptones, or they may be attacked directly by
the erepsin. This preliminary work saves, therefore, much work in
the intestine. In studying the products of pancreatic digestion it will
appear that some of them are identical with those formed in the
stomach, or by pepsin-hydrochloric acid action in general. Others
appear at first sight quite distinct and their existence leads to the long-
accepted notion that the peptic action is incapable of carrying the con-
version of proteins through to the final stages. The conclusions
which may be drawn from the most recent of the long investigations
which have been carried out on this question are somewhat conflicting,
but in the main they show that with a sufficiently long time allowed the
end products of peptic and tryptic digestion are essentially the same.

Some idea of the extent of the changes taking place in reasonably prolonged
peptic digestion may be obtained from a study of the rapidity of combination with
hydrochloric acid which has been already referred to in speaking of digestion
exp-riments. A digestive mixture was made with 90 gm. of coagulated and finely

I42 PHYSIOLOGICAL CHEMISTRY.

divided white of egg, 900 cc. of approximately 0.2 per cent hydrochloric acid and
150 mg. of commercial pepsin. Two portions of this mixture, of 25 cc. each, were
titrated at once, one with use of phenol-phthalein and the other with dimethyl-
aminoazobenzene. The remainder of the mixture was poured into a large flask
which was maintained at a temperature of 40° in a thermostat through a number
of days. Titrations were made from time to time with the following results, 25
cc. of the mixture being always taken. The phenol-phthalein titration was made
warm, the other cold.

Cc. oiN/10 Cc.oiN/10

Time. NaOH with NaOH with Dimethyl-

Phenol-phthalein. aminoazobenzene.

at once 14 9.0

10 hours 14.5 8.5

24 " 14-5 8.0

40 " 14-7 7-5

06 " 16.0 6.9

168 " 16.6 6.5

It appears, therefore, that the " total " acidity as measured in the phenol-phthalein
titration undergoes a slight increase. The hydrochloric acid remains, and added
to it are some digestive products of amino-acid character, and strong enough to
show in this way. On the other hand in the course of the week's digestion there
is a decrease in the "free" hydrochloric acid as measured by aid of the dimethyl-
aminoazobenzene indicator. The titration here is not as sharp as with phenol-
phthalein, but close enough to indicate the facts. An amount of acid correspond-
ing to 9 cc. of the N/10 alkali was " free " immediately after mixing. About 5 cc.
had evidently combined with the egg albumin to form the acid albumin. As
digestion progressed, and smaller molecules were formed, more acid was required
to unite with these. Finally the perfectly uncombined acid amounted to the equi-
valent of 6.5 cc. of alkali only. Before the end of the digestion bodies were formed
which acted as both acids and bases with the proper indicators. The amino acids
are of this character.

Some similar results were obtained in the author's laboratory in a very prolonged
digestion of casein with pepsin and hydrochloric acid. A mixture was made
containing in 1000 cc. 9.6 grams of pure casein, 2.33 grams of hydrochloric acid
and 500 milligrams of commercial pepsin. This was incubated at 380, and from
time to time portions were withdrawn for titration with N/10 NaOH. The fol-
lowing data were obtained. The original acid was of such strength that 25 cc.
required 16 cc. of the alkali for titration with phenol-phthalein or methyl orange.
The first titration after mixing with the casein was made at once.

Time.

at once
24 hours
48 hours
13 days
29 days
38 days
54 days

As the digesting mixture leaves a residue of so-called pseudo-nuclein the titra-
tion is not quite as sharp as in the other case.

Cc. of N/10

Cc. of N/10

NaOH with

NaOH with

Phenol-phthalein.

Methyl Orange.

l6.2

14.6

194

134

19.8

13.2

20.0

12.5

20.I

11.8

21.2

11.5

21.5

ii-S

THE GASTRIC JUICE AND CHANGES IN THE STOMACH. 143

The Milk Curdling Ferment. As has been intimated in this and
earlier chapters, two distinct views are held concerning the coagulation
of the casein of milk. Hammarsten studied this reaction very care-
fully and ascribed it to the presence of a peculiar ferment which he
called rennin. It has been explained that the adherents of the Pawlow
school consider this phenomenon as merely one of the varied manifes-
tations of peptic digestion, in which casein, as the substratum, becomes
first coagulated and then dissolved in part. The small portion which
is left in this peptic digestion is known as paranuclein.

It has frequently been observed that a coagulum is formed when a rennin solu-
tion is added to a crude proteose product from peptic digestion. This coagulum
is called a plastein. But as the precipitate is formed in other ways, as well, it can
no longer be referred to as a true rennin reaction. This plastein was at one time
assumed to be a step in the synthesis of larger groups from the proteoses, in
other words a reversed digestive process. How the product is formed, or what it
actually is, is not yet clearly known.

The Digestion of Fats. It is a discovery of comparatively recent
date that a lipase of considerable power is found in the gastric secre-
tion, but as to the extent of the action of this ferment in the normal
stomach but little is yet known. The ordinary lipase is an intestinal
enzyme which works in a slightly alkaline medium, whereas this lipase
is said to be active in a weak acid medium.

Absorption from the Stomach. Among other newer observations
on the functions of the stomach, it has been shown that the absorption
of certain digestive products and soluble salts follows to an appreciable
extent. Water is not absorbed here, but under certain conditions
sugars, peptones, alcohol and bodies soluble in alcohol. It has usually
been assumed that no absorption of any importance is possible before
the chyme passes into the intestines, but the later investigations on
dogs seem to show that this view can no longer be maintained, and
that the stomach may, in large measure, play the part in digestion
which the earliest investigators ascribed to it.

CHAPTER IX.

THE PRODUCTS OF PANCREATIC DIGESTION.

After leaving the stomach where the food is subjected to the influ-
ences described in the last chapter it passes into the small intestine,
where it comes in contact with other agents of change. The work in
the stomach is largely preliminary and serves to bring the food into a
finely divided homogeneous semi-liquid condition, in which it may be
readily attacked by the new digestive enzymes. As explained, the
chemical actions in the stomach are comparatively simple, and, leaving
out of consideration the continuation of the salivary digestion, are due
essentially to the combined effect of pepsin, hydrochloric acid and
protein substances. In the upper part of the small intestine, however,
the work of the pancreatic enzymes is much more complicated; at least
three kinds of reactions take place here, due to the three distinct types
of ferments in the pancreatic secretion. The protein digestion begun
in the stomach is completed, the carbohydrate digestion begun by the
saliva is continued or completed, while the fats, not yet attacked, are
brought into a condition for absorption through the intestinal walls.
These three groups of changes will be taken up in detail, but something
must be said first about the pancreatic juice as a whole.

COMPOSITION OF PANCREATIC JUICE.

For obvious reasons it was not possible to give any fair analysis of
the gastric juice. But something more is possible in the case of the
pancreatic secretion which may be collected by means of a fistula.
Most of the experiments have been made with dogs, and the flows, col-
lected under conditions to give a secretion as nearly normal as possible,
show that it contains in the mean over 95 per cent of water, and solids
consisting of salts and organic substances. Our knowledge of the
pancreatic secretion has been greatly increased by the work of Pawlow,
who, by specially devised surgical methods, succeeded in securing a
product with little disturbance to the animal, and which probably rep-
resented the normal liquid sufficiently well. As in the case of the
stomach secretion it was clearly shown that the pancreatic flow is
excited by certain stimuli, some of which may be clearly followed,
while others are beyond explanation, at present. The entrance of the
acid chyme from the stomach into the intestines seems to be the most

144

THE PRODUCTS OF PANCREATIC DIGESTION. 145

important of these stimulating factors; the hydrochloric acid appar-
ently aids in the production of a peculiar ferment which enters the
blood and finally reaches the pancreas. Other theories have been
advanced to explain the manner of action of the acid, but the fact is
clear that the acid is the most active of all the stimulants. Various
foods have also marked effects, and the character of the secretion fol-
lowing the consumption of milk, bread and meat has been reported by
Pawlow and his pupils. In one set of experiments these figures are
given for the percentage amounts of organic and inorganic solids in
the juice :

Inorg. Org.

Meat 0.907 1.558

Milk 0.869 4.399

Bread 0.925 2.298

In general, it has been noticed that the amount and nature of the
pancreatic flow seem to be adjusted to meet the requirements of the
peculiar chyme furnished by the stomach. Of the mechanism of this
adjustment practically nothing is known. The juice is always alka-
line, and the alkalinity is about that of a sodium hydroxide solution of
0.5 per cent strength. Of the volume of the secretion in man little
is accurately known; from fistulas several hundred cubic centimeters
daily have been collected, but this may not represent the normal flow.
The important enzymes present are trypsin, lipase and amylopsin.

THE BEHAVIOR OF TRYPSIN.

In an earlier chapter a few words were said about the function of
this important pancreatic enzyme and it remains to discuss its practical
relations to food digestion. In view of the discoveries of Pawlow, it
seems probable that the trypsin is not secreted by the pancreas as such,
but in the form of trypsinogen, which is activated by the intestinal
ferment, to be later described, known as enterokinase. The acid
chyme from the stomach passing into the intestine is neutralized by the
alkaline pancreatic fluid and the bile. In this neutralized condition
the trypsin is able to continue the breaking down process begun by
the pepsin, and the proteoses formed in the stomach are carried further
to the peptone stage and made ready for absorption or further cleavage
by erepsin. From what was said in the last chapter it is evident that
the trypsin could effect the preliminary changes also; that is, it is not
really necessary that the food proteins should be brought into the
proteose condition before the action of trypsin may begin. This en-
zyme is able to effect the complete digestion from the beginning, and

I46 PHYSIOLOGICAL CHEMISTRY.

rather rapidly too, which may be illustrated by experiments, using
either the minced gland from some animal or an extract made by the
aid of a proper solvent. Such an active extract may be secured in sev-
eral ways. The following methods answer very well.

DIGESTIVE EXTRACTS.

Experiment. Mince a hog's pancreas fine and weigh out about 10 gm., which
cover with absolute alcohol in a small bottle. Cork and allow to stand over night.
Then pour off the alcohol, which is added to remove water, and squeeze out the
residue. Return to the bottle, add 10 cc. of glycerol and allow the mixture to stand
about a week with frequent shaking. At the end of this time pour or strain off
the glycerol which is now a fairly strong pancreatic extract, and able to act on the
three classes of food stuffs. It has been found by experience that the extracts
from beef and hog glands are not quite the same in digestive activity, but the hog's
pancreas yields a product suitable for all practical purposes, and which keeps a long
time when made in this manner.

Experiment. An active pancreas powder which keeps indefinitely is also very
useful and may be made in this way. Remove the adhering fat as carefully as
possible from a hog or beef pancreas and mince it fine in a meat chopping mill.
The disintegrated substance is treated as above with an excess of absolute alcohol
to remove water. The alcohol is poured off and the residue pressed dry. This
residue is mixed with ether, allowed to stand an hour, and then freed from ether
by pouring, pressing and air evaporation. This treatment removes practically all
the water, traces of fat remaining and other substances soluble in alcohol and
ether. What is left is thoroughly air dried, ground to a fine powder and sifted
through gauze with 20 to 30 meshes to the inch. The powder so secured may be
kept in a stoppered bottle. In digestion experiments the powder may be used
directly, or an extract may be employed. This is best obtained by soaking a few
grams of the powder with fifty times it's weight of thymol water through 24 hours.

Some of the conditions of pancreatic digestion may be illustrated by
very simple experiments.

Experiment. Pour 25 cc. of a 1 per cent solution of sodium carbonate (crys-
tallized salt) into each of several small flasks or test-tubes. Add to each half
a cc. of the glycerol extract of pancreas and about a gram of finely divided hard
boiled white of egg. (The white of egg can be easily prepared according to the
method given under the pepsin test.) Make one of the tubes slightly acid by the
addition of dilute hydrochloric acid, enough to amount to 0.2 or 0.3 per cent. Now
place all of them in water kept at 400 C. At the end of half an hour remove one
of the alkaline tubes, and note that it still contains unaltered coagulated albumin.
Test the liquid for albumoses and peptones as given above. After another half
hour, test a second tube (after filtration). It will be observed that as the coagu-
lated protein disappears peptones become more abundant.

Allow one of the alkaline tubes to remain several hours at a temperature of 400 C.
In time it develops a disagreeable odor, due to the presence of indol formed. The
tube containing the hydrochloric acid kept several hours at 400 C. does not show the
effect of digestion, indicating that an acid medium does not suffice for the con-
verting activity of the pancreatic ferment. A minute trace of acid, below about
0.05 per cent, does not appear to check the action.

To readily recognize the final products of the pancreatic digestion of proteins
it is necessary to start with larger quantities of materials than are given above.

THE PRODUCTS OF PANCREATIC DIGESTION. 147

An experiment made as above shows at first digestion and finally
bacterial putrefaction as disclosed by the indol odor. A better idea of
some of the products formed in digestion may be secured by operating
as follows :

Experiment. Mince 50 gm. of fresh fibrin and 25 gm. of pancreas, mix and
cover the mixture with 250 cc. of alkaline thymolized water, the thymol being added
to check too rapid putrefaction. Keep the mixture at 40° two or three days in a
closed vessel, the mass being frequently shaken or stirred. At the end of the
digestion the alkali of the mixture is neutralized with a faint excess of acetic acid,
after which it is boiled in a porcelain dish and filtered. Some of the fibrin may
remain and there will always be some fat to separate by the filtration. The filtrate
is used for the identification of important products, some of which are readily
recognized, while others are not.

PRODUCTS OF DIGESTION.

Of the albumose stage in pancreatic digestion little is known, as peptones seem
to be the first recognizable products. The formation of peptones is greatly facilitated
by the previous activity of the stomach ferments. The peptones of trypsin forma-
tion are speedily followed by other products, the most important of which are
amino acids. Different proteins break down with very different degrees of readi-
ness, some quickly, others very slowly. Among the important cleavage products
the following may be referred to. Tryptophane and tyrosine seem to split off in a
very early stage, and may appear even with the peptones.

Tryptophane. This name is given to a peculiar product or mix-
ture of products found in a pancreatic digestion like the above. It is
characterized by giving a marked violet red color when mixed with a
little chlorine water or bromine water. The composition of the trypto-
phane is not yet known, but on treatment with alkalies at the fusion
temperature a mixture of several complex aromatic products, including
indol and pyrrol, is obtained. Quite recently the name tryptophane has
been given to one of the constituents of this mixture which has the
formula CnH12N202 and which has been shown to be indol amino
propionic acid.

In a concentrated solution the addition of bromine or chlorine pro-
duces a precipitate. This may be redissolved only in a very consid-
erable excess of water. The solution does not yield the protein reac-
tions at all, from which it follows that the body is an advanced decom-
pose ion product. The substance is sometimes called protein chromogen.

Experiment. To recognize the chromogen or tryptophane use two or three cc.
of the above lilt rate from the digestion experiment. Add to the liquid some bro-
mine water, drop by drop, shaking after each addition. Finally the desired color

appears.

Tyrosine and Leucine. These important amino acids have already
been referred to when the decomposition products of proteins were

I48 PHYSIOLOGICAL CHEMISTRY.

described. They are formed abundantly in a prolonged digestion like

the above and may be easily recognized. Tyrosine is paraoxyphenyl-

a-aminopropionic acid,

•OH

4^CH2CH(NH2)COOH

and is formed from most of the protein bodies on digestion. It is not
formed in appreciable quantity from gelatin. Leucine is regarded as
a caproic acid derivative, or possibly as a-aminoisobutylacetic acid
(CH3)2CH.CH2.CH(NH2)COOH. It is one of the most common
of the protein cleavage products, and is formed from gelatin also.
Both of these substances are but slightly soluble in cold water and may
be easily separated in crystalline form.

Experiment. To recognize the two amino acids in the digestion mixture pro-
ceed as follows : Concentrate the bulk of the liquid to a volume of 25 cc. and allow
it to stand in a cold place several days. At the end of this time filter through
fine muslin or a coarse filter paper. The granular mass so collected contains some
tyrosine while the bulk of the leucine remains in the filtrate. Examine the residue
first. Wash it into a beaker with a little cold water, allow to settle, decant and
wash again by decantation. Then add a large volume of water and enough am-
monia to give a marked odor. Heat to boiling and filter hot. The tyrosine dis-
solves in the alkaline liquid. Concentrate the filtrate until the odor of ammonia
has disappeared and allow to cool; crystals of tyrosine separate.

Examine some of these under the microscope. The appearance is that of bunches
or sheaves of fine needles. These needles may be dissolved in alkalies and also in
hydrochloric acid on the slide, which behavior distinguishes them from other some-
what similar crystalline deposits.

Millon's Test. A very distinctive test is by the use of Millon's reagent, which
has been already illustrated.' Mix a little of the crystalline deposit with some water
and Millon's reagent in a test-tube and apply heat. A red precipitate forms after a
time if much tyrosine is taken. With only a minute amount a red color only may
result. It will be remembered that this reaction is not confined to tyrosine alone,
but is given by many benzene derivatives containing a hydroxyl group attached
to the nucleus. Hence phenol gives the test distinctly.

By heating a little of the crystalline residue with 2 or 3 cc. of strong sulphuric
acid solution follows. On adding a drop of formaldehyde solution a red color is
produced which becomes green on heating further with addition of some glacial
acetic acid.

The solution left after filtering off the tyrosine is concentrated still more, which
finally causes a separation of leucine. The concentration is continued until a
volume of about 5 cc. is reached. Crystals which have separated may be examined
under the microscope. To the concentrated liquid about 20 cc. of alcohol is added,
the mixture heated on the water-bath to the boiling point and then allowed to stand
until cold. It is then filtered. The filtrate contains most of the leucine present.
In the precipitate there is peptone. Evaporate the alcoholic liquid slowly to dryness,
take up the residue with water, add some lead hydroxide (lead oxide with a little
alkali), boil and filter. From the filtrate remove the excess of lead by means of
hydrogen sulphide, filter again and concentrate the liquid to a small bulk for the
crystallization of the leucine.

THE PRODUCTS OF PANCREATIC DIGESTION. 149

Examine some of the leucine crystals under the microscope. They appear as
spherical bunches of very fine needles. Often the needle structure is not visible.
Hydrochloric acid and weak alkali solutions dissolve the needles on the slide.

Leucine gives some marked chemical tests. Dissolve some of the crystals in
water, add sufficient sodium hydroxide to give a good alkaline reaction and then
a few drops of copper sulphate solution. The precipitate of copper hydroxide which
forms at first redissolves, giving place to a blue solution containing a compound
of leucine and copper.

Leucine may be oxidized to yield valeric acid. On this behavior a test is based.
To some of the crystalline residue containing leucine add 3 drops of water and 2
or 3 grams of solid potassium hydroxide. Heat in a test-tube until the alkali
melts. The leucine decomposes, giving off ammonia. Allow the mass to cool, add
enough water to dissolve the residue and then enough dilute sulphuric acid to give
a sharp reaction. On applying heat the odor of valeric acid becomes evident.
Through the alkaline oxidation carbon dioxide is split off.

The Hexone Bases and Other Bodies. In recent years much
attention has been paid to the more complex residues left on tryptic
digestion. In this mixture the hexone bases, arginine, lysine and his-
tidine, are important components. These are all amino acids with six
carbon atoms, and, because of their constant occurrence in digestive
mixtures and other products of protein decomposition, they must be
looked upon as essential factors in the protein structure. Leucine and
tyrosine always seem to accompany the hexones in these decompositions.

Although by prolonged digestion products are reached which do
not give the biuret reaction, it is shown by Fischer and others in recent
work that residues remain which are still relatively complex. The
name polypeptides has been given by Fischer to such residues, and their
relations to chemical substances of definite composition pointed out.
But even these may be finally broken down into simpler amino acids.

Synthesis of Polypeptides. In the last few years a number of these polypeptides
have been produced by several synthetic processes. Among such bodies described by
Fischer the following may be cited as illustrations :

Diglycylglycine, XH.CH.CO ■ XHCHXO- NHCH.COOH. This is a tripeptide
and, as the formula shows, is formed by a condensation of three groups of
aminoacetic acid.

Alanylglycylglycine, CH^HNH^OXHCH^O-NHCH.COOH. In this com-
pound alanine, a-aminopropionic acid, is one of the groups brought into the com-
bination with glycine.

Phewylalanylglycylglycine, C,H,CILCH\TH2CO-NHCH2CO-NHCH2COOH.
This body is of interest because of the occurrence of phenyl alanine among the
commoner protein cleavage products, where reagents are used. Residues contain-
ing this group appear t<> be much more resistant toward tryptic fermentation.

LeUCYLPBOLINE. Proline = ^-pyrrolidine carboxylic acid.

' 1 1 ;\ yCH, — CH-.

>CIICH.,CHCON<
CH/ N If -CH,

•Ml, I

COOH

15° PHYSIOLOGICAL CHEMISTRY.

In this case the synthesis of leucine and the pyrrolidine carboxylic acid has been
made. In trypsin digestion residues containing the latter body along with phenyl-
alanine s.eem to be characteristic, especially where casein is used. But these residues
are easily decomposed by hydrochloric acid with separation of the constituent
amino acids.

These four artificial polypeptides are among the earlier products of laboratory
synthesis. In recent studies by Fischer and his coworkers the number has been
greatly extended.

Besides the hexone bases many simpler amino acids are always
found in the digestive residue; glutaminic acid, aspartic acid, alanine,
amino valeric acid, glycocoll and others have been separated. The
hexone bodies as end products of definite composition are of great
theoretical importance because of their relation to the protamines
referred to in a former chapter. Some of these protamines break
down almost quantitatively into arginine and the other hexones, so
that the latter may well be looked upon as nucleus structures which
unite, with loss of water, to form the more complex molecules. These
diamino acids seem to bear about the same relation to the peptones and
proteins that sugar bears to dextrin and starch. As in the hydrolysis
of starch the nature of the end product depends on the nature of the
agent of cleavage, so in proteolysis the same thing is true; acids and
enzymes work nearly in the same way, but not absolutely.

In this connection it should be pointed out that Siegfried has separated by a some-
what peculiar method of treatment a number of bodies which he calls trypsin-
fibrin peptones and pepsin-fibrin peptones which may be represented by the following
formulas :

trypsin antipeptone a Ci0H17N3O5

trypsin antipeptone £ CnH^NaOs

pepsin peptone a C21H34N609

pepsin peptone j8 C21H36N6O10

The pepsin peptone a seems to be related to the antipeptones in this way :

C21H34N609 + H20 = C10H17N3O5 + CuH„N,05

It is urged by Siegfried that the constant optical rotation of these various products
is a satisfactory evidence of their constant composition. In connection with these
formulas the formulas of the hexone bodies may be recalled:

histidine, C6H9N302
arginine, C6H14N402
lysine, C6H14N202

These compounds are relatively much simpler than the Siegfried peptones and might
readily be derived from them, with separation, at the same time, of still smaller
molecules.

The conception of " end product " in tryptic digestion is evidently a
somewhat indefinite one. In the last edition of this book the follow-

THE PRODUCTS OF PANCREATIC DIGESTION. 151

ing sentence occurs : " Certainly in the animal body the digestive cleav-
age cannot extend to the production of these small molecules which
would doubtless be useless for nutrition. What is obtained in artificial
digestions depends largely on the time given and the activity of the
enzyme employed; the term 'end product' is therefore wholly relative."
In a few short years our views have been materially changed, and
largely through the results of the investigations of Cohnheim and
Abderhalden. It has been shown that these advanced cleavage prod-
ucts are sufficient to maintain the body in nitrogen equilibrium through
long periods, and that they may play a very important part in nutri-
tion. This point will be taken up again presently.

At one time a great deal was written about the toxicity of these
digestive products. A toxic effect was certainly observed on injection
of the commercial peptones into the circulation, but this action seems
to be due to the presence of impurities, and to residues of the ferments
left, rather than to anything inherent in the amino acids themselves.
Since their behavior in nutrition has been shown the notion of toxicity
has been abandoned.

Indol and Skatol. In a prolonged pancreatic digestion, especially
in the absence of the protecting thymol or chloroform, these bodies
are always formed. Their appearance has nothing to do, however,
with the enzymic fermentation which gives rise to the other products.
They are always products of bacterial decomposition and seem to be
produced by the bacteria from some of the enzymic products, most
probably from tryptophane. Indol has the composition,

/CH>

c.h.C yen

Skatol is the methyl derivative,

C ^CH

nh/

Pure indol is a crystalline substance melting at 520. Skatol melts at
95 . Indol is oxidized in the body to indoxyl, which appears in part
in the urine as indican or potassium indoxyl sulphate,

CgH„Nv

Skatol suffers a similar change. More will be said about these reac-
tions later. Although these bodies arc not true pancreatic products, it

152 PHYSIOLOGICAL CHEMISTRY.

may be well to illustrate their production in this place, since they fre-
quently appear in pancreatic digestions. An experiment will show this.

Experiment. Chop fine 500 grams of meat and 25 grams of pancreas and allow
the mixture to stand exposed a day. Then mix with 2 liters of water and 50 cc.
of a saturated solution of sodium carbonate, place in a flask and keep at a tem-
perature of 400 through about 10 days. Then transfer the whole mass to a large
tin or copper can and distil off most of the liquid. For a complete separation 500
cc. of water should be added at this stage and this distilled also. The whole of
the distillate is now acidified with hydrochloric acid and divided into portions of
300 cc. each, which are shaken out thoroughly in a separatory funnel with ether.
For the first 300 cc. of acid liquid about 200 cc. of ether should be used. The
extracted aqueous layer is drawn off and a new portion of 300 cc. added to the same
ether. About 50 cc.of fresh ether must also be added. The mixture is thoroughly
shaken, separated as before, and the operation repeated until all the acidified
distillate is extracted. The ether is mixed with an equal volume of water and
enough sodium hydroxide to give a strong reaction. The alkali combines with and
holds the volatile acids which are present while indol and skatol remain in the
ether layer. Separate as before, transfer the ether to a flask and distil at a low
temperature. Drive off three-fourths of the ether and allow the remainder to
evaporate spontaneously. It will not be necessary to purify the residue in any
way. Dilute it largely with water and apply the following tests :

Transfer 10 cc. of the dilute indol solution to a test-tube and add 1 cc. of a
dilute sodium nitrite solution, mix thoroughly by shaking and then pour carefully
a few cc. of strong sulphuric acid down the side of the tube so as to form a layer
below the other liquid. At the junction of the two liquids a purple red color is
formed, which changes to bluish green on neutralization with alkali. This test
is similar to the one commonly employed in water analysis to detect the presence
of indol-producing bacteria. The nitrite solution used must be very weak, prefer-
ably not over 0.02 per cent in strength.

Another test is performed in this way. A splinter of soft pine wood is moistened
with strong hydrochloric acid and then dipped in a weak aqueous solution of indol.
The wood gradually becomes red. With much indol the color becomes deep and
characteristic.

A characteristic test of value depends on the formation of a salt of nitroso-
indol. Acidify the indol solution to be tested with nitric acid and then add a few
drops of a 2 per cent solution of sodium nitrite. The nitrate of nitroso-indol,
C16H13 ( NO )N2HN03, forms and produces a red precipitate if much indol is present.
If the indol solution is weak a red color only forms. By adding some chloroform
and shaking, the indol may be concentrated in the junction layer between the two
liquids.

By adding a weak solution of sodium nitroprusside to an indol solution a yellow
color is first obtained. The addition of weak sodium hydroxide changes this to
violet, which, in turn, becomes blue by acidifying with acetic acid. This is known
as Legal's test.

Skatol fails to give the above tests.

THE CARBOHYDRATE DIGESTION.

The pancreas furnishes an enzyme called amylopsin or pancreatic
diastase which acts on starch or dextrin to form sugar. Beginning
with starch we have the gradual formation of maltose by hydrolysis.

THE PRODUCTS OF PANCREATIC DIGESTION. 1 5 3

It has been already pointed out that this is not a simple process but one
which takes place in several stages, various kinds of " dextrins "
coming in between the original starch and the final sugar. In addition
to the enzyme which forms the malt sugar the pancreas furnishes, in
small amount, a " maltase " which converts this malt sugar into glucose.
The action may be very well shown by means of the glycerol extracts
of pancreas described some pages back under the head of tryptic
digestion.

Experiment. Prepare a starch paste with 5 gm. of starch to 100 cc. of water.
Mix 10 cc. of this paste, after cooling, with 5 cc. of the pancreatic extract, warm
to a temperature of 35°-40° C. and notice that the paste soon becomes thin and
nearly clear. After a time test for sugar. Repeat the experiment, using pancreatic
extract which has been boiled before mixing with the starch. The sugar reaction
now fails to appear, showing that high temperature destroys the activity of the
enzyme, as in the case of saliva. Note in the solution of the starch the dextrin
stages which may be followed by the iodine test. For the complete conversion of
the amount of starch here taken some hours may be required. This depends on
the strength of the pancreas extract.

It is well to vary the experiment by employing some fresh minced gland in place
of the extract. The pancreas powder may also be used.

We have then the two principal reactions here, the formation of
malt sugar and the inversion of the same. It is not possible to isolate
the enzyme which produces the one reaction from that which gives
rise to the other, but that both are products of the cells of the pancreas
has been satisfactorily shown against the view that the inverting
enzyme is furnished by the so-called intestinal juice. It may be recalled
that both reactions are hydrolytic.

It is possible that the living gland does not contain the active fer-
ment itself, but a pro- ferment or zymogen, which becomes active after
the secretion has passed into the intestine, but a zymogen action has
not been clearly proven as in the case of trypsin. In the minced gland
the change appears to take place through the agency of air and mois-
ture. There is a marked difference in the activity of the glands of
different animals, which fact is practically recognized by the manu-
facturers of the commercial products. The pancreas of the hog fur-
nishes an enzymic mixture richer in the starch digesting agents, while
the beef pancreas seems to be most active in the digestion of proteins.
At the present time some very active " pancreatic diastases " are pre-
pared by several firms in this country.

As the proteins are prepared practically for final absorption from
the intestine by the action of trypsin, so the remains of the carbohy-
drates are brought into the proper final condition by the amylopsin
and maltase ; at any rate the starches are so prepared, and maltose from

154 PHYSIOLOGICAL CHEMISTRY.

any source also. But as to cane sugar and milk sugar there appears
to be some little doubt, several authors claiming that the pancreas does
not contain lactase or invertase, but that the changes in these sub-
stances, when not already accomplished by the acid gastric juice, take
place through the agency of the enzymes of the intestine.

THE ACTION OF THE PANCREAS ON FATS.

In the general discussion of the subject of enzymes it was shown
that a certain product of the pancreas called steapsin or lipase is active
in splitting neutral fats into glycerol and acid. This is a true change
by hydrolysis and in effect is similar to the splitting by water alone
at an elevated temperature. In the pancreas the reaction may not be
complete, but may extend only to the separation of one-third of the
acid as illustrated by this equation for stearin :

rC18H3502 rOH

C3HB«j C18H3502 -f- HOH = C3H5-J C1SH3502 -f- HC18H3502
I C18H3502 I QsH3502

This amount of liberated acid combining with the sodium carbonate of
the intestinal juices produces a soap which in turn aids in the emulsi-
fication of the rest of the fat and thus prepares for its passage through
the intestinal walls. On the other hand, certain writers maintain that
the fat must be essentially all split before absorption is possible. The
fatty acid and glycerol pass through the intestinal walls directly and
recombine. The change in this case would follow through the typical
equation :

C3H5 ( C1SH3502) s + 3H20 = C3H5 ( OH ) , + 3C18H3602

The behavior of the pancreas may be shown by experiment, but for
this purpose it is much better to use the fresh pancreas than to depend
on extracts. The lipase seems to be soluble in glycerol to some extent,
but unless the fresh gland is employed for the extraction the result
may be unsatisfactory. These points may be tested by the student :

Experiment. Rub up a part of a pancreas with some fine, clean sand in a mor-
tar to bring . it into the condition of a creamy paste. Add some water, mix
thoroughly, and use this for the tests. Next melt some butter to allow the curd and
salt to settle. Collect the clear butter fat. Mix a few grams of the fat with an
equal volume of the pancreas paste, add some water and a few drops of chloro-
form or toluene as preservative. Keep the mixture at a temperature of 400
through a period of several hours or over night, and observe that it gradually
becomes acid through the liberation of butyric acid from the butyrin. This may
be shown qualitatively by means of the reaction with rosolic acid, a slightly
alkaline solution with red color changing to yellow on addition of some of the
pancreas-fat mixture. It may also be shown by adding a few drops of phenol-
phthalein to some of the mixture and then gradually very weak sodium hydroxide

THE PRODUCTS OF PANCREATIC DIGESTION. 155

until the alkaline reaction is secured. With a standard alkali solution the volume
used becomes a measure of the amount of acid set free.

In this form of the experiment the butter fat is more readily decomposed than
are the more solid neutral fats. Indeed the lighter esters, such as ethyl butyrate,
are frequently used to detect vegetable lipase through the same general reaction.
If in the experiment the butter fat used is not neutral to begin with, it is best to
add a few drops of rosolic acid and then very weak alkali until the color just
changes to red. Lipase is destroyed by heat as are the other enzymes.

Experiment. The emulsifying power of the pancreas may be shown also. Grind
some fresh pancreas to a thin paste with a little water. Add several grams of
this mixture to some perfectly neutral refined cotton-seed oil, about 10 cc, in a
warm mortar and rub thoroughly with a pestle. After a considerable time an
emulsion forms which will bear dilution with much water. With common oil con-
taining a little free fatty acid the emulsion forms more rapidly, but in this case
the reaction may be largely due to the formation of soap first, from the combina-
tion of the fatty acid and alkali of the pancreatic secretion. The experiment
would therefore fail to show the presence of an enzyme as fat splitter. For suc-
cess here a fresh pancreas is necessary.

A pure neutral fat suitable for such experiments may be obtained by adding
a little caustic soda solution to some refined cotton-seed oil and then ether. On
shaking thoroughly the neutral fat dissolves in the ether, leaving the soap formed
and excess of alkali undissolved, practically. The fat-ether layer is poured off,
shaken several times with water for the removal of traces of soap or alkali, and
then slowly evaporated. Neutral fat is left after the volatilization of the ether.

In these emulsification reactions the pancreatic secretion is assisted
by the alkaline bile. According to the theory of the formation of
soaps, as a preliminary to absorption from the intestines, the bile must
act as a very important factor, as its alkali would be needed for the
purpose. The bile acids have been shown to be activators for the
steapsin, and to assist materially in the cleavage of the glycerol esters.
In addition the bile has a distinct solvent action on fatty acids which
may be of help in the ultimate passage of the fat products from the
intestine. The general nature of the bile products will be discussed later.

THE FUNCTION OF THE INTESTINAL JUICE.

Closely related to the action of the pancreatic diastases is the beha-
vior of certain enzymes entering the small intestine from other sources,
especially from the glands of Lieberkiihn. As these enzymes seem to
follow up and complete the pancreatic digestion, they may be briefly
mentioned here. It should be said first, however, that any specific
digestive action due to ferments in the secretion of these glands was
for a long time denied, but there appears now to be no further question
as to the actual behavior of the secretion in this respect.

Character of the Secretion. The flow into the intestine from the
Lieberkiihn glands consists of a thin serum-like liquid, holding in solu-
tion protein bodies and salts. The reaction is strongly alkaline because

I56 PHYSIOLOGICAL CHEMISTRY.

of the presence of sodium carbonate. The amount of this is sufficient
to give rise to an evolution of carbon dioxide when an acid is added
to the secretion collected by a fistula. This alkali is doubtless impor-
tant in two ways; it aids in the emulsifkation of fats, and also helps
in the neutralization of the remaining hydrochloric acid from the gas-
tric juice carried into the intestine with the chyme current.

The ferments appear to have little or no action on fats or proteins,
but work on the residues of carbohydrates only. Some chemists claim
to find in the intestinal juice a slight starch-digesting power; others
deny that such a behavior is possible and limit the activity of the secre-
tion to the inversion of certain sugars, especially cane sugar and malt
sugar. Indeed some authors go so far as to urge that all of the inver-
sion processes taking place in the intestine are brought about in this
way, while the pancreas can produce malt sugar only. Investigations
of this kind are attended with considerable difficulty, which fact must
be kept in mind when attempting to draw conclusions from apparently
contradictory statements, such as are quoted above. All recent inves-
tigations have shown this, that while the intestinal juice may not be
the sole agent of inversion, it is certainly an important agent in this
direction. The ferments present are evidently of two types; one
resembling the invertase of cane sugar already described, while the
other is of the maltase type.

The Secretion from Brunner's Glands. The collection of the
product from these small glands offers considerable technical difficulty,
-and until recently no very clear statements were found in the literature
as to the exact nature of the secretion. By taking special precautions,
however, Glaessner succeeded in securing the secretion free from other
fluids, and has found that it possesses marked proteolytic properties in
solutions of all reactions. The digestion of protein is carried to the
stage where tryptophane may be easily recognized. The name pseudo-
pepsin may be given to the active enzyme. A lipase and an inverting
enzyme are also present.

Erepsin. Comparatively recently a ferment called erepsin has been
described by Cohnheim in the intestinal juice. It does not digest the
true proteins but has the power of splitting albumoses and peptones as
far as the mono and diamino acids. While trypsin has this power, it
is very weak as compared with erepsin, which seems to be the impor-
tant agent concerned in the last change in the proteins. Under the old
view of protein digestion there was no place for a ferment of this char-
acter, as extensive cleavage of proteins was assumed to take place only
in artificial media. But the newer views of protein metabolism and

THE PRODUCTS OF PANCREATIC DIGESTION. 157

protein synthesis are perfectly consistent with the profound hydra-
tion of the digesting material, which erepsin is able to effect. In all
discussions, then, of protein splitting in the body erepsin must be con-
sidered as one of the most active factors.

Enterokinase. This name has been given to a ferment-like body
which occurs in the intestinal juice and which has the power of acti-
vating trypsinogen. Without the presence of this activator, it is held
by some recent writers, trypsin is not formed and therefore cannot
digest protein. The enterokinase is not a digestive agent itself, but a
co-ferment of great importance. This ferment is one of a class much
discussed recently. In many reactions two enzymes seem to be con-
cerned, or perhaps, better, an enzyme and an activator. These acti-
vators are sometimes called kinases, and in some cases they are not
actual enzymes themselves.

In the brief discussions of the last few chapters it has been shown
in a general way how the important classes of food stuffs through the
action of enzymes in different parts of the body are gradually brought
into a condition suitable for assimilation and absorption. They have
undergone digestion and are ready to be carried through the intestinal
walls into the blood stream to be used as food for the building up of
the body or as oxidation material for the production of mechanical
energy and heat. These various digestive processes differ in many
ways, but they have this important element in common which must
be kept in mind : they are essentially hydrolytic in character, the addi-
tion of water by the enzymes being the essential feature in all of them.
We have next to consider some changes in the intestines in which
hydrolysis does not play an important part.

CHAPTER X.

CHANGES IN THE INTESTINES. THE FECES.

In an ideally normal condition of the alimentary canal after the com-
pletion of the digestive processes described in the last chapters, there
should be practically nothing left finally in the intestines but residues
of non-nutritive value, along with broken down products from the
digestive agents themselves. Every trace of sugar or starch should
have been brought into the form of a monosaccharide and absorbed ;
every particle of fat should have been hydrolyzed or emulsified and
then carried into the lacteal circulation ; while the proteins should have
reached the form of higher albumoses or peptones and have been like-
wise absorbed. The actual situation approaches this ideal condition
only approximately. In the first place the foods we consume are not
absolutely pure fats, carbohydrates or proteins. They all contain some
mineral matter which may escape the various digestive actions, and
they usually contain certain organic substances whicji are only partially
digestible. Some vegetable foods, for example, contain relatively
large quantities of cellulose, which is a body related to the carbohy-
drates but which is not attacked by the weak digesting enzymes. In
the foods of animal origin there are likewise substances which are very
difficult of digestive hydrolysis. This is true of some of the albumi-
noids; horn-like substances, for example, are practically not attacked,
while the cartilaginous and similar bodies are but slowly changed.
From foods containing portions of such compounds a residue would
always be left therefore, and in the case of poor, cheap meat this residue
might be considerable.

OTHER FERMENTATIONS.

Bacterial Processes. But the case is complicated by other consid-
erations. Our foods carry hosts of acid and putrefactive ferments
with them; and some of these at times work through the stomach into
the intestine, where they start reactions of their own. Just what
changes take place in the small intestine depends on the character of
the food. Following the alkaline zone where the pancreatic secretion,
the bile and intestinal juice rapidly effect the changes already described,
there is a zone of acid or neutral reaction where certain fermentation
processes of bacterial origin take place. If the food is rich in carbo-
ns

CHANGES IN THE INTESTINES. THE FECES. 159

hydrates this fermentation may be considerable, resulting- in the forma-
tion of appreciable quantities of lactic, butyric and acetic acids. The
liberation of these acids at this stage is a matter of very considerable
importance, since it prevents the breaking up of not yet absorbed pro-
tein by bacterial putrefaction. If the acids were not present bacteria
would reach the small intestine in enormous numbers from the large
intestine and greatly modify the conditions there. While, along with
the acid-forming bacteria, a few others are always present in the small
intestine, the real putrefactive changes do not begin to a marked extent
until later, when what remains of the food passes down into the large
intestine. Ordinarily the small intestine is devoid of disagreeable
odor, showing the absence of putrefactive changes.

It is evident therefore that the chemical nature of the food is a
factor of great importance in determining the character of the complex
reactions which follow the real digestive processes in the upper part
of the intestine. Here we have normally the work of enzymes, and
this is always followed by bacterial destruction of what is left. But
we must distinguish between fermentation changes and putrefactive
changes, the former being characteristic of carbohydrate food and the
latter of protein food. As one or the other of these predominates, the
chemical processes taking place must vary. Throughout the length of
the small intestine, and in the beginning of the large intestine active
absorption takes place, but between the enzymes and the bacteria a
struggle for the possession of the field is in progress all the time.
Theoretically, without the bacteria, the foods would undergo complete
digestion and be practically all absorbed, but before this ideal condition
can be reached the parasitic bacteria begin their work and rob the body
of part of its food.

Acid Fermentation. Just when this competition on the part of the
acid-producing bacteria begins is hard to say. Through the upper
third of the small intestine the reactions are essentially those of true
pancreatic digestion, and there is at no time a sharp line of demarka-
tion between this zone and the following one. The point in the intes-
tine where the acid fermentations begin is a fluctuating one and must
vary with the time which has elapsed since the beginning of the diges-
tion as well as with the character of the food. The enzymic and acid
fermentation zones must besides overlap each other; that is, in the
central part of the tract the two kinds of changes must go on simul-
taneously. Lactic and butyric fermentations are favored by a nearly
neutral medium, and this is for a time secured by the slow neutraliza-
tion of portions of these acids formed through the alkali of the pan-

l6o PHYSIOLOGICAL CHEMISTRY.

creatic, the intestinal and the bile secretions. As the foods push farther
down the neutralizing action of the alkali becomes less and less marked,
and finally the characteristic acid decomposition becomes the principal
feature.

In some animals this acid fermentation, to the almost complete
exclusion of putrefactive changes, is easily recognized. The food of
the herbivora contains an excess of pentoses, starches and other carbo-
hydrates, and these produce sugar enough to furnish a large portion
for intestinal fermentation. The feces of these animals have not the
disagreeable odors of those of carnivorous animals, where the putre-
factive reactions are very marked and the fermentations of very minor
importance. In animals with a mixed diet this condition can be very
largely changed at will by causing a variation of the food given them.

With the disappearance of the larger part of the carbohydrates
through absorption and acid fermentation, the products of fermenta-
tion being themselves partly absorbed, the activity of the putrefactive
organisms gains the upper hand and large numbers of complex reac-
tions follow. The nature of some of the bodies produced in this way
has been already referred to and further facts may now be given. In
laboratory experiments on pancreatic digestion it will be recalled that
two general results are obtainable. In working with the pancreas or
pancreatic extract plus fibrin or casein we add thymol or chloroform
water if it is desired to secure the maximum enzymic effect, but if, on
the contrary, the bacterial as well as the enzymic decompositions are
desired this protective addition is omitted and putrefaction soon be-
comes apparent. In the animal body the acid fermentation products
take the place in a measure of the chloroform or other substances used
in the laboratory experiments. Indol and skatol have been already
referred to as characteristic disintegration products resulting from the
action of bacteria on proteins; there are many others in addition to
these, and most of them are compounds of the aromatic group. Under
the conditions of their appearance in the intestines these disintegration
residues must be largely formed from the albumoses, peptones, leucine
and tyrosine of the previous enzymic digestions ; in comparatively rare
cases it is possible that the putrefaction may take place with portions
of left-over original proteins which for some reason escaped digestion
proper. Phenol, paracresol, phenylacetic acid, phenylpropionic acid,
para-oxyphenylacetic acid, glycocoll, methyl mercaptan, hydrogen sul-
phide, marsh gas and still other substances, including various volatile
fatty acids and carbon dioxide, have been found here along with the
indol and skatol. These various products are produced mainly in the

CHANGES IN THE INTESTINES. THE FECES. l6l

large intestine, and here again we find certain limitations to the extent
of the reactions. Through the small intestine the contents have re-
mained very soft and liquid, but in the large intestine normally a
marked absorption of water takes place, from which the contents
become thick and at times almost hard. This loss of water interferes
greatly with the progress of putrefaction. In addition to this the work
of the bacteria is hindered by the accumulation of the products of
their own production. Some of these products have to a certain degree
a bactericidal action and tend to check the more rapid bacterial devel-
opment. It follows therefore that when the rectum is reached in the
downward progress of the intestinal contents there may still be present
remains of putrescible matters which might have been broken down if
all the conditions had been favorable.

This brings us to a consideration of the final remains or the feces,
but first a word must be said about the absorption of certain products
in the lower stretches of the intestine. Not only are the normal diges-
tive products taken up from both intestines, from the small intestine
mainly, but various products of the bacterial reactions referred to
follow the same course. The importance of this fact is very great
from two directions at least. The excessive production of such a body
as indol is always a consequence of increased bacterial activity which
is often a pathological phenomenon. The indol may escape partly
with the feces, but a large portion is always absorbed and is oxidized
in the tissues, or in the liver mainly, from which it passes into the
blood and later into the urine, where it is recognized in the form of
indican. The amount of indican and certain similar substances de-
tected in the urine is a measure then of the extent of putrefactive
changes going on in the intestine. But it must be remembered that
other putrefactive processes, besides those of the intestine, may furnish
a small portion of the indol. In this oxidation an atom of oxygen is
taken up and indoxyl is formed :

C8HTN + O = C8H0(OH)N.

This indoxyl, like other basic substances, always finds sulphuric acid
to combine with to yield indoxyl sulphuric acid or a salt of the form

C.H.N-
K'

>SO„

or indican.

Phenol is another product of intestinal putrefaction and in great
part passes also into the circulation from the lower intestine to reach

12

l62 PHYSIOLOGICAL CHEMISTRY.

the urine finally in the form of ethereal sulphate. Certain aromatic
oxy-acids are also formed in the putrefactive processes, and are likewise
absorbed. Other substances referred to above as putrefactive products
follow the same course; in part they escape with the feces, and in part
they suffer absorption to be more or less changed and finally eliminated
by the urine. The importance of the two substances for our purpose
is mainly diagnostic. They are not absorbed in sufficient quantity to
be poisonous, but if found easily in the urine this points to a more than
normal intestinal disintegration of protein substances or their deriva-
tives. If the lower intestine becomes for any reason clogged with
fecal products which prevent the easy downward passage and escape
of the contents of the small intestine, time is given for the more pro-
longed action of the bacteria, resulting in the accumulation of these
disintegration products. In nearly all conditions of high fever the
same thing is observed. The urine test is frequently therefore a sug-
gestion of an approaching pathological condition, or of an aggravated
condition.

In another direction these bacterial products have interest and im-
portance. While the traces of indol, phenol, etc., found may be quite
harmless, it does not necessarily follow that other things produced in
the same way may be equally harmless. On the contrary, some of the
putrefactive products found in the intestine are violent poisons and
their absorption constitutes an element of danger to the body as a
whole. In laboratory experiments it is an extremely simple matter to
obtain from certain bacterial cultures soluble products which are very
toxic. These are the toxins formed by the bacteria and when injected
into the circulation of animals are capable of producing poisonous
effects. Similar bodies are undoubtedly formed in the intestines if the
bacteria there present become excessive in number. Sometimes the
microorganisms themselves penetrate the intestinal walls and pass to
other parts of the system, being collected finally by the urine. But the
peculiar poisons produced by them are much more likely to be absorbed
into the circulation and give rise to special symptoms at points far
removed from the infected intestine. No one of these intestinal toxins
has been isolated and definitely recognized, but with them other bodies
are formed which are readily detected in the urine, as is the indican
referred to above. In the urine of typhoid fever, and of other patho-
logical conditions also, certain complex aromatic products are always
present which give rise to the well-known reaction designated as the
diazo reaction of Ehrlich. When a mixture of weak solutions of

CHANGES IN THE INTESTINES. THE FECES. 1 63

sodium nitrite and sulphanilic acid is added to this pathological urine
under certain conditions it strikes a carmine to garnet red color, due
to the formation of an azo compound of some kind. The urine must
add an aromatic body, different from those normally present, to aid in
the formation of this azo coloring1 substance. With normal urine an
orange color is usually obtained, but this deeper red is characteristic of
some bacterial product probably, of the exact nature of which we are
still in ignorance.

THE FECES.

Composition. In the lower intestine the absorption of water is
one of the most important of the changes taking place and this leaves
what remains in a semi-solid condition ready for final discharge. The
amount of water left in the feces is quite variable, and although the
fecal mass may appear hard the water content is usually 70 to 85 per
cent. In the thinner pathological discharges it may be much higher.

At first sight it might appear that the feces should consist mainly of
undigested residues, and this was long held to be the case ; but we now
know that such substances may make up the least important part of the
discharge. The several kinds of products present may be roughly
divided as follows :

1. Bacteria.

2. Products formed by bacteria.

3. Remains of the digestive ferments.

4. Epithelium and mucus from the intestinal walls.

5. Food residues partly or wholly undigested.

In the normal fecal discharge all these groups are represented, but
incidentally there may be many other things present. The bacteria may
make up one third of the whole weight of the feces at times. There
are often substances which become accidentally mixed with the food
and which are not attacked by the digestive secretions. There may be
remains of various substances taken into the body as remedies, for
example, oxide or sulphide of bismuth from bismuth subnitrate, or
chalk or other insoluble substance taken in the same way.

NORMAL FECES.

For comparison it is necessary to have something as a standard, and
as such a fecal discharge from a condition approaching starvation
might be taken. In such feces there are no food residues, but the other
things are abundantly represented. Many analyses of feces have been
made from persons who for a period of several days had consumed no

164

PHYSIOLOGICAL CHEMISTRY.

food and these give some idea of the character of the discharges which
might be expected when the minimum of food is consumed and no
more. It has been calculated in this way that about 10 to 12 grams
daily is the average normal discharge from a man of 70 kilograms
weight, due to other sources than the remains of food. Numerous
attempts have been made to find the average composition of feces
from a diet which contains just enough protein, fat and carbohydrate
to keep the body in normal condition. Some of the results are given
in the table below, in per cent.

Water. Dry Subst. Fat. Nitrogen.

Mixed diet 76.5 23.5 6.2 1.0

Mixed diet 85.0 15.0 4.0 0.9

Milk diet 71.2 28.8 4.8 1.4

Milk diet 77.0 23.0 2.7 0.9

A better idea of the composition of normal feces from a full and
varied diet is shown in the following table, obtained from the analyses
of the feces of six men under observation in the author's laboratory
through a period of four months. The total feces were collected daily,
mixed, in periods of about a week each, and analyzed. The figures
below are the means of sixteen analyses, and give a good view of the
general composition. The results are given in grams for each 24
hours, and the so-called " crude fat " refers to the ether extract of the
dry feces, not including the fat combined as soaps.

Subject.

I
II

III

IV

V

VI

Moist

Dry

Nitrogen,

Crude fat,

Nitrogen

Crude fat

weight.

weight.

per cent.

per cent.

in grams.

in grams.

in moist.

in moist.

2.49

178

33-5

1.4

2.7

4.8l

I40

37-3

1-7

3-9

2.38

5-46

234

40.9

I.I7

2.32

2.74

5-43

112

25.6

1.20

3-04

1-34

3-37

197

32.1

I.I7

1.98

2.30

3.90

157

33-8

1.34

3-25

2.10

5-10

Crude fat,
per cent,
in dry.

144
14-6
13.3
13-2
12. 1

I5-I

The moist feces in the adult may weigh from 50 grams about to 400
or 500 grams daily in health, or even more. The average weight is
about 150 gm., as illustrated by the above table. The variations in the
values from which these averages were calculated were between 70 and
309 grams daily. The variations depend on the individual and also
largely on the character of the food. This last is illustrated in the fol-
lowing table from Konig's " Nahrungsmittel," where for certain foods
the daily consumption is given, and also the weight of the moist and
dry feces in grams.

CHANGES IN THE INTESTINES. THE FECES. 165

Food. Feces, Grams.

Fresh. Dry. Fresh. Dry.

Roast beef 884 366.8 65.3 17.7

Eggs, boiled 948.1 247.4 42-7 13.0

Milk 2438.0 315.0 96.3 24.8

Milk , 4100.1 529.7 174.0 50.0

Milk 2291.0 296.0 ) .

Cheese 200.0 123.8 } 3

Milk 2209 285.4

273.7 66.8

Cheese 517 320.0 ) ' ° '

Meat 614 135-9 S

Bread 450 303.3 V 299.1 46.5

Bacon 95.6 )

Cornmeal (mush) 750 641.4 108.0 49.3

Potatoes 3077.6 819.3 635.0 93.8

Rice 638.0 551.9 194.6 27.2

Flour (as bread) 500 438.8 95.2 23.5

Carrots 2566 351-6 1092.6 85.1

Peas 959.8 835.6 927.1 124.0

It will be noticed here that the highest weights of feces correspond
to the high weights of certain vegetable foods which are rich in cellu-
lose. Meat and milk in proper amount yield feces which are not exces-
sive, but with milk and cheese in excessive amounts the weight of feces
becomes large.

The mixed diets consumed by the subjects of the experiments from
the author's laboratory contained considerable amounts of the so-called
breakfast foods, as well as fruits and vegetables, and the presence of
these always tends to hinder the complete utilization of protein. The
apparently high loss in protein is not all waste, however, as suggested
above. It is not possible to consume a diet which is satisfactory
through a long period, and still show no loss of nitrogen.

In what may be called normal feces certain relations exist between
the nitrogen, the fat and the ash, if we understand by the term " normal
feces " a product containing no excess of unabsorbed food, as explained
above. Such feces contain as nitrogen compounds only those sub-
stances that are left over from the digestive secretions or bacterial fer-
ments, or are produced from the intestines themselves, while the " fats "
are ether-soluble products of similar origin, rather than the original
complex glycerides. In some cases recently reported the following
figures were obtained which will serve as illustrations. Three dogs of
similar character were fed on a meat diet through a number of days,
two receiving just enough to keep them in nitrogen equilibrium, while
the third received an excess of meat. The results of analyses of the
feces were, from the dried substance, in per cent amounts as follows:

1 66 PHYSIOLOGICAL CHEMISTRY.

Dog.

N.

Fat.

Ash.

I

8.59

I3.I8

19.24

II

8.8s

II.46

22.09

III

10.56

IO.I2

14.14

The high nitrogen of No. Ill indicates an excess of protein; this
being high, the fat and ash must be correspondingly low. The differ-
ence in the two kinds of feces becomes more apparent when it is
remembered that a much higher factor must be used in multiplying
the N values to obtain original substance in III than is probable for I
and II. Since in III an excess of protein is known to be present the
factor approaches 6.25, while for I and II it is probably not over 4.5
or 5.0. Something will be said below about the general character of
the important fat and nitrogen substances present in the feces.

THE ANALYSIS OF FECES AND INTERPRETATION OF RESULTS.

This may extend to the recognition of a large number of products,
but usually includes the detection and determination of a few impor-
tant ones only. Fat of some kind is always present, hence, a quali-
tative test is of little value. The total amount of fat must be deter-
mined by some kind of an extractive process. Nitrogen is determined
generally by the Kjeldahl method and special tests are made for pro-
teins or their more immediate derivatives. Occasionally unchanged
starch and other carbohydrates are present which may be recognized
by methods given below. The amount and character of the ash is
sometimes of value, likewise the reaction. Below a few details will be
given about some of these tests.

Separation. Ordinarily tests are made on feces of mixed diets, but
frequently it is desirable to observe the character of feces following
special diet and not modified by the product from a previous or later
diet. To separate the feces for such tests several schemes have been
proposed. It is best to give some inert substance at the beginning of
the period which will pass through the stomach and intestines un-
changed, and at the conclusion of the period of dieting the same sub-
stance may be given. Fine precipitated or floated silica, powdered
charcoal, carmine and other things have been used in this way. The
feces to be examined are collected between the discharges of the inert
and insoluble limiting substances. Various devices are also used to
collect the feces apart from the urine, which is essential for exact tests.

Reaction. In health the reaction of feces with litmus is practically
neutral in most cases, but at times either acid or alkaline reaction may
be found. With excessive putrefaction in the lower intestine various

CHANGES IN THE INTESTINES. THE FECES. 1 67

aromatic products and ammoniacal compounds are formed which may-
show alkaline behavior with indicators sensitive in this direction. On
the other hand free fatty acids may occasionally be present in sufficient
quantity to give a distinct acid reaction. In speaking of the reaction
in the intestine the conditions for the formation of light fatty acids
were explained. Putrefaction on the one hand and fermentation on
the other are the important factors in this connection, and these depend
in turn largely on the diet. Meat diet gives usually neutral or alkaline
reaction; with excessive carbohydrates the reaction may turn to acid.
With infants on mother's milk the reaction is commonly acid, while
with cow's milk it is neutral or alkaline. These tests refer to the
behavior with litmus, but with phenol-phthalein, which is not very sen-
sitive with weak alkalies, an acid reaction due to carbonic acid is often
obtained. This fact should be kept in mind, since the question of reac-
tion is often a question of indicators.

In testing for the reaction some of the mixed feces is spread on one
side of the test paper by means of a glass rod; the color effect is looked
for on the other side of the paper. If the feces are not quite moist it
will be necessary to rub up with a little water.

Dry Residue or Solids. As explained above, the larger part of the fecal dis-
charge is always water. The amount of solid matter is best obtained by drying
a weighed portion, at a relatively low temperature, in a current of hydrogen or air.
By evaporating over a water-bath there will be always some loss of volatile sub-
stances besides water. It is very difficult to obtain a perfectly dry product on the
water-bath in most cases, especially if fat is present. For most purposes it is safest
to evaporate a relatively large amount to moderate dryness on the water-bath,
after mixing the weighed feces with a little alcohol. This air-dry product is
weighed and finely powdered and a new portion is weighed out for the final com-
plete drying, at a temperature of 105° in the air bath. There will be some little
loss by volatilization of light acids and other substances.

For this kind of work a vacuum drying apparatus which can be heated to a
moderate temperature renders good service. It is also possible, where time is not
an object, to finally dry the air-dried product in a desiccator under sulphuric acid;
that is, in the form of drying apparatus in which the acid is above and the sub-
stance below. For this purpose the air-dried feces must be thoroughly powdered,
or distributed in a very thin layer. In some pathological stools there is an abun-
dance of fat, even to one-half of the total solids. In such cases a perfect drying
is always difficult with any process.

Specific Gravity. An exact determination of this datum is not
easily made, as the occluded gases interfere greatly with the test.
The normal specific gravity is about 1.045 to 1-070, but may be much
lower pathologically. Fatty stools may have a specific gravity as low
as 0.935.

1 68 PHYSIOLOGICAL CHEMISTRY.

THE TOTAL FATS.

In the analysis of feces a number of substances are included under
the term " fat." In the extraction of dried feces with some solvent
everything which goes into solution is classed as crude fat, to be more
fully identified by special tests later. Besides the fats proper feces
may contain fatty acids and their soaps, traces of lecithin, cholesterol,
cholalic acid and other bodies soluble in ether or chloroform. In the
acidified feces these substances go into solution, the acids of the soaps
being taken up also. In the feces of adults the fatty acids combined
as soaps may make up 30 to 40 per cent of the total "crude fat,"
obtained after acidification.

For this extraction it is customary to add enough acid to impart a faint acid
reaction to the feces and then evaporate to dryness with addition of sand or other
inert insoluble substance. The dry residue may be transferred to a paper tube and
extracted with anhydrous ether in the Soxhlet apparatus. A better plan is to
spread a weighed portion of the mixed and acidified feces over paper such as is
employed in the well-known milk fat extraction process. The test is completed by
drying the paper and extracting in the Soxhlet tube, as in the case of milk. The
results so obtained are higher than those from the ordinary process and the time
required for extraction much shorter. But it is not easy to obtain in this way
enough fat for further study, as not much more than 10 gm. can be easily worked.

The amount of crude fat in the dry feces is variable, but may make
up in the mean about 25 per cent if the acids combined as soaps are
included. Much of it under normal conditions must be derived from
other sources than the unutilized original fat of the foods; a portion
is always derived from residues from some of the intestinal secretions,
and from organized elements thrown off from the walls of the intes-
tines. The extent of the utilization of the food fat depends largely
on its physical character, especially its melting point. The solid fats
with high melting point are but poorly utilized as the following figures
illustrate, in which the amount of loss in the feces from different kinds
of fat is given, (v. Noorden.)

Melts. Loss.

Olive oil liquid 2.3 per cent.

Goose fat 250 2.5

Lard 34 2.5

Bacon 43 2.6

Mutton tallow 49 7.4

Stearin plus almond oil 55 10.6

Pure stearin (and palmitin) 60 90.0

The free fatty acids do not appear to be as well absorbed as are the
neutral fats, and in general from mixed vegetable foods the fat loss in
the feces is much greater than from the animal foods.

CHANGES IN THE INTESTINES. THE FECES. 1 69

Pathologically there may be a very great increase of fat in the feces,
so great in fact as to be readily recognizable by the eye. This is espe-
cially true in cases where the flow of bile into the intestine is diminished
or altogether hindered. The fat in the dry feces may then amount to
50 per cent of the whole. Any derangement of the normal pancreatic
functions leads also usually to an increase of fat in the feces. In the
last case protein and carbohydrates would suffer also in absorption.

ANALYSIS OF THE CRUDE FECES FAT.

The method of separating or extracting the crude fat has been
briefly referred to above. An extract obtained in this way may be
used for a number of tests after having been weighed. The recogni-
tion of all the substances in it is practically out of the question, but
there is no difficulty in making an approximate separation if enough
fat be used. A simple heating test will show the presence of light and
volatile fats, which, however, are not usually present in more than
traces. The following scheme will be sufficient for the recognition of
the more important constituents. The extraction is completed in the
Soxhlet apparatus, the ether distilled off and the residue dried and
weighed.

Cholesterol. Cholesterol is not a fat chemically and therefore does not undergo
saponification. This behavior makes it possible to recognize it. Add to the crude
fat some alcoholic potassium hydroxide, for 1 gm. of fat about 1.5 gm. of the stick
alkali in 25 cc. of alcohol. Boil under a reflux condenser half an hour and then
drive off the alcohol. From the dry residue extract the unchanged cholesterol by
use of an excess of ether. The substance is rather slowly soluble and a little soap
may be dissolved at the same time. The result is fairly accurate. The ethereal
solution of the cholesterol is evaporated and the residue weighed. By evaporating
a little of an ethereal solution of the substance on a glass slide a residue is secured
which will serve for microscopic identification. Cholesterol crystallizes in large
thin plates.

Cholalic Acid. Cholalic acid from the bile is an important constituent of the
crude fat, provided this is obtained from slightly acidified feces. This acid may
be detected in the soap left after extraction of the cholesterol as just explained.
The soap is mixed with a little water and acidified with dilute sulphuric acid to
free all the organic acids. The mixture is extracted with ether in a separatory
funnel. After completed extraction the ether is evaporated, leaving the free fatty
and other acids. To these a slight excess of barium hydroxide solution is added
and heat applied to form barium soaps. While warming, the mixture must be
well stirred or shaken. The separation to be made depends on the fact that the
barium soap of cholalic acid is soluble in about 25 parts of hot water while the
true fatty soaps are not. Therefore on washing with plenty of hot water the bile
acid soap dissolves. By evaporating the solution to a small volume, acidifying with
dilute sulphuric acid and shaking with ether the cholalic acid will pass again into
ethereal solution, from which it may be recovered on evaporation. The acid may be
recognized by mixing with strong sulphuric acid. A yellow solution results which
soon shows a green fluorescence. The acid may be identified also by mixing with

17° PHYSIOLOGICAL CHEMISTRY.

a small amount of water and a little cane sugar, and adding then a few drops of
strong sulphuric acid. A red color develops which becomes purple. The sulphuric
acid must be added in just sufficient quantity to warm the mixture to about 70°
or 750 C. This test is the Pettenkofer bile test.

Fatty Acids Proper. After separating the cholesterol and cholalic acid as just
described the true fatty acids are left in the form of insoluble barium soaps. By
acidifying with a little hydrochloric acid and shaking with ether these acids go into
solution and may be recovered by evaporation of the ether. The fatty acids of
any lecithin originally present are included, as the lecithin would be decomposed in
the first saponification. The acids of soaps as well as of neutral fats are also in-
cluded if the original extraction was made, as assumed, on acidified feces.

Lecithin. The separation of lecithin as such from feces is not practicable but
the amount may be estimated from the phosphoric acid separated in the saponifica-
tion. In the above tests the glycerophosphoric acid would go as barium salt along
with cholalic acid into the hot water solution. The phosphate could be recognized
or determined in this. But it may be estimated much more accurately by using
some of the original crude fat. This is mixed with some sodium carbonate and
ashed carefully. Then a little saltpeter is added to complete oxidation; the fused
mass is dissolved in water. The phosphoric acid may be determined by titration
with uranium nitrate, or, better, with molybdic acid by the Pemberton method. In
this way it is usually possible to secure enough phosphate to make an accurate
titration, in most cases, by starting with a gram of crude fat.

Soaps. The above tests give the total acids. It may be desirable to measure
the amount present in the form of soaps. For this purpose a double extraction is
necessary. In one case the feces are dried and extracted with ether without pre-
liminary acid treatment. The soaps, not being ether soluble, remain behind. Then
a second extraction, after acidification, is made ; the result gives the total fats
and acids and the difference between the two extractions shows the acid due to
soaps.

For those extractions the paper coil method is very satisfactory as sufficient
extract may be obtained from about 10 gm. of moist feces for satisfactory
weighing.

CARBOHYDRATES.

Under this term starch, sugar, gums and cellulose must be included.
With a vegetable diet the last named is always present, while on a
purely animal diet not one of the group can be found in the feces.

Starch. This substance is found commonly in feces, under normal
as well as pathological conditions, and especially when the diet has con-
tained starch in the form of coarse meal, which is difficult of digestion.
This is readily shown with cornmeal and other products containing
much cellulose. On the other hand with fine, well-sifted flours in
which the starch granules are easily turned into paste by boiling or
baking, the utilization of the starch is usually much more complete.
For the identification of starch several methods have been applied,
some direct, others indirect.

The recognition of starch by the microscope usually fails, as the outline of the
granules is destroyed by the process of cooking. When the cooking has been im-
perfect, however, the granules may be intact and in a condition suitable for
identification. The iodine test is frequently satisfactory. For this the feces are

CHANGES IN THE INTESTINES. THE FECES. 1 7 1

boiled with water and filtered. In the filtrate the iodine solution is added as for
other tests. To facilitate the separation of the starch from cell structures a little
hydrochloric acid may be added to the boiling water.

The amount of starch is best determined after conversion into sugar. This may
be accomplished by prolonged heating with a little hydrochloric acid, which changes
the starch into glucose. This may be measured by one of the copper reduction
processes, but not without some difficulty as the solution is always highly colored.
The necessary precautions can not be given here.

The normal starch content of the feces is always small, but in dis-
orders of digestion, especially with diminished activity of the pancreas,
more starch may be found. In very young children the consumption
of starchy foods is very often followed by the appearance of starch
in the feces.

Sugar. The presence of traces of sugar in the normal feces has
been frequently affirmed and as often denied. The larger part of the
more recent evidence on the question goes to show that sugar is not
normally present even in traces. All forms of sugar are very soluble
and easily absorbed from the intestine. The ability of sugar to escape
absorption through the whole length of the intestinal tract would there-
fore appear very problematical. Even in disease sugar is of rare
occurrence in the feces as far as has been determined by experiment.
But the detection of traces is not an easy task.

Pathologically sugar seems to pass through the intestine and escape
with the feces only when the conditions for absorption through the
intestinal walls are reversed. This is the case in diarrhoea because of
the more rapid movement downward in the intestine and because of the
diminished or interrupted flow from the intestine to the blood. There
may be an increased loss of proteins at the same time.

The detection of traces of sugar calls for a preliminary extraction, and purifica-
tion of the extract from substances which might interfere with the copper or
analogous tests. A fermentation test is sometimes made and without preliminary
treatment. This depends on the spontaneous decomposition of the sugar by bacteria
with liberation of gas which is collected and measured. Starch present gives the
same result, however, but not so rapidly.

Cellulose. This is a common constituent of feces after the con-
sumption of vegetable foods. Practically no digestion of cellulose
takes place in the small intestine of man, but in the large intestine there
is sometimes a bacterial destruction. The detection of cellulose is not
difficult, although the methods are somewhat complicated. The best
of the methods depend on the solution of other carbohydrates by treat-
ment with weak acid and then with alkali and finally with water. The
mixture is filtered and I lie residue washed with alcohol and ether; it is
crude cellulose contaminated with a little ash and protein substance,

17 2 PHYSIOLOGICAL CHEMISTRY.

both of which may be determined. The appearance of cellulose in the
feces has of course no pathological significance.

Gums. As these are not common articles of food they do not occur
usually in the feces. When they are consumed in pastry and confec-
tionery they may be found later in the feces since they are not digested
with readiness in many cases. Some of the gums are but slightly
soluble and undergo pancreatic digestion slowly. Experiments have
shown that gum trag'acanth and gum arabic may be found in consid-
erable quantity after their consumption in bon-bons.

NITROGEN AND THE PROTEINS.

Nitrogen is found in the feces in many combinations. Some of
these represent residues from the digestive operations and some are
found in secondary products formed by bacterial or chemical action.
Some of the molecular combinations are large, while others are rela-
tively small. No conclusion, therefore, as to the weight of the nitroge-
nous bodies can be drawn from the nitrogen found, but the datum has
value from other standpoints.

Total Nitrogen. The total nitrogen in the feces may be accurately determined
by a combustion process, but most readily by the Kjeldahl process which is now
everywhere employed. This depends on the conversion of the nitrogen into
ammonia by prolonged heating with sulphuric acid to which a very little metallic
mercury is added. Often a mixture of pure sulphuric acid and potassium sulphate
is employed. At the end of the digestion the mixture is made alkaline with a slight
excess of ammonia-free sodium hydroxide and distilled. The ammonia formed is
collected in standard acid and measured in this by titration of the excess of acid
with standard alkali.

Even in condition approaching starvation, when no food proteins can
possibly be present, the feces always show some nitrogen, which, as
pointed out above, must come from the secretions thrown into the
intestines and from the remains of bacteria and their products. A part
of this nitrogen therefore has once been absorbed to be later thrown
back into the intestine, which fact must be kept in mind in making
deductions from the nitrogen found as to the loss of nitrogen in assimi-
lation. Although usually overlooked, the nitrogen existing in the
bacterial cells is an appreciable quantity and often makes up a good
fraction of the whole. It has recently been shown that in normal feces
nearly one third of the dry weight may often be made up of the bacteria.

The amount of nitrogen excreted increases with the food consumed
in general, and especially if this food contains a large amount of indi-
gestible substance. The nitrogen of a meat diet is always more com-
pletely utilized than is the nitrogen of beans, for example, where there

CHANGES IN THE INTESTINES. THE FECES. 173

is considerable cellulose to disintegrate. The nitrogen in this case is
largely in the form of protein residues, and may be detected as will be
pointed out below. In pathological conditions of the digestive tract
there may be a great increase of unutilized nitrogen. This is more
especially true of failures in the pancreatic digestion than it is of
failure in the work of the stomach.

Proteins. The most important question to consider here is that of
proteins themselves in the feces. Nitrogen in other forms has a far
different meaning since it may represent bodies which have been
already digested and absorbed, and then thrown into the intestine again.
But nitrogen as protein represents practically waste in most cases.
Among the protein substances which may be found sometimes in feces
these may be mentioned : albumins proper, casein, nucleo-proteids, albu-
moses, peptones and naturally more or less of certain albuminoid bodies
which are digested with difficulty. The certain detection of all these
bodies under all conditions is not always possible with our present
knowledge. Some of the simplest of the tests employed will be briefly
mentioned. The soluble substances only are considered here.

Albumins. Acidify the fresh feces with dilute acetic acid and extract with dis-
tilled water. The acid prevents casein and mucin from going into solution at the
same time. Filter through good Swedish paper and apply tests to the filtrate.
Albumose and peptone go into solution with this treatment.

Albumins proper are coagulated by heating the filtrate, while the derived proteins
do not respond to this test. The albumins give also the biuret test and are pre-
cipitated by solution of potassium ferrocyanide in presence of acetic acid. But
albumose, not peptone, responds to the same test.

Albumoses and Peptones. By extracting as above, coagulating any albumin
possibly present and filtering, the filtrate may be used for albumose and peptone
tests. In the filtrate free from albumin, zinc sulphate or ammonium sulphate may
be used to precipitate albumose. In the filtrate from this peptone may be recognized
by the biuret test.

Casein. This is sometimes found in the feces of children on a milk diet. To
recognize it these tests may be made. The fresh feces may be extracted first with
rather weak sodium chloride solution to take out soluble albumins, then with weak
acid to complete the removal of such bodies. The casein may next be brought into
solution with sodium hydroxide, not too strong, and obtained in the filtrate. In such
a filtrate acetic acid produces a voluminous precipitate if casein is present, but
mucin is also precipitated. Casein, however, redissolves in an excess of acetic
acid while mucin does not. After filtering the casein may usually be thrown out
again by cautious addition of alkali to the neutral point, but the precipitate is not
as characteristic as in the first instance.

Mucin. This was formerly supposed to be a common and abundant product
in normal feces, but this is not the case. Pathologically mucin may be present so
as to be recognizable by the eye. A good chemical test for small amounts is still
lacking.

Nucleo-proteid. By extracting normal feces with lime water and acidifying
with acetic acid, a bulky precipitate is obtained usually, which was supposed to be

174 PHYSIOLOGICAL CHEMISTRY.

mucin. It, however, contains phosphorus and belongs to the proteid group. The
substance is a normal product in traces and can be found in feces following a diet
free from nucleins. It is therefore likely that traces of this protein are brought into
the intestines from the breaking down of the intestinal walls. Pathologically much
more may be found, but without having a distinct diagnostic indication.

Of all the protein substances mentioned, the casein, if it occurs in
large quantities in infants' feces, has perhaps the greatest importance
as pointing to imperfect digestive power. There can be no question,
of course, as to its origin. Serum or egg albumin as such could rarely
be present because such proteins are ordinarily consumed in the coagu-
lated condition. When the tests point to the presence of a true soluble
albumin the result shows probably the entrance of albumin from the
blood by a reversal of the normal osmotic process. It must be remem-
bered, however, that true albumin is very rarely found in the feces.
Occasionally a reaction due to presence of pus or blood may be obtained,
but the albumose or peptone reactions are much more frequent. In
diarrhoea stools, for example, where insufficient time is given for
absorption, these bodies may be found.

Insoluble Proteins. The detection of coagulated proteins and of
partly disintegrated albuminoids is practically impossible. Remains
of muscle fibers or other complex substances essentially protein may
sometimes be recognized by the microscope but they are beyond chem-
ical identification.

OTHER NORMAL AND ABNORMAL SUBSTANCES.

It will not be necessary to discuss the occurrence of the various putre-
factive bodies of bacterial origin which are always found in the feces.
We have here indol, skatol, various phenols and aromatic acids. Leu-
cine and tyrosine are occasionally found, but their presence is generally
pathological, if in quantity more than traces.

Among products of distinctly pathological origin blood and pus may
be mentioned ; both yield albumin and the corpuscles of each may fre-
quently be recognized by the microscope. It is also possible to recog-
nize the coloring matter of the blood by the spectroscope. It has been
mentioned that cholalic acid, a derivative of the two characteristic acids
of the bile, may be found with the fats of the feces. The bile acids
themselves, glycocholic and taurocholic, are also found; likewise the
bile pigments or their disintegration products. Most of the bile color-
ing matters fail to be reabsorbed from the intestine into which they are
discharged, and must be excreted therefore by the feces, and only in
small part by the urine.

SECTION III.

THE CHEMISTRY OF THE BLOOD, THE TISSUES AND
SECRETIONS OF THE BODY.

CHAPTER XI.

THE BLOOD.

How Supplied. The conversion of food-stuffs into absorbable
products has been discussed in the chapters of the last section. It
must be shown now how these products are utilized. Sooner or later,
by absorption from the stomach or the intestines, mainly from the
latter organs, they enter the blood stream through two principal chan-
nels, the portal vein and the lacteal lymph vessels leading to the thoracic
duct. Ordinarily the amount of absorption from the walls of the
stomach is not great ; only when a very large quantity of easily digested
food is present in this organ or under the influence of special stimuli
is the passage of digested substances into the circulation here appre-
ciable. The small intestine with its very considerable surface gives up
the bulk of the absorbable products to the blood or lymph stream.

The digested fats pass essentially into the minute lymphatic vessels
known as the lacteals. At the time of digestion the contents of these
vessels consists of a milky fluid termed chyle, but at other times the
lymph flowing here is nearly clear. Minute capillary vessels leading
to the portal vein take up the larger portions of the carbohydrates and
protein bodies from the small intestine and thus convey them to the
liver, where a number of important changes take place, the most pro-
nounced being the conversion of the sugar more or less perfectly into
glycogen. These reactions will receive attention later. Beyond the
liver the hepatic veins lead to the general circulation. In this general
way the nutriments reach the blood which is the main channel of distri-
bution, but this fluid is far from being a simple solution or mixture
of these nutriments in the condition in which they leave the alimentary
canal. The most important of the blood constituents are, in fact,
chemically quite distinct from anything produced in the course of diges-
tion. Certain organs of the body have the important function of
working over these digestive products and converting them into the
things required in the blood. To do this several synthetic reactions

'75

I76 PHYSIOLOGICAL CHEMISTRY.

are necessary ; how these are carried out we do not know, and in some
cases we are ignorant also of where they take place. In what follows
some of the main facts in this connection will be given.

COMPOSITION OF THE BLOOD.

Quantitative Variations. It is evident that only an average com-
position can be in general considered since the fluid is in a state of
constant change. Soon after a meal certain constituents would nat-
urally be found increased, and after a period of fasting a deficiency in
the same would follow. From what has been said it is further appar-
ent that the blood of the portal vein would be found much richer in
some substances than that of the hepatic vein or the arteries. It must
also be remembered that the blood is not a homogeneous fluid but con-
sists of a true solution in which are suspended certain cell structures.
We may therefore consider the average composition of the blood as a
whole, or of the corpuscles on the one hand and the fluid portion or
plasma on the other. The specific gravity of normal blood varies
between 1.05 and 1.07; the average specific gravity of the serum is
about 1.03.

Approximately the blood makes up 7 to 8 per cent of the body
weight; therefore in an individual weighing 70 kilograms the blood
weight would be 4.9 to 5.6 kilograms. Of this blood weight about 60
per cent belongs to the plasma and 40 per cent to the corpuscles.
Among the various recorded analyses of human blood as a whole the
following may be taken as best illustrating the mean composition, in
1000 parts.

BLOOD ANALYSES.

Men. Women.

Mean of 1 1 Mean of 8

Analyses. Analyses.

Water 779 791

Solids 221 209

Fibrin 2.2 2.2

Hemoglobin 134.5 121 .7 •

Albumin and globulin 76.0 76.0

Cholesterol, fat, lecithin 1.6 1.6

Salts and extractives 6.8 7.4

The individual analyses from which these means are taken show
rather wide variations. Some more recent analyses made in Bunge's
laboratory show the distribution of the mineral matters and may be
quoted, the figures here given referring to the blood as a whole.

THE BLOOD. 177

Man of Woman of

25 years. 30 years.

Water 789 824

Solids 211 176

Fibrin 3.9 1.9

Hemoglobin and albumins 199-5 164.8

Salts 7.9 8.6

The salt content was made up in each case as follows :

Man. Woman.

Sodium chloride 2.701 3417

Sodium oxide 921 1.862 -\- potassa

Sodium phosphate 457 .267

Potassium chloride 2.062 1.623

Potassium sulphate 205 .193

Potassium phosphate 1.202 .835

Calcium phosphate 193

Magnesium phosphate 137

}_

7.878 8.615

These figures do not show the distribution of the salts between the
plasma and corpuscles. In the original analyses from which they are
calculated by far the larger part of the sodium salts was found in the
plasma, while the potassium salts were found largely in the corpuscles.
The calcium and magnesium salts occur mainly in the plasma. In the
blood the excess of alkali shown exists probably mainly as carbonate.
All analyses seem to indicate a difference between the blood of men and
women. The male blood is richer in solids. The female blood on the
other hand appears to be slightly richer in the mineral salts.

Blood is characterized particularly by the peculiar compound con-
taining iron present, known as hemoglobin. Many of the tests for the
recognition or identification of blood depend on this substance, which
is found nowhere else. As a whole blood is distinguished by the phe-
nomenon of coagulation which is connected with the fibrin present.
Because of the great importance of this phenomenon it will be briefly
discussed here; the details of the subject belong to physiology rather
than to chemistry and are not yet sufficiently worked out for clear
elementary presentation.

FIBRIN AND THE COAGULATION OF BLOOD.

As has been already pointed out fibrin is the product resulting from
a certain reaction in which a forerunner or parent substance called
fibrinogen is concerned. As it exists in the blood vessels normally this
fibrinogen is soluble and stable, but when the vessel is pierced and the
contents allowed to come in contact with the air the soluble fibrinogen

'3

I 78 PHYSIOLOGICAL CHEMISTRY.

becomes the insoluble fibrin, which is the well-known stringy substance
described in an earlier chapter. A great deal has been written on the
subject of this spontaneous coagulation, which is now generally believed
to be brought about by the action of a peculiar ferment formed by the
breaking down of the white blood corpuscles. From these cells it
appears that a special zymogen which has been called prothrombin is
first formed; this in the presence of calcium salts yields the true fibrin
ferment, or enzyme, called thrombin. It may be easily shown that the
addition of ammonium oxalate or some other precipitant of calcium
salts to freshly drawn blood will prevent its coagulation. It was
formerly held that the calcium compounds enter into a chemical com-
bination as part of the fibrin molecule, but Hammarsten's researches
seem to show clearly that the part of the calcium is in the formation
of the ferment.

In this coagulation it appears that a portion of the original fibrin-
ogen is split off, yielding a product known as fibrin-globulin, which
remains in solution ; that is, the whole of the fibrinogen does not coagu-
late as such. The coagulation may be prevented or greatly retarded
by addition of oxalates as just referred to, and also by addition of
several other foreign substances, as acids, alkalies, strong solutions of
alkali salts, sugar, gum, albumose solutions, glycerol, etc. An excess
of carbon dioxide delays coagulation, as shown by the slower coagula-
tion of venous blood. Blood collected from a vein in a polished vessel
of porcelain or in a vessel whose sides have been covered with oil or
vaseline coagulates slowly. On the other hand collecting in a vessel
with a rough surface hastens coagulation, as does any mechanical agi-
tation. It has been shown that a polished platinum wire may be passed
through a vein without inducing coagulation, while a thread in the
same position will collect a layer of fibrin.

The various observations which have been made, while not affording
a full answer to the question why the blood does not coagulate sponta-
neously in the living veins or arteries, suggest several important reasons
to account for this absence of the reaction. One of the factors evi-
dently present in all ordinary coagulations is contact with a rough
foreign substance. The foreign substance need not be larger than the
specks of dust which blood can gather from the air. In leaving a vein
or artery blood naturally comes in contact with such particles, and these
serve as nuclei for the beginning of coagulation ; much as a minute dust
particle may be sufficient to start crystallization in a strong solution
of alum. In the body the blood is normally in contact with vessels
with very smooth walls. If such a vessel be ligatured at two points

THE BLOOD. I 79

and the sac thus formed be cut out it will be found that the contained
blood will remain fluid some hours or days even. This shows that
contact with living walls is not the element preventing coagulation.

Apparently blood exists normally in a very peculiar condition of
equilibrium, in which not one but several factors are concerned. The
same may be said of the equilibrium of many salt solutions. Changes
of temperature, the addition of foreign bodies in traces even, stirring,
pouring from one vessel into another, or contact with the dust particles
of the air in the one case as in the other may induce a change. In the
living vessels of the body as well as after leaving the body the equi-
librium may be destroyed and a coagulation take place. This is illus-
trated in the intravascular clotting after wounds in which the vessels
as a whole may not be impaired ; injury to the lining endothelium
results in throwing foreign particles into the blood stream sufficient to
induce clotting or coagulation.

EXPERIMENTAL ILLUSTRATIONS.

Some of the simpler phenomena connected with the coagulation of
blood may be readily shown by experiment.

Experiment. Have ready two test-tubes. Pour into the first one cc. of a cold
saturated solution of sodium sulphate, the other is left clean and dry. Decapitate
a rat and allow two cc. of the escaping blood to flow into the tube containing the
sodium sulphate. The rest of the blood is collected in the dry tube. In a very few
minutes coagulation takes place in the latter tube, while it is prevented by the
sodium sulphate in the former.

Allow both tubes to stand at rest a day or two. In the salted tube it will be
noticed that most of the corpuscles have settled to the bottom, leaving a clear and
lighter colored liquid, while in the other tube the coagulum has begun to shrink into
a smaller mass, from which droplets of yellowish serum ooze. The corpuscles in
this remain with the fibrin.

Experiment. Collect a quantity of slaughter-house blood by running two vol-
umes of the latter into one volume of saturated solution of sodium sulphate. Shake
the mixture and allow it to stand at a low temperature several days. Coagulation
does not occur, but a gradual precipitation of the corpuscles is observed, leaving a
yellowish liquid known as salted plasma, which may be poured off and used for
various experiments.

Experiment. Pour a few cc. of the salted plasma into a test-tube and dilute it
with several times its volume of water. On slight warming of the mixture, coagu-
lation follows. The effect of the sodium sulphate is to prevent coagulation. In this
case dilution favors it.

Experiment. Pour some fresh blood into a clean vessel and stir it thoroughly
with a glass rod, if a small quantity in a beaker is taken, or with a stick if a
larger volume, as of slaughter-house blood, is used. The fibrin gradually separates,
and entangles most of the corpuscles. Save the serum for tests to be explained and
wash the crude fibrin thoroughly under running water to remove the corpuscles and
coloring matter. The well-washed fibrin is white and stringy. Fibrin so pre-

l8o PHYSIOLOGICAL CHEMISTRY.

pared is employed in many experiments, especially in illustrating digestion phe-
nomena. On the large scale it is used in the manufacture of peptone.

Experiment. To illustrate the ready digestion of fresh fibrin use about half a
gram with 10 cc. of 0.25 per cent hydrochloric acid. Keep the mixture some hours
at 40° C. The fibrin gradually dissolves to form acid albumin, which may be ob-
tained in solution by filtering from any undigested residue. The careful addition
of a little sodium carbonate solution produces a precipitation of the acid albumin.

Time of Coagulation. It has long been observed clinically that the time required
for the coagulation of a drop of blood withdrawn by a needle is not constant but
varies, and in a marked manner in certain diseases. Based on this observation
several forms of apparatus have been devised in which the rapidity of coagulation
may be followed and measured. One of the best known forms it that of Wright,
which consists of a number of small glass tubes of uniform bore, and open at both
ends, into which definite volumes of the blood in question may be drawn. After
being filled with blood the tubes are immersed in warm water of body temperature,
or in some cases at a lower definite temperature. From time to time a tube is
removed from the bath and tested by blowing. As soon as coagulation begins the
blood can no longer be blown out easily, and the time required for this is noted.
In health this time may be four or five minutes usually, but in jaundice and some
other diseases it may be much longer. The time varies somewhat with the form of
apparatus used. In the Boggs coagulometer the time required for the clotting of a
drop of blood of definite size and shape is followed under the microscope.

BLOOD TESTS.

The serum left after separation of the fibrin by stirring, contains
much of the blood coloring matter and may be used as well as the fresh
blood for many tests, some of which will be illustrated here.

Experiment. Guaiacum Test. To a little blood solution in a test-tube add some
fresh tincture of guaiacum and then a few drops of an ethereal solution of hydrogen
peroxide. Shake the mixture and observe that the precipitated resin has assumed a
blue color, more or less marked. In this test turpentine oil, which has been shaken
with air in a bottle, or which has been exposed to the air, can be used instead of
the solution of peroxide. Hydrogen peroxide is developed by the action of oxygen
on turpentine. In this test the hemoglobin seems to act as a carrier of oxygen to
the resin. The oxidation product of the resin is blue.

Experiment. Hydrogen Peroxide Test. A reaction somewhat similar to the
above in principle is observed on mixing 2 cc. of the blood with 10 cc. of the com-
mercial hydrogen peroxide solution. The hemoglobin brings about the decomposition
of the peroxide with liberation of oxygen, which escapes, producing froth.

Reaction of Blood. The normal reaction of blood is alkaline,
which cannot be observed, however, in the usual way because of the
marked color of the pigment. It may be readily seen by working in
the following manner :

Experiment. Prepare some small plaster of Paris surfaces by pouring the
well-known plastic mixture of plaster of Paris and water on glass plates and allow
it to harden several hours at least. The prepared plates are removed from the
glass and soaked in a neutral solution of litmus and are then allowed to dry. The
test proper can now be made by putting a few drops of the blood on the smooth
plaster surface and allowing it to remain there five minutes. It is then washed

THE BLOOD.

181

off with pure water, when it will be found that the part of the plate which has been
covered by the blood has become blue from the action of the alkali of the blood on
the neutral litmus.

Experiment. Heat Test. Heat the solution of blood until it is near the boiling
temperature and note that the red color is largely destroyed and that a brownish
precipitate forms which contains albumin and decomposed coloring matter. Add
now a small amount of sodium hydroxide solution and observe that the precipitate
disappears while the blood solution becomes red again by reflected light, but greenish
by transmitted light.

Experiment. Hemin Crystals. When acted on by acids or strong alkalies
hemoglobin of blood is broken up into globin and a characteristic compound
called hematin. Hematin in turn is acted upon by hydrochloric acid yielding
the hydrochloride, hemin, which appears in crystalline form. From the name
of their discoverer, these crystals are called " Teichmann's crystals." Their appear-
ance constitutes one of the best tests we have for blood, and can be illustrated by
the following: Evaporate a drop of blood on a slide, add two or three drops of
glacial acetic acid, and boil. Put on a cover glass and allow to cool. Minute
(microscopic) plates or prisms separate out. If old blood, a stain, for instance, is
examined, it is necessary to add a small crystal of sodium chloride to the acetic
acid, by which means sufficient hydrochloric acid is liberated for the test. The
crystals have a dark brown color and are very characteristic. The usual forms as
found in human and other blood are shown below.

Fig. q. Hemin crystals. i is from human

blood ; 2 from a seal ; 3 from a calf ; 4 from a

pig; 5 from a lamb; 6 from a pike; 7 from a

rabbit. (Landois.)

x'K-»'

v» V.v

V

\ »

\

Fig. 10. Hemin crys-
tals from stains of hu-
man blood. (Landois.)

The most certain means of identifying blood, however, depends on
the peculiar behavior of hemoglobin toward light, which will be shortly-
explained.

HEMOGLOBIN.

Composition. In the systematic classification of the protein bodies
hemoglobin is grouped among the proteids or compound substances,
inasmuch as it may readily be broken up into a fraction containing iron
called hematin, and a histone substance called globin. This cleavage
is very easily effected by the action of weak acids and in the mean the
hematin fraction is found to amount to about 4.3 per cent. In some

182

PHYSIOLOGICAL CHEMISTRY.

experiments as much as 94 per cent of globin has been recovered. It
is therefore likely that only the two substances are present. The prop-
erties of hemoglobin are not quite constant, inasmuch as from different
bloods products of slightly different composition have been obtained.
It is possible to secure the hemoglobin in crystalline condition suitable
for analysis. A number of such determinations have been made and
from them formulas have been calculated. These formulas can be at
best only more or less close approximations, but they are interesting as
illustrating the great molecular weights here concerned.

Hemoglobin is dextro-rotatory. By an ingenious method Gamgee
and Hill have found ac= 10.40. The globin from it is levorotatory.

Analyses of Hemoglobin. Several results obtained by different
observers are here given. The variations must be partly due to differ-
ences in methods of preparation and analysis.

C

H

N

s

Fe

0

Author.

Horse.

51-15

6.76

17-94

0.39

o-335

23.42

Zinnofsky.

"

54-40

7.20

17.61

O.65

0.47

19.67

Huefner.

Dog.

54-57

7.22

16.38

0.57

Q-33D

20.43

Jaquet.

( (

53-85

7-32

16.17

0.39

o.43

21.84

Hoppe-Seyler.

Hen.

52.47

7.19

16.45

O.86

o-335

22.5

Jaquet.

In the first analysis the ratio of the sulphur atoms to the iron atoms
is 2:1; in the third analysis it is 3:1. On the assumption that the
molecule contains but one atom of iron the minimum molecular weight
which may be calculated from this analysis is :

^758-H-:i203-N las'-^is-t1 e^>3

It is interesting to note that the molecular weights found in this way
are practically confirmed by the determinations made on the combining
power of hemoglobin for carbon monoxide. Assuming that one mole-
cule of carbon monoxide is held by one molecule of hemoglobin, obser-
vations of the volume of the gas absorbed by a given weight of the
blood pigment lead to practically the same result as was obtained by
the iron method.

Combinations of Hemoglobin. The great importance of hemo-
globin depends on its power of forming several more or less stable
combinations with certain gases. Of these combinations that with
oxygen is by far the most important; we distinguish therefore between
hemoglobin and oxyhemoglobin. The common form of the substance
is really the latter, although it is usually referred to by the simple
term — hemoglobin. The oxygen of oxyhemoglobin is very loosely
held and may be driven out from its union by the aid of a current of

THE BLOOD.

I83

other gases, or by the pump. The amount so held corresponds to two
atoms of oxygen for each molecule of hemoglobin. This oxygen com-
bining power in some way depends on the presence of the iron of the
hematin.

Oxyhemoglobin. By various methods this substance may be ob-
tained in crystalline form, the crystals being often 2 mm. or more in
length. From blood of different animals different crystalline forms
have been observed. In all cases the crystals are red and soluble in
water ; they are more easily soluble in water containing a little sodium
carbonate. They are insoluble in ether, benzene and chloroform, and
the water solubility varies with the nature of the blood from which
they were made, that from the blood of man and the ox, for exam-
ple, being easily soluble, while the oxyhemoglobin from the blood of
the horse or dog is rather slowly soluble. Because of their solubility
it is very difficult to secure crystals from human blood, but from dog's
blood they may be made as follows :

Experiment. Beat up 100 cc. of the
blood thoroughly, cool to a low tem-
perature and add 10 cc. of ether and a
little water. Shake this mixture thor-
oughly and allow it to stand on ice over
night. Filter on porous paper, squeeze
out the mother-liquor as far as possible,
dissolve in a little water, filter again
and to the new filtrate add one-fourth
its volume of alcohol, meanwhile stirring
constantly. Allow the mixture to stand
to crystallize. An illustration is given
of the usual forms.

A simple test may be also made by
mixing a drop of dog's blood with a
drop of water on a slide and allowing it
to partly evaporate. A cover glass is
then put on and the crystals are looked
for with the microscope.

The conditions of combination
between hemoglobin and oxygen
have been studied by several au-
thors. It has been found that by
exhausting blood under the air
pump the greater part of the oxy-
gen becomes free. It has been
found further that 1 gm. of hem-
oglobin may be made to take up or give off something over 1.3 cc. of
'^en. This reduces to 2 atoms of oxygen for 1 atom of iron pres-

Fig. II. Crystals of hemoglobin, a and
b from human blood ; c from the cat ; d
from the guinea-pig ; e from the marmot ;
/ from the squirrel.

1 84 PHYSIOLOGICAL CHEMISTRY.

ent in the hemoglobin. Various chemical agents have the same effect.
In the case of certain solutions the action is a chemical one, while
with several inert gases the action is physical.

These reactions are accompanied by a change of color in the oxyhe-
moglobin or blood solution experimented upon. Oxyhemoglobin solu-
tions show a brighter red color than do those containing the reduced
hemoglobin. This difference is well illustrated in the contrasting
shades of arterial and venous blood, the former containing plenty of
oxygen in combination while the latter is deficient. The loss of oxygen
is illustrated by some simple experiments :

Experiment. Shake about 10 cc. of diluted defibrinated blood with a few drops
of ammonium sulphide solution or with Stokes' reagent. (Stokes' reagent is a
solution of ferrous sulphate, to which a small amount of tartaric acid has been
added, and then ammonia enough to give an alkaline reaction.) Warm gently, and
observe that the bright color of arterial blood gives place to the darker purple of
venous. On shaking the mixture now with air the bright red color returns. For the
success of this experiment where Stokes' reagent is employed it should be freshly
prepared before use. Various other substances behave in similar manner.

Experiment. Generate some hydrogen gas in the usual manner, and allow it to
bubble through diluted defibrinated blood. A change of color follows after a time,
due to the mechanical loss of oxygen. The same .result may be accomplished by
exhausting the oxygen of the blood by means of an air pump. Exposure to the
air restores the color in a short time, as before.

The color change may be noticed readily with the unaided eye, but
is much more marked when observed in the spectroscope, as will be
pointed out below.

The maximum amount of oxygen which may be held by the hemo-
globin was given above as i molecule for I molecule of the pigment.
This holds only for strong oxygen pressure, however. Under lower
atmospheric pressure a part of the oxygen becomes dissociated, as
illustrated by these figures given by Huefner for a 14 per cent hemo-
globin solution :

Atmospheric

Per cent of

Atmospheric

Per cent of

Pressure in Mm.

Oxygen Free.

Pressure in Mm.

Oxygen Free.

760.O mm.

1.49

238.5 mm.

4.60

715-6

1-58

1 19-3

8.79

620.8

1. 8l

47-7

19.36

524.8

2.14

23.8

32.51

477-1

2-15

4.8

70.67

357-8

3-II

The loss of oxygen does not become marked until comparatively
low pressures are reached.

Carbon Monoxide Hemoglobin. When a current of carbon
monoxide is led into a blood solution it displaces the oxygen and forms

THE BLOOD. 1 85

a very stable compound. One molecule of the monoxide takes the place
of the molecule of oxygen combined as oxyhemoglobin. This reaction
is accompanied by a change of color, not as marked, however, as the
change from reduced to oxyhemoglobin. The combination with car-
bon monoxide is the reaction which takes place in cases of poisoning
with illuminating gas, which contains 10 to 25 per cent of the monox-
ide. The addition of pure air does not displace the combined gas
except where a great excess is used.

Experiment. Lead a current of illuminating gas, best the so-called " water gas,"
into 50 cc. of blood in a flask. Continue the passage of the gas until a distinct
cherry red color is produced. When the combination appears to be complete treat
a few cc. of the liquid with Stokes' solution, which fails to effect a reduction. With
portions of the mixture further tests should be made to illustrate methods of dif-
ferentiating between normal blood and blood containing much monoxide. The dif-
ferentiation in each case depends on the greater stability of the monoxide hemo-
globin with the reagent in question.

Experiment. Add some strong solution of sodium hydroxide to ordinary blood.
This gives a brownish green precipitate at first and then a red solution. Treat
blood saturated with carbon monoxide in the same manner. This gives a red pre-
cipitate and finally a red solution.

Experiment. Dilute some of the monoxide blood with four volumes of water
and to the mixture add an equal volume of a 3 per cent tannic acid solution. The
red color persists much longer than it would in the case of a simple oxyhemoglobin
solution, which should be tried for comparison.

Experiment. To about half a cubic centimeter of the monoxide blood add 20
cc. of water and 10 drops of strong yellow ammonium sulphide solution. Shake
thoroughly and then add enough dilute acetic acid to give a faint acid reaction. A
rose red color appears, while with normal blood decomposition products are formed
which have a dirty gray color.

Experiment. Mix 1 cc. of the monoxide blood with 5 cc. of basic lead acetate
solution and shake well. The mixture remains red, while with normal blood under
the same conditions a brown color results.

Experiments have been carried out to determine what portion of the
hemoglobin must be combined with monoxide to have death follow.
It appears that if about half the pigment in the blood is still unchanged
recovery may be expected by free use of air. The mere action of a
great excess of air may gradually displace the combined monoxide.
The spectroscopic appearance of the monoxide hemoglobin will be
referred to below.

Nitric Oxide Hemoglobin. Under certain conditions nitric oxide,
NO, may be combined with hemoglobin to form a very stable com-
pound. The union takes place molecule with molecule and may be
obtained by treating carbon monoxide hemoglobin with the nitric
oxide, which has the power of breaking up the monoxide combination.
The direct action of nitric oxide on oxyhemoglobin does not lead to
the desired result, as oxidation of the NO follows and the acid formed

I 86 PHYSIOLOGICAL CHEMISTRY.

destroys the hemoglobin. In presence of urea, however, the direct
union is possible. The substance forms crystals like those of oxyhem-
oglobin and gives a very similar spectrum.

Sulphohemoglobin. When hydrogen sulphide is led into a solution
of oxyhemoglobin in presence of air a compound is formed which,
however, is not permanent. Decomposition soon follows and a green-
ish brown mixture results.. With reduced hemoglobin away from the
air a true compound is formed which gives a characteristic spectrum,
and which is much more stable.

Other Combinations. Several authors have described other com-
pounds formed by the union of hemoglobin and gases. Of these,
so-called carbohemoglobins, acetylenehemoglobin and cyanhemoglobin
are the best known. These combinations are but slightly stable and
have no special importance. As acetylene was formerly prepared on
the laboratory scale it was poisonous, and this property was assumed
to be due to its action on hemoglobin, which was thought to resemble
that of carbon monoxide. Since the manufacture of acetylene from
calcium carbide was begun this notion has been dispelled. The action
of acetylene on the -blood is very weak. In the early laboratory
product impurities present were probably responsible for the observed
effects.

DERIVATIVES OF HEMOGLOBIN.

Some of these are practically identical with hemoglobin, while others
are products of complete decomposition. The first to be considered is :

Methemoglobin. Oxyhemoglobin in solution or in crystal form,
alone or in presence of certain reagents, shows a great tendency to pass
over into this modification which contains just as much oxygen as the
original, held, however, in stable combination. Under the air pump
methemoglobin does not give up any oxygen, while from oxyhemo-
globin nearly the whole of the extra molecule may be abstracted.
While this formation of methemoglobin takes place spontaneously it is
greatly hastened by the action of several substances, some of which are
oxidizing agents, while others are reducers. Of the oxidizing sub-
stances ozone, potassium permanganate, potassium chlorate, iodine,
potassium f erricyanide and nitrates have been used, while such reducing
agents as pyrogallol, pyrocatechol, hydroquinol and hydrogen even,
acting on the blood have brought about a formation of the stable met-
hemoglobin. Certain substances given as remedies have the power of
converting the oxyhemoglobin into methemoglobin. Amyl nitrite,
acetanilid and nitrobenzene may be mentioned here. The poisonous

THE BLOOD. 1 87

action of large doses of potassium chlorate has long been supposed to
be due in part to the same reaction.

Solutions of methemoglobin are not bright red but reddish brown,
and the crystalline substance is also brown. The color of an alkaline
solution is red, but this is not due to a reconversion into oxyhemo-
globin. Certain reducing agents have the power of gradually chang-
ing the methemoglobin back into oxyhemoglobin and then into reduced
hemoglobin. Ammonium sulphide and Stokes' reagent work in this
way. The conversion may be followed by aid of the spectroscope.

A product known as acid hemoglobin is formed by the action of
weak acids on hemoglobin. This appears to be a step in the formation
of methemoglobin, the spectrum of which it resembles. With strong
acids decomposition takes place and hematin results.

Hematin. It has been already explained that hemoglobin breaks up
readily into globin, about 96 per cent, and hematin, about 4 per cent.
This decomposition follows, as just mentioned, by the treatment with
strong acids, and also by various other reactions. The product from
reduced hemoglobin is known as hemochromogen, while from oxyhem-
oglobin oxyhematin or common hematin is obtained. The relations
may be. thus illustrated :

Hemoglobin { Jlobin, ( + O = hematin

Hemoglobin j hem0chroinogen j _ Fe = hematolin

( globin r + HC1 = hemin
Oxyhemoglobin j hematin J _ 0 = hemochromogen

(^ — Fe = hematoporphyrin

Hematin is usually obtained as an acid combination or ester. In one
process frequently followed blood is warmed with an excess of glacial
acetic acid. Crystals containing acetic acid separate on cooling. In
another process the hydrochloride is obtained ; in either case the free
hematin is secured by saponification with weak sodium hydroxide solu-
tion. Several formulas have been given for hematin; the one most

commonly accepted is

C«H«N«FeO„

while for hemin crystals the formula

C32H30N4FeO3HCl

has been gi ven. Kiister has recently given the formula C34H34N4Fe05.
It is possible that different analysts have obtained, not the same, but
closely related products. Hemin is secured in minute brownish crys-

1 88 PHYSIOLOGICAL CHEMISTRY.

talline plates, hematin as an amorphous bluish black insoluble powder.
The spectrum, which is important, will be referred to later.

Hematoporphyrin. This is a derivative obtained by the action of
acids on hematin. In this treatment the iron is removed, as illustrated
by the following reaction, when hydrobromic acid is employed as the
decomposing agent :

C32H32N4Fe04 + 2H20 + 2HBr = 2C1GH18N203 + FeBr2 + H2.

The substance appears to be related to and isomeric with bilirubin.
The alkaline solutions of hematoporphyrin are deep red, the acid solu-
tions incline to deep violet or purple. The acid and alkali spectra are
very different and characteristic.

The relation of the blood coloring matter to the bile pigments is
illustrated by these formulas :

C32H32N404Fe hematin

C32H36N406 hematoporphyrin

C32H36N406 bilirubin

C32H3eN4Os biliverdin

Hematolin is the name given to an iron-free compound obtained by
decomposing hematin in absence of air.

Hemochromogen. This is obtained by reducing a hematin solu-
tion with ammonium sulphide or with zinc dust and alkali. It forms
a dark red powder insoluble in water but soluble in alkalies. The solu-
tion exposed to the air absorbs oxygen and appears to regenerate
hematin.

Hematoidin is a red-colored pigment which has been found in old
blood extravasations. It seems to be identical with bilirubin.

THE OTHER PROTEINS OF THE BLOOD.

In addition to fibrin and hemoglobin blood contains serum albumin
and serum globulin, which have been described already in a previous
chapter. The combined substances make up about 7 per cent. They
may be approximately separated by precipitating the globulin from
blood serum by addition of a large volume of water and also by salt
precipitation, which may be illustrated in this way :

Experiment. Prepare blood serum as free as possible from corpuscles as already-
shown and mix about 100 cc. with an equal volume of cold saturated ammonium
sulphate solution. A separation of the globulin follows. Filter; the filtrate con-
tains practically all the serum albumin which may be coagulated by boiling. The
albumin may be purified by long dialysis. To recognize the globulin in the pre-
cipitate, first wash the latter with more half-saturated ammonium sulphate and
then dissolve in slight excess of water. It may be necessary to add a little common

THE BLOOD. 1 89

salt to assist in the solution. On heating a portion of this solution coagulation
follows. On diluting some of it very largely with water precipitation of the globu-
lin takes place. From the first water solution most of the salts may be separated
by long continued dialysis.

Magnesium sulphate, added in powder form to saturation, is sometimes used in
the place of ammonium sulphate to effect the separation of the albumin and
globulin. The reaction in both cases depends on the fact that serum albumin may
be salted out only with great difficulty.

It is an interesting fact that other proteins do not appear to be
present in the blood. The various proteins consumed as food suffer
peculiar changes somewhere in the body and are converted into these
two. These in turn serve for the preparation of the various other
related bodies found in the several tissues of the organism. Gelatin
may be formed in this way from the proteins of the blood, but it does
not appear that gelatin can replace other proteins as a food since it is
deficient in one of the essential protein component groups, viz. : the
tyrosine group.

It is not yet known how constant the relation of serum albumin to
serum globulin is or whether this relation is the same in all kinds of
blood. Egg albumin is not equivalent to serum albumin physiolog-
ically, since if injected into the blood it appears soon unchanged in the
urine. The albumins of related animal species seem to be nearly alike,
but this does not hold absolutely true for animals of widely different
species.

THE SUGAR OF THE BLOOD.

This is found in the plasma and has generally been assumed to be
glucose, C6H120G, although good reasons may be assigned for the
assumption of other sugars as well. Ordinarily the simple sugar
finally formed in the digestive process is glucose and the possible pas-
sage of other sugars into the blood has commonly been overlooked.
As the amount of sugar in the blood is small, about 0.15 per cent in
the mean, its certain identification is a matter of extreme difficulty; it
must be remembered that separation from the large amounts of proteins
present must be complete before any accurate identification of the
remaining trace of sugar may be thought of. The older observers
depended almost solely on the common reduction tests which are not
very sensitive in dealing with traces. Recent investigators have shown
that a left-rotating sugar is present and apparently, also, pentoses in
traces. As glucose and fructose yield the same osozone this simple
reaction cannot be applied to detect a fructose content. Occasionally
small amounts of disaccharides appear to be present. Of these maltose
passes into dextrose by inversion, while saccharose and lactose would

190 PHYSIOLOGICAL CHEMISTRY.

be eliminated as such by the kidney. Glucoronic acid in combination
is also present and this may be confounded with a sugar in some of the
tests. More will be found on this point in a following chapter.

SALTS OF THE BLOOD.

The total mineral matters of the blood, exclusive of the iron of the
hemoglobin, amount to a fraction of one per cent only, but still are of
very considerable importance. These salts are largely the chlorides,
phosphates and carbonates of the alkali metals, the potassium salts
being most abundant in the corpuscles, while the sodium salts are most
characteristic of the plasma. It is believed that the variations in this
salt content are very small normally. The nearly constant osmotic
pressure of the blood points to this. Slight changes are speedily cor-
rected by the kidneys.

Experiment. The presence of reducing carbohydrate and salts in the blood may
be demonstrated in this way. Mix about 50 cc. of fresh blood with 300 cc. of water
and boil vigorously a few minutes. A drop or two of acetic acid may be added
during the boiling to maintain a nearly neutral reaction. Filter and divide the
filtrate, which should be perfectly clear, into two parts. Concentrate one-half to a
volume of about 10 cc. and apply the Fehling test for sugar. Concentrate the other
half likewise and use portions for tests for phosphates and chlorides. The sulphate
test usually fails with the volume of blood taken. Evaporate a small portion of
this concentrate nearly to dryness on a glass slide, allow what is left to cool and
crystallize. Sodium chloride crystals may be recognized by the microscope. To
some of the evaporated residue apply the flame test (with spectroscope) for
potassium.

GASES OF THE BLOOD.

The blood holds several gases in loose combination. These are
principally oxygen, nitrogen and carbon dioxide. Minute traces of
argon seem to be present also, which like the more abundant nitrogen
must exist in a condition of simple solution. The methods of accurate
gas analysis as applied to blood were developed by Lothar Meyer, who
made a number of determinations in blood from different sources.
These methods have been further improved by others who have placed
many results on record.

The mean amount of nitrogen is about 2 volume per cent. The
oxygen and carbon dioxide are variable. In arterial blood the oxygen,
which is held mainly through the agency of hemoglobin, amounts to
about 2,2 per cent by volume ; that is, from 100 cc. of arterial blood 22
cc. of oxygen in the mean may be obtained by aid of the vacuum pump.
The venous blood always contains less oxygen, probably not over 15
per cent by volume. These amounts are far larger than could be
absorbed from the air through the partial pressure of the oxygen pres-

THE BLOOD. I9I

ent. In fact it may be shown that only a fraction of i volume per
cent may be held by the blood plasma perfectly free from corpuscles.

The loosely combined carbon dioxide may vary from 30 to 40
volume per cent in the arterial blood, while in venous blood it is much
higher, reaching nearly 50 volume per cent in the mean. This gas is
held partly in the form of bicarbonate and partly through the agency
of the proteins, especially the hemoglobin. Most of the carbon dioxide
is, however, held by the serum and may be largely drawn out by aid
of the vacuum pump. On withdrawal of the gas other weak acid
bodies are able to take its place in alkali combination. It is held by
some observers that the globulins have this power. In acid intoxica-
tions where mineral or organic acids increase in the blood the carbon
dioxide rapidly decreases and may fall to a tenth or twentieth even of
its usual value. These stronger acids take the alkali and there is there-
fore nothing left to hold the carbon dioxide.

Some of these points will be taken up in a later chapter in discussing
respiration phenomena.

Other Substances. Besides the substances mentioned above the
blood always contains a number of other bodies of more or less impor-
tance. Among these may be mentioned the fats, soaps, cholesterol,
lecithin and jecorin. The total fats amount ordinarily to about 0.2
per cent, but after a meal may be temporarily much increased. Minute
traces of fatty acids as soaps may be also present. Cholesterol appears
to be present in free form in traces and also as an acid combination or
ester. Lecithins are present in very small amount in both corpuscles
and plasma, but anything like a quantitative determination does not
appear to be possible. The name jecorin is applied to a peculiar sub-
stance containing phosphorus, described by several observers as occur-
ring in blood. It is soluble in ether, like lecithin, and seems to exist
in combination with a carbohydrate group or similar reducing residue.
The substance has never been obtained in form pure enough for
analysis, and it is even possible that it may be a mixture of several
compounds, one of which is a combination of glucuronic acid.

Variations in Disease. In disease the normal proportions of the various sub-
stances may suffer marked changes. A decrease in the normal number of corpuscles
(about 5 millions to the cubic millimeter for men, 4 to 4.5 millions for women) may
follow to the extent of 10 per cent or more in certain anemic conditions. There may
also be a change in the proportion of hemoglobin without a change in the number of
corpuscles. The methods of estimating the amount of hemoglobin will be given
later. The salts in the blood suffer a percentage decrease after consumption of large
quantities of water, but only temporarily. An actual decrease may occur in cholera
and inflammatory diseases. Tlie normal minute amount of sugar is increased in
diabetes, but not greatly, because of the eliminating power of (he kidneys. It may

I92 PHYSIOLOGICAL CHEMISTRY.

be temporarily increased by the use of certain drugs such as curare, amyl nitrite,
chloral, or by inhalation of chloroform vapor. After meals rich in fats there is
a temporary increase of fat in the blood, but a persistent increase is noticed in the
blood of drunkards and of corpulent individuals. In diseases where there is rapid
breaking down of proteins there is usually observed an increase of fat.

A loss of blood to the extent of one-third is not necessarily dangerous if it be
withdrawn slowly. If one-half the blood is lost there is great danger of death.
Blood may be added by transfusion, but for safety should be from an animal of the
same species. The serum of one animal has usually a destructive action on the
corpuscles of another. Transfused blood then may be a source of danger rather
than a means of saving life. This peculiar action of serum will be referred to later
in some detail.

CHAPTER XII.

THE OPTICAL PROPERTIES OF BLOOD. USE OF THE SPECTRO-
SCOPE AND OTHER INSTRUMENTS.

Solutions of hemoglobin and the various modifications and deriva-
tives described in the last chapter absorb light from certain regions of
the spectrum. The character and extent of this absorption are such
as to afford a ready means of identifying blood or its pigments through
the aid of the spectroscope.

THE SPECTRUM FIELD.

The absorption spectra with which we are here concerned are all
found in the middle portions of the spectrum between the Fraunhofer
lines C and F, that is in a region easily observed. For practical pur-
poses an elaborate instrument is not necessary. Excellent service is
rendered by many of the smaller direct vision spectroscopes. For
quantitative tests, however, much more complete apparatus is required.
The blood spectrum differs from that of all other red solutions and is
very easily recognized with a little practice. As the absorptive power
of hemoglobin is very great dilute solutions only are used and these
must be observed in a rather shallow cell, preferably in one with par-
allel sides about I centimeter apart. For illumination a good oil lamp
flame is excellent ; a steady gas flame may also be employed.

The general arrangement of the essential parts of the spectroscope is shown
by the following figures. Fig. 12 illustrates, diagrammatically, the path of the
light rays through the instrument. From the source F the light enters the colli-
mater tube through a narrow slit and reaches the prism P, where it suffers refrac-
tion and dispersion. Beyond the prism the rays are received by the double con-
vex lens of the ocular tube and thrown to the eyepiece at E. A magnified virtual
image is formed as shown by the dotted lines. The third tube carries a scale, the
image of which is reflected into the ocular and shows with the spectrum. In
absorption spectrum analysis, with which we are concerned here, the light at F
must be white and between this and the collimator slit a cell must be placed to
hold the colored solution or diluted blood. This is shown in the next figure, where
B is an ordinary kerosene lamp with flat wick. The edge of the flame is turned
toward the absorption cell and slit. The apparatus here figured is arranged for
absorption analysis and, with parts to be described later, may be used for quanti-
tative work.

For most simple blood examinations the small direct vision spectroscope shown
below may be used. With proper combination of crown and flint glass prisms it
is possible to practically correct the refraction and leave a field with satisfactory
dispersion.

H '93

194

PHYSIOLOGICAL CHEMISTRY.

Variation in Spectra by Dilution. In all dilutions the positions
of the absorption bands remain the same, but their density and width

Fig. 12. Diagram of simple spectroscope.

vary with the concentration. In a relatively strong blood solution, for
example, there appears to be but one broad oxyhemoglobin band be-

Fig. 13. Spectroscope arranged for absorption analysis.

tween D and E, but on proper dilution this breaks up into two charac-
teristic bands. The question of dilution is therefore important and

A»

Fig. 14. Direct-vision spectroscope.

for any given instrument and light the observer should settle this by
a few preliminary experiments.

THE OPTICAL PROPERTIES OF THE BLOOD. 195

Spectrum of Oxyhemoglobin. This consists of two bands in the
yellowish green portion of the spectrum between D and E. The bands
have not the same width, the one near E being slightly broader than the
other. The preparation of proper dilutions may be illustrated in this
way:

Experiment. Measure out 5 cc. of defibrinated blood and dilute it accurately
with 120 cc. of water. Filter into a clean flask and use the clear filtrate for tests
to follow. Mark this mixture Solution No. I.

Dilute 50 cc. of No. I with 50 cc. of water and mark the mixture Solution No. II.
and continue this until seven dilutions in all are secured, the first one being I in
25, as above, and the last 1 in 1600. This is almost colorless.

Take seven test-tubes of thin, colorless glass, and as uniform as possible in
diameter. Number them 1 to 7 and two-thirds fill each one with the dilute blood
solution corresponding to its number. Place each tube before the narrow slit of
the spectroscope and adjust the flame of an oil or gas lamp so that its light may
pass through the solution into the slit. Pull out the draw tube until the light is
properly focused and observe that the bright field is traversed by two black bands
which cut out portions of the yellow and green. With strong blood solutions all
light except red is shut out, but with solutions of the dilutions 2 to 7 the field is
obscured only by the two bands. In solution No. 2 they are very dark and well
defined. With increasing dilution they grow fainter and are scarcely visible in
solution No. 7. In all the solutions examined note the position of these bands
with reference to the characteristic colors. Note also that the bands grow narrower
with increasing dilution, and that it becomes more and more difficult to locate the
edges of the bands sharply. This fact has some bearing on questions of quanti-
tative determinations to be referred to later. Some of the common absorption
spectra are illustrated.

With instruments furnished with a simple scale it soon becomes an
easy matter to fix approximately the limits between which each band is
found and also the point of deepest absorption in each band. These
data may be expressed in arbitrary scale divisions, in fractions of the
distance from D to E, or most definitely, in wave lengths of light, if
the instrument has been graduated in that way. In all accurate com-
parisons of spectra some such method of recording the observations
must be adopted.

Spectrum of Reduced Hemoglobin. It was pointed out in the
last chapter that the spectrum of reduced hemoglobin is very different
from that of the ordinary oxyhemoglobin. In place of two bands we
have after reduction a single broad band filling three fourths of the
space between D and E. This is the simple effect observed with
Stokes' solution. If ammonium sulphide is employed in place of
Stokes' solution the same broad band appears and in addition a single
narrow band, the center of which is in the red to the left of D. This
narrow band may be due to some sulphohemoglobin formed at the

196

PHYSIOLOGICAL CHEMISTRY.

same time. It will be recalled that the reduced hemoglobin solution
is purplish red in place of deep bright red.

Experiment. To a dilute solution of blood, about 1 part to 50 of water, add a
few drops of strong ammonium sulphide solution and warm gently in a test-tube
until the change of color noted above is reached. Now place the tube before the

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slit of the spectroscope and observe the bands referred to, especially the narrow
one in the red. Hydrogen sulphide gives practically the same result.

Experiment. Repeat the above experiment, using Stokes' solution instead of the
sulphide. A single broad band appears now; if the liquid is shaken briskly the air
acts on the reduced coloring matter with oxidizing effect, as shown by a division

THE OPTICAL PROPERTIES OF THE BLOOD. 197

of the band, but only temporarily. On standing a short time the single broad band,
not very sharply defined, returns. For these tests it would be well to employ
several dilutions, beginning with No. II of the series given above.

Spectrum of Carbon Monoxide Hemoglobin. It has been already-
mentioned that the spectrum of carbon monoxide hemoglobin is very-
similar to that of oxyhemoglobin. This maybe found by a simple test.

Experiment. Into diluted blood, as before, pass a stream of common illuminat-
ing gas until the liquid is saturated, which requires but a few minutes. On placing
the tube in front of the spectroscope the two dark bands described will be seen, and
slightly farther from the yellow than is the case with oxyhemoglobin.

These bands do not change in extent or position by agitation of the liquid with
air, as follows with reduced hemoglobin, and when the liquid is treated with
ammonium sulphide or with Stokes' reagent no reduction takes place. The two
bands persist.

Spectrum of Methemoglobin. This is characterized by a band in the yellowish
red and by a broad band in the blue. To some moderately dilute blood add a few
drops of fresh solution of potassium ferricyanide. The mixture becomes brown. It
has been already explained that by careful reduction with a small amount of am-
monium sulphide hemoglobin is regenerated. This may be followed by the spec-
troscope. Add a few drops of the sulphide solution, allow the mixture to stand a
short time and then shake vigorously in the air. Oxyhemoglobin bands now appear.

The Hematin Spectra. Of these the spectrum of the pigment in weak acid mixture
is the most characteristic. This may be obtained by first coagulating 10 cc. of blood
and 50 cc. of water by vigorous boiling, enough weak sulphuric acid being added
to maintain an acid reaction. The coagulum is separated, pressed dry and rubbed
up in a mortar with 25 cc. of absolute alcohol and 1 cc. of strong sulphuric acid
gradually added. The mixture is then transferred to a flask and heated half an
hour on the water-bath. After cooling the filtered liquid may be examined. A
strong dark band in the red is plainly seen.

Different spectra are found after alkaline treatment. Warm some diluted blood
with a few drops of sodium hydroxide solution until a brownish-green color results.
The absorption spectrum of this liquid is not characteristic. The whole of the
field from red to violet is dark. On careful reduction with a little ammonium
sulphide or Stokes' solution the spectrum of hemochromogen or reduced hematin
may be obtained. This consists of two sharp bands between D and E, somewhat
like those of oxyhemoglobin, but nearer E.

QUANTITATIVE SPECTRUM ANALYSIS.

The amount of hemoglobin in a solution may be very accurately estimated by
the aid of the spectroscope, but an instrument with special attachments for the
purpose is required. Several distinct methods have been applied for the purpose,
but the methods now followed involve a simple direct comparison between light
which has been weakened by passing through a blood solution and light passing
into the spectroscope directly. Such a comparison is easily made by means of
instruments with double collimator slit, as first introduced by Vierordt. The
arrangement of the apparatus made by Kruess is shown in Fig. 13, while the
double collimator slit and ocular and reading scale are shown in Fi^s. 16 and 17.

The method of measurement depends on the principle that there is a simple
relation between the amount of light absorbed by a solution and t lie concentration,

198

PHYSIOLOGICAL CHEMISTRY.

that is the number of absorbing molecules in the same. By finding therefore the
fraction of the original light absorbed we can arrive at the amount of absorbing

substance in solution. The loss of light in
passing through solutions of increasing
concentration follows the law worked out
by Lambert for the loss in passing a series
of glass plates of same thickness and color.
Each new layer absorbs the same fraction
of the light reaching it, and in the same
way each unit of added concentration ab-
sorbs the same fraction absorbed by the
first unit.

Supposing the increased absorption of
light to follow through the addition of new
layers of absorbing substance, the relation
between the original and residual inten-
sities may be reached in this manner.
Calling the original intensity / and the in-
Fig. 16. Symmetrical double slit for tensity after passing the first layer (or
the absorption spectroscope. first unit of concentration) /' we have

I' = I-

the original intensity being reduced to i/n by the first layer,
and following layers we have

By a second, third

n n n n 11

7-1.

nm

The last expression shows the intensity after passing m layers. For purposes of
calculation this can be put in another form, taking the original intensity as unity :

P

gives log F = — m log 11. log n =

log/'

Fig. 17. Ocular and reading scale of the Kruess spectrophotometer.

THE OPTICAL PROPERTIES OF THE BLOOD. 1 99

In comparing the light-absorbing powers of solutions some arbitrary basis must
be taken. Practically the thickness of layer which will reduce the original in-
tensity to tV its value is so taken. The light-extinguishing power of a substance
or its coefficient of extinction, has been defined as the reciprocal value of the
thickness of a layer of the substance necessary to reduce the intensity of the
transmitted light to tb its original value.

Representing the extinction coefficient by E and the reduced intensity by /' we
have from the above formulas :

log —

E = and F = — , log n = = E.

m 10 & I

E

Therefore

E.

log/"

In practice m may be given a constant value and called I (the thickness of cell, for
example). The formula becomes

E — — log r.

It was said above that increasing the thickness of a layer of absorbing substance has
the same effect as increasing its concentration in the same degree. From this it
follows that the extinction coefficient must be directly proportional to the con-
centration. Let E and E' represent two extinction coefficients and C and C the
corresponding concentrations, then

E:C::E':C.

The relations

E Ef E"
-£> c?> -q, >

etc., must be all equal and constant for the same substance. This constant ratio is
a characteristic which connects the light-absorbing power of a solution with its
strength; it may be represented by the letter A and be termed the absorption ratio.
Hence, for a given color

The value of the constant A must be found for a given spectrum region by em-
ploying a series of suitable concentrations. The determination of E consists in
finding the value of the reduced light as compared with the original ; from the
formula given above the extinction coefficient is equal to the negative logarithm of
this diminished intensity. In the various forms of photometers employed in this
kind of work the peculiar measuring mechanism permits the direct and simple esti-
mation of the intensity of the light after absorption as compared with the light
before absorption. We find /' then as a fraction, and E is the negative logarithm
of this. For example, suppose we have in a liter 0.25 gm. of an absorbing substance.
The concentration, or C, is 0.00025, which represents the value per cubic centi-
meter, taken as the unit of volume. Next, suppose we find with our special meas-
uring instrument that the value of the light after absorption is only 0.0436 of the
original, that is about one twenty-third.

200

PHYSIOLOGICAL CHEMISTRY.

Substituting in our formula we have

and finally

E = — log /' = — log 0.0436 = 1. 3605 1

C 0.00025 0

A = — = — ^ — - = 0.000184.
E 1. 36051

In this way, by repeating the observations with a number of different strengths of
solution of the substances, we find the value of the constant. As the individual
observations may differ a little the mean must be taken. A becomes thus fixed once
for all for a given spectral region, and its value may be employed to determine the
concentration of unknown solutions since

C = EA.

Quantitative spectrum analysis by absorption is based on these very simple prin-
ciples. Blood or other substance to be examined is placed in a cell with plane
parallel sides, preferably exactly 1 cm. apart. The cell should be half filled and is
brought into proper position in front of a spectroscope with a double slit, the level
of the liquid just reaching to the top of the lower slit. The light from the
illuminating lamp enters the upper slit directly and the lower one through the

Cfr

I

J

Fig. 18.

Fig. 19.

Absorption cell and Schulz glass prism as used in quantitative analysis by absorption.
The position of the prism is shown in Fig. 19.

colored liquid. If the two slit openings were the same to begin with the upper
one must now be narrowed until the light passing through it is reduced to the
intensity of the light through the blood and the lower slit. The width of each
slit may be measured on a micrometer screw head or in some other convenient
way. In these observations but a small portion of the spectrum is brought into
the field of view. The eyepiece in the spectroscope is therefore furnished with a
screen which can be opened or narrowed at will and symmetrically, that is from
both sides, so as to expose some definite small portion of the spectrum. The
instrument should be so constructed as to permit any desired portion of the spec-
trum to be quickly and accurately brought into the field.

In place of using a simple cell it is much better in practice to employ a cell with
so-called Schulz glass prism. With the simple cell the meniscus formed at the top
of the liquid projects a broad dark band across the field horizontally, which makes
the comparison of the upper and lower spectra very difficult. With the cell fur-

THE OPTICAL PROPERTIES OF THE BLOOD.

20I

nished with a Schulz prism this difficulty is largely overcome, but the details of the
arrangement can not be explained here. They will be easily understood by use of
the instrument. When the Schulz prism is employed light enters the upper slit
through i.i cm. of solution and the lower slit through o.i cm. of solution and the
i.o cm. of the clear glass prism.

Name of Substance.

Spectral Region.

K =

Oxyhemo-

Hemo-

Methemo-

CO-Hemo-

Bilirubin in

Bilirubin in

globin.

globin.
O.OOI22

globin.

globin.

Chloroform.

Alcohol.

569.3-555-5

O.OOI33

0.00260

O.OOI3I

549-9-540.0

O.OOIOO

0.00150

O.OOI99

O.OOII5

558-1-534-3

O.OOII3

O.OOO215

Soi. 2-494.3

O.OOOO598

O.OOOI42

494.3-486.1

O.OOOO356

O.OOOIl6

486.1-480.6

0.0000209

0.0O0IO2

480.6-474.4

O.OOOOI48

O.OOOO842

474.4-4684

O.OOOOI26

0.0000700

468.4-461.7

O.OOOOII8

O.OOOO667

The absorption ratios for a number of physiologically important substances are
shown in the above table. The spectral regions are given in the usual wave lengths.

CLINICAL METHODS OF ESTIMATING OXYHEMOGLOBIN.

The spectrophotometric estimation of oxyhemoglobin as described
above is not simple enough for quick clinical determinations, which
have to be made in the course of daily
practice by medical men. Other forms
of apparatus have been devised for
this purpose and are in common use.
In all of these, comparison is made
between the blood under examination,
properly diluted, and a standard color
assumed to represent normal blood
correspondingly diluted. Some of these
appliances give pretty good results,
but others are very faulty and the val-
ues they furnish quite untrustworthy.
In the following pages several of
the commoner forms will be briefly
described.

Fig. 20. Fleischl hemometer,
showing divided cell for blood and
water and reflecting mirror to se-
cure uniform illumination.

Fleischl's Hemometer. This instrument consists essentially of a circular cell
with glass hottom divided by a vertical partition into two equal compartments
as shown below. In one of these the accurately diluted blood is placed in given
volume. The other compartment is filled with pure water to the same level. The
cell rests on a stage below which there is mounted a white reflecting mirror by
means of which light may be thrown upward to illuminate the two compartments
of the cell uniformly. Immediately under the water compartment a long colored

202

PHYSIOLOGICAL CHEMISTRY.

glass wedge is placed in such a manner that light must pass through it into the
water. By a rack and pinion mechanism this wedge may be moved to the right
or left under the water cell so as to bring a thinner or thicker portion of the
glass below the water. The glass is colored by means of purple of Cassius to
resemble diluted blood as nearly as possible, and the light shining through it into
the water imparts a more or less perfect blood color to it. The wedge is moved

Fig. 21. Dare's hemoglobinometer. R,
milled wheel acting by a friction bearing on
the rim of the color disc ; S, case inclosing
color disc, and provided with a stage to which
the blood chamber is fitted ; T, movable wing
which is swung outward during the observa-
tion, to serve as a screen for the observer's
eyes, and which acts as a cover to inclose the
color disc when the instrument is not in use ;
U, telescoping camera tube, in position for
examination ; V, aperture admitting light for
lllumination of the color disc ; X, capillary
blood chamber adjusted to stage of instru-
ment, the slip of opaque glass, W, being
nearest to the source of light ; Y, detachable
candle-holder ; Z, rectangular slot through
which the hemoglobin scale indicated on the
rim of the color disc is read.

Fig. 22. Horizontal Sec-
tion, of Dare's Hemoglobin-
ometer, in which the. arrange-
ment of parts is clearly
shown. L is the standard
wedge-shaped color disc.
The blood is inclosed between
the glass plates O and P and
is illuminated by the flame /.
The eye observes the blood
and the color scale through
the apertures M and M' .

until the liquids in the two compartments of the cell appear to have the same color.
The color in a certain portion of the wedge is intended to correspond to normal
blood, or blood with ioo per cent of the normal oxyhemoglobin, and degrees placed
at proper intervals along the wedge represent corresponding higher or lower per-
centages. For example, if when the colors in the two compartments are matched
the reading on the wedge scale is 97, it means that the blood contains color due to
97 per cent of the normal or average oxyhemoglobin content.

This Fleischl instrument is one of the best in principle but it must be carefully
used to furnish good results. The color of the wedge does not correspond to blood
color unless a certain kind of white light is employed. A wedge made for candle
light cannot be used with sunlight. In addition to this difficulty the wedges them-

THE OPTICAL PROPERTIES OF THE BLOOD. 203

selves are often at fault. They sometimes fail to produce a blood red with any kind
of light.

Miescher has suggested several improvements in the Fleischl instrument which
render it much more accurate. Readings may be made from several different
dilutions from which a mean value may be taken.

The Hemoglobinometer of Gowers. This instrument has been made in several
forms. The construction is essentially this. Two narrow glass tubes of the same
diameter are used; one receives as a standard a 1 per cent solution of normal blood,
while the other is graduated from below from o to 100 degrees and is intended to
receive the blood under examination. A measured portion of this blood, usually
20 cubic millimeters, is poured into the tube and diluted with distilled water, a
little at a time. After each addition of water the tube is shaken thoroughly to
mix and a comparison made with the standard tube. When the colors are finally
the same, as read horizontally, that is across, not down through the tubes, the
degree of dilution reached in the graduated tube is noted. This indicates the per-
centage of color present as compared with the standard. A blood which can be
diluted to 100 degrees (100 times the original small volume taken) contains 100
per cent of the normal hemoglobin content or is normal, while if it can be diluted
to 75 degrees only, the comparison shows that this blood contains but 75 per cent
of the average hemoglobin.

In place of using blood as a standard a gelatin solution stained with picrocarmine
or other stain is frequently employed. But in time such color standards always
fade, and an abnormally high result is recorded as a consequence.

Dare's Hemoglobinometer. In this instrument the principle employed in the
Fleischl apparatus is used, but the comparison is made between the colored glass
standard and undiluted blood. The possible error due to dilution is thus avoided.
Some idea of the apparatus is given by the illustrations above. A drop of per-
fectly fresh blood is placed over the opening in a capillary flat cell into which it
is immediately drawn, much as a drop of water is drawn in between a slide and
cover glass not in absolute close contact, when the water is put on the edge of the
cover. The capillary observation cell is mounted at the end of an eyepiece through
which it may be clearly seen. A small portion of the colored glass standard may
be seen at the same time. The blood cell and red glass are evenly illuminated by
a candle flame placed in fixed position in front of the apparatus. The colored glass
standard is given the form of a circular disk, which may be rotated by a screw
motion. This disk is beveled from one side to the other, giving a wedge effect as
in the Fleischl apparatus. The rotation of the disk brings therefore thicker or
thinner portions of the edge in the field of the eyepiece along with the cell holding
the blood. When the colors are matched the corresponding hemoglobin value is
read off on a scale. In practice this instrument is somewhat more convenient than
the Fleischl. The accuracy is about the same.

Tallquist's Chart. This consists of a small book containing blank sheets of a
special fine grained filter paper and a colored chart with a number of shades
corresponding to blood stains with different hemoglobin content. To make the
test a small drop of blood is drawn and placed on one of the sheets of filter paper,
into which it soaks and spreads. The color produced is compared with one of the
ten shades in the color scale.

The test is extremely simple, but its accuracy is dependent on the accuracy with
which the colors in the chart are printed, and their permanence. Unfortunately
the colors change as the chart, in use, is exposed to light and air.

CHAPTER XIII.

FURTHER PHYSICAL METHODS IN BLOOD EXAMINATION. FREEZ-
ING POINT AND ELECTRICAL CONDUCTIVITY. THE
HEMATOCRIT.

OSMOTIC PRESSURE.

In many of the phenomena of the body the osmotic pressure of dis-
solved substances plays an extremely important part. This is espe-
cially true in the study of the blood as a whole, and it is therefore
proper at this point to enter upon a short explanation of what is meant
by osmotic pressure and what its relations are.

Nature of Osmotic Pressure. Solids in solution exert a pressure
in all directions quite analogous to that observed with gases, and in
general the laws connecting increase in pressure with concentration and
temperature are the same as for gases. With many solids, however,
dissociation in solution or separation into ions takes place and each
separate ion behaves as a whole molecule as far as pressure is concerned.

Some of the simple effects of this pressure are easily observed.
When a drop of a strong solution of blue vitriol is placed carefully on
the surface of a weak solution of potassium ferrocyanide a precipitate
of copper ferrocyanide forms as a sheath or membrane around the
vitriol drop and holds it in nearly spherical form. If the drop is prop-
erly deposited, which requires some care, it will gradually enlarge by
the entrance of water, which dilutes the enclosed copper sulphate.
None of the latter passes out and the ferrocyanide solution evidently
does not enter since no more precipitate forms within the drop. The
copper ferrocyanide membrane must possess therefore some interesting
properties ; it is permeable for water, it is not permeable for either of
the salt solutions. Similar membranes may be made with a number of
substances and their impermeability for many salt or other solid mole-
cules may be shown. Some membranes are permeable for certain salts
but not for others. The fact of the existence of pressure within such
a membrane may be shown by the following well-known experiment,
in which a copper ferrocyanide sheath or membrane is made in a dif-
ferent manner.

Experiment. Procure a small fine grained porous battery cell, about 3 to 4 inches
long and 1 inch in outside diameter, and clean it thoroughly. This may be done
by washing the cell with water, then with weak hydrochloric acid and finally with

204

PHYSICAL METHODS IN BLOOD EXAMINATION.

20t

water very thoroughly. Close the cell with a perforated rubber stopper, pass a
glass tube through the perforation and connect the outer end of this with a suction
pump. On dipping the cell in water or the acid it may be drawn through the
pores of the cell to effect the cleaning. When the cell is clean it is placed in a
potassium ferrocyanide solution containing about 150 gm. per liter and solution
drawn through by means of the pump until the pores are thoroughly filled. Then
the cell is washed, inside and out, with distilled water
and immersed in a blue vitriol solution containing about
250 gm. per liter. A precipitate is thus formed within
the pores of the cell, which is allowed to remain some
hours in the solution. The cell is then removed,
washed with water and is ready for use. Fill it with
a 5 per cent cane sugar solution, close with the rubber
stopper and long narrow glass tube and immerse the
cell in a beaker of distilled water the temperature of
which should be the same as that of the sugar solution.
After a short time liquid begins to rise in the glass
tube which serves as a kind of manometer. This is in
consequence of the entrance of water to the sugar
solution. Sugar can not pass out in the other direction
as the precipitate membrane is not permeable for it, but
it is readily permeable for the water. The sugar in
it's effort to pass out to the water exerts a pressure on
the retaining membrane, and it is because of this pres-
sure that the water is able to enter the cell. The flow
of the water continues until its hydrostatic pressure
exactly balances the sugar or osmotic pressure. In
some cases mercury manometers attached to such cells
register pressure, of several atmospheres.

The pressure actually observed in such an apparatus is just short of that required
to press the solvent, water, through the membrane in the opposite direction. Theo-
retically it should amount to 22.4 atmospheres for a solution containing a gram
molecular weight dissolved per liter, since it has been found that the osmotic pres-
sure of a body is the same it would possess if it existed in the condition of a gas
at the same temperature and in the same volume. A gram molecular weight of
hydrogen (2.014 gms.), of oxygen (32 gms.), of nitrogen or other gas occupies
a volume of 22.4 liters under normal temperature and pressure conditions. If con-
densed into 1 liter their pressure would be 22.4 atmospheres. Experiment has
shown that a gram molecular weight of sugar or similar solid in water to make
a liter volume exerts a pressure of 22.4 atmospheres. In the case of salts which
break up into component parts or ions the pressure becomes correspondingly greater.
In very dilute solutions a molecule of sodium chloride, for example, exerts prac-
tically double the pressure observed for a molecule of sugar. In this dilute con-
dition the component parts, or ions, of sodium and chlorine seem to exert a pres-
sure corresponding to whole molecules.

The above experiment is a somewhat crude one and is intended
merely as an illustration of the development of pressure. For accu-
rate measurements much more elaborate apparatus must be employed
and numerous precautions observed. Practically, however, osmotic
pressure is always measured by indirect methods to be explained later.
A familiar illustration of a semi-permeable sheath or membrane is

Fig. 23. Apparatus for
observing and measuring
osmotic pressure.

206 PHYSIOLOGICAL CHEMISTRY.

found in the red blood corpuscle. Normally this holds its hemoglobin
and certain salts because it is suspended in a liquid which has the same
osmotic pressure. But if the corpuscles be placed in pure water they
are seen to swell and finally break because of the passage of water
through the cell sheath which is not permeable for the solid contents.
By means of the hematocrit, as will be explained, it is possible to find
the average volume occupied by the corpuscles in a given sample of
blood. When mixed with water or solutions with lower osmotic pres-
sure the corpuscle volume increases ; in stronger salt solutions, on the
other hand, the individual corpuscles shrink in size and their total
volume becomes less. The hematocrit may therefore be used to measure
or compare osmotic pressures in certain cases.

INDIRECT METHODS. CRYOSCOPY.

Although the blood contains about 20 per cent of organic substances
and about 1 per cent of mineral matters its osmotic pressure depends
largely on the latter. This is because of the simple fact that the large
gross weight of organic matter represents relatively but a small number
of molecules, and the actual pressure is measured by the total number
of molecules or ions present. This osmotic pressure in health remains
practically constant; even after great loss of blood when the total
volume is restored by drawing on the lymph serum, although the rela-
tive number of corpuscles may be much reduced, the osmotic pressure
of the new blood is practically unchanged. This is due to the fact that
the blood and lymph serum are isosmotic.

The Freezing Point Method. The measurement of the osmotic
pressure of blood or any other solution by the direct method suggested
by the experiment given above is extremely difficult. Several indirect
methods may be followed, two of which are in common application.
One of these only is suitable for the examination of blood. In the
first of these methods the elevation of the boiling point of the solution
is observed; in the second the depression of its freezing point. Com-
paratively simple relations obtain between the three phenomena. In a
solution the tension of the vapor is decreased in proportion as the
osmotic pressure of the dissolved substance increases and more heat
must therefore be applied to actually lift the atmosphere or boil off
the solvent. For each gram-molecule per liter dissolved this elevation
of the boiling point of water is about 0.520. This method, by noting
the elevation of the boiling point, cannot be applied to blood, because
of its coagulation, but there is no drawback to the method depending
on the separation of the solvent by freezing. With increase in amount

PHYSICAL METHODS IN BLOOD EXAMINATION.

207

of salt or foreign substance dissolved in the water, the lower must its

temperature be brought to effect a partial separation by freezing out

a portion of the solvent. The lowering of the

freezing point is accurately proportional to

the number of molecules (or ions) present.

The molecular freezing point depression for

water is 1.850 ; that is, the freezing point of a

solution containing one molecular weight in

grams of a substance, such as sugar or urea,

dissolved in a liter, is 1.850 below the freezing

point of water. The osmotic pressure of a

substance of which 1 gram molecule per liter

is dissolved in water, is 22.4 atmospheres.

Therefore a freezing point depression of i°

C. corresponds to an osmotic pressure of 12. 1

atmospheres.

Apparatus. Various forms of apparatus have been
devised for the experimental determination of freez-
ing point. The Beckmann apparatus is most com-
monly employed. It consists essentially of a strong
test-tube to contain the substance to be examined.
This is suspended in a somewhat larger tube which
serves as an air bath. The large tube finally is sup-
ported in a strong beaker or battery jar which receives
the freezing mixture to reduce the temperature of
the substance under experiment. The freezing mix-
ture may consist of ice, water and salt, which must
be stirred up frequently to maintain a uniform degree
of cold. A very delicate thermometer passes down
into the substance in the inner tube, which is also
furnished with a stirrer of platinum wire. The blood
or other liquid is stirred until coagulation begins, the
thermometer being meanwhile carefully watched. The
temperature goes down at first a little below the
normal freezing point, because of overcooling, but
soon rises and remains stationary. In experimenting
with aqueous solutions a known weight of pure water
is taken and its freezing point with the thermometer
used is accurately found. Then the salt or other body,
which has been accurately weighed, is added and a new
determination made. While the principle is simple the
details c;dl for some skill in manipulation. Full de-
scriptions of the method may be found in works on
physical or organic chemistry, as it finds applications
in many directions, and especially in the determination
of molecular weight. The general appearance of the simple apparatus is here shown.

The freezing point of normal human blood is about — 0.560. As a
reduction of [° corresponds to an atmospheric pressure of 12.1 atmos-

Fig. 24. Beckmann freez-
ing point apparatus. D is
a fine thermometer, C the
containing jar, B the out-
side or air mantle tube and
A the tube in which the
mixture to be observed is
placed. Two stirrers are
shown ; one for the cooling
mixture in the jar and one
for the experimental mix-
ture.

208 PHYSIOLOGICAL CHEMISTRY.

pheres, the normal osmotic pressure of the blood is about 6.8 atmos-
pheres. It makes but little difference here whether we consider the
whole blood or the plasma free from corpuscles and fibrin. The result
is mainly due to the small molecules present, and these are inorganic.
A solution of 20 gms. of serum albumin in water to make 100 cc.
would have a freezing point of about — 0.03 ° ; the effect of the other
proteins would be practically the same. A solution of urea containing
10 gm. in 100 cc. has a freezing point of — 3.08°, one of glucose with
10 gm. in 100 cc. a freezing point of — 1.03°, while a solution of
common salt with the same weight dissolved would show a depression
of about 50.

Variations. This observed freezing point depression is normally
constant and nearly the same for the blood of all the common animals.
But temporary variations may occur. After consumption of large
quantities of water it may sink to — 0.51 °, while following a meal rich
in salty food a further depression to — 0.620, or even lower, may be
observed. But these changes are very speedily rectified through the
elimination of proper quantities of salts and water by the kidneys. If
an examination of the blood shows a greater depression than that
which may be accounted for by absorption of food constituents a failure
of some kind in the functions of the kidneys is indicated. Through
injury to the mechanism of these organs the osmotic pressure of the
blood may rise to over 12 atmospheres, corresponding to a depression
of the freezing point of a whole degree or more.

Because of these observed facts the determination of the freezing
point of the blood has become a test of practical importance in the
diagnosis of disorders of the kidney. With proper facilities the ex-
periment may be quickly made and will serve to detect an abnormality
in the blood more readily than this may be accomplished by chemical
analysis. It is customary at the present time to designate this freezing
point depression by A. Thus, normally, for human blood

A = — 0.56°.

ISOTONIC COEFFICIENT.

When a few drops of blood are mixed with an excess of salt solution
of a certain strength and the mixture allowed to stand at rest the cor-
puscles gradually settle and leave a colorless liquid above. If the same
volume of a certain weaker salt solution is taken with the blood the
mixture after shaking is found to leave no longer a colorless liquid
above the settled corpuscles, but a somewhat reddish liquid. This color
shows that the corpuscles have been broken and that a little of the

PHYSICAL METHODS IN BLOOD EXAMINATION. 200.

hemoglobin has escaped. An experiment will illustrate the fact; it is
due to Hamburger.

Experiment. Prepare a series of common salt solutions of the following strengths :
0.7 per cent, 0.65 per cent, 0.60 per cent, 0.55 per cenf, 0.50 per cent and 0.45 per
cent. Measure out accurately 20 cc. of each into test-tubes and add to each 5 drops
of defibrinated bullock's blood. Shake and allow to stand. Notice that in some
of the tubes the corpuscles have settled, leaving the salt solution practically clear
and colorless ; in others there is color, which is greatest in the tube with the least
salt. In the tube with 0.60 per cent of salt there should be no color, while in the
tube with the next weaker solution some appears. There must be therefore some
solution between these two in which the corpuscles just fail to give up color. Ham-
burger found this to be one with 0.58 per cent of salt.

Osmotic Tension. Hamburger made a large number of experi-
ments of this description and found the limiting value of the strength
of solutions for which no loss of color follows. He spoke of these
solutions as being isotonic, or as having the same osmotic tension as
the content of the corpuscles. The numerical values found bear a
close relation to the molecular weights of the salts used. Thus, the
following values were noted :

Molecular Weight. Isotonic Value.

NaCl 58.5 0.585 per cent.

NaBr 103.0 1.02 "

Nal 149.9 i-55

KNO3 101.2 r.oi "

KBr 119.1 1. 17

KI 166.0 1.64

These values, while isotonic and isosmotic with each other, are not,
however, quite isosmotic with the blood. A common salt solution
having a percentage strength of 0.9 per cent has practically the same
freezing point as the blood. Blood corpuscles (human) in such a solu-
tion do not swell or shrink, consequently lose no hemoglobin. But if
placed in weaker salt solutions water is gradually absorbed to make the
outside and inside osmotic pressure the same. After a time, however,
with decreasing strength of the salt solution, so much water is absorbed
that the limit of strength of the corpuscle sheath is reached and a break
follows. The escape of hemoglobin shows this point. With salt solu-
tions this break takes place with practically corresponding osmotic pres-
sures, but there are many substances which do not follow the rule at
all. This is particularly true of solutions of urea, glycerol, ammonium
carbonate, sodium carbonate and ammonium chloride. Even with
rather strong solutions of these bodies the corpuscles fail to hold their
hemoglobin. A satisfactory explanation of the abnormal behavior of
these bodies is not known. Blood so changed is said to be lake-colored.
Following the Hamburger designations normal blood was said to be
»5

2IO PHYSIOLOGICAL CHEMISTRY.

hyperisotonic, since it contains more than enough salts to hold the cor-
puscle intact.

HEMATOCRIT METHODS.

It has been shown above that the red blood corpuscles maintain their
normal volume in liquids which have the same osmotic pressure as the
blood. In liquids with a lower pressure they swell, while in solutions
possessing a higher osmotic pressure than the blood they contract.
The corpuscles are extremely sensitive to such influences, and changes
in volume follow with even very trifling changes in the osmotic pres-
sure of a liquid with which the blood may be mixed. The blood cor-
puscle may be used then as a kind of indicator to disclose variations in
osmotic pressure, and substances may be compared as to the osmotic
pressure they exert by noting their behavior with the corpuscles.

It would of course be very difficult to prove anything by measure-
ments on a single corpuscle, but it is possible to make the observation
on a large volume. If blood is drawn up into a narrow tube of capil-
lary dimensions, placed in a centrifuge and rapidly rotated the cor-
puscles are thrown to the outer end of the tube, which must be closed
of course. The volume occupied by the corpuscles compared with the
original blood volume may be easily seen.

Koppe's Hematocrit. An instrument in which such an observa-
tion may be accurately and easily made was devised by Hedin and
called the hematocrit. A special form of this apparatus was con-
structed by Koppe and is used for the purpose of comparison of cor-
puscle volumes. The essential part of the apparatus, as shown in the
figure, is a graduated capillary pipette about 7 cm. in length, which may
be closed at both ends by small metal plates. At the upper end the
capillary bore is widened out so as to form a small mixing vessel. The
pipette proper has a graduation of 100 divisions. By means of a
syringe attached to the pipette by a bit of rubber tubing blood may be
drawn up into the capillary and its volume accurately noted. The
pipette may be closed and rotated now rapidly in the centrifugal
machine, which throws the corpuscles to the outer end. To prevent
coagulation it is best to moisten the pipette first with a layer of cedar
oil which does not interfere with the reading of the blood volume.
The relation between blood volume and corpuscle volume may thus be
read off on the graduation. A drop of similar fresh blood is next
drawn into the capillary and its volume noted; following this some
solution is drawn in also, and then with the blood up into the wider
mixing part, where by means of a bright, fine wire the two liquids may
be stirred together. The plates are then put on the ends of the pipette,

PHYSICAL METHODS IN BLOOD EXAMINATION.

21 I

where they are held by springs. The pipette may be rotated as before
in the centrifugal machine, the rotation being continued until the
volume occupied by the corpuscles becomes constant. By using a
number of pipette tubes it is possible to employ different mixtures and
soon find one in which the corpuscle volume remains normal. If a
series of sugar or salt solutions of known osmotic pressure are em-
ployed, that of the blood must be taken as equivalent to the pressure in
the solution for which no change in the volume of the corpuscles occurs.

d.

Fig. 25. The essential part of the Koeppe hematocrit. The measuring tube a is
closed by two plates, b and c, which are held fast by the springs d. The tube is filled
by means of a peculiar syringe shown at the right.

Conversely the apparatus may be, and is frequently employed to find osmotic
pressures of solutions. The volume of the corpuscles is found in some solution,
of cane sugar for example, which has about the same osmotic pressure as the blood,
but which must be accurately known. Then other solutions of a new substance are
tested until two are found which give volumes, one greater and the other less than
that with the sugar. A simple calculation will then give the concentration of the
solution of the substance under comparison which has the same osmotic pressure as
the standard sugar solution. The method would naturally fail for any substance
which acts chemically on the blood or which destroys the corpuscles, such as urea
or glycerol.

CLINICAL USES OF THE HEMATOCRIT.

On the assumption that the volume occupied by the corpuscles varies
with the number of cells, attempts have been made to use the hematocrit
in place of the cell counter. With normal blood cells the relation is
practically constant and a volume of 50 per cent in the hematocrit cor-
responds very closely to the average 5,000,000 cells per cubic milli-
meter. But unfortunately where such a simple method of making a
blood cell count is the most desirable it is at the same time the least

212 PHYSIOLOGICAL CHEMISTRY.

reliable, since in disease the corpuscles do not always retain their
normal size. A factor of perhaps greater importance, however, is
obtained by taking the ratio of the volume as found by the hematocrit
to the corpuscle count as made by a hemacytometer. With undiluted
blood the hematocrit may be used to determine whether or not pig-
mentation has taken place. If the corpuscles are intact a nearly color-
less serum is secured ; a more or less reddish serum points to disintegra-
tion of the corpuscles.

THE ELECTRICAL CONDUCTIVITY OF BLOOD.

Electrolytes. It has been found by experiment that certain solu-
tions conduct the electric current while others do not. Pure liquids do
not conduct at all, as a rule. Thus absolutely pure water, glycerol,
alcohol, anhydrous sulphuric acid and similar substances are practically
non-conductors. Solutions of many organic substances are likewise
non-conductors, practically. The sugars, for example, belong to this
class. But organic acids and salts and many so-called basic bodies are,
like the corresponding inorganic substances, conductors. In general,
liquid conductors or electrolytes are compounds which in solution sepa-
rate or dissociate into component parts or ions more or less perfectly.

Fig. 26. Diagram of Wheatstone bridge connections. A represents a cell or induc-
tion coil, ac the bridge wire, 6" the standard resistance with which comparison is made,
R the conductivity cell containing the substance under examination. In most con-
ductivity experiments A is a small induction coil, a telephone, as shown, being employed
as the current indicator.

The mineral salts and inorganic acids and alkalies are in general good
conductors, as they "ionize" to a considerable degree.

Blood serum has the power of conducting the current and mainly
because of its content of salts. The proteins in absolutely pure con-
dition, salt free, are probably non-conductors. Some of them, how-
ever, because of their acid character exist in combinations resembling

PHYSICAL METHODS IN BLOOD EXAMINATION. 213

salts and these have a weak conducting power. But, because of their
high molecular weight, the part which this conductivity plays in the
total conductivity of the blood is very small. As applied to the blood,
therefore, conductivity measurements give us an idea of the number of
salt molecules present, or inorganic concentration.

In practice conductivity is the reciprocal of resistance, which is the factor actually
measured. Resistance is expressed in terms of some standard arbitrarily chosen, and
comparisons are usually with the " ohm " as the final standard. The legal ohm is
the resistance of a column of pure mercury 106.3 cm. long and 1 mm. in section at

Fig. 27. Simple Wheatstone bridge. The bridge wire of exact length is stretched
over the graduated scale. The several wire connections are made at the binding posts
lettered.

a temperature of o°, but for practical use resistance standards of wire are employed.
A series of standard wire resistances running from a tenth, or hundredth of an
ohm even, to 1000 ohms or more is generally employed in the form of a resistance
" set " or " box."

In dealing with solutions the unit of conductivity is taken as the reciprocal of the
resistance of a substance which, in the form of a column 1 cm. square and 1 cm.
long (a symmetrical cubic centimeter), has a resistance of 1 ohm. That is, the con-
ductivity, k, is measured in terms of that of an ideal liquid, one symmetrical cubic
centimeter of which has a conductivity of 1 between opposite faces, or which offers
between the same faces a resistance of 1 ohm. The resistance of liquids is always
found in small vessels of glass made in different shapes and sizes according to the
character of the liquid. Small platinum plates are mounted in the vessels and it is
the resistance of the column between these which is measured. Before use the
resistance capacity of the vessel must be found. This is done by measuring in it,
with the plates in fixed position, the resistance of some liquid the conductivity of
which has been previously determined by some standard method. The data for
several solutions have been very accurately determined and are everywhere used
for purposes of graduation of conductivity vessels. With such a standard liquid
with conductivity k we find in our cell the resistance, R. The resistance capacity
C, is given by the relation :

C = Rk.

That is, C is the resistance which would be found in the vessel if it were filled with
a liquid of unit conductivity, and is used as a constant in all following calculations
with the same vessel when we wish to find «. R we always find by direct measure-
ment in ohms and with C known we have now:

C
K = R

The resistance of liquids cannot be found as is that of a solid by means of (he
Wheat-tone bridge combination and a galvanometer, since under such circumstances
liquids suffer hydrolysis with rapid 1 hang< of re (Stance. In place of tli<' direct cur-

214

PHYSIOLOGICAL CHEMISTRY.

rent and galvanometer Kohlrausch suggested the use of a weak induction current,
with a telephone as current indicator. With this arrangement, which is illustrated by
the annexed diagram, it is possible to measure the conductivity of the serum or
other liquid, very readily; a simple form of Wheatstone bridge is shown also.

ac represents the graduated wire of the Wheatstone bridge, 5" the standard resis-
tance with which comparison is made, R the cell containing the serum or other liquid
under investigation, T the telephone which ceases to buzz when no current passes
through it to or from b. This gives the " null " point in the combination and when
this is found the following proportion holds :

ab : be : : S : R.

ab and be are read off directly as bridge wire lengths, 6" is the known comparison
resistance. Hence the unknown cell resistance is given by

R = S

be
ab

As 5" in practice is always taken as io, ioo or iooo ohms and ac is always divided
decimally, tables are constructed giving directly the value of R for any value of ab
read off. In practice the cell R is always kept at a constant temperature, as the
conductivity of liquids varies greatly with temperature changes. To maintain this

Fig. 28. Simple form of
Kohlrausch conductivity cell.

Fig. 29. Conductiv-
ity cell for poor con-
ductors or small quan-
tities.

constant temperature the cell is usually immersed in a large water thermostat, so
constructed that it may be readily controlled. Forms of cells are illustrated.

The electrical conductivity of urine is also an important factor which may be
found by the same kind of apparatus, and which will be discussed later.

Value of the Conductivity for Blood. Expressed in the terms just
explained, the value of the conductivity of blood serum is about
k = 0.012, or expressed in another form very convenient for calcula-
tion, 120 X io-4. A good part of this conductivity is due to the sodium
chloride present. If the chlorides be accurately determined by one of
the usual methods of quantitative analysis and the proper conductivity

PHYSICAL METHODS IN BLOOD EXAMINATION. 21 5

corresponding to this salt content be calculated, which is possible with
a considerable degree of accuracy, and subtracted from the total or
observed conductivity a remainder is obtained which measures the
" achloridic " conductivity, that is the conductivity due to the sulphates,
phosphates and carbonates present.

The conductivity of the salts in the serum is somewhat less than in
pure water, but it is possible to make a correction for this interference
of the proteins and obtain satisfactory values. The conductivity deter-
mination coupled with a few simple chemical tests gives probably a
better view of the inorganic combinations in the serum than would be
found by an examination of the ash of the blood, since the ash must
contain sulphur and phosphorus salts resulting from the oxidation of
the organic compounds of these elements. The general method of cal-
culating conductivities in a mixed fluid like the blood will be discussed
under the head of conductivity of the urine. The information fur-
nished by conductivity measurements is, it will be seen, an extension
of that furnished by the osmotic pressure determinations. By a com-
bination of the two processes it is possible to distinguish approximately
between the concentrations of several classes of molecules present, and
to follow variations in these concentrations rapidly. As yet the clinical
value of the method is somewhat uncertain, however.

CHAPTER XIV.

SOME SPECIAL PROPERTIES OF BLOOD SERUM. BACTERICIDAL
ACTION. PRECIPITINS, AGGLUTININS, BACTERIOLYSINS,

HEMOLYSINS.

SELF PRESERVATION OF THE BLOOD.

In earlier experiments on transfusion of blood to supply a loss
brought about by excessive bleeding it was recognized that the added
blood sometimes seemed to act as a poison to the individual to whom
it was given. It was found later that this toxic action followed the
passage of blood from one species of animal to another, but that the
transfusion from man to man, from dog to dog or from rabbit to
rabbit was not accompanied by the same danger. Such observations
were frequently made and gradually led to the conclusion that the
plasma or serum of the blood of each animal contains a something
which has a destructive action on the corpuscles of other bloods and
which may be designed to protect the blood from the action of any
foreign substance. Various theories have been put forward to explain
this recognized property of the serum. As yet our knowledge in this
field is largely of the empirical order, and scarcely suitable for clear
elementary presentation. But the importance of the subject as thus
far developed is so great that a short chapter on what seems most satis-
factorily established may not be out of place. The phenomena in
question are certainly chemical and from this side must receive their
final explanation. Numerous related phenomena are found to call for
the same kind of consideration.

It has long been known that the large white cells of the blood, the
so-called leucocytes, have the peculiar power of destroying bacteria or
other foreign cells which find their way into the blood stream. Hence
these corpuscles have been called phagocytes or devouring cells. This
destruction of small invading organisms they seem to accomplish by a
kind of digestive process which in a general way may be followed
under the microscope. To some extent the destruction of one kind
of blood by another may possibly be accounted for in this way. But
the chief action is certainly of a different character. While the white
cells are in a measure protective agents, active in destroying elements
which would be harmful if left in the blood, it seems altogether likely
that the most important conserving forces in the blood are soluble com-

216

SPECIAL PROPERTIES OF BLOOD SERUM. 21J

pounds, possibly of the nature of enzymes. This view has been grad-
ually developed and rests on a basis of experiment, and observation.

Harmful foreign bodies entering the blood may be in the nature of
cells, as of bacteria, or they may be the poisons called toxins produced
by bacteria. Anything in the blood which resists or overcomes the
force of this invasion is called an anti body. Normal serum seems to
contain a number of anti substances, which have received different
names, depending on how or against what they act. Some are called
precipitins, others agglutinins, cytotoxins, etc., which terms will be
explained later. In addition to the anti bodies normally present in sera
in variable amounts, and which confer a certain degree of immunity,
there may be produced artificially a greatly increased specific immu-
nity against some particular invasion. It was the discovery of this fact
which in reality led to the systematic study of the whole phenomenon.

The castor oil bean contains a peculiar poisonous principle known
as ricin, which if given in relatively large doses is fatal, but against
which an animal may be immunized by treatment with gradually in-
creasing small doses. An experiment made by Ehrlich, to whom much
is due in this field of investigation, showed that the serum of the treated
animal must contain now a specific anti body capable of neutralizing
the physiological action of ricin. He found that if the ricin poison
and the serum of the immunized animal are mixed in vitro in certain
proportion, and then injected into a fresh non-immunized animal no
toxic action follows. The serum of the first or immunized animal has
acquired the property of chemically combining with or in some manner
neutralizing the action of the poison. That something akin to a chem-
ical action is here in question is shown by the fact brought out by
further experiments that certain proportions must be observed in the
mixing of the serum and toxic substance just as in the complete neu-
tralization of an acid by an alkali. It was further found that this com-
bination may be hastened by heat and retarded by cold, which is true
of most chemical reactions. The behavior is also specific; that is, the
animal immunized against the castor bean poison is not immunized
thereby against other vegetable toxic substances, as abrin, for example,
and the serum of the animal will not, in vitro, neutralize the toxic abrin
solution.

The same general condition has been recognized in connection with
other immunizations and the characteristically specific nature of the
anti body produced in the serum has been shown beyond question.
The anti bodies protective against diphtheria have no effect against the
toxins of tetanus or other disease and vice versa. With such facts

2l8 PHYSIOLOGICAL CHEMISTRY.

established, inquiry was naturally directed toward the question of the
chemical nature of these substances and to the question of their mode
of action. In the voluminous discussions which have been carried on
over these points it is not always easy to distinguish between observed
facts and stoutly maintained theories.

GENERAL CHARACTER OF THE ANTI BODIES.

Antitoxins Proper. Foreign harmful agents gaining access to the
blood may be of several kinds. Some of these are soluble toxic com-
pounds, products of cell action, which in their behavior bear some rela-
tion to strong alkaloidal poisons. Many of these toxins are produced
by bacteria in the animal body during the progress of disease and the
symptoms observed are often due to the action of these poisons rather
than to mechanical disturbances brought about by the bacteria directly.
The toxins as soluble products have the power of wandering with the
blood stream and thus reaching particularly vulnerable or susceptible
organs. The soluble serum constituent which is normally present in
small amount or which may be developed there to neutralize the toxin
in some manner is called an antitoxin in the restricted sense. There is
reason to believe that the two things combine with each other in a true
chemical union and leave a soluble inert product.

Precipitins. The serum of normal blood contains constituents
antagonistic not only to toxic substances but to other sera as well.
The serum of one animal tends to precipitate or render cloudy the
serum of another. This effect may be greatly increased by a kind of
cultivation, which may be illustrated in this way. Rabbits' blood has
normally some antagonism for ox blood, but if sterile ox serum be
injected into the rabbit, intraperitoneally or intravenously, beginning
with small doses and increasing through a number of days, a condition
is finally reached in which the rabbit blood serum shows a very strong
precipitating power for ox serum. The small amount of anti body in
the rabbit's blood has evidently increased enormously through this
treatment. The organism through the attack of the foreign serum
gradually develops a protective agent which acts through exclusion or
precipitation. This serum constituent is called a precipitin.

A vast number of experiments have been made in this field and the
subject has importance in different directions. We recognize not only
the normal effort of the blood to protect itself in this way, but also the
remarkable power of development in the peculiar anti body here con-
cerned. From another standpoint, however, the phenomenon has
assumed even greater importance and that is in the identification of

SPECIAL PROPERTIES OF BLOOD SERUM. 219

blood. This precipitin reaction like the others is specific and the serum
of the rabbit immunized with ox serum will react only with the ox
serum. But precipitins do not seem to be formed in the blood of
animals which are closely related. The serum of a rabbit which has
been treated with pigeon serum will not react with chicken serum; an
anti rabbit serum cannot be secured by treating a guinea-pig with the
serum of rabbit's blood. These general facts have been confirmed by
many observations.

Blood Tests with Serum. The method of utilizing these generalizations is
essentially this. Rabbits are the animals commonly used for experiments, since they
bear the treatment in general well and yield a fairly large quantity of immunized
blood later. Each rabbit is treated by injection with the blood serum of one animal,
these injections being repeated a number of times, through several weeks. Then
the rabbit is killed, bled and the blood allowed to stand for separation of clot.
The clear serum is preserved in sealed tubes for future use, sometimes with, some-
times without addition of an antiseptic, as putrefaction does not appear to impair
the reaction. By immunizing rabbits separately with the blood of man, the ox, horse,
pig, dog, sheep, goat, chicken, etc., a whole series of test anti sera will be obtained,
with which it is possible to identify most of the common bloods. Not much blood
is required in the tests. A drop or two of blood, from a dried clot for example,
is soaked in water or normal salt solution, the liquid obtained filtered to clarify it
and treated in a small test-tube with two or three drops of the test-serum. The
liquid to be tested need not be strong. In practice it should be divided into a
number of small portions in test-tubes and each portion should receive a few
drops of an immunized rabbit serum. Precipitation or clouding will occur in the
tube to which the corresponding anti serum is added. For example, if the original
clot of blood was human blood the extracted dilute serum in all the tubes, except
the one to which rabbit blood immunized with human blood was added, will remain
clear; other tubes with portions of the extracted clot show no reaction with the few
drops of rabbit sera immunized with the blood of other animals.

The medico-legal importance of this reaction has already been recognized and
tested in many ways. The blood of certain monkeys seems to react as does human
blood, but those who have practiced the test most testify as to its certainty and
wide applicability in distinguishing between hu"man blood and the blood of the
common domestic animals.

The Cytotoxins. This name is given to certain anti compounds in
blood which are destructive of form elements. The anti bodies before
considered deal with soluble substances, but here we have to consider
something whose power extends to the breaking down of cell struc-
tures, whether of the blood corpuscle or of bacteria. In the one case
the term hemolysin is used to describe the anti body; in the other case
the term bacteriolysin is employed. In their mode of action these
agents appear to be much alike and both are found in normal bloods.
Both also may be greatly increased artificially.

The hemolytic action of one blood on another was first observed in
experiments on blood transfusion which have been referred to already.

220 PHYSIOLOGICAL CHEMISTRY.

A foreign blood introduced into the circulation of an animal of a dif-
ferent species brings about a variety of changes; clots are sometimes
formed and from resultant changes in pressure serous exudations may
follow. Hemoglobinuria is a general consequence and this of course
results from a breaking down of blood corpuscles in quantity. It has
been assumed by some writers that this hemolytic effect is possibly due
to altered osmotic pressure in the blood, as similar phenomena are
brought about by the admixture of blood with weak solutions. But
the peculiar specificity of artificial hemolysis shows that this explana-
tion is not satisfactory.

If the blood of man, for example, receives an injection of human
blood under proper condition no harm results, but if ox blood be used
the case is different. A large transfusion of the ox blood might be
at once fatal, the hemolysins of that destroying the human corpuscles.
On the other hand transfusion of small amounts of ox blood would
have different effects varying with the manner of transfusion. With
but little ox blood added the human hemolysins would be greatly in
excess and by their chemical mass action would bring about a relatively
great destruction of the ox blood corpuscles, while the corpuscles of the
human blood would suffer but little change. But more than this would
probably take place as illustrated by what has been observed with cer-
tain lower animals. The serum of the eel is especially destructive of
the corpuscles of rabbit's blood and a large injection of eel serum into
the rabbit would produce death. With repeated small doses, however,
the rabbit's blood is stimulated to develop the antitoxin or antihemo-
lysin which protects against the eel serum poison. A condition of
immunity is thus reached, and what has been shown for the rabbit has
been shown for other animals. An explanation of the origin of the
hemolysins will be offered below, but now we are concerned only with
the fact.

In the above cited experiment the eel serum develops gradually an
antihemolysin which works to prevent further destruction of the rabbit
corpuscles. But the action does not stop here. By injection of blood,
hemolysins for the corpuscles of this blood are also formed. Numer-
ous experiments of this kind have been made with animals. For ex-
ample, when rabbit's blood is gradually injected into the dog the pro-
duction of hemolysins is stimulated and the serum of the dog so treated
is found to be far more toxic for the rabbit than was the original
serum. This toxic action, by test-tube experiments, has been found
to be parallel to the hemolytic action of the dog serum on the rabbit
corpuscles, thus showing that the toxicity may depend on the destruc-

SPECIAL PROPERTIES OF BLOOD SERUM. 221

tion of the corpuscles. The hemolysins produced as just explained
are also in general specific in their character, which can be followed by
experiments in vitro as well as in corpore.

In general the bactericidal action of serum resembles its hemolytic
action, although control experiments in vitro cannot be as readily per-
formed. We have therefore the bad erioly sins to consider along with
the other cell destroyers. These bodies exist to some extent in normal
blood and other body fluids and serve to protect the organism against
the attack of bacteria which in any way gain admission to the body.
Milk is relatively rich in bacteriolysins and hence the well-known
germicidal action which has been long recognized. In this respect the
behavior of mother's milk is more marked than that of cow's milk.
Besides the cytotoxins of this class normally present in blood, specific
bodies may be developed by the general methods followed in other
cases, that is by the gradual introduction of cultures of specific bac-
teria, beginning with cultures of relatively little virulence. In this
way the blood of the treated animal becomes immune for some one
bacterium species and develops the power of destroying that bacterium
only for which it was specially immunized. The same animal may be
immunized against several kinds of bacteria at the same time and the
different specific bacteriolysins do not appear to have any destructive
action on each other. They exist together in the blood just as the
different proteins may exist side by side.

Through the process of immunization the blood of the animal
acquires not only the power of attacking the specific bacterium, but
also the toxins of this bacterium. At least two kinds of anti bodies
are therefore produced and there are conditions in which only one of
these may be active. A serum may be active in the breaking down of
bacterial cells, but inert as against the poisons produced by such cells.
The complexities of the phenomena, however, cannot be detailed here.
It should be said further that bacteria produce hemolysins, which are
probably part of the toxins secreted. At any rate some of the toxins
found in cultures are strongly hemolytic.

Agglutinins. Among the several modes of defense observed in
sera of various animals that of agglutination of invading cells must
next be briefly considered. We have seen that blood cells and bacterial
cells may suffer a kind of dissolution through the action of hemolysins
or bacteriolysins, and that a foreign serum is attacked by the precipi-
tins. In addition to these defensive anti bodies there are present others
which work by agglutinating or precipitating cells. A certain simi-
larity exists between these bodies and the precipitins, but investigations

222 PHYSIOLOGICAL CHEMISTRY.

appear to show that they are distinct. The agglutinating power is
found in normal serum, and like the other anti agencies it may be
greatly increased artificially and by the same general means. Agglu-
tinins as precipitating agents enter into a loose kind of combination
with the cells which they throw down. There is here a suggestion of
combination -in some kind of chemical proportions.

Bacteria agglutinins and blood cell agglutinins are to some extent
specific, but apparently less so than are the precipitins. Because of this
specificity the phenomenon has been applied in a method of diagnosis.
Following observations of Gruber and others, Widal suggested a test
which is now commonly employed in diagnosis of typhoid fever. It is
essentially this :

Widal Test. A small amount of the blood or serum of the sus-
pected typhoid fever patient is mixed on a slide with a bouillon culture
of typhoid bacilli. After a time the mixture is examined with the
microscope. If the suspected blood contains the agglutinins developed
in the disease, "clumping" or precipitation of the bacteria from the
culture must follow. (It is held as characteristic that loss of motility
must also be observed.) The intensity of the agglutinin reaction may
be estimated by noting the degree of dilution in which the blood serum
will still agglutinate the bacteria from the bouillon culture.

Opsonins. There has been much discussion as to the manner in
which the phagocytic power of the leucocytes, already referred to, may
he increased. By one school of observers it is held that increased
phagocytosis is due to a modification in the leucocytes themselves, which
modification is produced by a variable element in the serum in question.
The name stimulin has been given to this agent which is able to increase
the activity of the white cells in the destruction of bacteria. Another
and more generally accepted view is that increased phagocytic action is
due, not so much to these cells themselves, but to a peculiar change in
the bacterial organisms, which are to be destroyed. Wright and others
following him hold that the serum contains a specific ferment-like body
whose function it is to prepare or so modify invading bacterial cells
that they may be readily engulfed and destroyed by the phagocytes.
This specific agent is called an opsonin, and it has been shown that the
opsonic power of a serum is subject to great fluctuation. In disease
it may be greatly diminished ; on the other hand, it may be artificially
stimulated in certain directions so as to develop a marked bactericidal
action. From this point of view bloods may be compared through the
so-called opsonic index, which, briefly, is the ratio of the bacteria-
engulfing power of ioo washed white cells in contact with the serum

SPECIAL PROPERTIES OF BLOOD SERUM. 223

of a certain blood, to the power possessed by ioo similar cells in contact
with the serum of a normal blood, taken as a standard, the cells and
sera in each case being brought in contact with the same number of
bacteria. The observation is made by the aid of a high-power micro-
scope. The opsonic index observation has become of such importance
that it is frequently used in diagnosis, and the opsonic treatment of
disease is directed toward increasing this function or power of the
blood of a patient, by properly graduated injections of tuberculin, for
example, that a higher index is gradually developed. It is but fair to
state that the doctrine of opsonins has its active critics as well as
adherents.

Other Anti Bodies. In the above brief survey of the subject of
anti bodies present or developed in the blood only those which have
been the object of most frequent investigations have been mentioned.
Bacteriologists have called attention to numerous other varieties or sub-
divisions, but it is not the purpose of this chapter to take up the dis-
cussion of details. What has been given is sufficient to call attention
to the broader principles concerned.

CHEMICAL NATURE OF THE ANTI BODIES.

On this topic much has been written, but as yet no satisfactory
answer can be given to the question : What are they chemically ? As
formed in the serum of blood or in milk it may reasonably be assumed
that they must bear some relation in composition to the protein bodies.
On this basis attempts have been made to separate them by fractional
precipitation reactions such as were developed by Hofmeister and
others for the proteins and which have been detailed in former chapters.

It appears from the evidence thus far offered that some of these
bodies, at least, must be classed among the globulins. Pick and others
have recently been able to separate the active substances from several
kinds of immune sera and establish pretty accurately the limits of pre-
cipitation. The active fractions separated contained the real anti
bodies in minute amount only, probably. In some cases they were
found in the euglobulin fraction, and in other cases in the pseudo-
globulin fraction of the precipitate.

There has been much speculation as to the part of the blood which
gives rise to these various anti bodies. They are soluble and may not
be separated by filtration but on dialysis they behave as other sub-
stances of very high molecular weight. In many respects they resemble
highly active proteolytic ferments or enzymes, as the characteristic
phenomena are exhibited even in dilutions of the active serum of

224 PHYSIOLOGICAL CHEMISTRY.

I : 20,000, and heat and chemical reagents interfere with the active
properties much as in the case of the enzymes. But there are appar-
ently some exceptions which have led certain authors to deny their
enzyme-like character. From the sum of the facts observed in the
occurrence and action of the anti bodies several writers have been led
to think of them as derived from the breaking down of the highly com-
plex polynuclear white corpuscles of the blood. The behavior of these
in the "living" condition has been already referred to; in their disin-
tegration it is possible they may give off more and more of the groups
on which their activity depends. But there are other possibilities and
these will be referred to in the following section.

ORIGIN AND MODE OF ACTION OF THE ANTI BODIES.
EHRLICH'S THEORY.

In the early days of observations on blood serum. immunity the doc-
trine of the phagocytes received considerable attention. With increase
of knowledge this theory was seen to be inadequate to account for
accumulating facts, and the assumption of the soluble proteolytic fer-
ments, the alexins, was next to attract attention. These may be formed
from the polynuclear leucocytes or more remotely in the organs where
these cells may have their origin, in the spleen, for example, and in the
marrow of bones. As the name indicates the alexins are protective
substances, but the simple assumption of these bodies acting alone, as
a chemical reagent would for example, in the annihilation of intruding
bacteria was soon seen to be too narrow to accord with experience.
The phenomenon of immunity through the alexins or other bodies is a
complex one, but has received much elucidation through numerous
observations of recent years. One of the most important of these is
concerned with the so-called Pfeiffer experiment.

Pfeiffer's Phenomenon. In his well-known experiments on the
behavior of cholera bacteria Pfeiffer found that the serum of an animal
which had been immunized against cholera, when tested in vitro against
the vibrios, seemed deficient in bacteriolytic power, possessing no
greater activity, evidently, than that due to the proteolytic ferment of
the normal serum. Activity may be given to the immunized serum,
however, by injecting it back into the peritoneal cavity of the animal;
cholera vibrios injected at the same time are quickly destroyed. The
action is strictly specific, since typhoid bacilli injected in the same way
remain active. It was found also that the same result can be reached
with the vibrios by adding, in vitro, fresh normal serum to the latent
immunized serum. The vibrios succumb as they did in corpore, show-

SPECIAL PROPERTIES OF BLOOD SERUM. 22 5

ing that a vital action is not in play. Numerous similar observations
were made by Bordet and others. From all these experiments it is
evident that at least two things are concerned in the bacteriolytic action
and the analogous hemolytic phenomena. The immune body devel-
oped in the course of immunization does its work as a cell destroyer,
whether a blood corpuscle or a bacterium, only by the aid of something
normally present in fresh serum. The specific immune body was found
to be thermostable, that is it withstands a temperature of 550— 70 °
without loss of its special properties. If after being warmed to this
extent the immune serum is cooled to below 55 ° and mixed with fresh
normal serum the full cytolytic activity appears. On the other hand
the ferment in the normal serum is thermolabile ; a temperature of 550
or above destroys it permanently. For this thermolabile normal fer-
ment Ehrlich proposed the name addiment. This is the same as the
alexin body. The term complement is now more commonly used to
describe the same thing which seems necessary to make the specific
immune body really active.

The Side Chain Theory. Thus far we have been concerned with
the results of experiments, with facts about which there cannot be
much question. But a comprehensive theory to correlate all these gen-
eralizations became necessary. Many attempts have indeed been made
to establish a theoretical basis for the doctrines of immunity, but it
remained for Ehrlich to suggest something which is really tangible
from the chemical standpoint. To follow the Ehrlich notions some
other matters must be explained first.

Years ago, in attempting to explain some of the properties of large
organic molecules, Pasteur introduced the notion of molecular asym-
metry into chemical science. He showed the value of the notion of
configuration in dealing with certain classes of chemical problems.
This general idea was greatly advanced by Emil Fischer in his papers
on the chemistry of the sugars. Certain ferments were found to
decompose one sugar of an isomeric group, but to leave the other
almost identical sugars untouched. In other words the ferments were
found to observe a specific selection, and to work as ferments, the
enzymes in question must possess a certain stereochemical structure
bearing some relation to the stereochemical structure of the sugar.
Without this relation fermentation cannot take place. In order to
make his meaning plain Fischer employed a figure which has since
become famous. He said, in speaking of certain glucosides, " Enzyme
and glucoside must fit into each other as a key into a lock in order that
the one may be able to exert a chemical action on the other." In one
16

226 PHYSIOLOGICAL CHEMISTRY.

of his papers Fischer suggested that the idea of related molecular con-
figuration of ferment and fermentable body may prove of value in
physiological investigations as well as in chemistry, and in the develop-
ment of the theory of Ehrlich this prediction has been verified. Toxins
and anti bodies combine with each other only when they possess corre-
sponding atom groups, and specificity is regarded as dependent on this
relative configuration.

Without going into minute details the chemical part of the Ehrlich
theory is briefly this : Bacteria, animal cells and toxins are all complex
aggregations of more or less complex molecules. The latter have cer-
tain configurations dependent on the presence of side chains or side
groups, to borrow an expression from organic chemistry. These side
chains are directly or indirectly the points of attack or defense in the
action of the several bodies on each other. In order that a substance
may act as an enzymic poison or toxin to cells of the body both cells
and toxins must therefore possess certain reciprocal configurations. It
has been suggested further that these side groups are concerned in all
the actions of the cells and that it is through them, for example, that
the latter absorb their necessary nutriment and elaborate new struc-
tures from it. Some of the side chains may be constructed to combine
with fats, some with carbohydrates and some with proteins, but in the
presence of toxins or bacteria with the right kind of side chains com-
bination with these may take place instead. Many of these combina-
tions, perhaps all, take place not directly, but indirectly, through the
presence of an intermediary body or group which itself must possess
two linking complexes or groups with proper configurations.

To describe these various groups certain special names have been
suggested. Immune body is the specific substance formed in the im-
munizing process against cells and is known also by several other
names, among which amboceptor and intermediary body are the most
commonly used. The complement, addiment or alexin is the ferment-
like body found in normal fresh serum, and which added to the immune
body makes up the real cytotoxin. It is not specific and, as intimated
above, is sensitive to heat, and also to light and air (oxygen). The
various groups of the large cell complex, whether of a body cell or of
a bacterium, which have the power of uniting with other groups are
called receptors. The part of the receptor which is free to combine
with a food molecule or analogous substance is called its haptophorous
group. Ehrlich pictures the part played by the immune body and the
complement in this way. Assuming that a bacterial cell enters a me-
dium where these complexes are present the immune body attaches

SPECIAL PROPERTIES OF BLOOD SERUM. 227

itself to the haptophorous group of the cell. In this condition no
action follows immediately; but the immune complex has itself two
haptophorous groups or side chains, through one of which the union
with the bacterial cell is effected, while with the other it joins on to the
addiment or complement through its haptophorous group. In this way
the complement, which alone is inactive or unable to attack the cell, is
brought into the immediate neighborhood of the latter, where its pro-
teolytic efforts are more effective. Every fresh normal serum seems
to have present enough of the complement groups; the question of
destroying the invading cell depends then on the number of immune or
intermediary groups in the field. Another part of the Ehrlich theory
attempts to account for these.

The Immune Group. Ehrlich traces the development of the im-
mune body to the spontaneous effort on the part of the cell to protect
and regenerate itself in case of partial destruction. The various cells
of the body exist in a kind of equilibrium with each other. An injury
to one, that is the loss of some of its side chains, immediately leads to
an effort at compensation. The hyperplasia observed in an organ may
extend to the single cells and in consequence of this we have over-
compensation. The cell's efforts at regeneration lead to the produc-
tion of more side chains than are actually necessary and some of these
combine with the aid of their receptor groups with toxin or with com-
plement. Many are formed in excess and are thrown off into the circu-
lation. These free receptors constitute the various anti or immune
bodies. Combining with complement groups they form the true cyto-
toxins. The larger the number of free receptors thrown off into the
blood by the over-compensating efforts of the attacked cells the stronger
is its cytotoxic or antitoxic character since these receptors hold either
the toxin or foreign cell and thus protect the parent native cell from
attack or destruction.

The fundamental point then in the Ehrlich theory of serum immunity
is this formation of side chains in excess by over-compensation, and is
founded on the somewhat earlier Weigert doctrine of cell regeneration
and over-compensation in general. Ehrlich has added the chemical
conceptions of side chain groups and has drawn numerous illustrations
from organic chemistry to show how they may act. Large molecules
holding amino, sulphonic acid or halogen addition groups, for example,
may lose these or take them on again or take others like them without
losing their identity. Reagents acting on such a large molecule attack,
not the nucleus, but these side chains in general. The simple organic
molecule has not the power of self regeneration, but the cell, which is

228 PHYSIOLOGICAL CHEMISTRY.

a collection of many such molecules, has the power of forming new
materials from the nutritive substances furnished to it. If whole new
cells are formed why not parts of cells or the outlying side groups as
well, and this is the Ehrlich assumption, which is not unreasonable.

As explained above these side chains or receptors are of various
kinds. Three distinct types or orders are easily recognized. Recep-
tors of the First Order have one haptophorous group and form anti-
toxins. That is, they combine chemically with the soluble toxins in the
serum and in a sense neutralize them. Receptors of the Second Order
have one haptophorous group with which a foreign molecule or group
may be held and one special group which performs the function of an
agglutinin or precipitin. Receptors of the Third Order or amboceptors
have two haptophorous groups with which two things may be united.
One of these is the foreign cell (through its corresponding hapto-
phorous group) and the other the complement. In this way the com-
plement or alexin is able to work on the invading cell and attack it
through its " zymotoxic " group. These amboceptors are in themselves
inactive and can behave as cytotoxins (hemolysins or bacteriolysins)
only when joined to the complement or ferment group. They are
formed in the serum by immunization with foreign cells, and in turn
combine with cells.

Another product of immunization with cells is the agglutinin recep-
tor, while immunization with toxins leads to the formation of receptors
of the First Order. Cytotoxins produced by one animal species A,
brought into the serum of another animal species B, lead to the forma-
tion of anticytotoxins which may be either anti complements or anti
amboceptors.

Toxin molecules on standing or by heating seem to lose some of
their activity or toxic power, while their power of combining chem-
ically with or neutralizing antitoxins is not diminished. In this con-
dition they are called by Ehrlich toxoids, and he explains the behavior
by assuming that the toxins have two characteristic groups, one of
which, a haptophorous group, persists and combines with the antitoxin
while the other is less stable and may be lost; this he calls the toxo-
phorous group. By warming to 55°-6o° the complement bodies of
serum become converted into active complementoids, which retain the
haptophorous group but lose the zymotoxic group. Amboceptors may
lose in the same way one of their combining groups and become
amboceptoids.

In the further development of the Ehrlich nomenclature the term
toxon was introduced to describe another form of modified toxin.

SPECIAL PROPERTIES OF BLOOD SERUM. 229

The toxons are bodies of relatively slight toxicity, and exist with the
toxin from the start, in place of being developed on standing. They
have the power of combining with antitoxins.

It would not be proper in this place to go more fully into the details
of the Ehrlich theory ; enough has been given to furnish the student an
outline of the most important points in the theory. It is of course true
that much of the present view is artificial and tentative and, with closer
fixation of facts, must be modified. This has been the history of the
development of all chemical theories. In its main features the Ehrlich
doctrine gives us a tangible picture of how the serum may act toward
foreign bodies. For the ultimate reasons for the formation of immune
side chains by stimulation we have no more explanation than we have
for many of the manifestations of chemical affinity. In its outlines
this theory of the action of immune serum appears wholly fanciful, but
in reality it makes no greater claim on the imagination than do some of
the oldest accepted theories of general chemistry.

CHAPTER XV.

TRANSUDATIONS RELATED TO THE BLOOD.

THE LYMPH.

The capillary vessels convey the arterial blood with its store of nutri-
ment to the various tissues, which by transudation receive the required
amount of nourishing matter. The communication between the blood
vessels and the tissue cells is not a direct one; on the contrary, this
transudation takes place into the multitudes of star-shaped spaces
which break the continuity of the cells, and which communicate with
each other by means of fine canals. A liquid passes from the blood
into this network of spaces which form the beginning of a new vascular
system. The transuded liquid is the lymph which serves the double
purpose of nourishing the tissues and draining them also, since this
liquid not only gives up large molecules of absorbable matter, but takes
up at the same time various products of metabolism. What is left
over after this contact with the tissues collects in the minute lymph
capillaries and then into the larger lymphatic circulation proper.

Composition. Being thus related to the blood the lymph must have
a composition not greatly different from that of the plasma. The nor-
mal lymph is a nearly clear fluid with a specific gravity somewhat less
than that of the serum as a rule. It contains salts and organic sub-
stances as does the serum of the blood, but is naturally poorer in protein
elements since a portion of these has been taken up to nourish the neigh-
boring cells. The lymph contains a small amount of fibrinogen. Very
few red corpuscles are present, but as the formation of leucocytes or
white corpuscles takes place in the so-called lymphatic glands these
form elements are abundant in the final flow.

It has been already shown that potassium salts are common to the
corpuscles of the blood, while the salts of sodium are abundant in the
plasma. We naturally find the same thing in the lymph which con-
tains, in addition to proteins, fat, sugar, cholesterol, etc., inorganic
salts, having about the following composition, according to some old
analyses by Schmidt :

Sodium chloride 5.67 per 1000

Sodium oxide 1.27

Potassium oxide 0.16

230

TRANSUDATIONS RELATED TO THE BLOOD. 23 1

Sulphuric acid, S03 0.09 per 1000

P:05 as combined with alkalies 0.02

Calcium and magnesium phosphates 0.26

This result is approximately what one would expect from an analysis
of blood serum and is in fact about what has been found. The two
fluids have the same osmotic pressure, due largely in both cases to
salt content.

Function of the Lymph. The amount of metabolic substances
returned finally to the venous circulation through the lymphatics does
not appear to be great. The chief product of oxidation, COo, seems
to be thrown back directly from the lymph spaces to the smaller vessels
leading to the venous system. The lymph spaces into which the trans-
uded serum flows have apparently two ways of discharge. The bulk
of the liquid with some of the absorbed metabolic products passes, as
intimated, into the gradually enlarging lymphatic system, but certain
other complexes, and among them possibly the most abundant oxida-
tion products, evidently find their way immediately into the capillary
beginnings of the venous circulation. It is not possible to give exact
figures as to the relative amounts of these products going the two
courses, but it is accurately known that the carbon dioxide pressure in
the lymph is much less than in the venous circulation, and the urea
appears also to be less.

It appears to be pretty well established that the leucocytes are active
in hastening the destruction of complex products of tissue waste.
These cells seem to possess a marked chemical activity which is mani-
fested in a kind of digestion of the grosser complexes separated in the
tissue metabolism, but, unfortunately, our knowledge here is meager.
The normal end products of this breaking down process are not formed
at once. Possibly the leucocyte is one of the assisting agents. It has
been therefore held by many writers that the formation of these lymph
cells is probably the most important part of the work in the lymphatic
system.

The amount of lymph which is formed daily is relatively large and
possibly equal to the volume of blood. A large part of this comes
from the flow through the lacteals. Certain substances have the power
of stimulating the flow of lymph. Various salts, sugars and urea have
this property; they are called lymphagogues. Muscular exertion also
increases the production of lymph, because it is needed to supply tissue
waste, and a more rapid flow is called for to carry off the products of
disintegration. Of the manner in which the lymph gives up its content
of nutrients to the tissues absolutely nothing is known; but the object

232 PHYSIOLOGICAL CHEMISTRY.

of this intermediary system is readily seen. It serves as a regulating
mechanism to prevent too rapid changes in the blood composition which
would follow if it should come in direct contact with the tissues.

CHYLE.

During the digestion of fatty foods the lymph absorbed from the
intestinal walls contains numerous minute fat globules in the form of
an emulsion. This portion of the lymph is known as chyle and is
carried along by the lacteals and finally discharged into the lower part
of the thoracic duct.

In composition chyle differs from the lymph from other sources
mainly in its fat content. In the periods when digestion is not in
progress the lacteal lymph is also clear. These vessels are then partly
collapsed and hard to see.

TRANSUDATIONS PROPER.

The lymph has sometimes been considered a transudation of the
blood, but the term is now more commonly used to describe the flow
of liquid from the blood into certain cavities of the body under patho-
logical conditions. A transudation proper is then a modified lymph
and results often from an imperfect elimination of water by the kid-
neys, or from some disturbance in the circulation. Inflammatory
transudations are sometimes distinguished as exudations, and in these
the cell elements are much increased. If they are excessive the dis-
charge is known as pus.

For example, the pleural and peritoneal cavities contain but little
fluid. The serous surfaces are moist, but it would not be possible to
collect enough fluid substances for satisfactory analysis under normal
conditions. In the advanced stage of pleurisy a considerable quantity
of fluid collects in the pleural cavity and its composition resembles that
of the lymph, but it is poorer in solids ordinarily. In some forms of
acute peritonitis a collection of similar fluid may take place in the peri-
toneal cavity and this may amount in bad cases to several liters.

The various forms of dropsy described by physicians are essentially
characterized by analogous transudations of serous fluid without in-
flammation. Ascites, or dropsy of the abdomen, hydrocele, or dropsy
of the testicle, and hydrothorax, dropsy of the pleura, are illustrations.
Some analyses are given below, showing the general nature of the fluids
collected in such cases. It must not be supposed, however, that exactly
similar results would always be obtained by analyses of fluids from the

TRANSUDATIONS RELATED TO THE BLOOD. 233

same organs. The composition of pus serum is somewhat similar; it
contains, however, more products of protein disintegration.

Hydrocele

Fluid Pus Serum

(Hammarsten). (Hoppe-Seyler).

Water 938.85 Water 909.63

Serum albumin 35-94 Proteins 70.22

Globulin 13.25 Lecithin 1.03

Fibrin O.59 Fat O.27

Ether extractives 4.02 Cholesterol 0.70

Soluble salts 8.60 Alcohol extractives 1.13

Insoluble salts 0.66 Water extractives 9.22

Salts 7.75

Pleural Peritoneal

Transudate Transudate

(Scherer). (Hoppe-Seyler).

Water 935-52 Water 969.64

Albumin 49-77 Albumin 19.29

Fibrin ." 0.62 Urea 0.31

Ether extract 2.14 Ether extract 0.43

Alcohol extract 1.84 Alcohol extract 1.37

Water extract 1.62 Water extract 0.98

Salts 7.93 Salts 7.98

Amniotic Fluid. This may be considered as a kind of transudate.
A number of analyses have been made which show about 98.5 per cent
of water, 1 per cent of salts and 0.5 per cent of organic solids, largely
proteins.

THE LYMPH CELLS.

These large cells or leucocytes have already been referred to as
formed in the lymph glands. They are also formed in large numbers
in the spleen and the thymus gland. From whatever source produced
they are supposed to have the same general composition and chemical
function. The following analysis by Lilienfeld gives an idea of their
general composition. The dry substance of the cells amounted to 1 14.9
parts per 1000 and was made up of the following constituents, the
figures referring to per cent amounts of the dry matter. The cells
analyzed were from the thymus of the calf.

Leuco-nuclein 68.79

Albumins 1.76

Histone 8.67

Lecithin 7.51

Fat 4.02

Cholesterol 4.40

( i!;,< ogen 0.80

2 34 PHYSIOLOGICAL CHEMISTRY.

In addition to these organic substances mineral matters are present,
with salts of potassium characteristic. The substance described as
leuco-nuclein is apparently the nucleic acid complex which in the orig-
inal cell is combined with the histone as a nucleate. The lecithin is an
important fraction in these cells, and may exist in part as a protein
combination. The methods of separating lecithin are far from exact.
Pus Cells. In their origin and characteristics these may be consid-
ered as very similar to the leucocytes, if not indeed identical with them.
The few analyses made show a general agreement when reduced to
the same terms. It must be remembered that such analyses are far
from simple operations, especially in the separation of the several pro-
tein constituents. The following figures by Hoppe-Seyler should be
compared in that light with the above. The numbers refer as before
to the dry matter :

Nuclein and albumin 67.40

Lecithin 7.56

Fat 7.50

Cholesterol 7.28

Cerebrin and extractives 10.28

Cerebrin is the name given to a body containing nitrogen in small
amount, but which is not a protein. It is usually found in products
derived from the brain. As to its exact nature but little is known.

The pus cells float in a fluid known as the pus serum, which closely
resembles the other transudates, as shown by the analysis above. The
cells may be separated from the serum by the centrifuge, and if mixed
with a little strong alkali yield a gelatinous slime, which is character-
istic. This test is sometimes applied for the detection of pus in urine.
On bacterial decomposition pus yields a number of products easily rec-
ognized as derived from the nuclein fraction of the protein.

The Spleen. While our knowledge of the functions of the spleen
is very imperfect, a few words may be said in this connection, since as
far as is known the lymph glands in producing leucocytes do about the
same kind of work. The one thing most apparent about the spleen is
that in this organ large numbers of white cells are formed and given
to the blood ; it is also known that these cells suffer destruction there,
as the spleen pulp contains considerable quantities of the xanthine
bases, which are among the common products of cell nuclei destruction.
The cells so destroyed may possibly be those which have already served
their purpose in the blood as the disintegrating agents concerned in the
breaking down of other bodies. Uric acid, as derived from the xan-

TRANSUDATIONS RELATED TO THE BLOOD. 235

thine bases, is known to result when blood is rubbed up with the spleen
substance. The spleen is enlarged in many cases of infectious diseases.
This is possibly from the abnormally great production of leucocytes
needed in the blood in overcoming the effects of the toxic agents or
invading bacteria.

Of the chemical nature of the spleen substance little is known, as it
is practically impossible to free it from blood for analysis. In addition
to the xanthine bodies and other decomposition products there seems
to be present an albuminous substance containing iron, which is con-
sidered as an albuminate; but of its uses nothing definite is known,
beyond the possibility that it may be concerned in the production of red
corpuscles.

The chemical work performed by the spleen may evidently be done
by other organs, as it may be completely extirpated without leading to
fatal results. In its absence the production of great numbers of leuco-
cytes falls to the lymph glands, and the red marrow of bones may take
over the work of generating red blood corpuscles.

CHAPTER XVI.

MILK.

The qualitative composition of milk as produced by the mammary-
glands of different animals is nearly the same whatever the species of
animal. But in quantitative composition very great differences obtain.
Cow's milk has always been taken as the type with which comparisons
are made, as it is the kind everywhere in general use. The essential
differences between it and mother's milk will be pointed out in what
follows.

COW'S MILK.

In an earlier chapter an analysis of cow's milk is given which repre-
sents a general average of composition of good market milk. But the
normal milk of individual cows may be very different from that there
described. The qualitative composition is always the same, but in the
amounts of fat, sugar and protein present the greatest divergences are
noticed. These variations depend on the race of the animal, the period
of lactation and especially on the feed. It is also a well-known fact
that the richness of milk varies during the time of milking, the first
portions of milk withdrawn from the udder being poorer in fat than
the last part or " strippings." In speaking of normal milk, then, these
facts must be kept in mind ; a milk may be normal but not necessarily
rich or good, from the standpoint of food value. The following table
illustrates the variations found in the analyses of milk from a large
number of cows. The mean specific gravity is from 1.029 to 1.033.

Mean. Maximum. Minimum.

Water 87.4 91.5 84.0

Fat 3.5 6.2 2.0

Sugar 4.5 6.1 2.0

Proteins 3.9 6.6 2.0

Salts 0.7 1.0 0.3

When the water of milk is found as high as 91.5 per cent of the
whole the sum of the fat, protein, sugar and salts can be only 8.5 per
cent in place of 12.5 per cent, which should be expected in the mixed
market milk.

Market Milk. Experience has shown that the mixed milk from a
herd of well-kept cows should have a composition not far from that

236

MILK. 237

given in the table above under "mean." Laws have been passed in
most of the large cities of the United States and Europe requiring that
milk sold as pure must be of a quality not inferior to this mean value.
Indeed, in some places a market milk of still higher standard is required.

PHYSICAL COMPOSITION OF MILK.

The exact nature of the mixture of the component parts of milk has
long been a debated question. When taken from the udder the fats,
proteins, sugar and salts are mixed homogeneously, and no immediate
tendency is observed toward a separation of the light fat from the
other and heavier solids. In time, however, such a separation takes
place and the fat rises in the form of cream. Milk cannot be looked
upon as a transudation from the blood because it contains substances
not found in that fluid; the casein of milk and the lactose are different
from the proteins and sugar normally existent in the blood, and the
fat of milk is more complex probably than the blood fat. It is neces-
sary to admit, then, that some of the milk components are produced in
or from the substance of the mammary glands themselves. It is held
by some authorities that the nucleo-proteids of the gland cells are
similar to or identical with the casein, which therefore has its origin
in the gradual breaking down of those cells. In regard to the milk
fat it is known that certain fats can pass but little changed from the
food through the blood and appear finally in the milk, imparting pecu-
liar properties. But, on the other hand, milk fat is produced when the
food of the animal contains no fat whatever, and certainly no fats
resembling the characteristic volatile fats of the milk. In the car-
nivora, confined to an essentially protein diet, milk fat is formed, and
in the herbivora on a diet containing largely pentoses and other carbo-
hydrates milk fat is likewise produced normally and in quantity.

These facts, then, seem to be clear, that while under some circum-
stances fats as such pass from the blood into the milk, and this is further
evident by the experience of feeding cows with certain foods rieh in
fats, the milk glands have the power of producing the several individual
fats as occurring in milk from compounds which are not fat to begin
with. In discussing the chemistry of proteins in an earlier chapter it
was shown that in the breaking down of these bodies under the influ-
ence of various agents fatty acids are found among the decomposition
products. The complex protein molecule may be all that is necessary
to give rise to the milk fats if other things are not available.

The origin of milk sugar is not at all clear. Lactose is not a con-
stituent of our ordinary foods and at best the blood contains probably

238 PHYSIOLOGICAL CHEMISTRY.

only inverted sugars or monosaccharides. In some cases the forma-
tion of milk sugar may be traced indirectly to> the carbohydrates of the
food ; but this will not explain the production of sugar in the carnivora.
Here as before we are probably obliged to fall back on the behavior of
the complex proteins. Among the groups they contain, or at any rate
yield in decomposition, the presence of sugar groups has been certainly
shown. This was explained in a former chapter. The milk lactose
probably results from a synthesis of these simpler sugars.

From what is in general known of the nature of complex protein
matter such as exists in the milk glands it seems therefore possible to
trace the origin of the milk proteins, sugar and fats to the disintegra-
tion of this original protein substance. But of the agents of disin-
tegration, and following necessary syntheses, we know absolutely
nothing. The presence of certain enzymes has been assumed, but as
they have not been isolated or identified, their part in the reactions
remains speculative.

CHEMISTRY OF THE MILK COMPONENTS.

Fats. In the older literature milk fat was given a comparatively
simple composition. It was assumed to consist of stearin, palmitin,
olein and butyrin essentially, the last named volatile fat imparting the
flavor to the separated butter. At the present time we must admit
that our knowledge is far from exact on the subject, but we know
that the composition of milk fat is by no means as simple as once
assumed. In the chapter on the fats an analysis is given which con-
forms better to our modern notions. We find, then, besides butyrin
several glycerol esters of the same series of comparatively volatile
acids. Among the heavier fatty acids myristic acid seems to have
some importance, as disclosed by a number of analyses. A small
amount of lecithin appears to be also present.

Although produced from a variety of materials in feeding experi-
ments, milk fat, as butter, maintains a rather constant composition as
disclosed by both chemical and physical tests. The melting point of
the fat is usually between 31 ° and 32. 5 ° C. and its specific gravity at
380 is about 0.912. Butter fat is easily saponified and from the sapon-
ified mass the fatty acids which are non-volatile and practically insol-
uble in hot water may be separated. These insoluble acids amount in
the mean to about 87.5 per cent of the weight of the original butter
fat. That is, 10 grams of average butter fat should yield 8.75 gm.
of insoluble acids, the difference representing the lighter soluble fatty
acids and glycerol. In the fat from very rich milk the insoluble acids

MILK. 239

may be somewhat lower than this, while in poor milk they would be
higher. These facts are important in distinguishing between butter
and its substitutes.

Fat Globules. In milk the fat exists in the form of minute globules
of different sizes. The diameters of these globules vary between about
0.0016 mm. and 0.01 mm. A cubic centimeter of normal milk con-
taining 3.5 per cent of fat contains 100 millions or more of these
globules. In the milk they are described as existing in the form of
an emulsion, but of the exact nature of this emulsion our knowledge is
imperfect. It has been held also that a membrane of casein encloses
the fat globules and that this prevents the ready extraction of fat
when ether or similar solvent has been added to milk. If the milk is
previously shaken with a little acid which is supposed to break or
destroy this membrane the ether added will now dissolve it. But this
membrane cannot be directly detected with the microscope, and experi-
ments on the formation of fat emulsions by the aid of casein and weak
alkalies have shown that the presence of a membrane is not necessary
to account for the round form or the failure to dissolve readily in
ether. The surface of the globule is not the same as the interior por-
tion, as it appears to take a stain by certain agents which does not pene-
trate. But in the conflict of views advanced it is not yet known what
the surface actually is.

Casein and Lactalbumin. These compounds have been mentioned
in the chapter on proteins and their place in the general scheme of
classification pointed out. In the free, pure state the casein is a dis-
tinctly acid body which neutralizes alkali and forms salts with rather
sharply defined properties. Casein may be easily separated from milk
in this way :

Experiment. Dilute 500 cc. of skimmed milk with about 2 liters of water in a
large jar; add enough dilute acetic acid to make not over 0.1 per cent of the whole.
This causes a precipitation of the casein in fine white flakes which soon settle, leav-
ing a nearly clear whey. After some hours decant this whey and add a greater
volume of distilled water and stir up well. Allow this mixture to settle and pour
off the water. Add a liter of water and enough weak sodium or ammonium
hydroxide to dissolve all the casein and produce an opalescent solution. This in
turn is reprecipitated with dilute acetic acid after adding considerable water, and
these operations are repeated several times. In this way a casein nearly free from
calcium salts is obtained. It is washed well with water by decantation, then poured
on a Buchner funnel, drained, washed with alcohol, until the water is removed and
finally several times with ether to take out the fat. On drying a fine white powder
is obtained with which the important properties of casein may be shown.

Experiment. Weigh out 5 to 10 gms. of casein into a beaker or flask and add
distilled water. Note that it appears to be quite insoluble (This might be shown
by filtering and testing the filtrate by evaporation.) Add a few drops of phenol-

24O PHYSIOLOGICAL CHEMISTRY.

phthalein reagent and run in standard sodium hydroxide solution until a permanent
pink appears. In this way the equivalent or combining weight of the casein may
be found very closely. It is over 1000.

The alkali salt of the casein forms a somewhat viscid solution. If exposed to
the air it dries down to a gummy mass which is very adhesive and acts like a
mucilage. In the arts similar, but crude, solutions are used as sizing material and
as a constituent of certain paints. These products are made from the cheap whey
from the creameries.

Experiment. While the alkali salts of casein are readily soluble in water the
heavier metal combinations are not. This may be shown by adding to the alkali
solution as obtained above solutions of salts of other metals. Precipitates are formed
readily with most of them. The calcium salt is moderately soluble, as may be shown
by rubbing up some casein with calcium carbonate and water. On filtering and
adding a drop of acetic acid to the filtrate a casein precipitate comes down.

Experiment. The lactalbumin may be shown by boiling the decanted liquid from
the first acetic acid precipitation given above. A coagulum forms as in a dilute
white of egg solution to which a little acid had been added.

The phosphorus in casein appears to be combined in at least two
forms. On digesting casein with pepsin and hydrochloric acid a
product known as pseudo-nuclein is separated because of its failure to
digest. In long continued digestion some phosphorus seems to pass
into the form of orthophosphoric acid, while another portion remains
in the albumoses formed, in the organic condition. The digestion
residue, however, is not nucleic acid, which distinguishes the casein from
the true nucleo-proteids. In the precipitation of casein from milk by
the treatment given above the combined phosphorus, whether in acid
or organic combination, does not appear to be touched. The mineral
phosphates are separated, however, but not completely, as the finally
washed and dried casein always contains a trace of ash, a part of which
is calcium phosphate. This ash probably has nothing to do with the
true casein, but is present because of imperfect separation.

Milk Sugar. This crystallizes with one molecule of water,
C12H2201:l -f- H20, and yields glucose and galactose on inversion. It
is separated in large quantities from the whey of cheese factories and
is employed in the manufacture of invalid and infant foods. The
general properties of the sugar have already been given.

The Mineral Substances in Milk. In the analyses quoted at the
beginning of the chapter the ash of the milk is given as about 0.7 per
cent in the mean. This amount appears small, but still it is of the
highest importance, as it makes up between 5 and 6 per cent of the
total solids of the milk. The composition of milk ash has been the
subject of many investigations. While it cannot represent exactly the
condition of the inorganic substances in the original milk, the agree-
ment is an approximate one and is probably near enough for practical

MILK. 24I

purposes. In obtaining ash for analysis, sulphur and phosphorus in
organic combination are thrown into oxidized form and combined as
salts, sulphates and phosphates, in which form we find them in our
subsequent tests. The following figures from Konig represent the
composition of milk ash as the mean of 9 analyses :

Per Cent.

K20 24.06

Na20 6.0s

CaO 23.17

MgO 2.63

Fe203 0.44

P263 27.98

S03 126

CI I34S

Accepting these figures as fairly accurate, and they agree pretty well
with the results of all analysts who have dealt with the question, 1 liter
of average cow's milk would contain the following amounts of the
several constituents:

K20 1-74 gm.

Na20 0.44 gm.

CaO 167 gm.

MgO 0.19 gm.

Fe,03 0.03 gm.

P,03 2.02 gm.

S03 0.09 gm.

CI 0.97 gm.

7.15 gm.

Noteworthy here are the relatively large amounts of the phosphates
of calcium and potassium. These salts represent all the mineral mat-
ters needed in nourishing the body. As found in the milk they exist
in the combinations from which they are most readily assimilated.

The Colostrum. This is the milk secreted before and for a few
days after parturition, and is characterized by higher specific gravity
and content of solids. It contains a large amount of coagulable pro-
teins and therefore thickens on boiling. Some idea of the general
composition is given by the following figures which represent the means
of a number of analyses :

Per Cent.

Water 74-05

Casein 4-66

Albumin 13-62

Fat 343

Sugar 2.66

Salts 158

17

242 PHYSIOLOGICAL CHEMISTRY.

Whey is the fluid left after separation of the larger part of the fat and casein
in the cheese industry or by analogous coagulation. The sugar and salts remain
practically unchanged, while the fat and casein are reduced to traces. The lact-
albumin left averages about 0.5 per cent.

Buttermilk differs from ordinary milk essentially in its lower content of fat. It
is usually sour because of being separated from ripened cream, and contains there-
fore an appreciable amount of lactic acid formed at the expense of some of the
sugar.

Skimmed milk is in composition similar to buttermilk but is usually sweet. In
the modern methods of separation by centrifugal machines the fat may be reduced
to less than half of one per cent; the protein is also somewhat reduced.

SOME EXPERIMENTS WITH MILK.

A few simple tests may be made to illustrate the composition of milk.

The Test for Fat. Pour about 20 cc. of milk in a porcelain dish, add an equal
volume of clean, dry quartz sand and evaporate, with frequent stirring, about an
hour on the water-bath. Then loosen the dry mass as well as possible by means
of a spatula, or glass rod, and pour over it 25 cc. of light benzine. Stir up well
and cover with a sheet of paper and allow to stand 15 minutes. Then pour the
liquid through a small, dry filter into a small, dry beaker, and place this in hot
water to volatilize the benzine.

A residue of fat will be left. Do not attempt to evaporate the benzine over a
flame, or on a water-bath under which a lamp is burning. Heat the water, then
extinguish the flame and immerse the vessel containing the benzine in the hot water.

The Test for Sugar. Measure out about 10 cc. of milk, and dilute it with water
to make 200 cc. Add to this 5 cc. of a copper sulphate solution, such as is used in
making the Fehling solution (69.3 gm. per liter), and then enough potassium or
sodium hydroxide solution to produce a voluminous precipitate containing copper
with all the proteins and fat. For this purpose about 3.5 cc. of a 1 per cent sodium
hydroxide solution will be required. Allow the precipitate to subside, pour or filter
off some of the supernatant liquid, and boil it with Fehling's solution. The charac-
teristic red precipitate forms, showing presence of sugar.

Protein Test. The presence of proteins in milk can readily be shown as fol-
lows : Mix equal volumes of milk and Millon's reagent in a test-tube, and boil. The
bulky red precipitate which forms proves the presence of the body in question.

Action of Rennet on Milk. The mucous membrane of the stomachs of most
animals, and especially that of the young calf, contains an enzyme known as the
" milk curdling ferment," the " rennet ferment," or rennin, the nature of which has
already been explained in the chapter on the ferments.

A crude extract of the mucous membrane of the stomach from the calf is com-
monly called rennet and has long been in use for the curdling of milk in the produc-
tion of cheese. This curdling consists essentially in the coagulation or precipitation
of the casein, which, it will be recalled, is not readily thrown down by the usual
methods.

An active rennet can be readily obtained by digesting the stomach of the calf
with glycerol or brine. If a brine extract is precipitated by alcohol in excess a
white powder separates, which when collected and dried, has very active properties.
Several powders of this description are now in the market. Let the student try the
following experiment with such a product :

Experiment. Warm some fresh milk to a temperature of 380 to 400 C. in a test-
tube or small beaker, then add about half a gram of commercial " rennin," and after
stirring it well keep for 15 minutes at a temperature not above 400. Then as the

MILK. 243

milk cools it assumes the consistence of a firm jelly. It is essential in this experi-
ment that the temperature be kept within the proper limits, as the enzyme is not
active at low temperature and it is, like others, destroyed by high temperature. The
casein or cheese which is obtained in this way is not the same as that precipitated
by acids as it contains much calcium in combination. This form of casein is
usually called para-casein.

Repeat the experiment by adding about 5 drops of a concentrated sodium car-
bonate solution to the milk and then the rennet. Coagulation now fails or is partial.

The Action of Pancreatic Extract on Milk. The behavior of milk with extract
of pancreas is somewhat complicated because of the complex nature of the milk;
the sugar, the fat, and the protein bodies all suffer some change under the influence
of the several pancreatic enzymes.

The most interesting of these changes, however, is that produced in the proteins,
and is commonly called peptonization.

At the present time the digestion, or peptonization of milk, is a very common
practice in the preparation of food for the sick room, and can be illustrated by the
following experiment :

Experiment. Dilute about 10 cc. of milk with an equal volume of water, and add
half a gram of sodium bicarbonate. Next add a few drops of a liquid extract
of pancreas, or a very small amount (10 to 20 mg.) of one of the concentrated
" pancreatin " powders on the market. Shake the mixture and keep it at a tem-
perature of 40 degrees on the water-bath half an hour. At the end of this time
filter and apply the peptone test — potassium hydroxide and dilute copper sulphate —
and observe the pink color. As the action of the pancreatic extract is continued
the liquid resulting becomes very bitter from the formation of digestion products
other than " peptone." The reaction should therefore be checked by cooling before
this very bitter stage is reached.

It will be observed that these experiments illustrate the conditions
in two kinds of digestion. The pancreatic digestion of proteins in
milk is favored by a neutral or slightly alkaline reaction. Alkali inter-
feres with the rennet coagulation. In the stomach the clotting of the
milk is favored by the combined action of the acid and ferment. In
the normal stomach coagulation the presence of calcium salts seems to
be essential. If milk be treated with a small amount of sodium oxalate
solution and then rennet, coagulation fails. Calcium chloride solution
added later, the proper temperature being meanwhile maintained, brings
it about.

THE ANALYSIS OF MILK.

The above experiments suggest some of the steps in the quantitative
analysis of milk, a brief outline of which follows :

Water and Total Solids. Weigh out about 5 grams in a small platinum dish
and evaporate to dryness over a water-bath which requires some hours. Then
transfer the dish to a hot air oven and maintain at a temperature of 1050 through
half an hour. Cool the dish in a desiccator and weigh. The loss of weight repre-
sents the v. practically nothing else of consequence is volatile.

Ash or Mineral Matter. After weighing the dry residue or total solids above
place the dish on a triangle over a clear Bunsen (lame and heat until all the organic
matter, and finally the excess of carbon, i- driven off. The ash left must be per-

244 PHYSIOLOGICAL CHEMISTRY.

fectly white. Cool and weigh as before. There is some slight loss of volatile
salts in this ignition.

Fat. Where many analyses are made as a routine operation, as in the control
of market milk, fat is generally now determined, by separating it from the milk in
a centrifugal machine and reading off the volume. A definite quantity of milk is
measured out, mixed with a little acid to facilitate the breaking up of the fat
globules, placed in a special bottle with graduated neck or stem and rapidly rotated.
The liberated fat collects in the stem and is read off. With the Babcock machine
in common use the method is rapid and very accurate

Fat is very frequently determined by evaporating milk mixed with broken glass
or quartz sand to dryness and extracting with a good solvent, preferably light
petroleum benzine or perfectly anhydrous ether. A better method is to distribute
about s to io grams of milk from a pipette over the surface of a strip of specially
prepared absorbent paper. This is coiled up somewhat loosely, placed in an air
oven, dried thoroughly and then transferred to a Soxhlet extraction apparatus,
where it is treated with the solvent by percolation through two or three hours. The
solvent carries the fat down into a small weighed flask. On evaporation of the
solvent the dry fat is left and may be so weighed.

Sugar. To determine the sugar, the fat and proteins must be first separated,
which may be conveniently done by the copper process as illustrated above. 25 cc.
of milk is diluted with water to 400 cc. and 10 cc. of the Fehling copper solution
added. Then from a corresponding sodium hydroxide solution (containing 10.2
gm. to the liter) alkali is added in amount just sufficient to throw down a bulky pre-
cipitate containing all the proteins and fats with the copper. This requires about
7 cc. of the alkali. The mixture is diluted to 500 cc. and a portion is filtered off for
tests. If the precipitation was properly made a clear filtrate is secured which con-
tains only a trace of copper, and not enough to appreciably affect the accuracy of
titration by the Fehling solution as described in an earlier chapter. The proper
factor for lactose must be used in the calculation.

The protein and fat may be precipitated by use of a solution of lead acetate or
mercuric nitrate without dilution. On filtering a clear filtrate is obtained which may
be tested by the polariscope. 50 cc. of milk should be used, and after precipitation
and filtration made up to 100 cc. for the polarization test. The details cannot be
given here.

The Proteins. If the sugar, fat and ash are accurately found the proteins may
be estimated by difference; that is by subtracting the sum of these from the total
solids found. But this plan should not be followed except as a control. A direct
determination of casein may be made in this way: 10 cc. of milk is diluted to 50
and mixed with dilute acetic acid to produce complete precipitation. Something
less than 1.5 cc. of 10 per cent acid will be needed for this. The precipitate is
collected on a Gooch funnel, washed with water, hot alcohol and finally enough
ether to remove all the fat. What is left is dried and weighed as casein. From
the first filtrate plus the wash water the albumin may be precipitated by boiling. The
coagulum is collected on a Gooch funnel, washed with water and alcohol and dried
as before.

It is also possible to determine the total nitrogen by the Kjeldahl method, and
multiply this by the factor 6.25 to obtain corresponding total protein. This gives
a fairly good control.

MILK PRESERVATIVES.

Milk shippers and dealers often attempt to keep milk from spoiling — turning sour
usually — by the addition of some anti-ferment substance. The propriety of such an
addition has been much discussed. In general the use of food preservatives should

MILK. 245

be kept within certain defined limits, as the consumer has the right to know what
he is using. The chemical substances employed in this way possess different degrees
of activity. Boric acid and formaldehyde have been most frequently added for
the purpose, but they have been rather generally condemned for this and other foods.
In the case of milk it is sometimes a question of the lesser evil; the trace of for-
maldehyde required to effectually preserve it from acid fermentation is very small.
If no more than this minimum is used it is not likely that the harm from using
it would be very great, if at all noticeable. The use of such milk is probably pre-
ferable to that of the sour, unpreserved milk often used by children in the poorer
quarters of our cities.

In many of our large cities attempts are now made to pasteurize

a good portion of the milk supply. The degree of safety afforded by

this operation, as carried out in practice, is, however, very illusory.

MOTHER'S MILK.

We turn now to a short discussion of the chief points of difference
between mother's milk and cow's milk, which is a subject of great
practical importance. Success in substituting cow's milk for mother's
milk in the feeding of small children depends very largely on the
extent and accuracy of our knowledge here. It is a singular fact that
we know much less about the chemistry of human milk than we know
of other milks, and this is in part due to the difficulty in securing a
perfectly normal secretion for analysis.

Because of the presence of certain salts milk has a so-called ampho-
teric reaction, that is, it shows an acid behavior with blue litmus and
an alkaline with red. In mother's milk the alkaline reaction is stronger
than in cow's milk, but the attempts to determine it by titration with
the usual indicators lead to results of relatively little value because of
the disturbing action of the proteins present. The salts of mother's
milk are lower than in cow's milk.

Analyses. The analysis of human milk seems to present several
points of difficulty and the published results do not show very good
agreement. The separation of the proteins offers the greatest diffi-
culty, as the simple and accurate methods employed in the analysis of
cow's milk fail to give equally satisfactory results when applied to
mother's milk. The explanation of this will be given below. There
are variations in the composition of human milk as in that from other
species, but average values are about as given below :

Water 87.5

Fat 3-8

Casein 1 .6

Albumin 0.5

Sugar 6.2

Salts 0.4

100.0

246 PHYSIOLOGICAL CHEMISTRY.

This analysis must be accepted as representing the facts only in a
general way. Indeed, some authors go so far as to assert that no mean
value for woman's milk is possible, as the variations from individual
to individual are too great to permit an average result to have any
legitimate meaning. This much, however, is well established : the fat
in woman's milk is not greatly different in amount from that in cow's
milk; the sugar is about fifty per cent higher in the mean; the salts
are lower, sometimes as little as 0.2 or 0.3 per cent of ash being found;
the total proteins are about half as much as in cow's milk. But as to
the relation of the casein to the albumin and as to the nature of the
casein itself, the greatest divergence of views exists. Some analysts
have actually found more albumin than casein as a result of experi-
ments. This is probably due to the employment of a faulty method
for the precipitation of the casein; it has been pretty well established
that the conditions of precipitation or coagulation are entirely different
from those obtaining for cow's milk. It is indeed likely that the pro-
tein called casein in woman's milk is quite distinct from that of cow's
milk. Under the action of rennet the former coagulates in fine flakes
while the curd of cow's milk as at first produced is in very large flakes.
The two caseins have apparently different contents of sulphur and
phosphorus and give up their nitrogen in digestion experiments in
different ways. It has been recently suggested that the product coagu-
lated as casein from human milk may contain other proteins in sufficient
amount to give it the peculiar properties noticed.

It must be remembered that the salts put down in the analysis of
milk are always obtained as ash from the incineration of an evaporated
residue. In the original milk they do not occur in this form, but in
part, at least, in organic combination. Most of the sulphur and phos-
phorus occur in this condition. The lower casein content of mother's
milk must be responsible for part of the salt difference.

Modified Milk. From all this it is evident that attempts to modify
cow's milk so as to make it resemble mother's milk must be more or
less abortive, as we are not able to duplicate the unknown proteins in
the human secretion. However, many suggestions have been made in
this direction and the line followed is essentially this: Cow's milk is
first diluted with an equal volume of water or whey to reduce the
proteins to the proper percentage amount. Then a certain volume of
cream is added to restore the fat, and enough milk sugar or cane sugar
to bring that constituent up to about 6 per cent. Unfortunately, the
addition of fat is uncertain because of the great variations in market
cream. Good cream should contain at least 20 per cent of fat, but is

MILK. 247

usually much inferior to this standard. The cream sold in cities often
contains from 5 to 10 per cent of fat only. Assuming, however, a
cream containing 20 per cent of fat, 3.5 per cent of casein and 3.5 per
cent of sugar, and taking the gram and cubic centimeter as equivalent
for our purpose, the following illustration will serve as an example
of such a modification. Starting with average market milk of the
composition given some pages back, 500 cc. may be mixed with water,
cream and sugar to give a result as follows :

In 500 cc. of Market
Milk.

1000 cc. contains, approximately, after addition of 400 cc.
of Water, 100 cc. of Cream, 35 gm. of Milk Sugar.

Fat

17-5 gm.
22.5

19-5
3-5

37-5 gm-
61.0

23.0
4.0

3.8 per cent.
6.1

Sugar

Proteins

2-3

0.4

Salts

This mixture has a percentage composition pretty close to that of
mother's milk. Sometimes the dilution is made with whey in place of
water ; the final result in this case is a product containing a little more
protein because of the content of albumin in the whey.

Another important distinction, however, must not be lost sight of.
While the milk of the cow is sterile when it leaves the udder it takes
up from the hands of the milker or from the air a large number of
bacteria which speedily increase to give a content of millions to the
cubic centimeter. Most of these are doubtless harmless and have no
bad effect on the milk ; of others this cannot be said, as their presence
soon leads to changes in the milk which may render it absolutely unfit
for use as an infant food. Mother's milk is and remains sterile and
is therefore free from this danger. It must be recalled further that
the bactericidal behavior of human milk is relatively very strong.
While all milks seem to have a certain content of bacteriolysins, these
anti bodies in mother's milk are most potent as far as the destruction
of the ordinary bacteria is concerned. It is quite likely that no small
portion of the superiority of human milk as infant food is due to this
observed fact.

All kinds of milk are affected to some extent by peculiar flavoring
or other accidental substances in the food of the parent animal. It is
a well-known fact that cows having access to certain weeds yield a
milk with characteristic taste and odor. In the same way many sub-
stances given as remedies pass to some extent into the milk of the
mother and may have an effect on the nursing child. Bay rum used
for bathing the breasts of a nursing mother has been known to pass,
in part at least, into the milk and give to it a very strong odor and taste.

248 PHYSIOLOGICAL CHEMISTRY.

THE MILK OF OTHER ANIMALS.

In some countries the milk of the goat and the ass have economic
importance, and mare's milk is used by certain Asiatic peoples in pro-
ducing a fermented beverage. Analyses of several kinds of milk are
on record; some of these are given in the following table, taken mainly
from the Konig compilation :

Goat.

Ass.

Mare.

Sow.

Bitch.

Cat.

Sheep.

Elephant.

Water

86.9
4.1

4.4

3-7
0.9

90.O
1-3
6.3
2.1

0.3

90.0
I.I
6.7
1.9
0.3

82.4
6.4
4.0
6.1
1.1

75-4

9.6

3-i

11. 2

0.7

8l.6
3-4
4-9
9-4
0.7

81.3

6.8

4-7
6.4
0.8

67.O

Fat

22.0

Sugar

7-4

Proteins

3.0

Salts

0.6

Bunge has called attention to a relation which exists between the
composition of a milk and the rapidity of growth of the animal feeding
on it. In the case of the young of the dog, cat and sheep, for example,
the rate of growth immediately after birth is very rapid and the milk
of the mothers correspondingly rich in proteins and calcium phosphate.
The young of the horse and ass are slow growers, that is, a relatively
long period is required for them to double in weight. These mothers'
milks are low in proteins and salts, but relatively high in sugar. In
the human species these relations are even more pronounced.

CHAPTER XVII.
THE CHEMISTRY OF THE LIVER. BILE. CELLS IN GENERAL.

From the earliest days of physiological chemical investigation the
composition of the liver cells and the nature of the processes taking
place there have been the subject of many studies. It is well known
that the liver has a certain definite work to do in the animal organism
and of some of the functions we have fairly accurate ideas. Of other
functions there is much yet in dispute, but it may be said that a number
of synthetic reactions are unquestionably carried out through the
activity of cell enzymes there formed. Before taking up the special
work of the liver cells something should be said of the composition of
animal cells in general.

COMPOSITION OF CELLS.

In structure all animal cells agree in consisting of two essential parts,
a nucleus and surrounding protoplasm. In young cells these two parts
are usually easily recognized, but in the old cells of complex structures
they assume various forms, bearing apparently little resemblance to the
original type. Cells are in general the center of the various chemical
reactions taking place in the body. Some of these reactions take place
in the fluids outside the cell, but by the aid of ferments of cell origin;
most reactions, however, seem to be carried on within the cell, which
may be illustrated by the familiar conversion of sugar into alcohol and
carbon dioxide by the yeast cell. The enzyme which does this work
may, however, be extracted, as has been already shown.

Of the chemical nature of nucleus and protoplasm not a great deal
is known. It is extremely difficult to isolate original cells from their
modified products or tissues in general, and a sharp chemical differ-
entiation between the two component parts of the cells is not yet pos-
sible, however simple the microscopic differentiation may be. But
some points have been worked out and these will be briefly referred to.
Our information here comes mainly from analyses of the simplest cells,
as in cells of the more complex tissues the true cell characteristics are
obscured.

The Nucleus. The most important chemical constituent of the
nucleus is the complex protein substance known as nuclcin, already

249

25O PHYSIOLOGICAL CHEMISTRY.

referred to in an earlier chapter. Nucleins of different character are
obtained from different sources. These nucleins exist in combination
as nucleo-proteids, and in turn break up into nucleic acids and a protein
fraction, which was explained in the chapter referred to. The cell
nucleus appears to consist very largely if not wholly of the nucleo-
proteid. On digestion with pepsin and hydrochloric acid the nuclein
is separated and may be purified by washing with water, dissolving in
very weak alkali and reprecipitating with acid. By digestion with
pancreatic extract the nucleic acid is left. The pure nuclein is a white
amorphous substance which gives the Millon test and the biuret test.
The various nuclein substances in cells are characterized by a strong
affinity for dye stuffs, especially for some of the coal tar dyes; this
property is utilized in the microscopic examination of tissues. Nuclein
fused with sodium carbonate and nitrate yields phosphate, but heated
without the alkali an acid residue (metaphosphoric acid) is left. Of
other constituents of the nucleus but little is known. Lecithin may be
present.

By various decompositions nuclein substances yield a number of
peculiar basic bodies known as the xanthine or purine bases, which will
be considered in a following chapter. The cell nucleus contains in
combination a number of metallic elements among which iron is perhaps
the most important. Potassium salts are present, while those of
sodium are present only in traces, if at all.

The Protoplasm. This soft spongy portion of the cell consists
largely of water. The solid part, making up 10 to 20 per cent usually,
contains several albumins proper and nucleo-proteids. Lecithin is an
important and relatively abundant constituent of the protoplasm. Its
presence seems to be intimately associated with phenomena of repro-
duction and building up of new tissues. The chemistry of lecithin, as
a phosphoric acid fat, has been explained already, and it must be re-
called that this term includes a number of phosphatides. In the cell
protoplasm they doubtless exist in part as a complex lecitho-protein.
This may account for the fact that the separation of the lecithin is
sometimes difficult.

In all cases the protoplasm seems to contain the complex alcohol
substance cholesterol, glycogen and ordinary fats. How these various
complexes exist, to what extent they are necessary or essential in the
cell structure, we can not say. As cells have various functions to per-
form, they have the power of producing different ferments for the
purpose and such products can not be distinguished by our present
methods of analysis from the material of the cell itself.

THE CHEMISTRY OF THE LIVER. 25 1

FUNCTIONS OF THE LIVER CELLS.

The anatomical location of the liver gives it a most important rela-
tion to the other organs of the body. With the exception of the fats
most of the products of the digestion of foods pass through the portal
vein into the liver and there undergo certain preparatory changes.
Substances not true foods take the same course and many toxic bodies,
metallic and alkaloidal, find a resting place in the liver. In toxico-
logical examinations the liver, after the stomach, is the most important
organ for analysis.

Xot only are the fundamental food stuffs, the proteins and the carbo-
hydrates, worked over and more or less altered in the liver, but partly
metabolized products seem to be further changed in passing through
this organ and are there brought into a condition for final excretion.
The evidences that such reactions take place have been worked out in a
number of cases experimentally and will be referred to below.

Composition of the Liver. We have here the materials found in
cells in general and also others having special functions. The protein
substances separated belong to several groups ; albumin, globulin and a
nucleo-proteid have been recognized. Iron exists in combination with
several of these protein bodies. One of these is known as ferratin and
contains the iron in complex combination; others appear to be albu-
minates in which the iron is more readily recognized.

Next to the proteins the fats are relatively abundant in the liver and
may amount to 3 or 4 per cent by weight normally. Pathologically, by
fatty degeneration, or by filtration from other tissues, the fat may be
greatly increased, even to 30 or 35 per cent of the weight of the whole
organ. The liver fat is usually comparatively soft, but that formed in
some degenerations is harder.

The proportion of lecithins in the liver is variable and is ordinarily
below the true fats. The average amount is from 2 to 3 per cent. It
has a more important function to perform than have the fats proper,
since it is found by experiment that in starvation the lecithin fat is the
last to disappear. The ether extract of the organ in this case is largely
lecithin.

Much has been written of the functions of the lecithin bodies, and
part of their behavior is possibly physical. Recent investigations have
suggested that in certain ferment phenomena they may play the part of
activators for the pro-ferments. In the liver, where a multiplicity of
such reactions occur, the presence of such large quantities of these
phosphatides may have a special meaning.

The most important substance found in the liver is probably glyco-

252 PHYSIOLOGICAL CHEMISTRY.

gen, which is a transformation product and variable in quantity. The
amount present at any one moment depends on the carbohydrate con-
sumption and the time which has elapsed since a meal. It may be
as high as 15 per cent of the whole weight of the organ or may run
down to a fraction of 1 per cent, after fasting or after the performance
of hard work. The formation of glycogen will be discussed below.

The liver, consisting largely of cells in rapid state of change, fur-
nishes a relatively large amount of the so-called nitrogenous extractives.
These include the xanthine and related bodies, urea, uric acid, leucine,
cystin and other substances representing certain stages in metabolism.
The total amount of these compounds present at any one time is very
small, and probably not over 0.5 per cent of the dried organ; but even
this small amount is important, as will appear below.

We have finally several mineral substances present. These include
essentially the chlorides and phosphates of the alkali and alkali earth
metals with some iron compounds. Of the latter those with proteins
have been mentioned above, but other iron salts are present and the
quantity may be increased by the administration of inorganic substances
as remedies. In normal conditions the iron content is extremely var-
iable. The amount may be accurately determined only after washing
out the blood (containing hemoglobin) by aid of salt solution of proper
strength, about 0.9 per cent. Recent investigations have shown that
in the livers of women the iron varies from 0.05 per cent to 0.09 per
cent of the dry substance, while in men the content is more irregular,
running from 0.05 per cent to 0.37 per cent. The amount seems to
increase with age, but no explanation for the variations can be given.
In children and very young animals the content is also high. It sinks,
and rises again, later in life. In addition to the iron a trace of copper
is said to be always present and may have some physiological function.
Other metals occasionally found are probably of accidental occurrence,
as the liver retains such foreign substances through a long period.

CHEMICAL CHANGES IN THE LIVER.

In recent years much has been written on this obscure but highly
important topic. Many of the changes taking place in the liver come
under the head of fermentations, enzymic reactions. Hofmeister
pointed out, a number of years ago, that there are at least eleven of
these in play. He mentioned a proteolytic and a nuclein-splitting fer-
ment, one which splits off ammonia from amino acids, a rennet ferment,
a fibrin ferment, an autolyzing ferment, a bactericidal ferment, an
oxydase, a lipase, a maltase and a glucase. But since then our views

THE CHEMISTRY OF THE LIVER. 253

have been much broadened. We have, in addition to these reactions,
which result in general in the breaking down of molecules, a number
of others which are synthetic in their nature. A brief study of what
is known of all these changes is sufficient to indicate the immense
importance of the liver in the metabolic phenomena of the body.

CARBOHYDRATE CHANGES.

These reactions will be considered first because they have been the
most thoroughly studied and also because of their intrinsic importance.

Glycogen Formation. It was long ago established that the food
carbohydrates after digestion reach the circulation almost exclusively
by way of the portal vein and the liver. In the normal food of man
and the herbivora the carbohydrate food is usually starch and this
becomes dextrin, maltose and finally glucose before absorption. As no
marked accumulation of the sugar takes place in the blood after a meal
it must follow that it or some derived reserve product must be tem-
porarily retained somewhere. The place of this retention is the liver
and the form in which the sugar is held is glycogen. The chemical
reactions of glycogen have been discussed in the chapter on the carbo-
hydrates, but in this place other relations must be considered. No
simple answer can be given to the question as to the method of forma-
tion of glycogen from sugar. Although the formula is commonly
written C6H10O5, it is, like common starch, certainly a multiple of this.
Hence a simple equation connecting glucose and glycogen of the form

CeH1206 — H2O = C6H,0O5

is not strictly correct. Besides, several other facts appear which com-
plicate the problem. While glucose is ordinarily the sugar which
passes through the portal vein, other sugars are also consumed and in
the digestive process do not become changed to glucose. From cane
sugar we have some fructose and from milk sugar some galactose, and
with these in the food it appears that glycogen is still formed. More-
over, it has been shown that substances not carbohydrate at all may
give rise to glycogen. Animals have been starved until the liver was
practically free from glycogen (as known by previous trials with other
animals) and then fed on fibrin or washed out lean meat. On killing
the animals a short time later a store of glycogen was found in the
liver, indicating its formation from something in the protein. With
such facts in mind it is not possible to form any simple theory of the
production of the reserve substance. From the sugars it is likely that
some such reaction takes place as occurs in the formation of starch in

254 PHYSIOLOGICAL CHEMISTRY.

plants. The carbohydrate built up in the plant from water and car-
bonic acid is a sugar and this is transformed by some enzymic reaction
into starch as a reserve material. The mechanism of this change, how-
ever, is quite obscure.

Attempts have been made to connect the formation from proteins
with the sugar group of the gluco-proteids, but casein and gelatin fed
to animals lead also to production of glycogen, and these bodies in pure
condition do not furnish a sugar complex by laboratory treatment. In
addition to this it is impossible that the sugar group could be abundant
enough in the other common proteins to account for the large amount
of glycogen which may be formed by protein diet. These facts lead
to the view that a synthesis must be concerned in the reaction. Such
protein derivatives as leucine, the hexone bases and other bodies have
been thought of as leading possibly to the end, but direct experiments
with animals have given no satisfactory proof of such a hypothesis.
For the present, therefore, the method of production from proteins
must be left without explanation.

A diet of fat leads also to glycogen accumulation or formation in
small amount, according to some recent observations. This latter
reaction requires some kind of an oxidation and is more difficult of
explanation than the other. It must be remembered that an accumu-
lation of glycogen may follow from diminished destruction as well as
from increased production, and where the amount in question is small,
an apparent increase may be traced to errors of observation or experi-
ment. In a mixed diet it is practically impossible to trace the effect
of any one substance. The behavior of pentoses is an illustration;
according to the statements of some authors these carbohydrates in-
crease glycogen. It may be, however, that they simply behave as
sparers of glycogen by undergoing oxidation, which otherwise the
glycogen would have to undergo.

Not all the carbohydrate reaching the portal vein is transformed in
the liver; apparently only a certain portion is so changed, while the
excess is stored up temporarily in other organs. This is evident from
the fact frequently observed in animal experiments that the amount of
glycogen in the liver is below what should be expected from the food
when this is excessive in carbohydrates. With ordinary or deficient
feeding the liver doubtless is able to store as glycogen all the sugar con-
veyed to it, but an excess must find lodgment elsewhere. The muscles
undoubtedly receive the greater share of this excess. In extreme cases
the liver may hold 200 grams of glycogen, which would correspond to
the same weight of starch.

THE CHEMISTRY OF THE LIVER. 255

Glycogen Destruction. This stored up glycogen disappears in
normal conditions gradually after its accumulation; the disappearance
is hastened by work or by lowering of temperature, showing that it
may be called upon for supply of heat as well as for direct mechanical
work. Before being utilized, however, for these purposes the glycogen
must be thrown back into the form of sugar; how this is done is still
a matter of discussion. According to one view the action is a " vital "
one, depending on the life of the cells of the liver themselves; by
another view this conversion is wholly enzymic, a peculiar ferment
bringing about the change during life as well as post-mortem. This
question has lost much of its importance since the work of Buchner on
the zymase, or enzyme of yeast active in alcohol formation, as we now
know that enzymes are present where, by earlier methods of experi-
ment, they were supposed to be absent.

The more recent careful experiments seem to show beyond question that a true
glycogen-splitting ferment is present. By proper manipulation the cell effect may be
excluded, while that of the ferment is left intact. This may be accomplished in
the following way : The fresh organ is washed free from blood by forcing water
through the portal vein until that escaping by the hepatic veins is clear and colorless.
The liver is then chopped fine and allowed to stand a day in a large excess of
alcohol for dehydration. The alcohol is poured off, the residue pressed, dried at
a low temperature and ground to a powder. In this form it is suitable for extrac-
tion with something which does not interfere with enzymic power, but which pre-
vents bacterial or other cell activity. For this purpose chloroform water, or solu-
tions of sodium fluoride have been used. A good extracting mixture may contain
in 100 cc. of water 0.2 gm. of sodium fluoride and 0.9 gm. of sodium chloride.
The liver powder is exhausted with such a solution at a temperature of 380 and the
filtered liquid obtained may be used- in two ways. On standing, the sugar in the
solution increases while the glycogen decreases. In addition, if pure glycogen be
added to such an extract it is found also to diminish with corresponding increase
of sugar. It is further found that boiling the fluoride extract destroys all con-
verting power, which fact speaks likewise for enzyme action.

The glycogen-converting power of solutions made as above is considerable and
sufficient to fully account for the post-mortem increase of sugar always found in
the liver. By extracting not the whole liver but portions it is possible to compare
the distribution of the ferment. Experiments made with this end in view have
shown that this is pretty uniform. By following the same general method Pick
has compared the ferment activity of the liver with that of other organs where
glycogen may be stored. In such experiments the diastatic action of the liver has
been found to be in excess as should be expected, since this is the organ where
the greatest accumulation normally takes place. This normal conversion of gly-
cogen by the liver ferment is interfered with by various substances which may be
taken as remedies; quinine salts seem to be especially active.

AUTOLYTIC FERMENTATION.
The liver, or other organ, removed from the body and left to itself
speedily undergoes a change. Unless precautions are taken to prevent

256 PHYSIOLOGICAL CHEMISTRY.

it the bacterial decomposition may become pronounced and obscure
other reactions. Some years ago Salkowski gave the name auto-
digestion to the fermentations taking place in the liver, in which a
change in the nitrogenous constituents is mainly involved. Other
chemists followed the subject further, taking precautions to exclude
all bacterial influences, and have brought to light a number of very
peculiar reactions which follow from the presence of ferments in the
organs. The name autolysis has been given to these self -digestion
reactions in general. They are not confined to the liver, but are
observed in all organs. An enormous literature has accumulated
already on this topic, because it has great practical as well as scientific
importance. In these spontaneous digestions various products are
formed, some of which are volatile; a general softening of the tissues
concerned may also take place and the sum of these changes is impor-
tant in bringing about the difference between fresh meat, and stored,
" ripe " or " hung " meat, for example. While bacteria play an impor-
tant part in curing meat it is well known that changes go on within the
tissues which cannot be due to bacterial action. These are the autolytic
changes which were first clearly followed in the liver, and which will
be here briefly discussed.

The Production of Organic Acids. This is one of the simplest
phenomena observed. If the livers of dogs or other animals are care-
fully removed and kept under chloroform or toluene a gradual gain in
acidity is observed. The liver must be minced before being covered
with the protecting liquid. The autolysis in this case is slow, weeks
or months being required to show any large amount of acid. The best
temperature for the experiment is 38-400 C. Instead of employing
antiseptics it is possible with care to remove and store the liver in sterile
jars aseptically. Under these conditions the spontaneous change is
very rapid, more acid being formed in one day, ordinarily, than after
a month of the antiseptic treatment. By making several pieces of the
liver on removal from the animal, putting each in a separate jar and
testing one from time to time, it is possible to follow the course of the
autolysis. Among the acids produced formic, acetic, fermentation
lactic and paralactic, butyric and succinic have been recognized. In
experiments described by Magnus-Levy the total acid formed in one
day in 100 grams of liver, by the aseptic treatment, may correspond to
over 20 cc. of normal alkali. If this were calculated as lactic acid it
would amount to 1.8 gm. The relation between the volatile and non-
volatile acids varies with the animal, but not regularly.

It is not possible to trace exactly the source of all these acids, but

THE CHEMISTRY OF THE LIVER. 257

apparently they come in part from a decomposition of the sugar of the
liver, since this is found to decrease as the acid increases. Lactic acid
may be formed first from sugar and butyric acid from the lactic as in
the bacterial fermentations. The appearance of hydrogen and carbon
dioxide at the same time favors this view.

The Alteration in the Proteins. When subjected to aseptic auto-
digestion, or to the same digestion with chloroform or toluene, the
protein substances gradually break down into simpler products. Among
these the amino acids may be most readily recognized ; there is also an
increase in the nitrogen which may be distilled off with magnesia. The
behavior here is somewhat similar to that which follows in acid hydrol-
ysis of the proteins, or which occurs in prolonged boiling with water
under pressure; in both cases a kind of hydrolysis results and this may
be what takes place in auto-digestion.

In prolonged aseptic auto-digestion of the liver very considerable
quantities of leucine and tyrosine are formed; on the outer surfaces,
where evaporation can take place, the latter may even separate in crystal-
line bunches easily recognized. The hexone bases and bodies of the
xanthine group also result but not always in very great quantities.
The greater number of these reactions are those of hydrolytic cleavage,
and that they follow spontaneously is one of the best proofs of the
character of the ferment agents present. While in a general way sim-
ilar, it has been found that certain organs yield amino acid and other
groups not liberated in the autolysis of other organs. This is an
extremely interesting fact, as it points to the specificity of function
suggested also by other reactions. It has been pointed out, further,
that the corresponding organs in different animals show certain differ-
ences in this respect.

Pathological Importance. This possibility of self-digestion in the
liver and other organs may help explain some of the phenomena
observed in pathological conditions. The acids found sometimes in
the urine as well as the leucine and tyrosine have usually been traced
to the liver. These experiments show the rapidity with which such
products may be formed by a degenerative process. Pathologically the
urine sometimes shows a very high reducing power which cannot be
associated with sugar or uric acid or creatinine. The liquid formed in
the liver autolysis is always strongly reducing in action and this may
suggest an explanation for the observation of the urine.

Bactericidal Products. It is worthy of note that in these autolytic
decompositions substances are formed which have a marked bacteri-
cidal action. This has been shown in many ways and the suggestion
18

258 PHYSIOLOGICAL CHEMISTRY.

appears reasonable that in the continuous breaking down processes
going on in various organs we have some of the factors of natural
immunity. These autolytic products must not be confounded with
the alexins already referred to. In a few experiments on record injec-
tions with pressed out juice from autolyzed organs have been sufficient
to prevent death in small animals infected with virulent cultures. The
bactericidal action of the fresh liver or other organ is comparatively

slight.

OTHER FERMENT ACTIONS.

Other ferments present in the liver have not been very thoroughly
studied. The presence of a fat-splitting ferment or lipase has been
shown, but, as yet, little is definitely known of the extent of its action
in the body. The oxidase ferments are better known and the action
of liver extracts in bringing about oxidations of various organic sub-
stances has been studied with the object of throwing some light on
normal oxidations in the body. How many of these oxidizing fer-
ments the liver may contain is of course not known. The reaction thus
far the most carefully studied is that between water extracts of the
liver and salicylic aldehyde. In the process this becomes salicylic acid.

The action of a liquid obtained by pressing the minced liver ground
up with sand has been studied with reference to its power of hydro-
lyzing certain esters. Ethyl butyrate seems to be readily split by this
liver juice. The reaction points to the presence of a lipase-like fer-
ment which doubtless has the power of splitting other bodies of this
type. The boiled liquid is without the ester-splitting power. It has
been found further that the active element can be completely salted
out by addition of ammonium sulphate to saturation, and it may be
precipitated by addition of a strong solution of uranium acetate.

THE BEHAVIOR OF THE LIVER WITH POISONS.

The fact has been referred to already that many metallic and some
organic substances combine with the liver cells. All this has a prac-
tical bearing on toxicological investigations, in which experience has
shown the importance of including the liver in the analytical tests.
Recent experiments have thrown some light on the question of the
manner of combination of poisons. Corrosive sublimate, for example,
fed in very small portions to dogs was found later by post-mortem
examinations in the globulin fraction of the liver extract. The fixa-
tion of arsenic is different ; it combines with a nuclein substance and in
very stable form, which explains the practical difficulty of separating
this substance in forensic investigations.

THE CHEMISTRY OF THE LIVER. 259

Experiments have also been published showing the behavior of small
doses of morphine sulphate and strychnine sulphate in the liver. It
appears that the retaining power of the liver for these poisons is rela-
tively large when they are administered by the mouth or injected into
the portal vein. The retention of the alkaloids by the organ has been
experimentally shown. Such observations have an important bearing
in explaining the fact that many poisons are far more active when
injected hypodermically than when given through the stomach. This
seems to be true of many substances besides the metallic poisons and
the alkaloids. The phenols, for example, are likewise retained to a
marked extent by the liver.

SYNTHETIC PROCESSES IN THE LIVER.

It has long been known that the liver is the seat of the formation of
a large number of metabolic products, some of which involve syntheses.
Several of these reactions may be briefly explained in this place, but
nothing like a full discussion will be attempted. A few illustrative
cases only will be taken to show in a general way what is best known
in this field. The reactions mentioned take place in other organs, as
well as in the liver, but as the latter seems to be mainly concerned, this
is a good place to discuss them.

The Formation of Urea. Of all the synthetic reactions known to
occur wholly or in part in the liver this one has been the most thor-
oughly studied. The older notion of the formation of urea exclusively
from the more complex uric acid is no longer held ; the belief that the
latter complex represents a portion of the protein residue which in
some manner escaped its normal and proper fate, that is, conversion
into urea, has long since been abandoned in view of much accumulated
evidence to the contrary. Indeed, at the present time it appears more
likely that a part of the uric acid excretion may be traced to a synthesis
from urea.

A great many observations unite in suggesting the liver as the organ
in which urea is most abundantly produced, and certain ammonium
salts as being largely or mainly concerned in this production. These
observations have been made in the laboratory as well as clinically. In
diseases in which the liver is involved there has frequently been noticed
a marked reduction in the portion of the excreted nitrogen appearing
as urea. It is also known that the administration of ammonium salts
is not followed by an increase of ammonia in the urine. Parallel witli
this observation we have the further one made on the blood, which has
shown that the fluid of the portal vein is far richer in ammonia than is

260 PHYSIOLOGICAL CHEMISTRY.

that from the hepatic vein. Such observations have been followed up
by experiments in which fresh blood is forced through a living or a
recently removed liver by means of specially constructed apparatus.
The same blood may be caused to pass the liver many times. After
passing a few times and reaching uniformity in composition various
ammonium and related compounds are added to the blood and the cir-
culation then continued. In this way the abundant transformation of
ammonium carbonate into urea is readily shown. It has also been
found that certain amino acids are converted rather readily in going
through the liver. Experiments have shown that in the course of a
few hours several grams of leucine, glycocoll or aspartic acid may be
transformed into urea under these unfavorable conditions.

The importance of this observation will be recognized. It is well
known that the amino acids are among the most important of the dis-
integration products of the proteins ; by hydrolytic and other cleavage
reactions these amino complexes result, and we see here the possibility
of further destruction with ultimate formation of urea. It is possible
that in this reaction carbamates are concerned, as the formation of urea
by alternate oxidations and reductions of ammonium carbamate has
been shown by Drechsel. These reactions illustrate the relations

NH40 • CO • NH2 + O = NH20 • CO • NH2 + H20

NH20-CO-NH2 + H2=NH2-CO-NH2 + H20

It has been shown that the carbamic acid salt frequently appears in
urine, and perhaps normally. This relation is also apparent:

NH4 — O NH4 — O NH2

I I I

CO -* CO+H20 -» CO + H20

NH4 — O NH2— I NH2

There is one ferment reaction which is known to lead to the forma-
tion of urea under definite conditions, and this is the production from
the diaminic acid arginine. The liver and other organs contain an
enzyme, known as arginase, which has the property of splitting argi-
nine into urea and ornithine, or diamino valeric acid. As arginine is
known to be produced normally by the erepsin digestion we have here
a source, for a small part at least, of the urea formation.

The Synthesis of Uric Acid. The mode and place of the forma-
tion of uric acid in the animal organism have been the subjects of
numerous investigations. In birds, serpents and some of the mammals
the excretion of nitrogen is largely in the form of uric acid, and experi-
ments have shown that it is, in part at least, of synthetic origin. The

THE CHEMISTRY OF THE LIVER. 26 1

excretion of uric acid in birds is increased by doses of ammonium
salts ; with the livers extirpated there is a decrease in the elimination
of uric acid and increase in excretion of ammonium compounds. In a
number of such observations the liver has been connected with uric
acid formation, and transfusion experiments, in which blood containing
ammonium lactate and certain other compounds has been forced
through the livers of geese, pointed to the same kind of a synthetic
conversion. For the higher animals, however, a different formation
has usually been assumed, the oxidation of the purine bodies coming
from the breaking down of nucleins being looked upon as the principal
formative reaction.

Later, in a chapter on the urine, the relations of the purines to uric
acid will be pointed out. It is sufficient to state here that the enzymic
production of uric acid from other purines has been clearly shown by
recent observers. These enzymes are contained not only in the liver,
but in the spleen and elsewhere, and it seems likely that other enzymes,
which have been called nucleases, must begin the cleavage of the
nucleins or parent substances.

Comparatively recent experiments by several authors suggest syn-
thetic reactions as likewise possible. Wiener, for example, mixed
chopped beef liver with physiologic salt solution and allowed the mix-
ture to stand at the body temperature an hour. The liquid was then
pressed out and the uric acid in it determined after some time in a
given volume. To the same volume of liver extract definite weights
of urea and various ammonium and sodium salts were added and the
mixture allowed to stand as before. In certain cases a very marked
increase in the uric acid resulted, pointing to the presence in the liver
extract of some agent capable of effecting the combination. The best
results were obtained with dialuric acid salts and tartronic acid and urea.

It is fair to state that another interpretation of these results has been
given. While admitting the formation of uric acid in this way it is
claimed by other physiologists who have repeated the experiments that
the purines in the organic mixture are alone converted, the non-
nitrogenous bodies used acting merely as accelerators in the reactions.
The organs used are all rich in the parent substances of the purines.

The Formation of Ethereal Sulphates. Another reaction of far-
reaching importance in the body is the production of organic sulphates.
The oxidation of the sulphur of proteins leads finally, mainly, to the
formation of sulphuric acid which is eliminated in the urine in the
form of the ordinary mineral sulphates and the ethereal sulphates.
The mineral sulphates are readily formed directly by combinations in

262 PHYSIOLOGICAL CHEMISTRY.

the blood, but for the union of the organic groups with sulphuric acid
some active agent is required. The addition seems to take place in
the liver where it is probable that the oxidation of the sulphur-contain-
ing complex, furnished by protein disintegration also occurs. This
complex seems to be cystin, C6H1204N2S2, which undergoes nearly
complete oxidation to yield sulphuric acid from the sulphur. A small
portion reaches the urine finally in other forms, the so-called " neutral "
sulphur.

Several attempts have been made to determine the seat of the reac-
tion by irrigation tests, and comparatively recently it has been shown
that blood containing phenol and cystin and led through the liver,
freshly dissected, discloses a very considerable oxidation of the sul-
phur compound with production of aromatic sulphate. It appears that
other organs are not much concerned, if at all, in the reaction.

The aromatic radicles which join with sulphuric acid in this way are
mainly products of intestinal putrefactive changes, and by absorption
finally reach the liver. In addition to sulphuric acid glucoronic acid
acts to hold the phenol bodies ; it is usually present in traces in normal
urine and is often greatly increased pathologically. The glucoronates
may be formed in the liver along with the aromatic sulphates. In
experiments which have been carried out on the passage of the blood
through a liver the conjugate phenol bodies produced have frequently
been in excess of the amount called for by the sulphuric acid found;
this excess may correspond in the main with the glucoronic acid.

It has been shown recently that the aromatic complex from the intes-
tine and the sulphur body unite in the liver or other organ only when
the sulphur group is not yet completely oxidized. In other words,
sulphite sulphur and not sulphate sulphur is here concerned. After
the union the final oxidation takes place. More will be said about these
combinations under the head of urine products.

THE BILE.

The formation of bile is one of the important functions of the liver
and the amount secreted in man is several hundred grams daily. Some
of the uses of the bile have been referred to in earlier chapters under
the head of digestion phenomena. Other functions will be discussed
presently.

AMOUNT AND COMPOSITION.

The volume of the bile secreted seems to be subject to variations
which are not well understood. Through the aid of a biliary fistula

THE CHEMISTRY OF THE LIVER.

263

it is possible to collect the total excretion in dogs and other animals
which are easily experimented upon and determine the rate of flow and
the whole amount. The volumes reported by different observers are
not in good agreement. The amounts secreted by different animals
in 24 hours for each kilogram of body weight vary between 12 grams
for the goose and 137 grams for the rabbit. For man the amounts
observed have varied between about 150 and 1,000 grams daily.

The flow of the bile is increased, as far as volume is concerned at
any rate, by the administration of certain remedies. These are known
as cholagogues and among them calomel, certain resins, rhubarb and
oil of turpentine are perhaps best known. That the solids of the secre-
tion are increased is a disputed question. It is proper to state here
that many of the older data on this subject were obtained by methods
which are open to serious objection.

Composition of Bile. Qualitatively bile is characterized by the
presence of certain acids and coloring matters which are not found
elsewhere in the body. The acids are taurocholic and glycocholic, and
the coloring matters are bilirubin and biliverdin, which have been
referred to already in their relation to the coloring matter of blood
from which they are derived. In addition to these substances several
others are present which, while important, are not characteristic. These
include cholesterol, fats, soaps, inorganic salts and mucin. The quanti-
tative composition is extremely variable as shown by the analyses below,
by Hammarsten, which are frequently quoted. The results are in
parts per 1,000:

1

2

3

Water

Solids

974.80
25.20
5-29
3-03
6.28
1.23
0.63
O.22
0.22
8.07
0.25

964.74
35-26
4.29
2.08
16.16
1.36
1.60
0-57
0.96
6.76
0.49

974-60
25.40

Coloring matters and mucin.

5.15
2.18
6.86

1. 01

Lecithin

1.50
0.65

Fat

Soluble salts

0.61

7-25

Insoluble salts

0.21

.Many of the older analyses quoted were made from bile from the
gall bladder. The solids in the bladder bile are always much higher
than those given above, because of a concentration which takes place
in that receptable. Some results for bladder bile are given below :

264

PHYSIOLOGICAL CHEMISTRY.

Water

Solids

Biliary salts

Mucin and pigments

Cholesterol

Lecithin

Fat

Soaps

Inorganic salts

860.0

140.0

72.2

26.6

1.6

3-2

£5

859-2

140.8

91.4

29.8

2.6

9.2

7-7

822.7

177-3

107.9

22.1

47-3

10.8

898.1

101.9

56.5

14.5

30.9

6.2

Analyses have been made of bile from different animals with the
object of connecting composition with the food of the animal or its
habits. The results are not very definite. Human bile contains more
glycocholic than taurocholic acid, while in carnivorous mammals, birds
and fishes taurocholic acid is the more abundant. Hog bile contains
largely glycocholic acid, but in ox bile the relation is variable. The
amounts of the pigments are small and not accurately known.

Glycocholic Acid. This is a complex substance made up of a com-
bination of glycocoll or glycine with cholalic acid. The constitution
of the acid is not known, but the empirical formula C26H43N06 has
been given to it. In the bile it exists in the form of a sodium or
potassium salt, which is readily soluble in water or alcohol. The free
acid is but slightly soluble ; hence the addition of mineral acids to bile
produces a precipitate. On boiling a solution of glycocholic acid with
weak acids or alkalies a cleavage follows, and glycocoll and the nitrogen-
free cholalic acid separate. Water is taken up at the same time. This
is a reaction analogous to the separation of glycocoll and benzoic acid
from hippuric acid by the same manner of treatment. There appear
to be several cholalic acids, but with the common one the reaction
would be represented, probably, in this way :

C6H43NOe + H20 = C2H302NH2 + C24H4o06.

Taurocholic Acid. To this substance the empirical formula
C26H45NS07 is given. With weak acids it undergoes likewise a
hydrolytic cleavage from which taurin and cholalic acid result. Taurin
appears to be aminoethylsulphonic acid, C2H4NH2-HS03, and the
cleavage would be represented in this way :

C26H«NS07 + H20 = C24H4»05 + C,ILNH2HS03.

The free acid has a bitter-sweet taste ; it is much more soluble in water
than the glycocholic acid and somewhat soluble in alcohol. The free
acid has the property of holding glycocholic acid in aqueous solution,
which is shown by the difficulty in precipitating the mixed acids from

THE CHEMISTRY OF THE LIVER. 265

ox bile. The free acid is but slightly soluble in ether. The alkali salts
are soluble in water and alcohol.

Cholalic Acid. Although many investigations have been carried
out with this substance its constitution is not clear. The above em-
pirical formula, C24H40O5, is that of a monobasic acid to which Mylius
has given this possible structure,

fCHOH

c»n»1 CH2OH
[cOOH

The free acid is very slightly soluble in water, but the alkali salts are
readily soluble. The free acid is somewhat soluble in ether; hence it
is found as a decomposition product of the bile acids in the crude fat
extracted from feces. By oxidation cholalic acid yields several new
acids which have been much studied with the hope of gaining an insight
into the structure of the original acid. Among the various derived
acids these may be mentioned : Choleic acid, C25H4204, dehydrocholeic
acid, C24H3404, cholanic acid, C24H34Os. Fellic acid, C23H40O4, and
lithofellic acid, C20H36O4, are found in some kinds of bile.

Preparation of Acids from Ox Bile. This may be illustrated by the following.
Evaporate 200 to 300 cc. of the bile to dryness, or as near to dryness as possible, on
the water-bath with the addition of about 60 grams of bone-black. After cooling
the mass rub it up thoroughly, transfer to a flask and extract with alcohol by
heating over a water-bath. The two bile salts are soluble in the alcohol, while
the mucin and inorganic salts present are not. Therefore cool the extracted mix-
ture and filter. The filtrate contains the bile salts along with cholesterol, some fat
and traces of other substances. There is also some water present. Evaporate the
filtrate to dryness, take up with absolute alcohol and filter again. Taking advan-
tage of the practical insolubility of the bile salts in ether they may be precipitated
in this way : Add to the strong alcoholic solution an excess of dry ether, or enough
to cloud the mixture, and allow to stand. After some hours or days a crystalline
precipitate of the bile salts separates. The crystals may be used for preparation of
other substances, or for tests. In the mother liquor cholesterol may be detected by
the tests given in an earlier part of this work.

Preparation of Glycocholic Acid. Use the larger part of the above described
crystalline precipitate for this purpose. Dissolve in water and add enough dilute
sulphuric acid to produce a marked turbidity. Add a little ether, shake the mix-
ture well and allow to stand in a cold place. The glycocholic acid separates in the
form of fine silky needles. Press out the mother liquor, redissolve in hot water
and allow to crystallize a second time. A nearly pure product may be so obtained.

Preparation of Taurocholic Acid. The separation of this acid from the glyco-
cholic acid is extremely difficult, hence in preparing it, it is best to start with a bile
which contains essentially only the one salt. Dog's bile should therefore be em-
ployed. Treat it as described for the mixed salts, and decompose finally with dilute
sulphuric acid in presence of ether.

Preparation of Cholalic Acid. Dissolve 200 grams of barium hydroxide in 6
liters of water. In this solution saponify 50 gm. of glycocholic acid, by boiling

266

PHYSIOLOGICAL CHEMISTRY.

ten to twenty hours, replacing the water lost by evaporation. Filter hot and to
the cooled liquid add enough hydrochloric acid to decompose the barium salt.
The cholalic acid separates in the form of a granular precipitate. Wash with water
and crystallize from hot, strong alcohol.

Optical Properties of These Acids. The three acids and their sodium salts
are characterized by rather strong rotating power, which under some circumstances
may be used for measurement or identification. The following specific rotations
have been found :

For Aqueous Solution.

For Alcohol Solution.

c

Wj,

c

Wa

Taurocholic acid, sodium salt

Cholalic acid, anhydrous

24.928
8.856

I9.O49

+20.80
+ 21.5

+26.0

9-5°4
20. 143
9.898
2.942
2.230

-I-29.00

+ 25.7

424.5
447.6

431-4

Chemical Test for the Bile Salts. The three acids are characterized by giving
a certain reaction with furfuraldehyde, or sugar yielding furfuraldehyde, in presence
of acid. The test is commonly made by adding to a dilute solution of the salts,
say 5 cubic centimeters, a few drops of a dilute cane sugar solution and strong
sulphuric acid in volume about half that of the mixture. Let the acid flow down
the side of the test-tube so as to form a layer below the lighter liquid. A deep
purple band appears at the line of contact. On slowly mixing the liquids in the
test-tube the color becomes purple throughout. In this test any excess of sugar must
be avoided.

With a trace of pure furfuraldehyde in place of sugar the reaction is sharper,
but certain proportions must be observed. A good mixture is 1 cubic centimeter
of weak alcoholic solution of the bile acid, 1 drop of 0.1 per cent furfuraldehyde
solution and 1 cubic centimeter of strong sulphuric acid. The original test was
devised by Pettenkofer; later it was recognized that the reaction belongs to the
group of " f urf urol " reactions, and the aldehyde was recommended in place of
the sugar. The test cannot be used with bile directly because of the presence of
other substances, which would give a strong color with the sulphuric acid.

Preparation of Taurin. Use several hundred cubic centimeters of ox bile. Add
to it an excess of strong hydrochloric acid, about one-third of the volume of the
bile, and boil on the water-bath. A resinous mass separates and when this be-
comes stringy enough to solidify, when a little is taken up on a rod and allowed to
cool, the reaction has gone far enough. Decant from this mass and evaporate the
liquid resulting until a crystallization of salt forms. Filter and evaporate to a
small volume. If salt separates filter again and pour the liquid finally into a large
excess of alcohol. This causes the taurin to separate; wash the crude substance
with strong alcohol, and recrystallize from hot water. In a successful separa-
tion large plates or prisms of taurin are obtained. The substance may be recog-
nized by several tests. On heating it chars and gives off an odor of sulphurous
acid. When fused with sodium carbonate the sulphur is converted into sulphide,
from which hydrogen sulphide may be separated and identified by the usual tests.

THE BILE PIGMENTS.

The two substances, biliverdin and bilirubin, are related to hematin
from hemoglobin, as pointed out above, and as may be illustrated by
these formulas :

THE CHEMISTRY OF THE LIVER. 267

Hematin C32H32N404Fe

Hematoporphyrin C16HiSN203, or C32H3cN406

Bilirubin C16H1SN203, or C32H3eN406

Biliverdin C]6H18N204, or C32H36N408

The two bile pigments are formed in the liver and normally, appar-
ently, only in the liver, but by what kind of reaction is not clearly
known. Hematoporphyrin may be produced from hematin and it is
isomeric with bilirubin, though not identical. The relation of bilirubin
to blood is perhaps best shown by this observation : in old blood extrav-
asations the blood color appears to be gradually decomposed and in its
place the new coloring matter is found, which was called hematoidin
by its discoverer. Later studies have apparently shown the identity
of this with bilirubin.

Bilirubin is practically insoluble in water, but it seems to act as an
acid, the alkali salts of which are soluble. In this form it exists in bile.
The solution is reddish yellow and in the air, or by treatment with
oxidizing agents, it takes up oxygen and becomes biliverdin, which
gives a green solution. The bile always contains the two pigments,
from which the greenish yellow color follows. The amount of the
two substances in the bile is normally very small, but as the reactions
are sharp recognition is easy. The total weight of the two pigments
produced in one day is not over 200 milligrams probably; the physio-
logical meaning of the formation is not known. The iron of the orig-
inal hematin is largely retained by the substance of the liver cells.

Preparation of Bilirubin. The pigment cannot be easily obtained from bile
because of the small amount present, but may be obtained from the pathological
concretions known as gall-stones, which will be described later. Powder several
grams of these stones from cattle very fine and exhaust thoroughly with ether, then
repeatedly with boiling water to take out cholesterol and bile acids. In the residue
the bilirubin exists as an insoluble calcium compound ; this is decomposed by the
addition of a little dilute hydrochloric acid, after which what is left is washed
thoroughly with hot water, and then with alcohol to leave the pigment in a still better
condition for extraction. Finally extract with chloroform in which the substance
is relatively soluble. On evaporating the chloroform crude bilirubin is secured,
which after washing with alcohol may be recrystallized from hot chloroform or
from dimethylaniline, in which it dissolves in the proportion of about 1 to 100
cold or 1 to 30 hot. By several crystallizations it is possible to obtain a product
pure enough to employ as a standard for spectroscopic measurements.

I'.y exposing an alkaline solution to the air or by treating with a little acid and
sodium peroxide, bilirubin is converted into biliverdin. The latter free substance
is not soluble in water, chloroform or ether.

The Bile Pigment Tests. Some of these are extremely delicate and have long
been used for the recognition of bile, especially in urine. For the test to be
givf-n the bilirubin alkali in very dilute solution may be used, or a diluted bile.

i.in's Test. In a test-tube take a few cubic centimeters of nitric acid con-
taining some nitrous acid. Over this pour carefully the weak bile solution to be

268 PHYSIOLOGICAL CHEMISTRY.

tested. At the junction point colored rings appear which result from the forma-
tion of oxidation products of the bilirubin. The colors appear in this order from
above down : green, blue, violet, red and yellowish. Of these the green is the most
characteristic; the other shades represent more advanced stages in the oxidation.
For success in the test the bile solution must not be too strong, and the amount of
nitrous acid in the nitric acid must be small.

Hammarsten's Test. Use as reagent a mixture of strong nitric acid and strong
hydrochloric acid in the proportion of about I to 50 by volume. This mixture must
stand some time before use, or until it becomes yellow. It keeps a long time. For
the practical test mix i cubic centimeter of the acid with 4 cubic centimeters of
alcohol and add a drop or two of the bilirubin solution to be tested. A perma-
nent green color appears, but if strong oxidation is secured by adding more of the
acid mixture the colors change as in the Gmelin test. The reaction can be well
applied to urine.

FUNCTIONS AND BEHAVIOR OF BILE.

The bile as a whole has a number of functions to perform in the
body, some of which have been referred to in the discussion of diges-
tive processes. It represents also the avenue of escape of a number
of by-products formed by the katabolic processes in the liver. Many
of these processes are doubtless very complex and in them a variety of
secondary or side reactions occur which furnish matters of no further
use apparently in the body. These are collected in the gall bladder
and finally discharged into the small intestine, where escape from the
body with the feces is possible for the constituents having no further
value. If the escape of these products from the liver is hindered, some
form of icterus results, as the bodies in question must pass more or
less directly into the blood.

In part, therefore, the bile must be regarded as an excretion like
the urine, but that the parallelism is not complete is shown by the fact
that a considerable absorption takes place from the intestine, and
products are returned which find further application in the organism.
There is evidence to show that this portion returned from the intestine
serves as a cholagogue to stimulate new secretion in the liver. It is
likely that this free secretion and flow of bile in the liver is necessary
for the successful completion of certain metabolic processes going on
there, so that it may be regarded not merely as an end but also as a
means toward an end.

The one digestive process in which the bile seems to play a practically
necessary part is in the splitting and absorption of fats ; here its action
is partly mechanical as in some way it aids the passage of the finely
divided fat through the intestinal walls. The general behavior of bile
in this respect may be illustrated by a simple experiment.

THE CHEMISTRY OF THE LIVER. 269

Experiment. Moisten two similar filter papers in funnels, one with water and
the other with bile. Into each filter pour some fatty oil, such as cotton-seed oil
or olive oil. Note that while the oil will not pass through the paper moistened
with water a small amount passes slowly through the bile-moistened filter. Similar
experiments have been made with animal membranes.

A more important action with fat, however, is shown in the power
of bile to form fat emulsions, which depends on the behavior of the
bile salts with the fat splitting ferment, as already pointed out, and on
the formation of soaps directly. This is now looked upon as the one
reaction in the intestine in which the presence of bile is actually prac-
tically essential, since the old views of the antiseptic value of the bile
in preventing excessive intestinal putrefaction have been shown to be
without foundation. In a diet rich in fats the emulsifying behavior
of the bile unquestionably comes into play as a leading factor in the
final absorption. Experiments have been made on animals in which
the flow of the bile could be diverted from the natural outlet into the
intestine by means of a fistula. In such cases the digestion of proteins
and carbohydrates seemed to suffer no change but the digestion of fats
was always imperfect and a large portion ultimately escaped with the
feces. Indirectly there may be also a loss in protein if the fat in the
food in such cases has a rather low melting point and is abundant. A
fatty layer encloses portions of the partly digested proteins and pre-
vents access of the digestive fluids until the lower stretches of the intes-
tine are reached, where bacterial changes soon get the upper hand and
rob the protein of any further food value. The action of bile in pro-
ducing an emulsion with fatty oils may be illustrated by experiment.
In an earlier chapter the formation of emulsions by other methods was
shown.

Experiment. In a slightly warmed mortar pour about 5 cc. of bile and add to
it one cc. of cottonseed oil. Rub the two thoroughly together for several minutes,
and then add another small portion of the fatty oil. An emulsion forms slowly,
and becomes more persistent as the working with the pestle is prolonged. The
amount of oil which can be brought into the form of a stable emulsion with the
5 cc. of bile depends largely on the character of the oil. The presence of a small
amount of free fatty acid in the cottonseed oil aids materially in producing the
emulsion. The weak alkalinity of the bile is doubtless an important point here, as
through the alkali a little soap is formed and this may be the chief factor in pro-
ducing the emulsion.

In the intestines the stimulating action of the bile salts is probably
more important than this last reaction. At the present time these salts
are prepared in comparatively pure form as medicinal agents.

Bile contains a large amount of mucin as the analytical table above
shows. The stringy character of the secretion is due to this substance

270 PHYSIOLOGICAL CHEMISTRY.

which may be recognized by several precipitation tests. The addition
of alcohol in excess throws down a flocculent mass which may be sepa-
rated by the centrifuge. The addition of a little acetic acid produces
likewise a precipitate. It is, however, practically impossible to secure
pure mucin in this way as other bodies are carried down with the pre-
cipitates and their subsequent separation is difficult. The mucin of
human bile is said to be nearly pure, while that of other animals is
mixed with nucleo albumins.

BILE CONCRETIONS. GALL STONES.

Under conditions not well understood a precipitation of certain con-
stituents of the bile may occur in the gall bladder. These precipita-
tions take the form of solid masses which sometimes grow to consid-
erable size, by gradual surface additions. In every case the deposited
material is built up in layers, often well defined, around some body as
a nucleus. Three general classes of such calculi are recognized. In
man balls of cholesterol, more or less pure, are the most abundant while
pigment stones are also frequently found. These pigment stones con-
tain essentially bilirubin in combination with calcium, the alkali earth
salts of the pigments being insoluble. The center of the cholesterol
stone may be a nucleus of bilirubin calcium. Pigment stones are com-
mon in the gall bladders of cattle. Finally we have stones consisting
of calcium phosphate or carbonate, which, however, are not usual in
man.

The following analyses made of gall-stones of very different appear-
ance illustrate the composition of the cholesterol stones in man :

Water (at ioo°) 4.60 4.50

Cholesterol (and trace of fat) 90.87 90.08

Bilirubin (CHC13 extraction) 0.81 ~l H 0.19 ~j

Biliverdin (C2H,0 extraction) 2.24 J3-05 1.58 J I-77

Mucin and soluble extractives 0.14 1.53

Total ash 0.88 2.72

Total P205 0.20 1.00

These concretions frequently give rise to serious pathological condi-
tions and they must then be removed by surgical operations. In addi-
tion to the above constituents the stones contain small amounts of iron
and often traces of copper. But the iron found is far from accounting
for the amount which must be separated from the hematin in the for-
mation of bilirubin. In a former chapter the preparation of choles-
terol from gall-stones was described, also the general chemical behavior
of the substance. The character of a stone is most easily recognized

THE CHEMISTRY OF THE LIVER. 27 1

by its behavior toward boiling alcohol, in which cholesterol is rather
readily soluble, to crystallize in large thin plates on cooling.

The solutions of cholesterol have a marked action on polarized light,
which property may be employed sometimes in the identification and
estimation. The specific rotations below have been found.

Ether solution c = 2 [a]ols = — 31.12°

Chloroform solution " 2 " — 37-02°

" 5 " - 37.8i °

" 8 " -38-63°

In feces a modified cholesterol is found which has been called koprosterin and also
stercorin. This new substance is a reduction product with the probable formula
QrH^O and is dextrorotatory, [a] = -f 24°.

Besides the two principal pigments several derived substances have been obtained
from the gall-stones. The following have been described: bilifuscin, biliprasin, bill-
hum in, bilicyanin. These substances exist in small amount and are without practical
importance. Their relations to the others are not clearly established.

CHAPTER XVIII.

CHEMISTRY OF THE PANCREAS AND OTHER GLANDS. MUSCLE,
BONE, THE HAIR AND OTHER TISSUES.

In this chapter a number of substances will be briefly discussed, the
chemical relations of which in some cases are unimportant, or some-
times, when important, not well understood. In regard to the pan-
creas, it will be recalled that in the discussion of digestive phenomena
the behavior of active enzymes in the liquid secreted by the organ was
rather fully considered. In the so-called pancreatic juice the three
most important enzymes are active in the digestion of carbohydrates,
fats and proteins, but in addition to these functions others must be
mentioned.

THE PANCREAS.

The organ is relatively poor in solids, containing only about ioo
parts per 1,000. The solid substance consists largely of nucleo-proteids
with but comparatively small amounts of the other protein bodies.
Besides producing the digestive enzymes, or their zymogens, the pan-
creas cells have an important function to perform in connection with
the oxidation of sugar in the body. It has long been known that a
kind of diabetes results on the extirpation of the pancreas. Something
seems to be produced there which is apparently essential in the oxida-
tion process. Experiments with animals have shown that the oxida-
tion takes place if even a small portion of the organ is left. Of the
nature of the active ferment here or of its mode of action practically
nothing is known; but it has been pointed out recently by several
writers that in this sugar oxidation, taking place in the muscles prob-
ably, two things are concerned. The pancreas may furnish one of
these and an enzyme formed in the muscle cells is the other. Cell-free
extracts from the organs taken separately have been found to be prac-
tically inert toward sugar, while in presence of a mixture of the two
extracts oxidation follows readily. It has* been suggested that one of
these organs furnishes an enzyme which is the catalyzer for the other,
and attempts have been recently made to produce the pancreas enzyme
on a large scale for use therapeutically.

Autolysis. The pancreas readily undergoes autolytic digestion
under the aseptic treatment or when preserved by toluene. A large

272

CHEMISTRY OF THE PANCREAS AND OTHER GLANDS. 273

number of products may be separated from the altered mass, which in
a general way resemble those produced in the liver, as already referred
to. Ammonia, leucine, tyrosine, aspartic acid, glutaminic acid and the
hexone bases have been recognized; also, the somewhat unusual oxy-
phenylethylamine, HO-C6H4-CH2-CH2NH2, which may be derived
from tyrosine by splitting off of C02.

On account of the relatively high content of nucleo-proteids, and the
constituent nucleic acids, a marked amount of sugar in the form of pen-
tose is liberated. No other organ subjected to prolonged autolysis
seems to yield as much. In certain pathological conditions involving
the pancreas, the urine contains a complex which yields a pentose on
treatment with acid at the boiling temperature. The pentose is iden-
tified through its phenyl hydrazine compounds.

THE SUPRARENAL BODIES.

A soluble substance contained in the capsules, because of its impor-
tant property of raising the blood pressure, has attracted a great deal
of attention in the last ten years. This soluble substance was first
recognized as a chromogen which, on account of its oxygen-absorbing
power, was assumed to be related to pyrocatechol. An aqueous extract
of the capsules becomes dark on exposure to the air and produces a
dark green color when treated with ferric chloride. It also reduces
Fehling's solution strongly and shows the same behavior toward other
metallic salts. The oxygen-absorbing power of the extract had been
known about thirty years before the important relation to blood pres-
sure was discovered. It was soon found that the two properties seem
to reside in the same constituent of the extract, since the destruction of
one is followed by the disappearance of the other. Numerous investi-
gations have been carried out on the isolation of the active principle,
especially by Abel, v. Fiirth and Takamine, who have given the names
epinephrin, suprarenin and adrenalin respectively to active extracts
which they have separated by different processes. Some idea of the
nature of the substance may be obtained from considering a method
given by Takamine for separating it.

The minced capsules are extracted by weakly acidulated water in an atmosphere
of carbon dioxide to prevent oxidation. The temperature of the extraction is at
first 5o°-6o° and finally 90°-95° to coagulate proteins. The extract is con-
centrated in vacuo and precipitated with strong alcohol; the filtrate is concen-
trated— the alcohol distilled off — in vacuo, and to the aqueous residue ammonia is
added. This produces a precipitate of the active principle in crude form, which crys-
tallizes in time. The precipitate is redissolved with a little acid in alcohol, and
certain impurities are thrown out by addition of ether. The filtrate is concen-

19

274 PHYSIOLOGICAL CHEMISTRY.

trated in vacuo again and a new precipitation effected by ammonia. By repeating
this treatment several times a much purer product is obtained.

A light yellowish crystalline powder is secured, which is somewhat
soluble in water. It combines with hydrochloric acid to form the stable
salt commonly used in medicine. The empirical formula is C9H13N03
and for this several constitutional formulas have been suggested.

The other constituents of the suprarenal capsules have no impor-
tance at the present time that can be clearly denned, but as is well
known, complete removal of the bodies is usually attended with fatal
results, and Addison's disease is associated with certain pathological
conditions in the organs. Lecithin bodies and a glucose-furnishing
complex are present in small amount, as well as the mass of protein
substance which has not yet been fully investigated.

THE THYROID GLAND.

The relation of this gland to certain pathological conditions which
sometimes appear in man and which may be induced in animals has
been a subject of study for many years. Attempts to isolate the active
principle or principles on which the functions of the gland depend have
been in a measure successful. In the course of investigations a number
of basic bodies have been separated, but these may have no connection
with the observed physiological behavior.

From the investigations of Oswald, who has made the fullest con-
tributions to the literature, there are two peculiar protein bodies present,
one of which is a globulin and the other a nucleo-proteid. To the first
he has given the name thyreo-globulin; this exists frequently combined
with iodine, and it is the latter complex which is assumed to be theo-
retically and practically important. It has been called iodothyreo-
globulin and appears to be found only in those glands which contain
colloid, and the amount of iodine present is proportional to the amount
of colloid. The normal gland weighs usually 30-45 grams, in which
the thyreoglobulin fraction is in the mean about ten per cent. The
amount of iodine is usually less than one tenth of one per cent of the
whole. In case of enlarged glands — goitre — the whole organ may
weigh up to several hundred grams. If the goitre is rich in colloid
the iodine appears to be absolutely, but not relatively, increased. In
the thyreo-globulin from a normal gland over 0.3 per cent of iodine
has been found, while in the preparation from colloid goitres the
amount in the mean is 0.06-0.07 Per cent.

By treatment with acids the gland, or the thyreo-globulin from it,
undergoes a cleavage in which a residue rich in iodine remains. The

CHEMISTRY OF THE PANCREAS AND OTHER GLANDS. 2J5

organic iodine compound so obtained which may be the true active
principle is called iodothyrin or thyroiodine. In earlier experiments
Baumann, the discoverer of this compound, found an iodine content of
about 9 per cent, but Oswald, starting with pure iodothyreo-globulin
which was secured by a salting-out process with ammonium sulphate,
obtained finally iodothyrin with over 14 per cent of combined iodine.
This iodothyrin is not a protein substance; the analyses of different
preparations are not in very good accord, from which it appears that
the pure substance has not yet been actually secured. The crude
product at present known has been used in medicine and attempts have
been made to duplicate or replace it by other iodine compounds.

It is now generally recognized that the physiological activity of the
dried thyroid on the market in powdered form is proportional to the
iodine content. No exact method of valuation is known.

The smaller glands associated with the thyroid and known as the
parathyroids are possibly even more important. Both sets of glands
have apparently much to do with the general metabolic functions of the
body, and the complete removal of the parathyroids is usually followed
by death. How they act is not clearly known.

THE REPRODUCTIVE GLANDS.

Of the chemical composition of the testicles and their secretion not
much can be said. The testicles contain several proteins and extrac-
tives, but their investigation has been extremely limited. The most
complete examinations of the spermatic fluid are probably those re-
ported by Slowtzoff, from whose work the following figures are taken.
The specific gravity of the fluid varies from 1.02 to 1.04; the reaction
is alkaline and as measured by the aid of rosolic acid corresponds to
0.15 per cent sodium hydroxide. As a mean of five analyses the fol-
lowing results may be given :

Spermatic Fluid.

Specific gravity 1.0299

Water 90.32 per cent.

Dry substance 9.68

Salts 0.90

Proteins 2.09

Ether extract 0.17

Water and alcohol extracts 6.1 1

The tables below show the calculations for dry substance and the
character of the ash :

276 PHYSIOLOGICAL CHEMISTRY.

For Dry Substance. Ash.

Organic 90.81 per cent. NaCl 29.05 per cent.

Inorganic 9.19 " KC1 3-12

Proteins 24.48 " S03 11.72

Ether extract 2.15 " CaO 22.40 "

Water and alcohol ex- P205 28.79

tract 59.36 "

The ash is peculiar in containing a large amount of sodium chloride
and calcium phosphate. The phosphoric acid is present in larger
amount than corresponds to the nuclein substances.

The proteins are made up approximately as follows :

Albumins 68.5

Albumose-like bodies 21.6

Nucleins < 9-9

A characteristic basic body known as spermine is present in small
amount. The empirical formula C2H5N has been given to it. This
substance forms a combination with phosphoric acid which sometimes
separates in crystalline form on evaporation of the fluid. The charac-
teristic odor of the discharged secretion is said to be due to partial
decomposition of the base.

The spermatozoa are relatively stable bodies and resist the action of
chemical reagents to a remarkable degree. The heads of spermatozoa
consist largely of nuclein compounds while the tails contain other pro-
teins, cholesterol, fat and lecithin. The ash content of the whole is
relatively high and is rich in potassium phosphate.

BRAIN AND NERVE SUBSTANCES. CEREBRO-SPINAL

LIQUID.

These tissues contain several peculiar compounds of which our
knowledge is limited, largely because of the great difficulty in separa-
tion. The solid matter of the brain contains globulins, nucleo-proteids,
cholesterol, lecithin, fatty bodies and complex compounds not found
elsewhere. Various soluble extractives, somewhat similar to those
from muscular tissue, are also present.

Protagon. This has been assumed to be an important constituent
of the white substance of the brain, which has this elementary compo-
sition, according to Gamgee: C 66.4, H 1.07, N 2.4, P 1.07. But as
others writers report rather widely different figures it is likely that the
pure substance has not yet been isolated. As extracted by means of
85 per cent alcohol at 45 ° from the minced brain, and purified by
crystallization and washing with ether, it is obtained as a white powder

MUSCLE AND ITS EXTRACTIVES. 277

practically insoluble in cold ether or alcohol and not properly soluble in
water. With much water it finally yields a gelatinous liquid, which
suffers decomposition readily.

Notwithstanding the bulky literature which has accumulated in the
discussion of this substance, its exact nature is not yet known. All
recent investigations seem to show that it is a mixture of a number of
bodies. By treatment with certain solvents, or by gentle cleavage, it
is possible to separate a group of phosphatides, similar to some of the
lecithin bodies, and a group of substances free from phosphorus, but
containing nitrogen. Cerebrin and cerebron are names given to two
of these products. Of the functions of these little is known.

In the white substance of the spinal marrow the so-called protagon
is abundant. In degeneration changes in the tissues of the nervous
system it is probably this compound which suffers the greatest altera-
tion, with the production of neurine with marked toxic properties. It
is likely that the neurine comes from a lecithin body as one of the
groups in the protagon complex, and that these reactions will prove of
great importance in pathological study.

It is also known that complex sulphur compounds are present in the
brain tissue, but little is known of their reactions.

Cerebrospinal Liquid. This is a thin, watery liquid of which only
a few partial analyses have been recorded. Its general character is
shown by these figures recently given by Zdorek :

1,000 parts by weight contain

Dry substance 10.45

Organic 2.09

Inorganic 8.36

Proteins 0.77

Chlorine 4.24

Sodium oxide 4.29

The organic substance includes traces of fats, lecithin, cholesterol
and, pathologically, choline or neurine. Common salt, however, is the
main solid substance in solution.

MUSCLE AND ITS EXTRACTIVES.

A large part of the solid portion of the body is made up of muscular
tissue. A knowledge of the composition of this tissue is of the highest
importance, especially since some of the fundamental chemical reactions
of the animal organism take place within the cells of the muscles. For-
tunately we have fairly satisfactory information on some of the points

278 PHYSIOLOGICAL CHEMISTRY.

of interest here, as numerous analyses have been made of the muscles
and of the liquid which may be extracted in various ways from them.
The dry part of the muscle is made up largely of proteins of which
several are present ; in the muscle plasma there are at least five accord-
ing to Halliburton. In addition to these bodies there are a number of
so-called extractives which play an important part.

GENERAL COMPOSITION OF MUSCLE.

The following figures represent approximately the average compo-
sition of the fresh muscle dissected free from visible fat.

Water 76 per cent.

Solids 24

Proteins (true) 17-6

Collagen substance 3-o

Fat, interstitial 1.5

Flesh bases 0.2

N-f ree extractives 0.4

Salts 1.3

The Muscle Proteins. It is not possible to give a perfectly clear
account of all these bodies at the present time, as the products obtained
by different investigators vary with the details of the extraction
methods employed. The more important constituents commonly rec-
ognized are indicated in the following paragraphs. By washing out
the blood from living muscle by physiological salt solution (transfu-
sion), dissecting it, grinding it to a pulp and pressing very strongly a
clear yellowish liquid is obtained which is called muscle plasma. The
ordinary dead muscle treated in the same manner yields a different
liquid which may be called muscle serum. The plasma has an alka-
line reaction and is distinguished by the property of spontaneous
coagulation.

The term myosin was formerly applied to the solidified or coagulated
body as a whole, but experiment shows that two things at least are
here present. One of these is called musculin, or by some authors,
myosin proper, while the other product is known as myogen. The
musculin, or myosin, coagulates at about 470, while for myogen the
coagulating temperature is about 560.

The two substances, musculin and myogen, differ also in their pre-
cipitation properties. The first is precipitated from solution by adding
ammonium sulphate to make up 28 per cent; from the filtrate the
myogen may be thrown down by adding the sulphate to saturation,
and is found to make up about 80 per cent of the plasma protein.

MUSCLE AND ITS EXTRACTIVES. 279

The serum left after the formation of the plasma coagulum usually
contains a little soluble albumin. This may be normal to the muscle
substance, or it may be due to the blood not perfectly removed by the
preliminary washing. At any rate the plasma consists essentially of
the two myosin bodies.

After separation of the plasma what may be called the stroma re-
mains. This is mainly albuminous, but its exact nature is not known.
The sarcolemma portion of the muscle fiber, which by weight makes
up but a small part of the whole, appears to belong to the albumoid
group of proteins, resembling elastin. It has been shown in an earlier
chapter that from ordinary dead muscle, as represented by lean meat,
a considerable amount of " myosin " may be separated by extracting
with a weak solution of ammonium chloride. What remains does not
agree fully with the stroma left on pressing out the plasma of the
fresh muscle, but contains approximately the same substances. By
this method of separation the insoluble stroma portion is much larger
than the soluble or " myosin " portion. The latter may amount to 7
or 8 per cent of the weight of the muscle in the mean.

Collagen. As given in the above table this refers to the binding
substance holding the muscle fibers together and includes the sarco-
lemma. It is insoluble in cold water, but swells and disintegrates
finally in boiling water.

Fat. After removing all visible fat from the dissected muscle,
analyses still show a small amount remaining. This must therefore
be associated with the minute structure of the fibrils.

Flesh Bases. A number of very remarkable substances are
included here. They are sometimes described as the nitrogenous
extractives. The most abundant of these bodies is creatine or methyl-
guanidine acetic acid ; some of the purine bases are also present. A
brief description of these substances may be given.

Creatine, C4H9N302, may be represented structurally by the
formula

H-N=C< CH,

wCH,COOH

It is found in all muscles and is probably a product of metabolism, but
the method of its formation is not yet known. Being readily soluble
in warm water, and in about 75 parts of water at the ordinary tem-
perature its extraction from muscle is easy. When the solution is
boiled with dilute hydrochloric acid, through a long period, a molecule
of water is split off and the anhydride creatinine is left. This is a

280 PHYSIOLOGICAL CHEMISTRY.

normal urinary constituent and will be described later. When boiled
with alkali solution, especially baryta water, creatinine undergoes a
complete cleavage into urea and sarcosine, which relation is an inter-
esting one and has suggested a possible derivation of the urinary urea.
Creatinine may be readily crystallized from water solution. It was
formerly made for experiment directly from meat. It is best secured
from certain crystalline residues occurring as by-products in the manu-
facture of " beef extract," referred to below.

Carnine. The amount of this in muscle is very small, but it may
be recognized in beef extract. It bears some relation in structure to
hypoxanthine, and has been given the formula C7H8N4Os.

Comparatively recently several other crystalline products have been
isolated from meat extracts. Among these carnosine and carnitine
are perhaps the most important.

The Xanthine Bodies. These constitute a peculiar group of
great importance because of their relation to uric acid and other products
of metabolism. Traces of several of them have been recognized in the
muscular juices ; in a later chapter the structure and properties of the
substances will be discussed in connection with uric acid. Traces of
urea are also found in the muscles.

The Nitrogen-Free Extractives. The muscular juices hold dis-
solved a number of compounds which contain no nitrogen, some of
which are very important. The chief of these are glycogen, inosite,
glucose and lactic acid.

Glycogen. The chemical relations of glycogen have been dis-
cussed already in earlier chapters. The glycogen as found in the
muscles comes from the liver, being transported there by the blood, and
in part is probably formed in the muscles by the same kind of an
enzymic action which leads to its synthesis in the liver. The liver is
capable of storing up a large weight of the reserve substance in a small
space. The amount stored in an equal weight of muscle is small, but
taking the muscles of the body as a whole the glycogen content is con-
siderable, reaching a hundred grams or more.

It is probably through this glycogen that the muscle is capable of
doing its work. Through enzymic hydration the glycogen becomes
sugar, possibly maltose and then glucose, and the potential energy of
this is liberated by oxidation to water and carbon dioxide ultimately.
The oxidation may not be direct; in all probability there are several
transformation products before the final stages are reached. But the
energy transformation is the same whatever the intermediate steps may
be. The importance of the glycogen and related bodies in this direc-

MUSCLE AND ITS EXTRACTIVES. 251

tion will be pointed out in a following chapter. It may be recalled that
in these oxidation processes, where sugar is concerned, a muscle enzyme
and a pancreas enzyme seem to be both necessary.

While glycogen in the muscles must come mostly from sugars, either
directly or through the liver, there is also some evidence that it may
come in part from other substances, especially from proteins. Animal
experiments have shown apparently a storing of glycogen from a pro-
tein diet after previous starvation had exhausted the reserve in store.
In the breaking down of some proteins it has been shown that certain
carbohydrate groups are liberated ; it is doubtless these which undergo
synthesis to form at least part of the glycogen, and from this stand-
point the behavior of protein as a glycogen factor is not so hard to
understand.

The glycogen content of the muscles of different animals is var-
iable; in the flesh of the horse it is relatively high, amounting often
to over i per cent. As the muscle glycogen is not altered rapidly in
the dead organ, as is the liver glycogen, the presence of the substance
in horse-flesh sausage may be quite readily recognized. Methods have
been devised for the identification of horse-flesh, sold for food, based
on these facts. Glycogen may be extracted from the muscles by the
general method given for the liver in an earlier chapter; the chemical
and optical properties may be used for the final identification.

Inosite. This substance has the empirical formula C6H1206 -f- H20
and was long spoken of as muscle sugar. It is not a true carbohydrate,
however, but an aromatic product C6H6(OH)G, that is, hexahydroxy-
benzene. The amount found in muscle is very small and how it is
derived is not known ; but it is not peculiar to these tissues, as it occurs
in other organs of the body and also in many vegetable substances. It
may be extracted from muscles without much trouble and when pure
is found to be a white crystalline powder melting at about 2200. It
is very soluble in water, to which a sweetish taste is given, and in pres-
ence of alkali is not a reducing agent for metallic solutions. Although
the usual structural formula does not show an asymmetric carbon atom
the substance is optically active and exhibits a strong rotation, both
right and left forms being known.

Glucose. From what was said above about the transformation of
glycogen it is not surprising that a small amount of sugar should be
found in the muscles ; both maltose and glucose have been detected.

Lactic Acid. Several forms of this acid are known, but that occur-
ring in the muscle is the dextrorotatory paralactic or sarcolactic acid,
O.I [«08. It is one of the a-hydroxypropionic acids. There has been

282 PHYSIOLOGICAL CHEMISTRY.

much speculation as to the source of this acid in the body, but it seems
most rational to regard it as derived from the glycogen or sugar by a
comparatively simple cleavage. It is also possible that in the katabolic
reactions of proteins lactic acid may result from a splitting of the
carbohydrate group. The acid is not very readily detected in the living
muscle because it is probably oxidized or removed too rapidly by the
fluid circulation. In the dead muscle, however, it may accumulate to
the extent of half a per cent or more. The living muscle shows a
neutral or slightly alkaline reaction, while in the dead muscle the
increase of lactic acid changes the reaction.

The lactic acid of the muscle probably results from an enzymic
cleavage. In the aseptic autolysis of liver paralactic acid has been
recognized among the products, and this fact shows, at least, the possi-
bility of such a formation. The amount of lactic acid formed in the
muscle seems to be greatest during working periods, which is true also
of the final products of katabolism. The acid may simply represent
a stage in the gradual breaking down, whether we consider a carbohy-
drate or protein as the parent substance. We should expect therefore
an increase in the muscle acid if the oxidation processes of the body
are hindered or retarded, while at the same time protein or sugar
decomposition is increased, or, at any rate, not diminished. In the
dead muscle the enzymic formation of lactic acid doubtless continues
long after the oxidation reaction ceases, and this is probably the main
reason for the ready detection in the muscle after death.

The pure acid occurs as a thickish liquid miscible with water. It
forms salts which are mostly readily soluble. The zinc and calcium
salts crystallize well and are hence prepared for identification. The
pure liquid shows a right hand optical rotation, with [a]D = about 30.
The result is not constant because of the difficulty of preparing con-
centrated solutions free from anhydride or lactide. The rotation of
the salts, on the contrary, is to the left.

The Inorganic Salts. Although making up not much over 1 per
cent of the weight of the moist muscle, these salts are extremely im-
portant. Of dry substance the salts constitute 5 per cent or more.
The salts are usually estimated from the ash left in burning the mus-
cle; this gives of course no correct idea of how they are combined in
the living muscle, but is the only method available. In the living
muscle many of the inorganic elements are doubtless in chemical union
with proteins or other organic groups, while in the derived ash we
have chlorides, phosphates, sulphates or carbonates. A carbonate is
probably formed during the combustion of organic acids and corre-

MUSCLE AND ITS EXTRACTIVES. 283

sponds to no simple preexisting compound. Phosphorus and sulphur
of proteins furnish phosphates and sulphates. The analyses of ash
made disclose very different results, but mean values may be given to
show the general approximate composition. In the calculation car-
bonic acid is not considered. The table below is from the Konig
collection.

K.0 37-04

Na,0 10.14

CaO 2.42

MgO 3-23

Fe203 0.44

P,05 4120

S03 0.98

CI 4-66

Si02 0.69

From the table it appears that potassium phosphate is the most
abundant substance in the ash. Much of this doubtless preexists in
the muscle juices, while a small portion is of oxidation origin. The
small sulphate content is probably due to protein sulphur fully oxidized
in the combustion. In the past too little attention has been given to
the mineral constituents of the body, it being commonly assumed that
they represent "waste" or "ash" only. But the newer applications
of chemistry, especially physical chemistry, to physiology have dis-
closed the fact that the inorganic salts are especially concerned in the
proper maintenance of many of the body functions. The balanced
osmotic pressure of the body fluids is largely a function of the salt
content, and variations here are of great importance. The mineral
salts are the carriers of electric charges in the body and as such seem
to have important duties to perform.

EXTRACT OF MEAT.

By boiling lean meat with water the soluble constituents are dis-
solved, producing an extract. When this is concentrated to a paste
the article known commercially as " Extract of Meat " results. The
article was first made in quantity in South America to utilize the car-
casses of cattle slaughtered for the hides, but later the manufacture
was introduced elsewhere, and generally to utilize certain waste or
by-products in the meat industries. At first the extract was assumed
to possess food value in a high degree, but after a time, as the chem-
istry of the proteins and their derivatives became better understood,
this notion was gradually abandoned. Lean meat, muscle, is employed
practically in the process ; hence little or no fat can be present. At the

284 PHYSIOLOGICAL CHEMISTRY.

boiling temperature nearly the whole of the proteins are coagulated
and are filtered out. A little gelatin remains, but the food value of
this is of minor importance. Unless the boiling is greatly prolonged
the extract must therefore contain essentially the meat bases and other
extractives referred to above, and the actual nutritive value of these is
low, in the case of the bases being nil. On prolonged boiling, how-
ever, a small portion of the original protein seems to pass over into
the soluble form of albumose, which is therefore found in some ex-
tracts. Finally, the phosphates and other inorganic salts, being largely
soluble, pass into the extract and constitute a considerable part of the
finished pasty product.

In this country " extract " is made by concentrating the broth resulting from the
boiling of beef as a step in the canning process. Large quantities of meat being
boiled in the same water, it becomes rich in the " extractives " and is finally boiled
down to the usual pasty condition. Before the concentration is complete the liquid
is filtered and skimmed and therefore leaves a residue free from fat or fiber
Roughly speaking the paste extract has about this composition:

Water 20

Salts 20

Organic substances 60

Numerous analyses have been made of some of the commercial extracts, but the
methods employed have not always been delicate enough to furnish trustworthy
information. This is especially true as regards the amounts of so-called peptone
and albumose present, for which the definitions have not been fairly uniform until
comparatively recently. The recognized relations of these substances are explained
in the chapter on protein compounds. Analyses made by the older methods were
generally reported as showing more or less " peptone " when, according to the
present views, " albumose " is meant. The following figures may be taken as repre-
senting approximately the average composition of typical samples of American meat
extract :

Water 20.0

Inorganic salts (ash) 22.5

Albumose (and gelatin) 16.5

Flesh bases, etc 26.4

N-f ree extractives 14.6

According to these results the food value of the extract would be measured by
the nitrogen-free extractives and the albumose and gelatin fractions. In some kinds
of extract the flesh bases and related bodies are much higher than here given, with
corresponding diminution in the other organic constituents. The real value of these
extracts lies mainly in other directions, however. They contain the flavoring and
stimulating portions of the meat, and should not be considered so much as foods
as additions to foods. Added to vegetables they impart an agreeable taste and
doubtless serve a very useful purpose in stimulating appetite for substances not in
themselves possessing much flavor. In their action the basic and similar substances
in the meat extracts may be perhaps fairly compared with the alkaloids in tea and
coffee, which, experience shows, have a real value. Large amounts of the extracts

BONE AND GELATIN. 285

cannot be used, however, as foods, because of the presence of the large percentages
of alkali phosphates and other salts. A few simple experiments may be made to
show some of the properties of the common commercial extracts.

Experiment. Heat a little of the solid extract on a piece of porcelain until it
is reduced to a char. Extract this with dilute nitric acid, filter and divide the
filtrate into two portions. In one test for phosphates by the addition of ammonium
molybdate and in the other for potassium salts by the flame test. Both reactions
should be very distinct.

Experiment. Dissolve 20 grams of extract in water to make about 200 cubic
centimeters. A nearly clear solution should be obtained, showing absence of fat or
coagulated protein. To a few cubic centimeters add enough weak acetic acid to give
a slight reaction, and boil. If a precipitate forms, which is rarely the case, albumin
is shown.

With 50 cc. of the liquid make the albumose test. Add to it finely powdered zinc
sulphate as long as it dissolves on stirring. On saturating the solution completely
a flocculent precipitate gradually settles. This is essentially the " albumose " frac-
tion and may contain a little gelatin. After 24 hours filter, and test the filtrate for
peptone by the biuret reaction ; this is generally negative.

Use the remainder of the original solution for the recognition of creatine. Add
to it carefully a solution of basic acetate of lead as long as a precipitate forms.
This will carry down phosphates, sulphates and other compounds forming insoluble
combinations with it, but not creatine. A slight excess of the lead must be added
to insure complete precipitation. This can be determined by allowing the first
formed precipitate to settle and adding more reagent as necessary. Finally filter,
and remove the excess of lead by passing in hydrogen sulphide. Filter again, and
remove as much as possible of the excess of sulphide used, by shaking. Then con-
centrate the liquid to a small volume by slow evaporation on the water-bath and
allow it to stand a day or more in a cool place for crystallization of the creatine.
Pour off the supernatant liquid and wash the fine crystals obtained with a little
strong alcohol in which creatine is but slightly soluble.

Experiment. Dissolve the creatine in a little hydrochloric acid and evaporate
the solution slowly to dryness on the water-bath. This action converts creatine into
creatinine. Dissolve the residue in a little water and divide the solution into two
parts. To one add a solution of zinc chloride, which produces a white crystalline
precipitate containing the creatinine-zinc chloride, (C4H7N30)2ZnCl2. The character
of the crystals can be seen under the microscope. To the other part of the solu-
tion add a few drops of a dilute solution of sodium nitroprusside and then, drop
by drop, dilute solution of sodium hydroxide. This gives a ruby red color which
fades to yellow. Add enough acetic acid to change the reaction and warm. The
color becomes green and finally blue. This is known as Weyl's reaction. The blue
color finally obtained is Prussian blue.

A further very delicate reaction for creatinine is given later, in the chapter on
urine analysis.

Experiment. The mother liquor left after crystallizing the creatine contains
traces of xanthine bases. Add enough ammonia to give an alkaline reaction and
filter. Then add a few drops of ammoniacal solution of silver nitrate which pre-
cipitates the several substances in flocculent form.

BONE AND GELATIN.

In the moist bone as it exists in the body the water and solids are,
in the mean, in about the proportion of one to two. In very young

286 PHYSIOLOGICAL CHEMISTRY.

persons, however, the water is in greater excess, while with age the
solids increase. The solid matter consists roughly of I part of organic
matter to 2 of mineral.

THE ORGANIC MATTER OR OSSEIN.

The crude organic substance in the bone is commonly called ossein ;
it may be extracted with hot water and forms a gelatinous mass on
cooling. But fuller investigations show that this ossein is not a single
substance, as several different constituents may be separated by proper
solvents. These are, however, closely related substances and for our
present purpose they may all be considered as practically identical with
the collagen or glue-forming substance of the connective tissues. The
conversion of the ossein or collagen into gelatin appears to be a hydra-
tion process, as at a higher temperature the reverse operation takes
place. The preparation and properties of bone gelatin may be illus-
trated experimentally:

Experiment. Clean a long, slender bone (best, a rib), and immerse it in dilute
hydrochloric acid of about ten per cent strength. Let it remain several days. At
the end of this time remove the bone from the acid and observe that it has lost
its rigidity and has become very flexible. It may be even possible to tie it in a
knot. Wash the elastic mass several times in fresh water to remove all the
hydrochloric acid, then with a little dilute sodium carbonate solution followed by
more water, and finally boil it with a small amount of pure water. By heating it
long enough the ossein becomes converted into gelatin, which solidifies, on cooling,
to a jelly.

By boiling the bone ossein under pressure the formation of the gelatin is very
much hastened.

The solution as obtained above may be used for tests such as were described in
Chapter V, under Gelatin.

THE MINERAL MATTER IN BONES.

We are not able to say exactly how the mineral elements are com-
bined in the moist fresh bone. Our knowledge of these combinations
is practically limited to what we can learn by a study of the residue
left on burning the bone completely, known as boneash. This is a
white powder containing the non-volatile compounds, of which calcium
phosphate is the most important. The following table shows the aver-
age composition of human boneash :

Calcium phosphate 85.7 per cent.

Magnesium phosphate 1.5 "

Calcium carbonate 11.0 "

Calcium fluoride and chloride 1.0 "

Ferric oxide 0.8 "

100.0

BONE AND GELATIN. CARTILAGE. 287

The presence of calcium, magnesium and phosphoric acid may be
shown in the weak hydrochloric acid extract of the bone described
above.

Experiment. To a few cubic centimeters of the filtered solution add some
ammonium molybdate solution. In a short time a yellow precipitate appears, indi-
cating presence of a phosphate, as familiar to the student from the reactions of
qualitative analysis.

Experiment. To a few cubic centimeters of the solution add solution of sodium
acetate until a distinct odor of acetic acid persists. Then add some solution of
ammonium oxalate, which produces a white precipitate of calcium oxalate.

Experiment. To another portion of the hydrochloric acid solution add am-
monia until a good alkaline reaction is obtained. A white precipitate of calcium
and magnesium phosphates settles out. Filter, and to the filtrate add some am-
monium oxalate solution. A further precipitate appears. This is calcium oxalate
and proves that the original solution contains calcium in excess of that combined
as phosphate. The calcium of the carbonate, fluoride and chloride appears here.

Experiment. To detect the small amount of magnesium requires greater care.
To another and relatively large portion of the acid solution add enough ammonia
to give an alkaline reaction, and then acidify slightly with acetic acid. This dis-
solves everything except ferric phosphate, which may be filtered off and tested for
iron. To the filtrate add enough ammonium oxalate to precipitate all the calcium
as oxalate. Separate this after long standing by means of close-grained filter
paper. In the clear filtrate the magnesium may be thrown down with the phos-
phoric acid still present, by the addition of ammonia water in slight excess.

Bone Marrow. The pure marrow consists largely of fat in which
olein is abundant; cholesterol is present and some nitrogenous extrac-
tive substances, which, however, have not been very thoroughly
examined.

CARTILAGE.

Collagen is probably the most abundant substance in the cartilagi-
nous tissue where it exists mixed or combined with several other
bodies, of which these have been described : chondronmcoid, chon-
droitin-sulphuric acid and an albuminoid. The nature of crude col-
lagen has been explained, and in Chapter V the somewhat obscure
chemistry of the chondroitin-sulphuric acid has been outlined. Of the
nature of the chondromucoid little is known definitely; it has been
held by some writers to be merely a combination of part of the collagen
with the salts of the complex ethereal sulphuric acid mentioned, while
Morner, who first described it, held it for a distinct body somewhat
allied to mucin. His analyses showed C 47-3°> H 6.42, N 12.58,
S 2.42, O 31.28. The sulphur is probably all in the ethereal combi-
nation and on incineration of the cartilage the ash is found to contain
a very large amount of alkali sulphate.

Chondromucoid as separated is insoluble in water alone, but with a
little alkali forms a thick solution, which is precipitated by acids.

288

PHYSIOLOGICAL CHEMISTRY.

Stronger acids bring about a cleavage with separation of the chon-
droitin-sulphuric acid. The weak alkali solutions are precipitated by
metallic salts, but most of the other protein reactions fail. The
ethereal sulphate group seems to prevent the ordinary precipitations.
The albuminoid substance is not well characterized but is insoluble
in water, and in weak acids or alkalies. It undergoes gastric diges-
tion. This protein is said to be found in old cartilage only, and is
absent in young cartilage.

KERATIN BODIES.

Compounds of the keratin group occur in hair, the finger 'nails and
horn. They resemble the proteins but contain rather large amounts
of sulphur, as shown by these analyses, which are of keratin from
several sources :

Hair.

Nails.

Horn.

c

50.65

6.36

17.14

20.85

5.OO

51.OO

6.94

17-51

21-75

2.8o

6.80

H

N

16.24

22.51

3-42

0

S

The sulphur in hair is in part loosely combined and may be split off
easily by reagents, alkalies for example. The ash of hair is rich in
sulphates and contains also silica and other mineral substances. Much
of the ash may be removed by washing the hair with weak acids, fol-
lowing treatment with ether and alcohol to remove fatty and other
soluble substances. The purified " keratin " thus secured gives results
like the above on analysis.

Horn and nails contain along with the insoluble keratin insoluble
salts, mainly phosphate of calcium, which stiffen them. From very
fine horn shavings these salts may be dissolved out by acids, leaving a
soft flexible keratin.

SECTION IV.

THE END PRODUCTS OF METABOLISM. EXCRE-
TIONS. ENERGY BALANCE.

CHAPTER XIX.

THE EXCRETION OF NITROGEN, SULPHUR AND PHOSPHORUS.

THE URINE.

Having considered in the foregoing pages the substances used in the
nutrition of the body, the agencies of nutrition, and the general char-
acter of the products formed, we come now to a short study of the
waste products rejected by the body after it has assimilated and used
the nutrients furnished to it. The food-stuff's which the animal can
utilize are comparatively complex, but consist essentially of the mem-
bers of the three groups, the fats, carbohydrates and proteins. The
theoretically simplest waste or oxidation products of these are nitro-
gen, carbon dioxide and water, but in the animal organism the breaking
down does not go so far. While from fats and carbohydrates essen-
tially only water and carbon dioxide are formed, the protein metabo-
lism is not carried to the elimination of nitrogen, but ends with the
formation, largely, of urea, a body in a way related to the theoretical
end products, but which would call for three more atoms of oxygen
to complete oxidation.

The nitrogen metabolism involves some extremely interesting prob-
lems which are still far from complete solution. From the older point
of view urea was considered the one normal end point in the chain of
katabolic reactions, and the other nitrogenous bodies found in the
urine, such as uric acid and creatinine, were looked upon as substances
which in some way had accidentally escaped the fate due them. This
view is doubtless incorrect, as we have good reason to believe that uric
acid is not a step in the ordinary protein metabolism, but is a derivative
of certain substances only, which break down to a limited degree.
The amount of uric acid which could be formed in this way would not
be very large at most. In the metabolism of nitrogen, therefore, a
number of normal end products must be considered and these will be
discussed in the next few pages.

The question of the fundamental changes in protein before the
20 289

29O PHYSIOLOGICAL CHEMISTRY.

recognizable end products are reached is one in which there has been
a great deal of discussion. In a general way Pflueger assumed that all
protein actually katabolized must first be built up into a part of the
living tissues, from the absorbed products of protein digestion. The
cells of this living tissue must, therefore, undergo constant and far-
reaching changes, since the body is able to dispose of some hundreds
of grams daily of protein in forced feeding. The somewhat older
theory of Voit assumes that the absorbed protein, in the form of com-
plex molecules, from the intestinal tract, is carried along by the blood
in dissolved or suspended condition to certain cells or tissues, and is
then broken down through the influence of forces residing in, or ema-
nating from, these tissues. This protein is described as circulating
protein, and before destruction does not become an integral part of the
actual tissues of the body. Both of these theories, following the older
views of the conditions under which protein is absorbed after diges-
tion, assume that only the highly complex protein structures are
capable of beginning the katabolic change. But in late years the facts
brought out by the investigations of Cohnheim, Abderhalden and
others, on the fate of protein in the digestive operations, have sug-
gested very different views regarding the general course of this nitrog-
enous metabolism. It appears probable that the greater part of the
protein of the food, broken down, as it largely is, into the component
amino acid complexes, and absorbed as such, may not be built up again
into structures like the original, but may be at once hydrolyzed and
oxidized. A nitrogenous fraction may be separated in the form of
ammonia by a hydrolytic cleavage, to be further converted into urea,
while the residue, rich in carbon and hydrogen, would suffer ultimate
oxidation like a fat or sugar.

- This general view, which has found expression notably by Cohnheim
and Folin, does not call for the building up of great masses of tissue
protein, or even for the circulating protein of Voit. There is recon-
struction of protein only insofar as it is needed for the repair of wasted
or worn out tissues, and of the extent of this we know but little. It
is probable that the protein of the tissues, in its final katabolism may
yield some products different from those produced in the hydrolysis
of the simply absorbed complexes. A study of the urine gives us
some ideas on this subject, which will appear in what follows.

It will be well to begin with the consideration of the urine as a whole,
as all these substances are eliminated through that channel.

THE URINE. 29I

THE GENERAL COMPOSITION OF URINE.

The work of the kidneys in the discharge of the urine, or more prop-
erly the separation of its constituents from the blood, is usually spoken
of as one of excretion. But something more than simple elimination
of worthless products is here concerned ; the work done by these organs
is in part secretory, as certain synthetic reactions are beyond question
carried out here. Years ago Bunge and Schmiedeberg demonstrated
the synthesis of hippuric acid from benzoic acid and glycocoll in the
kidney, and since then other changes have been brought to light.
Further than this, the peculiar mechanism of the kidney accomplishes
another very remarkable thing. The blood circulating through the
kidney contains valuable material to be saved as well as worthless sub-
stances to be rejected. Toward all these constituents the epithelial
cells of the kidney tubules exercise a sort of selective treatment. The
proteins, which are colloids, are retained by the blood, but the sugar,
which is a crystalloid, and very soluble, is retained also unless its con-
centration passes a certain limit. The soluble salts are in part passed
through the kidneys and in part retained by the blood, with the final
result of maintaining a very nearly constant osmotic pressure in that
fluid. How this is done we cannot say. It is indeed a problem of
physiology and histology rather than of chemistry. We know only
this, that the selective absorption and control of the blood concentra-
tion are perfectly automatic. When the osmotic pressure of certain
constituents is increased beyond a pretty definite limit, the filtering
mechanism in the kidney for those constituents becomes active and the
excess is allowed to pass. The simple laws of diffusion and osmotic
pressure do not help us greatly in explaining the actions of the kidneys
where the flow of excreted substances is usually from a level of low
concentration to one of higher. Attempts have been made to compare
the separating medium between the urine and the blood to a semi-
permeable membrane, but the comparison is very imperfect unless the
degree of impermeability be specially limited for each substance passing
from the blood to the urine. The limitation would have to account
for a concentration of salt from about 0.6 per cent in the blood to over
1.0 per cent in the urine, while for urea the concentration would change
from about 0.05 per cent or lower to over 2.0 per cent, that is, forty
fold. Limitations as wide as these render the comparison of little
practical service.

Percentage Variations. It is not possible to speak of the mean
strength of normal urine since the variations are extremely irregular,

292 PHYSIOLOGICAL CHEMISTRY.

depending in health on a great many factors. The volume excreted
daily, as stated in the books, is usually given much too high for the
conditions obtaining in the United States. In place of the 1,500 cc.
as found in most of the foreign works we should take 1,150 to 1,200
cc. as nearer the average excretion for 24 hours. In some hundreds
of examinations made by the writer in the last few years on people of
both sexes engaged in various occupations the average volume comes
within these limits.

A number of complete analyses of urine are found in the literature, but in most
of them the uric acid content is placed too low because of the faulty methods of
determination formerly employed. In the following table are given some results
obtained in the author's laboratory in which the recognized sources of error have
been avoided as far as possible. It expresses the mean values obtained in the
analysis of the urine of six well nourished men. The daily excretion is taken as
1200 cc, with a specific gravity of 1.023, at 200 referred to water at 40 as 1.000.
In grams per 24 hours we have :

Potassium, K 2.82

Sodium, Na 4.87

Calcium, Ca 0.13

Magnesium, Mg 0.15

Ammonium, NH4 • • 1.13

Chlorine, CI •• 8.90

Phosphoric acid, (P04) '" 2.41

Sulphuric acid, (S04)" 2.73

Urea, CON2H4 3372

Uric acid, (CBH2N403)" 0.88

Creatinine, C4H7N30 1.98

. Hippuric acid, (C9HSN03) ' 1.00

These figures are merely suggestive, as diet makes, naturally, a great change in
the excretion.

Color. In health the straw-yellow color of the urine is characteristic, the depth
of shade depending largely on the concentration. With the same solid excretion
in 24 hours the color may be light if the volume of water consumed is large, or
it may be a deep yellow if the water consumption is deficient. These facts must
be kept in mind.

Various darker shades of the urine may be observed after consumption of certain
foods or certain chemical substances. Rhubarb, senna, santonin, salicylates and many
other aromatic bodies produce highly colored urines. In some cases a marked
smoky shade is observed, and this is usually due to the oxidation of more or less
complex phenols. With a number of fruits and berries a bright yellowish or yel-
lowish-red color is noticed in the urine.

In diseases the urine may be colored from the presence of substances from the
blood, the bile, or from absorbed products of intestinal putrefaction.

Odor. The odor of urine in health is aromatic and absolutely characteristic. On
standing it usually changes rapidly from the action of bacteria, and then an am-
moniacal odor is ordinarily developed, through the alteration of the urea. Later,
other organic matters begin to break down, resulting in the development of putre-
factive or other disagreeable odors.

THE URINE. 293

Certain remedies impart very peculiar odors to the urine, and the same is true
of several vegetable foods. The behavior of asparagus and turpentine in this regard
is marked.

In disease a great variety of organic substances may be carried into the urine
in traces, and the presence of these is often accompanied by some peculiar odor.
This may be marked enough to be of importance in diagnosis.

Reaction. The urine for the 24 hours is normally acid to litmus paper. This
acidity is due ordinarily, to the presence of acid salts, rather than of free acid;
among the acid salts the di-hydrogen sodium phosphate is probably the most
important.

Under normal conditions the urine may become temporarily alkaline, usually from
the elimination of traces of alkali carbonates due to the combustion of certain
organic salts of the diet. This occasional alkalinity must not be confounded with
that which is very commonly observed in urine which has been passed some time.
In this case the alkaline reaction is due to the presence of ammonium carbonate
coming from the bacterial decomposition of the normal urea.

In the practical examination of urine litmus papers are commonly used in pref-
erence to other indicators. The measurement of the degree of acidity is uncertain.
Occasionally urine shows the so-called amphoteric reaction ; that is, it turns blue
litmus paper red, and red litmus paper blue. Very sensitive paper is necessary
to show this.

The Excretion of Alkali Salts. The alkali salts found in the urine
come from the sodium chloride consumed as such in salted food, and
in part from potassium salts in the juices of meat and in vegetables.
In the analysis of the ash of muscle given some pages back chlorine
as well as potassium is shown. Chlorine is found, although usually in
small amount, in the ash of all vegetable substances. In the latter,
however, especially in the cereals, potassium phosphate is the charac-
teristic constituent of the ash. On a cereal diet we should expect the
urine, in consequence, to show a relatively high potash and phosphoric
acid content. The ash of potatoes contains in the mean over 60 per
cent of potassium oxide while the chlorine is in excess of the sodium.
With a mixed diet, therefore, the composition of the alkali salts in the
urine must be variable and difficult of explanation. As the alkali com-
pounds are practically all soluble, they are excreted almost solely by
the urine and to a small extent only by the feces. The analysis of the
urine gives us then, in ordinary cases, a fairly accurate measure of the
alkali metals taken in with our food and drink; in normal condition
there is no accumulation of alkali salts in the body.

Calcium and Magnesium Compounds. The full significance of
these in the urine we can not explain, since without complete analyses
of the feces we do not know the relation of the excreted to the ingested
alkali-earths. Our natural waters contain usually appreciable amounts
of these salts, with those of calcium in excess as a rule. In Lake
Michigan water, for example, we have about 125 milligrams per liter

294 PHYSIOLOGICAL CHEMISTRY.

of these salts as carbonates, but in our common animal and vegetable
foods we consume daily much greater quantities than we could get
from water. The ash of wheat contains about 12 per cent of magnesia
and 3 per cent of lime, while in the ash of muscle we have over 3 per
cent of magnesia and between 2 and 3 per cent of lime. Five hundred
grams of lean meat would furnish us then with over 150 milligrams
of magnesia and with something less than that amount of lime.

But only fractions of these compounds find their way into the urine.
In the original foods they exist, in part at least, in insoluble forms.
While some of these substances may be dissolved in the stomach, the
conditions are reversed in the intestines, and insoluble phosphates, car-
bonates and sulphates are lost with the feces. There has been much
discussion as to the exact nature of the calcium and magnesium salts
excreted. In a measure the discussion is fruitless, as we must cer-
tainly admit the free exchange of ions in solution. Under ordinary
conditions the acid ions of the urine appear to be in slight excess of
the metals, which prevents precipitation of insoluble phosphates, for
example. Temperature plays a very important part in the problem of
the stability of the calcium and magnesium compounds in the urine,
and the problem is further complicated by the presence of uric acid,
the peculiar behavior of which will be touched upon below.

THE NITROGEN EXCRETION.

For many reasons this excretion is the most important which we
have to consider in connection with the urine, as it gives us an insight
into some of the fundamental problems in metabolism. The largest
part of it leaves the body as urea, but the proportion excreted in other
compounds cannot be neglected. We have pretty accurate methods
for the estimation of urea, ammonia, uric acid, creatinine and purine
nitrogen as they are found in the urine. Hippuric acid, which is
found in urine, is not as readily measured, and for several other com-
pounds which contain nitrogen our methods are far from exact. The
following table shows the distribution of the nitrogen in the urine of
six men on whose complete excretion daily tests were made in the
author's laboratory through a period of four months. The figures are
the mean values for the whole period, and are in percents of the total
nitrogen excretion, as measured by the Kjeldahl process. The general
mean represents 720 determinations for each constituent.

Under the head of undetermined nitrogen, shown in the table, there
is included the nitrogen of hippuric acid, oxyproteic acid, alloxypro-

THE EXCRETION OF NITROGEN.

295

No.

Urea
Nitrogen.

Ammonia
Nitrogen.

Purine
Nitrogen.

Uric Acid
Nitrogen.

Creatinine
Nitrogen.

5.38

5-52
5-64
5-50
6.29
4.94

5-54

Undetermined
Nitrogen.

I
2
3

4
5
6

83.26
84.50
82.43
85.05
81.46
84.17

4-39
3.56

5-55
4-56
4.71
4.26

4-50

0.67
0.6l
O.36
0.41
0.6l
0.51

I.70
I.69
I.63
1.23

1.94
I.69

4.60

4.12

4-39
3-25
4.99

4-43

Mean.

8348

0.53

1.65

4.30

teic acid and traces of other bodies of obscure composition. A brief
discussion of each one of the important constituents will follow.

UREA.

The relation of this substance to ammonium carbonate has been
referred to many times, but especially in discussing the enzymic proc-
esses of the liver. The nutrient proteins contain many amino groups
which seem to be split off in the general combustion or hydrolytic
processes going on in the body; also a great excess of groups which
oxidize more completely and yield carbon dioxide. The large part of
this escapes by way of the lungs, while another part is evidently taken
care of in the liver through combination with the amino groups to
form urea. It is also true that normally some of this amino nitrogen
fails to take this simple course, because of the presence of strong acid
radicles, which have great tenacity in their combining reactions. The
ammonium salts so formed are stable and cannot be worked over
into urea.

It appears, also, that the nitrogen of some other groups in addition
fails to reach the urea stage. Creatinine and uric acid nitrogen are
not included here, as these substances seem to have an independent
origin which will be discussed below. But there are obscure com-
pounds in the urine in small amount of which we know but little, and
some of these contain nitrogen. The oxyprotcic acid referred to above
is an illustration. What the relation of this is to urea we cannot say,
but an idea of this kind suggests itself: the original protein complex
may contain certain groups which do not fall an easy prey to the work
of the oxidation enzymes in the body; they do not break down to
amino compounds and carbon dioxide, but remain intact as very resist-
ant residues, and hence when the liver is reached they are not in
condition to pass into the urea stage. In the katabolic changes of
protein it is possible that a number of such resistant groups may be
produced, and it is likely that the amount of nitrogen or other element
which so escapes the normal end reaction depends largely on the

296 PHYSIOLOGICAL CHEMISTRY.

strength of the enzymic functions. These must vary in different indi-
viduals, and hence sometimes more and sometimes less of these resist-
ant, or left over, residues will find their way into the urine.

From this point of view urea represents that part of the original
body nitrogen, aside from the creatine and nuclein derivatives, which
takes the normal course. It represents no store of practically realiz-
able energy, while with some of the other bodies which escape in the
urine this is not the case ; under more favorable conditions they might
be expected to suffer further oxidation with liberation of more heat.
Such ideal conditions are realized in some individuals more than in
others.

Urea may be built up outside of the body by many synthetic proc-
esses, but is most easily prepared by the conversion, of ammonium
cyanate, NH4OCN, into the isomer. On evaporation of a solution
of this salt the transformation into urea is complete. Urea is very
soluble in water, from which it may be obtained easily in crystalline
form. Its solutions are easily decomposed by many oxidizing agents
with formation of water, carbon dioxide and free nitrogen, on which
behavior several of the processes for determining it are based. This
change is brought about by hypochlorites, for example, in this manner :

CON.H* + 3NaOCl = 3NaCl + 2H20 + C02 + N2.

The amino groups in urea may be completely converted into ammo-
nia in many ways, and this reaction, also, is applied in estimating urea,
as will be shown in the next chapter.

On the other hand, urea may take part in synthetic reactions and
may be combined to form complex substances, in certain cases, as will
be shown below.

AMMONIA.

This represents a portion of the protein disintegration which for a
number of reasons has not been converted into urea. The ammonia
passing into the urine takes that course ordinarily through combination
with mineral or other acids, which are not destroyed, or may not be
destroyed, by oxidation. In any pathological increase of such acids,
if there is not enough fixed alkali in the blood to combine wifh them,
ammonia is split off from protein derivatives in quantity sufficient to
complete the neutralization. This may be shown also by the injection
of free mineral acids either directly or with the food; an increased
elimination of ammonia results. It should be expected, therefore, that
the proportion of ammonia in the urine would be subject to marked
fluctuations, which is indeed the case. Taken with other determina-

THE EXCRETION OF NITROGEN. 297

tions the estimation of ammonia may possess considerable diagnostic
value, as it measures to some extent the excessive acid excretion. In
advanced stages of diabetes, with marked elimination of acid, the
ammonia content of the urine may increase to several grams daily.
The normal amount is usually a gram or less.

Ammonia must be determined in fresh urine only, since in old urine
fermentation changes soon produce large quantities of the substance
from the breaking down of urea.

URIC ACID AND THE PURINE BODIES.

Few topics in physiological chemistry have attracted more attention
than the relations of uric acid to other nitrogenous products excreted
in the urine, and its behavior in relation to disease. The importance
of the substance in this point of view has undoubtedly been very fre-
quently over-estimated and even at the present time clinicians are much
divided as to the part it plays in certain diseases. This much may be
said with truth, however, that many of the fine-spun theories which
have been advanced by medical men on the uric acid question, and
which have held our attention for a longer or shorter period, have
been founded on very weak chemical evidence, and this, it should be
mentioned, is the real factor in the case.

Under the older view, as explained already, uric acid was supposed
to be but a step in the formation of urea, the normal end product in
protein metabolism, and numerous disorders were attributed to the
accumulation of uric acid in the blood through some failure in the
final oxidation processes. But it appears now from the evidence avail-
able that uric acid is not a natural step in the oxidation of the simple
proteins; it does result, however, from the breaking down of the
complex nucleo-proteids which are represented to a limited extent only
in the body, as compared with the muscle proteins, for example. The
glandular organs rich in cells furnish the chief amount of the nuclein
complexes. In the katabolism of these, true proteins and the residues
rich in phosphorus known as nucleic acids result ; the proteins undergo
the usual further oxidation probably, while the nucleic acids break
down into a variety of products of which the purine bases, the pyrimi-
dine bases, phosphoric acid and carbohydrate groups are the most im-
portant. The purine bodies in turn doubtless give rise to uric acid.
As pointed out in Chapter V several nucleic acids exist; their struc-
tural formulas are not known, but empirically these formulas have been
given to acids from different sources.

298 PHYSIOLOGICAL CHEMISTRY.

C40H52N14O:,5P4 Salmon milt

C40H5eN14O26P4 Salmon milt

C36H4SN14O30P4 Yeast cells

C41H61N16031P4 Wheat embryo

The cleavage products of these acids are not constant, since from dif-
ferent acids different purine bases have been made. Those found in
the animal body are the following: xanthine, hypoxanthine, guanine,
adenine, heteroxanthine, paraxanthine and epiguanine. In order to
show the relations of these compounds to uric acid, E. Fischer pro-
posed to consider them all as derivatives of a nucleus group which he
called purine.

As the chemistry of these bodies is complex it may be well to illus-
trate their relations by the structural formulas worked out or con-
firmed by Fischer. Starting with the assumed purine nucleus we have
these formulas, with the nucleus atoms numbered, as suggested by
Fischer :

T n — C 6 N=CH HN— CO

I I 7 I I H ||

2 C 5 C— Nv HC C— Nv OC C— NH

I I >C8 || || )CH ; !! \

3 n — C— W N— C— N^ ^CH

4 9 HN-C-N/

Purine nucleus, C5N4 Purine, CEH4N4 Xanthine, CsH^NiOa

HN— CO N=C— NH2 HN— CO

OC— C— NH HC C— N HC C— NH

)C0 jl I ^CH || I ^CH

HN— C— NH N— C— N— H N— C— N

Uric acid, C5H4N403 Adenine, C5H5N6 Hypoxanthine, CbILN^O

Employing the Fischer nomenclature these bodies have the follow-
ing names :

Adenine 6-aminopurine

Hypoxanthine 6-oxypurine

Xanthine 2, 6-dioxypurine

Uric acid 2, 6, 8-trioxypurine

Guanine 2-amino-6-oxypurine

As their relations have been shown by various syntheses and other
transformations, and as further, the xanthine and hypoxanthine, ade-
nine and guanine have been directly derived from the nucleic acids,
the relation of uric acid to the latter bodies is not far to seek.

Not all of the nucleic acid destroyed can be assumed to come from
body cell structures; many of our foods contain nucleins and these
must give rise to the same derivatives on oxidation without passing

THE EXCRETION OF NITROGEN. 299

through, becoming part of, the cells of the glandular organs of the
body. Accordingly we distinguish between endogenous and exoge-
nous purines and uric acid. With the food nucleins eliminated as far
as possible, it has been found that the uric acid excreted becomes nearly
constant and bears a more uniform relation to the urea. This indi-
cates that the destruction of cell substance in the body leads as regu-
larly to uric acid as does that of muscle proteins to urea. The use of
rich protein foods does not necessarily occasion greater elimination of
uric acid. It is only when they contain appreciable amounts of the
nucleins that this is the case. In addition to these facts it has been
found experimentally that the oxidation of nucleins outside the body
leads to the production of uric acid in small amount.

Uric acid may be obtained synthetically by combining urea with
glycocoll, and at a high temperature it may be decomposed with pro-
duction of urea, ammonia, prussic acid and other bodies, under dif-
ferent conditions. But little importance is attached to these facts at
the present time, but formerly they were supposed to support the view
that uric acid is a stage in the urea formation through which all the
katabolic nitrogen should pass. Of greater interest is this fact that
when uric acid is introduced into the circulation of certain animals
some of it appears to be destroyed, and with the production of a little
urea. Such observations suggest that possibly a small part of our
urea may come from uric acid, but they have no bearing on the propo-
sition that the acid in turn has its origin in the nucleins and not in
the common proteins.

According to the structural formula above given uric acid appears
to have four hydrogen atoms of equal value in the formation of salts.
But apparently only two classes of salts may be formed : neutral salts,
in which two hydrogens are replaced, and acid salts, in which but
one hydrogen is replaced. We have therefore salts of the types
MC5H3N403 and M2C5H2N403. In addition to these, so-called
quadrinrates are known as urine sediments. These salts are of the
type MC5H3N403C5H4N403. The pure acid requires nearly 40,000
parts of water for solution; the neutral salts of the alkali metals are
much more soluble ; while the acid salts are but slightly soluble. The
data given by different observers are very contradictory. The salts
of barium, strontium and magnesium are nearly insoluble in water.

Solutions of urates in presence of alkali exhibit a reducing action
toward copper, silver and certain other salts, which fact possesses an
importance which will be explained later.

3<X) PHYSIOLOGICAL CHEMISTRY.

XANTHINE AND THE OTHER PURINES.

The amount of these purines not changed to uric acid is small, the sum of all of
the nitrogen so held not being over one-third of the uric acid nitrogen, according
to most observers. They are but slightly soluble in pure water, but dissolve readily
with alkali hydroxides to form compounds like the urates. With the heavy metals
the combinations are mostly insoluble.

PYRIMIDINE DERIVATIVES.

Among the decomposition products of nucleic acids the so-called pyrimidines
should be referred to, as they must bear some relation to the urinary nitrogen.
Pyrimidine itself represents a nucleus like purine from which various derivatives
may be formed. The relations as outlined by Kossel are these:

i N=C— H 6 NH— C=0 NH— CO

II II II

2 CH C— H s C=0 C— H C=0 C— CH3

II II I II II

3 N— C— H 4 NH— C— H NH— CH

Pyrimidine, C4H4N2 Uracil, C4H4N202 Thymine, C5H6N202

2, 6-dioxy pyrimidine 5-methyl uracil

We have no definite knowledge of the occurrence of these bodies in urine, but
they are among the compounds which should be expected to result from the nuclein
metabolism and possibly form a part of the nitrogen residue not fully accounted
for. Thymine has been obtained as a well-crystallized compound, soluble readily in
warm water. It is identical with the body formerly described as nucleosin.

CREATININE.

Creatinine, C4H7NsO, is the anhydride of the creatine described
under the head of the muscle extractives, and is always present in
normal urine. The amount in which it occurs is indicated by the
analyses given above. Much has been written on the question of the
relation of creatine to urea, but the evidence for the view held by some
writers that the latter is derived from the former is not very con-
vincing. In a laboratory way by boiling creatine with baryta water,
urea, sarkosine and several other things result. But no corresponding
reaction appears to take place in transfusion experiments with crea-
tine, and it has been held until recently that creatine introduced with
the food appears in the urine, not as urea, but as creatinine.

Of this transformation there is now considerable doubt, and we have
at present no very satisfactory theory concerning the origin of the
urinary creatinine. Much of our information on this subject has come
from the investigations of Folin, who pointed out a few years ago that
the elimination of creatinine is fairly constant for any given individual
from day to day, but varies in different individuals. The total elimi-
nation seems to be independent of muscular exertion, and varies with
the body weight, amounting to about 16 to 20 milligrams per kilo
daily. The excretion is not essentially changed by a high protein

THE EXCRETION OF NITROGEN. 30 1

diet, and is not lowered by fasting. It may be, then, the measure of
some peculiar phase of nitrogenous metabolism characteristic for each
individual.

The conversion of creatine into creatinine by boiling with weak
acids follows quantitatively according to the following reaction, but

the change is not rapid, as formerly assumed.

NH
yNH2 / \

\T CHS -» H-N=C\M CH2 • CO + H20

NCH2-COOH iNCH3

But in alkaline solution the reverse reaction takes place, creatine being
slowly formed. Creatinine may be separated from the urine most
easily with zinc chloride as a crystalline double salt which is yellow
because of the coprecipitation of pigments; when purified the crystals
are colorless. Creatinine is found in normal amount in the urine of
vegetarians. Toward copper and some other metallic solutions it be-
haves as a reducing agent when alkali is present.

OTHER NITROGENOUS BODIES.

It has been intimated that other compounds of nitrogen, besides
those just mentioned are excreted with the urine. Of the nature and
amount of these much remains to be shown by experiment. It was
pointed out that 4 to 5 per cent of the total urine nitrogen in the table
given above was "undetermined." It did not belong to the urea,
uric acid or other purines, ammonia or creatinine. Among the bodies
which contain some of this nitrogen the following may be mentioned
as the most important.

Hippuric Acid. This is benzoyl glycine, C6H5CO • NHCH2COOH,
and is always present in some amount in normal urine. It is formed
by synthesis from the glycine group of metabolism, and the benzoic
acid ingested with many aromatic food substances, certain acid fruits
and spices, for example. Some aromatic products of protein metabo-
lism may also give rise to it. Moderate doses of benzoates, or benzoic
acid, appear in the urine wholly in the form of hippuric acid, as the
glycine groups seem to be always present in sufficient amount to com-
plete the synthesis. These groups, otherwise, go to form urea, appar-
ently. The hippuric acid content of the urine is far from constant, as
it depends so largely on the diet, but a gram or more daily is often
present. The nitrogen in the acid amounts to 7.8 per cent.

Oxyproteic Acid. This product was first described by Bondzynski
and Gottlieb, and has been studied by others. The exact formula is

302 PHYSIOLOGICAL CHEMISTRY.

not known, but the percentage composition is about C, 39.62; H, 5.64;
N, 18.08; S, 1. 12; O, 35.54. Under the names of antoxyproteic acid
and alloxyproteic acid Bondzynski and other colleagues have described
somewhat related substances. As these, and still other bodies which
have been found in urine contain both sulphur and nitrogen, it is evi-
'dent that they represent a peculiar small fraction of the original protein
which has failed to be hydrolyzed and oxidized in the general metabo-
lism. It has been estimated that 2 or 3 per cent of the total urine
nitrogen is found in these compounds, and the sulphur in them would
be included in the so-called " neutral " sulphur, to be referred to later.
Pathologically the relations in the nitrogen excretion may be very
much changed, and protein, as such, may occur in the urine in con-
siderable amount. The detection of this protein will be discussed in
the following chapter, along with the methods for the measurement of
the normal constituents.

THE SULPHUR EXCRETION.

The sulphur in the urine is found in several compounds and comes
from different sources. A small part has its origin in traces of sul-
phates taken directly in food and natural waters; some comes from
the sulphur existing in peculiar combinations in certain vegetables,
while the largest part has its origin in the sulphur of proteins, which
undergoes more or less complete oxidation before elimination through
the kidneys. Most of this sulphuric acid of oxidation combines with
alkalies for elimination; if fixed alkalies are deficient ammonium sul-
phate is formed and this ammonia therefore escapes the natural oxida-
tion to urea.

It has been explained in an earlier chapter that in the putrefactive
changes taking place in the intestines certain phenol bodies are split
off from proteins there remaining, or perhaps in most cases from the
unabsorbed protein derivatives. A very considerable part of these
phenol bodies escapes with the feces, but another portion, often much
increased in disease, is absorbed by the blood vessels from the lower
intestine and carried to the liver where combination with sulphuric
acid is effected, probably through some kind of enzymic action. From
recent observations it appears likely that the combination is effected
before the sulphur is completely oxidized, that is, while it is in the
sulphite condition. The final oxidation then follows. The ethereal
sulphate so formed is discharged finally with the urine. The fraction
of the sulphuric acid so voided is extremely variable, reaching some-
times 20 per cent of the whole. The most abundant of these combi-

THE EXCRETION OF SULPHUR.

303

nations are salts of phenyl-, cresyl-, indoxyl-, and skatoxyl-sulphuric

acid, the structural relations of which are shown by the following

formulas :

CH

//\
HC C — C.O.SCUDH

CeH50 -1 c;o CH3C„H40 \ qo

r bU= • HO / bU=

HO

Phenyl-sulphuric acid Cresyl-sulphuric acid

HC C CH

\/\/

C N
H H

Indoxyl-sulphuric acid

Skatoxyl-sulphuric acid is the methyl derivative of indoxyl-sulphuric
acid. Phenyl- and cresyl-sulphuric acids are frequently called phenol-
and cresol-sulphuric acids, but the former names are preferable. The
alkali salts of indoxyl-sulphuric acid are known as indican, the appear-
ance of which in the urine in quantities above minute traces is an indi-
cation of the existence of excessive putrefactive reactions in the intes-
tines. The observation is therefore of value in diagnosis. Accurate
methods are known for the recognition of the substance in the urine.

In addition to the oxidized or sulphate sulphur in the urine there are
always traces of so-called " neutral " sulphur compounds present. Ac-
cording to various authorities the sulphur in these compounds, which
are all complex organic bodies, may make up 12 to 25 per cent of the
total sulphur. The sulphur bodies here in question are not easily
recognized in most cases and the existence of several of them described
in the journals is yet to be demonstrated. The fact of the excess of
sulphur over that contained in the mineral and ethereal sulphates may
be easily shown by determination of the total sulphates directly, and
then after complete oxidation of the urine by sodium peroxide, or
other agent. This unoxidized or neutral sulphur is found in such
bodies as the oxyproteic acid mentioned above, and occasionally in
cystin or taurin present.

The following table gives a good idea of the sulphur excretion

No.

Inorganic Sulphur.

Ethereal Sulphur.

Neutral Sulphur.

I

72.30

9.71

17.98

2

74.96

9.42

15.62

3

75-93

8.48

15 59

4

78.13

6-55

I5-32

5

71.84

9-13

1903

6

76.80

6.39

16.81

Mean.

74-99

8.28

'6.73

through a long period, in the urine of six men in normal health. The
values represent fractions of the total sulphur, and in each case are the

304 PHYSIOLOGICAL CHEMISTRY.

means of daily observations on the whole urine, through four months.
The observations were made in the author's laboratory.

THE PHOSPHORUS EXCRETION.

As mentioned in an earlier chapter, some of our foods are very rich
in phosphates, or phosphate-furnishing material. This is especially
true of the cereals, of a few animal proteins and of the lecithins. The
phosphates formed by oxidation of these substances are mainly elimi-
nated with the urine, and in traces with the feces. A very consider-
able portion of the urinary phosphoric acid comes from this source,
which may be described as the exogenous phosphoric acid. Another
portion comes from the breaking down of the nucleic acids of the cell
substances and from the so-called phospho-globulins or nucleo-albumins
also found in the body. These are the endogenous sources. How the
phosphorus is held in these last-named bodies is not known, but in the
nucleic acids it appears to be in oxidized form, according to some
authorities as metaphosphoric acid. The excreted product is ortho-
phosphoric acid, combined to form salts of the type MH2P04 or
M2HP04. The alkali salts are soluble readily, while those of cal-
cium and magnesium are only in part soluble in water. The condi-
tions of solubility in urine are complicated by the presence of other
salts.

The amount of phosphoric acid, as P205, excreted daily varies
within wide limits; according to the above analysis the mean may be
about 2.5 gm. If the urine becomes alkaline through the fermenta-
tion of urea a very considerable part of the phosphate may be precipi-
tated in the form of calcium and magnesium salts. One of the com-
monest of these is the so-called triple phosphate, NH4MgP04-6H20.
The determination of the amount of phosphoric acid in the urine will
be discussed in the next chapter.

CARBOHYDRATES.

Normally no large amount of any carbohydrate passes from the
blood into the urine, but with increased sugar concentration in the
blood from any cause, the urine may contain sugar in more than traces.
When sugars are consumed in more than the "assimilation limit" a
part of the excess may be found in the urine. This is true of glucose
as well as of cane sugar and maltose.

But under normal conditions there is evidence that traces of several
carbohydrates and of bodies related to them pass also into the urine.
It is well known that the oc-naphthol reaction is given with ordinary

THE EXCRETION OF CARBOHYDRATES AND PROTEINS. 305

normal urines, and this points to some carbohydrate derivative which
can yield furfuraldehyde. The other delicate sugar tests give the
same indication. Some of the carbohydrate doubtless appears as such,
but a portion of it probably exists in the form of glucoproteids or
other complex groups. Part of the carbohydrate can be readily fer-
mented, while another portion resists the action of yeast. Such
observations may be interpreted as suggesting the presence of other
carbohydrates than the common glucose. More will be said about
this below.

Pathologically, the passage of even large quantities of sugar into
the urine is a common phenomenon. This is most frequently observed
in diabetes mellitus, a disease in which the power of oxidizing sugar
in the muscles seems to be wholly or partly lost. It has been already
mentioned that this oxidation is possibly an enzymic operation in which
certain muscle and pancreas enzymes are at the same time active. In
this form of diabetes several hundred grams daily of sugar may be
excreted.

In another, " artificial," form of diabetes sugar may be caused to
appear in considerable quantity temporarily in the urine. The admin-
istration of small doses of phloridzin, a glucoside, is followed by the
appearance of sugar in the urine, but this condition is now believed to
depend on a disturbance of the proper functions of the kidneys rather
than on any alteration of the sugar-oxidizing power. The power of
retaining the sugar in the blood from which it should be taken up and
oxidized by the muscular tissues depends on the maintenance of the
integrity of the membranous structures of the kidneys. If these suffer
strain, which seems to be the case after administration of phloridzin,
a little sugar more than the normal may be able to diffuse or pass
through in some manner. It is possible that many other substances
may have a somewhat similar action, but in much smaller degree, and
so occasion sugar excretions still within what may be called " normal "
limits.

PROTEINS.

As in the case of the sugars, so with the proteins; there is good evi-
dence that traces of these nitrogen compounds are normally present in
the urine. The amount which may be present is, however, very small,
in the mean not over 40 to 50 milligrams per liter. With these traces
it is not possible to say how many different kinds of proteins may
occur, but the serum albumin is doubtless the most abundant. With
greatly increased amounts of albumin in the diet the excreted albumin
is also increased, with no visible impairment of the kidney mechanism.

306 PHYSIOLOGICAL CHEMISTRY.

It is also true that foreign albumins injected into the blood circulation,
white of egg, for example, are speedily eliminated by the kidneys.

Pathologically, however, the serum albumin and serum globulin of
the blood may pass through the kidneys in considerable quantity.
This is occasionally due to disturbances in the circulation which may
bring about an increase of blood pressure, but ordinarily is due to
structural changes in the kidneys themselves, through which the power
of perfectly retaining albumins of the blood is lost. A discussion of
the nature of these changes is not within the scope of this book, and
for an explanation of the tests which are employed in recognizing the
proteins of the urine the reader is referred to the next chapter. Many
of these tests are but modifications of the delicate protein tests de-
scribed in one of the earlier chapters.

URINARY SEDIMENTS.

The urine when passed is usually clear, but frequently it soon be-
comes cloudy and deposits a precipitate. This precipitate may consist
of a variety of constituents and may be due to several causes. Sooner
or later all urines, unless specially protected by preservatives, undergo
the so-called ammoniacal fermentation in which urea is converted into
ammonium carbonate by certain bacteria. When this alkaline condi-
tion is reached the condition of equilibrium in which the various salts
exist together is destroyed and insoluble products are commonly
formed which appear as precipitates. The alkali-earth phosphates are
the bodies usually thrown out in this way.

But disturbances in the equilibrium may result from changes of
temperature also, and precipitation occur because of the simple cooling
of the warm voided urine. Many of the peculiar urate sediments are
formed in this way. Sometimes the separation of sediment begins in
the bladder or ureters and this sediment may take the form of hard
concretions or calculi. In years past numerous attempts have been
made to explain the formation of these deposits, especially as they
occur within the body. The theoretical explanations given have been
in general far from satisfactory, and the most recent studies have only
gone to show the complexity of the problem. It is now coming to be
recognized that we have here one of the most difficult problems of
physical chemistry, which like other questions of chemical equilibrium
in solution may be approached only through elaborate studies. The
beginning of such studies may be seen in some of the valuable papers
which have been published in the last few years on the solubility of
uric acid and its salts, and the degrees of dissociation which obtain

REDUCING POWER OF NORMAL URINE. 3O7

in the various solutions. As yet these matters are scarcely in condition
for elementary presentation.

The recognition of the general character of the sediments from
urine is most readily effected by the aid of the microscope, which is
explained in the next chapter.

THE REDUCING POWER OF NORMAL URINE.

On account of the presence of some of the bodies described in the
last few pages, normal urine exhibits a certain reducing action toward
metallic solutions. At one time this was ascribed to traces of carbo-
hydrates present, but later doubt was thrown on this conclusion and
the presence of even traces of sugar-like bodies was denied by most
writers concerned with the question. With the development of
greater accuracy in the methods of detecting sugars, especially through
the aid of the phenylhydrazine combination and the formation of ben-
zoic esters, the question of the passage of traces of carbohydrates into
the urine seems to be finally settled in the affirmative, but the amount
of sugar which may be so identified is too small to account for the
total reduction easily measured by copper solutions. This normal
reduction cannot be quantitatively followed with the ordinary Fehling
solution, but if a dilute Pavy solution, as described in Chapter III, is
used, a very satisfactory determination may be made. This modified
Pavy solution is given such a strength that I cc. oxidizes i mg. of
glucose in very dilute solution, approximating 0.2 per cent strength
or less.

In a large number of tests made in the author's laboratory a few
years ago it was found that to reduce 50 cc. of such a solution urine
volumes ranging between 14.9 cc. and 58 cc. were required. The
mean of all the determinations on these normal urines was 23 cc. In
hundreds of normal urines examined since similar results have been
obtained. In all these urines the creatinine and uric acid present were
accurately determined and an attempt was made to connect these bodies
with the reduction.

The Reducing Power of Creatinine. The reducing power of creatinine may be
easily found by the method referred to, using the weak ammoniacal copper solution.
A liter of this solution contains 8.166 gm. of CuS04-5H20, corresponding to 2.6042
gm. of CuO. A measured volume of this, usually 25 or 50 cc, is reduced by a
creatinine solution of definite strength and the volume required noted. The details
me experimenti are «ivcn in the following tabic. The copper solution was
diluted to 100 re. before the titration:

3o8

PHYSIOLOGICAL CHEMISTRY.

Creatinine
in ioo cc.

Copper Solution
Taken

CuO Equivalent.

Creatinine Solu-
tion Used.

Creatinine to
130.2 mg. CuO.

Mols. CuO

to 1 Mol.

C4H7N30.

50 mg.

50
I20
I20

25 CC.
25

50

65.I mg.

651
I3O.2
I3O.2

92.5 CC.
94.O
76.O
77.0

92.5 mg.
94.O
QI.2
92.4

I.998
I.967
2.026
2.000

Mean, 1.998

It appears, therefore, that in this kind of solution 2 molecules of copper oxide are
required to oxidize 1 molecule of creatinine. The 2 molecules of copper oxide
yield 1 atom of oxygen. It will be recalled that 5 molecules of copper oxide are
used up in oxidizing 1 molecule of dextrose. For equal weights the reducing
power of dextrose is about twice as great as is that of creatinine.

Reducing Power of Uric Acid. With the same reagent the reducing power
of uric acid may be measured, the uric acid being dissolved with a little alkali to
form a soluble urate. The table below illustrates the relation of the reducing and
oxidizing solutions :

Uric Acid in
100 cc.

Copper
Solution
Taken.

CuO Equivalent.

Uric Acid

Solution

Used.

Uric Acid to
130.2 mg. CuO.

Mols CuO
to 1 Mol.
C5H4N403.

80 mg.

80

120
120

15.3 cc.

25
5°
25

39.8 mg.
65.I
130.2
65.1

36 CC.

58

76.8

37-8

94.2 mg.
92.8
9I.8
90.8

2.92
2.96
2.99
3-°3

Mean, 2.98

From this it appears that 1 molecule of uric acid requires 3 molecules of copper
oxide for oxidation under the conditions of the test, or 1.5 atoms of oxygen.
This is a larger amount of oxygen than would be required for the oxidation of uric
acid to urea and alloxan. This requires 1 atom :

C5H4N403 + O + H20 = CON2H4 + C4FLN204.

But secondary reactions also take place, and a partial oxidation to parabanic
acid may be represented in this way :

2C6H4N403 + 30 + 2H20 = 2CON2H4 + QH2N204 + C3H2N203 + C02.

This possibly represents the course of the reaction with such a solution.

With these reducing values established it is possible to estimate the fraction of
the total urine reduction which is not due to these two most important substances
besides sugar. To do this it is necessary to find the amount of uric acid and
creatinine in a given volume of urine and determine its reducing value in terms
of CuO with the same ammoniacal solution. If the reducing power of the crea-
tinine and uric acid be subtracted from the total, the reduction due to glucose and
other bodies is arrived at. To illustrate, in the examination of a large number of
urines these values were found :

Total reducing power of 1 cc. of urine in mg. of CuO = 6.204
Reducing power of the creatinine present in same terms = 1.961
Reducing power of the uric acid present in same terms = 0.935
Reducing power of the sum of uric acid and creatinine = 2.896

THE ELECTRICAL CONDUCTIVITY OF URINE. 309

Nearly one-half of the total reduction then corresponds to the action of these
two nitrogen bodies, while the reduction of the other substances is equivalent to
3.308 mg. of CuO per cc. If this is calculated as glucose it represents 1.27 mg.
per cc. As several other bodies contribute to this reducing action the value of
any saccharine substance present must be still smaller. It has been found by
earlier investigations that on concentration urine loses a part of its reducing power.
This suggests that some volatile substances may be responsible for part of it.
It should be mentioned in addition, that the relative extent of the reducing action
varies with the reagent employed. Knapp's mercury solution has been used for
the purpose but it is not as convenient as the ammoniacal copper solutions.

A knowledge of this normal reducing action is not without value,
since very frequently the mistake has been made of assuming the pres-
ence of sugar in suspicious quantities in the urine merely from a reduc-
tion test. In some of the extreme cases quoted in one of the state-
ments above, the normal reduction was equivalent to over 0.3 per cent
of glucose, when in reality it was largely due to uric acid and creati-
nine. This point has importance in clinical observations. In another
direction also the question is important, as the reducing power of the
urine is a measure of the extent of certain kinds of excretion. Crea-
tinine and uric acid are probably perfectly normal end products of
metabolism, and when large in amount the reduction is high. It will
be recalled further that, as mentioned in a former chapter, strongly
reducing substances are produced in the autolytic changes taking place
in the liver and pancreas. In cases then, this " normal " reduction may
become excessive.

THE ELECTRICAL CONDUCTIVITY OF URINE.

In one of the chapters on the blood the nature of electrical con-
ductivity in the fluids of the body was explained and the methods of
determination outlined. As this conductivity depends mainly on the
sum of the inorganic constituents present, and as sodium chloride is
the most abundant of these, the determination in itself has but a lim-
ited importance. In some cases the value of the conductivity would be
merely a rough measure of the salt consumed with the food, and the
salt consumption is extremely variable.

Nearly all the other substances found in the urine have a significance
very different from that of the salt. The latter is consumed and
excreted as such, while the other important urinary constituents are
products of metabolism, that is, of the breaking down of the digested
and absorbed food materials. The organic products of metabolism
are practically non-electrolytes or bodies with a very low conducting
power; indeed the conductivity of a weak salt solution is materially
lowered by the addition of urea and the effect of the purine bodies is

3io

PHYSIOLOGICAL CHEMISTRY.

practically in the same direction. Aside from the chlorides, the inor-
ganic salts of the urine are mainly phosphates and sulphates of the
alkali or alkali-earth metals, and these are made up largely from the
oxidation of sulphur and phosphorus of protein foods. We consume
a certain amount of phosphoric and sulphuric acids in complex organic
combination, in the lecithins and chondroitins, for example, and small
amounts of mineral sulphates and phosphates are also found in some
of our foods, but these amounts are not large enough to vitiate the
truth of the general proposition that the sulphuric and phosphoric acids
as detected in the urine are results of certain kinds of metabolism.
Now, the conductivity measures the combined effect of these products
of oxidation and, if it could be determined apart from the effect of the
chlorides, a factor of considerable practical importance would be
secured.

Approximately, the conductivity due to metabolic products may be
found by subtracting from the observed conductivity that due to
sodium chloride in the same solution. The chlorine may be accurately
determined and calculated as chloride; then from tables the conduc-
tivity for a solution of this concentration may be found and used as a
correction to be taken from the total observed conductivity, leaving

B

Period.

A

B

A

Volume

Passed,

cc.

Specific
Gravity
200

Conduc-
tivity, K.

Volume

Passed,

cc.

Specific
Gravity
200

Conduc-
tivity, K.

ervations of first

y:

retion = 1,162 cc.
uid consumption
1,750 cc.

a
. 0
o'S
0 a,

»!

II C 0

II 0 0

E U m

O H

si? 11

6- 9 A. M.
9-12
12- 3
3-6

6- 9 P. M.
9-6

145

178

13s
118

1 28
458

1.024

1.023

1.024
1.027
1.026

1.023

O.02589
O.O302I
O.O2834
O.O279O
O.02694
O.O242I

112
220

77

70

198

184

I.022
I.OI9
I.026
I.027
1. 02 1
I.026

O.02264
O.O2519
0.02226
0.02614
O.02178
0.02008

-S -S S .2* 11

C WiJ

Means, 3-hr. period

1.024

O.O2650

1.0 4

0.02228

ervations of sec-
d day :

retion = 1,265 re.
uid consumption
2,100 cc.

• S

H 0.

B

M 3

II §

0 ° *"

■£•0 "i

85*11

6- 9 A. M.
9-12
12- 3
3-6

6- 9 P. M.
9-6

192
245
155
"3
155
405

1.020
1.020
1.026
1.026

1.025
I 025

O.02645
O.O2926
O.02803
O.02702
O.O2702
0.02I22

410
122
96
138
1 80
285

I.OO7
I. OI7
I.O23
I.O24
1. 02I
I.O24

O.OIO39
O.O2408
O.02422
O.O2355
O.02144
O.02369

.a 0 x .2" II

Means, 3-hr. period

1.024

O.025I8

I.020

O.02184

ervations of third

y.

.retion — 1,324 cc.
uid consumption
1,670 cc.

g;S.
0 £

m3

II gg

'Z— m
<uI2 J"
u B m

a 1-11

6- 9 A. M.
9-12
12- 3
3-6

6- 9 P.M.
9-6

230
260
160

134
146

394

1.022
1. 021
1.026
1.026

1.025
1.023

O.O2683
O.O2939
O 02792
O.O2755
O.02580
O.OI997

278

158

92

7i
112
388

I-OI5
I.022
I 029
I O3O
I.O27
I. OI4

O.O2629
O.03037
O.02720
O.O2566
O.02577
O.OI492

0 aj

Means, 3-hr

period

1.024

O.O2468

I.O^I

O.O225 1

THE ELECTRICAL CONDUCTIVITY OF URINE.

311

the desired residual or metabolic conductivity. In this plan, however,
an error is involved, because the conductivity of the chloride taken
from the tables directly is that found in pure aqueous solution in
absence of other salts, and is larger than the true conductivity of the
chloride as it exists in the urine. It is necessary, therefore, to use as
a correction the value of the salt conductivity, not in aqueous solution,
but in a solution of a concentration corresponding to that of urine.

Several attempts have been made to measure the conductivity of mixtures of
electrolytes and formulate the results. Most of the experiments have been made
with dilute solutions and can not be used well in cases like the present one. The
author has investigated the question with special reference to mixtures like the
urine and has considered the effect of urea as a non-electrolyte also. The con-
ductivity varies from individual to individual, and from hour to hour according
to the kind and amount of food metabolized, but is seldom above ^ = 0.03. The
table (page 310) illustrates the variations in the urine of two men, both well
nourished on mixed diet, with the water consumption intentionally high.

By making complete urine analyses and duplicating the results by mixing the
inorganic and organic ions and the non-electrolytes in proper proportion, it is pos-
sible to reach almost exactly the corresponding urine conductivity as shown, for
example, with six urines, the complete analyses of which were employed in reach-
ing the mean values given at the beginning of the chapter.

No. of Urine.

1

2 3

4

5 6

k as observed.
k from mixtures.

0.02372
0.02332

O.02402
O.024OO

O.O2793
O.02768

O.OI984
O.OI944

O.O2898 I O.O225I
O.02886 O.O2158

But if an attempt were made to calculate the urine conductivity by adding together
the individual conductivities of the various salts for the concentrations as found
by analysis the result would be too high, as the conductivity of each substance
is lowered somewhat by the presence of the others. It is possible, however, by
experimenting with known artificial mixtures, to determine the extent of this modi-
fication and so be able to introduce a correction. For the present purpose the
disturbing action of the other urinary constituents on sodium chloride is all that
is called for. This has been done, but the details of the investigation can not be
given here.

In a long series of experiments it was found that a mean correction for the
effect of sodium chloride may be made in this way. The chlorine is accurately
determined in the urine and calculated as sodium chloride. From conductivity
tables the value of « for the calculated concentrations is found and this value is
diminished by 3 per cent which is the average correction due to the presence of
other substances, organic and inorganic, in the urine. This corrected salt con-
ductivity in turn must be subtracted from the observed total conductivity to find
the fraction due to metabolic products. This correction gives a result which is near
the truth in ordinary normal urines and which may be taken as furnishing a
peculiar kind of measure of the total metabolism, that will be found to have a
value in certain calculations.

312 PHYSIOLOGICAL CHEMISTRY.

THE FREEZING POINT OF URINE. CRYOSCOPY.

In the thirteenth chapter the application of cryoscopic methods to
blood examinations was discussed, and the apparatus used described.
In urine investigations also freezing point determinations have become
important and a very considerable literature has accumulated. The
Beckmann apparatus may be employed, as with the blood, but the Zikel
modification has been found to give good results and is somewhat
simpler in manipulation.

Conductivity and cryoscopic methods do not yield exactly parallel results ; the
conductivity power of the urine depends essentially on the number of inorganic
molecules or ions present, while the freezing point depression depends on the sum
of all the dissolved substances. Urea is therefore important in the one instance,
but not in the other, as it is practically a non-conductor. This being the case it
is evident that valuable information may be obtained by a combination of the two
methods, as it is possible to determine the fraction of the osmotic pressure of
the urine due to electrolytes and non-electrolytes. Such applications are fre-
quently made.

While the osmotic pressure of the blood is nearly constant, that of the urine
is extremely variable. Ordinarily the limits are between A = — 1-3° and — 2.00,
but after great water consumption on the one hand, or consumption of much
nitrogenous food, or salt, without sufficient liquid, on the other, the freezing point
of the urine may vary from A = — o.r° to — 3.00. That is, the urine concen-
tration may range from one-fifth that of the blood to over five times the blood
concentration, expressed in active molecules. It will be remembered that a freez-
ing point depression of i° C. corresponds to an osmotic pressure of 12.1 atmo-
spheres.

The applications of this cryoscopic method to urine are mainly in the direction
of diagnosis. Since it is possible to collect the urine from each kidney separately,
by ureter catheterization or equivalent means, a test of the two portions, will dis-
close any difference in the performance of the two organs. Normally, the secre-
tions from the two kidneys, for a given time, should be the same. A freezing
point determination is easily made and will show if one kidney is doing more work
than the other. By including a conductivity test it may be found that the diffi-
culty in excretion is more pronounced for one class of substances than for
another.

CHAPTER XX.

SOME PRACTICAL URINE TESTS.

In this chapter brief directions will be given for the routine exami-
nation of urine, such as is called for in clinical observations. In such
work the color and reaction, already referred to, are always noted, and
the specific gravity.

SPECIFIC GRAVITY.

For very accurate work the specific gravity of the urine is determined by means
of the pycnometer or weighing bottle, but in the ordinary clinical practice it is
customary to employ the urinometer, which may furnish very accurate results if
the instrument is properly made and graduated. Formerly the reading was based
on a temperature of about 15. 5° in the urine, referred to water of the same tem-
perature as unity. A much more rational plan is to assume a standard tempera-
ture of 25° C. in the urine and refer the reading to water at 40 C. as the unit.

From a test of the specific gravity it is possible to make an approximate estimate
of the total solids present. From a large number of observations made in the
author's laboratory it has been found that by multiplying the last two figures of
the specific gravity at 250 C, by 2.6 a product is secured which represents the
weight of urinary solids in 1000 cc. This 2.6 is Long's coefficient, and takes the
place of the Haeser coefficient based on tests at a lower temperature. Thus, if
the specific gravity at 25° C. is 1.023 (Sp. gr. -" = 1.023) we have 23X2.6 = 59.8,
or the number of grams of solids per liter. The direct determination of total
solids by evaporation and weighing is not perfectly accurate, because of losses
through partial decomposition of some of the constituents.

The specific gravity of the urine varies, naturally, with the food consumption
and volume of liquid consumed. It may, normally, be as low as 1.010 and as high
as 1.030 or perhaps higher. A diet rich in meats gives rise to a urine with high
specific gravity because of the resultant urea excretion ; much salt in the food has
the same effect. On the other hand, a diet rich in fats and carbohydrates, with
low proteins, gives rise to a urine with low specific gravity, because in such cases
little is formed to be excreted by the kidneys. With ordinary mixed diet a spe-
cific gravity of 1.020 to 1.025 is generally observed.

In diabetes mellitus the specific gravity of the urine may be very high, from the
excretion of sugar, as will be pointed out below. But it must be kept in mind
that a specific gravity high enough to suggest sugar may be reached through high
salt and protein and low water consumption.

The clinician is not so much interested in the normal constituents of
the urine as he is in the possible presence of bodies pointing to a patho-
logical condition in his patient. A marked variation in some of the
normal constituents of the urine may also have an important bearing
on the diagnosis, and he must know how to make these tests. Follow-
ing the usual routine, we shall take up albumin and sugar first.

3'3

314 PHYSIOLOGICAL CHEMISTRY.

THE TESTS FOR ALBUMINS.

Albumins are present in normal urine in traces only, and such traces are not
detected by the usual reagents described below. Albumin in the urine in any
appreciable amount is indicative of a pathological condition, and may be due to a
number of causes. A great number of tests have been suggested for the recogni-
tion of this pathological albumin, and these all depend, practically, on the fact
that the soluble, invisible protein may be easily coagulated in the urine and so
rendered visible. The ordinary serum albumin will be considered first.

Heat Test. At a temperature near 70° C. the serum albumin becomes coagulated
and opaque. Heat a few cc. of the urine in a test-tube nearly to the boiling point.
If the liquid becomes cloudy albumin is suggested. But the urine usually contains
enough so-called earthy phosphates to become cloudy on heating, and this cloud
resembles the albumin coagulation very closely. To avoid a mistake here the
urine must be very weakly acidified with dilute acetic acid before warming. This
prevents the precipitation of phosphates but does not interfere with the albumin
coagulation. Avoid using much acid.

Nitric Acid Test. In contact with strong nitric acid the albumin of the urine
is immediately coagulated. To make the test pour two or three cc. of strong nitric
acid into a narrow test-tube, warm slightly and by means of a dropping-tube
introduce over the acid a layer of the clear urine (filtered previously, if not
clear). In presence of albumin a cloudiness, or even a heavy precipitate, appears
at the junction of the two liquids. This is an extremely delicate test and is very
commonly employed clinically. The nitric acid is warmed to prevent the possible
precipitation of urea nitrate, which occasionally happens with cold, very con-
centrated urine. A simple change of color at the junction layer is not due to
albumin. The latter yields an actual coagulation.

Picric Acid Test. Many reagents besides nitric acid have the power of forming
precipitates with albumin. A saturated aqueous solution of picric acid is very
useful for this purpose, and when added to albuminous urine produces a yellowish
white cloud or precipitate. It is best to acidify the urine slightly with acetic
acid first. If this causes a precipitate, of mucin possibly, filter it off and then
add the picric acid, drop by drop. The test is extremely delicate, but is not
better than the preceding one.

Double Iodide Test. This is known also as Tanret's Test and the Mercuro-
Potassium Iodide Test. The reagent used is made by dissolving 33.1 gm. of
potassium iodide in distilled water and adding to it gradually 13.5 gm. of mer-
curic chloride. The mixture is stirred until all is dissolved, and diluted to make
800 cc. To this 100 cc. of pure, strong acetic acid is added. If a slight pre-
cipitate remains decant carefully and dilute to one liter with distilled water. The
solution contains approximately 4 KI to HgCl2.

To make the test filter the urine, if not clear, and add enough acetic acid to
give a sharp reaction. If a precipitate or turbidity now appears, possibly due
to mucin bodies, filter again and to 10 cc. of the clear filtrate add a few drops
of the reagent. In presence of proteins a white cloud is formed, or even a pre-
cipitate if much of the albumin substance is in the solution.

The test is extremely delicate, but it must be remembered that the same reagent
is employed in testing for alkaloids. If the urine happens to contain traces of
those bodies, from previous medication, a reaction will certainly follow. But
such precipitates may be distinguished by their solubility in alcohol. A precipitate
may be formed also in presence of excess of urates. Such a precipitate is dissi-
pated by heating ; it may be avoided in the first place by proper dilution of the urine.

The Amount of Albumin. It is not alone sufficient that we are able to detect
the presence of albumin in urine; we often need to know its amount to determine

SOME PRACTICAL URINE TESTS. 3 I 5

the practical value of a line of treatment pursued from day to day. To be of
the greatest possible service, a method must be so easy of execution that approxi-
mately correct results may be obtained by it by the use of simple apparatus and in
a short time. Several methods are known by which the amount of albumin in
urine can be found. One of these, and the best, may be called the gravimetric
method, as by it the albumin is precipitated, collected, and weighed. In another,
the albumin is precipitated and its volume measured, while in a third process the
amount of albumin is estimated from the degree of turbidity caused by its pre-
cipitation in the urine.

The gravimetric method consists essentially in coagulating the albumin, collect-
ing it on some form of filter, and, after proper washing, weighing the pre-
cipitate. This method may be made to give very good results but is too difficult
and tedious for the clinical laboratory.

Volume Methods. One of the simplest of these is the one proposed by Esbach.
In this a special tube is used, called the Esbach albuminometer, and a special solu-
tion or reagent made by dissolving 10 grams of pure picric acid and 20 grams of
pure citric acid in a liter of distilled water. The solution must be filtered if it is
not perfectly clear, and is the same as the one used for the qualitative test. The
principle involved in the employment of the method is this : The precipitate of
albumin and picric acid settles in coherent manner and in a compact volume pro-
portional to its weight, provided certain definite amounts of the reagent and urine
are taken. The albuminometer, or measuring tube used, resembles a test-tube of
heavy glass about six inches long and is graduated, empirically, to show how much
urine and reagent to take and the amount of albumin obtained in grams per liter,
or tenths of 1 per cent.

The test is carried out in this manner:

Urine is poured in, to the mark, and then the reagent, described above, to its
proper level. The tube is closed with the thumb and tipped backward and for-
ward eight or ten times until the liquids are thoroughly mixed. It is then closed
with a rubber stopper and allowed to stand in a perpendicular position twenty-
four hours. This will give the precipitate time to settle thoroughly after which
the amount can be read off on the scale. The results are accurate enough for
clinical purposes and by practice can be made to agree moderately well with those
found by the gravimetric method. But the tube must not be violently shaken, and,
as the test is an empirical one, it should not be allowed to stand much longer than
one day before reading the result.

In any case in applying this test the urine should not be highly concentrated.
The best results are obtained with urine of low specific gravity, and with the
albumin not over 0.3 per cent. When the test shows an amount in excess of this,
the urine should be diluted and a new trial made.

A yellowish-red precipitate which sometimes separates on long standing must
not confuse the analyst. It consists of uric acid.

The Tests for Globulins. These protein bodies resemble the albumins in many
respects, and like them occur only pathologically in the urine. In all the pre-
ceding tests, globulins react as do the albumins. Among the distinctive tests one
only need be mentioned here.

Dilution Test. Globulin is insoluble in water, but soluble in dilute salt solutions;
— hence its solubility in urine. If the latter is diluted until the specific gravity is
1.002 or 1.003 the globulin may separate out. At any rate the addition of a few
drops of dilute acetic acid will produce the desired result. A current of carbon
dioxide passed into the diluted liquid for several hours accomplishes the same
end.

The test may be modified in this manner. Filter the urine if it is not perfectly

316 PHYSIOLOGICAL CHEMISTRY.

clear, and then pour it, drop by drop, into a tall, narrow beaker of distilled water.
If globulin is present it is thrown out as a white cloud which shows as the drops
pass down through and mix with the lighter, clear water. The globulin may
afterward be confirmed by adding a small amount of salt solution which will cause
the precipitate to disappear.

PEPTONES AND PROTEOSES.

True peptones are very rarely found in urine. The bodies described as peptones
are probably highly converted proteoses, or albumoses of the deuteroalbumose type.

The recognition of albumose is not a matter of difficulty, as it is distinguished
from the other protein compounds sometimes found in the urine by several well-
marked characteristics. It is not coagulated by heat or by the addition of acetic
or warm nitric acid, and is very soluble in hot water. It is much less soluble in
cold water, but the presence of small amounts of salts seems to increase its solu-
bility here in marked degree.

In presence of albumin or globulin it can be found by the following process
unless it is in very small amount. The urine is saturated with pure sodium
chloride, which will precipitate albumose if present, and then enough acetic acid
is added to give a very strong acid reaction. The mixture is boiled and filtered
hot. This treatment throws out both albumin and globulin, and redissolves a pre-
cipitate of albumose which may have formed. The latter would therefore be found
in the clear filtrate and sometimes in amount sufficient to precipitate as this cools.
The filtrate should therefore be allowed to remain at rest until quite cool. If much
albumose is present it will appear as a white cloud. Sometimes, however, it will
be necessary to concentrate the filtrate before looking for this reaction, and this
is done by evaporating slowly on the water-bath to half the volume. On now
cooling, salt will quickly settle out while the albumose precipitates later in floc-
culent form. After the salt has separated the liquid may be tested by the biuret
reaction, described in earlier chapters.

Another test is this : Separate the albumin and globulin by boiling with a small
amount of acetic acid without the salt. Filter while warm and concentrate the
filtrate to a volume of one-third. Allow to cool thoroughly and add a large excess
of saturated solution of ammonium sulphate. This gives a white flocculent pre-
cipitate of albumose, if present. The precipitate can be collected on a filter and
washed with the saturated ammonium sulphate solution and then dissolved in a
little distilled water, poured on the filter. This filtrate gives tests with picric acid,
potassium ferrocyanide and acetic acid, and other albumin reagents.

If the original urine shows no reactions for albumin or globulin the albumose
tests can be applied directly after concentration. The method by precipitation by
means of picric acid gives good results. The biuret test is also delicate if the
urine is clear and of light color.

THE TESTS FOR SO-CALLED MUCIN.

Much confusion exists at the present time as to the exact nature' of the protein
bodies in the urine which have long been known as mucins. The true mucins
belong to the group of gluco-proteids and are seldom found in the urine, but
certain other protein bodies are practically always present normally, and with
them several acid substances which, under the proper conditions, are able to
precipitate these normal traces of protein. Among these acids chondroitin-sulphuric
acid is the most important, and in acetic acid solution it combines with and pre-
cipitates the normal protein. Sometimes the trace of protein normally present is
so small that no precipitate forms on slightly acidifying. In such cases the

SOME PRACTICAL URINE TESTS. 3 17

addition of a little albumin solution to the acidified urine usually produces a pre-
cipitate, as the albumin-precipitating agent is ordinarily present in sufficient amount.

The kind of albumin which may be present and give this reaction is not clearly
defined. In some cases it appears to be a nucleo-albumin, and occasionally a gluco-
proteid, which yields a reducing substance on boiling with acids. In working with
a large volume of urine it is sometimes possible to separate from the so-called
mucin precipitate a reducing substance suggesting the gluco-proteid; in other
cases this precipitate may be free from sulphate or reducing substance, but contain
a phosphate-yielding complex which can point to a nucleo-albumin or nucleo-proteid.
These reactions are too complex for ordinary clinical demonstration.

Acetic Acid Test. The addition of an excess of acetic acid, that is enough
to give a strong acid reaction and make up about 0.2 per cent, of the whole volume,
produces a flocculent precipitate in presence of the " mucin " substance. Heat is
not applied in this test, and its delicacy may be increased by dilution of the urine.

Citric Acid Test. In this test a layer of the urine is poured over a concentrated
solution of citric acid. The "mucin" appears as a cloud at the junction of the
two liquids.

In pouring urine over nitric acid a mucin cloud or band sometimes makes its
appearance about a centimeter above the junction point. In this test albumin
shows as a cloud at the junction point. In these acid tests the precipitation of
uric acid is prevented by the dilution with two or three volumes of water.

THE TESTS FOR SUGARS.

Normal urine contains a trace of carbohydrate, probably glucose, which may
be recognized by very delicate tests. Pathologically glucose, and sometimes other
sugars, occur in quantity and may be identified by a great number of tests. We
have to distinguish then, between what is known as a physiological glycosuria,
and a pathological condition most commonly characteristic of diabetes mellitus.
In the normal urine the trace of sugar may not be over one-twentieth of one per
cent., while in advanced diabetes it may amount to five per cent., or even more,
with a volume of five or six liters daily.

The most convenient of our sugar tests are based on the fact that the usually-
occurring glucose is an aldehyde body and responds to certain characteristic tests.
Fructose, sometimes present, is a ketone sugar, and responds to the same tests in
general. Among these we have, first, the so-called reduction tests, the best of
which are as follows :

Moore's Test. This depends on the reaction between reducing sugars and strong
alkali solutions. When a solution of sugar or diabetic urine is mixed, without
heating, with a solution of sodium or potassium hydroxide, no change is at first
apparent unless the amount of sugar present is large or the alkali very strong.
But on application of heat, even with weak sugar solutions, a yellow color soon
appears which grows darker, becoming yellowish brown, brown, and finally almost
black, while an odor of caramel is quite apparent. The strong alkali-sugar solu-
tion absorbs atmospheric oxygen, giving rise to a number of products among which
lactic acid, formic acid, pyrocatechol and others have been recognized. The brown
color is due to other unknown decomposition products.

This is a good reaction for all but traces of sugar, as the intense dark brown
color and strong odor are not given by other substances liable to be present in
urine.

But traces of sugar cannot be recognized by this test with certainty, as the
color of normal urine even is darkened to some extent by the action of alkalies.

Urine containing much mucin becomes perceptibly darker when heated with
sodium, potassium, or calcium hydroxide solutions.

3.1 8 PHYSIOLOGICAL CHEMISTRY.

The Trommer Test. This is one of the oldest and best known of the tests
for the recognition of sugar in urine, and is a typical reduction test. It is per-
formed by adding to the urine an equal volume of 10 per cent, solution of sodium
or potassium hydroxide and then a very few drops (three or four to begin with)
of dilute solution of copper sulphate as described in Chapter III.

The test must be used with certain precautions. If albumin is present it must
be coagulated and filtered out. The amount of copper sulphate used must be small,
because if only a trace of sugar is present and much copper is used the latter will
give a blue precipitate which can not redissolve, and which turns black on boiling,
thus obscuring a sugar reaction which may be given at the same time. In mak-
ing this test if a black precipitate is thus found it must be repeated, using less of
the copper.

The active body in producing the reaction is copper hydroxide, but this must
be in solution to act as a good oxidizing agent with sugar; and the test, therefore,
becomes uncertain or unsatisfactory if so much copper is added that the hydroxide
formed cannot be dissolved by the sugar which may be present. In doubtful
cases it becomes necessary to make several trials before the right proportion between
urine, alkali, and copper solution is found. In the solution, on completion of the
reaction, several oxidation products of sugar are found, among which are formic
acid, oxalic acid, tartronic acid, etc. But the complete reaction is obscure. In
order to avoid the indicated uncertainty of the Trommer test when used for small
amounts of sugar the next one was proposed.

The Fehling Solution Test. Fehling suggested the use of a solution containing
along with the copper sulphate and alkali a tartrate to dissolve the copper hydroxide
formed by the first two. Many substances besides sugars have the power of dis-
solving copper hydroxide with a deep blue color. Among these may be mentioned
tartaric acid and the tartrates, glycerol, mannitol and others of less value.

A solution prepared by mixing certain quantities of alkali, copper sulphate, and
either one of these bodies, with water in definite proportions, remains perfectly
clear when boiled. But if a trace of glucose (or several other sugars) is present
the usual yellow precipitate forms.

In performing the test with the Fehling solution described in Chapter III three
or four cubic centimeters of the solution are poured into a test-tube, diluted with
an equal volume of water, and boiled. The solution must remain clear. Then the
urine is poured in, a few drops at a time, and the mixture is boiled. If sugar is
present in the urine in an amount above one-fifth of one per cent, it should show
at once with this treatment. If but a trace of sugar is present, more urine must
be added and the boiling repeated.

When normal urine is heated with Fehling solution a greenish flocculent pre-
cipitate usually makes its appearance. This has no significance, as it is due to the
phosphates normally present, which come down when the reaction is made alka-
line. Many urines produce a clear dark green solution when heated with the Feh-
ling solution. This is a partial reduction reaction and like the other has no spe-
cial importance, as urines free, from sugar give it. At other times urines free
from sugar yield an almost colorless mixture when boiled with the Fehling solu-
tion. These peculiar reduction effects are due to the presence of uric acid, crea-
tinine, pyrocatechol and several other substances and are generally characterized by
discharge of the deep blue color of the solution without precipitation of the copper
suboxide. Certain substances taken as remedies give rise to products in the urine
which exert a similar action. Occasionally, however, the amount of uric acid is so
large that the reduction is accompanied by actual precipitation of the copper as
red oxide. This fact is of interest as it makes the test, at times, somewhat uncertain,
but it is a very simple matter to determine whether or not a great excess of uric

SOME PRACTICAL URINE TESTS. 3 1 9

acid is present, as will be pointed out later. The liability to error in the Trommer
test from these causes is less than in the Fehling test, but notwithstanding this
the latter must still be regarded as the better test practically, because of its great
convenience and the sharpness of the reaction with even traces of sugar. The
ingredients of the Fehling test are best kept in separate bottles closed with rubber
stoppers, as the mixed solution does not keep well, as ordinarily prepared. It is
customary to keep the copper sulphate in one bottle and the alkali and tartrate in
another.

Several other copper test solutions are in use; the Loewe solution contains
glycerol in place of sodium-potassium tartrate of the Fehling solution, while man-
nitol is employed for the same purpose in the Schmiedeberg solution. The reduc-
tion is the same as in the Fehling test.

The Bismuth Test. Bottger found (1856) that in the presence of alkali, bis-
muth subnitrate is reduced to the metallic condition by the action of glucose in
hot solution. As a urine test he recommended to make it strongly alkaline with
sodium carbonate, and then add a very small amount, what can be held on the
point of a penknife, of the pure bismuth subnitrate. On boiling the mixture the
insoluble bismuth compound, which settles to the bottom, turns dark if sugar is
present.

The test is at present carried out by adding to the urine in a test-tube an equal
volume of 10 per cent, solution of sodium or potassium hydroxide, and then the
subnitrate. Boiling gives the reaction as before. In absence of sugar (or albu-
min) the bismuth compound remains white.

In performing this test only a very small amount of the subnitrate should be
taken. This is absolutely necessary in the detection of traces of sugar. In this
case the reduction is but slight, and not much black powder of bismuth or its
oxide can be formed. If a great excess of the white subnitrate is taken it may
be sufficient to completely obscure the reduction product. It is frequently well to
use not more than four or five milligrams of the subnitrate.

The black precipitate formed was at one time supposed to be finely divided
metallic bismuth. Later investigations seem to show that it consists essentially
of lower oxides of bismuth. This test has certain advantages over the copper
tests. It is easily made, and with materials everywhere obtainable in condition of
sufficient purity. Furthermore, the reaction is not given with uric acid, which it
will be remembered may act on the Fehling solution if excessive.

Albumin, however, interferes with the test, as it gives, also, a black precipitate
when boiled with alkali and the bismuth subnitrate. In this case the albumin
gives up sulphur and forms bismuth sulphide ; if it is present in a urine it should
be coagulated and filtered out before trying the test.

Fallacies in the Reduction Tests. It has been shown above that several bodies
normally found in urine are able to reduce the alkaline copper solutions. Some
of these interfere with the bismuth reactions also, but not to the same degree; but
attention must be called to another source of error which is very important. In
warm weather it is often desirable to add something to urine to prevent its rapid
decomposition, and several substances have been suggested for the purpose. The
best known are chloral, chloroform, salicylic acid, phenol, and formaldehyde. Un-
fortunately all of these except phenol have a rather marked action on the copper
solutions. As a preservative phenol is objectionable from other standpoints. Urine
intended for sugar tests should be tested as soon as possible after collection, and
no foreign substances should be added as a preservative. Neglect of this very
simple and obvious precaution has caused many serious blunders, especially in the
examination of urine from applicants for life insurance.

The Phenylhydrazine Test. — In this test a reaction discovered some years ago

320 PHYSIOLOGICAL CHEMISTRY.

has been applied by v. Jaksch to the examination of urine. Add to about 10 cc.
of urine 0.2 gram of phenylhydrazine hydrochloride and a slightly greater amount
of sodium acetate. Warm the mixture gently, and if solution does not take place
add half the volume of water and heat half an hour on the water-bath. Then cool
the test-tube by placing it in cold water and allow it to stand. If sugar is present
a yellow precipitate settles out, which consists of minute needles generally arranged
in rosettes, visible under the microscope. Albumin does not obscure this test, but
if much is present it is best to coagulate it as well as possible by heating, and then
filter. The yellow precipitate is phenyl-glucosozone. The reaction is not much used
in urine analysis, but is of great value in the identification of sugar under other
conditions.

The Fermentation Test. — When yeast is added to urine containing sugar and
the mixture left in a moderately warm place the usual fermentation soon begins
which is shown by two principal changes. Carbon dioxide is given off, which may
be collected and identified, and the mixture becomes lighter in specific gravity.
When only traces of sugar are present the test by collection and identification of
the carbon dioxide frequently fails because of the solubility of the gas in the liquid.

The variation in the specific gravity is an indication of greater value, as it can
be readily observed with proper appliances. The test has practical value, however,
only as a confirmation of some other one. If by the copper solutions, for instance,
a strong indication is obtained which it is suspected may be due to an excess of uric
acid, the reaction by fermentation may be resorted to because only sugar will
respond to it.

The Amount of Sugar. — It is not always sufficient to be able to detect the pres-
ence of sugar in urine. A knowledge of the amount is frequently of the greatest
importance. A number of methods have been proposed by which a quantitative
determination can be made, some of them crude and of little practical value, while
others give, when properly carried out, results which are accurate. The methods in
general may be divided into four groups, depending on the

(1) Reduction of solutions of heavy metals, and measurement of the amount of
reduction.

(2) Change of color produced in organic solutions, by action of sugar, the depth
of final color being proportional to the amount of sugar.

(3) Results of fermentation, with measurement of change in specific gravity of
the urine, or measurement of evolved carbon dioxide.

(4) Observation of rotary polarization of light.

The reduction methods, which alone need be considered here, are illustrated in
the use of the Fehling solution as a qualitative test and in the bismuth tests.
The general principles involved in making a quantitative determination of sugar by
aid of the Fehling solution are the same as those involved in making other volu-
metric analyses with standard solutions, and are fully explained in Chapter III.
A measured volume of the properly prepared Fehling solution is poured into a flask
and brought to the boiling point. Then from a burette the urine is run in slowly,
a few cubic centimeters at a time, until the deep blue of the copper solution is just
discharged, leaving a pale yellow. At this stage the copper is all reduced to the
condition of insoluble red oxide, Cu20, and the volume of urine added from the
burette contains the amount of sugar measured by the oxidizing power of the
Fehling solution taken. The details of the titration are as follows : Use the stand-
ard quantitative Fehling solution as described, and dilute an accurately measured
volume with exactly 4 volumes of water. That is, 50 cc. should be diluted to 250
cc. or 25 cc. to 125. One cubic centimeter of this diluted liquid will oxidize almost
exactly one milligram of glucose, as was shown, provided the sugar is in approxi-

SOME PRACTICAL URINE TESTS. 321

mately i per cent, solution. For all practical purposes of urine analysis the oxidiz-
ing power may be considered the same in a solution of one-half per cent strength,
and only very slightly increased in still weaker solutions. Therefore, before begin-
ning the test dilute the urine, if necessary, accurately with four or nine volumes
of water. This can be done by making 50 cc. up to 250 or to 500 cc. and mixing
well by shaking.

With the diluted urine so prepared proceed as follows : Measure out 50 cc. of the
dilute Fehling solution, pour it in a flask and heat to boiling on gauze. Fill a
burette with the diluted urine and when the solution in the flask is actively boiling
run in about 3 cc. Boil two minutes, remove the lamp, and wait half a minute to
observe the color. If blue is still visible, heat to boiling again and run in 3 cc. more.
After boiling two minutes as before, wait a short time and observe the color near
the surface of the liquid in the flask. If still blue repeat these operations until
on waiting it is found that the blue has given place to a yellow. The urine should
be so dilute that at least 10 cc. must be run in to reduce all the copper hydroxide.

When the volume required is found to within 2 or 3 cc. a second experiment must
be made, the urine being added very gradually now, without interrupting the boil-
ing longer than necessary, until the first of the limits between which the correct
result must lie, as shown by the former test, is reached. From this point the
addition of the urine is continued, with frequent pauses for observation of color,
until the reduction is complete. The volume of urine used contains 50 milligrams
of sugar.

If the preliminary experiment shows that the urine is strong in sugar and that
the reduction is easy, that is, that the cuprous oxide separates and settles readily,
the second test may advantageously be made with 50 cc. of a stronger Fehling solu-
tion. With many strong diabetic urines it is possible to use the undiluted copper
solution with the oxidizing power of 4.75 milligrams of sugar to each cubic centi-
meter. The difficulties in this test have been very much overestimated ; with a little
practice any one can make a good sugar determination in urine. The important
point is to find by a few simple preliminary tests the best conditions of dilution of
Fehling solution and urine to give a precipitate which settles readily. With this
information, and it can be acquired in a few minutes, the actual quantitative experi-
ment can be easily made.

As an illustration of the calculations involved let it be assumed that 50 cc. of
the dilute Fehling solution is reduced by 11 cc. of urine. Each cubic centimeter
of the urine must therefore contain 4.54 milligrams of sugar. If the urine was
undiluted this corresponds to 4.54 grams to the liter. If it had been diluted with
9 volumes of water the result must be multiplied by 10, giving as the original
strength 45.4 grams per liter. If the specific gravity of the urine were found to be
1.032, the percentage strength would be

4-54
1.032

Method by Use of Ammoniacal Copper Solution. This is described fully in
Chapter III. The solution can be used with dilute urines only, as it has about
one-fifth of the oxidizing value of the standard Fehling solution. For urine work
one cc. is considered the equivalent of one milligram of glucose.

The great advantage in the use of this solution rests in the fact that the end point
is easily recognized by the disappearance of color of the standard solution when
the copper is all reduced. Cuprous oxide; dissolves in ammonia without color while
the slightest trace of cupric oxide leaves a marked blue in the liquid. To use
this solution with advantage the student should learn to judge, from the results of
22

322 PHYSIOLOGICAL CHEMISTRY.

a qualitative test, about how far the urine should be diluted. A very strong dia-
betic urine can be accurately titrated only after marked dilution.

Other Sugars in Urine. Besides the glucose other sugars are occasionally found
in the urine. Fructose and lactose have been frequently described, and also a
pentose. The identification of these sugars calls for certain precautions which can
not be detailed here. The significance of the pentose has been the subject of much
discussion. Fructose is found, as a rule, only when glucose is present.

In addition to the sugars a few other bodies are occasionally found in urine
which exhibit some of the reduction and similar reactions. One of the most char-
acteristic of these is glucoronic acid, C6H10O„ which in structure bears some rela-
tion to glucose. The acid is a strong reducing agent, reacts with phenylhydrazine
and possesses a marked dextro-rotating power. When distilled with hydrochloric
acid it yields f urf urol, recognized by its reaction with aniline. In urine the glucoronic
acid usually occurs in ester-like combinations with phenols and other bodies.
These exhibit a levo-rotation, which changes to the other direction on boiling the
urine with a little acid. This optical property is of importance in the recognition
of the acid.

ACETONE, ACETOACETIC ACID AND OXYBUTYRIC ACID.

The first of these is frequently found in urine in small amount. Indeed, it may
be true, as has been asserted, that it is normally always present in traces. This
physiological acetonuria has no clinical significance. Under some circumstances,
however, it may be found in larger quantity, sometimes in amount sufficient to be
detected by the odor alone, which fact first called attention to it. At one time it
was supposed to be related to the sugar found in urine, but it is now established that
it more generally accompanies albumin and is frequently observed in many febrile
conditions.

Acetone in urine is believed to be a decomposition product of albumins, or of
bodies which may in turn be looked upon as resulting from protein disintegration,
the fatty acids, for example. It has been shown that in health, even, it can be
much increased by a diet rich in nitrogenous materials.

But, occurring as it does in fevers and in advanced stages of diabetes mellitus,
a certain interest attaches to its detection, and numerous methods have been pro-
posed by which it may be identified in small amount. Those which depend on its
direct recognition in the urine are mostly uncertain. It is always safer to distil
the liquid and apply the test to a portion of the distillate. Half a liter, or more,
of the urine is poured in a retort attached to a Liebig's condenser, and, after ad-
dition of a little phosphoric acid, is subjected to distillation, ioo cc. of distillate
will be enough. A portion of this can be taken for each test as follows :

Legal's Test. — Add to 25 cc. of the liquid a small amount of a fresh solution of
sodium nitroprusside, and a few drops of a strong potassium hydroxide solution.
If a ruby-red color appears which slowly gives place to yellow, and if the addition
of acetic acid changes this to purple or violet-red, the presence of acetone is
indicated.

Creatinine gives a ruby-red color as does acetone when the nitroprusside reaction
is directly applied to urine, but after adding acetic acid a green or blue color results.

Lieben's Test. — This depends on the production of iodoform, and is carried out
in this manner. To about 5 cc. of the distillate add a few drops of a solution
of iodine in potassium iodide (the "compound solution of iodine," Lugol's solution),
and then enough sodium hydroxide to make distinctly alkaline. Warm gently. If
acetone is present a yellowish white precipitate soon appears, which, on standing,
becomes crystalline and more deeply colored. The test is said to be sharper and
more characteristic if ammonia is used instead of the fixed alkali. The liquid is

SOME PRACTICAL URINE TESTS. 323

first made strongly alkaline with ammonia, and then the iodine solution is added
until the brownish precipitate formed at first dissolves very slowly. In a short
time the yellowish iodoform precipitate makes its appearance. A rough quantita-
tive measure of the amount of acetone present is given by noting the smallest
volume of the distillate with which a distinctive iodoform reaction can be seen.
It is said that 0.0001 milligram in 1 cc. can be detected; 0.5 milligram in 10 cc. can
be recognized by the nitroprusside reaction.

Acetoacetic Acid. This acid is frequently found with acetone in the urine of
fevers and diabetes, and also in many other pathological conditions, but its rela-
tion to these disorders is not clearly understood. While acetone may be normally
present, the acetoacetic acid is probably always pathological.

As it is extremely unstable tests for it must be made in the fresh urine. As
it yields acetone by decomposition, tests for this substance must be made first. If
these are negative there is no need in going further, but if they are positive the
following direct test may be made for the acid.

Ferric Chloride Test. Add this reagent, a few drops at a time, and look for
the formation of a reddish color. Ordinarily there can be nothing in the urine to
give a similar reaction, and the color test is a strong indication of the presence of
acetoacetic acid. But several coal tar products given as remedies furnish residues
in the urine which also give a red or purple color with ferric chloride when added.
To detect the acetoacetic acid with certainty under these conditions it is necessary
to proceed with greater care. To this end add to the urine, which should be fresh,
a few drops of ferric chloride or enough to precipitate the phosphates present.
Filter and add a little more of the chloride. A red color indicates the acid. Divide
the liquid into two portions ; boil one and allow the other to stand a day or more.
In the boiled portion the color due to acetoacetic acid should disappear within a
few minutes, while in the other it should remain about twenty-four hours.

Acidulate another portion of the urine with dilute sulphuric acid and extract it
with ether, which takes up acetoacetic acid. Remove the ethereal layer and shake
it with a very dilute aqueous solution of ferric chloride. The red color in the
new aqueous layer should appear as before and disappear on boiling, which be-
havior distinguishes the acid from other substances likely to be present.

^g-Oxybutyric Acid. The detection of this acid in the urine by chemical methods
is by no means simple, but if present in amounts not too minute it may be found
by the aid of the polariscope, inasmuch as its solutions possess a strong negative
rotation. In dilute solution the specific rotation is approximately [oi]d = — 23.40.
As this acid is found associated with sugar in diabetes it is necessary to destroy the
sugar by fermentation before making the test. Amounts as high as 200 grams in
the day's urine have been reported, but usually, where present at all, the amount is
far below this, 15 to 20 grams being nearer the average.

If the urine does not give a test for acetoacetic acid it is useless to look for the
/3-oxybutyric acid. To test for the latter evaporate about 50 cc. of the urine, freed
from sugar if necessary, to a syrup and distil the residue, mixed with an equal
volume of strong sulphuric acid, from a small flask. Collect the distillate without
a cooler in a test-tube. If the oxybutyric acid was present in the urine the dis-
tillate will contain a-crotonic acid, which on strong cooling yields a crystal mass
melting at about 72° C. To get characteristic crystals it may be necessary to
extract the distillate with ether and allow this to evaporate.

NORMAL COLORING-MATTERS.

Although many investigations have been carried out on the subject of the normal
urinary pigments we are yet unable to give a very definite account concerning them.
This is partly due to the fact that the coloring substances exist in the urine in

324 PHYSIOLOGICAL CHEMISTRY.

minute traces only, which makes their separation and recognition exceedingly diffi-
cult, and partly to another fact that some of them are easily altered or destroyed
by the action of the reagents employed in their investigation. By proceeding accord-
ing to different methods, physiologists have obtained very different results indicating
the existence of several colors, or at any rate modifications of colors. The diffi-
culty of detecting the normal colors in urine is sometimes increased by the pres-
ence of traces of accidental coloring-matters having their origin in peculiar or
unusual articles of food consumed. Some of these will be referred to below.

It seems to be settled, however, that in health not merely one but several coloring-
bodies must be present. It has not yet been found possible to separate these in the
free state.

Uroerythrin is the name given by Thudichum and others to a common reddish
coloring-matter which often precipitates with urates and other substances. It is
colored green by solution of potassium hydroxide, but the color is not restored by
addition of acid.

Urochrome. This seems to be the most important and characteristic coloring
matter of normal urine. It is responsible for the ordinary yellow color of the
secretion, and its decomposition products appear on treatment of the urine with
strong acids.

Urobilin is a coloring matter found in much smaller amount than the last one.
When separated it is a reddish brown amorphous substance. In the urine the fol-
lowing test is sufficient for identification. Precipitate 200 cc. of urine with basic
lead acetate, collect the precipitate on a filter, wash it with water and dry it, and
then wash with alcohol. Finally, digest with alcohol containing a little sulphuric
acid, and filter. The filtrate is usually fluorescent. Make it strongly alkaline with
ammonia, and add solution of zinc chloride. This will give the fluorescence re-
ferred to above if but little is added, while if an excess of the zinc chloride is
added, a reddish precipitate falls.

Urophain. This is the name given by Heller to a substance identical with, or
similar to, urobilin. Heller gives this test: Take a few cubic centimeters of strong
sulphuric acid in a conical glass and pour on it, drop by drop, about twice as much
urine. As the two mix, a deep garnet-red is produced.

This reaction is not, however, characteristic, as several other matters may give it.

Urohematin is the name given by Harley to a coloring-matter similar to the
above. He applies this test: Dilute or concentrate the urine so that it is equivalent
to 1,800 cc. for the twenty-four hours. Take a few cubic centimeters in a test-tube
or wine-glass, and add one-fourth of its volume of strong nitric acid. No change
of color can be observed if the urohematin is present in normal amount, but if
in excess various shades from pink to red may appear.

Indican. This is a normal constituent of urine in small amount, but in many
intestinal diseases may be greatly increased. Its detection in the urine is then a
matter of considerable clinical importance. It is formed from the oxidation of
indol which is a common product of bacterial origin in the intestines. The oxida-
tion product, indoxyl, combines with sulphuric acid and the potassium salt of this
is excreted as indican. The formula of the indican is KS04C8H6N. Through fur-
ther oxidation this yields indigotin, a blue coloring-matter which is the substance
finally identified in the test. For further relations consult earlier chapters.

Indican is found in normal urines in very small amount only. It may, under
favorable circumstances, be detected as here given : Take about 4 cc. of pure hydro-
chloric acid in a test-tube and add about half as much urine, shaking well. A
blue or violet color shows indican. This test depends on the conversion of the
indoxyl compound into indigo, but the oxidizing action of the acid is not always
strong enough to bring about the change in presence of other organic bodies in the
urine.

SOME PRACTICAL URINE TESTS. 325

A more generally applicable method is this : To 10 cc. of urine and the same
volume of strong pure hydrochloric acid, add 2 or 3 cc. of chloroform. Then
add, drop by drop, solution of sodium hypochlorite, shaking after each addition.
The hypochlorite acts as an oxidizing agent, liberating the coloring-matter, which
is then taken up by the chloroform. The oxidation must not be carried too far;
that is, too much hypochlorite must not be added, as it would then destroy the color
as fast as formed. In fact, small traces of the product sought might be completely
overlooked in the process, as the hypochlorite is a very active oxidizer, the effect
going far beyond the production of indigo. It has therefore been proposed to use
nitric acid as the oxidizing agent. The urine is boiled with an equal volume of
hydrochloric acid and then a few drops of nitric acid are added. The mixture is
cooled and to it a little chloroform is added and well shaken. In presence of indigo
the blue color appears. Bromine water in small amount may be employed in the
same manner.

More recently hydrochloric acid containing a little ferric chloride has come into
use as an oxidizing agent. This is less destructive than the nitric acid or hypo-
chlorite. The reagent may be made by dissolving 3 or 4 grams of the ferric chloride
in a liter of strong hydrochloric acid. It is known as the Obermeyer reagent.

As a comparative quantitative test the following may be employed : To a hundredth
part of the whole day's excretion add an equal volume of the above reagent and
5 cc. of chloroform. Shake a minute or more, and allow to settle. Compare the
color with that of a strong Fehling solution, taken as 100, as a standard.

ABNORMAL COLORS.

We have here a number of pathological substances which are best recognized
through certain color reactions. These substances may not have much significance
in themselves but they are often important in pointing to certain conditions, indi-
rectly.

The- Bile Pigments. Several coloring-matters originating in the liver may find
their way into the urine. Their recognition is frequently important, and several
reactions are available for this.

Biliary urine has generally a characteristic greenish yellow color sometimes tinged
with brown. The froth from such urine is readily recognized by its yellow color,
which is often a sufficient test in itself. Among the chemical tests the following are
the best known.

Gmelin's Test. This is easily performed and depends on the oxidation of bili-
rubin, the pigment commonly present in fresh jaundice urine, by nitrous acid. Pour
in a test-tube about 5 cc. of the urine under examination and by means of a pipette
introduce below it an equal volume of strong nitric acid mixed with nitrous. This
should be carefully done so as to avoid mixing the liquids much. At the junction
of the two liquids, if bile is present, several colored rings appear of which the green
due to biliverdin is most characteristic. The bands or rings appear above the acid
in this order, yellowish red, red, violet, blue, and green. The last is essential. It
must be remembered that nitric acid gives the other colors at times with urine free
from bile, but green is characteristic of the latter.

Fleischl modified this test by mixing the urine with a strong solution of sodium
nitrate and then adding strong sulphuric acid carefully. This settles below the
urine and decomposes the nitrate at the point of contact, liberating the necessary
nitric and nitrous acids for the oxidation as before. This method is a very good one.

Trousseau's Test. Add to some urine in a test-tube a few drops of tincture
of iodine, allowing this to float over the urine. If the bile pigments are present a
greenish color appear! at the junction of the two liquids and may remain some
hours. An excess of iodine must not be used.

326 PHYSIOLOGICAL CHEMISTRY.

The Diazo Reaction. Ehrlich and others have called attention to the behavior
of many urines with solution of diazobenzene sulphonic acid, which often has im-
portance in diagnosis. Normal urine treated with a weak solution of this reagent
shows no marked change, but in several pathological conditions after adding the
acid and saturating with ammonia a deep carmine or scarlet-red color appears, fol-
lowed by greenish or violet precipitation. After the addition of ammonia the foam
should show a distinct pink color.

This reaction depends on the combination of the sulphonic acid of diazobenzene
with some aromatic compound found in the urine in pathological condition. At one
time it was supposed to have special significance in the diagnosis of typhoid fever,
but it now appears that in many diseases of the intestinal tract the urine receives
traces of complex aromatic products of bacterial origin which respond to the test.
It has, therefore, general rather than special significance.

As the diazobenzene sulphonic acid is not very stable, it is not convenient to use,
and a reagent is made which, in its application, is its chemical equivalent. The
reagent is prepared by dissolving 1 gram of sulphanilic acid in 200 cc. of water
with the addition of 10 cc. of pure hydrochloric acid. Another solution is made by
dissolving 1 gram of sodium nitrite in 200 cc. of water. To make the test take 50
cc. of the first solution, add 5 cc. of the nitrite solution and then 50 cc. of urine.
Ammonia is then added in sufficient quantity to impart a strong alkaline reaction
after thoroughly shaking. A scarlet-red color is the result if the urine in question
contains the abnormal products referred to.

It must be remembered that normal urines usually give some color. It is only
the strong reactions which have significance.

Ehrlich has introduced another test which discloses the presence of products of
intestinal origin. This is a 2 per cent solution of dimethyl amino benzaldehyde in
15 per cent hydrochloric acid. A few drops of this reagent added to 5 cc. of urine
strikes a red color, weak in normal urine, but deep in certain pathological urines.
The exact significance of the reaction is not known.

BLOOD COLORING MATTERS.

These may appear in the urine from several sources. We may have color due to
the presence of corpuscles themselves and we may have the color due to dissolved
hemoglobin. It is important to distinguish between these conditions. The presence
of the corpuscles, which are best recognized by the microscope, points to a lesion of
the kidney or some part of the urinary tract, and to a fresh lesion if the corpuscles
are sharp in outline. The term hematuria is applied to the condition when cor-
puscles themselves are present, while hemoglobinuria is the condition in which few
or no corpuscles are found, but the free pigment is present. Hemoglobinuria is the
result of the disintegration of corpuscles within the tissues or blood vessels, per-
mitting the oxyhemoglobin or some derivative to pass through the kidney with the
urine. With much of the coloring matter present, the urine may have a very dark
color. Sometimes the hemoglobin suffers further decomposition, giving rise to
hematin and methemoglobin.

The following are the best chemical tests 'for the recognition of these bodies :

Heller's Test. Treat the urine with solution of sodium or potassium hydrox-
ide, and heat to boiling. This produces a precipitate of the earthy phosphates which
in subsiding carry down coloring-matters. If a precipitate does not separate readily
it may be hastened by adding two or three drops of magnesia mixture. Hemo-
globin, when present, is decomposed by this treatment with separation of hematin,
which in turn settles down with the phosphates, imparting a red color to the
precipitate.

Struve's Test. Make the urine slightly alkaline with sodium hydroxide solu-

SOME PRACTICAL URINE TESTS. 327

tion, and then add enough solution of tannic acid in acetic acid to change the re-
action. If hemoglobin is present a dark brown precipitate of hematin tannate
settles out. The test is a good one, and easily performed, but is not sufficiently
delicate for the detection of small traces of hemoglobin directly. By collecting
the precipitate on a filter and washing it, it may be used for two confirmatory tests.
One of these depends on the formation of hemin crystals and is made in this
manner: Place a small portion of the precipitate on a microscopic glass slide and
add a minute crystal of sodium chloride. Then add a large drop of glacial acetic
acid and cover with a cover glass. Warm very gently over a small flame. When
the acid, salt, and precipitate have become thoroughly mixed allow the slide to cool.
Small rhombic crystals of hemin should now appear, which are best seen under a
microscope.

The washed precipitate may also be ashed and used for an iron test. The ash
should be dissolved in a little pure hydrochloric acid in a porcelain dish and tested
by the addition of potassium ferrocyanide and ferricyanide to give the well-known
reaction. This test presupposes purity and freedom from traces of iron in the
reagents used.

Almen's Guaiacum Test. In a test-tube mix equal volumes of fresh tincture
of guaiacum and ozonized turpentine — 2 or 3 cc. of each will suffice. The mixture,
if made of proper materials, must not show a green or blue color after thoroughly
shaking. Now add a few cubic centimeters of the urine to be tested, a drop at a
time, and agitate after each addition. If hemoglobin is present it causes the oxidiz-
ing material of the ozonized turpentine (probably hydrogen peroxide) to act on the
precipitated guaiacum resin, imparting to it first a greenish, and finally a blue color.
Old and alkaline urine must be made faintly acid before performing the test. Pus
in the urine gives a somewhat similar reaction, and a few other bodies, very seldom
present, interfere. The test is very delicate, and if it gives a negative result it is
safe to conclude that blood is absent.

Vegetable and Other Colors. It has long been known that many peculiar col-
oring-matters enter the urine from substances taken as remedies, or with the food.
Santonin from wormseed, chrysophanic acid from some kinds of rhubarb, the
coloring substances from blueberries, carrots and other fruits and vegetables, are
all occasionally met with in the urine, where they may lead to confusion. In most
cases the addition of acid produces a yellowish tinge, while alkalies give rise to a
red color.

Many aromatic compounds given as remedies pass into the urine in small amount,
where they may often be recognized by the peculiar color reactions they yield with
certain reagents. Salicylic acid is an illustration, and it may be recognized by the
blue color it strikes on addition of ferric chloride.

TOTAL NITROGEN.

In all complete examinations of the urine a determination of the total nitrogen
present is necessary. This may be easily made by means of the simple Kjeldahl
method, as follows :

Measure accurately 5 cc. of urine into a large Kjeldahl digesting flask and add
25 cc. of pure sulphuric acid and 10 grams of pure potassium sulphate. Heat over
a free flame about an hour, or until the mixture has become perfectly colorless.
Complete hydrolysis is effected and the nitrogen is all left as ammonium sulphate.
Cool the flask, dilute largely with distilled water, neutralize with an excess of pure
alkali solution and distill off the ammonia.

This ammonia is caught in 25 cc. of N/4 sulphuric acid, which is titrated after-
wards with corresponding alkali, using alizarin red or methyl orange as indicator.
The results are accurate.

328 PHYSIOLOGICAL CHEMISTRY.

The total nitrogen determination should always be made as a check on the sum
of the nitrogen factors found in the following paragraphs. It will be remembered
that the urea nitrogen makes up a large fraction of the total nitrogen.

UREA.

The physiological importance of urea was discussed in the last chapter. As it
makes up a large fraction of the nitrogen excretion, which in turn varies with the
protein of the diet, the total amount excreted may vary between a few grams and
fifty grams daily. By bacterial agency it is rather quickly converted into ammonium
carbonate in the voided urine. Quantitative tests should be made, therefore, on
fresh urine only.

Recognition of Urea. Because of its extreme solubility urea cannot be easily
obtained by evaporation of urine. It may be shown, however, by a simple experi-
ment that by concentrating the urine slowly to a small volume — to one-third or
one-fourth — cooling and adding strong nitric acid, a crystalline precipitate of plates
of urea nitrate separates, which is characteristic. From this precipitate pure urea
can be obtained.

Clinically, this test has no importance, as we are concerned only with a measure-
ment of the amount of urea. This determination can be made in several ways,
but in actual practice we employ three essentially different methods. The first
depends on the fact that solutions of urea precipitate solutions of certain metals in
a definite manner, from which a volumetric process has been derived. The second
depends on the fact that solutions of certain oxidizing agents decompose solutions
of urea with the liberation of its nitrogen (and carbon dioxide) in gaseous form.
From the known relations between weight and volume of the gas, and weight of
nitrogen and weight of urea, the absolute amount of the latter may be calculated.
The third method depends on the fact that when the urea of urine is heated in
solution under certain conditions it is converted quantitatively into ammonium salts
from which the ammonia may be distilled and measured.

In the practice of the first method a standard solution of mercuric nitrate is
employed, as this precipitates urea quantitatively, but as the reaction is complicated
by the presence of other bodies it is seldom used practically at this time. This is
the old Liebig method. Ordinarily for clinical purposes we measure urea by the
second process; that is from the nitrogen liberated in some oxidation reaction, as
will now be described.

Method by Liberation of Nitrogen. A solution of urea is decomposed by a
solution of a hypochlorite or hypobromite as illustrated by this equation.

CON,H4 + 3NaOCl = C02 + N2 + 2H20 + 3NaCl.

That is, nitrogen and carbon dioxide gases are given off. If the reaction is
allowed to take place in an alkaline medium the carbon dioxide will be held and
the nitrogen alone given off. The volume liberated is a measure of the weight of
urea decomposed. From the above equation it is seen that 28 parts by weight of
nitrogen correspond to 60 of urea, from which it follows that 1 cc. of pure nitro-
gen gas, measured at a temperature of o° C. and under the normal pressure of 760
mm. corresponds to 0.00269 gram of urea. One gram of urea furnishes 371.4 cc.
of nitrogen gas.

In employing these principles in practice it is simply necessary to bring together
a measured volume of the urine or urea solution and the hypochlorite or hypo-
bromite reagent under such conditions that all of the nitrogen liberated may be
collected and accurately measured.

As a reagent, a solution of sodium hypobromite is very commonly employed.
As it does not keep well it must be made fresh for use, which is inconvenient

SOME PRACTICAL URINE TESTS.

329

unless many tests have to be made at one time. The reagent may be prepared
in this manner:

Dissolve 100 grams of good sodium hydroxide in 250 cc. of water. When cold
add 25 cc. of bromine by means of a funnel tube carried to the center of the solu-
tion. The bromine must be poured into the funnel a little at a time, with constant
agitation, the containing vessel being kept cold. There is an excess of alkali
here to hold the carbon dioxide liberated.

A very convenient form of apparatus used in this test is shown in the annexed
cut.

The tall jar is filled with water which must stand until it has the air tempera-
ture. A 50 cc. burette is inverted in the jar, the
delivery end being connected with a bottle holding
about 150 cc, by means of a piece of firm rubber
tubing. The rubber tube is slipped over a short
glass tube passing through the hole in a rubber
stopper which must close the bottle accurately.
In the bottle is a short stout test-tube, or vial,
holding about 10 cc. and which contains the urine
to be tested. Into the bottle itself is poured the
reagent as above described, which must not reach
to the top of the test-tube. On mixing the liquids
the urea decomposes, liberating the gas as ex-
plained, which passes through the rubber tube and
displaces water in the burette so that the volume
can be readily determined.

The test is made practically in this manner :
Pour about 20 cc. of the strong hypobromite or 40
cc. of the weaker hypochlorite reagent into the
bottle. With a pipette measure some exact vol-
ume of urine, usually 5 cc, into the small test-
tube, and by means of small iron forceps place
the latter carefully in the bottle containing the
reagent. Insert the stopper which connects the
bottle with the burette standing in the jar of
water. Allow the apparatus to stand about ten
minutes to get the proper air temperature, and
then adjust the levels of the liquid in the burette
and jar. Note the reading in the burette, and the

temperature. Incline the bottle to mix the urine and the reagent, and shake to com-
plete the reaction. The liberated nitrogen, or its equivalent volume of air, passes
over into the burette and depresses the water. When there is no change in the gas
volume allow the whole apparatus to stand until the contents of the bottle and
burette have cooled down to the air temperature again. Then lift the burette,
with the clamp as before, to restore the levels and read the gas volume. From this
subtract the volume at the first reading. The difference is the volume of nitro-
gen gas liberated in the reaction, at the observed temperature and atmospheric
pressure. If, as sometimes happens, more gas is liberated than the burette will
hold, repeat the experiment, using urine diluted with an equal volume of water.

Reduce the gas volume to standard conditions by the usual formulas, and for
each cubic centimeter calculate 2.7 milligrams of urea as present in the volume
taken.

Exact investigations have shown that the whole of the nitrogen is not liberated
in the reaction, as at first assumed, but falls short between 7 and 8 per cent. It

330 PHYSIOLOGICAL CHEMISTRY.

appears that under some conditions a small part, possibly 3 or 4 per cent., of the
nitrogen of the urea is oxidized to nitric acid in the reaction and escapes meas-
urement. Another small portion is left in the ammoniacal condition. Attempts
have been made to prevent this abnormal oxidation by adding to the urine a re-
ducing agent to destroy nitric acid, as fast as formed. Dextrose has been used
for the purpose, also cane-sugar, and apparently with success. But a great excess
of sugar must be added. The results are therefore only approximately correct.

Doremus has introduced a small apparatus for the quick and approximate esti-
mation of urea in which 1 cc. of urine is used. This apparatus is often employed
clinically. The same reagent is used.

In the more exact methods of estimation ammonia is formed by hydrolysis re-
actions, usually aided by hydrochloric acid. Folin has simplified these methods
greatly.

The Folin Method. This conversion is accomplished by boiling the urine in a
flask with magnesium chloride and hydrochloric acid, the boiling point of the mix-
ture being high enough to effect the hydration. The determination is carried out
practically as follows : Measure accurately 5 cc. of urine into a 200 cc. Erlenmeyer
flask, add 5 cc. of strong hydrochloric acid and 20 gm. of crystallized magnesium
chloride. Add also a small piece of paraffin, about half a gram, and a few drops
of alizarin red as indicator. Attach a small reflux condenser to the flask, or the
special condensing bulb tube, which Folin recommends, and heat to boiling. Con-
tinue the ebullition until the drops returning from the condenser produce a sharp
sound when they strike the liquid, now concentrated, remaining in the flask. After
this the boiling may be continued more gently through 45 minutes or an hour. But
the reaction in the flask must remain strongly acid, as shown by the indicator. The
condensing bulb permits the return of evaporated acid, as necessary. Then the
mixture is diluted with water, washed into a liter flask with water enough to give
a volume of 700 cc, made alkaline with 10 cc. of 20 per cent, sodium hydroxide and
distilled. This carries over the ammonia which is best caught in N/10 sulphuric
acid, of which about 50 cc. should be taken. At the end of the distillation, which is
carried on nearly to dryness, the excess of acid is titrated back, and the difference
corresponds to the ammonia. For 17 milligrams of ammonia calculate 30 milli-
grams of urea.

As urine contains a little ammonia, this must be deducted in the final cal-
culation. It should be determined by a special process. The distillation of the
urea ammonia requires about an hour and in the final titration alizarin red is used
as indicator. Uric acid and creatinine are not appreciably attacked in this hydroly-
sis. With practice the method gives very good results. As much of the mag-
nesium chloride on the market contains traces of ammonia this must be separately
determined and subtracted from the amount found.

Benedict and Gephart have recently recommended a method in which the hydroly-
sis of the urea is effected by heating the urine with dilute hydrochloric acid in an
autoclave. 5 cc. of urine is mixed with 5 cc. of 8 to 10 per cent acid in a test-
tube, which is heated an hour and a half to about 150° in the autoclave. Many
tubes may be heated at one time. The results are a trifle higher than in the
Folin method, as other constituents appear to be slightly hydrolyzed. The contents
of the test-tubes are diluted to about 400 cc, made alkaline, and distilled as in the
Folin method.

AMMONIA IN URINE.

The normal amount excreted varies usually between 500 milligrams and one gram
daily, and makes up about 5 per cent of the total nitrogenous excretion as shown
in the last chapter where, also, the conditions of formation are explained.

Determination of Ammonia. As ammonia is always present in urine in some

SOME PRACTICAL URINE TESTS. 331

amount, qualitative tests have little value, and we proceed immediately to quanti-
tative methods. As the ammonia present is in combination as a salt it must be
liberated by the action of a strong alkali, but in the choice of one for this pur-
pose we are limited by the fact that the hydroxides of sodium and potassium
have a decomposing action on urea and other nitrogenous bodies in urine and
cannot, therefore, be used. Milk of lime is free from this objection and may be
employed. The experiment is so arranged that the liberated ammonia may be
absorbed by a measured volume of standard sulphuric acid, the amount of this
neutralized being the measure of the ammonia absorbed:

H2S04 + 2NH3= (NH4)2S04.

98 parts by weight of the acid correspond to 34 parts of ammonia, NH3. The
old Schloesing method is based on these principles. But it is by no means as
accurate or convenient as the later processes in which the ammonia is drawn out
from a measured volume of urine by an air current or through the aid of a vacuum
pump, absorbed in standard acid, and so titrated. One of the best of these absorp-
tion processes is the following.

The Folin Ammonia Determination. In this method 25 cc. of urine is meas-
ured into a tall narrow cylinder with about a gram of dry sodium carbonate.
Enough light petroleum is poured into the cylinder to form a layer about 5 mm.
deep. This is to diminish the frothing in the following operation. The tall
cylinder, furnished with a doubly perforated stopper, is connected with an absorp-
tion apparatus to wash the air current to be drawn through, and free it from
ammonia, and is followed by another absorption apparatus containing a measured
volume of N/10 sulphuric acid. The system is connected with a large Chapman
pump, capable of drawing several hundred liters of air through in an hour. With
a sufficiently rapid air current bubbling through the urine, the ammonia may be
completely drawn out in an hour and a half or two hours, and absorbed in the
standard acid, of which 25 cc. should be taken and diluted to 200 cc. with water.
To aid in the absorption of the ammonia carried along by the air current Folin
suggests a special bulb tube through which the air must pass into the standard acid.
It is also well to put a Reitmair or Hopkins bulb over the tall cylinder holding
the urine, to catch any liquid which may be carried up by the froth produced by the
rapid air current. The time required in the determination depends on the air cur-
rent available, and this should be found by a few preliminary experiments. The
results are extremely exact.

With a good air current it is possible to work six or eight of these combinations
in series, and complete the tests in the same time. One protecting washing flask
at the end of the series is sufficient for the whole, and the tube from this leads to
the bottom of the liquid in the first urine cylinder. The cylinder may be 30 to
36 cm. high and 4 or 5 cm. wide, with advantage.

URIC ACID.

The relations of uric acid to the whole nitrogen excretion were explained in
the last chapter. The normal variations are between about 0.2 gm. and 1.2 gm. daily.

In the recognition of the acid the following points may be noted: When present
in large amount it frequently precipitates from the urine in free form, or as an acid
urate ; in both cases the precipitate is yellow. When the amount present is small it
may be found by acidifying with hydrochloric acid and then allowing the urine to
stand some hours in a cool place; uric acid crystals separate. In mixed sediments
it may \><-. recognized by this test:

Murexid Test. Throw the sediment on a filter and wash once with water.
Place the residue in a porcelain dish, add a drop of strong nitric acid, and evaporate

332 PHYSIOLOGICAL CHEMISTRY.

to dryness on the water-bath. A yellow or brown mass is obtained, and this
touched with a drop of ammonia water turns purple.

Unless the uric acid or urate is present in the sediment in fine granular form
its recognition by the microscope is very simple. Illustrations of the forms of uric
acid and certain urates are given in the paragraphs on the sediments.

THE AMOUNT OF URIC ACID.

For determination of the amount of the acid in the urine we have the choice
of several methods, not one of which is very convenient or of the greatest accuracy.
The first of these depends on the fact referred to above, that hydrochloric acid
liberates uric acid from its combination, precipitating it in crystalline form.

Precipitation Test. Measure out 200 cc. of urine and add to it 20 cc. of strong
hydrochloric acid. Mix thoroughly and set aside in a cool place for about forty-
eight hours. At the end of this time collect the reddish-yellow deposit on a
weighed filter, wash it with a little cold water, dry, and weigh. Not over 30 or 40
cc. of water should be used in the washing. The precipitated uric acid is not pure,
holding coloring and other substances which increase its weight. On the other
hand, it is soluble to some extent in cold acidulated water so that not the whole
of it is obtained on the filter and a correction must be made. It is usually recom-
mended to add to the weight obtained 4.8 mg. for each 100 cc. of filtrate and
washings.

If the urine under examination contains albumin, the latter must be coagu-
lated by heating with a drop or two of acetic acid and filtered out, before the test
is made. If the urine is very cold to begin with and has a sediment of urates, the
latter must be brought into solution by warming before beginning the test. To
prevent precipitation of phosphates during the warming a few drops of hydro-
chloric acid may be added. This method gives only approximate results.

Ammonium Sulphate Precipitation. It has been found that the addition of
certain ammonium salts to urine produces a precipitate of ammonium urate which
is practically complete. Fokker and Hopkins recommended the chloride. Folin sug-
gested the sulphate, which possesses some advantages, as follows :

A special precipitating mixture is employed which in 1000 cc. contains 500 gm.
of ammonium sulphate, 5 gm. of uranium acetate, 6 cc. of absolute acetic acid and
water to make 1 liter. Measure out 300 cc. of the urine and add 75 cc. of the above
reagent. Mix well and allow to stand five minutes. A precipitate containing phos-
phate and some protein substance, which would interfere with subsequent work if
left, separates. Filter off two portions of 125 cc. each, equivalent to 100 cc. of the
original urine, and to each add 5 cc. of strong ammonia. Allow the mixtures to
stand 24 hours, in which time a complete precipitation of ammonium urate takes
place. Collect the precipitates on a small filter and wash practically free from
chlorine by aid of 10 per cent, ammonium sulphate solution. Next dissolve the
precipitates in 100 cc. of hot distilled water, add 15 cc. of pure strong sulphuric
acid, and while still hot titrate the mixture with N/20 potassium permanganate
solution, each cubic centimeter of which oxidizes 0.00375 gm- of uric acid. A re-
duction of the reagent, with loss of color, follows. When the uric acid is fully
oxidized a further addition of permanganate leaves a pink tinge in the liquid. The
addition of the standard reagent from the burette should cease as soon as a pink
tinge is reached, which is permanent two seconds after good shaking. By waiting a
longer interval the color fades and more solution must be added from the burette.
If the reaction is stopped with the first decided tinge obtained, as explained, for
each cubic centimeter of permanganate used from the burette, 3.75 milligrams of
uric acid may be calculated as present. In illustration, suppose we start with 300
cc. of urine and precipitate, wash and dissolve as described. If now we run 12.5 cc.

SOME PRACTICAL URINE TESTS. 333

of the twentieth normal permanganate solution into the hot uric acid solution to
obtain the pink color, the amount of this acid present is 12.5 X 3-75 — 46.87 milli-
grams in the 100 cc. As ammonium urate is slightly soluble in the mother liquor,
a small correction must be added. This amounts to 3 milligrams for each 100
cubic centimeters of the urine carried through to the titration. Urine contains
traces of other bodies which are precipitated with the uric acid, but in amount so
small that their effect may be practically neglected. The duplicates should agree
closely.

THE PURINE BODIES.

These compounds were discussed in the last chapter. Leaving uric acid out
of consideration the amount present in urine is not large, but because of their
relations to certain diets a determination is often a matter of importance. It is
not practically possible to make an accurate separation of the individual purines,
and this is, besides, not necessary. Several methods have been suggested for the
group precipitation, and of these the following gives good results and is generally
employed.

This method depends on the precipitation of the purine bodies as cuprous
salts, and may be carried out in this way : measure out 200 cc. of urine into a large
casserole, slightly acidify with acetic acid, add 10 grams of sodium acetate and
boil about a minute. Add 50 cc. of saturated sodium bisulphite solution, 40 cc. of
10 per cent copper sulphate solution, boil again and filter. Wash the precipitate
with hot water on the filter. Return the precipitate to the same casserole by aid
of a stream of hot water, add 25 cc. of strong solution of sodium sulphide, and then
enough acetic acid to give a distinct acid reaction. Boil five or six minutes to
expel the liberated hydrogen sulphide, filter and wash the precipitated copper
sulphide with plenty of hot water. Save the filtrate and washings in the same casse-
role in which the process was begun. This solution contains an approximately
pure mixture of the purines, free from other nitrogenous bodies of the
original urine. By repeating the precipitation the traces of foreign substances
may be removed. Therefore add to the liquid in the casserole copper sulphate and
the bisulphite solutions as before, boil several minutes and filter. Wash the1 copper
salts thoroughly with hot water, and finally work down into the bottom of the
filter, which should not be large. Allow the precipitate to drain thoroughly, and
throw it, with the filter paper, into a Kjeldahl flask for determination of nitrogen
by the process already given. It may be well to add 35 to 40 cc. of strong sul-
phuric acid, 15 gm. of potassium sulphate, about half a gram of copper sulphate
and some small flakes of feather tin to aid in the oxidation, which under these
conditions follows easily. A clear solution is obtained in less than one hour. This
is neutralized with pure alkali and distilled as before into 50 cc. of N/4 sulphuric
acid.

In the process as outlined the uric acid is precipitated with the other purines,
and the nitrogen determined is the nitrogen of all the purines, including the uric
acid. Therefore, the uric acid nitrogen, as determined above, must be subtracted
from this result to find the true purine nitrogen. It is absolutely essential to
employ a fresh acid sulphite solution in the original precipitation process in order
to secure a proper reduction and precipitation of the cuprous salts. The acid sul-
phite solution may be made by leading a good current of sulphurous oxide (from
copper and sulphuric acid) into sodium hydroxide solution until saturation is
reached.

CREATININE.

This product, having the formula GH7N.O, occurs normally in urine and is
excreted to the amount of 1.5 to 2 grams daily. It is, therefore, more abundant
than uric acid, as already pointed out. As it is readily soluble in water and acids

334 PHYSIOLOGICAL CHEMISTRY.

it escapes detection, except when looked for by special reagents. In weak solu-
tions it is precipitated by phosphotungstic acid, phosphomolybdic acid, and especially
by solutions of several heavy metallic salts. The precipitate given with a neutral
solution of zinc chloride is the most characteristic. It gives certain color reactions
also. The following test may be applied to urine. If acetone is present it must be
expelled by heat. To about 25 cc. of this urine add half a cubic centimeter of a
dilute solution of sodium nitroprusside made alkaline with caustic soda. With
this the urine gives a ruby-red color, fading to yellow. Then add acetic acid in
slight excess and warm. A green color soon appears, deepening finally to blue.

With picric acid creatinine gives a very characteristic reaction, suggested by
Jafre. Add to the urine an aqueous solution of picric acid and a few cc. of dilute
sodium hydroxide solution. A deep red color is formed almost immediately, which
persists a long time. The reaction is characteristic, as other bodies in the urine
do not give it. Folin has made the reaction the basis of a quantitative colorimetric
test as follows :

Determination of Creatinine. This is accomplished by aid of a colorimeter
in which the color of the above Jafre reaction is compared with the color from a
known creatinine solution, or from a standard dichromate solution. Folin suggests
the Duboscq colorimeter for the purpose, but other forms may be used, and employs
a half-normal potassium dichromate solution, with 24.54 grams to the liter as the
color standard.

Measure 10 cc. of urine into a 500 cc. flask, add 15 cc. of saturated picric acid
solution and 5 cc. of 10 per cent, sodium hydroxide solution. Allow the mixture
to stand 5 or 6 minutes and dilute to the mark with distilled water. Meanwhile
pour dichromate solution into one of the cylinders of the colorimeter and adjust
to a depth of exactly 8 millimeters. Pour some of the urine solution into the
other cylinder and adjust the depth until the shades are the same, leaving the dichro-
mate side at 8 millimeters. An exact reading of the depth of the urine-picric acid
layer is noted, and a calculation of the strength is made from this basis : Folin found
that 10 mg. of pure creatinine in 500 cc, under the same conditions, gave such a
color that a layer of 8.1 mm. was equivalent to the 8 mm. of the dichromate. The
concentration of the solution is inversely proportional to the depth of layer re-
quired to match the constant standard. Suppose this is a millimeters. Then
10 X (8.1/a) = x. If, for example, we read off a depth of 7.5 mm., x= 10.8 mg.
of creatinine in the dilute solution. If the calculation shows over 15 mg. or below 5
mg. it is necessary to make a new dilution with a smaller or larger volume of urine,
to get the most accurate results, as the comparison is based on a mean content of
10 mg. of creatinine to 500 cc. after the dilution. The colorimetric reading should
always be made without delay.

Creatine may be determined in the same manner after conversion into crea-
tinine by prolonged boiling with dilute hydrochloric acid, and subsequent neutra-
lization.

HIPPURIC ACID.

The amount of this acid in the urine is not large, ordinarily, but may be
increased by the consumption of certain fruits and vegetables, especially by those
containing benzoic acid. The methods of determination proposed are not very
exact. The following is the best one.

Measure out 200 to 300 cc. of urine, make it alkaline with sodium carbonate
and filter. Evaporate the filtrate nearly to dryness and extract it four or five times
by shaking with alcohol. Unite the alcoholic extracts and distill off all the alcohol.
The aqueous residue is acidified with hydrochloric acid and extracted by shaking
with five or six portions of pure acetic ether. The hippuric acid is dissolved in
this way, and the united extracts are partially purified by washing with a little

SOME PRACTICAL URINE TESTS. 335

water. The acetic ether is evaporated to dryness at a moderate temperature. A
residue of hippuric acid with traces of other substances is left. Most of these
impurities may be removed by washing this residue with light petroleum ether, in
which fats, benzoic acid and certain other things are soluble.

The hippuric acid left after this treatment may be still further purified by
dissolving it in a little hot water, heating with well-burned animal charcoal, filtering
hot and evaporating the colorless filtrate slowly to dryness. A crystalline residue
should now be obtained. When properly carried out 90 per cent of the hippuric
acid in the urine may be removed by this general method.

THE PHOSPHATES IN URINE.

Phosphoric acid occurs normally in the urine combined with alkali and alkali-
earth metals, of which combinations the alkali phosphates are soluble in water,
while the earthy phosphates are insoluble. In the urine, however, they are held
in solution through several agencies. The larger part of the earthy phosphates
appear to be held here normally in the acid condition; that is, as compounds of
the formulas CaH^PCX)* and MgH«(PO*)a. The salts of the type CaHPO* are
present, also, in small amount. As long as the urine maintains its acid reaction
these bodies may be expected to remain in solution, but if it becomes alkaline by
fermentation, or by the addition of the hydroxides or carbonates of ammonium,
sodium, or potassium, the acid phosphates are converted into insoluble, neutral
phosphates and precipitated. Most urines contain along with the acid phosphates
'traces of neutral phosphates which precipitate on boiling. It has been suggested
that these phosphates are held by traces of ammonium compounds or by carbonic
acid, both of which are driven off by heat, allowing the phosphates to precipitate.
It is well known, however, that some urines can be boiled without showing any
sign of precipitation. In such cases it is probable that the neutral phosphates are
not present.

Part of the phosphoric acid of the urine comes directly from the phosphates
of the food and another portion results from the oxidation of the phosphorus-
holding tissues. In health, the rate of such oxidation is practically constant, or
nearly so, but in disease it may be greatly increased or diminished. Variations in
the amount of excreted phosphates may therefore become of considerable clinical
importance.

Various statements are found in the books regarding the mean excretion of
the alkali and earthy phosphates. Different observers have reported between 2 and
5 grams of phosphoric anhydride (P2O0), while 3 grams may be taken, perhaps as
the mean.

The recognition of the phosphates is an extremely easy matter. The presence
of earthy phosphates may be shown by adding to the urine enough ammonia water
to give a faint alkaline reaction and then warming. A flocculent precipitate, re-
sembling albumin, appears and is usually white, or nearly so. But sometimes color-
ing-matters come down with it in amount sufficient to give it a brownish or reddish
shade. It will be recalled that the color of this precipitate was referred to under
the head of blood tests.

The alkali phosphates can be detected in the filtrate after separation of the
earthy phosphates. To this end, add to the clear alkaline liquid a little more am-
monia and some clear magnesia mixture. A fine crystalline precipitate of am-
monium-magnesium phosphate separates and settles rapidly. This is very char-
acteristic. The qualitative tests for phosphates have, however, little value in ex-
amination of the urine. We are chiefly concerned with the amount, the measure-
ment of which will now be described.

Determination of Phosphates. It is customary to measure the total phos-

336 PHYSIOLOGICAL CHEMISTRY.

phoric acid, not the alkali or earthy phosphates, separately. We have at our dis-
posal several methods, gravimetric and volumetric, of which the latter are accurate
and most convenient. A volumetric process will be described which serves for the
measurement of the phosphoric acid as a whole, and which can be used for the
separate measurement of the earthy and alkali phosphates by dealing with the pre-
cipitate and filtrate described in the qualitative test above. This method depends
on the fact that solutions of uranium nitrate or acetate precipitate phosphates in
greenish-yellow colored, flocculent form, and that in a solution holding in sus-
pension a precipitate of uranium phosphate any excess of soluble uranium com-
pound may be recognized by the reddish brown precipitate which it gives with a
solution of potassium ferrocyanide. The latter substance serves, therefore, as
an indicator. If to a phosphate solution in a beaker a dilute uranium solution be
added precipitation continues until the whole of the phosphates have gone into
combination with the uranium. If, during the precipitation, drops of liquid from
the beaker are brought in contact with drops of fresh ferrocyanide solution on a
glass plate, no reddish brown precipitate of uranium ferrocyanide appears until the
last trace of uranium phosphate has been formed. The production of uranium
ferrocyanide is the indication, therefore, of the finished precipitation of the
phosphate.
The reaction between uranium and phosphates is shown by the equation :

U02(N03)2 + KH2P04 = U02HP04 + KN03 + HN03

From this it follows that 238.5 parts of uranium are required for 71 parts of*
P205. In order to have the reaction take place as above it is necessary to neutralize
the nitric acid as fast as formed, or dispose of it in some other manner. The best
plan is to add to the solution some sodium acetate and acetic acid. The latter
brings the phosphates into the form of acid salts while the acetate decomposes
with formation of sodium nitrate and free acetic acid, which does not interfere
with the reaction.

We need the following reagents :

(a) Standard Uranium Solution. This is made by dissolving 36 gm. of the
pure crystallized nitrate, U02(N03)2-6H20, in water to make one liter.
The solution is standardized as below :

(b) Standard Phosphate Solution. This is made by dissolving 10.087 gm. of
pure crystals of sodium phosphate, HNa2P04i2H20, to make one liter.
50 cc. of the solution contains 100 mg. of P205.

(c) Sodium Acetate Solution. Dissolve 100 grams in 800 cc. of distilled
water, add 100 cc. of 30 per cent, acetic acid and then water enough to
make one liter.

(d) Fresh Ferrocyanide Solution. Dissolve 10 grams of pure potassium
ferrocyanide in 100 cc. of distilled water. The solution should be kept in
the dark.

The actual value of the uranium solution is determined by the following ex-
periment. Measure out 50 cc. of the phosphate solution, (&), add 5 cc. of the acet-
ate solution, (c), and heat in a beaker in a water-bath to near the boiling temper-
ature. Place several drops of the ferrocyanide solution on a white plate. Fill a
burette with the uranium solution and when the solution in the beaker has reached
the proper temperature run into it from the burette 18 cc. of the uranium standard.
Warm again, and by means of a glass rod bring a drop of the liquid in the beaker
in contact with one of the ferrocyanide drops on the plate. If the uranium solu-
tion has been properly made no red color should yet appear. Now run in a fifth of
1 cc. more from the burette, warm and test again, and repeat these operations until

SOME PRACTICAL URINE TESTS. 337

the first faint reddish shade begins to show on bringing the two drops in contact.
With this test as a preliminary one make a second, adding at first one-fifth of a
cubic centimeter less than the final result of the preliminary, and finish as before.
Something less than 20 cc. should be needed to complete the reaction. Supposing
19.8 cc. are required for the purpose, the whole solution should be diluted in the
proportion,

19.8 : 20 : : a : x

in which a represents the volume on hand. Each cubic centimeter precipitates
exactly 5 mg. of P205. The liter contains 35.38 gm. of the true uranium nitrate,
which, if the salt were absolutely pure, could be weighed out directly.

The test of the urine is made exactly as above. Measure out 50 cc, add 5 cc.
of the acetate mixture and finish as before. The 50 cc. of urine, in the mean
contains about as much phosphoric acid as was present in the same volume of
standard phosphate solution. The titration must be made hot, because the reaction
is much quicker and sharper in hot solution than in cold. Make always two tests;
the first is an approximation, while the second gives a much closer result.

A separate test of the earthy phosphates may be made by adding to 200 cc. of
urine enough ammonia to give an alkaline reaction. The urine then must stand
until the precipitated phosphates settle out. The precipitate is collected on a
small filter, washed with water containing a very little ammonia, and then allowed
to drain. It is next dissolved in a small amount of acetic acid, the solution
diluted to 50 cc, mixed with 5 cc. of the sodium acetate solution and titrated as
before. The reaction here is not quite as accurate as with the alkali phosphate,
but the results are satisfactory for the purpose. The difference between the total
phosphates and the earthy phosphates, expressed in terms of P205, is the amount
combined as alkali phosphates.

It is also possible to determine the amount of phosphoric acid combined as
monohydrogen salt and that combined as dihydrogen salt, but the determination
has at the present time little clinical value.

Instead of finding the end point in the precipitation with uranium solution by
means of drops of ferrocyanide as explained, the following process may be fol-
lowed. Add to the urine the sodium acetate as before and then three or four
drops of tincture of cochineal. Heat to boiling and add the uranium solution to
the hot liquid. Just as soon as the phosphate is combined and a trace of uranium
left in excess it produces a green color or precipitate with the cochineal, which
thus serves as an indicator to show the end of the reaction. If the urine is quite
warm the color is sharp.

THE CHLORIDES IN URINE.

Practically all the chlorine consumed with the food, mainly in common salt,
is eliminated in the urine. The excretion, therefore, varies within wide limits in
different individuals, but in the mean amounts to 10 or 15 gm. daily of the salt.
A large increase in excreted chlorine points merely to increased consumption, but
a marked decrease may point to one of several pathological conditions. Quantita-
tive tests have sometimes considerable value, and are easily made through the
reaction between silver nitrate and a chloride, by a volumetric process.

Determination of Chlorine. The reaction between nitrate and chloride is
expressed as follows :

NaCl -J- AgNO, = AgCl + NaNO,

from which it appears that 5.85 mg. of sodium chloride require for precipitation
16.997 rng- of silver nitrate. A standard silver solution may be made then of this

23

338 PHYSIOLOGICAL CHEMISTRY.

strength, by dissolving 16.997 gm- of the pure fused nitrate to make 1 liter with
distilled water.

In one method of determination 10 cc. of urine is evaporated in a platinum
or porcelain dish to dryness, after mixing with 2 gm. of potassium nitrate, and
1 gm. of sodium carbonate. The dry residue is carefully fused, and the resulting
mass dissolved in water. After exactly neutralizing with nitric acid, and adding
a little potassium chromate as indicator the chlorine is titrated in the usual manner.
But the process is not generally followed, being replaced by the next one.

Volhard's Method. We have here a method by which the chlorine in urine
can be quickly and accurately determined without fusion. The principle involved
in the process is this. If to a chloride solution a definite volume of standard silver
solution be added, and this in excess of that necessary to precipitate the chloride,
the amount of this excess can be found by another reaction, subtracted and leave
as the difference the volume actually needed for the chloride. The reaction for
the excess depends on these facts. A thiocyanate solution gives with silver nitrate
solution a white precipitate of silver thiocyanate, AgSCN. It also gives with a
ferric solution a deep red color due to the formation of soluble ferric thiocyanate,
FeS3(CN)3. If the silver and ferric solutions are mixed and the thiocyanate added
the second reaction does not begin until the first is completed; that is, the silver
must be first thrown down as white thiocyanate before a permanent red shade of
ferric thiocyanate appears. The presence of silver chloride interferes but slightly
with these reactions. Therefore, if we have a thiocyanate solution of definite
strength we can use it with the ferric indicator to measure the excess of silver
used after precipitating the chlorine solution.

The reaction between silver nitrate and a thiocyanate is expressed by the follow-
ing equation :

AgN03 + NH4SCN = AgSCN + NH4N03.

For 16.99 milligrams of the silver nitrate we use 7.6 milligrams of the thiocyanate.
In this method the standard solutions required are

(a) Standard Silver Nitrate Solution, N/10. — Made as before with 16.997
grams of the fused salt to the liter.

(b) Standard Thiocyanate Solution. Weigh out about 7.7 gm. of ammo-
nium thiocyanate and dissolve to make 1 liter. Adjust the exact strength
as below:

(c) Ferric Indicator. Use for this a strong solution of ferric alum, free
from chlorine.

To find the exact strength of the thiocyanate solution proceed as follows :
Measure into a flask or beaker 25 cc. of the N/10 silver nitrate, and add about
3 cc. of the strong ferric alum solution. Add also enough strong, pure nitric acid
to make a perfectly clear mixture, for which not over 2 or 3 cc. should be needed.
From a burette run in the thiocyanate solution a little at a time, shaking after each
addition. A red color appears temporarily, but vanishes on shaking. After a
time this color disappears very slowly, which shows that the end point is near.
The burette solution is therefore added more carefully, best by drops, until at
last a single drop is sufficient to give a permanent reddish tinge. Something less
than 25 cc. should be used for this. Repeat the test and if the same result is found
dilute the thiocyanate solution so as to make 25 cc. of the volume used in the titra-
tion. For instance, if 24.2 cc. were required 900 cc. of the solution may be diluted
in this proportion :

24.2 : 25 :: 900 : x .'.x = 929.8.

We have now a standard thiocyanate solution corresponding exactly to the

SOME PRACTICAL URINE TESTS. 339

silver solution. To test it further and illustrate its use with chlorides measure
out 25 cc. of an N/10 sodium chloride solution, very accurately prepared, and add
to it, from a burette, exactly 30 cc. of the silver nitrate solution, then the ferric
indicator and the nitric acid as given above. Shake the mixture and filter it
through a small filter into a clean flask or beaker. Wash out the vessel in which
the precipitate was made with about 20 cc. of pure water, pouring the washings
through the filter. Then wash the filter with about 20 cc. more of water, allowing
the washings to mix with the first filtrate. This mixed filtrate contains all the
silver used in excess of the chloride. Now bring it under the thiocyanate burette
and add this solution until a reddish tinge becomes permanent. Exactly 5 cc.
should be necessary for this.

The chlorides of the urine may be treated in about the same manner. To a
measured volume of the urine, usually 10 cc, an excess of silver nitrate solution
is added ; 25 cc. with most urines is enough, and then the indicator and acid.

But as the coloring matters in urine interfere somewhat with the sharpness
of the titration it is best to destroy them by partial oxidation with sodium peroxide
or potassium permanganate. The latter is preferable. To 10 cc. of urine add 3 cc.
of pure, strong nitric acid, then the ferric alum. Add also 3 or 4 drops of a
saturated, chlorine-free, solution of potassium permanganate. The red color which
forms at first, soon disappears. Then add 25 cc. of the N/10 silver nitrate, stir
up, filter and titrate the excess of silver with thiocyanate, as above. If the first
drops run in from the thiocyanate burette produce a red color it is evidence that
there is no silver in excess, and that the urine was very strong in chloride. In
this case start a new test with urine diluted one-half.

To illustrate the calculation, if we use 10 cc. of urine, 25 cc. of silver nitrate
and finally 3.4 cc. of thiocyanate, 25 — 3.4 = 21.6 cc, the amount of silver nitrate
actually needed for the chloride. Then, 21.6 X 3-55 = 76.68 mg., the amount of
chlorine in the urine. This is equivalent to 126.36 mg. of sodium chloride, or
12.636 gm. per liter.

THE TOTAL SULPHUR AND SULPHATES IN URINE.

It was shown in the last chapter that sulphur appears in the urine in the
ordinary mineral sulphates, in the organic or ethereal sulphates and in the so-called
neutral or unoxidized form. The total sulphur is determined by oxidizing every-
thing to the condition of mineral sulphate, and precipitating as barium sulphate.
As oxidizing agents which may be used with urine the following have been tried :
fuming nitric acid, sodium peroxide, potassium or other nitrate and chlorates.
The method given below, which was worked out by Benedict in the author's
laboratory yields good results.

Total Sulphur. An oxidizing reagent is made by dissolving 200 grams of
pure copper nitrate and 50 grams of potassium chlorate in water to make 1 liter.
To 10 cc. of urine in a small porcelain dish add 5 cc. of the above reagent and
evaporate to dryness over a low flame, then increase the heat gradually and bring
up to a high temperature with a good Bunsen flame. Continue the heat five to ten
minutes after the mass has fused and solidified. Finally allow the dish to cool,
add 10 cc. of 10 per cent hydrochloric acid, and warm until solution takes place.
Filter into a small Erlenmeyer flask, wash the filter thoroughly and make the
filtrate up to 150 cc. Add now, slowly, 10 cc. of 5 per cent barium chloride
solution, best drop by drop, -lir gently and allow to stand an hour. At the end of
this time collect the precipitate on a weighed Gooch crucible in the usual manner.
The increase in vreighl gives the barium sulphate from the total sulphur.

Total Sulphates. The method recommended by Folin gives uniform results.
It is as follows: Measure into an Erlenmeyer flask of 250 cc. capacity, 25 cc. of

340 ' PHYSIOLOGICAL CHEMISTRY.

urine and 20 cc. of 8 per cent hydrochloric acid. Boil gently half an hour, with
a watch glass or small beaker over the neck of the flask. At the end of this time
cool and dilute with cold water to 150 cc. Add 10 cc. of 5 per cent barium chloride
solution, without agitating more than is necessary to mix the liquids. If the
barium chloride is added very slowly from a dropping tube no further agitation
is necessary. At the end of about an hour filter through a weighed Gooch crucible
and wash with 250 cc. of cold water. Dry and weigh as usual.

In this method of determination the long boiling of the urine with the hydro-
chloric acid effects the splitting of the ethereal sulphates, so that in the following
barium chloride precipitation they come down with the inorganic sulphates. The
latter may be found separately, as recommended by Folin.

Inorganic Sulphates. Dilute 25 cc. of urine with 10 cc. of 8 per cent hydro-
chloric acid and water to make 150 cc. in an Erlenmeyer flask of 250 cc. capacity.
Add slowly, from a dropping pipette, 10 cc. of 5 per cent barium chloride solution,
without shaking. Allow the mixture to stand an hour and then filter through a
weighed Gooch crucible. Wash the precipitate with 250 cc. of cold water, dry and
ignite in such a manner that the barium sulphate is protected from reducing gases.

By the conditions of the precipitation the ethereal sulphates are not decom-
posed, as the liquid was not heated during the operation. From the weight of the
barium sulphate the sulphur should be calculated in each case for a given volume.
The difference between the two results is the sulphur in the form of ethereal
sulphates.

If the difference is taken between the sulphur of the "total sulphur " test and
the " total sulphate " test the result is the " neutral " or unoxidized sulphur.

THE SEDIMENT FROM URINE.

Urine is frequently cloudy when passed and on standing deposits a sediment
of the substances imparting the cloudiness. Other urines which may appear per-
fectly clear at first also throw down deposits after a time. This is always the
case with urine allowed to stand long enough to undergo alkaline fermentation,
when a precipitate of phosphates forms. The deposit is frequently caused by a
change of temperature. Warm voided urine holding an excess of urates may be
perfectly clear, but becomes cloudy as its temperature goes down with the forma-
tion of a light reddish sediment. This is a perfectly normal action, and indeed
most sediments may be considered in the same light. Urine containing a deposit
is not necessarily pathological.

There are conditions, however, in which the sediment is an indication of ab-
normality, and its examination becomes important clinically. Certain sediments
are pathological because of their origin, others because of their amount. For in-
stance, blood and pus corpuscles, casts of the uriniferous tubules of the kidney
and a few other forms are not found normally in urine, and their presence is of
importance, whether observed in large or small quantity. Sediments containing
phosphates, uric acid and urates, calcium oxalate and other salts, are common
enough and usually attract no attention, but if the amount of these deposits is
very large there may be attached to them clinical significance and they deserve
study.

In the examination of a sediment it is necessary to allow the urine to stand
long enough to deposit the important forms it may contain, which may require
twenty-four hours or more. For the deposition of a sediment the urine should be
left in a place with an even temperature, preferably not above 150 C. A low
temperature favors the precipitation of urates, while decomposition may begin if
the temperature be allowed to go up. Some of the light organic forms have a
specific gravity so little above that of the urine that they may remain a long time

SOME PRACTICAL URINE TESTS. 341

in suspension. It is important, therefore, to allow plenty of time for these to
settle. If the weather is warm and there is no good means at hand for keeping
the temperature of the urine down until the examination can be made, or if for
any reason this must be delayed for some days, it is well to add some preservative
to the urine; i. e., something to prevent fermentation. Many substances have
been suggested for this purpose, some of which are very objectionable inasmuch
as they form precipitates which often obscure what is sought for. Chloroform is
the simplest and at the same time one of the best substances which can be added.

To ioo cc. of the urine to be set aside for tests add three or four drops of
chloroform and dissolve by shaking. It is not well to add more than this, as there
is danger of leaving minute droplets undissolved, and these are confusing in the
subsequent examination. The chloroform may be applied in the form of aqueous
solution. Add about 10 grams of chloroform to a liter of distilled water and shake
thoroughly; about three-fourths will dissolve at the ordinary temperature; 25
cc. of this saturated solution may be added to 100 cc. of the urine to be examined,
which is then allowed to stand as before.

Recently, formaldehyde has come into use as a urine preservative and is applied
as is the chloroform. It must be remembered that both of these substances are
reducing agents, and therefore should not be used with urine to be tested for sugar.

All of these methods of preservation are unnecessary if a centrifuge is at hand,
with which a deposit from about 10 cc. of urine may be secured in a few minutes.
This plan is always preferable, and is now generally followed.

After the deposit has settled pour off the supernatant liquid very carefully and
by means of a small pipette with a coarse opening transfer one or two drops
to a perfectly clean glass slide. Clean a cover glass with great care and by means
of small brass forceps lower it on the drop of liquid in such a manner as to ex-
clude air bubbles. This can be done by lowering it inclined to the slide, not
parallel with it, so as to touch the liquid on one side first. In settling down, the
cover now pushes the air in front of it and gives a field generally free from bub-
bles. The slide is then examined under a microscope with a magnifying power of
250 to 300 diameters. Either natural or artificial light may be used, but it must not
be very bright. A very common mistake in the examination of urinary sediments
by the microscope is to employ so high a degree of illumination that the lighter
and nearly transparent bodies are completely overlooked.

Sediments from urine are commonly classed as organized and unorganized, these
divisions being then subdivided according to various plans. The important forms
under each division are shown in the following schemes :

Organized Sediments. Unorganized Sediments.

Blood corpuscles. Uric acid.

Mucus and pus corpuscles. Various urates.

Epithelium from various locations. Leucine and tyrosine.

.Mucin bands, or threads. Cystin.

Casts of the uriniferous tubules. Cholesterol.

Spermatozoa. Fat globules.

Fungi. Hippuric acid.

Certain other parasites. Calcium carbonate.

Calcium phosphate.
Calcium oxalate.
Magnesium phosphates.
In addition to these there are often found in the urine certain bodies whose
presence must be called accidental; for instance, hairs, fibers of cotton, silk or
wool, starch granules, bits of wood, mineral dust, etc. Some of these will be
referred to later.

342 PHYSIOLOGICAL CHEMISTRY.

ORGANIZED SEDIMENTS.

Blood Corpuscles. Urine containing blood presents a characteristic appearance
easily recognized, unless it be present in very small quantity. If the reaction of
the urine is acid the color is generally dark; but if alkaline the shade is inclined
to reddish. Blood corpuscles enter the urine from several different sources and
their presence is usually a pathological indication, but not always, as they may come,
for instance, from menstruation. The kidneys, or their pelves, the ureters, the
bladder, the urethra, the vagina, or the uterus may be the seat of the lesion from
which the blood starts, and its appearance sometimes gives a clue to its origin.

Fresh blood corpuscles are clear in outline and show distinctly their bicon-
cavity. But corpuscles which have been long in contact with the urine become
much swollen, less distinct in outline, often biconvex, or nearly spherical even,
and lighter in color. As long as the reaction of the urine is acid the corpuscles

'•-'"-> ® %®®o

J® * ° o 0

o°o o

Fig. 31, Human blood corpuscles, 400 diameters.

remain comparatively fresh in appearance, but with the beginning of the alkaline
reaction disintegration and loss of color soon set in.

The microscopic recognition of blood in urine is easy enough if it is not too
old. The fresh, red corpuscles of human blood have a mean diameter of about
0.0077 mm., but when swollen by absorption of water they are somewhat larger.
When seen on edge they appear as shown at the left in the figure above. If pre-
senting the flat side to the eye, they appear as disks whose centers grow alternately
light and dark by changing the focus of the instrument. In old urine, especially
with alkaline reaction, they appear as granulated spheres, shown at the left of
the figure. In all cases the color is more or less yellowish. It is generally assumed
that the paler washed-out corpuscles come from lesions higher up, from the pelvis,
or kidney even, while the brighter fresh blood suggests a lesion nearer the point
of discharge; that is, from the bladder or urethra. This is pretty certain to be
the case if the blood is discharged but little mixed with the urine and settles
rapidly as a distinct mass.

The crenated appearance of the corpucles, shown at the right and above in the
figure, is due to contact with a denser medium, to partial drying and sometimes
to certain pathological conditions, while the swollen appearance, from the absorp-
tion of water, is illustrated below.

Mucus and Pus Corpuscles. These are white corpuscles somewhat larger than
the red blood corpuscles and spherical in outline. The term leucocyte is frequently
applied to these as well as to the so-called white corpuscles of blood. Their size
varies greatly but the average diameter may be given as 0.009 mm- All these
corpuscles present, when fresh, a slightly granular appearance and occasionally
show one or more nuclei. The addition of a little acetic acid to the sediment

SOME PRACTICAL URINE TESTS. 343

brings the nucleus out distinctly so that it may be seen under the microscope as a
characteristic appearance.

Mucus corpuscles in small number are normally present in urine, but pus cor-
puscles enter the urine as a constituent of pus itself which is an albuminous product
discharged from suppurating surfaces and not normal. It has been pointed out
that the reactions of mucus and albumin are distinct, but urine containing pus
always affords reactions for albumin. Pus in urine tends to form a sediment
at the bottom of the containing vessel and may be recognized by the following
method :

Donne's Test. Pour the urine from the sediment and add to the latter an
equal volume of thick potassium hydroxide solution, or a small piece of the solid
potassa. Stir with a glass rod. The strong alkali converts the pus into a thick
viscid mass closely resembling white of egg. Sometimes this is so thick that the
test-tube containing it can be inverted without spilling it. In alkaline urine this
glairy mass is sometimes spontaneously formed.

The appearance of the mucus or pus corpuscles in urine depends largely on the
concentration of the latter. In urine of low specific gravity the corpuscles absorb
water and swell to larger size than normal, while in a highly concentrated urine
they may give out water and become reduced in size and shrunken in appearance.

To recognize them under the microscope transfer a few drops of the sediment
to a slide, and cover as usual. If the nuclei are not distinct place a drop of
diluted acetic acid on the slide at the edge of the cover glass. Part of the acid
will flow under the cover and mix with the urine. As it does this the clearing
up of the corpuscles, with appearance of the nuclei, can be very easily followed.
Urine containing much pus is white and milky. The same appearance is often
noticed with an excess of earthy phosphates, but the latter clear up with acids
while the pus does not.

Epithelium Cells. Epithelium cells from different sources may appear normally
in the urine, and the light cloud which separates from normal urine on standing
consists chiefly of these cells. When present in small amount this epithelium
has usually no clinical importance, as it easily finds its way into the urine from
the bladder, vagina, or urethra. An abundance of cells from these organs would,
however, be considered pathological, pointing to a catarrhal condition.

Unfortunately, it is not possible in all cases to determine the source of the cells,
as found in urine, partly because cells from different localities have frequently
the same general appearance, and partly because, owing to immersion in the urine,
they become greatly changed from what they are in the tissue as shown by the
microscopic study of sections. It is customary to make three rough divisions of
the cells as found in the urine:

i. Spherical cells. 2. Columnar or conical cells. 3. Flat or scaly cells.

The spherical cells are probably normally much flattened, but by absorption of
water they become swollen and globular. These cells may be derived from several
sources, as from the uriniferous tubules or from the deeper layers of the lining
membrane of the pelvis of the kidney, or the bladder, or the male urethra.

These cells have a well-defined nucleus resembling that of a pus cell. But they
are much larger, and besides show the nucleus without addition of acid. In
nephritis, or other structural diseases of the kidney, these round cells are found
along with albumin, and their recognition is then a matter of importance as
indicating a breaking down of the tubular walls. Sometimes these cells form a
variety of tube cast, to be described later. But it must be remembered that we
cannot distinguish with certainty between the cells from the tubules and those from
the other localities mentioned.

Conical cells come generally from the pelvis of the kidney, from the ureters

344 PHYSIOLOGICAL CHEMISTRY.

and urethra. Some of these cells are furnished with one or two processes, and are
broad in the middle and taper toward each end, while the others are broad at the
base and taper to a point.

The large flat cells come from the vagina or bladder, and it is generally im-
possible to distinguish between them. Sometimes they are very nearly circular,
sometimes irregularly polygonal in outline. Sometimes the vaginal epithelium is
found in layers of scales, which appear thicker and tougher than the cells from the
bladder, which occur singly.

' What was said about the decomposition of blood or pus cells in urine obtains
also for the various epithelium cells. In acid urine they may maintain their dis-
tinct outlines many days, but in alkaline secretion they soon undergo disintegra-

W^ ..,,». ^7^h \ *- "^ A-vw:-:? ^.m.

Ik ill fli \

'■L:~> '

•■•y^y

t

90 w%f^m

Fig. 32. Common forms of epithelium scales.

tion, which makes their recognition practically impossible. In general the greatest
importance attaches to the cells from the tubules of the kidney. The presence of
albumin in more than minute traces in the urine would suggest that any smaller
spherical cells present may have had their origin in the kidney rather than in the
bladder or male urethra. In general it may be said that urine containing large
numbers of the smaller, round tubule cells with albumin will also show casts.

Mucin Bands. Urine containing much mucus sometimes exhibits a deposit
consisting of long threads or bands, curved and bent in every direction. These
bands are important because they are sometimes confounded with the tube casts
to be described next. They can be produced in urine highly charged with mucus
by the addition of acids, and appear therefore sometimes spontaneously when the
urine becomes acid. These threads are sometimes covered with a fine deposit of
granular urates and then bear some resemblance to granular casts. In general,
however, they are relatively longer and narrower than the true casts of the
uriniferous tubules. The mucin threads can occur, and frequently do occur, in
urine entirely free from albumin, while true tube casts are usually associated with

SOME PRACTICAL URINE TESTS. 345

albumin, although not always, as will be explained below. The length and shape
of the mucin threads may generally be relied upon to distinguish them from
true casts.

Casts. The structures properly termed casts are seldom found in urine which
does not contain albumin. They are formed in the uriniferous tubules, and, to a
certain extent, are " casts " of portions of the same. Their specific gravity differs
but little from that of the urine, for which reason they remain long in suspension.
It is therefore necessary to allow the urine to stand some hours at rest, over
night or longer, before attempting an examination, if a centrifuge is not at hand.

True casts of the uriniferous tubules rarely appear in normal urine and their
recognition is therefore a matter of the highest importance in diagnosis. Much
has been written on the subject of the origin of these bodies in the kidney and
several theories have been advanced to account for their formation and chemical
constitution. Most of this discussion would be out of place in a work like the
present dealing mainly with questions of analysis, but enough will be given to aid
the student in his practical work. It must be said that few subjects are more per-
plexing to the beginner than that of their certain recognition, because of the fact
that some varieties are so transparent as to be almost invisible, while others are
closely resembled by formations of entirely different nature, not pathological.
With practice, however, these difficulties can be surmounted.

Most of the bodies termed casts are formed of organized structures or the
remains of such, but another and rather common form consists of crystalline mat-
ter, usually uric acid or fine granular urates.

These bunches of urates have no pathological significance and are of frequent
occurrence. Urine containing them clears up by heat, and the deposits themselves
are dissipated by weak alkali. While it is true that they resemble, to some degree,
the so-called granular casts referred to below, there are certain well-defined points
of difference. The bunches of urates lack the coherence which can be observed
in the true casts, and besides, the granulation is finer and more clearly defined.

The fact that mucin bands occasionally appear covered with a precipitate of
granular urates has been referred to. These aggregations are more compact than
the loose bunches of urates just mentioned and much longer generally. They are
also darker and therefore more easily seen than are the casts proper or the urates.

The true casts are made up of matter in which evidence of cell structure or
transformation is visible. An accurate classification of these bodies cannot yet be
made, and, as said, authors differ regarding the importance of several forms and
their origin. But for our purpose it will be sufficient to make the following rough
division, which accords in the main with what is found in the text-books of urine
analysis :

i. Blood casts. 4. Fatty casts.

2. Epithelium casts. 5. Waxy casts.

3. Granular casts. 6. Hyaline casts.

What are termed blood casts consist of or contain coagulated blood, recognized
by the corpuscles. Plugs of this coagulated matter are forced out from the tubules
by pressure from behind, and form one of the most characteristic varieties of casts.
They are generally very dark in color, and easily distinguished from other matter.
A representation of blood casts is given in the following cut.

In epithelium casts the characteristic substance is the lining epithelium of the
tubule. Sometimes this lining epithelium becomes detached in the form of a hol-
low cylinder, the walls consisting of the united cells. Again, the coagulated con-
tents of the tubule in passing out may carry the epithelium with it as a coating.
In either case a grave disorder of the kidneys is indicated, as acute nephritis, or

346 PHYSIOLOGICAL CHEMISTRY.

other disease in which a profound alteration of the internal structure of the organ
is involved.

What are termed granular casts, proper, appear in a variety of forms, produced
probably by the disintegration of blood or epithelium casts.

There is no uniformity in the fineness of the granulation; sometimes a high
amplification is necessary to disclose the structure. Occasionally blood corpuscles,
epithelium, fat globules and crystals can be detected in them, and when derived
from blood cast disintegration they usually have a yellowish red color, which
makes their recognition comparatively easy. In outline they are generally regular,
with rounded ends, one of which is somewhat pointed. Frequently, however, they
appear to be broken, the ends showing irregular fracture.

Fatty casts contain oil drops produced by some variety of fatty degeneration

Fig. 33. Blood casts and granular casts.

of the tissues of the kidney. These oil drops may form coherent bunches, or they
may be held by patches of epithelium. It also happens that epithelium or granular
casts may be partially covered by oil drops. The name, fatty cast, is applied to
those in which the fat globules predominate. Along with these globules the
microscope sometimes shows crystals of free fatty acids, and probably also of
soaps containing calcium and magnesium.

Waxy casts consist of the peculiar matter produced by amyloid degeneration
of the kidney. They have a glistening wax-like or vitreous appearance, and re-
fract light very strongly. Sometimes they reach a great length, and they fre-
quently are found with blood corpuscles or oil drops on the surface. They have
been detected in several renal disorders. Illustrations are given.

True hyaline casts are nearly transparent and hard to see unless the illumina-
tion is very carefully managed. To detect them it is often necessary to add a
few drops of a dilute solution of iodine in potassium iodide to the sediment. This
imparts a slight color which renders them visible.

The hyaline casts seem to be formed by the passage of homogeneous matter
from the tubules, leaving the epithelium behind. A cast is rarely perfectly hya-
line, as at least an occasional blood corpuscle, fat globule, or epithelium cell will
usually be found attached to it. Waxy casts may be looked upon as a special
form of hyaline casts. Very imperfect representations are given in the above cut.

In general, it must be said that the representation of these casts on paper is
a very difficult matter. Ordinarily they are drawn and printed much too heavy
and dark.

Hyaline casts do not necessarily indicate kidney disease, although this is usually

SOME PRACTICAL URINE TESTS. 347

the case. They have been found in urine free from albumin and under circum-
stances not connected with renal disorders.

The preservation of sediments containing casts is unusually difficult because
of the nature of the material to be preserved. In urine of the slightest alkalinity
their disintegration soon begins, so that the outlines are rendered indistinct, often
making identification impossible.

For temporary preservation the addition of chloroform renders as good service
as anything else. Many other sediments can be permanently mounted and kept
for future comparison but with casts this can rarely be done.

Beginners are apt to overlook casts in their first examinations. It must be
remembered that some of them are nearly transparent and unless brought into
proper focus they may not be seen at all. At the outset students usually employ
too bright a light in looking for casts. While no specific directions can be given
regarding the intensity of illumination best suited for the purpose, this may be

Fig. 34. Waxy and hyaline casts.

said, that the light commonly found necessary in studying ordinary histological
slides is far too bright to use in the search for casts.

Practise alone, first under the direction of the instructor, will indicate what is
proper here.

Spermatozoa. These minute bodies, as found in the semen of man, have a
mean length of about 0.050 millimeter. Nearly one-tenth of this is in the head
portion. When observed in recently discharged semen they have a characteristic
spontaneous movement by which they are propelled forward rapidly. This motion
is soon lost if the semen is diluted with water or similar liquid. Hence, as usually
seen in urine, they are entirely motionless. They are found abundantly in the
urine of men after coitus or nocturnal emissions, and also in spermatorrhea, when
their presence is continuous and characteristic.

Fungi. The urine sometimes contains certain fungus growths, the recognition
of which is important. These may have entered the urine after voiding, or they
may have come from the bladder.

Normal urine when passed is probably free from fungi of all kinds, but in a
short time certain organisms enter it from the air or from other sources and be-
come active in producing in it characteristic changes.

The three important groups of fungi, the schizomycetes or bacteria, the
hyphomycetes or molds, and the blastomycetes or yeasts are represented in the
organisms sometimes found in the urine. The conditions under which they are
found will be briefly explained.

348 PHYSIOLOGICAL CHEMISTRY.

Of the bacteria the following have been observed:

Micrococcus Ureae. This is the exceedingly common form found in urine un-
dergoing alkaline fermentation by which urea is converted into ammonium carbon-
ate. It is usually introduced from the air and multiplies very rapidly under ordi-
nary conditions. Nearly all old specimens of urine, unless containing some active
preservative, are found infected with this small organism. The micrococci are
minute spherical bodies belonging to the suborder spherobacteria and are found
separate or in chains. They are the smallest of the organized forms occurring in
urine and appear under a power of 250 diameters but little more than points.

While generally finding their way into urine after it has been voided they are

b

\

Fig. 35. Micrococci and other bacteria.

occasionally present in the bladder. It is usually held that under such circumstances
they have been introduced by a dirty catheter or sound, although cases are on record
where this has not been proved.

In the bladder they give rise to alkaline fermentation, so that the voided urine
may show ammonium carbonate directly.

It is now recognized that the production of ammonium carbonate from urea may
be brought about by several species of bacteria.

Streptococcus Pyogenes is a pathogenic form sometimes found in the urine in
cases of infectious diseases.

Sarcinae. The genus sarcina is frequently classed with the spherobacteria and
several species have been found in urine. The cells are larger than those of
micrococcus urese, and are arranged in groups of two or four usually. They are
not pathogenic.

Bacilli. Several species of the genus bacillus are found in urine in disease.
The most important of these are the typhoid bacillus, bacillus typhi abdominalis,
the tubercle bacillus, bacillus tuberculosis, and the bacillus of glanders, bacillus
mallei. These bacilli occur in urine only during the progress of the correspond-
ing diseases and their detection is of the highest interest. A description of the
methods to be followed for the certain demonstration of these bodies is not within
the scope of this book, but must be looked for in the laboratory manuals of
bacteriology. It may be said, in general, that in diseases characterized by the
presence of certain bacteria in the blood, they may be found also in the urine.

Spirilla. Certain species of the genus spirillum have been found in urine. The
best known of these is the spirillum of relapsing fever, spirillum obermeieri.
This is only found rarely and as its habitat is the blood of relapsing fever patients
it must enter the urine through a hemorrhage into the kidney. Its form is that of
a long, wavy spiral, which makes its detection somewhat easy.

Although not pathogenic it is well to call attention to certain molds which may
sometimes be seen in urine. The common blue-green mold, penicillium glaucum,

SOME PRACTICAL URINE TESTS.

349

is the best known of these, and is occasionally found in urine along with yeast
cells. Another mold which has been found in urine is the oidium lactis, commonly
occurring in milk and butter. It has been observed in fermenting diabetic urine.
Both of these fungi enter the urine after voiding. In urine which has stood some
time in a cool place the penicillium glaucum sometimes becomes covered with
an incrustation of urates or minute crystals of uric acid.

Finally we have yeast cells in urine and sometimes in great numbers. Like
other fungi they enter the urine from the air and when not very abundant have

Fig. 36. Yeast cells and common mold.

no significance. In great numbers the yeast cells suggest presence of sugar. The
ordinary yeast plant, saccharomyccs cerevisice, is shown, isolated and budding, in
the accompanying figure. These forms are described here because their presence
is often confusing to the beginner.

UNORGANIZED SEDIMENTS.

Uric Acid. Among the more common of the unorganized sediments found in
urine this must be mentioned first. As was explained in the last section uric acid
occurs normally in combination in all human urine.

Some time after its passage urine often undergoes what has been spoken of
as the acid fermentation by which a precipitate of urates and even free uric acid

Fig. 37. Uric acid.

may appear. This reaction is in no case due to a ferment process in the ordinary
sense of the term, but is probably brought about by a purely chemical double
decomposition. Urine contains acid sodium phosphate and neutral sodium urate
and it has been suggested that these react on each other according to the follow-
ing equation :

Na2CBH3N.O, + NaH2PO< = NaC.H.N.0, + Na2HPO«.

3 SO PHYSIOLOGICAL CHEMISTRY.

The precipitate of acid urate settles out and forms a light reddish deposit. If
the amount of acid phosphates present is excessive the reaction may go still further,
resulting in the precipitation of free uric acid. The well-characterized crystals
of uric acid are often found with the sediment of fine urates. Sometimes this
liberation and precipitation of the acid takes place in the bladder, and the urine, as
passed, shows the crystals or " gravel." If they are relatively large, which is
sometimes the case, their passage through the urethra may cause severe pain.

As the illustrations show, uric acid occurs in a great variety of forms. The
rosettes and whetstone-shaped crystals are probably the most common, while long
spiculated forms are frequently seen. Pure uric acid is colorless but as deposited
from urine it is always reddish yellow, because of its property of carrying down
coloring-matters. The crystals are often so large that their general form can be
seen by the naked eye ; usually, however, they are minute.

Uric acid crystals when once deposited are not readily redissolved by heat, but
they go into solution by the addition of alkali. If the urine contains extraneous
matter, as specks of dust, bits of hair, cotton or wool fibers, the crystals are very
apt to deposit on them.

Urates. The common fine sediments of urine are usually urates or amorphous
phosphates. They can be most readily distinguished by their behavior with acids
and on application of heat. Urates disappear on warming the urine containing

Fig. 38. Common crystalline and granular urates.

them, while a phosphate sediment is rendered more abundant. A urate sediment
is little changed by acids, while the phosphates dissolve completely if the urine
is made acid in reaction with hydrochloric or nitric acid. The acid urates of
sodium and ammonium are the most abundant and are shown in the cut. Acid
ammonium urate may exist in urine which has become alkaline from the decomposi-
tion of urea and formation of ammonium carbonate, and may therefore be seen in
company with the phosphate sediments. The other urates dissolve in alkaline urine.
Like uric acid the urates appear in a great variety of forms, and there is still
some uncertainty about the composition of some of their crystals which have been
found in urine.

Leucine and Tyrosine. These two substances are of rare occurrence in urine
and appear only under pathological conditions. Urine containing them shows
usually strong indications of the presence of biliary matters as they generally are
found in consequence of some grave disorder of the liver in which destruction
of its tissue is involved. They have been most frequently found, and associated,
in acute yellow atrophy of the liver and in severe cases of phosphorus poisoning.
In general they must be considered as products of disintegration and are pro-

SOME PRACTICAL URINE TESTS.

351

duced in the intestine in large quantity by bacterial agency in the last stages of
the digestion of proteins, as was pointed out in an early chapter.

As both bodies are slightly soluble they may not be seen directly, but only after
partial concentration of the urine. In pure condition leucine crystallizes in thin
plates but from urine it separates in spherical bunches made up of fine plates or
needles. These bunches are sometimes so compact that it is hard to distinguish
between them and other substances, particularly lime soaps and oil drops. Chem-
ical tests must therefore be applied. If mercurous nitrate is added to a leucine
solution and the mixture is warmed, metallic mercury precipitates. This test can
be carried out only when the substance is abundant enough to be purified by crys-
tallization from hot water. Pure leucine, when strongly heated with nitric acid
on platinum, forms a colorless residue, which when heated with potassium hydrox-
ide leaves an oil-like drop that does not wet the platinum.

Tyrosine is usually seen in long needles, which sometimes are bunched in the
form of sheaves, and is more readily recognized than is leucine. Tyrosine heated
with nitric acid on platinum turns orange-yellow, and leaves a dark residue which
becomes reddish yellow by addition of caustic alkali. Solutions containing tyrosine
when treated hot with mercuric nitrate and potassium nitrite, turn red and finally
throw down a red precipitate.

Cystin. This is a rare sediment, although it is found constantly in the urine
of certain individuals. It crystallizes in thin hexagonal plates, small ones some-
times resting upon or overlapping large ones. The crystals are regular in form but

Fig. 39. Leucine spheres, tyrosine needles and cystin plates.

variable in size and readily recognized. A rare form of uric acid crystallizes in a
somewhat similar manner but the two substances differ in their behavior toward
ammonia. To distinguish between them in the microscopic test place a drop of
ammonia water on the slide and allow it to pass under the cover glass. Cystin
dissolves but, unless heated, uric acid does not. When the ammonia evaporates
cystin precipitates.

Cystin is precipitated from urine by addition of acetic acid. Mucin and uric
acid may come down at the same time. The precipitate is collected on a filter,
washed with water and finally dissolved in ammonia. By neutralizing the am-
moniacal filtrate with acetic acid and concentrating a little, it comes down in the
characteristic form suitable for microscopic recognition.

Fat Globules. These are often seen in urine, but in most cases have not been
voided with it. They can come from several extraneous sources, as from a cathe-
ter, from vessels in which the urine is collected or sent for examination, from ad-
mixed sputum, etc., which facts should be borne in mind.

352 PHYSIOLOGICAL CHEMISTRY.

Fat has been found in cases of fatty degeneration of the kidney and more
abundantly in chyluria where communication seems to be formed between the
lymphatics and the urinary tract by the invasion of small thread worms.

Hippuric Acid. This acid is found normally in human urine in small amount,
It may be found in large quantity after taking benzoic acid and may even appear
in crystalline form in the sediment. It has no pathological importance, ordinarily.

Calcium Carbonate. This is sometimes observed as a coarse, granular sedi-
ment which dissolves with effervescence in acetic acid. It occasionally forms
dumb-bell crystals, and is devoid of pathological importance.

Calcium Sulphate. Crystals of this substance are rarely found in urine. They
form long, colorless needles, or narrow, thin plates.

Calcium Oxalate. We have here one of the commoner of the crystalline bod-
ies observed in urine.

This may be found in neutral or alkaline urine, but more commonly in that
of acid reaction. It occurs normally and sometimes is very abundant, especially
after the consumption of vegetables containing oxalic acid.

Two principal forms of the crystals are found, the octahedral and dumb-bell
crystals.

The octahedra have one very short axis which gives the crystals a flat appear-
ance. When seen with the short axis perpendicular to the plane of the cover

Fig. 40. Calcium oxalate.

glass, which is the common position, they appear as squares crossed by two bright
lines. Sometimes they are seen on edge, and then present a rhomb in section
with one diameter very much shorter than the other.

A form of triple phosphate bears a slight resemblance to calcium oxalate, but
it is soluble in acetic acid, while the oxalate is not.

The dumb-bells are much less common than the octahedra, and are found in
several modified forms, as shown in one of the figures.

The clinical significance of the oxalate is not clearly understood. It does not
seem to be characteristic of any disease even when occurring in quantity. It has
been found considerably increased in dyspeptic conditions, but not always, and
many of the statements found concerning its significance seem to have been based
on insufficient observations.

Urine may contain a large amount of oxalic acid, which does not show as a
sediment, but must be found by precipitation with calcium chloride in presence of
ammonium hydroxide. Acetic acid is then added in very slight excess and the
mixture is allowed to stand for precipitation.

SOME PRACTICAL URINE TESTS.

353

The constant or prolonged excretion of large amounts of oxalic acid is spoken
of as oxaluria.

The Phosphates. It has been explained that phosphates of alkali and alkali-
earth metals occur normally in the urine, and a method was given for their esti-
mation. As sediment we know several forms of calcium and magnesium phos-
phates and the microscopic detection of these will be here explained. In normal
fresh urine of acid reaction these phosphates are held in solution, but if the urine
as passed is alkaline it is often turbid from the presence of basic phosphates held
in suspension. Urine which has stood long enough to undergo the alkaline fer-
mentation always contains phosphates in the sediment. Finally, it must be re-
membered that a neutral or very slightly acid urine, containing ammonium salts
in abundance, may also deposit a crystalline precipitate of ammonium magnesium
phosphate. The common phosphate sediments are those consisting of ammonium

Fig. 41. Triple phosphate.

magnesium phosphate (triple phosphate), normal magnesium phosphate, neutral
calcium phosphate, and mixed amorphous phosphates of calcium and magnesium.

Triple Phosphate. Of the crystalline phosphate deposits this is the most abun-
dant and at the same time the most characteristic.

The crystals are the largest found in urine, and from their shape are some-
times spoken of as coffin-lid crystals. Ordinarily they are not found in perfectly
fresh urine, but after it has undergone the alkaline fermentation they are gen-
erally present in profusion.

Normal Magnesium Phosphate. Crystals having the composition, Mg3(POJ2.
22FLO, are sometimes found in urine of nearly neutral reaction. They consist
of thin, transparent, rhombic plates with angles approximately 6o° and 1200. If
urine containing this sediment becomes alkaline, triple phosphate forms.

Neutral Calcium Phosphate. This has the composition, CaHPOv2H20, and
is found in urine of neutral or slightly acid reaction. It crystallizes frequently
in rosettes formed of wedge-shaped, single crystals, uniting at their apices. The
cut shows some variations in the form.

Amorphous Phosphates. Finally we have the very common, finely granular,
earthy phosphates in amorphous condition. This sediment dissolves readily in
weak acetic acid and is colorless. The common amorphous urate sediment is col-
ored and does not dissolve in acetic acid. On addition of sodium carbonate or
hydroxide to urine, the precipitate which forms consists mainly of this phosphate.

These several phosphates can be produced artificially and should be made for
study and comparison. The normal magnesium phosphate can be made by dis-

24

354

PHYSIOLOGICAL CHEMISTRY.

solving 15 grams of crystallized common sodium phosphate in 200 cc. of water
and mixing this with 3.7 grams of crystallized magnesium sulphate in 2000 cc.
of water. Enough sodium bicarbonate is added to give an amphoteric reaction
and then the mixture is allowed to stand a day or more for precipitation.

Crystals of triple phosphate of peculiar form are often obtained by adding
ammonia to urine, and sometimes a trace of ammonia is sufficient to throw down
the crystals of neutral calcium phosphate. The latter can also be obtained by

Fig. 42. Neutral calcium phosphate and amorphous phosphate.

adding to a weak solution of crystallized sodium phosphate a trace of acid and
then a very little calcium chloride solution.

URINARY CALCULI.

Calculi, like the sediments just described, are formed by the precipitation of
certain substances from the urine, but in compact form. Occasionally a calcu-
lus consists of a single substance, as calcium oxalate or cystin, but in the great
majority of cases a mixture of bodies is present, these being deposited usually
in layers around a nucleus which serves as the foundation of the concretion.
Calculi are built up much as certain forms of crystals are by successive depositions
on a nucleus. Uric acid is a very common nucleus on which may be deposited
urates, phosphates, organic matters, etc.

Calculi are sometimes distinguished as primary or secondary. Primary cal-
culi may be traced to an alteration of the urine of such a nature that its reaction
is constantly acid. The foundation for the concretions in this case is found in
the kidney and they are built up of such substances as most easily deposit from
acid urine. Secondary calculi are generally formed in the bladder, and have for
nuclei matters precipitated from alkaline urine, as coagulated blood or other
organic substances. Sometimes fragments introduced into the bladder from with-
out serve as the foundation for these secondary formations. Bits of catheters,
remains of bougies, and other things have been found as the nuclei around which
concretions have formed. The recognition of the nucleus is a matter of the first
importance as this gives a clew to the determining cause active in the formation
of the calculus.

In making an examination, then, of a calculus, it is first cut in two by means
of a very sharp thin saw. This exposes the nucleus which may often be recog-
nized by the eye alone. If one of the halves be polished it is often possible to
discern distinctly the various layers grouped around the center.

In a large number of cases examined by Ultzmann about 80 per cent, were
found to contain uric acid as the nucleus.

Chemical Examination. In the chemical examination of a calculus several

SOME PRACTICAL URINE TESTS. 355

methods may be employed. We may begin by applying certain preliminary tests
designed to show the general nature of the stone.

Heat Test. Reduce some of the calculus to a powder and heat to bright red-
ness on platinum foil. Two cases may arise: (a) the powder is completely con-
sumed; (b) the powder is only partially consumed or not at all.

Case (a). If this is the result of the incineration the following substances
may be suspected :

Uric Acid, which may be recognized by dissolving a little of the powder in
weak alkali, precipitating by hydrochloric acid and examining the precipitate by
the microscope.

Ammonium Urate. This gives the above reaction under the microscope, and
is further recognized by the liberation of ammonia when heated with a little pure
sodium hydroxide solution.

Cystin. Dissolve some of the powder in ammonia, filter if necessary and allow
drops of the filtrate to evaporate spontaneously on a slide. Cystin is then recog-
nized by the microscope as already explained. Cystin contains sulphur which,
on burning on the platinum foil, gives rise to a disagreeable sharp odor. If a
little of the powder be heated with a mixture of potassium nitrate and sodium
carbonate the sulphur is oxidized to sulphate, which may be recognized by the
usual tests.

Xanthine. This is a rare substance in calculi. Those consisting wholly of
xanthine are brown in color and take a wax-like polish.

Organized Matter. Parts of blood cells, epithelium, precipitated mucin, pus
corpuscles and similar substances may become entangled with the growing stone
and even form a large part of it. On burning, these bodies are recognized by
the characteristic odor of nitrogenous matter.

Case (b). When an incombustible residue is left on the platinum foil the stone
may contain the following constituents :

Calcium Oxalate. Stones of this substance are very hard and break with a
crystalline fracture. They are often called " mulberry calculi." When the powder
is heated it decomposes, leaving carbonate, which may be recognized by its effer-
vescence with acids.

Calcium and Magnesium Phosphates. They leave a residue in which the metals
and phosphoric acid may be detected by simple tests of qualitative analysis. The
ignited powder is soluble in hydrochloric acid without effervescence. When am-
monia is added to this solution in quantity sufficient to give an alkaline reaction,
a precipitate of triple phosphate or calcium phosphate appears, which may be recog-
nized by the microscope.

The above tests are generally sufficient to tell all that is practically necessary
about the calculus. If more detailed information is desired a systematic analysis
must be made.

CHAPTER XXI.

THE GASEOUS EXCRETION. RESPIRATION.

In the last chapters the amount of nitrogen excreted with the urine
was discussed at some length. With the nitrogen certain correspond-
ing proportions of carbon, hydrogen and oxygen are excreted in the
urea, uric acid and other bodies described. But the larger amounts of
these elements are thrown off from the body in different form, and
especially in the carbon dioxide and water vapor eliminated in respira-
tion and perspiration. From certain classes of foods the end products
formed are these two only when the oxidation is ideally complete.
This is the case with the fats and carbohydrates, and supposing them
wholly burned in the body the final results are represented in this way,
taking typical substances for illustration :

CH^O, + 602 = 6C02 + 6H 20,

C3H5(C18H3502)3 + 163O = 57C02 + S5H20.

In the actual behavior of these compounds in the human body, how-
ever, the results are somewhat different. The oxidation is never quite
as complete as here indicated, as traces of both carbohydrates and fats
are left in more complex forms.

THE RESPIRATORY QUOTIENT.

In studying the completeness of oxidation of certain foods much
has been learned by a consideration of the factor known as the respira-
tory quotient which is simply the ratio of the carbon dioxide elimi-
nated to the oxygen absorbed, measured by volume. This quotient is
therefore given by the expression C02/02. For the sugar of the
above equation we require six molecules of oxygen, and the carbon
dioxide produced is also six molecules. Hence C02/02 = 1. For all
common carbohydrates the result is the same. For the fats, however,
the quotient is much smaller since 57 C02 is the carbon dioxide volume
excreted for an oxygen consumption of 81.5 02. In this case C02/02
= 57/81.5 = 0.7. For the protein bodies the factor cannot be as
easily calculated, since we are not able to assign a formula to these
substances, and moreover we are not familiar with all their oxidation
products. But from the percentage composition, and the known facts
regarding the elimination of urea, uric acid, ammonia and creatinine

356

THE GASEOUS EXCRETION. 357

it is possible to calculate an approximate quotient. This is about 0.8,
which factor may be used in calculations.

The use of these quotients is ordinarily based on the assumption
that the oxidation is a direct one, and that corresponding to the oxygen
absorbed there is almost immediately a liberation of carbon dioxide in
the right proportion. But this assumption does not hold absolutely
true; the breakdown of carbohydrate, for example, may yield at first,
in part, products with high oxygen content, from which C02 separates
later. In other words, there may be an apparent temporary storing
up of oxygen, which would make the quotient appear low. Later a
compensating excessive liberation of carbon dioxide would have the
opposite result. However, in observations carried out through a period
of proper length these variations would not affect the general mean.

The Carbon and Nitrogen Balance. The body is in carbon equi-
librium when just as much carbon is eliminated as is consumed in the
food, and a determination of this element in the various excreted
products and in the food is sufficient to show whether there is gain,
loss or equilibrium in body weight. All the food stuffs are organic, it
will be remembered, and contain carbon as the fundamental element.

A change in weight may result from gain or loss in fat or gain or
loss in protein. Nitrogen equilibrium exists when income and outgo
are equal ; in this case all the proteins consumed as foods are decom-
posed. A determination of nitrogen in the urine and feces, coupled
with a knowledge of the food protein, will decide this point, since the
excreted nitrogen multiplied by 6.25 gives a measure of the food pro-
tein. The most accurate method of reaching the value of the excreted
nitrogen is by Kjeldahl determinations on the urine and feces, but good
approximate results are secured by determination of urea alone, it
being remembered that about 85 per cent of the urinary nitrogen
appears in this form.

Respiration Apparatus. To determine the volume of oxygen in-
haled and carbon dioxide given off, the animal or person under experi-
ment is placed in a respiration chamber of some kind. In a form of
respiration chamber sometimes used, an accurately measured volume
of air with known content of moisture and carbon dioxide is forced
through. The air leaving the tight chamber is analyzed and the
amount of carbon dioxide, moisture and oxygen determined. This
last determination may be made directly, or the loss of oxygen by the
respiration of the person in the cage may be found by calculation from
this basis: The sum of all the factors consumed, that is the food and
the oxygen, plus the body weight, must be balanced by the weight of

3 5§ PHYSIOLOGICAL CHEMISTRY.

the body at the end of the experiment plus the various excreted matters.
HA represents the body weight at the beginning of the test and A'
the body weight at the end of the test, Ox the oxygen consumed, F the
food consumed, Ex the total excreta by weight, then

A + Ox + F = A' + Ex.

Ox = A' + Ex—(A + F).

In some of the recent forms of respiration apparatus, especially that
of Atwater and Rosa, extremely accurate results are possible in the
determination of carbon dioxide and moisture produced; but with
increase in size of the apparatus a direct determination of oxygen dif-
ference becomes more and more difficult.

In the Zuntz apparatus, which is often used for short experiments
on the gaseous excretion only, a peculiar mouthpiece is worn which
permits a collection of the carbon dioxide and vapor from the lungs,
and of the total expired air. A determination of the oxygen and
carbon dioxide is accurately made and this furnishes all the data nec-
essary for the calculation. The nose is closed in this experiment; the
mouthpiece is so arranged that air may be drawn in without allowing
the excretory products to escape.

DEDUCTIONS FROM RESPIRATION EXPERIMENTS.

These are undertaken to answer a number of important questions.
The weight of carbon dioxide excreted may reach fifteen hundred
grams or more daily and it is interesting to know under what circum-
stances it is increased and when diminished. Very simple observa-
tions show that the body at rest produces much less of the gas than
does the body at work. In the latter condition the destruction of food
stuffs is called for to liberate mechanical energy. This is practically
possible only through oxidation, and carbon dioxide is the first tangible
result of the oxidation.

The question also comes up, what kind of organic matter is most
readily or most commonly oxidized when work is done ? On this ques-
tion much has been written and our views have undergone various
changes through the years. Liebig considered the proteins as the
foods which must be burned to enable us to do mechanical work, but
in a famous experiment by Fick and Wislicenus, undertaken to throw
some light on this question, no great excess in the excretion of urea
was found in the work of ascending the Faulhorn, and the protein
oxidized was far too little to account for the work done. Other inves-
tigators reached the same conclusion, but it has been found that under

THE GASEOUS EXCRETION.

359

certain conditions the proteins may be consumed to do work. Ordi-
narily fats and carbohydrates are used in preference, and no large
amount of protein is used if the other substances are present in suffi-
cient quantity.

The question of what kind of foodstuff is oxidized through periods
of work and rest may be answered by experiment. As just intimated,
examinations of the urine give us information as to the nitrogen excre-
tion, and the extent of oxidation of fats and carbohydrates may be
measured by respiration experiments. In a fasting animal at rest the
respiratory quotient sinks to a value but little above 0.7, showing that
the substance metabolized is mainly fat ; as some proteins are also used
up the quotient cannot absolutely reach 0.7. If work is done by the
fasting animal, the carbohydrate bodies of the muscular juices, glyco-
gen essentially, are called upon and their effect is added to that of the
proteins in raising the respiratory quotient. On the other hand, it has
been found that a well-fed animal at rest, with abundance of carbo-
hydrates in the ration, will excrete a volume of carbon dioxide nearly
as great as that of the oxygen absorbed. In this case the ratio C02/02
shows that essentially carbohydrates are burned and that fat is allowed
to accumulate. When very hard work is done by the well-nourished
animal the quotient sinks to an intermediate value, showing that fats
are now consumed as well. This would be evident also from observa-
tions continued over a long period in which no accumulation of fat
could be recorded. With moderate work there is not much change.

Some of these results are illustrated by the figures in the following
table taken from observations published by Chauveau and Laulanie, in
which dogs were the subjects of experiment. These figures show
very clearly alteration in the respiratory quotient with work, and also
by diet.

Food Consumption.

CO

Observed Ratio 2 , or Respiratory Quotient.

o2

No.

mi*

Minutes of Work Before
Observations.

Minutes of Rest Follow-
ing Work.

30

45

60

90

120

180

45

60

120

240

I

2

3
4

!

24 hours fast.
6 days fast.

1 day fast.

2 days fast.

3 days fast.
After full meal.
After full meal.

0.790
0.750
0.874
0.740
0.685
1033
1. 000

0.943
O.819

I.OI7

O.905
O.84O
O.895
O.780
O.79O

I.042

O.9OO
I.O44

O.9OO

0.866

0.808

1.008

0.900
0.866
0.772

O.789

O.687
O.770
O.73O
O.681
I.052
I.032

O.77O
O.708
O.681

I. OI 7

O.756

Other experiments are in general good agreement with these. The
effect of work in the fasting animal is seen almost immediately. In

360

PHYSIOLOGICAL CHEMISTRY.

the last experiments the respiratory quotient is greater than unity.
This may be due in part to slight errors in observation, but it should
be remembered that there are classes of compounds in which such a
result would always follow. Such compounds are not common articles
of food, but often make a part of certain vegetable foods. The com-
plete oxidation of tartaric acid, for example, would yield a quotient
of 1.6.

The quotient may sometimes be high, as intimated above, if the
oxygen has been at first absorbed to form compounds relatively rich
in oxygen, which are later broken down rapidly, under working or
other conditions. If an observation is made just at this period the
excess of C02 liberated would present an abnormal result. For char-
acteristic results the observation periods should be as long as possible.

Illustrative Case. Some idea of the importance of the respiratory
coefficient determination may be obtained from a consideration of the
following assumed case in which the conditions are made somewhat
ideal for simplicity of calculation. The numerical values given are
such as might be obtained from the mean of several 24-hour experi-
ments in a large respiration chamber. The diet is assumed to be abun-
dant and the tests begun after a condition of practical nitrogen equi-
librium is reached.

Initial weight 75 kilograms

Final weight 75-OS kilograms

Income Observed.

Wt. in
Grams.

C

Per Cent.

N
Per Cent.

O
Per Cent.

H
Per Cent.

c

Total.

N
Total.

0
Total.

H

Total.

Proteins

I50

■ no

44O

35
2,000

53-5

76.5
44.2

16.O

23-5
II.4
49.6

7.0
12. 1

6.2

80.3

84.2

194-5

24.O

35-2

12.5

2l8.2

IO.5
13-3

27-3

Fats ...

Carbohydrates...
Salts

Water

Total

2,735

1

359-o

24.O

265.9

511

Outgo Observed.

Weight in Grams.

c.

N.

Salts.

Vol. co2.

Respiration, C02..
Respiration, H20. .
Urine, H20

932
904

i,35°

74

100

33

254.2

8.7

16.2

20.4
3-6

3°

'5

471 1-

Total

3,393

279.1

24.0

35

The weight of the various excreted products is greatly in excess of

THE GASEOUS EXCRETION. 36 1

the visible income, but the oxygen inhaled has not yet been calculated.
The formula given above may be applied to find this :

Oxygen = A' -f- Excreta — (A + F)
75>050 3,393 75,ooo 2,735

= 78,443 — 77,73s

= 708 grams
= 495.4 liters.

In the table above the volume of carbon dioxide eliminated is given as
471 liters. The respiratory quotient is therefore

495-4
This gives us the first clue as to the nature of the foods metabolized.
The factor is so much larger than that corresponding to the fats that
we may practically exclude these at once. In any event there is a large
protein metabolism since the original nitrogen of the food is all found
in the urine and the feces. In other words, we have nitrogen equi-
librium, with no storing up of protein in the tissues. The respiratory
quotient corresponds to the combustion of carbohydrates and proteins
mixed.

If we assume for the moment that no fat is oxidized, this calculation
may be made. The 254.2 gm. of carbon in the carbon dioxide of
respiration calls for 678 gm. of oxygen. The difference between this
and the calculated absorbed oxygen, 708 gm., amounts to 30 gm., which
must be used up in oxidizing hydrogen of protein substances. This
conclusion is drawn because the carbohydrates contain enough oxygen
to burn their own hydrogen, and the protein nitrogen appears as urea
and calls for no outside oxygen. The burning of fat hydrogen is
excluded in the assumption.

The nitrogen of the feces corresponds to 22.5 gm. of original pro-
tein (6.25 X 3-6). Not all of this nitrogen is actually in the form of
unchanged or residue protein ; a part of it represents products of
metabolism which are excreted in the feces, as explained in a previous
chapter. Probably a considerable fraction may be considered in that
form ; but it must be counted as a loss to the body, and we have there-
fore as net available protein (actually used) about 127.5 gm. In the
final metabolism of this the nitrogen appears in urine in several forms,
but mostly as urea. The per cent of nitrogen in this is 46.7. In some
of the other compounds the nitrogen is higher and in some lower. In
ammonia much hydrogen (relatively) is held, and in uric acid little.
In some cases there is an excess of carbon and in other cases relatively
little carbon is held with the same weight of nitrogen. The various

362

PHYSIOLOGICAL CHEMISTRY.

conditions balance each other pretty well, so that no great error will
be made if, for our special purpose, we count all the excreted nitrogen
as combined in the form of urea. We have then these relations :

C.

N.

H.

0.

68.3
8.7

20.4
20.4

8.9
2.9

29.9

In urea

11. 6

59-6

OO.O

6.0

18.3

To oxidize this remaining carbon requires 159.8 gm. of oxygen. The
18.3 gm. of protein oxygen will oxidize about 2.3 gm. of hydrogen.
The remaining hydrogen from the 6 gm. will call for about 29.6 gm. of
oxygen, which corresponds closely -to the amount calculated above.

The ingested carbon is seen from the table to be 359 gm. ; the
excreted carbon is 279 gm., from which it follows that the body has
gained 80 gm. If we assume this to be in the form of fat the latter
must amount to about 104 gm. This represents the true gain in body
weight ; the weighings showed a gain of only 50 gm. The discrepancy
may be accounted for by assuming an excessive excretion of urine.
No such discrepancy would appear if the urine were passed from the
bladder as fast as formed, but as it is collected at intervals it is not
possible to obtain exactly comparable results.

In the feces there must be some carbon derived from fats; but the
amount cannot be large, because for the nitrogen of the feces we must
calculate at least 10 or 12 gm. to correspond. This would leave about
5 gm. of carbon from other sources, accounting for the discrepancy
between consumed fat and deposited fat.

The above calculations illustrate the principles involved; in an actual practical
observation the method would be the same, but the interpretation of results might
not be as simple, especially with a low respiratory quotient found. In the above
tables the salts taken with the food are assumed to include those to be formed by
the oxidation of the protein, and the latter substance figured as income is assumed
to consist of the organic elements only. A slight error is introduced in the calcu-
lation in this way, but that is not considered. In actual practice, of the carbo-
hydrates some little would escape complete metabolism. The above results would
correspond to a completely burned carbohydrate.

In the above table of observations the total oxygen in the consumed substances,
including the water, is 2,044 gm- In the excreted products, allowing 30 gm; for the
solids of the urine and feces coming from bodies other than the original salts, the
oxygen appears to amount to about 2,800 gm. The difference shows an excess of
756 gm. while the calculation above gave 708 gm. of oxygen taken in. The dis-
crepancy is due to the excess of water excreted as urine. It will be noticed also
that there is a great excess of excreted water over the 2,000 gm. consumed. This
amounts to over 350 gm., of which 300 gm. would come from the combustion of the
carbohydrates and proteins metabolized.

THE GASEOUS EXCRETION. 363

SKIN RESPIRATION.

It is usually assumed that the gaseous exchange is wholly through
the lungs, but this is not quite correct. Experiments with men and
animals have shown an absorption of oxygen and an escape of carbon
dioxide through the skin. A number of observers have put results
for the latter on record which, however, are not in good agreement.
For 1.6 square meters of skin surface the results found in seven
observations varied from 2.2 gm. to 32 gm. in 24 hours The last
result is probably much too high. It has been noticed further that the
amount of carbon dioxide escaping through the skin is increased
greatly by temperature. The excretion at 30° seems to be several

times as great as at 200.

times as great asawu .

For the absorption of oxygen no exact figures are given, but the
amount is very small. In some of the lower animals, however a large
part of the absorbed oxygen, as well as of the excluded carbon dioxide,
may be by way of the skin. This has been shown especially in the
frog, where after removal of the lungs a nearly normal exchange may
be noted for a period of days.

The question of the excretion of other gases than carbon d.ox.de by
the skin, and the lungs also, has been much discussed. Formerly it
was held that a very appreciable quantity of organic gaseous bodies is
given off through the skin and this elimination was considered neces-
sary for the well being of the body. The unpleasant odor of the a r
of a crowded room was ascribed to these organic emanations. But
much doubt has been thrown on this notion by various «*«""*
some of which are of very recent date, which seem to show that these
odors come, not through the skin, but from decaying substances on the
surface of the skin or from the clothing, if it is old and soiled. Ex-
Z ments have been made of testing the air drawn through a small
Ration chamber, enclosing the body of a tnan to the neck, with
perfectly clean skin and clothed in fresh, clean garments. Such air is
pr c Jly without odor and has no action on solutions of perman
Late through which it is aspirated. It is free from ammonia. The
ooors of perforation are apparently largely due to the fermen a ion
changes of solid or semi-solid substances on the surface of the skin
ra her than to excreted gaseous products passing through the pore
vTh he water. It has been found also that the whole surface of the
Tody may be covered with varnish without harmful result if precau-
tions arc taken to prevent loss oi beat.

364 PHYSIOLOGICAL CHEMISTRY.

TIME AND PLACE OF OXIDATION.

The determination of the respiratory quotient through short inter-
vals shows considerable variations, as pointed out some pages back.
The human organism has not the power of storing up oxygen in the
free or combined form through a long period, as appears to be the
case with some cold-blooded animals, which are able to exist for a time
in an atmosphere free from oxygen. With man and warm-blooded
animals in general this is not possible; with these life without oxygen
may be maintained for but a few minutes at most. An exception
exists in the case of those animals which pass the winter in a dormant
condition (hibernating animals) and for human beings in trance.
Here the absorption of oxygen and excretion of carbon dioxide are
reduced to a minimum. But ordinarily man and the higher animals
require some inflow of oxygen all the time.

The extent to which this oxygen is used depends on the activity of
the muscles largely. In rest periods the amount of oxygen taken up
by the muscles is much greater than is the carbon dioxide given off,
but with the contracting or working muscle the reverse is the case. In
experiments in which the changes in the blood supply of individual
muscles may be followed it may be shown that for rest periods the
respiratory quotient for the muscle may fall far below 0.7 or even
below 0.5. From the rapidly contracting muscle, on the other hand,
the evolution of carbon dioxide is relatively great. A respiratory
quotient, for the muscle, of 1.5 or even 2 or more may be found. This
indicates that during rest oxygen may be taken up from the blood and
held or condensed in some manner by substances within the muscular
tissue, but of the mechanism of this reaction unfortunately but little is
known. In doing work tissue is rapidly oxidized at the expense of the
stored-up oxygen, and a great excess of carbon dioxide is given off
quickly. These changes follow one upon the other rather rapidly. In
the oxygen-absorbing stage some intermediate products are probably
built up from sugar or glycogen or other substances, which fall apart
with liberation of water and carbon dioxide in the succeeding active
condition of the muscle.

The problem of oxidation in the tissues is possibly somewhat like
that of etherification in which alcohol yields ether indirectly through
the intermediate ethyl sulphuric acid, and several suggestions have
been brought forward as to the character of complexes formed in one
stage of the oxidative metabolism to be decomposed in another. At
the present time these suggestions are practically wholly within the
realm of speculation, and not therefore suitable for presentation in this

THE GASEOUS EXCRETION. 365

place. It is likely that all these reactions, which seem to be carried on
in the tissues rather than in the fluids of the body, are incited by
enzymic ferments, and to-day certain classes of oxidases are often
assumed to be the agents active in the changes. For some of the
oxidations it has been pretty well settled that an enzyme produced by
the pancreas is necessary.

CHAPTER XXII.

THE ENERGY EQUATION.

We come now to a brief consideration of one of the most important
questions connected with the whole animal chemistry, and this is the
question of the liberation of energy from the consumption of various
foods. The function of the food we eat is a multiple one. It may not
only increase the body weight and maintain the various functions of
the body through oxidation, but in its combustion heat is liberated to
maintain also the body temperature, and energy is furnished to enable
us to perform external work. It is interesting to measure the effect
of the food in these several directions, which may be done with a fair
degree of accuracy. The following considerations will show the basis
of the calculations.

POTENTIAL ENERGY OF FOOD.

The food, consisting essentially of combustible substances, is the
source of a large amount of potential energy. In the complete com-
bustion of the fats, carbohydrates and proteins of the food a large
amount of heat is liberated and this in turn is the equivalent of a
certain amount of work. The potential energy of chemical substances
may be measured in various ways, but for purposes like the present it
is customary to measure this energy in terms of the units of heat lib-
erated in the combustion of the body in question with oxygen. Cer-
tain units are in common use :

Unit of Heat, Calorie. The unit of heat or calorie may be defined
as the quantity of heat required to raise the temperature of a gram of
water one centigrade degree, at a mean temperature. As the heat
absorption of a gram of water is not quite the same throughout the
scale, the calorie is perhaps more satisfactorily defined as the one hun-
dredth part of the quantity of heat required to raise the temperature
of a gram of water from o° to ioo° C. This gives the ordinary, or
small calorie. In dealing with large heat transfers a larger unit is
preferable and one just 1,000 times as large is frequently used. In
this the kilogram in place of the gram of water is warmed, and the
unit is called the large calorie. The first may be abbreviated cal. and
the second Cal.

366

THE ENERGY EQUATION. 367

Unit of Work and Unit of Force. The unit of force is called the
dyne and may be defined as the force which, acting for I second on a
mass of 1 gram, gives to it an acceleration of I centimeter per second.
The force of gravity at the sea level is about 981 dynes, since this adds
to a falling body an acceleration of 981 cm. per second.

The unit of work is the erg, and it may be defined as the work done
in overcoming unit force through unit distance. One dyne acting
through one centimeter gives us one erg of work. To lift 1 gram
through 1 centimeter requires 981 ergs of work.

Mechanical Equivalent of Heat. Work may be done by the proper
utilization of heat, and in turn work may be wholly converted into
heat. It is possible, therefore, to express one in terms of the other.
The mechanical or work equivalent of a unit of heat has been deter-
mined many times by very elaborate experiments. If a given quantity
of heat could be applied wholly to the lifting of a weight it would be
found, in accordance with the mean results of these experiments, that
1 calorie would be able to lift 423.5 gm. through 1 meter, or 1 gm. of
substance through 423.5 meters. Conversely, if a gram of water be
dropped from a height of 423.5 meters, and its energy of motion wholly
converted into heat, its temperature will be found to be increased i°
C. We have then these relations :

1 calorie = 42,350 gm. cm.
= 41,500,000 ergs.

Heats of Combustion. By means of calorimeter experiments the
following heats of combustion have been determined. Results found
by different workers show slight variations, but the values here are
mean values and sufficient for illustration. The number of calories
furnished by burning 1 gm. of substance in each case is given.

Table of Heats of Combustion.

Hydrogen 34,200 Cane sugar 4,000

Carbon 8,100 Starch 4,200

Ethyl alcohol 7,060 Casein 5,700

Glycerol 4,200 Egg albumin 5,7oo

Mannitol 4,000 Urea 2,500

Palmitic acid 9,300 Uric acid 2,700

Stearic acid 9,400 Leucine 6,500

Fats, average 9,400 Tyrosine 6,000

Hexoses 3,700 Creatine, anhyd 4,250

These values are for complete combustion, but as the proteins in the
body are not oxidized to leave water, carbon dioxide and nitrogen, we
must subtract from the given values the heats of combustion of the

Potential energy of
Food.

368 PHYSIOLOGICAL CHEMISTRY.

urea, uric acid, creatinine and other products found in the urine, in
order to secure the physiological heats of combustion, with which we
are practically concerned.

DISTRIBUTION OF FOOD ENERGY.

With these preliminary considerations we are able to look at the
manner in which the energy of the consumed food is distributed. On
the one side we have the substance burned, on the other the products,
which may be represented diagrammatically in this way :

Potential energy of

Flesh gained.
Feces.
Urine.

Perspiration
Kinetic energy of

Work.
Heat.

Experimentally, the whole of the kinetic energy may be made to take
the form of heat, which simplifies the observations materially. It is
practically possible to determine the heat liberation in the large respi-
ration calorimeters already referred to, and the use of such apparatus
will be explained below. First, however, a general method of calcu-
lating the energy liberated as heat will be given.

CALCULATION OF KINETIC ENERGY OF FOOD.

In illustration of this we may make use of the example given in the
last chapter, and employ a method which in principle is very simple.
The income of energy is due to the consumption of certain weights of
protein, fat and carbohydrates, the last of which we may assume is
made up of 9 parts of starch and 1 part of cane sugar, all weights
referring to the anhydrous condition. The effect of the oxidation of
sulphur and phosphorus will be neglected here, and the protein will be
assumed pure carbon, hydrogen, oxygen and nitrogen. We have then
as income :

From 150 gm. protein, 150X5700= 855,000

no gm. fat, 110X9400=1,034,000

440 gm. carbohydrate, 440X4180 = 1,839,200

3,728,200

In small calories the whole income is therefore equivalent to 3,278,-
200 cal.

We have next to calculate the potential energy of the food stuffs

THE ENERGY EQUATION. 369

not actually consumed, which are left in the feces and the urine, and
also the energy of any substance which may be put down as a gain in
weight in the body. Recalling the data of the experiment in the last
chapter we have

l33 HP*1- feces with 16.2 gm. C.
1,424 gm. urine with 20.4 gm. N.

Calculating the N of the urine as urea, which in practice would not be
quite accurate, we have 44 gm. of t.hat substance. The organic matter
of the feces corresponds approximately to 22.5 gm. of bodies resem-
bling protein and 5.5 gm. of bodies resembling fats, and these data we
can now employ in the calculation.

The illustration gave also a gain of 80 gm. of fat. The solid matter
lost in the form of perspiration is so small that it may be ignored for
the present purpose. We have then the following deductions to make :

Potential energy in 80 gm. of fat stored 752,000

!33 gm- of feces 180,000

1,424 gm. of urine 1 10,000

1,042,000
This leaves as a balance to be calculated as kinetic energy

3,728,200
1,042,000

2,686,200 calories

Another method of calculation deals with the carbon and hydrogen of the food
and feces only. The heat production was at one time assumed to depend on the
combustion of the carbon of the fats, carbohydrates and proteins and the hydrogen
of the fats and proteins. The hydrogen of the carbohydrates was not considered
because it was supposed to be closely combined with the oxygen present in the same
compounds in such a form as to yield no more heat on oxidation. In like manner
the combustion heats of the urine and feces may be calculated from the whole
carbon and hydrogen content of the organic substances. The total carbon of the
food in the experiment is 395 gm., of the hydrogen in fats and proteins 23.8 gm.
The carbon of the urine and feces is 24.9 gm., while the hydrogen of the urine and
feces is about 5.2 gm. We have then :

Heat units from 359 gm. of food carbon 2,907,900

Heat units from 23.8 gm. of food hydrogen. . .. 813,900

3.721,800 3,721,800

Heat units from 24.9 gm. of excreta carbon 201,600

Heat units from 5.2 gm. of excreta hydrogen. . 177.840

379-530 379,530

Net calories 3,342,270

From this result the value of the energy stored as fat would have to be subtracted
as before. This method of calculation gives a somewhat lower result than the other,
and largely because of the uncertainty in allowing for the excreted carbon and
hydrogen, but it has value as a comparison process.

25

370 PHYSIOLOGICAL CHEMISTRY.

Respiration Calorimeters. In experiments with men or large animals on the
combustion of food and liberation of heat some kind of respiration apparatus is
employed. Some modification of a type originally introduced by Pettenkofer is
generally used. In this the subject is placed in a chamber with double walls through
which a current of air may be forced and uniformly mixed inside. A known part
of the ingoing air and of the outgoing air may be diverted for analysis so as to
permit an exact determination of the amount of oxidation products liberated at any
time. The Atwater and Rosa calorimeter is the most complete of all such con-
structions. In this the heat liberated by the subject is taken up by a current of
cold water circulating through numerous toils of pipe inside the chamber and in
such a way as to maintain a perfectly uniform temperature in the chamber space.
The walls of the chamber are made of compartments containing two layers of
air and two layers of water maintained in such relations that they prevent gain or
loss of heat. The whole heat liberation is taken up by the circulating water and
may be accurately measured. The respiration chamber has a capacity of about
175 cubic feet and is large enough to contain a chair and small table for the con-
venience of the occupant and a couch to sleep on at night. The construction is
such that food may be passed in and the urine and feces removed without making
any appreciable change in the temperature or content of the air inside. With such
an apparatus it is possible to carry on a test of many days duration and obtain
extremely accurate and important results.

In work experiments in such a calorimeter a bicycle is mounted so that work is
done against friction. The final effect is increased heat liberation, measured as
before.

The construction of this large calorimeter suggested the building of still larger
ones of the same general type. Some of these are being used in agricultural experi-
ment stations in metabolism experiments on large animals, from which results of
great practical value may be expected.

DISTRIBUTION OF THE HEAT ENERGY.

According to the first calculation above we have from the 700 grams
of food consumed a balance of 2,686,200 calories. It remains to show-
about how this may be dissipated. If retained in the body it would
soon bring the latter to the boiling point. But the heat liberated in
the combustion of the food finds several outlets, the most important of
which will be now indicated. In the first place the urine and feces
leave the body at a temperature much higher than that of the water
consumed; the water of respiration and perspiration has to be vapor-
ized at the expense of heat ; the air inhaled is warmed to a temperature
of 370, which is in the mean 20 ° higher than when taken in. The
specific heat of the air (at constant pressure) is about 0.25. We have
then, approximately, the following relations, assuming 15 kilograms
of air to be inhaled in the 24 hours :

To warm 15,000 gm. air 200 7S,ooo cal.

To warm 1,557 &m- urine and feces 200 31,140

To evaporate 904 gm. of water (904 X 580) 524,320

630,460

THE ENERGY EQUATION. 371

This number of calories must be taken from the net produced calories
to obtain the heat radiated or otherwise lost by the body. We have
then this difference :

2,686,200
630,460

2,055,740
That is, something over 2,000,000 calories are dissipated by radiation.

HEAT RADIATION WHEN WORK IS DONE.

All these calculations are based on the assumption that no mechan-
ical work is being done by the person under observation, or if done it
is finally all converted into heat. In experiments in the respiration
calorimeter a very close agreement is found between the calculated
and observed heat or energy liberations. This is illustrated by the
results of one of the Atwater and Rosa experiments. The figures are
the daily means from tests running through 4 days :

Total energy of food, determined 3,6/8 Cal.

Energy of urine and feces 264

Net energy 3,4T4

Energy of fat lost 488

3,002
Energy stored as protein 38

Total energy of material actually oxidized 3,864

Heat actually measured 3,739

125

There is, therefore, a difference of only 125 large calories in this test,
which was one of the early ones with the new apparatus. In later
experiments described by Atwater and his colleagues much closer
results have been reported, which shows the general correctness of the
method of calculation followed.

Effect of Work. To maintain the individual at work a greater
expenditure of energy is necessary, and the total energy of substances
metabolized must be balanced by the heat liberated and external work
done, fn this connection it may be well to recall some relations first
pointed out by flirn, in which ;i comparison is drawn between the
work of man and an engine. In both cases the work is accomplished
through the expenditure of the potential energy stored up in carbo-
naceon- substances, food in the one instance, coal in the- other. As the
illustrations above show, the heat from the food is practically constant,
whether it be evolved through oxidation in the body or in a calorimeter.

372 PHYSIOLOGICAL CHEMISTRY.

Imagine now a small steam engine burning a constant amount of coal
inside a calorimeter. In one case let no work be done by the piston;
the heat of the steam is not employed in expansion, but is totally
absorbed by the water of the calorimeter, which takes up a certain
number of calories that may be accurately noted. In a second case
allow the same amount of coal to be burned under the boiler of the
small engine in the same time, but let the engine do work outside the
calorimeter, which may be accomplished, for example, by means of a
small shaft passing through the walls of the calorimeter in such a way
as to convey no appreciable amount of heat. It will now be found that
the gain in temperature in the water of the calorimeter is- less than
before for the same coal consumption, and that the difference is meas-
ured by the external work alone. One calorie less in the calorimeter
heat corresponds to 423.5 gram-meters of. work done through the
agency of the shaft.

With an animal the case is different. The doing of external work
necessitates always the burning of more food than is the case with the
fasting metabolism, when the energy requirement is for doing internal
work, as will be explained below. In the engine the amount of coal
burned may be constant whether work is done or not. However, with
the animal this result is noticed : Increased food consumption, with
increased oxidation, may not be accompanied by an increase in work;
in this case there must be an increased liberation of heat. If work is
done, there is still an increase in the liberation of heat, but, as with the
machine, we must subtract the heat equivalent of the accomplished
work. A part of the increased heat liberation is called for by increased
internal work also. It follows, therefore, that the working animal is
warmer than the passive animal, but the increase is not proportional
to the food consumed or oxygen absorbed.

External Work Equivalent. Although the animal is able to con-
vert but a limited portion of the potential energy of the food into
external work, as a machine it is still much more perfect than the steam
engine. This is especially true of man. In the best steam engines
not more than about 12 per cent of the potential energy of the fuel
can be recovered in the form of work. In animals, through a short
period, the transformation may amount to as much as 35 per cent
of the net available potential energy. In making such comparisons,
however, it must be remembered that the animal can work but a limited
time. In the rest periods of the animal the loss of heat, without any
corresponding mechanical gain, goes on.

THE ENERGY EQUATION. 373

The law defining the maximum conversion of heat into work through the steam
engine is well known. The extent of the limitations in the animal are not known.
In the steam engine the limitation depends on the relation of the highest heat of the
steam to the temperature of the condenser. If T is the absolute temperature of
the live steam and t the temperature of the condenser the maximum transforma-
tion of heat into work cannot be greater than

T — t

THE INTERNAL WORK OF THE ANIMAL.

Even when the animal appears passive a great deal of work is going
on which may be described as internal work. The nature and extent
of some of this is known with a fair degree of accuracy, while for the
extent of the metabolism corresponding to other kinds of work we
have not much beyond conjecture. It is possible to calculate approxi-
mately the work done in maintaining the circulation of the blood, and
in respiration, and some attempts have been made to estimate the work
of the other muscles at rest ; but to approximate the work done in mas-
ticating, digesting, transporting and transforming the food stuffs is
much more difficult. The work of the heart alone has been estimated
at from 20,000 to 60,000 kilogram-meters in 24 hours. Three thou-
sand Cal. of heat liberated would correspond to 1,270,500 kilogram-
meters of work; hence the heart work in forcing the blood through the
vessels may amount to as much as 5 per cent of the whole metabolism.

In respiration the work done is largely the expansion of the thorax
against the atmospheric pressure and the elastic tension of the rib
cartilages and the lungs. The conditions for estimation are perhaps
more favorable than in the other case. It has been calculated that 4
to 5 per cent of the whole metabolism is called for by this work.

In maintaining the tonus of the great mass of skeletal muscles of
the body it is likely that a large metabolism is required. The muscular
part of the body is not far from 10 per cent in the mean, or 40 per
cent of dry substance. In the body of a man weighing 75 kilograms
we have therefore about 7.5 kilograms of muscle substance. A large
fraction of this falls to the so-called skeletal portion which exists
always in a peculiar tense condition. In keeping up this condition
without doing outside work oxidation is necessary. Glycogen is split
up and water and carbon dioxide appear. The only visible effect of
this metabolism is the production of heat. When the muscle docs
outside work, as in lifting a weight, although the heat production may
be greater in the sum, it is relatively less in proportion to the oxygen
consumption. A pari of the energy is consumed in lifting the weight.

374 PHYSIOLOGICAL CHEMISTRY.

Heat Production Incidental. It is possible that the whole heat
liberation, at times, is but a result of the various kinds of internal work
done, and that no oxidation takes place for the simple production of
heat. This may be the case at relatively high temperatures. At cer-
tain lower temperatures, on the other hand, it is apparent that a part
of the heat liberation is called for independently of that transformed
in the internal work. A certain heat production is necessarily con-
nected with the performance of the various body functions and this
down to some particular external temperature limit is sufficient for the
heat demands of the body. Numerous experiments have shown that
for each animal species there is an external temperature at which the
general metabolism, as indicated by carbon dioxide excretion or oxygen
consumption, and the resultant heat liberation, reach a minimum value.
This temperature limit has been called the critical temperature; below
it the metabolism and heat liberation increase, evidently not in conse-
quence of a call for more work but because of the necessity for more

heat.

ISODYNAMIC RATIOS.

In metabolism, fats, carbohydrates and proteins all yield kinetic
energy and to a large extent each one is capable of replacing the
others, a certain minimum of protein being always, of course, neces-
sarily present. The proportions in which they may replace each other
may be found by a variety of experimental methods, which yield fairly
concordant results. The foods may be burned in a calorimeter and the
heats of combustion noted, or their values may be compared through
the amounts of oxygen required by calculation to oxidize them, or
finally animal experiments in the respiration calorimeter may be re-
sorted to to fix the relative values. The isodynamic relations are
given in this table calculated from results of animal experiments.

ioo gm. of fat =

Lean meat, dry 243 gm.

Cane sugar 234 gm.

Glucose 256 gm.

Starch 232 gm.

For such substances the calculated and observed values, or the com-
bustion calorimeter and the respiration calorimeter, give closely agree-
ing results, but it must be remembered that many compounds which
show considerable value as measured by combustion are absolutely
worthless as measured by nutrition. Creatinine and urea are illus-
trations ; both are products of metabolism. On the other hand, alcohol
shows about the same value in the respiration calorimeter as it shows

THE EXERGY EQUATION. 375

in the combustion calorimeter, and its metabolic value would therefore
appear high. However, the actual food value of alcohol is practically
low because limited by its toxic action.

FOOD CONSUMPTION IN SEVERE MUSCULAR EXERTION.

In the last chapter reference was made to earlier discussions on the
question of the kind of food which must be metabolized to enable the
animal organism to do work. By many authorities heat liberation was
looked upon as an end in itself, and hence foods were divided into the
two classes : those important in the production of heat and those impor-
tant in the production of outside work. The fats and carbohydrates
are found in the first group and the proteins in the second. One of
the earliest experimental investigations on the subject was the classic
one of Fick and Wislicenus, already referred to. These two men in
1866 made the ascent of the Faulhorn in the Swiss Alps, from a
known level, and determined the excretion of nitrogen, as urea, during
and following the ascent. As the elevation to which they ascended
was known, it was possible to make some calculations, approximately
accurate, of the work done in the ascent. Two things were shown
especially by the tests and calculations : there was not a great increase
in the urea excreted, and secondly the protein metabolized, as indicated
by the urea measured, was not at all sufficient to account for the work
done. Their own conclusions were that the protein consumed would
not furnish more than half or three- fourths the energy necessary to
lift their bodies through the 1956 meters of ascent, to say nothing of
the work done in the horizontal direction on a winding pathway, or
of the internal work of the body. This experiment attracted a great
deal of attention. Frankland made a new determination of the heat
of combustion of protein and showed that the value assumed by Fick
and Wislicenus was far too high, thus making the discrepancy still
greater.

Since then many similar observations have been made which show
pretty clearly that when fats or carbohydrates are abundant in the food
there is no excessive destruction of protein in the performance of ordi-
nary work. With the ingestion of a small amount of protein it is easy
to cover the normal metabolism. But the case is different when the
work is hard. Here, even with abundant food and plenty of protein,
there appears to be some loss of nitrogen by the body. In other words,
more tissue is broken flown than is formed new. This is brought out
clearly in the observations of Atwater on the work done by bicyclers
in a six-day race some year-, ago, in which the food consumed and

376

PHYSIOLOGICAL CHEMISTRY.

nitrogen eliminated were carefully watched. The following table
gives a summary of the most important observations, with the energy
in large calories :

Average

Miles

per

Day.

Protein Daily.

Energy Daily.

Rider.

In Total
Food.
Grams.

In Available

Food.

Grams.

Metabolized.
Grams.

In Total
Food.
Lai.

In Available
Food.
Cal.

Metabolized.
Cal.

A
B
C

334-6
3°3 -8

287.7

169
179
211

158
163
197

223
223
243

4,957
6,300
4,898

4,547
5,871
4,323

4,789
6,066
4,464

Riders A and B rode through six days. C rode three days. The
energy metabolized does not include that from body fat, which may
have been considerable.

DIETARIES.

The question of the proper amount of food and the character of the
food for different kinds of work has been very thoroughly studied in
the past few years and a large number of observations on individuals,
families and communities of soldiers, prisoners and paupers have been
collected. The diet in some cases is known to be sufficient, in others
insufficient. From present experience it is possible to say of many
dietaries that they are excessive, and probably objectionable in conse-
quence. The following table illustrates the dietaries of a great many
people living under different conditions. The figures are taken mainly
from the compilations of Konig and Atwater:

Occupation, Etc.

Italian laborers, Chicago

Bohemian laborers, Chicago

Russian laborers, Chicago

Laborers, crowded district, New York.

Laborers, low income, Pittsburgh

Mechanics, eastern and central U. S. . .

French Canadians, Chicago

French Canadians, Massachusetts

American professional men

Bavarian workmen, high class

Munich prisons, work

Munich prisons, no work

Bavarian soldiers, war

Bavarian soldiers, garrison

(J?

t >

Protein,

Fat,

Carbohy-

.a a

Grams.

Grams.

0.2

4

I03

Ill

391

8

115

103

36o

9

137

102

4l8

19

106

117

367

2

8l

97

311

14

103

150

402

5

Il8

158

345

5

III

193

485

14

I05

124

420

3

151

54

479

104

38

521

87

22

305

145

100

500

120

56

500

Calories.

3,060
2,885
3,232
3,030
2,5IO
3,465
3,365
4,235

3,335
3,o85
2,916
1,819
3,575
3,063

The above results are fairly representative and show that in general
over 3,000 Cal. per day must be provided in the food. In the figures
the available or net calories are calculated from 1 gm. protein or
carbohydrate = 4. 1 Cal., 1 gm. fat = 9.3 Cal. But many extreme

THE ENERGY EQUATION. 377

results are also found in the literature. For prisoners confined in
cells and not working, for paupers in asylums, and even with laborers
poorly paid, the foods consumed may not yield 1,500 Cal. On the
other hand, workmen in the American winter lumber camps, who as a
rule are well paid, workmen in the building trades on outside work in
the colder weather, teamsters and car drivers who are constantly
exposed to the weather, even when the work is not excessive, may con-
sume a diet yielding 4,000 or even 5,000 Cal.

Special Diets. With such facts as the above in mind it is not diffi-
cult to understand why nutrition with a single article of food is unsat-
isfactory. Assume, for example, the case of a diet of potatoes of
which the edible portion shows in the mean about these per cent values :
protein 2.2, fat 0.1, carbohydrates 18.4. One hundred grams of pota-
toes would yield then the following :

Protein 2.2 gm. 9.0 Cal.

Fat 0.1 0.9

Carbohydrate 18.4 75.4

A pound of potatoes would furnish, therefore, 386 Cal., and 6 to 8
pounds would have to be consumed to furnish energy for ordinary
work. The protein in this would be considerably below the amount
which has generally been held necessary, while the fat is scarcely
appreciable. The storage of energy on such a diet would be practically
impossible.

On the other hand, a diet of lean meat, round steak, for example,
would be almost as bad. This averages about 20.9 per cent of protein
and 10.6 per cent of fat, from which 100 grams would furnish:

Protein 20.9 gm. 85.7 Cal.

Fat 10.6 98.6

1843

A pound would furnish 835 Cal. and about 3 to 4 pounds would have
to be consumed for support of the body daily. While the fat in this
would be proper the protein would amount to 275 grams at least, and
make the work of excretion extremely difficult.

As a third case a diet of the small white beans may be considered.
We have here protein 22.5, fat 1.8, carbohydrate 59.6. In 100 grams,
therefore,

Protein 22.5 gm. 92.2 Cal.

Fat 1.8 16.7

Carbohydrate 59.6 244.3

353-2

37^ PHYSIOLOGICAL CHEMISTRY.

A pound would furnish about 1600 Cal. and 2 pounds would cover the
needs of the body practically. The proteins in this would be but
slightly excessive, while the same would be true of carbohydrates.
The trouble is with the deficiency in fat. Notice how easily this may
be corrected. A pound and a half of beans cooked with one fourth
pound of fat pork will yield over 3,000 Cal. and furnish a diet easily
assimilated by men at moderate work. What is said of the bean is
practically true of the pea ; each one approaches in value a mixed meat
and cereal diet.

Required Protein. A much debated question is that of the actual
protein requirement, supposing the other food elements sufficiently
abundant. The standards given above have not gone unchallenged.
Several observers have described metabolism tests in which less than
one half the 120 or more grams of protein, usually considered neces-
sary in the daily food, appears to be sufficient for all needs and able
to maintain the body in nitrogen equilibrium. Most of these experi-
ments have been, however, too short to really prove much definitely.

But recently very elaborate and long continued investigations on
groups of men have been described by Chittenden in which the evi-
dence in favor of a low protein requirement is put in an entirely new
light, and in which it is also shown that the 3,000 large calories of
energy in our food is more than necessary for ordinary practical needs.
In a group of five professional men Chittenden found an average nitro-
gen liberation corresponding to the metabolism of something over 46
gm. of protein daily and an average energy value in the whole food of
about 2,300 calories through a period of six to nine months. In a
group of thirteen soldiers, taking abundant exercise, there was a daily
consumption of food having an average value of about 2,600 calories,
and an average protein consumption of about 56 gm. through five
months, fall, winter and early spring. In a group of seven student
athletes a protein consumption of about 61.5 gm. daily with a total
food consumption equivalent to about 2,575 calories was observed.

In all these cases the tests were continued long enough to bring the
men into practical nitrogen equilibrium with good physical condition
and good general health. For this reason they deserve the fullest
attention and study. It is the opinion of the author of the experi-
ments that increased food consumption, so far from being necessary,
is even in most cases a detriment, since it calls for a large amount of
extra internal work, in the liver and kidneys especially, in metabolizing
the digestion products and in removing the waste. This is certainly
a consideration of some moment. How far these findings may be

THE ENERGY EQUATION. 379

applied to the case of men at hard work, in the open, in cold weather,
remains to be tried. Chittenden's results are especially interesting
with respect to the necessary nitrogen; but for the hard-working
man probably more fat and more carbohydrate will always be found
desirable.

INDEX

Abnormal colors in urine, 325
Absorption analysis, 194

cells for spectroscope, 200

from stomach, 143

ratios, 201
Acetic acid, 40

fermentation, 115
Acetoacetic acid in urine, 322
Acetone in urine, 322
Achroodextrin, 27
Acid albumin, yy

fermentation, 159
of urine, 349
Acid, acetic, 40

alloxyproteic, 302

amino-acetic, 61
caproic, 62
glutaric, 62
isobutylacetic, 62
propionic, 62
succinic, 62
valeric, 62

antoxyproteic, 302

arabic, 38

arabonic, 18

arachidic, 40

aspartic, 62

behenic, 40

butyric, 40, 119

capric, 40

caproic, 40

caprylic, 40

carbonic, 64

cholalic, 265

cholanic, 265

choleic, 265

chondroitin sulphuric, 89

cresyl sulphuric, 303

dextronic, 18

elaidic, 46

erythritic, 18

fellic, 265

formic, 40

glutaminic, 62

glyceric, 18

glycerophosphoric, 48

Acid, glycocholic, 264

glycollic, 18

hippuric, 301, 334

hypogseic, 41

indoxyl sulphuric, 303

lactic, 117

in stomach, 134

lauric, 40

linoleic, 41

lithofellic, 265

mannonic, 18

margaric, 40

myristic, 40

nucleic, 86

cenanthylic, 40

oleic, 41

oxalic, 18

oxyphenyl amino propionic, 63

oxyproteic, 301

palmitic, 40

parabanic, 308

paralactic, 281

pelargonic, 40

pentoic, 40

phenyl amino propionic, 63

phenyl sulphuric, 303

phosphoric, urinary, 304

picric, 56

propionic, 40

pyrrolidine carboxylic, 62

ricinoleic, 41

saccharic, 18

sarcolactic, 119

skatoxyl sulphuric, 303

stearic, 40

tartaric, 18

tartronic, 18

taurocholic, 264

thiolactic, 281

trioxyglutaric, 18

undecylic, 40

uric, 297

valeric, 40
Acidity of gastric juice, 137
Acids in fats, 40
Acid zone, 158

380

INDEX.

381

Acrose, 19
Activators, ioi, 157
Addiment, 225
Adenine, 87
Adipocere, 45
Adrenalin, 273
Agar-agar, 38
Agarose, 27
Agave sugar, 27
Agglutinins, 221
Air, 12

tests, 13
Alanine, 62

Alanylglycylglycine, 149
Albumin, 51
Albuminates, 77
Albuminoids, 53, 90
Albumin in urine, 314
Albumins proper, 66
Albumoses, 80, 81
Alcoholic fermentation, 112, 113
Alcohol in wine, 113
production, 113
test for, 113
Aldopentoses, 18
Alexins, 224
Alkali albumin, 77
Alkalies and protein, 61
Alkaline zone, 158
Alkaloid reagents and proteins, 56
Alloxyproteic acid, 302
Alpha naphthol test, 23, 58
Amboceptors, 226
Amino acids, 61

as digestive products, 150
Aminocaproic acid, 62
propionic acid, 62
valeric acid, 62
Ammoniacal copper solution, 29
Ammonia, determination of, 330
in urine, 296
in water, 10
Ammonium cyanate, 296
Amniotic fluid, 233
Amorphous phosphates in urine, 353
Amount of acid in stomach, 135

sugar in urine, 320
Amphopeptone, 83
Amygdalin, 106
Amylodextrin, 37
Amyloid degeneration, 92

substance, 92
Amylopsin, 103

Amylase, 103
Amylose, 33
Analysis, spectrum, 197
Analyses, ash of milk, 240, 241
ash of muscle, 283
bile, 263
blood, 176
bone ash, 286
cells of thymus, 233
cerebrospinal liquid, 277
colostrum, 241
feces, 164, 166
gall stones, 270
hydrocele fluid, 233
lymph, 230
meat extract, 284
milk of cow, 236
mother's milk, 245
muscle, 278

peritoneal transudate, 233
pleural transudate, 233
pus cells, 234

serum, 233
spermatic fluid, 275
urine, 292
Animal foods, 293

internal work of, 373
starch, 36
Animals and plants, 3
Anti bodies, chemical nature, 223

development of, 220
Anti body defined, 217
Anti group, 81
Antipeptone, 83
Antitoxins, 218
Antoxyproteic acid, 302
Apparatus for freezing point, 207
Arabinose, 18, 20, 38
Arabitol, 18
Arabonic acid, 18
Arachidic acid, 40
Arginine, 61, 84
Argon in blood, 190
Aromatic products from intestinal

putrefaction, 160
Artificial purification of water, 9
Ash in tissues, 14

of milk, 240
Aspartic acid, 62
Assay of pepsin, 131
Atwater, food standards, 376
Autodigestion, 256
Autolysis, bacterial products from, 257

382

INDEX.

Autolysis, importance of, 257

organic acids- from, 257

pancreas, 272

protein in, 257
Autolytic fermentation, 255

Bacteria in feces, 163

in urine, 348

lactic acid, 117
Bacterial process, 158

purification of water, 9
Bactericidal products of autolyses, 257
Bacteriolysins, 219
Bacteriolytic processes, 117
Bases in body, 16
Beckmann apparatus, 207
Beef extract, 280

composition, 93

pancreas, extracts from, 146
Beeswax, 49
Beet sugar, 25
Behavior of trypsin, 145
Behenic acid, 40
Benedict and Gephart, urea method, 330

total sulphur in urine, 339
Benjamin Thompson, 6
Benzoates and hippuric acid, 301
Benzoic acid, 106
Bernard, C. L., 5
Berzelius, 4

Bicycle rider, food and work, 376
Bile, 262

acids, 265

in feces, 169
optical rotation, 266

colors in feces, 174

composition, 263

concretions, 270

emulsification by, 269

pigments, 266
Bilicyanin, 271
Bilifuscin, 271
Bilihumin, 271
Biliprasin, 271
Bilirubin, 188, 263, 267
Biliverdin, 188, 265, 267
Biology, field of, 2
Bismuth test, 23
Bitter almonds, 106
Biuret, 57

reaction, 57
Blood, 175

albumin, 66

Blood, analyses, 176

and bile pigments, 185

anti bodies in, 218

ash of, 14

casts, 346

cholesterol in, 191

conductivity, 212

corpuscles in urine, 342

cryoscopy, 206

freezing point, 206

gases of, 190

in tissue, 191

in urine, 326

lecithins in, 191

optical properties, 193

osmotic pressure, 204

phagocytes in, 216

salts of, 190

self preservation, 216

serum tests, 219

sugar in, 189

tests, clinical, 201

transfusion, 192
Boas' reagent, 134
Body, bases in, 16

composition of, 7
Bogg's coagulometer, 180
Bone, 285

ash of, 14, 286

gelatin from, 90

glue from, 90

marrow, 287

ossein in, 256
Brain and nerve substance, 276
Bran, pentose in, 20
Bread, composition, 94

fermentation in, 118
British gum, 35

Brunner's glands, juice from, 156
Buchner, ferments, 99
Bunge, 248

Burchard-Liebermann test, 50
Butter, 46

composition, 238
Butter milk, 242
Butyric acid, 40, 119

fermentation, 117

Cadaver wax, 45

Calcium and magnesium salts in urine,
293

in body, 8

oxalate in sediment, 352 '

INDEX.

333

Calcium sulphate in sediment, 352

Calculation of food energy, 368, 369

Calculi, 354

Calves stomach, rennet in, 108

Calorie, definition, 366

Calories in foods, 93, 94

Cane sugar, 25

group, 25
Caproic acid, 40
Caprylic acid, 40
Carbohemoglobin, 186
Carbohydrates, 17

changes in liver, 253

digestion, 103, 152

group in proteins, 81

heats of combustion, 367

in urine, 304
Carbonates of body, 15
Carbon balance, 357

dioxide and plant-life, 2

in body, 8

monoxide hemoglobin, 184
Carnine, 280
Carnitine, 280
Carnosine, 280
Cartilage, 287

ash of, 14

gelatin from, 90

mucoid in, 89
Casein, 72

in feces, 173

preparation, 239
Caseoses, 81
Casts in urine, 345
Catalytic action, 98
Cat fat crystals, 43
Cell globulin, 69
Cells, conductivity, 214

epithelium in urine, 343

in general, 249

lymph, 233
Celluloid, 30
Cellulase, 104
Cellulose, 38

in feces, 171
Centrifuge, uses of, 341
Cereals, composition, 94
rospinal liquid, 276
Character of antibodies, 218
Charts, Tallquist's, 203
Cheese, composition, 93
Chemical nature of anti bodies, 223
Chemistry of milk, 238

Chittenden, 53

preparation of pepsin, 130

protein classification, 81

protein requirement, 378
Chlorides, determination of, 2>2>7

in water, 10

of body, 8, 15

tests for, 10
Chlorophyll-bearing plants, 2
Cholagogues, 268
Cholanic acid, 265
Choleic acid, 265
Cholesterol, 49, 270

in blood, 191

in feces, 169

in protoplasm, 250

optical rotation, 271
Choline, 48
Cholalic acid, 265
Chondroitin, 89

sulphuric acid, 89, 287
Chondromucoid, 89, 287
Chromophoric group, 136
Chyle, 232

Classification of proteins, 52
Clinical blood tests, 201

uses of hematocrit, 211
Clumping, bacteria, 222
Clupein, 75

Coagulated albumins, 76
Coagulating proteins, 69
Coagulation of blood, 177

tests, 54
Coagulometer, 180
Coefficient, isotonic, 208
Co-enzymes, 101
Cohnheim, 156

theory of protein metabolism, 290
Collagen, 90

from muscle, 279
Collodion, 39

Coloring matter in urine, 323
Color of urine, 292
Colostrum, 241
Combustion, heats of, 367
Commercial bile salts, 269

pepsin, 109, 130
Complement, 225
Complementoids, 228
Component groups in proteins, 60
Composition of body, 7

of cells, 249

of lymph, 230

3§4

INDEX.

Composition of milk, 237
Conductivity cells, 214
of blood, 212
of urine, 309
Congo red test, 133
Coniferin, 107
Conjugated proteins, 54
Conservation of energy, 6
Conversion of starch, 37
Corpuscles, number in blood, 211
Count Rumford, 6
Cow's milk, 236
Creatine, 279
Creatinine, 279

determination, 333
from urine, 300

reducing power, 307
Crude fat in feces, 168
Cryoscopy, 206, 312
Crystallin, 69
Crystals of fats, 43

of hemin, 181
Curtius, 64
Cystin, 92, 262

calculi, 355

in urine, 351
Cytase, 104
Cytosine, 87
Cytotoxins, 219
Dare's hemoglobinometer, 202
Dehydrocholeic acid, 265
Denatured proteins, 77
Derived products, proteins, 53

protein, 54
Despretz, 5

Destruction of glycogen, 255
Detection of free acid in stomach, 133
Determination of acid in stomach, 135

of albumin in urine, 314

of ammonia in urine, 330

of blood colors, 197

of chlorides in urine, 357

of creatinine in urine, 333

of digestive power, pepsin, 131

of electrical conductivity, 212

of fats, 47

of fats in milk, 244

of feces fat, 169

of freezing point, 207

of hemoglobin, 203

of hippuric acid, 334

of nitrogen in feces, 172

of nitrogen in urine, 327

Determination of osmotic pressure, 209
of pepsin, 139

of phosphates in urine, 335
of proteins, 59

of milk, 244
of purine in urine, 333
of specific gravity, 313
of sugar, 27
in milk, 244
in urine, 320
of sulphates in urine, 339
of total sulphur in urine, 339
of urea in urine, 328
of uric acid in urine, 332
Deutero albumose, 83
Dextrin, 35

from starch, 123
Dextronic acid, 18
Diabetes mellitus, 305
Diagram of spectroscope, 194 '

Wheatstone bridge, 212
Dialyzer, 67
Diastase, 97, 103

action of, 123
Diazo reaction, 162, 326
Diet and feces, 165
Dietaries, 376
Diets, special, 377
Digestion, 96

of fat's in stomach, 143
of starch, 122
pancreatic, 144
peptic, 109, 127
salivary, 121
tryptic, no
Digestive extracts, 146
Diglycylglycine, 149
Dilution test, 315

Dimethylaminoazobenzene test, 133
Diose, 18
Dioxyacetone, 18
Direct vision spectroscope, 194
Disaccharides, 17
Distearin, 41

Distribution of food energy, 368
of heat energy, 370
of nitrogen in urine, 295
Donne's test, 343
Dulong, 5

Edestan, 69
Edestin, 69
Edible fats, 41

INDEX.

3»!

Effect of work, 371
Egg albumen, 67

and pepsin, 131
Ehrlich reaction, 162
Ehrlich's theory, 224
Elaidic acid, 47
Elaidin, 47
Elastin, 92
Electrical conductivity of blood, 212

of urine, 309, 310, 311
Elements in body, 7, 8
Emulsin, 106
Emulsions, 42, 269
Endothermal reactions, 2
End products of digestion, 150

of metabolism, 289
Energy balance, 289
equation, 366
of food, distribution, 368
Enterokinase, 157
Enzymes, 96

as catalytic agents, 100

of stomach, 127
Epinephrin, 273
Epithelium in urine, 343
Erepsin, no, 156
Erg, definition, 367
Erythritic acid, 18
Erythrodextrin, yj
Erythrogranulose, 37
Erythrol, 18
Erythrose, 18
Esbach albuminometer, 139, 315

reagent, 139
Ethereal sulphates, 162, 261
Ewald test meal, 132
Examination of stomach contents, 132
Excretion by skin, 363

gaseous, 356

of alkali salts, 293

of calcium and magnesium, 293

of nitrogen, 289

of phosphorus, 304

of sulphur, 302
External work equivalent, 372
Extinction coefficient, igg
Extract of meat, 283
Extracts from yeast, 114
Extractives from muscle, 277
Exudations, 232

Fat and chyle, 232
crystals, 43
26

Fat from muscle, 279

globules, 239

in urine, 351

in feces, 164

in foods, 93, 94

of milk, 238
Fats, 40

from proteins, 44
sugars, 44

heats of combustion, 367

in blood, 191

in body, 44

in pancreatic digestion, 154

solubility, 44

splitting of, 107
Fatty acids, 40
Faulhorn experiment, 375
Feces, 161, 163

amount of, 164

bile acids in, 169

blood and pus in, 174

carbohydrates in, 170

cellulose in, 171

composition, 164

from various foods, 165

lecithin in, 168, 170

nitrogen in, 172

proteins in, 173

starch in, 170

sugar in, 171
Fehling reduction, 28

test, 22

urine, 318
Fellic acid, 265
Fermentation, acetic, 115

alcoholic, 113

autolytic, 255

butyric, 97, 117

in intestines, 158

lactic, 97, 117

mucous, 119
Ferments, 96

classification, 102
Ferric chloride test, 323
Fibrin, 70, 177

digestion of, 147
Fibrinogen, 70
Fibronoses, 81

Fick and Wislicenus experiment, 375
Fields of study, 2
Filter paper, 38
Fischer, 64, 225

nomenclature of purines, 298

386

INDEX.

Fish, composition, 93
Fission fungi, 112
Fleischl hemometer, 201
Flesh bases, 279
Flour, composition, 94
Fluorine in body, 8
Folin, ammonia in urine, 331

creatinine method, 334

sulphate method, 339

theory of protein metabolism, 290

urea method, 330

uric acid method, 332
Food and work, 6

consumption and muscular work, 375

of plants, 3
Foods, relation to feces, 165
Food stuffs, 93

Formaldehyde condensation, 2
Formic acid, 40
Formulas for hemoglobin, 187

spectrophotometry, 199
Fraunhofer lines, 193
Free acid in stomach, 132
Freezing point of blood, 206

urine, 312
Fructose, 18, 24
Fruit sugar, 20

Fuel value of foods, 93, 94, 367
Functions of bile, 268

liver cells, 251

lymph, 231
Fungi and fermentation, 112

in urine, 347
Furfuraldehyde, 20
Furoaniline, 20

Gadus-histone, 75
Galactose, 24, 105, 240
Gallstones, 49, 270
Gaseous excretions, 356
Gases in air, 12

of blood, 190
Gastric juice, 126
acidity, 137
titration of, 138
Gelatin, 90, 285

tests for, 91

uses, 91
General composition of urine, 291

relations, 1
Gland, thyroid, 274
Gliadin, 73
Globin, 74

Globulinoses, 81
Globulins, 68

in urine, 315
Glucase, 104
Gluco-proteids, 88
Glucosamine, 63
Glucose, 21

from starch, 21

in blood, 189

reducing power, 28
Glucoses, 18
Glucosides, 20
Glucoside reactions, 106
Glucoronic acid in urine, 322
Glue, 90

Glutaminic acid, 62, 84
Glutelins, 74
Gluten, 73, 94
Glutenin, 73
Glutin, go
Glyceric acid, 18
Glyceraldehyde, 18
Glycerol, 18, 47
Glycero-phosphoric acid, 48
Glycerose, 18
Glyceryl butyrate, 46

caproate, 46

oleate, 46
Glycine, 61
Glycocoll, 61, 264
Glycogen, 36, 280

destruction, 255

formation, 253

in flesh, 281

in protoplasm, 250

stored in liver, 254
Glycol, 18
Glycollic acid, 18
Glycocholic acid, 264
Gmelin, 4
Gmelin's test, 267
Goitre and iodine compounds, 274
Gower's hemoglobinometer, 203
Graham dialyzer, 67 >

Grain composition, 94
Granular casts, 346
Granulose, 33
Grape sugar, 21
Group, immune, 227

zymotoxic, 228
Groups in protein, 60
Guaiacum test, 180, 327
Guanine, 87

INDEX.

387

Guenzberg's reagent, 133
Gum arabic, 38

British, 35
Gums, 37
Gun cotton, 39

Hair, keratin from, 288
Hamburger, 209
Hammarsten's test, 268
Haptophorous group, 226
Hard water, 8
Heart, ash of, 14
Heat and food stuffs, 6

energy, distribution, 370

mechanical equivalent, 367

of friction, 6

production incidental, 374

radiation, 371

unit, definition, 366
Heats of combustion, 367
Hematin, 187

spectrum, 196
Hematocrit, clinical uses, 211

methods, 210
Hematogen, 73
Hematoidin, 188
Hematolin, 187, 188
Hematoporphyrin, 187
Hematuria, 326
Hemi group, 81
Hemin crystals, 181
Hemochromogen, 187, 188
Hemoglobin, 74, 181

analysis, 182

combinations, 182

crystals, 183

specific rotation, 182
Hemoglobins, 87
Hemoglobinometer, Dare's, 202

Gower's, 203
Hemoglobinuria, 326
Hemometer, Fleischl's, 201
Hemolysins, 219
Heteroalbumose, 82
Hexitols, 18
Hexone bases, 61

in digestion products, 149
Hexoses, 18, 20
Hippuric acid, 301

determination, 334
in sediment, 352
Hirn, comparison between man and
machine, 371

Histidine, 61

Histones, 74

Historical sketch, 4

History of fermentation, 97

Hofmeister, 64

Hog pancreas, extracts from, 146

Hoppe-Seyler, 5

Horn, composition, 93

keratin from, 288

substance, 92
Human fat, 47

milk, 245
Hyaline casts, 346
Hydrazones, 20
Hydrocele fluid, 233
Hydrochloric acid in stomach, 126
Hydrocyanic acid, 106
Hydrogen in body, 8

peroxide test, 181
Hydrolysis of proteins, 60

starch, 22, 122
Hydrolytic reactions, 102
Hypogaeic acid, 41
Hypoxanthine, 87

Ichthulin, 73
Immune body, 226

group, 227
Immunization, 219
Important early works, 5

fats, 45
Indestructibility of matter, 6
Index, opsonic, 222
Indicators and stomach contents, 136

theory of, 136
Indican, 151, 161, 324
Indol, 147, 151, 160
Indoxyl, 151, 161
Inorganic elements, 7
Inosite, 281
Insoluble ferments, 99
Intermediary body, 226
Internal work of animal, 373
Intestinal bacteria, 159

changes, 158

juice, 155
Inulase, 104
Tnulin, 24, 35
Invertasc, 25, 105
Invertin, 105
Invert sugar, 25

reducing power, 28
Investigations, early, 5

388

INDEX.

Iodine in body, 8

in thyroid, 274

test, 34
Iodothyreoglobulin, 274
Iodothyrin, 275
Iron in bile, 267

in body, 8

masked, 86
Isinglass, 91
Isocholesterol, 49
Isodynamic ratios, 374
Isolation of pepsin, 129
Isomalt'ose, 27
Isotonic coefficient, 208

Joule, 6

Juice, intestinal, 155
pancreatic, 144

Kelling's test, 135
Kephir, 26, 118
Keratin, 92, 288
Ketopentose, 18
Kidney, ash of, 14
Kinases, 101, 157
Kinds of ferments, 102
Kinetic energy of food, 368
Kjeldahl test, 327
Koeppe's hematocrit, 210
Koprosterin, 271
Kruess spectrophotometer, i(
Kuehne, 5

protein classification, 81
Kumyss, 118
Kyrine, 84

Laborers, dietaries, 376
Laccase, 116
Lactalbumin, 67, 239
Lactic acid, 281

bacteria, 117

tests for, 135
fermentation, 117
Lactase, 105

Lactose, 25, 26, 105, 240
Landwehr's animal gum, 88
Lanolin, 49
Laplace, 5
Lard, 46

Laurent polariscope, 30
Laurie acid, 40
Lavoisier, 4
Lead hydroxide test, 58

Lecithan, 48
Lecithin, 48, 68
Lecithins in blood, 191

in cells, 250

in feces, 168
Legumin, 74
Lehmann, 5
Leucine, 62, 92

as urine sediment, 350

tests for, 147
Leucocytes, 231, 233
Leucylproline, 149
Leuwenhock, 96
Levulose, 24
Lieberkuehn's glands, juice from, 155

jelly, 78
Liebig, 4

theory of fermentation, 98
Lignocellulose, 39
Linoleic acid, 41
Lipase, 107, 126, 154
Lithofellic acid, 265
Liver and poisons, 258

ash of, 14

autolysis, 256

chemical changes, 252

chemistry of, 249

ethereal sulphates, 261

fats in, 251

formation of urea, 259
uric acid, 260

glycogen in, 251

iron in, 252

lecithin in, 251

mineral substances, 252

protein in, 251

synthetic processes, 259

work of cells, 252
Loewe solution, 29
Loss of free acid in digestion, 142
Lymph, 230

amount, 231

composition, 230

functions, 231
Lymphagogues, 231
Lysine, 60, 84

Magnesium in body, 8

phosphate in urine, 353
Malondiamide, 57
Malt, 103
Maltase, 104
Malt extract, 123

INDEX.

;89

Maltodextrin, 37
Maltose, 25, 104

reducing power, 28
Malt sugar, 26, 123
Margarin, 45

Manufacture of starch, 33
Maple sugar, 25
Market milk, 236
Marrow, 287
Masked iron, 86
Mayer, 6

Margaric acid, 40
Meal, composition, 94
Meat, composition, 93

extract, 283
Mechanical equivalent of heat, 367
Melibiose, 27
Melitose, 27

Meyer, blood gases, 190
Metabolism experiments, 360-362

theories of, 290
Methemoglobin, 186

spectrum, 197
Metaproteins, 80
Methyl orange indicator, 136

violet test, 133
Microorganisms in fermentation, 9J
Milk, 236

albumin, 67

ash of, 14

composition, 95

curdling ferment, 143
of, 108

fat in, 238

flavors, 247

human, 245

modified, 246

mother's, 245

of ass, 248

of bitch, 248

of elephant, 248

of goat, 248

of mare, 248

of sow, 248

origin of, 237

preservatives, 244

salts of, 240

sugar, 24, 26, 240

reducing power, 28
Milton's reagent, 56

test in digestion, 14X
Mineral matter-, in blood, 177

residues of organs, 14

Mineral substances in milk, 240
Modified albumin, 76

milk, 246
Molds in urine, 348
Molisch reaction, 83

test, 23, 58
Mannitol, 18
Mannonic acid, 18
Monosaccharides, 17
Monoses, 17
Monostearin, 41
Moore's test, 317
Mother of vinegar, 115
Mucin bands in urine, 344

in saliva, 121

in urine, 316
Mucins, 88
Mucoid bodies, 89
Mucors, 112

Mucous fermentation, 119
Mucus in urine, 342
Murexid test, 331
Muscle, ash of, 14

extraction, 71

plasma, 71

sugar, 281

substance, 277
Musculin, 278
Mutton tallow crystals, 43
Mycose, 27
Myogen, 70, 278
Myosin, 70, 107, 278
Myosinogen, 70
Myosinoses, 81
Myricin, 49
Myristic acid, 40

Nails, keratin from, 288
Natural fats, 40

purification of water, 9

waters, 8
Native albumins, 53, 65
Nature of bile, 268
Neutral sulphur, 262, 303
Nicol prism, 31
Nitrates in water, 11
Nitric oxide hemoglobin, 185
Nitrites in water, 11
Nitrocellulose, 39
Nitrogen balance, 357

excretion of, 294

-free extractives from muscle, 280

in blood, 190

39°

INDEX.

Nitrogen in body, 8

in feces, 164, 172

of urine, distribution of, 295
Normal colors in urine, 333

feces, 163

reduction, 307
Nucleates, 86
Nucleic acid, 85, 86, 298
Nuclein, 85, 250
Nucleo-albumin, 71
Nucleo-histone, 75, 86
Nucleo-proteids, 85
Nucleus, 249

Number of corpuscles, 211
Nutrients, 7
Nutrose, 72
Nuts, composition, 94

Occurrence of metals in body, 8

Odor of urine, 292

Oil of bitter almonds, 106

Oleic acid, 41

Olein, 45

Oleomargarin, 46

composition, 93
Opsonins, 222
Opsonic index, 222

treatment, 223
Optical properties of blood, 193

rotation, 30

sugar tests, 30
Organic acids by bacteria, 141
from liver, 256
in stomach, 134

chemistry and agriculture, 4
pathology, 4
physiology, 4

matter of bones, 286
Organized ferments, 99

sediments, 341
Organs of body, ash of, 14
Origin of fats, 44
Osazones, 21
Osborne, 53, 69
Osmotic pressure, 204
cell, 205

tension, 209
Osones, 21
Ossein, 90, 286
Outline of topics, 6
Oxalate calculi, 355
Oxalic acid, 18
Oxaluramide, 57

Oxamide, 57
Oxidase enzymes, 115
Oxidases, 115
Oxidation reactions, 111

test's, 10

time and place of, 364

value of copper solutions, 28
Oxybutyric acid in urine, 322
Oxygen absorption by blood, 184

in blood, 190

in body, 8

liberation of by plants, 2
Oxyhemoglobin, 183

spectrum, 195
Oxyproteic acid, 295, 301
Oysters, composition, 93
Ozone in air, 13

Palmitic acid, 40
Palmitin, 45
Pancreas, 272

ash of, 14

autolysis, 272
Pancreatic diastases, 153

digestion, 144
of fats, 154

starch, 153

ferments, no

juice, 144
Pancreatin and milk, 243
Paracasein, 72
Paralactic acid, 281
Parasitic plants like animals, 3
Parathyroids, importance of, 275
Pasteur, 5, 97

theory of fermentation, 98
Pavy method, 321

solution, 29
Pawlow, 126
Payen, 97

Peas, composition, 94
Pectase, 104
Pectin, 104
Pectinase, 104
Pelargonic acid, 40
Pentitols, 18
Pentoic acid, 40
Pentosans, 20
Pentoses, 18, 19
Pepsin, 108, 126

amount of, 139

peptone, 150

preparation, 129

INDEX.

391

Pepsinogen, 108
Peptic digestion, 127

products of, 140
Peptones, 80, 81, 83, 108, 141

in urines, 316
Percentage variations in urine, 291
Peritoneal transudates, 233
Permanent hardness, 9
Permanganate test, 11
Peroxidases, 116
Persoz, 97

Pfeiffer phenomenon, 224
Pflueger theory of protein metabolism,

290
Phagocytes, 216
Phenylalanine, 63
Phenylalanylglycylglycine, 149
Phenyl glucosazone, 23
Phenyl hydrazine test, 21, 23

in urine, 319
Phenol in intestinal changes, 160
Phenol-phthalein indicator, 136
Phenomenon, Pfeiffer's, 224
Phosphate, amorphous, 353

sediments, 353
Phosphates, 14

determination of, 335
Phosphatides, 48
Phospho-proteins, 54, 72
Phosphorus excretion, 304

in body, 8
Physical blood tests, 204
Physiological chemistry and medicine, 5

Physiological chemistry, scope of, 2

Phytoglobulins, 69

Phytovitellins, 69

Pigments of bile, 266

Pioneer investigators, 5

Plants and animals, 3

Plasma of muscle, 70, 278

salted, 179
Plasmon, 72

Pleural transudates, 233
Poisons and liver, 258

from intestine, 162
Polarimeter, 31
Polar i scope, 30
Polarization tests, 30
Polypeptides, 64, 85, 149
Polysaccharides, 17, 32
Pork, composition, 93
Potassium in body, 8

Potassium indoxyl sulphate, 151
Practical urine tests, 313
Precipitation by salts, 55

limits, 55
Precipitins, 218

Preparation of bile acids, 265
Preservatives in milk, 244
Pressure, osmotic, 204
Primary albumose, 82

phosphates, 14
Products of peptic digestion, 140
Prolamins, 74
Proline, 62

Prolonged digestion, 142
Propepsin, 108
Propionic acid, 40
Protagon, 276
Protalbumose, 82
Protamines, 75
Proteans, 69
Proteids, 53, 85
Protein classification, 52

combination, 64

digestion, 128

in foods, 93, 94

metabolism theories of, 290

required, 378
Proteins, coagulating, 69

determination, 59

heats of combustion, 367

in autolysis, 257

in feces, 173

in urine, 305

of muscle, 278

pancreatic digestion, 146

substances, 51

synthesis, 64
Proteolytic reactions, 107
Proteoses, 81

in feces, 173

in urine, 316
Prothrombin, 178
Protones, 75
Protoplasm, 250
Pseudo acids, 56

bases, 56

cellulose, 39

pepsin, 156
Psychic stimulus, 126
Ptyalin, 97, 124
Purine, 298

bodies, 297
Purines, 87

392

INDEX.

Purines, determination in urine, 333
Pus, 232

in urine, 343
Pyrimidine bodies, 300
Pyrimidines, 87
Pyrrolidine carboxylic acid, 62

Quadriurates, 299

Quantitative composition of blood, 176

spectrum analysis, 197
Quotient, respiratory, 356

Raffinose, 27

Ratios, isodynamic, 374

Reaction, diazo, 326

of blood, 180

of feces, 166
Reactions, ferments, 97

of fats, 41

proteins, 54
Receptors, 226
Reduced hemoglobin, 195
Reducing power of sugars, 28

of urine, 307
Reduction tests, 22
urine, 318
Rennet, 108

action on milk, 242
Rennin, 72, 108, 126
Reproductive glands, 275
Required protein, 378
Resorcinol test, 24
Respiration apparatus, 357

experiments, 358

gases of, 13
Respiration in plants, 3

skin, 363
Respiratory quotient, 356
illustrations, 359
Reversible reactions with proteins, 77
Ricinoleic acid, 41
Riegel test meal, 133
Rotation, specific, 32

Saccharic acid, 18
Saccharodioses, 17
Saccharomycetes, 112
Saccharose, 25
Saccharotrioses, 17, 27
Salicin, 107
Saliva, 121
Salivary diastase, 97
digestion, 121

Salkowski's test for cholesterol, 50

Salmin, 75

Salmo-histone, 75

Salted plasma, 179

Salt, need of, 16

Salts, and proteins, 56

in body, 14

in blood, 177, 190

of casein, 72

of milk, 240

of muscle, 282
Saponification, 41
Sarcolactic acid, 281
Sauerkraut, acid in, 118
Scheele, 97
Schuetzenberger, 5
Schultz prism, 200
Schweitzer's reagent, 39
Scomber histone, 75
Scombrin, 75

Scope of physiological chemistry, 2
Secondary albumose, 82

phosphates, 14
Sediments from urine, 306, 340
Self preservation of blood, 216

purification of water, 9

regeneration of cells, 227
Semipermeable membrane, 205
Serine, 62
Serum albumin, 66

globulin, 66, 68

immunity, 227

pus, 233

tests, 219
Side chain theory, 225
Silicon in body, 8
Silver nitrate, uses of, 338

test, 10
Simple proteins, 54
Sizes of starch grains, 34
Skatol, 157, 160
Skimmed milk, 242
Skin, ash of, 14
Skin respiration, 363
Soap and hard water, 42
Soaps in feces, 168
Sodium in body, 8
Soft water, 8
Solids in feces, 167

of blood, 177

of body, 7

of milk, 243
Solubility of fats, 44

INDEX.

393

Soluble ferments, 99

starch, 33
Sorbinose, 25
Soxhlet extraction of fat, 244

sugar values, 28
Special diets, 377
Specific gravity of urine, 313
Specific rotation, 31

of arabinose, 20
of bile acids, 266
of cholesterol, 271
of dextrins, 38
of edestin, 69
of egg albumin, 67
of fibrinogen, 70
of fructose, 24, 32
of glucose, 24, 32
of hemoglobin, 182
of invert sugar, 32
of lactic acid, 282
of lactose, 26, 32
of maltose, 27, 32
of melitose, 27, 32
of saccharose, 26, 32
of serum albumin, 66
of serum globulin, 69
of xylose, 20
Spectroscope, 193
Spectrum analysis, 197
of blood, 193

of carbon monoxide hemoglobin, 197
of methemoglobin, 197
of oxyhemoglobin, 195
of reduced hemoglobin, 195
Spermaceti, 49
Spermatozoa in urine, 347
Spermine, 276
Spleen, 234

ash of, 14
Splitting of fats, 107
Starch digestion, 122
Starches, 33
Starch in feces, 170

su^ar, 21
Steapsin, 107, 154
Stearic acid, 40
Stearin, 45
Stercorin, 271
Stomach, acids in, 134
actions in, 126
contents, tests, 132
Sturin, 75
Substratum, ferment, 101

Sucrase, 105

Sudan III reagent, 48

Sugar from malt, 123

in feces, 171

in milk, 240

of blood, 189

of malt, 26
Sugars, 17

determination, 27

heats of combustion, 367

in urine, 304

determination, 320

reducing, 22

relations of, 18

synthesis of, 19

tests for in urine, 317
Sulphates, ethereal, 261

in body, 16

in urine, 303
Sulpho hemoglobin, 186
Sulphur compounds from proteins, 63

distribution of in urine, 303

excretion, 302
Sulphur in body, 8, 16

in keratin, 92

neutral, 262, 303
Supply of blood, 175
Suprarenin, 273
Suprarenal bodies, 273
Swedish filter paper, 38
Symbiotic processes, 118
Syntheses in liver, 259
Synthesis of ethereal sulphates, 261

of polypeptides, 149

of sugar, 19

of uric acid, 260
Syntonin, 79
Syrup, glucose, 22

Table of body elements, 8
Tallow, 46

crystals, 43
Tallquist chart, 203
Talosc, 24
Tartaric acid, 18
Tartronic acid, 18
Taurin, 266
Taurocholic acid, 264
Temporary hardness, 9
Tendons, mucoids in, 89
Tension, osmotic, 209
Tertiary phosphates, 14
Test, Almen's, 327

394

INDEX.

Test, biuret, 57
bismuth, 319
Boas', 134
congo red, 133

dimethylaminoazobenzene, 133
Donne's, 343
double iodide, 314
Fehling's, 22

for urine, 318
ferric chloride, 323
Gmelin's, 267
guaiacum, 180
Guenzberg's, 133
Hammarsten's, 268
Heller's, 326
hydrogen peroxide, 180
Kelling's, 135
lead hydroxide, 58
Legal's, 322
Lieben's, 322
methyl-violet, 133
Moore's, 317
murexide, 331
phenylhydrazine, 319
picric acid, 314
Struve's, 326
Tanret, 314
Trommer's, 318
Trousseau's, 325
Uffelmann's, 135
xanthoproteic, 58
Test meals, 132
Tests for acetoacetic acid, 322
acetone, 322
air, 13
albumins, 54 to 59

in urine, 314
alcohol, 113
ammonia in water, 10
bile colors, 267
bile salts, 266
blood, 180

in urine, 326
chlorides, 10
cholesterol, 50
colors in urine, 324
creatinine, 334
digestive products, 131
drinking water, 9
fats, 42, 47

in flour, 94

in milk, 242
free acid in stomach, 133

Tests for gelatin, 91

globulins in urine, 315

glycogen, 36

hemoglobin, 195

indol, 152

lactic acid, 135

leucine, 148

levulose, 24

meat extract, 285

milk constituents, 242

mucin in urine, 316

muscle extractives, 79

nitrates, 11

nitrites, 11

organic acids in stomach, 134
nitrogen, 54

oxybutyric acid, 322

pentose, 20

pepsin, 139

proteins, 54

in feces, 173

proteoses, 131

saliva, 121

starch, 34

sugar, 22

in milk, 242
in urine, 317

sulphur in proteins, 65

thiocyanates in saliva, 121

tryptophane, 147

tyrosine, 148

urea in urine, 328

uric acid in urine, 331
Tests on blood, 179
bones, 287
calculi, 354
pepsin, 331
Theories of fermentation, 98

indicators, 136

side chain, 225
Thiocyanates in saliva, 121
Thrombin, 178
Thymine, 187
Thymus cells, 233
Thyreoglobulin, 274
Thyroid gland, 274
Thyroiodine, 275

Time and place of oxidation, 364
Time of coagulation, 180
Tissue oxidation, 364
Tissues, ash in, 14

water in, 12
Titration of gastric juice, 138

INDEX.

395

Titration of stomach contents, 132
Total fat in feces, 168

hydrochloric acid in stomach, 134

nitrogen in urine, 327
Toxins, 227

from intestine, 162
Toxoids, 228
Toxons, 228

Transformation products, 53, 76
Transfusion of blood, 192
Transudations, 232
Treatment, opsonic, 223
Trehalose, 27
Triolein, 45
Triose, 18

Trioxyglutaric acid, 18
Tripalmitin, 45
Triple phosphate, 304
Trisaccharides, 17, 27
Tristearin, 45
Trommer test, 22
Trousseau's test, 325
True albumins, 53, 65
Trypsin, 83, no, 145

antipeptone, 150
Trypsinogen, 145
Tryptophane, 63, 147
Turanose, 27
Turkey, composition, 93
Tyrosine, 63, 92, 116

in urine, 350

group, 56

tests for, 147
Tyrosinase, 116

Uffelmann's test, 135
Unit of force, 367

of heat, 366

of work, 367
Unorganized ferments, 99

sediments, 341
Uracil, 87

Uranium solution, uses, 336
Urates, 299

in sediment, 350
Urea, 295

decomposition, 296

rmination, 328, 329

fermentation, 11 1

found in liver, 259

synthesis, 296
Urease, 11 1
Uric acid, 297, 331

Uric acid, calculi, 355

determination, 332

from spleen, 234

in liver, 260

reducing power, 308

sediment, 349
Urinary calculi, 354
Urine, acetoacetic acid in, 322
actone in, 322
albumin in, 314
ammonia in, 330
analysis, 313
bacteria in, 348
blood in, 326, 342
calculi from, 354
casts, 345
chlorides in, 22>7
color and odor, 292
coloring matters in, 323
conductivity, 309
creatinine, 300
cryoscopy, 312
epithelium, 343
fat globules in, 351
fermentation, in
freezing point, 312
fungi in, 347
general composition, 291
leucine and tyrosine, 351
molds in, 348
mucin, 316

bands, 344
mucus in, 342

nitrogen compounds in, 294
oxalate sediment, 352
oxybutyric acid in, 322
peptones in, 316
phosphate in, 335

sediment, 353
proteins in, 305
proteoses in, 316
purines in, 298
pus in, 343
reaction, 293
reducing power, 307
sediments, 306, 340
spermatozoa in, 347
sulphates in, 339
total nitrogen, 327

sulphur, 3yj
triple phosphate in, 353
urates in, 350
urea in, 295

396

INDEX.

Urine, uric acid in, 297

sediment, 349
xanthine bodies in, 298
Urobilin, 324
Urochrome, 324
Uroerythrin, 324
Urohematin, 324
Urophain, 324
Uses of chlorine in body, 15

Value of blood conductivity, 214
Variations in blood, 191
Vegetables, composition, 94
Vegetable proteins, 73
Vinegar, 115
Vitellin, 73

Vitreous body, mucoid in, 89
Voit, 5

theory of protein metabolism, 290
Volhard's method, 338

Water, distillation, 9

in body, 8

in tissue, 12

physiological importance, 12

purification, 9

tests, 9
Waxes, 49

Waxy casts, 347
Wheat flour, 73

starch, S3
Wheatstone bridge, 213
Whey, 240

sugar from, 26
White of egg, 67
Widal test, 222
Wood paper, 38

sugar, 20
Wool fat, 49
Works of Liebig, 4
Wohler, 4
Wright's coagulometer, 180

Xanthine, 87

bodies, 280

in urine, 298

calculi, 355
Xanthoproteic test, 58
Xylose, 20

Yeast, 112

action of, 95

Zymase, 99, 114
Zymotoxic group, 228

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