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THE PHYSIOLOGY OF THE AMINO ACIDS

Courtesy of Prof . L. B. Mendel

THE PHYSIOLOGY OF THE
AMINO ACIDS

By
FRANK P. UNDERHILL, Ph.D.

Profeaaor of Pathological Chemistry, Yale University

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New Haven: Yale University Press

London : Humphrey Milford

Oxford University Press

MDCCCCXV

and B show the contrast between two rats of the same
age, one of which, B, has been stunted. The lower two pictures
afford a comparison between two rats of the same weight, but
widely differing in age. The older, stunted rat, B, has not lost
the characteristic proportions of the younger animal, C.

THE PHYSIOLOGY OF THE
AMINO ACIDS

By
FRANK P. UNDERHILL, Ph.D.

Professor of Pathological Chemistry, Yale University

New Haven: Yale Univehsity Press

London: Humphrey Milford

Oxford University Press

MDCCCCXV

COPYRXGHT, 1915
BY

Yale University Press

First printed, November, 1915
Reprinted, June, 1916

PREFACE

During the past few years the physiology of the
amino acids has been subjected to much experimenta-
tion with the result that these protein cleavage prod-
ucts have assumed an ever increasing importance in
the problems associated with nitrogenous metabolism.
Owing largely to our too recent appreciation of the
significance of these substances in metabolic processes
there exists at present no compilation which fur-
nishes an adequate conception of the roles which may
be played by the amino acids. It has been, therefore,
the aim of the writer to gather together in one place
the results which have thus far been obtained in the
field of the biochemistry of the amino acids, thus
affording the busy practitioner, and others whose
resources for consulting original communications are
limited, an opportunity of gaining a knowledge of the
present-day problems in this field of nutrition. In the
accomplishment of this purpose the writer has made
no effort to include all the details or all the literature
available upon a given topic, but has sought rather to
indicate leading lines of thought. At the end of each
chapter are given references in which all the impor-
tant literature upon the topic discussed is cited.

It is assumed that the reader is familiar with the
fundamental principles of metabolism, hence, in gen-
eral, these have been omitted.

The author is deeply indebted to Professor Lafay-
ette B. Mendel for suggestions, criticisms of the
manuscript and for some of the plates presented.

CONTENTS

Chapter I. page

The Proteins and their Derivatives, the Amino
Acids ....... 1

Chapter 11.

Digestion, and Bacterial Activity in Relation
to the Amino Acids ..... 28

Chapter III.

The Absorption of Proteins and Amino
Acids 46

Chapter IV.

In What Form Does Ingested Protein Enter
the Circulation? . . . . .58

Chapter V.

Theories of Protein Metabolism ... 81
Chapter VI.

The Further Fate of Amino Acids . . 99

Chapter VII.

The Amino Acids in Relation to the Specific
Dynamic Action of Proteins . . . 120

Chapter VIIL
The Amino Acids and Simpler Nitrogenous
Compounds as Foodstuffs . . . 126

Chapter IX.

The Specific Role of Amino Acids in Nutrition
and Growth . . . . . .136

Index ....... 159

t

LIST OF ILLUSTRATIONS
Plate. Photograph of mice . . Frontispiece

PAGE

Figure 1. Survival periods of mice on diet of
zein and tyrosine and zein and
tryptophane .... 140

Figure 2. Growth curve of normal white rats 144

Figure 3. Growth curve with casein and milk

diets ..... 144

Figure 4. Growth curve with milk diet . . 145

Figure 5. Maintenance on casein and growth

after addition of protein-free milk 146

Figure 6. Resumption of growth after addi-
tion of protein-free milk to casein
diet 147

Figure ^. Failure of growth on gliadin plus

protein- free milk . . . 149

Figure 8. Recovery of the capacity to grow

after a period of stunting . . 150

Figure 9. Maintenance and fertility on a

gliadin diet . . . .151

Figure 10. Indispensability of lysine for

growth 153

Figure 11. Failure of zein to maintain or pro-
mote growth .... 154

Figure 12. Indispensability of tryptophane for

maintenance in nutrition . . 156

Figure 13. Growth on diets containing zein +

tryptophane -f- lysine . .157

THE PHYSIOLOGY OF THE AMINO ACIDS

CHAPTER I

THE PROTEINS AND THEIR DERIVATIVES—
THE AMINO ACIDS

The Proteins

The presence of nitrogen as a fundamental con-
stituent of protoplasm attests the supreme importance
of this element for the construction of living matter
and the continued existence of organized life. It is
well recognized, however, that all forms of nitrogen
are not equally available for the maintenance of
physiological rhythm. In support of this may be cited
the fact that although the animal organism is con-
tinually surrounded by an atmosphere rich in nitrogen,
little or none of this nitrogen can be utilized by the
body for nutritional purposes. The organism pos-
sesses discriminating powers and demands nitrogen
in a specific form, namely, such as that peculiar to
protein and its derivatives. Protein material con-
stitutes therefore an essential foodstuff and without
it life would be impossible for any considerable period
of time. "It is the chemical nucleus or pivot around
which revolves a multitude of reactions characteristic
of biological phenomena."

2 THE AMINO ACIDS

Viewed from the chemical standpoint protein is
seen as a huge molecule, complex in structure, labile
in character and therefore prone to chemical change.
So large and intricate is the make-up of the molecule
that chemists for generations have been baffled in
their attempts to gain any adequate conception of its
nature. At the present stage of our knowledge it is
impossible to form any satisfactory definition of a
protein based either on its chemical or physiological
properties. In general, proteins contain about 15 to
19 per cent of nitrogen, 52 per cent of carbon, 7 per
cent of hydrogen, 23 per cent of oxygen and 0.5-2.0
per cent of sulphur. Some also contain phosphorus
or iron. They act like amphoteric electrolytes, that
is, they are capable of forming salts with both acids
and bases. Proteins belong to that class of substances
known as colloids and as such do not possess the
power to pass through animal or vegetable mem-
branes. In a manner similar to colloids they may be
separated from their solutions by suitable treatment
with salts, such as sodium chloride, ammonium sul-
phate, etc. By a process known as "coagulation,"
which may be induced by the action of heat or the
long continued influence of alcohol the proteins lose
their colloidal characteristics which cannot be restored.

Many proteins are capable of crystallization and
indeed may occur in nature in crystalline form. It
has been found possible also to cause some to crys-
tallize although their presence in nature as crystals
is unknown. Some doubt has been cast upon the

THE PROTEINS 3

probability of proteins, as we differentiate them at
present, being chemical units, but since many of the
crystalline plant proteins show a constancy of proper-
ties and ultimate composition there is little reason
for the assumption that these at least are mixtures of
two or more individuals.

Concerning the size of the protein molecule some
idea may be gained when it is recalled that the molec-
ular weight has been calculated to be approximately
15,000.

The proteins possess the property of turning the
plane of polarized light to the left, the degree of rota-
tion for an individual protein varying with the solvent
employed.

Classification of Proteins

At present proteins are classified according to their
physical properties, as, for example, their solubility
in pure water, weak salt solutions and dilute acids and
alkalies. It is well recognized that such a classifi-
cation is far from ideal, but it is the most satisfactory
plan that has been offered. When more complete
knowledge is gained concerning the chemical make-up
of the protein molecule a classification will undoubt-
edly be framed which will be based upon the presence
or proportion of certain chemical groups in the differ-
ent proteins.

All albuminous substances may be divided into

4 THE AMINO ACIDS

three large groups, namely, the Simple Proteins, the
Conjugated Proteins and the Derived Proteins,
Simple Proteins may be defined as substances which
yield only a-amino acids or their derivatives on hydrol-
ysis. Conjugated Proteins are substances which con-
tain the protein molecule united to some other mole-
cule or molecules otherwise than as a salt. As their
name implies, the Derived Proteins are substances that
have been formed from naturally occurring proteins.
The various sub-divisions of these large groups, as
adopted by the American Physiological Society and
the American Society of Biological Chemists, follow:

Simple Proteins

Conjugated Proteins

1. Albumins. 1. Nucleoproteins.

2. Globulins. 2. Glucoproteins.

3. Glutelins. 3. Phosphoproteins.

4. Alcohol-Soluble Proteins. 4. Hemoglobins.

5. Albuminoids. 5. Lecithoproteins.

6. Histones.

7. Protamines.

Derived Proteins

A. Primary
Protein Derivatives.

1. Proteans.

2. Metaproteins.

3. Coagulated Proteins.

B. Secondary
Protein Derivatives.

1. Proteoses.

2. Peptones.

3. Peptides,

THE PROTEINS 5

Occurrence and Characteristics of Different
Classes of Proteins

A. Simple Proteins

Albumins are simple proteins that are soluble in
pure water and are coagulable by heat. Globulins,
on the other hand, are insoluble in pure water but
are readily soluble in dilute salt solutions. Albumins
and globulins are generally found together in nature
occurring, for example, in large quantity in the blood
serum, white of egg, in the substance of cells in gen-
eral, and in various seeds. Egg white may be divided
into two parts by dialysis against distilled water —
the globulin being precipitated owing to the diffusion
of the salts from the solution which originally were
present in quantity sufficient to hold the globulin in
solution.

Glutelins are simple proteins insoluble in all neu-
tral solvents but easily soluble in very dilute acids and
alkalies. Alcohol-Soluble Proteins are simple proteins
readily soluble in relatively strong alcohol (70 to 80
per cent), but are insoluble in water, absolute alco-
hol and other neutral solvents. These two groups of
proteins occur together as constituents of the cereal
grains. Glutenin and Gliadin, respectively, from
wheat, are the best known examples of these two
groups. They constitute the gluten of flour. The
elasticity and strength of the gluten, and therefore the

6 THE AMINO ACIDS

baking qualities of a flour are influenced by the pro-
portions of glutenin and gliadin.

Albuminoids may be defined as simple proteins
which possess essentially the same chemical compo-
sition as the other proteins, but are characterized by
great insolubility in all neutral solvents. Examples
of this group may be found as the organic basis of
bone (ossein), of tendon (collagen and its hydration
product, gelatin), of ligament (elastin) and of nails,
hairs, horns, hoofs, and feathers (keratins).

Histories are basic proteins which may be looked
upon as standing between protamines and the more
complex proteins. They are precipitated by other
proteins and yield a coagulum on heating which is
readily soluble in very dilute acids. The histones are
soluble in water but insoluble in ammonia. They have
been isolated from varied sources, as glohin from
hemoglobin, scombron from spermatozoa of the mack-
erel, gaduhiston from the codfish and arbacin from
the sea-urchin.

Protomines are the simplest natural proteins. They
are soluble in water, are not coagulable by heat, have
the property of precipitating other proteins from their
solutions, are strongly basic and form stable salts with
strong mineral acids. Examples of protamines are
salmin (from the spermatozoa of the salmon), sturin
(from the sturgeon), clupein (from the herring), and
scombin (from the mackerel).

THE PROTEINS 7

B. Conjugated Proteins

I

Nucleoproteins are compounds of one or more
protein molecules united with nucleic acid. The
nucleoproteins, as their name implies, are the proteins
of cell nuclei and give to the latter their character.
The nucleoproteins are therefore found in largest
quantity wherever cellular material is abundant, as
in glandular tissues and organs. By artificial hydroly-
sis or during treatment in the alimentary tract a nucleo-
protein is decomposed into protein and nucleic acid.
Nucleic acid, of which there are several types, may
be made to yield a series of well-defined compounds,
the purine bases (xanthine, hypoxanthine, adenine and
guanine), the pyrimidine bases (uracile, cytosine and
thymine), a carbohydrate group (pentose or hexose)
and phosphoric acid.

Glucoproteins are compounds of the protein mole-
cule with a substance or substances containing a car-
bohydrate group other than a nucleic acid. Particu-
larly rich in glucoproteins are the mucus-yielding
portions of tissues. They serve also as a cement sub-
stance in holding together the fibers in tendons and
ligaments. An amino-sugar, glucosamine, has been
isolated from some of the glucoproteins and it is gen-
erally regarded as constituting the carbohydrate radicle
of these conjugated proteins.

Phosphoproteins are compounds of the protein
molecule with some, as yet undefined, phosphorus-
containing, group other than a nucleic acid or lecithin.

8 THE AMINO ACIDS

Conspicuous foods containing phosphoproteins are
milk with its caseinogen and tgg yolk with its vitellin.
A trace of iron is also evident in these proteins and
although it is possibly present as an impurity there
is no evidence that it does not exist in combination
with the protein.

Hemoglobins are compounds of the protein mole-
cule with hematin or some similar substance. The
coloring matter of the blood is hemoglobin which
acts as oxygen carrier for the tissues and is charac-
terized by holding iron as a constituent part in organic
combination. Globin is the protein portion of hemo-
globin. In certain of the lower animal forms copper
enters into combination with protein forming hsemo-
cyanin imparting a blue color to the blood.

Lecitho proteins are compounds of the protein mole-
cule with lecithins. Lecithins are complexes charac-
terized by yielding glycerol, phosphoric acid, fatty
acid radicles, and a nitrogenous base, choline. The
lecithins are present in all plant and animal cells but
are especially abundant in the nervous tissues. They
belong to the group of essential cell constituents.

C. Derived Proteins

Certain of the native soluble proteins upon con-
tinued contact with water, or the influence of enzymes
or acid change their character and become insoluble.
Such insoluble substances are called proteans. After
repeated reprecipitation globulins may become insolu-

THE PROTEINS 9

ble, that is, they are changed to proteans, and it is
believed by some protein investigators that nearly
every protein may assume a protean state.

The metaproteins may be formed from simple
protein by the action of acids and alkalies. In this
instance, however, the change is undoubtedly more
profound than in the case of the proteans. Formerly,
metaproteins were termed albuminates, that formed
by acid being called acid albuminate, that from the
action of alkali being designated alkali albuminate.
These substances are insoluble in neutral fluids but
are readily soluble in an excess of acid or alkali. The
metaproteins are of interest when it is recalled that
the acid metaprotein arises as the first step in gastric
digestion of protein and that likewise alkali meta-
protein may be formed during pancreatic digestion.

The coagulated proteins can be produced from
simple proteins by the long continued action of alco-
hol, stirring or shaking of their solutions, or by the
influence of heat. In one instance, namely, the trans-
formation of fibrinogen into fibrin in shed blood, the
process has long been assumed to be induced by an
enzyme. More recent work, however, tends to show
that enzyme action is not concerned in the reaction.

The class of derived proteins called Secondary
Protein Derivatives represent a more profound change
from simple proteins than is true for the proteans,
metaproteins and coagulated proteins which are
grouped together as Primary Protein Derivatives.
Of the secondary protein derivatives the proteoses

10 THE AMINO ACIDS

and peptones are characterized chiefly by their greater
solubiUty and by the fact that, unUke most other pro-
/ teins, they are diffusible through suitable membranes.
They represent stages in gastric, pancreatic and bac-
terial digestions of protein and the peptones are
regarded as products of greater cleavage than the
proteoses. There are several proteoses, as protopro-
teose, heteroproteose and deuteroproteose and prob-
ably there may be several types of peptones. The
proteoses are distinguished from the peptones prin-
cipally in being precipitated from solutions by. satura-
tion with ammonium or zinc sulphate.

The peptides are "definitely characterized combina-
tions of two or more amino acids, the carboxyl
(COOH) group of one being united with the amino
(NH2) group of the other with the elimination of a
molecule of water." For example, if two molecules
of glycocoU (glycine) — amino-acetic acid — are con-
densed, a peptide, glycyl-glycine, will result. Thus —

NHH NH,

CH«.CO.NH

CH,.COOH

CH,.CO

OH

NH

H

CH2.COOH glycyl-glycine.

The peptides are designated di-tri-tetra-peptides, etc.,
according to the number of amino acids in combina-
tion. The name polypeptides is also applied to these

THE PROTEINS 11

substances. It is usually accepted at the present time
that the peptones are relatively simple polypeptides,
the line of demarcation between a simple peptone and
a complex peptide not being well defined.

The Amino Acids

For nearly a century chemists have been seeking
to establish the composition and structure of the pro-
tein molecule. Progress, which was slow and irregular
in the earlier decades of this period, has taken rapid
strides in the last twenty years, more intimate knowl-
edge of the problem being gained during this inter-
val than in all previous time. The investigation has
been pursued in three directions — first the demolition
of the molecule and the subsequent identification of
the resulting fragments; second, the determination of
the quantitative relationships of these fragments ; and
finally, attempts to unite the disintegration products
in such a manner as to reproduce the original molecule.

After a considerable period of investigation it was
established that, although the protein molecule may
yield different types of substances according to the
character of the means employed for disrupting it
thus indicating a variety of possible lines of cleavage,
hydrolysis furnishes the most promising types of
units. Latterly, this type of chemical reaction has
been employed exclusively and it has yielded the
important information now available concerning the
nature of the protein decomposition products. Each

12 THE AMINO ACIDS

protein investigated by this method was found to
yield relatively large molecules, such as proteoses and
peptones, and on further disintegration a series of
comparatively simple nitrogenous substances of low
molecular weight which belong to a definite group of
chemical compounds — namely, the amino acids. An
amino acid may be regarded as an organic acid in
which one hydrogen is replaced by the amino group
(NH2), or viewed from another standpoint, an
amino acid may be considered as a substituted am-
monia, one hydrogen of ammonia, NH3, being
replaced by an organic acid- A description of the
amino acids yielded by proteins follows.

... ^ NH2

Glycocoll or glycine, ammo-acetic acid. CH2. <[ p^w^tt

is the simplest of the products obtained from pro-
tein by hydrolytic cleavage and it was also the first to be
discovered. Its separation dates back to 1820 in which
year Braconnot obtained the substance by boiling gelatin with
sulphuric acid, and because of its sweet taste called it sugar
of gelatin. About twenty-five years later Dessaignes isolated
it after a hydrolysis of hippuric acid. It was shown by
Strecher in 1848 that glycocholic acid, then called cholic acid,
consists of a combination of cholalic acid and glycocoll, and
in consequence of its being a constituent of a bile acid, glyco-
coll assumed a position of some physiological importance. Its
presence in various types of albuminoids, such as elastin, etc.,
was later demonstrated and finally it was shown to be a
decomposition product of globulin. Glycocoll is not present
in all proteins for albumin, casein, and hemoglobin fail to
yield it, and from the vegetable proteins it is obtained in
small quantities only. On the other hand, albuminoids are
particularly rich in glycocoll. In an extract of the mollusc

THE PROTEINS 13

Pecten irradians Chittenden found glycocoll in a free state;
and it has been reported as occurring in the urine under vari-
ous pathological conditions. After administration of benzoic
acid to man and animals hippuric acid (benzoyl-glycocoll) is
found in the urine — thus demonstrating a synthesis of hip-
puric acid from benzoic acid and glycocoll.

NH2
Alanine — a- amino-propionic acid, CHs.CH «< p^^^^tt

was prepared synthetically previous to its isolation

from among the protein decomposition products and was

named by its discoverer, Strecher. Alanine has been shown

to be a constant decomposition product of proteins.

CH3 NH2

Valine — a-amino-isovalerianic acid. "> CH . CH <"

CH3 XOOH

In 1856 V. Gorup-Besanez Isolated a substance having the
formula C5H11NO2 from pancreas and because it possessed
properties similar to leucine he looked upon it as a homologue
of leucine and called it butalanine. Although a similar sub-
stance was isolated from certain seedlings by Schulze and
Barbieri, and from the protamine, clupeine, by Kossel, it was
not until 1906 that its identity was established by Fischer
who gave it the name of valine. Valine is obtained from most
proteins.

Leucine, a-amino-isobutylacetic acid.

CH3 NH2

>CH.CH2.CH<^^^ ^
CHs COOH

Leucine was described by Proust in 1818 and was called
oxide-caseux. Braconnot in 1820 obtained a substance from a
hydrolysis of meat which on account of its glistening white
appearance he called leucine. Liebig regarded it as one of the
constituents of the protein molecule and this was later proved
to be correct. Leucine is also a constituent of many organs
and tissues occurring in the free state. It is yielded by both

14 THE AMINO ACIDS

animal and vegetable proteins and with the possible exception
, of arginine is the most widely distributed amino acid found as
a protein cleavage product. Leucine has been found also in
the urine under pathological conditions.
Isoleucine, a-amino-/3-ethyl-propionic acid.

CHs NH2

C2H5>^^-^^<COOH

This amino acid was not described as a protein constituent
until 1903 when it was isolated as a decomposition product of
fibrin and other proteins by F. Ehrlich.

Norleucine. o-amino-normal-caproic acid. CH3.CH2.
CH2.CH2.CH.NH2.COOH. From the leucine fraction of
the decomposition of the proteins of nervous tissue this amino
acid has recently been isolated by Abderhalden and Weil. It
is probable that other proteins may yield it also.

Phenylalanine, jS-phenyl-a-amino-propionic acid.

NH2

Although it had been recognized for many years that a
substance having the composition of C9H11NO2 could be ob-
tained by cleavage of both animal and vegetable proteins, it
was Fischer who first proved the presence of phenylalanine as
a protein derivative. In those proteins lacking tyrosine, as
\/ gelatin, for example, the aromatic ring is supplied by phenyla-
lanine.

Tyrosine. /S-para-oxyphenyl-a-amino-propionic acid.

NH2
HO.C6H6.CH2.CH<^^^„
COOH

In 1846 Liebig isolated from a decomposition of cheese a
substance possessing the property of crystallizing in silky
needles. He named it tyrosine. Since then tyrosine has been
regarded as a protein cleavage product. It was not until 18S2,

THE PROTEINS 15

however, that the structure of tyrosine was positively deter-
mined. Tyrosine is absent from the gelatine molecule. In
acute yellow atrophy of the liver and in phosphorus poisoning
it is claimed that tyrosine may be present as a urinary con-
stituent.
Serine /S-hydroxy-a-amino-propionic acid.

NHa
Ca(OH).CH<^^^^

Cramer found serine among the decomposition products of
sericin (silk gelatin), and it was not obtained again until 1902
when Fischer isolated it from various proteins as a result of
hydrolysis. He also definitely established its structure.

Cystine, di-cysteine or di-jS-thio-a-amino-propionic acid.
HOOC.CH.NH2.CH2.S— S.CH2.CH.NH2.COOH.

Cystine has been known since 1810 having been first de-
scribed by WoUaston who separated it from a urinary calculus
and called it cystic oxide. From that period, although cystine
was repeatedly isolated from various organs of the body, as
the liver and kidney, its presence as a regular decomposition
product of protein was not established until 1899 when
K. A. H. Morner ohtained it by a hydrolysis of horn. Bau-
mann demonstrated the relationship of cysteine to cystine and
thus revealed the structure of cystine. Cysteine and cystine
bear the same relation to one another as does a mercaptan to
a disulphide, thus,

CH2.SH CH2 S — S CHj

I I I

CH.NH2 CH.NH2 CH.NHa

I I I

COOH COOH COOH

cysteine cystine

Cystine is of considerable importance in metabolism inasmuch
as it is the only known sulphur-containing amino acid in the
protein molecule.

16 THE AMINO ACIDS

Aspartic Acid — ^Amino-succinic acid.

CH2.COOH

I
CH.NH2.COOH

Asparagine, the amide of aspartic acid, has been known since
1806, having been isolated from asparagus juice by Robiquet
and Vanquelin. Upon boiling asparagine with lead hydroxide
Plisson in 1827 obtained aspartic acid. In 1868 aspartic acid
was shown by Ritthausen to be present as a product of hydro-
lytic cleavage of vegetable proteins. In a similar manner
Kreussler obtained it upon hydrolysis of animal proteins and
in 1874 it was isolated by Radziejeioski and Salkowski from
a tryptic digestion of protein. Its structure was established in
1887.

Glutamic Acid (Glutaminic Acid) a-amino-glutaric acid.

CH2

I
CH2.COOH

(
CH.NH2.COOH

Although glutamic acid was first separated from a hydrol-
ysis of wheat gluten in 1866 by Ritthausen its structure was
not shown until 1890. Ritthausen demonstrated that it was an
amino acid and from this fact together with its origin from
gluten gave it the name of glutaminic acid. Glutamic acid
was later shown to arise from hydrolytic cleavage of proteins
of animal origin as well as from those derived from the vege-
table kingdom.

Lysine, a-, e, -diamino-caproic acid.

H2N.CH2.CH2.CH2.CH2.CH.NH2.COOH.

Lysine is widely distributed as a protein constituent. It
was first isolated from casein by Drechsel in 1889. Ellinger

THE PROTEINS 17

first demonstrated its structure in 1900 by obtaining cadav-
erine from it by putrefaction.
Arginine. a-amino-5-guanidine-valerianic acid.

NH2

HN = C — NH.CH2.CH3.CH2.CH.NH2.COOH.

Among the products of a decomposition of casein Drechsel
found a substance which he called lysatinine. Later, in 1894,
Hedin demonstrated that this product was in reality a mix-
ture of lysine and arginine. Arginine had been obtained pre-
viously by E. Schulze and Steiger from the seedlings of
various plants. Urea and ornithine are among its decomposi-
tion products.

HisHdine. jS-imidazole-a-amino-propionic acid.

CH

^ \
N NH

I I

CH=C.CH2.CH.NH2.COOH

Histidine was discovered by Kossel in 1896 among the de-'
composition products of the protamine of sturgeon testes.
From the fact that histidine, arginine, and lysine each contain
six carbon atoms Kossel called these three substances the
hexone bases, and they were regarded as a very important
portion of the protein molecule. It was not until 1904 when
the structure of histidine was shown by Pauly and Wind-
haus and Knopp that it was recognized to belong to a group
of compounds entirely different from that including arginine
and lysine.

Proline, o-pyrrolidine-carboxylic acid,

CH2 CH2

CH2 CH.COOH

18 THE AMINO ACIDS

Proline was first isolated by Fischer from casein. Its pres-
ence in various other proteins was soon shown.

Oxyproline.

This amino acid was prepared from gelatin in 1902 by
Fischer. Its structure is not yet definitely established although
it undoubtedly possesses one of the following formulas.

HO.CH CH2 CH2 CH.OH

11 II

CH2 CH.COOH or CH2 CH.COOH

Tryptophane. /3-indole-a-amino-propionic acid.
C.CH2.CH.NH2.COOH

C6H4<' XH

It was shown in 1826 by Tiedemann and Gmelin that when
chlorine or bromine water is added to a tryptic digestion
mixture a violet color is produced. Stadelmann named the
substance giving this reaction proteinochromogen and Neu-
meister proved that any severe treatment of protein would
cause the production of this compound to which he gave the
name tryptophane. Hopkins and Cole in 1902 isolated from
a tryptic digestion of casein a substance which gave all the
reactions of tryptophane, namely, the violet coloration with
bromine or chlorine, the Adamkiewicz reaction, and the pro-
duction of indole and skatole as a result of putrefaction. In
this manner the origin of the substances characteristic of
putrefaction was made clear. The structure of tryptophane
was regarded by Nencki as indole amino acetic acid. Ellinger,
however, showed it to be an indole amino propionic acid.

Caseinic Acid, or diamino-trioxy-dodecanic acid. This
compound has been isolated by Skraup from casein only. Its
structure is still unknown.

THE PROTEINS

19

On inspection of these formulas it may be estab-
lished that certain of the amino acids are very closely
related; thus, glycocoll, the simplest of all, by intro-
duction of the group (CH3) becomes alanine. This
substance possesses interest because several of the
amino acids may be regarded as alanine derivatives.
By the replacement of an (OH) group alanine be-
comes serine, or by substitution of an (SH) group
alanine is changed to cysteine. If the phenyl group
(CeHs) is introduced phenylalanine is obtained, and
the additional substitution of an (OH) group leads to
tyrosine.

CH,

CH5.OH CH,.SH

CH.NH,

CH.T^H, CH.NH,

COOH

COOH COOH

Alanine

Serine Cysteine

CHa.CeHg

CHs.CeH^.OH

CH.NH3

CH.NH,

COOH

COOH

Phenylalanine

Tyrosine

The introduction of the indole or iminazole group
leads to the formation of tryptophane or histidine
respectively.

20

THE AMINO ACIDS

CH3

CH.NH,

I
COOH

Alanine

CH,

CH.NH,
COOH

CH

/\
-C CH

CH C CH

\ /\/
NH CH

Tryptophane

CH— NH
\

CH

CH — C N-^ •

CH.NH,

I
COOH

Histidine

Again valine, leucine and isoleucine are closely re-
lated structurally as may be seen from the formulas
following.

CH3 CH3

\ch/

I
CH.NH,

I
COOH

Valine

CH3 CHj

\ch/

I

CH,
CH.NHj

COOH

Leucine

CHs C^Hb

\ch/

I

CH.NH,

I
COOH

Isoleucine

THE PROTEINS 21

Viewed from another standpoint the amino acids
may be divided into mono-amino acids, — ^glycocoll,
alanine, valine, leucine, isoleucine, phenylalanine, tyro-
sine, serine, aspartic acid, and glutamic acid, — each
containing, as the name implies a single amino (NH2)
group — diamino acids, containing two amino groups,
as arginine, and lysine and finally the heterocyclic com-
pounds as histidine, proline, oxyproline, and trypto-
phane.

The Quantitative Relationships of Amino Acids

IN Proteins

The most serious obstacle to the quantitative estima-
tion of amino acids in hydrolysis mixtures has been
that of inadequate methods of separation. By means
of the ester method of E. Fischer this difficulty has
been obviated in large measure. In Table I below are
presented figures showing the yield of individual amino
acids obtained by various investigators from repre-
sentative simple proteins. The figures have not all
been derived from use of the most exact methods of
isolation, hence it is probable that they may not repre-
sent maximal values or be strictly correct. Neverthe-
less, they are sufficiently suggestive to demonstrate the
distinct differences that exist between the simple
proteins.

Table II undoubtedly gives the most accurate figures
obtainable at present for the quantitative yield of

K

to

§

H

o

P

Pi

O

i^

O

Q

fe

W

t^

'A

o

M

^

H

^

n

o

O o

CO

Q

«

^

M

<)

<

en

o

^

w

M

H

S

O

<

P^

H(

h

o

w

Hi

tn

PU

W

M

§

M

M

CO

H

>^

w

<

>

P

H

a

-u

H

u

!zi

W w
CO

(z) aoiivos

iZ) inqiiOE:

{Z) ud:^oti{Q

{z) /99a"

uttunqiY

(z) S3a

(e) uRBpo

piouxtunqiY

21UW s.Avoo

(I) upsBO

U'i9j.oj,dO'qdsoiu

(azrepi)
(2) IIX9Z

lonooiv

puoinxv
too J J uipuBtav

(z)

tuoaj uisxaoxg
pazinBt^SifjQ

■ (2)
«nnqoi£)

(S) (T)
tinnqoio

muinqxv uinaag
(S) (T)

(pooia ssaoH)
niqoxSomsq
uiojj uiqoxf)

VOCM -tH

VO 00 O T-H

O O i-H o

O Cvj r-t 00

fO (M"r00

(tj (T)

O CO

rH CM
COCV)

ooo"

•OTh+t

(M 0"r VO t^ <M

»-i 00 O rH^

CO vo~rvor^ev)

N t^ CT\ VO

I- Ti-iD t^

evi os~r'^coTH

CvlO voov vOOOtJ-

>o CO o o o ^^ cvj o

CO VO
CM O

CO'*OOCOO»-(»Hr-l'^lOINOr-5

•Tt-CM tH ■*
■ VO • »H 00 O »H O O

<M iH O Ol^

coco

00 lO
CO <M

t^oo

OCvj

»H ^ VOIOO
CO fvj O CMrH

^•+

Ot^TcOOO

CM T^ lo o 00
■«^ rH oot^«o

ioco~r'Ho,-<

CM 1-1

OOOV iH VOIO
COCM + vdr-ir-J

cvioo~r

1-1 (^
cot^+

'S-t-H OO CM

»H Tt- i-I + lO ■* O

a

o).a ca

c

§l»llfl2lSf|EB-2s

•ill

W Oi3 g

0

0

en

to

<J

•O

rt

h

0)

rfl

0

.•0

MS

^^

,*^

iii

«

M

0

V

OS

-M

<J

0

Oi

n

ns

r/l

0

ri

tn

W

c3

<:

<

s

Tf

(U

n

•d

t3

U

fl

-3

a>

c8

a

^1
0)

(U

0

id

/3

5^

<i 0 fe

^--^

^^

,,-^

iH

CM

THE PROTEINS

23

amino acids obtained by hydrolysis from proteins
representing various groups of these substances.

Table II.

Quantitative Comparison of Amino Acids Obtained
BY Hydrolysis from Proteins

(Compiled by T. B. Osborne, 1914)* (After Mendel)

Casein

Oval- ^T ,.
bumin Gliadin

Zein

Edestln

LeRumiQ
(Pea)

GlycocoU

Alanine

Valine

Leucine

Proline

Oxyproline

Phenylalanine

Glutammic acid

Aspartic acid

Serine

Tyrosine

Cystine

Histidine

Arginine

Lysine

Tryptophane, about
Ammonia

0.00
1.50
7.20
9.35
6.70
0.23
3.20
15.55
1.39
0.50
4.50
?

2'. 50
3.81
5.95
1.50
1.61

0.00
2.22
2.50
10.71
3.56

?

5.07
9.10
2.20

?
1.77

?

1.71
4.91
3.76
present
1.34

0.00
2.00
3.34
6.62

13.22

?

2.35

43.66
0.58
0.13
1.61
0.45
1.49
2.91
0.15
1.00
5.22

0.00
13.39

1.88
19.55

9.04
?

6.55
26.17

1.71

1.02

3.55
?

0'.82
1.55
0.00
0.00
3.64

3.80
3.60
6.20

14.50

4.10

?

3.09

18.74
4.50
0.33
2.13
1.00
2.19

14.17
1.65
present
2.28

0.38

2.08

?

8'. 00

3.22

?

3*. 75
13.80

5.30

0.53

1.55
?

2.42
10.12

4.29
present

1.99

65,49 48.85 84.73

88.87 82.28 57.43

♦These analyses are combinations of what appear to be the best de-
terminations of various chemists.

It may be seen from these tables that certain pro-
teins, as serum albumin and casein contain no glyco-
coll, whereas serum globulin contains a small amount
and gelatin a large quantity. Alanine presents vari-
able figures but is usually present. The same may be
said of leucine, phenylalanine, proline, and aspartic
acid. Tyrosine may be absent as in gelatin. Glutamic

24 THE AMINO ACIDS

acid may show very wide variations being present to
the extent of nearly 44 per cent in wheat gliadin where-
as gelatin contains less than 1 per cent. Tryptophane
may be absent as in zein and gelatin. Arginine shows
great variation being present in largest quantity in the
protamines (salmine). On the other hand, lysine is
absent in salmine as well as in zein. In the protamine,
salmine, histidine is not present but may be isolated
from all other examples of simple proteins shown here.
It is clear that in general the various proteins are made
up of the same units and it undoubtedly follows that
the individual protein characteristics are bestowed by
the relative proportion of the units or by their absence.
In the tables given it will be observed that in most
instances the total amino acids fall far short of the
theoretical yield, a deficit of 40 to 50 per cent being in
order. Previously it has been assumed that only a
portion of the amino acids was known. At present,
however, it seems very probable that the deficit is to
be explained on the hypothesis of inadequate methods
of analysis.

Synthetic Proof of the Structure of Protein

Since the time of Liebig it has been assumed that
the protein molecule consisted of a huge complex of
amino acids linked together in some unknown manner.
There are many possibilities for such combinations
and certain of them have been subjected to experimen-

THE PROTEINS 26

tation without, however, yielding any very far-reaching
conclusions. It remained for Emil Fischer and his
associates in 1901 to conceive of a combination which
undoubtedly will ultimately lead to a clear under-
standing of the structure of the protein molecule.
These combinations of amino acids were termed
polypeptides. Just as we have mono-, di-, or tri-
saccharides so there may be di-, tri-, etc., -peptides.
According to Fischer's method the amino acids are
linked together by dehydration of their hydroxy 1 and
amino groups, the carboxyl group of each acid being
condensed with the amino group of its neighbor in the
molecule, thus

NHH

R. CH.CO

I

NH

OH
H

R .CH.COOH

By continued union of amino acids infinite possi-
bilities of complexes are presented. Actually com-
pounds containing as many as eighteen amino acids
have been synthesized by Fischer and some of the
products obtained have shown properties similar to
those of the native protein.

After demonstration of the possibility of forming
protein-like compounds by synthesis Fischer next
attempted to determine whether similar simple com-
plexes could be derived from proteins by suitable treat-

26 THE AMINO ACIDS

ment. For this purpose he employed a mild hydrolysis
which only partially broke up the large aggregates
formed and he succeeded in isolating from the pro-
ducts peptides identical with those made synthetically.
Since then other investigators have separated similar
compounds. One of the best proofs that proteins are
built up of these amino acid complexes is that, also
furnished by Fischer, of the action of various enzymes
upon the synthetical products. It was found that with
the exception of pepsin the various enzymes of the
^ body are quite capable of hydrolyzing the polypeptides
into amino acids.

Although these investigations prove beyond doubt
that amino acids are linked together in protein in the
form of polypeptides, there are possibilities of other
forms of combination which will be revealed only by
future research. For the present we are justified in
accepting the hypothesis of the protein molecule as a
huge complex polypeptide.

References to Literature

Abderhalden: Text Book of Physiological Chemistry. 1914.

Ahderhalden and Weil: Zeitschrift fiir physiologische chemie.
1913, 88, p. 272. [Norleucine.]

Hammarsten: Text Book of Physiological Chemistry. 1914.

Kossel: The Chemical Composition of the Cell. The Harvey
Lectures. 1911-1912.

THE PROTEINS 27

Kossel: The Proteins. Johns Hopkins Bulletin. 1912, 23,
p. 65.

Mann: Chemistry of the Proteins. 1906.

Mendel: Nutrition and Growth: Harvey Lectures 1914-15.
Journal of the American Medical Association. 1915, 64,
p. 1539.

Osborne: The Vegetable Proteins, 1909.

Osborne: Chemistry of the Proteins. The Harvey Lectures,
1910-1911.

Plimmer: Chemical Constitution of the Proteins, 1908.

Van Slyke: The Proteins. New York Medical Journal. 1912,
August 10 and 17.

CHAPTER II

DIGESTION, AND BACTERIAL ACTIVITY IN
RELATION TO THE AMINO ACIDS

Concerning the nature of protein digestion Schaefer
in 1898 wrote : "The products found toward the end of
a proteid digestion in vitro are distinguished from the
proteids from which they originate by being slightly
diffusible. To this fact great importance was at one
time attributed, because it was thought that only pro-
teids in a diffusible form were capable of absorption,
and hence that peptonization was in all cases a neces-
sary preliminary. It is now generally admitted that
many forms of native proteid are capable of entering
the epithelial cells (of the intestine) without previous
change by digestion or otherwise ; and in those cases in
which a proteid is incapable of direct absorption a
much less profound change than peptonization is suffi-
cient to render it so, namely, conversion into acid or
alkali albumin." With regard to the extent of amino
acid formation in digestion Schaefer says: "It is not
known with certainty to what extent amino acids are
formed from proteids, in the natural course of intes-
tinal digestion. The experimental evidence is some-
what conflicting, but the majority of observers are of
the opinion that but little proteid is absorbed as leucine
or tyrosine, being nearly all absorbed as albumose or

DIGESTION 29

peptone, or even at a still earlier stage. The only posi-
tive evidence as to the formation of leucine and
tyrosine in natural digestion, rests on the amounts
found in the intestinal contents during protein diges-
tion." It is then stated that in general the quantities
of amino acids present during digestion are small.

In the few years since the above was written the
advances made in the chemistry of the proteins and of
digestion have made necessary a radical revision of our
ideas of the nature and extent of the alimentary treat-
ment of protein. No longer tenable is the view that
digestion stops with the transformation of insoluble
and non-diffusible substances into compounds soluble
and diffusible, nor can the idea be accepted of a dis-
tinction between directly assimilable and non-assimi-
lable proteins. The change to "peptone" is now held to
be merely an intermediate stage in digestion, not the
end, as was once assumed. According to the latest con-
ception of protein digestion a profound disintegration
occurs, the ultimate products formed being a variety of
polypeptides and amino acids. Digestion, in accord-
ance with this idea, consists in a series of hydrolytic
cleavages which are induced through the agencies of
the enzymes present in the gastro-enteric tract. The
products formed by these enzymes undoubtedly are
identical with those produced outside the body by
means of the action of acids. Amino acids therefore
must be looked upon as the ultimate nitrogenous food-
stuffs— it is to these substances that the organism must
look for its essential requirement of nitrogen.

30 THE AMINO ACIDS

Are Amino Acids Formed during Gastric Digestion

OF Protein?

Protein digestion is initiated in the stomach through
the action of gastric juice — the active constituents
being pepsin and hydrochloric acid. In investigating
the nature and extent of gastric digestion three general
methods have been employed — as follows: (1) the
stomach tube method, the only procedure applicable to
man, whereby the stomach contents are withdrawn at
intervals after a meal, (2) animals fed definite diets
are quickly killed at varying periods of time and the
stomach contents examined ; or animals are killed with
the stomach empty, food introduced and analyzed at
intervals, (3) the polyfistulous method — ^fistulas being
inserted in the stomach and at various points in the
intestine, the food products being withdrawn through
these openings.

What products are formed in the stomach under the
influence of peptic digestion? It is self-evident that
experiments carried out under artificial conditions, as
in beakers, can afford no positive assurance that the
products are identical with those formed in the stom-
ach. Kiihne was the first to demonstrate that pepsin
digestion in vitro leads only to the formation of pro-
teoses and peptones. Oa the other hand, numerous
recent investigations have shown by the polyfistulous
method that under normal conditions also proteoses and
peptones represent the final stages in gastric digestion
of protein. All the protein does not of necessity reach

DIGESTION 31

the peptone stage. Indeed, in general the process goes
only as far as the proteose stage as may be seen from
the following table from London. In these experi-
ments dogs were fed different types of proteins and
from a fistula below the pylorus the products were
collected.

Kind of Protein Fed Percentage of Proteoses Found

Egg Albumin 72.5

Gliadin 67.7

Edestin 60.3

Casein 59.1

Gelatin 50.6

Serum Albumin 46.1

London and his co-workers also found that upon
feeding varying quantities of the same protein a defi-
nite proportion of proteoses was always formed, thus —

Quantity of Gliadin Fed Percentage of Proteoses Found
in grams

25 80.8

50 86.1

75 86.5

100 84.9

It is therefore probable that ingested protein enters
the duodenum largely in the form of proteoses and to
a smaller extent as peptones^.

By long continued action of both artificial and nor-
mal gastric juice various investigators have observed
the gradual formation of amino acids. These results
obtained from digestive mixtures allowed to stand for
months cannot be regarded as applicable to normal

32 THE AMINO ACIDS

stomach digestion which is at most a matter of hours.
They may be explained in several ways, as for instance
in those cases where extracts of the stomach were
employed amino acids may arise from autolytic proc-
esses, or perhaps in all cases from the action of hydro-
chloric acid alone. Protein is a labile molecule which
apparently needs slight inducement to start on the
downward path to its demolition into amino acids.
The formation of amino acids in gastric digestion
under normal conditions seems hardly probable.
Against such an idea may be set the fact that pepsin-
hydrochloric acid is utterly incapable of breaking
down artificial polypeptides thus far tested. On the
other hand, they are readily split by pancreatic juice.
It would seem therefore that in peptic digestion
neither amino acids nor relatively simple polypeptides
are normally found in significant amounts.

Gastric digestion, however, has the very important
function of preparing protein for the later action of
trypsin and the intestinal juices. Fischer and Abder-
halden have shown that tryptic digestion is much more
rapid and complete when protein has been previously
acted upon by pepsin-hydrochloric acid. If casein is
first digested with an artificial gastric juice and then
subjected to the influence of trypsin amino acids like
proline and phenylalanine could be isolated. Treated
with trypsin alone casein failed to yield the free amino
acids ; instead a corresponding polypeptide was present.

One may conclude therefore that although gastric
digestion fails to yield amino acids directly it aids in

DIGESTION 33

their rapid formation indirectly by facilitating the
action of trypsin.

Intestinal Digestion

Kiihne made the important discovery that there is
an essential difference between the digestive action of
trypsin and that of pepsin. He stated that the influ-
ence of the former does not cease with the formation
of peptone but is carried to a stage where crystalline
products appear — the amino acids. As late as 1900,
however, these substances were regarded as by-pro-
ducts in natural digestion — of little significance and
formed in relatively small quantities. At that time the
cleavage products recognized were leucine, tyrosine,
aspartic acid, glutamic acid, lysine, arginine and histi-
dine and proteinochromogen (see Chapter I). With
the growth of knowledge concerning protein chemistry
most of the characteristic amino acids have since been
isolated from intestinal contents.

In 1906 Cohnheim gave a new meaning to intestinal
digestion by his discovery of an enzyme capable of
splitting proteoses and peptones into simpler products.
Cohnheim was of the opinion that synthesis of protein
from peptones occurred in the intestinal wall. While
endeavoring to determine this point he noted that the
characteristic peptone reaction disappeared. Its dis-
appearance was not due to protein synthesis as was
early assumed, but because crystalline decomposition
products were formed from it. This chemical trans-

s

34 THE AMINO ACIDS

formation was shown to be enzymatic in nature and to
the enzyme Cohnheim gave the name erepsin. Later
investigators showed that erepsin is quite specific in
its action — it has no influence upon native proteins
with the exception of casein and gelatin — but is capable
of completely transforming proteoses and peptones into
amino acids, such as leucine, tyrosine, lysine, histidine,
and arginine. In intestinal digestion, therefore, two
agencies are to be considered in protein disintegration,
namely, trypsin and erepsin. From these two differ-
ent types of activity one may perhaps draw the con-
clusion that there is a purposeful function for each.
It may be imagined for instance that trypsin may per-
form a twofold function, the degradation of the
protein molecule which may have escaped gastric diges-
tion to the proteose or peptone stage, or completely to
amino acids. Erepsin on the other hand is present to
guarantee that all complicated structures as proteoses,
peptones, or polypeptides are reduced to their simplest
terms. It is apparent, therefore, from the distribution
of enzymes in the intestinal tract that there is a natural
provision for ingested protein to be subjected to a
series of hydrolytic cleavages whereby only relatively
simple amino acids are finally present.

Although it was generally admitted that protein di-
gestion may proceed to the stage of amino acids it was
exceedingly difficult to prove the fact when applied
to the alimentary tract under normal conditions. The
difficulty was twofold in nature. In the first place,
demolition of the protein molecule is not of the nature

DIGESTION 35

of an explosion resulting in a large number of frag-
ments scattered about, but instead it may be looked
upon as a kind of slow erosion whereby certain pro-
jecting pieces are rubbed or broken off. Secondly,
absorption takes place rapidly and the erosion products
have a tendency to disappear from the alimentary
canal. A knowledge of the thorough character of
intestinal digestion has been made possible through the
employment of the polyfistulous method devised by
London. Animals with a series of fistulas along the
intestinal tract were fed gliadin and from successive
openings the enteric contents were examined for the
quantity of tyrosine and glutamic acid present. It was
shown that in the duodenal contents 0.75 gram tyrosin
and 2.5 grams of glutamic acid were present, in the
jejunum were 1.1 gram tyrosin and 20.9 grams of
glutamic acid w^hile the ileum yielded only a trace of
tyrosin and 33 grams of glutamic acid. Similar experi-
ments with casein and meat yielded comparable results.
From these observations it is quite evident that the
processes of digestion in the intestine are gradual in
nature but the rate of disintegration is much greater
than obtains in artificial digestion mixtures. The
apparent explanation for the slower rate of hydrolysis
in in vitro experiments is that the digestion enzymes
form compounds with the amino acids split off and thus
are rendered inactive. This inactivation probably does
not occur to any extent in the intestine because the
amino acids do not accumulate therein, undoubtedly
being absorbed almost as soon as they are split off.

36 THE AMINO ACIDS

The small intestine, therefore, may be regarded as
the seat of profound protein digestion, the products
arising being the amino acids typical for hydrolytic
cleavage of protein. Undoubtedly all digestible pro-
teins are ultimately reduced to the condition of amino
acids. From this it follows according to present views
that nitrogenous metabolism is concerned mainly with
the amino acids and the transformations which they
undergo.

Intestinal Bacteria and the Amino Acids

In the early days of the history of protein digestion
great difficulty was experienced in the determination of
the actual products formed because of the accompani-
ment of putrefaction. This was especially true for
tryptic digestion where it is desirable to maintain an
alkaline medium, an environment also favorable for
bacterial growth. Kiihne was the first to demonstrate
the activity of trypsin in the presence of antiseptics
and through the employment of antiseptic digestion
mixtures a sharp division line was soon drawn between
the products of tryptic digestion and those formed by
bacterial agencies.

In general the products of putrefaction are identical
whether formed outside the body or within. The type
of action is similar to other kinds of digestion activity.
Indeed, there is little doubt that the same kind of
agencies are at work in the two instances, namely,
enzymes. In the one case they are present in a secre-

DIGESTION 37

tion, as in intestinal juice, in the other instance they are
contained within an organism. In bacterial digestion
the first stages of digestion are very similar to those
induced by trypsin. If the protein is insoluble solution
is first effected which is not a rapid process as in the
case of trypsin. The proteoses and peptones are next
formed but are quickly transformed into lower decom-
position products. Proteoses and peptones are much
more readily attacked than are the native proteins,
which may not begin to undergo a profound change
until the former have been broken up to smaller mole-
cules.

Putrefaction may be regarded as causing a different
type of cleavage than occurs in ordinary tryptic or in-
testinal digestion as exemplified by the specific sub-
stances produced. It would appear much more likely,
however, that the early stages of tryptic digestion and
those induced by bacteria are identical in both instances,
amino acids being the final products. On the other
hand, little or no putrefaction occurs in the small intes-
tine and there is little reason to assume that under
ordinary circumstances any unchanged protein or
perhaps even proteoses or peptones succeeds in pass-
ing the ileo-caecal valve. It is therefore probable that
normally putrefactive bacteria act upon the amino acids
rather than upon their precursors, the complex protein
molecules. It is even doubted whether pure solutions
of native proteins will putrefy directly. Accepting the
hypothesis that it is the amino acids which are con-
cerned primarily in putrefactive processes the forma-

38

THE AMINO ACIDS

tion of the substances characteristic of putrefaction is
readily understood. The amino acids which are espe-
cially susceptible to bacterial action are tyrosine and
tryptophane. From tyrosine a whole series of com-
pounds may be formed and are regularly present as
putrefactive products, as for example, paroxyphenyl-
propionic acid (hydro-paracumaric acid), and paroxy-
phenylacetic acid (also phenylpropionic and phenyl-
acetic acids), as well as paracresol and phenol. The
relationships are readily seen from the following
formulae :

OH

CH,

OH

CH,

OH
/\

CH,

CH.NH,

CH,

COOH

COOH

Tyrosine, or

p. oxy-phenyl

a-amino-propionic

acid

COOH

p. oxy-phenyl

propionic

acid

p. oxy-phenyl

acetic

acid

OH

CH3
p. cresol

OH

Phenol

DIGESTION

39

From tryptophane the malodorous bodies indole and
skatole may be produced, thus :

C.CH2CH.NH2.COOH
CH

C.CH2CH2.COOH
CH

Indole-amino-
propionic acid
Tryptophane

C.CHs

NB.

Indole-
propionic acid

CH

CH

CH

Indole-acetic
acid

NH
Indole

In the explanation of these changes in both instances
it is seen that the types of chemical reactions are iden-
tical. First deamination or splitting off of ammonia,
NH3, occurs. This is followed by a cleavage of carbon
dioxide, oxidation, and finally demethylation. The
chemical transformations therefore are quite varied
and extensive.

When putrefaction is mentioned one invariably
thinks of indole, skatole, the oxy acids, etc. These
compounds, however, by no means represent all of the
substances actually formed for a type of chemical
compound has been isolated which is also peculiarly

40

THE AMINO ACIDS

characteristic of putrefaction — namely, the amines.
Dixon and Taylor in 1907 aroused considerable interest
by the publication of their observation that alcoholic
extracts of the human placenta when injected intro-
venously caused a marked rise in blood pressure and
contractions of the pregnant uterus. It was later
shown that these phenomena failed to appear in placen-
tal extracts free from putrefaction. Evidence was soon
produced showing that putrefaction of the placenta
caused the production from tyrosine of a new body,
namely p-oxyphenylethylamine. This substance was
isolated from a pancreas digestion several years pre-
viously by Emerson and its production as a product of
tryptic action was regarded as unique. In the light of
present knowledge there is little doubt that here also
it was formed through bacterial agency. This new
substance is produced by the liberation of CO2 from
tyrosine, thus:

OH

OH

/

CH,
CH.NH,

|COO|H

Tyrosine

CH,
CH,
NH,

p. oxyphenyl-ethylamlne

DIGESTION

41

To this compound has been given the name tyramine.
It is of special significance both from the chemical and
pharmacological standpoints because of its resemblance
in both respects to epinephrine.

OH

CH,

I
CH,

NH,

Tyramine

OH

lOH

\/
CH.OH

I
CH,

I
NH.CH3

Epinephrine

Tyramine acts upon the sympathetic nervous system
as does epinephrine. Its action, however, is somewhat
weaker. Its effects are produced whether absorbed
from subcutaneous tissues or from the alimentary
canal. A further interest attaches to tyramine in that
it is one of the substances that confers upon ergot its
characteristic action on the uterus.

Not only is tyramine found in putrefaction mixtures
without the body, but it has been isolated from the
contents of the large intestine and it may, therefore,
be looked upon as a product formed regularly in the
body. On the other hand its presence in the alimentary
canal does not necessarily imply that it was formed

42

THE AMINO ACIDS

there for it has been shown quite recently that it may
be ingested with certain food products. Thus tyramine
occurs in such varieties of cheeses as the Camembert,
Roquefort, Emmenthal and even the American cheddar
cheese is not free from it.

In a manner similar to the formation of tyramine
we may have amines produced from other amino acids
by bacteria. From leucine may be formed isoamyla-
mine, thus :

CH3

CH,

CH.

CH.

CH

CH

CH«

CH,

CH.NH,

CH,

COOH

NH,

Leucine

Isoamylamine

From tryptophane a corresponding amine may be
produced, thus :

x\

C.CH2.CH.NH2.COOH

/X

C.CH2.CH2.NH2

/ \

rNcH

fNcH

\/ NH

\/ NH

Tryptophane

Indo

le-ethylamine

When histidine is subjected to the action of putre-
factive bacteria it is transformed to )8-iminazolylethyl-

DIGESTION 43

amine or as it has been called histamine. The reaction
occurring follows.

CH

HN ^

CH
HN ^

HC C

HC C

CH,

CH,

CH.NH,

CH,

COOiH

NH,

Histidine

/S-iminazolylethyla

This substance besides possessing an action upon
the nervous system is capable of producing symptoms
identical with those of anaphylactic shock. Its pres-
ence in the alimentary canal has also been demon-
strated.

The diamines, cadaverine and putrescine, arise in
the alimentary through the action of bacteria. Cadav-
erine is produced in the following manner, lysine serv-
ing as the mother substance. Lysine, which has the
following formula :

CH,.CH,.CH,.CH,.CH.COOH

NH, NH,

by cleavage of carbon dioxide yields cadaverine which
has the structure belo\\i mriArkw -.^ — .^

^IBf^ARY OPTUS

MuTflVm'^' ■ASSOCIATION

WtUMBIA UNIVEksn V

44 THE AMINO ACIDS

CHj.CHa.CHa.CHa.CHj

NHa NH,

Cadaverine or Pentamethyldiamine

Arginine, another amino acid, is the mother sub-
stance of putrescine. Arginine under suitable condi-
tions yields urea and ornithine, thus :

CHa.CHa.CHa.CH.COOH

NH

NH, ^

NH,

Arginine •

NH,

CH,.CH,.CH,.CH.COOH

C = O +

NH, NH,

NH,

Urea

Ornithine

Ornithine by cleavage of carbon dioxide yields
putrescine or tetramethyldiamine.

CH,.CH,.CH,.CH.COOH CH,.CH,.CH,.CH,

NH, NH, -CO, NH, NH,

Ornithine Putrescine or

Tetramethyldiamine

DIGESTION 45

It is exceedingly probable that the purpose of pro-
tein digestion is the reduction of these complex mole-
cules to the form of crystalline products, the amino
acids. Putrefaction is also concerned with these sub-
stances forming from them compounds which may
exert perhaps at times a more or less deleterious
action as, for example, indole or skatole, but also trans-
forming the amino acids into products, as tyramine or
histamine, which time may show to have distinct physi-
ological activities in keeping normal the adjustment
of nutritional rhythm.

References to Literature

Barger: The Simpler Natural Bases. 1914.

Cathcart: Physiology of Protein Metabolism. [Digestion.]

Hammarsten: Text Book of Physiological Chemistry. 1914.
[Digestion.]

London: Handbuch der Biochemie. Oppenheimer. III. 1909.
[Digestion.]

Retiger: Journal of Biological Chemistry. 1915, 20, p. 445.
[Putrefaction of pure proteins.]

Schaefer: Text Book of Physiology. 1898. [Digestion.]

Underhill: Middleton Goldsmith Lecture for 1911. Archives
of Internal Medicine, 1911, 8, p. 356. [Putrefaction.]

Winterstein and Trier: Die Alkaloide. 1910.

CHAPTER III

THE ABSORPTION OF PROTEINS AND
AMINO ACIDS

The views which have been held from time to time
relative to the character of the protein material or
products capable of absorption have been greatly-
influenced naturally by the ideas concerning the nature
and extent of protein digestion prevalent at a partic-
ular period. It is obvious that in the days of Liebig
and his contemporaries when digestion was assumed
to be little more than a process whereby proteins were
rendered soluble that the conception of the absorption
of unchanged protein should hold sway. Later, after
Kiihne had added his contributions to the knowledge
of digestion, theories of absorption were correspond-
ingly modified. Since the extent of formation and sig-
nificance of the amino acids have become better appre-
ciated our present-day views as to absorption are like-
wise undergoing modification.

Absorption from the Stomach

A great deal of discussion has taken place regarding
the question of gastric absorption of protein. It has
been asserted by Tobler that as much as 22 to 30 per
cent of the material in the stomach after a protein

THE ABSORPTION OF PROTEINS 47

meal is absorbed. London and his co-workers, on
the other hand, deny that any absorption takes place.
Abderhalden with Prym and London have apparently
decided the question in the negative for they have
demonstrated that amino acids fed to a dog with
several fistulae almost completely pass the pylorus, the
first absorption occurring in the duodenum. Under
normal conditions, therefore, gastric absorption so
far as protein is concerned may be regarded perhaps
as a negligible factor.

Looked at from another viewpoint, that of the
present with its modified ideas of the purpose of diges-
tion, one would naturally expect little or no absorption
from the stomach. If the view is correct that the pur-
pose of alimentary treatment of protein is to hydrolyze
this substance to either a polypeptide or amino acid
stage then it is reasonable to assume that these are the
products absorbed, rather than the proteoses or pep-
tones. Inasmuch as gastric digestion fails to decom-
pose protein to the stage of products naturally absorbed
it is reasonable to assume that the stomach is not an
organ adapted for extensive absorption under ordinary
circumstances. On the other hand, Folin and Lyman
have shown conclusively that amino acids, Witte pep-
tone and urea, may be absorbed from the stomach when
a ligature is tied around the pyloric opening. In view
of these conflicting facts one is hardly justified in
making a positive statement as to the importance of
the stomach in the absorption of nitrogenous decom-
position products.

48 THE AMINO ACIDS

Intestinal Absorption

From extensive experimentation it would appear that
the small intestine is capable of absorbing unchanged
native proteins and their decomposition products the
proteoses peptones and amino acids.

Absorption of Undigested Protein

It was pointed out by Voit and Bauer in 1869 that
undigested proteins such as serum or natural egg albu-
min may be absorbed by the small intestine, an obser-
vation which has been repeatedly confirmed by others.
It has been suggested that this is not the manner in
which most of the absorption takes place, only enough
protein being absorbed to replace worn-out tissue, the
excess being oxidized without ever having entered into
the tissue metabolism proper. Various explanations
have been offered to account for the fact of absorption
of undigested protein. The most obvious assumption
to make is that enzymes must have been present in the
intestine resulting in hydrolysis to amino acids and
hence their absorption. This point, however, is not
valid since the absorption was too rapid to admit the
possibility of such an hypothesis. Again, it has been
assumed that the experimental conditions rendered the
intestinal wall unusually permeable, thus allowing
protein to pass. It is possible that these results may
later be explained on grounds other than that of ab-
sorption, for Abderhalden, Funk, and London after
introducing excessive amounts of protein into the

THE ABSORPTION OF PROTEINS 49

intestine failed to obtain any reaction with the pre-
cipitin test. If protein were actually absorbed un-
changed in its natural form it is almost incredible that
the precipitin test failed to demonstrate its presence
when the extreme delicacy of this reaction is recalled.

What Happens to Protein Parenterally Introduced?

If it is possible for unaltered native protein to be
absorbed by the intestinal epithelium is it capable of
supplying the nitrogenous needs of the body? Or
what changes does it undergo after absorption? In
attempts to answer these questions endeavors have
been made to follow the fate of native proteins intro-
duced into the organism with avoidance of the gastro-
enteric tract. For many years it has been accepted
that protein introduced parenterally may be utilized in
part at least. This view was initiated from the investi-
gations of Zuntz and v. Mering and Neumeister. Since
then it has been repeatedly confirmed by a long list
of observers. Although it is generally admitted that
parenterally introduced protein reappears in the urine
only in small measure, there is not a unanimity of
opinion as to its fate. Even though the intravenous
injection of ^gg albumin fails to lead to a large output
of protein in the urine it has been agreed that its
failure to be eliminated by the kidney is no evidence
of its utilization in the tissues. In such an argument
one might assume with reason that the protein could
be excreted through the bile, be poured into the intes-

50 THE AMINO ACIDS

tine, undergo intestinal digestion and eventually be
absorbed in the form of protein decomposition pro-
ducts. Experiments to test this point have been carried
out. It has been shown that when a solution of casein
is introduced directly into the blood stream a small part
may reappear in the bile

On the other hand, when egg white or serum are
injected subcutaneously into dogs and goats a goodly
portion of the protein may be eliminated in the form
of non-coagulable protein. This observation would
tend to demonstrate the non-utilization of the injected
protein as such and points out that it undergoes a
change in its transit through the organism. In the
blood also a non-coagulable protein, perhaps a proteose,
is detectable, and a marked increase in nitrogen of
the urine — in the form of urea — is apparent. Indeed,
nitrogen equilibrium may be maintained under these
circumstances when animals are given a sufficiency of
carbohydrates.

In most of the work on parenteral absorption of
protein the material introduced did not possess enough
differentiation from other body proteins to distinguish
it from them. Borchardt conceived the idea of inject-
ing a protein with peculiar properties and chose hemi-
elastin ; after intravenous injection this substance was
present in the wall of the small intestine, and Borchardt
concluded that the protein was either on its way for
excretion by the gut or further changes by the intes-
tinal juices to prepare it for utilization by the tissues,
or finally had found its way into the intestine by way

THE ABSORPTION OF PROTEINS 51

of the bile. The last hypothesis did not appear likely
however, since none of the introduced protein could
be found in the liver.

From the foregoing arguments it is clear that the
apparent utilization of parenterally introduced protein
may be disposed of in at least three ways : 1. The
direct utilization by the tissues, for which there is little
or no evidence. 2. The excretion of the injected mate-
rial into the intestine where it is subjected to the
action of digestive enzymes, finding its way either
directly into the intestine or indirectly through the
bile. There is little doubt that a certain portion of
the injected material is treated in this manner. 3. The
transformation of the native protein in the tissues
into smaller fractions such as proteoses, peptones or
amino acids. Heilner has suggested that the utilization
of parenterally introduced protein is induced by the
generation of a specific enzyme capable of bringing
about a hydrolysis.

In the last suggestion it is probable that we have
the true explanation for the phenomenon under dis-
cussion for Abderhalden and his co-workers have
demonstrated that the parenteral introduction of native
protein or of peptone gives to the blood serum of the
animal the power of decomposing these substances, and
this power is destroyed by heating to 60° to 65° C.
Over-feeding by mouth confers upon the blood serum
the same property. The acquisition of this power on
the part of the blood serum may be regarded, as
Heilner suggested, as a generation of an enzyme or

52 THE AMINO ACIDS

it may be possible that the transport of the foreign
material through the tissue has carried with it into the
blood enzymes already existing in other parts of the
organism. Be this as it may, it is very probable that
protein retained in the body after parenteral intro-
duction or even perhaps after absorption from the
intestinal canal without profound disintegration even-
tually undergoes decomposition into simpler products
after reaching the blood stream. It would appear from
this statement, therefore, that the tissues must prefer,
to say the least, their pabulum in the form of relatively
simple compounds rather than as complex molecules
like the native proteins.

Absorption of Proteoses and Peptones

It was early discovered that peptone left in contact
with the living intestinal wall disappeared or at least
failed to show its characteristic reactions. From these
observations Hofmeister formulated the theory that
the peptones were taken up by the leucocytes of the
intestine after absorption and by them transformed
into protein and distributed to the tissues. This
hypothesis failed to meet with the approval of Heiden-
hain, who, although believing in the conversion of pep-
tone to protein, assigned to the intestine itself the
important role of this transformation. In confirmation
of the correctness of this idea may be cited the experi-
ments of Hofmeister and Neumeister who demon-
strated that peptone introduced directly into the blood

THE ABSORPTION OF PROTEINS 53

was treated as so much waste material being eliminated
by the kidneys. Moreover, he also failed to find any
trace of peptone in the blood or lymph of animals at
the height of digestion.

The experiments cited above failed to throw any
light upon the fate of the peptone, beyond its dis-
appearance. In later experiments Neumeister showed
the presence of leucine and tyrosine in the intestine
after introduction of peptone, thus indicating a further
decomposition of the peptone. Even though it might
be accepted that peptone placed in the intestine under-
goes a further breakdown to amino acids there still
existed no proof that the amino acids were absorbed
as such. It is possible to assume a synthesis of the
amino acids back into protein in the act of absorption
through the intestinal wall. An example of such a
reaction is found in the digestion of fat where fat is
split into fatty acids or soap and glycerol and regener-
ated as fat during the process of absorption.

It was Cohnheim's attempt to isolate this hypotheti-
cal protein from the intestinal wall which led to his
discovery of the enzyme erepsin already considered the
action of which has had a tendency toward filling in
some of the gaps in our conception of the nature of
intestinal digestion and absorption. Although it had
been recognized for many years that amino acids are
formed in intestinal digestion they were regarded as
by-products and quite unimportant. As a result of
the discovery of erepsin the purpose of the formation
of amino acids first received its due recognition.

54 THE AMINO ACIDS

That all of the older investigators did not regard the
direct absorption of protein or even of such large
molecules as peptone as essential for nutrition may be
seen from the view formulated by Salkowski and
Leube. According to this suggestion leucine may be
regarded as a substance capable after absorption of
being built up into protein and therefore leucine might
be looked upon as a stage in protein regeneration.
Against such a view, however, stood the fact that the
amino acids were not at that time demonstrable in the
blood or lymph.

From the numerous researches carried through con-
cerning the absorption of proteoses and peptones from
the intestines, the conclusion may be drawn that
although these proteins disappear when placed in con-
tact with the intestinal mucosa, there is no evidence of
their absorption as such for they can be found neither
in the blood nor in the lymph. On the other hand,
inasmuch as their disappearance from the intestine is
coincident with the appearance of amino acids, an
enzyme being furnished which specifically effects such
a transformation, it is probable that these proteins are
absorbed only in the form of amino acids.

The Absorption of Amino Acids

As has been stated previously the presence of amino
acids in the small intestine has long been known. Their
absorption therefrom, however, has been a matter of
conjecture. Inasmuch as their presence in the blood

THE ABSORPTION OF PROTEINS 55

or lymph could not be detected the theory of their
synthesis to protein coincident with their absorption
was promulgated. To Folin we owe the first indirect
proof of the absorption of amino acids from the intes-
tines. By a set of delicate methods adapted for the
partition of different forms of nitrogen he has suc-
ceeded in demonstrating an appreciable increase in the
"non-protein" portion of the blood, after introduction
of amino acids into a loop of intestine. Although the
amino acids themselves were not isolated the non-
protein fraction of the blood was so significant as to
leave no doubt of amino acid absorption. Van Slyke
and Meyer strongly fortified the view by the use of a
different method. The quantity of amino acids in the
circulation at one time is so small as to have escaped
detection by methods previously in use. It remained
for Abderhalden to demonstrate the presence of amino
acids in the blood under normal circumstances and
actually to isolate them. This he accomplished by
employing fifty liter lots of blood. From such large
volumes he succeeded in separating and identifying
proline, leucine, valine, aspartic acid, glutamic acid,
alanine, glycocoU, arginine, histidine, and lysine. In
no instance did he obtain more than 0.5 gram of any
one substance. The absorption of amino acids as such
is, therefore, an assured fact.

Absorption from the Large Intestine

Recalling to mind the heterogenous mixture of sub-
stances that may reach the large intestine one at once

66 THE AMINO ACIDS

realizes the great number of compounds that may find
their way into the blood stream. Leaving out of con-
sideration any residue of undigested protein we may
confine our attention to the possibilities of proteose and
peptones, the amino acids and the derivatives of the
latter. The evidence of the absorption of proteose and
peptone derivatives is decisive since Folin and Denis
have demonstrated an increase in the non-protein
nitrogen of the blood after placing Witte peptone in a
ligatured loop of the large intestine. The absorption
from the large intestine, however, is much slower than
obtains in the small intestine. In a similar manner
Folin and Denis have observed the absorption of dif-
ferent amino acids and urea. Throughout the entire
intestinal canal, therefore, the absorption of amino
acids may be regarded as a normal process.

The absorption of the well-known typical products
of putrefaction needs only brief description since their
fate has long been recognized. Absorption of indole,
skatole, phenol, cresol, etc., is certain since their
addition products are found in the urine. Thus indole
is absorbed, carried to the liver through the portal
vein, oxidized to hydroxy indole (indoxyl), combined
with sulphuric acid and eventually is eliminated as the
potassium salt, indican — its amount being indicative
of the extent of intestinal putrefaction. Or indole may
be combined in part after oxidation with glycuronic
acid and be excreted as a glycuronate. Phenol and
cresol may likewise be eliminated in the urine as
ethereal sulphates.

THE ABSORPTION OF PROTEINS 57

The fate of the amines formed in putrefaction is also
fairly well established, at least in certain instances.
Thus, for example, it is known that the amine formed
from tyrosine, p . oxyphenylethylamine in passing the
organism is transformed to and excreted as p.oxy-
phenylacetic acid.

References to Literature

Ahderhalden: Zeitschrlft fiir physiologische Chemie. 1913,
88 y p. 478. [Amino acids in blood.]

Cathcart: The Physiology of Protein Metabolism. 1912.

Folin and Denis: Journal of Biological Chemistry. 1912, ii,
p. 87 and p. 161; 1912, I2, p. 141 and p. 253. [Fate of
digestion products.]

Hammarsten: Text Book of Physiological Chemistry. 1914.

Van Slyke and Meyer: Journal of Biological Chemistry.
1912, 12, p. 399; 1913-1914, i6, p. 187, p. 197, p. 213, and
p. 231. [Fate of digestion products.]

CHAPTER IV

IN WHAT FORM DOES INGESTED PROTEIN
ENTER THE CIRCULATION?

Our conception of the nature of metabolic processes
in the tissues will be more or less modified by the view
accepted concerning the degree and character of dis-
integration of protein induced in the alimentary tract
and the form of the products absorbed. This being so
a consideration of the hypotheses that have been ad-
vanced relative to the fate of protein after its dis-
appearance from the gastro-enteric tract is now in
order. This fate of ingested protein has been ex-
plained in at least four different ways, namely :

1. The proteins are absorbed with little or no
chemical change and are taken up by the tissues and
incorporated into them. In a previous chapter it has
been noted that native protein may be absorbed as
such at times and fail to reappear leading to the infer-
ence of utilization by the tissues. This, however,
cannot be accepted as the usual procedure for all the
food protein. Once in the blood stream as shown by
parenteral introduction protein utilization apparently
occurs through the intervention of enzymes which sud-
denly make their appearance in the circulation.

PROTEIN AND CIRCULATION 59

2. The proteins of the food are hydrolyzed and the
products are absorbed and carried to the tissues.

3. The digestion products are synthesized into
serum protein by the intestinal wall during the act of
absorption, and the serum protein serves as pabulum
for the tissues.

4:. Deamination of the digestion products occurs
previous to their entrance into the circulation. Since
it is impossible to accept the hypothesis that unchanged
protein is the form in which ingested protein is usually
absorbed the next natural inference is that the pro-
teoses and peptones are absorbed directly into the blood
and conveyed to the tissues. In attempting to deter-^*
mine the correctness of this hypothesis the query has
arisen

Are Proteoses and Peptones Present in the

Blood ?

In spite of the discovery of erepsin by Cohnheim
and the consequent improbability of proteoses and
peptone representing the usual form of final digestion
products, some modern investigators have clung to the
idea that it is in the form of proteoses and peptones
that protein is absorbed. This view is based upon a
number of investigations from which the assertion has
been made that proteoses and peptones are present in
the blood stream. The work of Neumeister led to the
conclusion that proteoses and peptones are not found
in the blood and it was not until 1903 through the

60 THE AMINO ACIDS

observations of Embden and Knoop that any doubt
of Neumeister's view was entertained. In experiments
designed to show the fate of proteoses and peptones
when brought into contact with the intestinal wall
Embden and Knoop were able to show the presence in
the blood of substances having the properties of pro-
teoses and peptones. They held that the mucous mem-
brane of the intestine neither synthesized these sub-
stances to a larger molecule, as for example to a coagu-
lable protein, nor were they hydrolyzed to the amino
acid stage but, on the contrary, absorption took place
directly. The results of Embden and Knoop were con-
firmed by some observers and discredited by others.
Schumm for example was always unable to find a trace
of proteose in the blood both under normal and ab-
normal conditions of health. Abderhalden and his co-
workers maintained that the substances giving
reactions for proteoses and peptones were present
because of imperfect methods employed in the separa-
tion of the coagulable protein from the blood.

It has been pointed out by others, however, that there
is a possibility of the presence in the blood of a protein
naturally non-coagulable which would still give the
reaction — the biuret test — significant under the experi-
mental conditions for the presence of proteose or pep-
tone. Zanetti described such a protein in the blood
and found that it belonged to the group of mucoids.
Among others, Howell believes in the existence in the
blood of a protein possessing some of the character-
istics of the proteoses and peptones, for example, non-

PROTEIN AND CIRCULATION 61

coagulability, which is not, however, identical with
these substances. Bywaters has concluded that this
body is a substance called by him sero-mucoid. In spite
of these views Bergmann and Langstein and Kraus
assert that small amounts of true proteose are present
constantly. After feeding elastin Borchardt claimed to
find elastin proteose in the blood stream. Upon repe-
tition of this work of Borchardt, Abderhalden and
Ruehl failed to give it confirmation. The explanation
of Abderhalden and Oppenheimer that imperfect sepa-
ration of coagulable protein is responsible for the
proteose test was denied by Freund who maintained
that the method employed by Abderhalden not only
precipitated the coagulable protein but the proteose
also.

From the foregoing brief review of only a few of the
investigations carried through for the decision of the
problem it is apparent that the whole question is in a
chaotic state of contradictions and that a positive
answer cannot be given. It may at least be said that
positive proof of the presence of proteoses and pep-
tones is still lacking. Perhaps one of the most con-
vincing arguments against the existence in the blood of
proteoses and peptones is derived from the work of
Abderhalden and Pincussohn. They have demon-
strated that just as with the parenteral introduction
into the body of native protein so with proteose injec-
tion there is a development in the blood plasma of an
enzyme capable of causing its disintegration to smaller
molecules. Such an enzyme is not present in the

62 THE AMINO ACIDS

blood under ordinary conditions. If proteoses were
present normally in the blood it is probable that this
enzyme would also be a normal constituent of the
blood.

The Synthesis, or Regeneration of Protein, by
THE Intestine

If it is accepted that protein is not absorbed in the
form of proteoses or peptones the query naturally
arises, In what form is it absorbed ? An answer to this
question must necessarily determine also the place of
protein regeneration so long as the conception of nutri-
tion obtains that metabolic changes in the organism
demand the formation of new material to replace that
broken down. If one maintains that protein gets into
the blood stream in the form of a molecule larger than
the amino acid, proteose or peptone molecule, it is self-
evident that the intestine must be regarded as capable
of synthesizing amino acids to protein. On the other
hand if amino acids are regularly present in the sys-
temic circulation, the place of protein regeneration
must be relegated to the cellular elements of the dif-
ferent tissues.

Ahderhalden. In the past various theories have been
maintained. In view of the failure to find amino acids
in the blood, Abderhalden put forward the view that
the intestinal wall possessed the power during the act
of absorption to synthesize the amino acids to proteins,
probably serum proteins, to meet the needs of the

PROTEIN AND CIRCULATION 63

organism's requirements. The necessity for prelimi-
nary extensive disintegration of the food protein has
been offered as follows : "Every species of animal — in
fact every individual — has its specifically constituted
tissues and cells. If the diet was always the same, the
formation of the tissues might bear some close relation
to the components of the food. The diet varies, how-
ever, and, especially in the case of human beings and
the omnivora, is exceedingly diverse in nature and to
make its organism independent of the outer world in
the matter of food taken, it disintegrates the nutrient
it receives, and utilizes those components which may be
of service to it in building up new complexes."

Objections to this theory have been summarized by
Cathcart as follows : "The view that the tissue proteins
differ from one another, that they are specific bodies
of definite constitution, and that, therefore, each re-
quires a different amount and supply of building mate-
rial is gradually being accepted. Abderhalden himself
accepts this. What end then is served in having a
single uniform pabulum formed when the demand is
so varied? This is all the more questionable when it
is remembered that there is no indubitable evidence
which shows that one amino acid can be converted
into another. Further, the belief is gradually gaining
ground, as regards the protein requirements of the
organism, that it is not so much the actual quantity as
the quality of the protein supplied in the food, which
is of importance. If the material supplied be uniform
it necessitates a fresh breakdown by each tissue,

64 THE AMINO ACIDS

perhaps by each individual cell. Although the tissues
all probably possess this power of breaking down pro-
tein material by means of their intracellular proteolytic
enzymes, still the extra work involved seems to nega-
tive the immediate resynthesis hypothesis, especially
when the hypothesis of the circulating digestion pro-
duct postulates the presence of the individual food
material in the blood. As already remarked, the mere
failure to detect these products in the blood does not
give adequate reason for concluding that they are not
present. The tissues certainly do not break dov/n in
regular sequence, nor are they left to fall to pieces for
lack of repair material. Repair is among the most
active functions of all tissues. Must, then, a tissue
of highly complex structure keep destroying and digest-
ing plasma, picking out from the debris the nuclei
which it requires and letting the rest go ? (Why, and
this destruction is admitted by Abderhalden, are the
superfluous amino acids not found in the blood?)
What happens, for instance, in the case of connective
tissues with their demand for, say, glycine, where the
food supply is not over-abundant as the circulation is
poor, and the tissues not very suited for lymph per-
fusion? It will not do merely to say that there is no
great breakdown of material here. Pfliiger, in an inter-
esting paper in which he combated this immediate
resynthesis hypothesis, ascribed to the cells of the
intestinal wall, with regard to the protein synthesis,
the same capacity as the cells of all tissues, but denied
that the synthesis of protein for the whole organism

PROTEIN AND CIRCULATION 65

was carried out there. He held that such a hypothesis
was contrary to all existent knowledge of physiological
assimilation."

One may query to what extent does immediate
resynthesis take place ? Are all the digestion products
transformed into coagulable protein or are some se-
lected and others rejected in part? The questions
cannot be answered by the supporters of Abder-
halden*s theory. On the other hand, the intestine evi-
dently is capable of exerting a marked selective action
as to the type and amount of amino acid it shall absorb.
The experiments of Abderhalden and his co-workers
have indicated this. They fed gliadin to polyfistular
animals and observed as the material traversed the
gastro-enteric tract that tyrosine disappeared from the
intestine whereas glutamic acid steadily increased in
amount.

Freund, The idea of Freund is somewhat similar
to the hypothesis advanced by Abderhalden except
that he ascribes to the liver an important role in the
subsequent breakdown of the protein. The protein
digestion products are assumed by Freund to travel
the portal circulation in the form of pseudo-globulin.
The liver is unable to properly decompose protein
unless it has first entered the blood stream by way of
the intestine. This hypothesis carries with it the sug-
gestion that the parenteral utilization of protein must
be carried out through aid from the intestine, that the
protein is excreted from the blood into the intestine,
undergoes digestion, the products are absorbed, and

66 THE AMINO ACIDS

during the act of absorption are polymerized,. This
suggestion has been tested in different ways. One
might expect that protein parenterally introduced into
dogs, the intestine of which had been removed, would
reappear in the urine. Korosy has failed to find more
than traces of protein after parenteral injections of
protein into animals without an alimentary tract. These
observations tend to show that intestinal preparation
of protein cannot be regarded as essential. The prob-
lem was attacked in another way by Abderhalden and
London. They attempted to determine the excretion of
protein into the intestine of polyfistular animals after
parenteral introduction but failed to obtain any evi-
dence of such a reaction. On the other hand, the excre-
tion of substances into the intestine after parenteral
injection is known. Thus, Abderhalden and Slavu
have shown that iodine may find its way into the
intestine when iodine-polypeptide combinations are
injected subcutaneously.

Hofmeister. It was the view of Hofmeister that the
leucocyte is intimately associated with protein re-
generation. The idea undoubtedly originated from the
marked leucocytosis which occurs after meals and
Hofmeister thought that peptone after absorption was
changed in some unknown manner into protein by the
leucocytes or else through the agency of adenoid tissue.
Later, the lymphocyte was selected as the specific form
of leucocyte responsible for protein synthesis. This
theory, however, finds few supporters today. Perhaps
the best criticism of the leucocyte synthesis theory has

PROTEIN AND CIRCULATION 67

been offered by Halliburton. "He pointed out that the
number of the lymphocytes was not commensurate
with the work to be done. He calculated that a man
of eighty kilos had about four kilos of blood of which
some 40 per cent was in the form of corpuscles, that is
about 1600 grams. Now as the ratio of white cor-
puscles is about 1 : 500 it means that about 3.2 grams of
leucocytes are present. Of this amount lymphocytes
form at most 30 per cent, and therefore in the blood
there would be about one gram of lymphocytes. If
this amount w^ere doubled during digestion *it is diffi-
cult to see how two grams of lymphocytes can tackle
the enormous burden which every meal must impose
upon them.' " (Cathcart.)

What Is the Evidence for the Synthesis of

Protein ?

The Synthetic Action of the Gastric and Intestinal
Mucous Membranes.

Hofmeister has ascribed to the stomach mucous
membrane the property of synthesizing protein from
proteoses. An outline of his experiment follows —
at the height of digestion a dog was killed and its
stomach and contents divided equally into two parts.
One part was immediately placed in boiling water to
stop all enzyme and cellular activity and the other
portion was placed in an incubator for a period of
two hours. The amount of proteose and peptone
present in each part was then determined. In the por-

68 THE AMINO ACIDS

tion placed in the incubator, there was an almost com-
plete disappearance of proteose and peptone, which of
course could not be ascribed to further decomposition
since gastric juice does not hydrolyze proteoses and
peptones to amino acids, at least during such a short
period. The conclusion drawn was that the proteoses
and peptones disappeared because of their synthesis
to protein. In other experiments Hofmeister demon-
strated that the intestine possesses the same property.
Glaessner repeated and confirmed Hofmeister's inves-
tigation. On the other hand, Embden and Knoop
failed to find any evidence of protein synthesis. They
employed the normal intestine and also the intestine
from which pancreatic juice was excluded by ligature
of the duct. The evidence for resynthesis of protein
in the gastric or intestinal mucous membranes is not
convincing and one must obtain other than negative
evidence before the idea of such a protein resynthesis
can be accepted.

Plasfein Formation. It has been observed repeatedly
that when solutions of proteoses are brought into con-
tact with rennin a precipitate called plastein forms.
Various views as to its formation have been held. It
has been assumed by some that plastein is a new syn-
thetic product formed from the proteoses — a new pro-
tein, by others a resynthesis of the proteoses to the
original protein from which they were derived, and by
still others as a digestion product on its way to com-
plete solution. The results of the most searching
investigations concerning the nature of plastein incline

PROTEIN AND CIRCULATION 69

one to the belief that this substance is of the nature
of proteose rather than that of a complete protein so
that plastein formation affords little or no evidence for
the support of the existence of protein synthesis.

The Synthetic Action of Pepsin and Trypsin

It has been demonstrated by A. E. Taylor that by
the long continued action of trypsin of the clam liver
upon concentrated protamine digestion products a
reformation of protamine takes place. The quantity
reformed is very small in comparison with the original
amount of protamine digestion products. In a similar
manner Robertson has found the synthesis of a para-
nuclein by the action of pepsin upon concentrated
casein digestion products. These results lead to the
suggestion that trypsin and pepsin may possess a two-
fold action, a disintegrative influence and a synthetic
action in accord with the idea of the reversibility of
enzymes. If the synthetic action in the intestine is as
slow as that shown in the experiments just cited little
value can be assigned to them as aids in the regenera-
tion of protein in the body for the influence could be
observed only after the influence had continued for
several months.

The evidence for the synthesis of protein in the
intestinal wall is all of an indirect nature. If the
adherents of that theory could demonstrate an increase
of protein in the blood after an ingestion of protein

70 THE AMINO ACIDS

their argument might be established. This they have
failed to do.

Deamination

The failure to demonstrate the presence of amino
acids in the blood of the higher animals during diges-
tion led to the conception that the amino acids are
deaminated, that is, ammonia is split off while passing
the intestinal wall, this deamination being regarded as
the first stage in the catabolism of the amino acids.
This possibility was suggested by the work of Cohn-
heim upon certain of the lower forms of animal life
in which he showed the giving off of ammonia by the
intestine after addition of amino acids, and derived
support from the older work of Nencki and others
who showed that the ammonia content of the portal
blood was greater than that of the arterial during
digestion. It has been assumed that as result of this
process of deamination the ammonia split off is trans-
formed by the liver into urea and so quickly eliminated
by the kidneys. Such a view has been adopted as
explanatory for the long known rapid rise in urea
excretion following protein ingestion.

It has been shown by Lang that deamination is a
property of a great many tissues of the body but it is
probable that certain of them possess a selective action
in this respect for some tissues deaminate certain of the
amino acids much more readily than others. In par-
ticular the intestine and liver seem to possess this
action in a high degree. To the liver a great import-

PROTEIN AND CIRCULATION 71

ance has been attached as a deaminating agent and
during recent years discussion of the so-called defect-
ive or insufficient deamination in a series of pathologi-
cal conditions has come into vogue.

In accordance with this idea amino acids have been
administered as a test for the functional activity of
the liver. Glaessner has shown that normal liver tissue
is capable of transforming definite amounts of specific
amino acids into urea. In a series of experiments he
has shown that in various diseased conditions of the
liver, such as fatty liver, in syphilis, cirrhotic liver, and
a phosphorus poisoned liver a failure to convert amino
acids into urea and a consequent output of amino acids
in the urine took place.

That deamination is undoubtedly an important intra-
cellular activity may be derived from a series of experi-
ments in which amino acids have been fed and their
fate determined. Thus with arginine most of the
nitrogen reappears as urea. Probably through the
intervention of the enzyme, arginase, a splitting of
arginine into urea and orinthine occurs and by deami-
nation of the latter more urea is formed. Again,
after administration of alanine, lactic acid in the urine
has been observed. With the purines also it may be
shown that a splitting off of ammonia occurs. One
may accept without hesitation that the function of
deamination is an important activity of cell life. The
contention that certain organs or tissues possess this
function more specifically than others has been a matter
of controversy. The role played by the intestine in

72 THE AMINO ACIDS

this regard is especially to be considered, correlated
as it has been with the explanation of the form in
which protein digestion products are absorbed.

The recent observations of Folin and Denis and
others have rendered untenable the hypothesis that
deamination by the intestine is the first stage in the
catabolism of amino acids. They demonstrated that
during the absorption of amino acids from the intes-
tine there was no increase in ammonia or urea of the
blood and they further showed that the ammonia of
the portal blood is produced in large measure by the
products of putrefaction in the large intestine. The
retirement of the theory of intestinal deaminization
to account for the apparent absence of amino acids in
the blood carries with it also the untenability of the
idea that the liver is specifically concerned in the
formation of urea. To quote the authors : "In the
absence of satisfactory proof that deaminization and
urea formation is localized we are not justified in
assuming that the process is a specialized process in
the sense of being confined to some particular organ.
In other words, so far as we yet know, the urea form-
ing process is a characteristic of all the tissues and by
far the greatest amount of urea is therefore prob-
ably formed in the muscles. The negative results,
so far as any localized urea formation is concerned, is
almost satisfactory proof that there is none, for if
there were one central focus from which all or nearly
all of the urea originated we could scarcely have
failed to find it."

PROTEIN AND CIRCULATION 73

Are Amino Acids Found in the Blood?

Opposed to the investigators advancing the regenera-
tion of protein immediately after absorption is a
second group of men who have long believed that
amino acids are absorbed into the blood. The great
difficulty has been to demonstrate their presence. A
large number of experiments have been devised in
various ingenious ways to overcome the difficulties
attendant upon such a procedure. Many investigators
have obtained partial evidence of the presence in the
blood stream but an actual isolation and identification
of individual amino acids was for a long time lacking.

The failure to obtain definite proof of the amino
acids in the blood has been due in large measure to
the inadequacy of the methods available. At any one
moment the quantity of these substances in a deter-
mined sample of blood must be exceedingly small.

Moreover, one must remember that the formation of
amino acids in the intestinal tract is a gradual process
and not of the nature of an explosion so that the
quantity of amino acids available for passage into the
blood during a given period must be relatively small.
The rapidity of circulation is another factor to be
taken into consideration. It has been shown that in
the portal vein of the dog the blood travels at the rate
of about 150 cc. per minute. Pfliiger has estimated
that for human beings a maximum rate of absorption
of protein may be represented at 1.14 gram protein
per kilo per hour. If the 1.14 gram protein absorbed

74 THE AMINO ACIDS

per hour is compared to the volume of blood in which
it would necessarily be dissolved we find that the
protein would be present in the concentration of 0.12
per cent. Another difficulty arises from the fact that
the blood is a fluid already containing about 3 per
cent of coagulable protein and also nitrogen to a
smaller extent in other forms.

Certain fairly definite indications, however, have led
many physiologists to maintain a belief that protein
is absorbed and is taken up by the cells in the form of
amino acids.

Determination of the "residual nitrogen" of the
blood, that is, the difference between the total nitrogen
and that representing coagulable protein has shown
that after meals there is a slight gain both in the portal
blood and that of the systematic circulation. This
increase of nitrogen may be attributed to the absorbed
amino acids or polypeptides, but in view of the possi-
ble existence of non-coagulable proteins in the blood
it cannot be accepted as proof positive. A second
method for the same endeavor has been the formation
of amino acid compounds by use of j8-naphthalene
sulphochloride. Shaken with the fluid obtained after
separation of the serum proteins either by coagulation
or by means of dialysis precipitates have been obtained
with this reagent, strongly indicating the presence of
amino acids but the failure of the precipitate to assume
a crystalline form has made impossible a positive
identification of amino acids. Cohnheim by observa-
tions with the isolated intestine of the octopus was able

PROTEIN AND CIRCULATION 75

to prove absorption and the existence in the blood of
certain amino acids but failed to detect these sub-
stances when experiments were carried out on the
intact animal.

By the elaboration of new methods, Folin and Denis
and Van Slyke and Meyer have been able to prove the
entrance of amino acids into the blood stream. Later,
Abderhalden, the chief opponent of the idea of amino
acid absorption, was successful in isolating from the
blood several of the individual amino acids by the
employment of great volumes of blood. The absorp-
tion of protein in the form of amino acids having thus
been established the question next arises what becomes
of them? It was soon proved that there was a rapid
disappearance of amino acids from the circulation and
this fact made pertinent the queries : "Are they decom-
posed in the blood: are they chemically incorporated
into the complex molecules of the tissue proteins ; or
are they merely absorbed by the tissues in general, or
by certain tissues in particular, without undergoing
any immediate change?" These questions have been
fully answered by Van Slyke and Meyer in experi-
ments designed to follow the fate of the amino acids
after absorption. It was found that the amino acids
are absorbed by the tissues without undergoing any
immediate chemical change. This absorption though
rapid is never complete, the blood always containing
a small quantity of amino acids. It would appear
from this that there is an equilibrium between the
amino acids of the blood and of the tissues. The way

76 THE AMINO ACIDS

in which amino acids are taken up by the tissues and
held by them is still undetermined.

In a later communication the same investigators have
attempted to determine the fate of amino acids after
absorption by the tissues and selected the changes
occurring in the liver. Amino acids absorbed by the
liver rapidly disappear. In explanation of this obser-
vation several possibilities exist: 1. The amino acids
may be excreted through the bile. This view, however,
is not probable since the quantities of amino acids in
the bile and urine were entirely too small to account
for the amount that disappeared from the liver. 2. A
second possibility is that the amino acids are trans-
ferred to other tissues. This hypothesis is also highly
improbable since none of the other large organs show
a greater avidity for amino acids, yet three or four
hours after injection of amino acids other organs
usually contain more amino acids than the liver. 3.
The absorbed amino acids are synthesized into body
protein in the liver. The possibility cannot be defi-
nitely decided at present. 4. The amino acids are
deaminated with formation of urea or ammonia. In
all probability a portion of the amino acids which
disappears from the liver reappears in the urine as
urea.

The disappearance of amino acids from the liver is
more rapid and complete than is true for other tissues
like the kidney, intestine, pancreas, and spleen. From
the muscles the amino acids disappear very slowly.
As a summary of the whole question one may quote the

PROTEIN AND CIRCULATION 77

words of Van Slyke and Meyer: "The amino adds,
with perhaps some peptides, from the intestine enter
the circulation, from which they are immediately
absorbed by the tissues. The power to take them up
from the blood stream is common to all the tissues,
but is limited. The muscles of the dog, for example,
reach the saturation point when they contain about
75 mgm. of amino acid nitrogen per 100 grams. The
liver, however, continually desaturates itself by metab-
olizing the amino acids that it has absorbed, and con-
sequently maintains indefinitely its power to continue
removing them from the circulation so long as they do
not enter it faster than the liver can metabolize them.
When the entrance is unnaturally rapid, as in our
injection experiments, or when the liver is sufficiently
degenerated, as observed clinically in some pathological
conditions, the kidneys assist in removing the amino
acids by excreting them unchanged. Death may result
when the above agencies for preventing undue accu-
mulation of protein digestion products are over-taxed.

"In regard to the synthesis of tissue proteins it
appears reasonable to believe that, since each tissue
has its o-wTi store of amino acids, which it can replenish
from the blood, it uses these to synthesize its own
proteins."

Concerning the manner in which the free amino
acids are utilized by the tissues two possibilities may
be assumed, and according to Van Slyke and Meyer
these are : 1. The amino acids serve as a reserve energy
supply, Hke glycogen, or as a reserve of tissue-building

78 THE AMINO ACIDS

material. In either case the supply would be depleted
if not renewed from the food. 2. The amino acids
are merely intermediate steps in both the construction
and breakdown of the tissue proteins. In this case they
could originate, not only from absorbed food products,
but also from autolyzed tissue protein: starvation
would not result in a disappearance of the amino acid
supply of the tissues, and might even increase it. To
determine the correctness of one or the other of these
hypotheses the authors mentioned above analyzed the
tissues of animals in various states of nutrition. The
results are in harmony with the second hypothesis, for
free amino acids of the tissues tend to increase in
starvation rather than to disappear. The investigators
have summarized their views regarding this in the fol-
lowing words : "The amino acids appear, therefore, to
be intermediate steps, not only in the synthesis, but in
the breaking down of body proteins. Otherwise, in
order to explain their maintenance in the tissues during
starvation, one would be forced, contrary to the con-
clusions of all experimental work on the subject, to
assume that they are inert substances lying unchanged
for long periods, even when most urgently needed to
build tissue or supply energy. The maintenance of
the amino acid supply by synthesis, from ammonia and
the products of fats or carbohydrates, seems excluded.
The supply of raw material in the form of fat and
carbohydrates nearly disappears during starvation, and
the ammonia could originate only from broken-down
protein, as the normal store of ammonia nitrogen is

PROTEIN AND CIRCULATION 79

only a fraction of that of the free amino acids. These
considerations, and the self-evident wasting of starved
tissues, point strongly to autolysis as the main source
of the free amino acids in the fasting body."

"The failure to increase the free amino acid content
of the tissues by high protein feeding indicates,
furthermore, that when nitrogen is retained in the
organism it is not to an appreciable extent, as stored
digestion products, but rather as body protein."

These results, and the consequent inference from
them, have made void all the older theories of metab-
olism and it is becoming more and more evident that
in any consideration of protein transformations within
the organism in health or in disease amino acids are
the substances which demand attention. This is the
age of amino acid metabolism and at present the inves-
tigations are being narrowed down to the point of the
determination of what actually occurs with the indi-
vidual amino acids and what special role in nutrition
each may play.

References to Literature

Abderhalden: Zeitschrift fiir physiologische Chemie. 1913,
88, p. 478. [Amino acids in blood.]

Abderhalden: Text Book of Physiological Chemistry. 1914
[Enzymes in blood.]

Cathcart: The Physiology of Protein Metabolism. 1912.

80 THE AMINO ACIDS

Folin and Denis: Journal of Biological Chemistry. 1912, ii,
p. 87 and p. 161; 1912, I2, p. 141 and p. 253. [Fate of
digestion products.]

Mendel: Theorien des Eiweissstoffwechsels nebst einigen
praktischen Konsequenzen derselben. Ergebnisse der
Physiologic. 1911, n, p. 418.

Van Slyke and Meyer: Journal of Biological Chemistry. 1912,
12, p. 399; 1913-1914, i6, p. 187, p. 197, p. 213, and p. 231.
[Fate of digestion products.]

CHAPTER V

THEORIES OF PROTEIN METABOLISM

Under the term protein metabolism are included all
the processes in the animal organism concerned with
the fate of protein whether introduced as food or
serving as tissue substance. The metabolic changes
are divisible into two distinct phases known as anabo-
lism, or building up processes, and cataholism, or de-
structive processes. Although it is definitely recog-
nized that metabolic activity is manifested in two dia-
metrically opposed directions, the successive stages in
either process are but vaguely understood. Only the
starting point and the final end products in each in-
stance can be stated with certainty, although here and
there individual stages in the processes under discus-
sion point in one or another direction, and thus give
indication of the type of activity that must have gone
before. For the unravelling of the mystery the first
requisite is a clear conception of the problem. In the
words of Liebig : "If we take the letters of a sentence
which we wish to decipher, and place them in a line,
we advance not a step towards the discovery of their
meaning. To resolve an enigma, we must have a per-
fectly clear conception of the problem. There are
many ways to the highest pinnacle of a mountain ; but

82 THE AMINO ACIDS

those only can hope to reach it who keep the summit
constantly in view. All our labor and all our efforts,
if we strive to attain it through a morass, only serve
to cover us more completely with mud; our progress
is impeded by difficulties of our own creation, and at
last even the greatest strength must give way when so
absurdly wasted."

The development of knowledge in science succeeds
best when an hypothesis is formulated as a basis for
investigation. By holding fast to that which is proven
as fact and discarding that which is shown to be con-
trary to fact is real progress made. This, indeed, has
been the case in the history of protein metabolism, as
may be seen in the following pages where is traced the
evolution of ideas concerning it. .

LlEBIG

The first clearly defined theory of protein metabo-
lism was that enunciated by Liebig who assumed that
protein material undergoes little or no chemical change
previous to its introduction into the blood stream and
its assimilation by the tissues. "According to this
theory, the plant holds a position intermediate between
the mineral and animal world. The animal is incapable
of assimilating the compounds stored up in inorganic
nature. To render these compounds subservient to
the purposes of animal life they may have to undergo
a preliminary preparation within the living organism of
the plant. The simple mineral molecules are thus con-

PROTEIN METABOLISM 83

verted into molecules of a higher order, fit to serve in
building up and maintaining alive the body of the
animal." "How admirably simple after we have
acquired a knowledge of this relation between plants
and animals, appears to us the process of formation
of the animal body, the origin of its blood and organs !
The vegetable substances, which serve for the produc-
tion of blood, contain already the chief constituent of
blood ready formed, with all its elements." "The true
starting point for all the tissues is, consequently,
albumen ; all nitrogenized articles of food, whether
derived from animal or from the vegetable kingdom,
are converted into albumen before they can take part
in the process of nutrition."

According to Liebig digestion is merely a process
whereby food becomes changed to a soluble condition
capable of absorption without transformation of its
identity. This soluble albumen is built up into organ-
ized tissue previous to its degradation (an idea later
adopted by Pfliiger). Thus we read: "There can be
no greater contradiction, with regard to the nutritive
process, than to suppose that the nitrogen of the food
can pass into the urine as urea, without having pre-
viously become part of an organized tissue; for albu-
men, the only constituent of blood which, from its
amount, ought to be taken into consideration, suffers
not the slightest change in passing through the liver or
kidneys ; we find it in every part of the body with the
same appearance and the same properties."

Liebig divided all foods into two groups, the nitro-

84: THE AMINO ACIDS

genous, or plastic, foods, and the non-nitrogenous or
respiratory foods. In accordance with this classifica-
tion plastic foods were tissue formers and supplied
energy for muscular activity; the respiratory foods,
on the other hand, were essential for the respiratory
act and the constant temperature of the body, but
could not be transformed into organized tissue.

VoiT

The fundamental conception of Voit (1867) was
that all protein in the body is not decomposed with
equal ease. In accordance with this idea he divided
the protein material of the body into two groups, the
organised or tissue protein, that built up into living
protoplasm and difficult of disintegration and, secondly,
circulating protein existing in the fluids and tissues of
the organism without being an integral part. The
circulating protein may be more easily and readily
destroyed than the organized or tissue protein. In his
classic experiment designed to show the difference of
metabolism between tissue protein and circulating
protein, Voit allowed a well-fed dog to starve for
several days. He demonstrated that under these cir-
cumstances there is at first an abundant decomposition
of protein material which is later followed by a period
during which very little protein is catabolized. His
interpretation of these facts was to the effect that dur-
ing the time when a large protein disintegration ob-
tained only circulating protein was destroyed, whereas

PROTEIN METABOLISM 85

in the later stages the greatly diminished uniform
protein metabolism was that of the organized or tissue
proteins.

In his theory Voit assigned to the cells the function
pf utilizing proteins, the older view that metabolism
took place in the blood having been discarded. From
the fluids bathing the tissues food or circulating pro-
■(ein is drawn within the cells and there transformed.
On the other hand, a certain small amount of tissue
protein is constantly dying and is replaced by circulat-
ing protein, thus becoming eventually living proto-
plasm. "The tissue-elements constitute an apparatus
of a relatively stable nature, which has the power of
taking proteins from the fluids washing the tissues and
appropriating them, while their own proteins, the tissue
proteins, are ordinarily catabolized to only a small ex-
tent, about 1 per cent daily." (Voit.) "By an in-
creased supply of proteins the activity of the cells and
their ability to decompose nutritive proteins are also
increased to a certain degree. When nitrogenous
equilibrium is obtained after an increased supply of
proteins, it indicates that the decomposing power of
the cells for proteins has increased so that the same
quantity of proteins is metabolized as is supplied to the
body. If the protein metabolism is decreased by the
simultaneous administration of other non-nitrogenous
foods, a part of the circulating proteins may have time
to become fixed and organized by the tissues, and in
this way the flesh of the body increases. During
starvation or with a lack of protein in the food the

86 THE AMINO ACIDS

reverse takes place, for a part of the tissue proteins is
converted into circulating proteins, which are metabo-
lized, and in this case the flesh of the body decreases."
(Hammarsten.)

Pfluger

In 1893 Pfluger severely criticised the theory of
Voit and offered another in its place. In its essence
the theory of Pfliiger rests upon the hypothesis that
food protein must become living protoplasm before
it can be utilized for the needs of the body. In accord-
ance with this idea he assumed that food protein is
catabolized with great difficulty whereas living proto-
plasm is in a state of continual unstable equilibrium
leading to any easy oxidation or decomposition of its
protein. Pfliiger's theory rests upon experiments
carried through by his pupil Schondorff . It was shown
by Schondorff that when the blood from a starving
dog was passed through the hind limbs and liver of
a well-fed animal the urea of this blood was increased.
On the other hand, no increase of urea could be ob-
tained when blood, whether of starved or well-fed
dogs, was passed through the hind limbs and liver of
a starved dog. From the results Pfliiger argued that
the determining factor in protein catabolism is the
state of nutrition in the tissue cells and not the cir-
culating protein.

Although in general Pfliiger appeared to disprove
many of the points in Voit's theory, one positive evi-
dence stands out clearly in favor of Voit's theory, and

PROTEIN METABOLISM 87

that is the fact of the rapidity with which large quan-
tities of protein are cataboHzed in the body. In a few
hours great quantities of protein may be disintegrated
as judged by the corresponding increase in urinary
nitrogen. It is hardly probable that living protoplasm
could be synthesized so rapidly and so much of it be
so quickly destroyed again. This is the more incredi-
ble since the same fact applies irrespective of the
previous state of nutrition of the organism.

In 1905 Folin subjected the experiments of Schon-
dorff to a searching criticism and pointed out that the
evidence furnished by them was by no means unassail-
able. Upon studying the details of one of Schondorif's
experiments, Folin found that the actual increase in
urea nitrogen in the transfused blood amounted to less
than one-tenth of 1 per cent instead of 125 per cent as
calculated by Schondorff. "Considering the numerous
sources of error and uncertainty necessarily attached
to an experiment of this kind, it seems very strange
that the extraction of 25 mgm. of urea-nitrogen from
the hind limbs of a dog killed while engaged in digest-
ing 700 gm. of meat should be accepted as proving not
only that protein catabolism did occur during the
experiment, but also that it occurred in the bioplasm
and not in the circulating protein."

No direct evidence has been obtained to prove or dis-
prove the one or the other of these last two widely
divergent theories. The distinction between tissue
protein and food protein is probably one of degree
rather than of kind.

88 THE AMINO ACIDS

ICASSOWI.TZ

Kassowitz in 1904 put forth the view that it was
scarcely probable that a substance would serve both as
reconstructive material for disintegrated cells and as
a source of energy. According to his ideas food pro-
tein is not merely transformed into living protoplasm
by some obscure rearrangement but there is an actual
synthesis with fat and carbohydrate to form living
bioplasm. Like Pfiiiger he adopts the view that only
^'organized" protein is oxidized.

In metabolism there are two types of protoplasmic
disintegration: the inactive, whereby the protoplasm
formed from food protein during rest is immediately
changed or broken down into non-nitrogenous storage
materials (glycogen and fat) and urea; the active, by
which under the influence of stimuli which induce
muscular contractions, the protein nucleus of the dis-
integrating protoplasm molecule is left intact so that
it may serve for the resynthesis of protoplasm with
fresh non-nitrogenous compounds. (Mendel.)

Speck

In the theory of Speck (1903) the view is held that
two forms of protein exist but that the catabolism of
organized protein is quite different from that of the
unorganized protein. That portion of food protein
(unorganized) not employed for the building up of
living tissue, is split into two portions, first, a nitro-
genous part, which is rapidly converted into urea,

PROTEIN METABOLISM 89

and a nitrogen free residue, serving as a ready source
of energy.

On the other hand, after death of cells, the tissue
or organized protein, although also broken down into
two parts, finds a destiny unlike the products of food
protein disintegration. Tissue protein splits into a
nitrogen-containing and a nitrogen-free portion. Under
normal conditions the nitrogen-free part is trans-
formed to glycogen or fat which may be utilized for
purposes of energy. The portion containing nitrogen
is not broken down at once into urea but it leads to
the formation of a variety of substances which play
important roles in metabolism but are finally excreted
as urea. In the decomposition of tissue protein Speck
assigned to oxygen deficiency an exceedingly important
part.

RUBNER

In Rubner's theory of protein metabolism it is main-
tained that a study of metabolism cannot be considered
separately from the study of heat production. Ac-
cording to Rubner, therefore, metabolism must be
studied in connection with the exchange of energy.
In all of the metabolic changes undergone by protein
in the body reference is made to the accompanying
production of heat. Rubner believes in a "store" pro-
tein which may be compared to Voit's "circulating"
protein, and in a "wear and tear quota" necessary for
the repair of tissue waste. He assumes that most of
the protein after absorption is rapidly split into two

90 THE AMINO ACIDS

parts, one nitrogen-free, the other containing nitrogen.
Inasmuch as the nitrogen-containing part plays Httle
role in energy exchange, its fate is left somewhat in-
definite. The part free from nitrogen forms the
dynamic quota of the protein ingested. When protein
is disintegrated into its two parts mentioned above,
there occurs a certain liberation of heat which is of no
value to the body cells and is therefore lost. This lib-
eration of energy has been called by Rubner the "spe-
cific dynamic action" of protein.

"A highly speculative hypothesis explained how the
various changes took place. All protoplasm was not
regarded as being of the same type, one kind might
be thermolabile, another thermostable, but all varieties
had in common a certain molecular grouping which
acted as a kind of nucleus to which other protein
groups (for example those which were thermostable
or thermolabile) could attach themselves. The mech-
anism of the energy exchange, which is characteris-
tic of activity, was effected by a distinct vibratory
movement of the whole or a definite part of the proto-
plasm. Owing to the specific oscillation, the proto-
plasm had the power of bringing about the brerl^'iown
of contiguous foodstuffs. The 'affinities' (specific
oscillations) must be of a specific nature for each
tissue and were probably somewhat akin to ferment
action. Thus, in diabetes, the 'affinities' ♦rhich
brought about the breakdown of carbohydrates, were
for some reason or other in a state of suspension,
inoperative or actually destroyed, whereas those which

PROTEIN METABOLISM 91

dealt with the catabolism of fat were active. The
direct effect of the approximation of the foodstuffs
to the 'affinities' resulted in an atomic rearrangement
and the entry of oxygen. The potential energy of the
foodstuff now became available and caused a complete
alteration in the 'affinities'; an absorption of energy
into the living substance took place at the moment of
the catabolism of the foodstuff. The internal oscilla-
tions and changes in the cells, however, gradually used
up all the energy, which was converted into heat and
lost, and there was a return to the original condition,
the 'affinities' being again ready to begin work. The
rate of the change depended on the nature of the
living substance, the temperature, nervous influences,
and the conditions of the organism itself." (Cathcart.)

FOLIN

It was Folin's conception that "the laws governing
the composition of the urine represent only the effects
of other more important laws governing the catabo-
lism of protein in the animal organism" which led
him to determine these laws under widely differing
conditions of diet. His interpretation of protein
metabolism on the basis of observed variations in the
percentage composition of the urine has stood as the
almost universally accepted theory of protein metabo-
lism of the present period.

Previous to his investigation only lengthy and none
too accurate methods were in use for the estimation

92

THE AMINO ACIDS

of the urinary constituents. He, therefore, first de-
vised a method, in each instance short and accurate,
for the estimation of every important nitrogenous
constituent of the urine, together with methods for the
determination of sulphur containing compounds, and,
secondly, with the aid of these methods made complete
analysis of normal urines. In order to make the factor
of food protein as evident as possible, diets rich in
protein were fed and were succeeded by rations
markedly deficient in nitrogenous substances although
containing a sufficiency of energy yielding substances.
As showing the wide range of variation on the two
diets a typical example of the urinary composition
follows :

Nitrogen rich diet Nitrogen poor diet

1170 c.c. 38Sc.c.

16.8 grams 3.60 grams

14.7 grams = 87.5% 2.20 grams = 61.7%

0.49 gram = 3.0% 0.42 gram =11.3%

0.18 gram = 1.1% 0.09 gram = 2.5%

0.58 gram = 3.6% 0.60 gram =17.2%

Volume of urine

Total Nitrogen

Urea-Nitrogen

Ammonia-Nitrogen

Uric acid-Nitrogen

Kreatinine-Nitrogen

Undetermined

Nitrogen
Total S03
Inorganic SO^
Ethereal SO^
Neutral SO^

0.85 gram = 4.9%
3.64 grams
3.27 grams
0.19 gram
0.18 gram

90.0%

5.2%
4.8%

0.27 gram
0.76 gram
0.46 gram
0.10 gram
0.20 gram

= 7.3%

= 60.5%
= 13.2%
= 26.3%

The general laws deduced by Folin as a result of
urinary analysis are :

1. Kreatinine. The absolute quantity of kreatinine
eliminated on a meat-free diet is a constant quantity,

PROTEIN METABOLISM 93

different for different individuals, but wholly inde-
pendent of quantitative changes in the total amount of
nitrogen eliminated.

2. Uric Acid. When the total amount of protein
metabolism is greatly reduced, the absolute quantity of
uric acid is diminished, but not nearly in proportion to
the diminution in the total nitrogen, and the per cent
of the uric acid nitrogen in terms of the total is, there-
fore, much increased.

3. Ammonia. With pronounced diminution in the
protein metabolism (as shown by the total nitrogen in
the urine), there is usually, but not always, and there-
fore not necessarily, a decrease in the absolute quan-
tity of ammonia eliminated. A pronounced reduction
of the total nitrogen is, however, always accompanied
by a relative increase in the ammonia-nitrogen, pro-
vided that the food is not such as to yield an alkaline
ash.

4. Urea. With every decided diminution in the
quantity of total nitrogen eliminated, there is a pro-
nounced reduction in the per cent of that nitrogen
represented by urea. When the daily total nitrogen
elimination has been reduced to 3 gm. or 4 gm. about
60 per cent of it only is in the form of urea.

5. Inorganic Sulphates. Decided diminutions in
the daily elimination of total sulphur are accompanied
by reductions in the per cent of the sulphur present as
inorganic sulphates. The reductions are as great as in
the case of urea.

6. Neutral Sulphur. The neutral sulphur elimina-

y

94 THE AMINO ACIDS

tion is analogous to that of the kreatinine. It repre-
sents products which in the main are independent of
the total amount of sulphur eliminated or of protein
catabolized.

7. Ethereal Sulphates. The ethereal sulphates rep-
resent a form of sulphur metabolism which becomes
more prominent when the food contains little or no
protein.

Folin concludes that neither the theory of Voit nor
that of Pfluger can be correct for these theories do not
harmonize with the above laws governing the compo-
sition of the urine. With respect to his own views he
says: "We have seen (from the tables) that the com-
position of urine, representing 15 gm. of nitrogen, or
about 95 gm. of protein, differs very widely from the
composition of urine representing only 3 gm. or 4
gm. of nitrogen, and that there is a gradual and regu-
lar transition from the one to the other. To explain
such changes in the composition of the urine on the
basis of protein catabolism, we are forced, it seems
to me, to assume that catabolism is not all of one
kind. There must be at least two kinds. Moreover,
from the nature of the changes in the distribution of
the urinary constituents, it can be affirmed, I think,
that the two forms of protein catabolism are essentially
independent and quite different. One kind is extremely
variable in quantity, the other tends to remain constant.
The one kind yields chiefly urea and inorganic sul-
phates, no kreatinin, and probably no neutral sulphur.
The other, the constant catabolism, is largely repre-

PROTEIN METABOLISM 95

sented by kreatinin and neutral sulphur, and to a less
extent by uric acid and ethereal sulphates. The more
the total catabolism is reduced, the less prominent be-
come the two chief representatives of the variable
catabolism."

"The fact that the urea and inorganic sulphates
represent chiefly the variable catabolism does of course
not preclude the possibility that they also represent to
some extent the constant catabolism."

In accordance with these two types of catabolism
Folin has furnished suitable names. The protein
metabolism which tends to be constant is tissue metab-
olism, or endogenous metabolism ; the other, the vari-
able protein metabolism, is the exogenous or inter-
mediate metabolism.

Instead of assuming, as did Voit and Pfliiger, that
the same type of decomposition, i.e., oxidation, occurs
with protein as with fats and carbohydrates, Folin
advances the view that the disintegration of protein
in catabolism is produced in large measure by a series
of hydrolytic splittings, nitrogen being split off as
ammonia.

It is further shown that contrary to the ideas of
Voit and Pfliiger, extensive formation of urea does
not occur in the muscles. Folin believed (1905) that
the nitrogenous cleavage products formed in the ali-
mentary canal from food protein are denitrogenized,
probably in the intestine, the ammonia split off, carried
to the liver, built up into urea and eliminated. The
non-nitrogenous residue is in part converted into

96 THE AMINO ACIDS

carbohydrates. "The chief reason why the nitro-
genous spUtting products produced by the digestive
enzymes are universally assumed to be reconverted
into albumin is the teleological one. The food proteins
are tissue builders and the organism must not waste
them. The fact that the muscle tissues of normal men
do not increase when the protein of food is increased,
but that all of the nitrogen of such protein is at once
eliminated, has not been sufficiently considered in this
connection. The only adequate teleological explana-
tion of this fact is that this nitrogen is not needed for
the building of new tissues. It is not needed because
the organism cannot enlarge indefinitely, and because
after it has attained its full growth the daily waste of
tissue is small. Yet when more nitrogen than the
organism needs is furnished with the food, we find
that the protein containing it is still absorbed up to
the limit of the digestive capacity." "The greater part
of the protein furnished with standard diets like Voit's,
i.e., that part representing the exogenous metabolism,
is not needed, or to be more specific, its nitrogen is not
needed. The organism has developed special facilities
for getting rid of such excess of nitrogen so as to get
the use of the carbonaceous part of the protein con-
taining it. The first step in this process is the decom-
position of protein in the digestive tract into proteoses,
amido acids, ammonia, and possibly urea. The hydro-
lytic decompositions are carried further in the mucous
membrane of the intestines, and are completed in the

PROTEIN METABOLISM 97

liver, each splitting being such as to further the forma-
tion of urea."

"In these special hydrolytic decompositions, the
result of which is to remove the unnecessary nitrogen,
we have an explanation of why and how the animal
organism tends to maintain nitrogen equilibrium even
when excessive amounts of protein are furnished with
the food. This excess of protein is not stored up in
the organism, as such, because the actual need of nitro-
gen is so small that an excess is always furnished with
the food. ..."

Present-Day Theory of Metabolism

The proof of the presence of amino acids in the
blood through the investigations of Folin, Van Slyke,
and others has rendered necessary some slight modifi-
cation of our views concerning metabolic processes.
Although Folin in his original theory foretold the
probable importance of the lower protein decomposi-
tion products it was not until the actual presence of
these substances in the blood and tissues was demon-
strated that acceptance of this idea was general. Since
it has been proven beyond question that amino acids
are normally absorbed directly into the blood from the
intestine and are distributed to the tissues, it is assumed
that each tissue rebuilds itself from the mixture of
amino acids thus received. That portion of amino
acids which is not necessary for synthesis is changed
into urea and carbonaceous residues presumably by a

98 THE AMINO ACIDS

process of deamination. The carbon remainders may-
be transformed into carbohydrate or in other ways
changed so as to yield energy and heat. Protein
material broken down within the tissues undoubtedly
suffers a series of hydrolytic cleavages, resulting in the
formation of amino acids and the latter presumably
undergo the same fate as those produced from food
protein.

According to this view protein synthesis is not
restricted to any one organ or tissue but all possess the
same property. Urea formation also can no longer
be assigned to the liver or some special urea-forming
organ, but on the other hand every tissue probably is
capable of forming this substance.

References to Literature

Cathcart: The Physiology of Protein Metabolism, 1912.

Folin: American Journal of Physiology. 1905, 13, p. 45.

Folin: Intermediary Protein Metabolism. Journal of the
American Medical Association. 1914, 63 j p. 823.

Hammarsten: Text Book of Physiological Chemistry. 1914.

Liebig: Complete Works on Chemistry. 1856.

Mendel: Theorien des Eiweissstoffwechsels nebst einigen
praktischen Konsequenzen derselben. Ergebnisse der
Physiologic. 1911, I J, p. 418.

CHAPTER VI

THE FURTHER FATE OF AMINO ACIDS

It is well recognized that a large part of the amino
acids of the food is eliminated from the body in the
form of urea, carbon dioxide, and water. The various
amino acids presumably undergo a variety of chemical
changes previous to their excretion as the simple pro-
ducts mentioned above. Also the ease with which these
transformations take place is different for the indi-
vidual amino acids. The steps leading to the ultimate
fate of some are quite clear, of others it is very ob-
scure or entirely unknown. The unlike ease of trans-
formation of amino acids into urea has been shown
by intravenous injection. Thus glycocoll and leucine
yield urea more or less completely whereas alanine,
cystine, aspartic and glutamic acids are not readily
catabolized.

In general the first step in the metabolism of amino
acids is that of oxidative deamination — a splitting off
of ammonia with an accompanying oxidation. For
any straight chain amino acid the reaction occurring
may be represented as follows :

R.CH2.CH.NH2.COOH -f- O =

R.CH,.COOH + CO, + NH3

100 THE AMINO ACIDS

The CO2 and urea are then synthesized to form urea.
This synthesis may occur according to our present
views in any active tissue or organ. Taking leucine as
a specific example of oxidative deamination we have
the reaction following:

CxioCH* CHaCHj

CH CH

I I

CH, + O, CH, + CO, + NH,

CH.NH, COOH

COOH
Leucine Isovaleric acid

It has been shown that under suitable conditions leu-
cine, for example, may yield acetone. In order to
explain the chemistry of this change it becomes neces-
sary to introduce the intervention of a type of acid
known as a ketone acid, that is, one possessing the
ketone group, C =: O. Leucine by oxidative deamina-
tion may be changed to a ketone acid.

CH3CH3

CH3CH,

\/

\/

CH

by oxidative

CH

CH,

deamination
becomes

CH,

CH.NH,

c = 0

COOH

COOH

Leucine

Ketone acid

FURTHER FATE OF AMINO ACIDS 101

The ketone acid is then transformed to a lower fatty-
acid, isovaleric acid, by cleavage of CO2.

CH3CH, CH3CH3 CH,

)8 CH , C = O COOH

I by

cleavage

I ^ becomes

a CHa

COOH

Isovaleric acid Acetone Acetic acid

By cleavagfe of isovaleric acid between the a + ^
carbon atoms acetone and acetic acid may be formed,
both of w^hich may finally yield COg + HgO.

Another possibility of the transformation of straight
chain amino acids is first the formation of hydroxy-
amino acids, then oxidative deamination with the sub-
sequent splitting off of CO2 from the nitrogen free
residue, or fatty acid, and the final direct change of the
latter to CO2+H2O, thus :

R.CH2.CH.NH2.COOH is first changed to
R.CH2.C(OH).NH2.COOH
Hydroxy-amino acid

then oxidative deamination follows with the formation
of a ketone acid.

102 THE AMINO ACIDS

R.CH2.C(OH).NH2.COOH
Hydroxy-amino acid
R . CH2 . CO . COOH + CO2 + NH3
Ketone acid

By cleavage of COg this becomes a fatty acid with less
carbon atoms.

R. CH2. CO. COOH — COgrrrR.CHg. COOH
"Ketone acid

By further cleavage this fatty acid is changed to
CO2 + H^O.

In an abnormal organism, such as that of the dia-
betic, leucine may yield beta-oxybutyric acid instead of
acetone. The reactions involved follow.

CH3CH3

CH

CH3CH3

CH

\

CH3CH3
\/

CH
_\

CH3
CHOH

CH,

'^ CH,

^ CH,

^ CH,

CH.NH,

CHOH

COOH

COOH

COOH

COOH

Leucine

Oxy-isobutyl
acetic acid

Isovaleric
acid

jS-oxy-butyric
acid

FURTHER FATE OF AMINO ACIDS 103

As a general rule substances containing the aromatic
or benzene nucleus do not readily undergo complete
oxidation in the organism, the benzene nucleus remain-
ing unchanged. The amino acids derived from protein
hydrolysis, and containing this nucleus, namely, tyro-
sine, phenylalanine and tryptophane do suffer complete
disintegration, the benzene nucleus being disrupted.
There are at least two ways in which the aromatic
amino acids may be destroyed. In the first place the
following series of reactions may occur — phenylalanine
being employed as a specific example. Phenylalanine
by oxidative deamination is first changed to phenyl-
pyruvic acid :

COOH

a ketone acid

COOH

CH.NH,

C = O

CH,

CH,

C

/\
HC CH

A

HC CH

HC CH

\/
CH

HC CH

\/
CH

Phenylalanine

P

'henyl-pynivic a(

104

THE AMINO ACIDS

If we assume that the next step is the simple spHtting
open of the benzene nucleus of phenyl-pyruvic acid its
formula may be written as follows :

COOH

COOH

COOH

C =

0

CH,

CH,

CH,

C=

c = o

C=

by cleavag-e

of CO3 this

becomes

CH

CH,

CH
CH

CH

CH

CH

#

CH

CH

C

C =

0

H

H

Phenyl-pyruvic
acid written in

Diacetic acid

open chain form

By the splitting in two of this chain and oxidation
aceto-acetic or diacetic acid is produced which in turn
may be directly oxidized to CO2 and HgO. Secondly,
employing tyrosine as a specific example we may follow
it through the following changes :

FURTHER FATE OF AMINO ACIDS 105

OH

OH

by oxidative
deamination

CH,

CHNH,

I
COOH

/

oxidation

and
rearrange-
ment

(Tyrosine)

p.oxyphenyl

amino-propionic

acid

(Ketone acid)

p.oxyphenyl

pyroracemic

acid

HO

/\

by
CO.

cleavage

\
/

CH,
C = O
COOH

\
/

H

pyr

ydroquino
oracemic j

ne
icid

106 THE AMINO ACIDS

HO

OH \

Acetone
Bodies

'^ CO, + H,0
CH,

COOH

Homogentisic acid

In these transformations homogentisic acid is an
important intermediary product which at times appears
in the urine. (See Alkaptonuria.) In harmony with
the idea that homogentisic acid is an intermediary
product in the decomposition of tyrosine and pheny-
lalanine is the demonstration that the liver may form
acetone from homogentisic acid.

Little definite is known concerning intermediary
stages in the fate of trytophane in the human body.

Arginine undoubtedly undergoes hydrolysis by the
enzyme arginase yielding urea and ornithine and the
latter may also yield urea.

NH,

C = NH

NH.CH,.CH,.CH,.CH.NH,.COOH + H^O =

Arginine

FURTHER FATE OF AMINO ACIDS 107

CH,.CH,.CH,.CH.COOH

NH,

C = O + NH, NH,

NH,

Omithine

Synthesis of Amino Acids

It has been demonstrated that alanine, phenylalanine
and tyrosine may be synthesized in the liver by per-
fusion of ammonium salts of ketone acids.

R.CH,.CO.COONH,

Ammoniuin salt of ketonic acid

OH

R.CH,.Cf COOH R.CH,.CH.NH,.COOH

NH

Imino acid hydrate Amino acid

The possibility of synthesis of amino acids in this
manner renders the interpretation of metabolic changes
in the tissues more complex than ever and confers upon
the organism a range of synthetic powers practically
unlimited.

Under ordinary circumstances, however, it is un-
likely that amino acids are synthesized to a great extent

108 THE AMINO ACIDS

in this way. The question has been tested experiment-
ally in an indirect manner. If protein with a certain
amino acid lacking is fed to animals it is reasonable
to assume that if nitrogenous equilibrium can be main-
tained a synthesis of the missing amino acid must have
occurred. From experiments planned to test this
hypothesis it has been shown that no amino acids with
the exception of glycocoll are ordinarily formed by
synthesis. For glycocoll the evidence is strongly indic-
ative of synthesis. As a rule in the body there is
about 5 per cent of glycocoll nitrogen in every 100
grams of protein nitrogen. It is well known that
benzoic acid ingested is united with glycocoll to form
hippuric acid — in other words, benzoic acid feeding
^- robs the body of glycocoll. If benzoic acid is fed in
sufficient quantities to exhaust the possible content of
glycocoll preformed in the tissues, the continued forma-
tion of hippuric acid must be provided for by glycocoll
newly formed or synthesized. Hippuric acid does con-
tinue to be formed under these circumstances and
hence glycocoll must be synthesized. It is possible, of
course, that glycocoll may be formed from the trans-
formation of some other amino acid, as by cleavage of
a long change amino acid. Another evidence in favor
of the synthesis of glycocoll is the following — milk
proteins are very poor in glycocoll, yet suckling ani-
mals are capable in a short time of building up in their
bodies proteins which contain far more of this amino
'^^ acid than can be accounted for by the ingestion of
glycocoll yielded by the milk.

FURTHER FATE OF AMINO ACIDS 109

The Relationship Between Carbohydrates and

Amino Acids

I. The Formation of Carbohydrate from Amino Acids

For a long time it has been accepted that carbo-
hydrate may be formed from ingested protein. To
determine the mechanism of this transformation many
experiments have been carried through upon animals.
In particular the formation of glycogen from ingested
protein has been subjected to experimentation and
although the consensus of opinion would indicate that
protein may give rise to glycogen formation, the
experimental conditions under which most of the inves-
tigations were made are not free from criticism. The
evidence of clinical experience, with diabetes, where
fat or carbohydrate ingestion cannot always be held
responsible for the large amounts of sugar passing
through the kidneys daily, points positively to protein
as the source of the carbohydrate excreted. In agree-
ment with this conception is the observation that the
urinary nitrogen and sugar excretion in the patho-
logical state mentioned run along parallel lines.

How may this sugar formation be explained? One
may assume, for instance, that protein contains groups
of a carbohydrate nature or groups closely allied to the
carbohydrates. Although it must be accepted that
certain proteins do contain carbohydrate groups, the
possession of such groups by proteins is by no means
universal, and, on the other hand, one is unwarranted
in stating that any specific protein will not lead to

110 THE AMINO ACIDS

sugar production. If we take a typical protein as egg
albumin, and then on the assumption that all the nitro-
gen present is eliminated from the body as urea, we
find left over a large carbon residue, the so-called
"carbon moiety," of the protein which may be regarded
as material capable of being transformed into carbo-
hydrate. This potential of carbohydrate forming
material must gain access to the blood stream, hence
to the tissues, in the form of amino acids, in accord-
ance with the present-day view of the processes of
metabolism. The problem under consideration, there-
fore, resolves itself in the question, "Are amino acids
capable of being transformed into carbohydrates?"
The most convincing work in this direction is that of
Lusk and his pupils. They have administered to dogs
rendered diabetic with phlorhizin various amino acids
and have observed that some yield sugar whereas
others fail to do so. In their experiments the relation-
ship between the dextrose and nitrogen of the urine,
the D : N ratio, was determined. Under suitable con-
ditions this becomes a constant. The ingestion of
sugar forming substances changes this constant and
any change serves as an index to the quantity of sugar
formed from a given amount of substance introduced.
It was found that the N-f ree parts of glycocoll, alanine,
aspartic acid, and glutamic acid, containing respectively
two, three, four, and five carbon atoms may be either
completely or partially transformed to dextrose. All
of the glycocoll and all of the alanine were converted

FURTHER FATE OF AMINO ACIDS 111

into glucose, whereas three of the carbon atoms con-
tained in aspartic and glutamic acids were so changed.
The stages through which these amino acids are
carried have been outlined by Lusk. In the first place
it is assumed that the initial change is a hydrolytic
deamination whereby ammonia is formed and a
hydroxy group is added to the denitrogenized amino
acid. According to this view glycocoll would first be
changed to glycolic acid, which on reduction would
yield glycolic aldehyde, three molecules of which will
form one molecule of dextrose. The chemical rela-
tionships are shown below :

CH,.NH, CH^.OH

3 I + H,0 = 3 I - 3 O =

COOH COOH

Glycocoll Glycolic acid

CH.GH
5 I
CHO

Glycolic
aldehyde

CeHaOs

Dextrose

For alanine the changes undergone are a direct trans-
formation into lactic acid which is well known to give
rise to dextrose production.

CHs CHg

2 CHNH, + H,0 = 2 CHOH = C.HeOe

COOH

Alanine

COOH
Lactic acid

Dextrose

112

THE AMINO ACIDS

With aspartic acid only three of the carbon atoms
are available for dextrose formation, the remaining
carbon atom being changed to carbon dioxide in ac-
cordance with the following reactions :

COOH

I
CH,

CHNH,

+ H,0

COOH
Aspartic acid

COOH

I
CH,

CH.OH

2 CO,
/3 lactic acid

C.H.O.

Dextrose

As with aspartic acid so also with glutamic acid only-
three of the carbon atoms are changed to dextrose, the
two remaining carbon atoms being liberated in the
form of acetic acid.

COOH

CH,

2 I
CH,

CHNH,

COOH

Glutamic acid

+ 4 H,0

COOH
CH3

CH.OH
2 CHOH

COOH

Acetic acid and
Glyceric acid

= C.H«0,

Dextrose

Dakin has demonstrated that serine, proline, orni-
thine and arginine are all capable of yielding large
amounts of sugar when given to glycosuric dogs.

FURTHER FATE OF AMINO ACIDS 113

Apparently arginine is the only amino acid with more
than five carbon atoms which furnishes glucose freely.
In this case it is probable that the sugar comes from
the ornithine moiety with five carbon atoms, into which
it may be converted by the action of arginase. Lysine
is the only straight chain amino acid derivative of
protein which fails to yield sugar. Although the rela-
tionship of the remaining amino acids to carbohydrate
metabolism is less definitely established, Lusk has
made the interesting calculation that in diabetes sugar
may arise from protein to the extent of nearly 60 per
cent. He says, "It becomes evident that there may
be a condition of nutrition in which protein is used
neither for repair nor for growth, but simply to be
diaminized and subsequently to act like fat or carbo-
hydrate as nutritive materials for the organism."

2. The Formation of Amino Acids from Carbo-
hydrates

The formation of amino acids from carbohydrate
material is a reaction less well known than the reverse
process. The close relationship existing between lactic
acid and carbohydrates on the one hand and lactic acid
and alanine on the other suggests the ready trans-
formation of glycogen to alanine presumably with
lactic acid and ammonium pyruvate as intermediary
products. With this suggestion in mind Embden per-
fused a liver rich in glycogen and found that alanine
was formed. When, however, perfusion of a glycogen-

114 THE AMINO ACIDS

free liver was carried through alanine was not present
in significant quantities. From experiments of this
nature it is evident that the metabolic processes con-
cerned in protein metabolism are intimately associated
with those of the intermediary metabolism of carbo-
hydrate, and further that at times at least protein may
serve for both sources of nitrogen and carbonaceous
material. Protein, therefore, should not be regarded
in the strict sense merely as a purveyor of the nitrogen
which is essential for life processes. It is much more
than that, as has been demonstrated.

Anomalies of Amino Acid Metabolism
Alkaptonuria •

In previous pages it has been pointed out that the
normal organism is capable of demolishing the benzene
ring as found in tyrosine and phenylalanine for under
normal conditions no evidence of these substances can
be found in the urine. For the successive steps as-
sumed to occur in this destruction see p. 105. How-
ever, there are certain individuals who apparently are
unable to break open the aromatic ring and in the urine
is found an intermediary decomposition product, homo-
gentisic acid. Urine containing homogentisic acid
exhibits a tendency to turn dark on exposure to the air
and may show a strong reducing action. This condi-
tion, known as alkaptonuria, is of rare occurrence and
may last through life without affecting the health of
the individual. It can scarcely be regarded as of

FURTHER FATE OF AMINO ACIDS 115

pathological nature, but should be looked upon rather
as an anomaly of metabolism and is generally con-
sidered as being hereditary in origin. It occurs oftener
in man than in woman and blood relationship, as first
cousins, predisposes to the condition.

Whenever homogentisic acid is present in the urine,
it is there in relatively large amounts for the anomaly
is an absolute one, that is, apparently homogentisic
acid formed is destroyed by the normal organism.
The relationship of tyrosine phenylalanine and homo-
gentisic acid are shown below :

OH

HO

OH

\X

CH,

CH,

CH,

COOH

CHNH,

CH.NH,

COOH

COOH

Pyrosine

Homogentisic acid

Phenylalanine

The significance of alkaptonuria in connection with
the metabolism of the amino acids is that the appear-
ance of homogentisic in the urine of alkaptonurics
gave the first hint as to the probable transformations ^
occurring in the demolition of the benzene radical
found in tyrosine and phenylalanine. That homogen-

116 THE AMINO ACIDS

tisic acid is a step in the degradations of these amino
acids has been doubted by Dakin who beUeves that
there is in these subjects an abnormal formation of
homogentisic acid as well as an inability to destroy it
once formed. In accord with this idea he has shown
how tyrosine and phenylalanine may be destroyed with-
out homogentisic acid as an intermediary product (see
p. 103). On the other hand, it has been demonstrated
by Abderhalden that normal individuals may eliminate
homogentisic acid when excessive quantities of tyrosine
are fed. Also the administration of tyrosine or phenyl-
alanine, or of foods rich in tyrosine, that is proteins,
causes a significant increase in the excretion of homo-
gentisic acid. If alkaptonurics live on a protein-free
diet for short periods of time the excretion of homo-
gentisic acid is markedly diminished, but does not dis-
appear entirely. Undoubtedly the aromatic amino
acids formed from tissue metabolism do not suffer
destruction to any greater extent than those introduced
into the blood from the food protein. Why an alkap-
tonuric individual fails to destroy the tyrosine radicle
is still a matter of conjecture.

Cystinuria and Diaminuria

, Under ordinary circumstances cystine, the sulphur
bearing amino acid fails to appear in the excreta, prob-
ably undergoing extensive destruction. The sulphur
is oxidized to sulphuric acid and eliminated as a sul-
phate in the urine. In certain individuals, however,
cystine appears in the urine and owing to its relatively

FURTHER FATE OF AMINO ACIDS 117

slight solubility is deposited as hexagonal crystals. It
may also form cystine concretions in the bladder. The
condition of cystinuria with that of alkaptonuria must
be regarded as an anomaly of metabolism. Cystinuria
appears to be a distinctly hereditary condition since
it may appear in families for many generations, and
apparently follows the Mendelian law of heredity.
Like alkaptonuria it is found oftener in males than in
females ; it seems to lead to no pathological symptoms
other than the formation of concretions. There is
probably no complete failure to destroy cystine since
only a portion of the cystine from protein ingested
reappears in the urine. Undoubtedly a part of the
cystine is catabolized in a normal manner. From the
fact that cystinuria persists in the absence of protein
intake and further that cystine fed to cystinurics fails
to appear in the urine, the conclusion may be reached
that the urinary cystine has its origin in that formed
during catabolism of the tissues. It may be possible
that in these subjects there is only a limited capacity
for destroying amino acids in general for, in addition
to cystine, leucine and tyrosine have been found in
some cases.

At times the diamines cadaverine and putrescine
formed by putrefaction in the intestine may also be
present in the urine of cystinurics. The diamines are
significant in that they are two of the so-called
ptomaines. Putrescine and cadaverine occur also in
diseased conditions of the intestinal tract, thus they
may be foimd in various infections, in cholera, dysen-

118 THE AMINO ACIDS

tery, gastro-enteritis, etc. Their origin in putrefaction
of protein decomposition products together with their
appearance in the urine of cystinurics led to the view
that the cystine in the cases mentioned had a Hke source.
This, however, has been shown to be incorrect. A
second view was that diamines interfered with sulphur
oxidation in the organism, hence the appearance of the
unoxidized cystine. This idea has been shown to be
untrue for cystinuria may occur in the absence of the
diamines, and the administration of diamines has no
influence upon the output of cystine in the urine. "In
intestinal disturbances, it is probable that these com-
pounds are the result of bacterial activity — indeed, they
may be the metabolic end-products eliminated by bac-
teria. In cystinuria, however, it is possible that a dif-
ferent explanation for diaminuria is pertinent. It may
be assumed, for instance, that in the beginning cysti-
nuria and diaminuria are brought about through a
similar, or indeed the same cause, or causes, for
example, a gradually changing type of metabolism
induced by some unknown agency, resulting in an
anomaly of metabolism. If the anomaly is slight in
character, cystine alone is eliminated as a result,
whereas if the change in metabolism is sufficiently pro-
nounced diamines are also excreted. If this assump-
tion is accepted it is easy to explain why in some cases
of cystinuria the diamines are absent, and that gradu-
ally one or both of these compounds disappear, that
cystinuria persists, but that cystinuria does not cease
and leave diaminuria."

FURTHER FATE OF AMINO ACIDS 119

References to Literature

Fate of Amino Acids

Dakin: Oxidations and Reductions in the Animal Body. 1912.
Dakin: Journal of Biological Chemistry. 1913, 14, p. 321.
Lusk: Journal of the American Chemical Society. 1910, 32,

p. 671.
Lusk: Ergebnisse des Physiologic. 1912, 12, p. 315. [Phlor-

hizin glycosuria.]

Alkaptonuria

Neuherg: Oppenheimer's Hanbuch der Biochemie. 1910, IV,

2, p. 353.
Wells: Chemical Pathology. 1914.

Cystinuria and Diaminuria

Neuherg: Oppenheimer's Handbuch der Biochemie. 1910, IV,

2, p. 338.
Underhill: Middleton Goldsmith Lecture for 1911. Archives

of Internal Medicine. 1911, 8, p. 7 and p. 17.
Wells: Chemical Pathology. 1914.

CHAPTER VII

THE AMINO ACIDS IN RELATION TO THE
SPECIFIC DYNAMIC ACTION OF
PROTEINS

The view has been held for many years that the
ingestion of protein increases the power of the body
cells to metabolize materials brought to them. More
recently it has been shown by Rubner that under
suitable conditions each foodstuff is capable of exert-
ing a specific accelerating influence upon the energy
metabolism. In order to maintain life Rubner believes
that a fixed requirement of energy is necessary. When
the organism is fasting, the essential energy require-
ment may be furnished from the tissues of the organ-
ism itself. After their ingestion the foodstuffs are
changed in various ways with the evolution of heat
until finally they are transformed into materials which
are capable of supporting vital phenomena. The final
products formed may be employed for the replacement
of the substances oxidized during fasting. The heat
produced in the formation of these compounds is
added to the heat produced for the maintenance of
vital processes, and the total heat production, there-
fore, exceeds that found in starvation. This increased

DYNAMIC ACTION OF PROTEINS 121

heat production induced by each foodstuff is different,
and is specific for each foodstuff. Rubner, therefore,
has named this effect "specific dynamic action."

It has been shown under the correct conditions that if
the energy metabohsm of a fasting dog be represented
as 100 calories, food must be given in the following
amounts to prevent body loss, 106 calories of sugar,
or 114 calories of fat, or 140 calories of protein. In
an experiment by Rubner it was found that a fasting
man metabolized 2042 calories. After the ingestion
of 2450 calories of sugar he metabolized 2087. When
given 2450 calories of meat 2566 calories were metab-
olism. Whatever the cause of the greater metabolism
of protein ingestion it is believed that there is produced
a liberation of energy which cannot be used by the
tissues in support of their activities but it is possible
that it may contribute to the maintenance of body
temperature. On a mixed diet this liberation of heat
unavailable for energy purposes is not of great sig-
nificance in the total metabolism since it increases the
metabolism of energy less than one tenth on a mainte-
nance diet above that when no food is eaten.

Another explanation for the increased energy metab-
olism after the ingestion of foodstuffs has been put
forward by Zuntz, who ascribes this effect to the
mechanical work of the intestinal canal (Darmarbeit)
performed during digestion and assimilation. Such a
theory would seem to fit in well with the greater
specific dynamic action of protein and its probably
greater difficulty of digestion in comparison with

122 THE AMINO ACIDS

sugar. To test the Zuntz hypothesis Benedict has
attempted to produce the effects of food ingestion
such as augmented mechanical work through increased
peristalsis produced by large doses of purgatives, as
sodium sulphate. In spite of greatly increased peris-
talsis the administration of sodium sulphate failed to
show any measurable increase in metabolism. "In the
belief that when the intestine is full of partly digested
food products and epithelial debris, the amount of
mechanical work thereby incurred might be greater
than that involved in several powerful peristaltic
waves, experiments were made in which relatively
large amounts of agar-agar were ingested, thus pro-
ducing a bulky, voluminous stool. The agar-agar
being practically non-oxidizable, there was no great
complication due to the combustion of carbohydrate
from the agar-agar. With the agar-agar it is reason-
able to assume that there must have been an extensive
segmentation process as well as peristaltic waves.
But even under these conditions on the ideally con-
trolled experiments there was absolutely no increase
in metabolism. In so far, then, as the experiments on
men show with controlled conditions, the work of
peristalsis and probably of segmentation is not suffi-
cient to be measured in the great daily energy trans-
formation of the body. It is impossible to think of
muscular activity of any kind taking place without
some slight increased metabolism, but the amount
involved in intestinal activity must be so small as to

DYNAMIC ACTION OF PROTEINS 123

be entirely negligible in the extensive energy trans-
formations for body maintenance."

These results are in accord with conclusions reached
earlier by Rubner who denied that the work of diges-
tion and assimilation could be held responsible for the
effects observed after food ingestion.

Benedict is of the opinion that from the food sub-
stances are absorbed which, carried by the blood, stimu-
late cells to greater activity, and he further indicates
that these unknown bodies are of an acid character.
From his extensive investigation on the influence of
amino acids upon metabolism, that is, their specific
dynamic action, Lusk believes that the explanation of
Rubner as to the cause of specific dynamic action must
be revised. "Amino acids act as stimuli upon the
cells, raising their power to metabolism. They may
act instead of nerve stimuli when increased heat pro-
duction is required in the presence of external cold —
the chemical regulation of temperature of Rubner.
The energy liberated in response to these stimuli may
be supplied by carbohydrate or fat. When fat and
carbohydrate are given separately or together there
may be an increased heat production on account of
the increase in the quantity of materials available for
the nutrition of the cells. With the cessation of
absorption and the return of the blood to the compo-
sition it possessed before food was taken the metabo-
lism falls to its basal value." When glucose and an
amino acid, as alanine, are given together the metab-
olism is increased to a point where the resultant effect

124 THE AMINO ACIDS

is nearly equal to the sum of the two individual influ-
ences. This indicates a distinct difference between the
cause of the specific dynamic action of glucose and
that of alanine. Two types of processes are here sug-
gested, namely, a metabolism of plethora and amino
acid stimulation. Carbohydrate or fat metabolites
which are being absorbed from the intestine into the
blood bring about a metabolism of plethora. In the
metabolism of plethora the influx of carbohydrate or
fat enables the cells to oxidize at a higher level through
the increased mass action of food particles which are
available. (Lusk.) A recent attempt by Lusk to ex-
plain "amino acid stimulation" of the cells has resulted
in the conclusion that some at least of the amino acids
even when they are not oxidized "yield products of
metabolism, either hydroxy or ketone acids which act
as stimuli to induce higher oxidation in the organism.
This is the conclusive proof of a true chemical stimu-
lation of protoplasm within the mammalian organism.
It explains the specific dynamic action of protein."

As has been shown repeatedly throughout this book
the effects characteristically produced by protein are
gradually being ascribed as a function of the amino
acids. The amino acids therefore may be regarded
not alone as pabulum for the restoration of depleted
cells but must also be looked upon as playing a distinct
and significant role in the rate or extent of cellular
metabolism.

DYNAMIC ACTION OF PROTEINS 125

References to Literature

Lusk: The Science of Nutrition. Second Ed. 1909.

Lusk: Journal of Biological Chemistry. 1915, 20, p. 555.

Rubner: Zuntz, Benedict, Lusk. Trans, of the 15th Inter-
national Congress on Hygiene and Demography. 1913,
Part 2.

Sherman: Chemistry of Food and Nutrition. 1911.

CHAPTER VIII

THE AMINO ACIDS AND SIMPLER NITRO-
GENOUS COMPOUNDS AS FOODSTUFFS

Value of Amino Acids as Foodstuffs

The dictum of Liebig that the animal organism is
endowed with very limited capacities for processes of
synthesis was accepted for a great many years with-
out serious question. A partial reason for this assump-
tion is to be found in the great difficulties to be over-
come in putting the question to experimental proof.
The discovery of erepsin by Cohnheim with the con-
sequent readjustment of ideas relative to the extent
and character of digestion processes and the form of
products absorbed casts doubt upon the inability of the
animal body to synthesize. It was reasonable to
assume that the disintegration of the protein molecule
to the stage of amino acids is with a purposeful object
and that the absorption of such relatively small com-
pounds as amino acids predicates the probability that
synthesis must occur if the organism is obliged to
reconstruct new protoplasm to replace that worn away
through the many metabolic activities.

About a dozen years ago the first attempt to deter-
mine the possibility of positive protein synthesis was

NITROGENOUS COMPOUNDS 127

made by Loewi. This investigator allowed protein to
digest until it no longer gave a reaction with the biuret
test an indication that products possessed of a protein
nature had all been reduced to a lower stage. Upon
feeding this mixture of amino acids together with
fat and carbohydrate to a dog, Loewi demonstrated
that nitrogen in the form contained in his digestion
mixture was not alone capable of maintaining the life
of the animal, but furthermore kept it in a state of
nitrogenous equilibrium, a retention of nitrogen to-
gether with an increase in weight being observed.
The experiments of Loewi were quickly followed by
those of Henderson and Dean, who were the first to
employ digestion products formed through the action
of acids rather than by ferments. They found nitro-
gen retention but were uncertain whether it signified
protein synthesis. As a result of many succeeding
investigations it soon became clear that the power of
the digestion products to replace body tissue depended
upon the manner in which such products were formed.
To put it differently, digestion products formed from
protein by the agency of enzymes were fully capable
of supplying to the body its necessary quota of nitro-
gen whereas those products obtained from proteins
through the action of acids could not take over so
completely this function but were regarded as of
value inasmuch as they could be looked upon as being
"protein sparers." In this connection it may be well
to cite some experiments of Abderhalden and Rona
with mice. To these mice were fed different prepara-

128 THE AMINO ACIDS

tions of casein together with sugar. To one series of
mice unchanged casein was fed, to a second, casein
that had been digested with pancreatin for a period
of two months, a third series received casein digested
for one month with pepsin-hydrochloric acid and then
for two months with pancreatin, the fourth series
were fed with casein hydrolyzed for ten hours with
25 per cent sulphuric acid. The results showed that
those animals fed with casein digested with pancreatin
for two months and those given unchanged casein
lived about the same length of time. Mice fed on the
other two preparations lived shorter periods of time.
Other investigators obtained similar results. An
interesting controversy now arose as to the reason
for the specific difference between products formed
by enzymes and those resulting from acid hydrolysis.
Abderhalden and Rona from their work cited above
put forth the hypothesis that the difference in the
two products lay in their content of polypeptides.
According to this view digestion by ferments results
in the presence of considerable amounts of fairly
complex polypeptides which serve as nuclei for the
synthesis of new protein material. Hydrolysis by acid,
however, carries the digestion beyond the stage of
polypeptides, hence, no nuclei for synthesis are present
and the inability of acid digestion mixtures to fully
serve as nitrogenous pabulum is explained. In sup-
port of their hypothesis they offer the observation that
the casein preparation formed by pancreatin action
contained only 16 per cent of polypeptides, that of the

NITROGENOUS COMPOUNDS 129

pepsin-hydrochloric acid mixture further subjected
to the influence of pancreatin contained only half as
much polypeptides, whereas from that formed by acid
hydrolysis polypeptides were entirely absent. Later,
however, it was shown by Abderhalden and his co-
workers that the varying content of polypeptides can-
not be the sole reason for the differences observed in
the two classes of products in their ability to supply
the nitrogenous needs of the body, for a dog was kept
alive for thirty-eight days and the only supply of nitro-
gen was in a digestion mixture containing only amino
acids. Again, a young dog gained weight and retained
nitrogen in completely digested meat and a bitch was
kept in nitrogenous equilibrium during lactation with
meat digested to the amino acid stage. Abderhalden
and London were able to maintain a dog with an Eck
fistula (the liver shunted out of the portal circulation)
on fully digested meat. From this experiment they
further concluded that the liver could play a small
role only in protein synthesis and used these results
as support for their view that protein synthesis occurs
during absorption.

From the work of Henriques, Abderhalden and
others it soon became evident that the difference in
nutritive value between ferment and acid hydrolysis
products could not be ascribed wholly to the presence
or absence of polypeptides. Upon closer investiga-
tion it developed that the failure of acid hydrolytic
products to meet nutritive requirements satisfactorily
could be explained by the fact that during acid hydroly-

130 THE AMINO ACIDS

sis tryptophane, unquestionably one of the most impor^
tant of the amino acids, is destroyed. The proof that
herein lies the true explanation was furnished by
Abderhalden and Frank, who succeeded in maintaining
dogs in nitrogenous equilibrium on meat completely
hydrolyzed by acid to which had been added a small
amount of tryptophane.

After the demonstration that the nitrogenous nutri-
tive requirements of the organism can be supplied by
a mixture consisting of the products of hydrolysis,
whether by acids or enzymes, an attempt was made
by Abderhalden to support a dog in nitrogen equilib-
rium on an artificial mixture of amino acids to which
were added carbohydrate and fat. The successful out-
come of the investigation led Abderhalden to declare
that the animal organism is capable of forming all
the tissue constituents out of the simplest derivatives
of the proteins. Inasmuch as carbohydrate and fat
may be prepared synthetically, as may some of the
amino acids, the problem of the artificial production
of foodstuffs is solved according to Abderhalden,
who says that such a possibility is limited only by the
question of sufficient funds. From these observations
it becomes evident that mixtures of amino acids are
fully capable of supplying the nitrogenous needs of
the organism when applied to the lower animals. An
opportunity was afforded Abderhalden and his co-
workers to extend this type of investigation to man.
A boy with a stricture of the oesophagus on whom
gastrotomy had been performed was the subject. To

NITROGENOUS COMPOUNDS 131

him was given per rectum a mixture of protein (meat)
digestion products obtained through the combined
action of trypsin and erepsin. The experiment was
continued for fifteen days and during this period
nitrogen equihbrium was maintained, the body weight
increased and the general condition of the subject was
excellent.

This brief review of the salient features of the
problem leads to but one conclusion, namely, that the
amino acids must be regarded as foodstuffs capable
of supplying the nitrogenous needs of the organism,
and that the chief factors to be taken into account
with regard to the nutritive value of any protein or
proteins are the character and the extent of the amino
acids contained therein.

The Value of Amides and Ammonium Salts as

Foodstuffs

The nutritive value of various simple nitrogenous
compounds has been a subject for investigation for
many years. This is especially true for such sub-
stances as the amides and has been of particular inter-
est to those concerned with agricultural problems
since in the food of herbivora amides may play an
important role. From the viewpoint of nutrition in
general, the proof that animals may thrive on amides
or other simple nitrogenous compounds supplied as
sources of nitrogen carries with it indirect evidence of

132 THE AMINO ACIDS

the transformation of these substances into amino
acids — in other words, amino acid synthesis occurs.

Particular attention has been paid to the deter-
mination of the value of asparagine as a source of
nitrogen, one of the first investigators being Mer-
cadente, who believed that protein formation could
take place from asparagine, especially in plants.
Sachse believed that protein was formed from aspara-
gine by the simple addition of fatty aldehydes. On
the other hand, Loewi thought that in the presence of
carbohydrates protein was formed from asparagine
by a series of condensations. Zuntz suggested that in
herbivora asparagine was built up into protein by
bacteria in the intestine previous to utilization. This
latter view has been supported by numerous investi-
gators, some of whom state that protein-forming bac-
teria are widely distributed in nature and may act
very efficiently and quickly when in suitable envi-
ronment. The evidence available seems to speak
strongly in favor of the view that asparagine may
serve as source of nitrogen or act at least as a protein
sparer, as much as two-thirds of the protein in the
diet of herbivora being replaceable by asparagine. On
the other hand, several investigators claim that aspara-
gine cannot take the place of protein at all, hence,
cannot be used as a source of nitrogen and that even
the degree of protein sparing action that may be
exhibited by this amide is extremely limited.

In a comparable manner it has been suggested that
various ammonium salts may also replace protein to

NITROGENOUS COMPOUNDS 133

a certain extent at least, or act as protein sparers.
The problem of the utilization of ammonium salts
subjected to much experimentation in the past has
recently been revived through the work of Grafe and
Schlapfer who have asserted that ammonium salts,
urea, and even nitrates may serve as sources of nitro-
gen for the animal organism, and they regard the
utilization as indicated by nitrogen retention as a
process of amino acid synthesis. These results have
been assailed by others on the ground that the observed
retention of nitrogen as a result of feeding the above-
mentioned compounds may be explained in other ways
than as a proof of amino acid synthesis. "There are
several ways in which they may be assumed to behave
in the organism. In the first and foremost instance
they may serve as pabulum for the alimentary bac-
teria, which in turn are destroyed in large numbers
in the digestive tract and can furnish a yield of per-
fect protein synthesized from simple compounds like
urea and the salts of ammonia. It is generally ad-
mitted that in certain species like the herbivora, in
which bacterial processes have a free play in the
gastro-intestinal tube, the contribution of dead bac-
terial bodies to the intake is by no means negligible."
"The feeding of urea or ammonium salts might lead
to an apparent nutritive advantage by depressing or
inhibiting the usual breaking down of nitrogenous
compounds in metabolism. This would accord with
the belief that the products of cellular waste them-
selves tend to impede cellular metabolism. Now that

134 THE AMINO ACIDS

the synthesis of amino acids from ammonia and car-
bohydrates has been accompHshed directly or in-
directly in the laboratory, the possibility of a similar
reaction in the body must be reckoned with. Finally,
the alleged utilization of urea and other simple nitro-
gen derivatives may merely be an instance of unsus-
pected retention and delayed excretion. Even so
soluble a salt as an iodide may not be entirely recov-
ered in the excreta until several days after its adminis-
tration has been stopped. Surely no one would look
on the temporary deficit as an indication of nutritive
'utilization' of the foreign salt."

Various possibilities therefore present themselves in
the interpretation of the alleged utilization of these
simple nitrogenous substances. The influence of
alimentary bacteria may be eliminated by parenteral
feeding of the compounds, which, however, has not
been feasible until recently when Henriques succeeded
in devising a method whereby a slow constant stream
of nutritive solution may be intravenously introduced
into the body. Subjecting utilization of urea and
ammonium salts to the test by means of this device,
Henriques and Anderson have demonstrated that no
permanent retention of these nitrogenous compounds
occurred. It is therefore exceedingly improbable that
the body itself is in a position to transform these sub-
stances into amino acids. Amino acid synthesis is
not an easy task for the organism nor is there evidence
that even the transformation of one amino acid to
another is accomplished to any extent. The organism

NITROGENOUS COMPOUNDS 135

must have ready formed amino acids supplied to it
in sufficient quantity and variety if it is to accomplish
its task of tissue building.

References to Literature

Amino Acids as Foodstuffs

Ahderhalden: Zeitschrift fiir physiologische Chemie. 1912, 77,

p. 22.
Cathcart: The Physiology of Protein Metabolism. 1912.
Henriques and Anderson: Zeitschrift fiir physiologische

Chemie. 1913, 88, p. 357.
Liithje: Ergebnisse des Physiologie. 1908, 7, p. 795.
Cathcart: The Physiology of Protein Metabolism. 1912.

[Amides.]

Ammonium Salts

Ahderhalden: Zeitschrift fiir physiologische Chemie. 1912,
78, p. 1, and 1912, 82, p. 1.

Grafe and Schldpfer: Zeitschrift fur physiologische Chemie.

1912, 77, p. 1.
Henriques and Anderson: Zeitschrift fiir physiologische

Chemie. 1914, 92, p. 21.
Pescheck: Biochemische Zeitschrift. 1912, 45, p. 244.
Underhill: Journal of Biological Chemistry. 1913, 15, p. 327

and p. 337.
Underhill and Goldschmidt: Journal of Biological Chemistry.

1913, 15, p. 341.

CHAPTER IX

THE SPECIFIC ROLE OF AMINO ACIDS IN
NUTRITION AND GROWTH

That the chemical differences in proteins as deter-
mined by their amino acid content must be of con-
siderable significance in metabolic processes has been
understood in a vague way for a long time. As soon
as recognition was gained for the view that the prob-
lems of nutrition are concerned with other factors

'than a mere sufficiency of nitrogen or an adequate
intake of potential energy the problems of interme-
diary metabolism forced themselves upon the atten-
tion of physiologists and led to a thorough apprecia-
tion of the value in nutrition of factors previously
entirely overlooked or considered of little or no
moment.

Reference to the table on p. 22 will bring out clearly
the differences that exist between a few of the typical
proteins. The most striking variations in amino acids
from a quantitative viewpoint are evident. Such

■ differences are undoubtedly of importance from a
nutritional standpoint, but of much greater signifi-
cance are the qualitative variations. To point out
briefly the most evident of these it may be seen that

NUTRITION AND GROWTH 137

albumin and casein are glycocoU-free. Gliadin from
wheat contains no glycocoll and only a trace of lysine.
Zein from maize yields no lysine nor tryptophane and
gelatine contains no cystine, tyrosine nor tryptophane.
The first appreciation that qualitative differences
in protein composition may be of importance in nutri-
tion was furnished by the classic experiments of Voit
and Munk, who showed that gelatin could not support
nitrogen equilibrium. The demonstration by Escher
that the addition of tyrosine improved the powers of
gelatin in establishing nitrogenous equilibrium gave
rise to a series of investigations, the results of which
have led to a much more complete understanding of
the problems intimately connected with metabolism.
Only a few of these, however, need be reviewed here,
Kaufman was able to show that when gelatin is fed
to man and dogs with the addition of the missing
amino acids, tyrosine and tryptophane, nitrogen equi-
librium could be maintained for short periods at least.
The work of Willcock and Hopkins with zein, which
it will be remembered is deficient in lysine and tryp^
tophane, is of great interest in the present discussion
as it attacked the problem from new viewpoints, in
entire accord with the conceptions of the present. In
their introduction these authors point out that: "We
are no longer bound to Liebig's view, or to any modi-
fication of it which implies that the whole of the
protein consumed is utilized as intact protein : nor are
we even compelled to assume that the whole of what
is broken down in the gut is resynthesized before

138 THE AMINO ACIDS

utilization. Protein products may function in other
ways than in the repair of tissues or in supplying
energy. It is highly probable that the organism uses
them, in part, for more specific and more immediate
needs. The discovery of substances absolutely essen-
tial to life, highly specific, and elaborated in special
organs, suggests that some part, at least, of the pro-
tein products set free in the gut may be used directly
by these organs as precursors of such specific sub-
stances. In adrenaline, for instance, we have an aro-
matic substance absolutely essential for the mainten-
ance of life, and it is probable that the suprarenal
gland requires a constant supply of some one of the
aromatic groups of the protein molecule to serve as
an indispensable basis for the elaboration of adrena-
line. If this be so, it is certain that the gland itself
could not, in starving animals, supply sufficient of such
a precursor to outlast the observed survival periods.
Since adrenaline must be produced at all costs, the
required precursor must, in starvation, be obtained by
tissue breakdown outside the gland. We may be sure,
however, that adrenaline is far from being the only
substance elaborated to which such considerations
apply. Similarly, in an animal upon a diet sufficient
to supply energy, but lacking in some essential group,
the minimal waste in the general tissues of the body
will be determined by the special need of the organs for
the missing group. On this basis we have a hypoth-
esis to account for the special protein-sparing prop-
erties of gelatin. It shares with protein certain

NUTRITION AND GROWTH 139

molecular groupings needed to satisfy specific needs,
and is thus superior to fats and carbohydrates as a
protein-sparer: it lacks, on the other hand, certain
necessary groupings, fails therefore to supply all such
needs, and thus cannot replace protein."

These considerations served as the basis for the*
experiments described by Willcock and Hopkins. Mice
kept under exactly similar conditions were fed with a
diet having zein as its source of nitrogen. In certain
instances small quantities of tyrosine or tryptophane
were added to the dietary. The results of the influ-
ence of such diets were measured by the "survival
period" — that is, th« period necessary to cause the
death of the animal. In Fig. 1 is reproduced a dia-
gram illustrating very clearly the influence of tryp-
tophane upon the survival period. With zein as the
only nitrogenous component of the diet young miQe
were shown to be unable to maintain growth. Tryp-
tophane addition does not make zein capable of main-
taining growth, but does greatly prolong the survival
period. In Fig. 1 the survival periods of mice fed
upon zein alone are not given, for they were identical
with those obtained with mice fed zein plus tyrosine.
Although added tyrosine exerted no influence upon
the survival period, it must not be inferred that this
amino acid is without specific effect on metabolism:
it Evidently played little role here because zein fed
supplied sufficient tyrosine, hence an excess was with-
out special influence. In reality tyrosine was added as
a control to tryptophane addition, in order to determine

A- 8 12 16 20 24 28 32 36 ^10 ^44 ^8 da

Figure 1. The thick lines show the survival periods (in days) of twenty-o
individual mice upon the zein diet with tyrosine added. The thin lines show t
same for nineteen mice upon the zein diet with tryptophane added. [From
Journal of Physiology^ volume 35.]

NUTRITION AND GROWTH 141

whether addition of any amino acid would produce
an effect, and hence, therefore, to find out directly the
specific action of tryptophane.

A prominent feature in connection with the mice
given zein alone was a condition of torpor; the mice
were very inactive and made no movement when
handled or touched, the ears, feet, and tail were cold,
the coat was glairy and the eyes were half-closed.
Those fed tryptophane With zein showed a strikingly
different behavior, being active and apparently healthy
even up to the end of life. In both instances death
was not caused by a lack of food intake, as all animals
gave evidence of appetite. Quantitatively, sufficient
food was received but qualitatively something essen-
tial to life was lacking. It is possible that had lysine,
the other amino acid lacking in zein, been fed also,
even better results would have been obtained. Tryp-
tophane undoubtedly is essential for the maintenance
of life, although the specific role it plays has not yet
been determined. As the authors mentioned above
point out, "If it [tryptophane] serves as a basis for
the elaboration of a substance absolutely necessary
for life — something, for instance, of an importance
equal to that of adrenaline — then, in starvation, or
when it is absent from the diet, a supply is likely to
be maintained from the tissue-proteins, the demand
for it would become one of the factors determining
tissue breakdown. In the case of young animals which
directly benefit from the addition of a protein con-
stituent otherwise absent from their diet, to the extent

142 THE AMINO ACIDS

of a well-nigh doubled life, and lose, instead of gaining,
weight, the utilization of the constituent would seem to
be of some direct and specific nature." These words
give the first definite suggestion that individual amino
acids may play a specific role in the maintenance of
nutritional rhythm.

The failure of zein as a suitable source for the
essential nitrogen requirement leads to the query
whether any single protein will suffice in this respect.
Attempts to answer this question have been many and
it is only recently that a satisfactory positive reply has
been given. In many of the older experiments lack of
success has been attributed to various factors other
than the character of the protein, and where appar-
ently successful results have been obtained criticism
has been pertinent in that, in most instances, the protein
or proteins employed have not been free from impuri-
ties. The general impression gained from this type
of investigation has been that sooner or later animals
die when kept for a prolonged period upon a con-
stant diet even though an abundance of energy
producing material may be present. A so-called
"pure" diet has been deemed impracticable. Lunin, one
of the early investigators of the problem, fed mice
with mixtures of casein, fat, cane sugar, and milk
ash. On this artificial diet death occurred in from
twenty to thirty days, a survival period greater than
when the ash of milk was omitted. Mice fed dried
milk were alive at the end of two months. Hall with
mice and Steinitz with dogs obtained comparable

NUTRITION AND GROWTH 143

results when a similar form of dietary was used. By
considerable variation in the non-nitrogenous portion
of the food Rohmann showed that mice will thrive
for weeks. A criticism of these experiments is that
the range of variation in the make-up of the dietary
resulted really in furnishing the animals an ordinary
mixed diet. The experiments of Jacob with pigeons,
of Falta and Noeggerath, and of Knapp with rats
demonstrated that variety in the dietary undoubtedly
tends toward prolongation of life but that death
eventually ensues.

After experiencing many failures, Osborne and
Mendel have succeeded in maintaining white rats for
long periods of time upon single, pure, isolated pro-
teins, growth also being at a normal rate. They
attributed their success to the addition to the dietary
of what they term "protein-free milk." This is pre-
pared by removing the protein and fat from milk,
leaving the milk sugar, inorganic salts and the un-
known components. "Protein-free milk" always con-
tains very small quantities of protein but not enough
to support life. They have also demonstrated that
by artificially imitating the composition of "protein-
free milk" by union of the various ions fairly success-
ful results have been obtained. It is therefore pos-
sible to construct a dietary in such a manner from
purified isolated foodstuffs and artificial salt mixtures
that animals may not only be maintained but normal
growth may also be induced.

In their work, Osborne and Mendel differentiate

144 THE AMINO ACIDS

sharply between a maintenance diet and one capable
of promoting growth. They have shown, for example,
that a young animal may be maintained on a certain
diet indefinitely without manifesting any tendency to
grow. From the work of Donaldson it has been dem-
onstrated that the life span of the white rat is about
three years. Sexual maturity is reached in sixty
days. The first year of life for the rat corresponds
to the first thirty years of human life, and the curve
of growth for this period is reproduced below. Fig. 2.
As an illustration of the influence of an isolated
protein, casein ( fed with . starch, sugar, agar, lard,
and a salt mixture), the chart. Fig. 3, is shown. It
is evident that casein as the sole source of nitrogen
was apparently incapable of allowing normal growth
in a young rat during a period of forty-six days. In
other words, stunting occurred. In period 2, casein
and sugar were replaced by milk. Growth was
resumed. The influence of changing the salt mixture
content of the food intake is quite evident in periods

3, 4, and 5. The ability of milk to furnish the necessary
nitrogen requirement is well shown in the chart, Fig.

4, the curve obtained being to all intents and purposes
identical with the normal growth curve.

If to the casein diet "protein-free milk" is added,
instead of whole milk replacing casein, normal condi-
tions obtain as is well illustrated in the chart. Fig. 5.

Casein alone was found to be unable to support
growth. In Fig. 6 is shown a curve in which, during
period 2, casein was the only source of protein and

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NUTRITION AND GROWTH

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as a result a decline set in, which could not be checked
by doubling the percentage of casein in the diet. That
lack of protein can not account for the decline is well
shown in period 4, during which the original amount

t«rm»nal6d

Figure 4. Growth Curve with
Milk Diet.

of casein was replaced and "protein-free milk" was
also added. An immediate response in appetite was
evidenced and speedy recuperation and growth were in
order. This experiment demonstrates that a rat unable
to maintain itself on an isolated protein may be caused

146

THE AMINO ACIDS

to speedily resume a normal condition by the addition
to the diet of "protein-free milk."

From these and many similar results it is apparent
that if suitable non-protein constituents of the dietary

20

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Figure 5. Maintenance on Casein and Growth
AFTER Addition of Protein-free Milk.

are supplied, such as are furnished by "protein-free
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normal. Emphasis should therefore be laid upon the
importance of the role played by the accessory food-
stuffs, as contained in "protein-free milk" the nature

SUJ9J0

148 THE AMINO ACIDS

of which remains obscure. It is also evident that the
establishment of a satisfactory non-protein dietary
aifords an opportunity for the study of any specific
influence which a peculiar type of protein, or one with
an unusual type of internal structure, may exert in
nutrition.

In addition to casein Osborne and Mendel have
demonstrated that perfectly satisfactory results may
be yielded when other types of pure proteins are
employed, a single one sufficing for all the nitrogen
requirements of white rats. Thus, adequate growth
has been secured with lactalbumin from cow's milk,
ovalbumin from hen's egg, ovovitellin from hen's egg,
edestin from hemp seed, cannabin from hemp seed, glu-
tenin from wheat, glycinin from the soy bean, globulin
from squash seed, globulin from cotton seed, excelsin
from Brazil nut, and glutelin from maize.

Taking advantage of the opportunity afforded them,
the above mentioned authors have studied the influ-
ence which a peculiar protein, for example, one lack-
ing one or more important amino acid, may exert in
nutritional processes. It soon became evident that all
proteins do not promote growth under otherwise
favorable conditions. Gliadins . of rye and wheat,
which are deficient in glycocoll and lysine and on the
other hand are very rich in glutamic acid, and hordein
of barley, which closely resembles gliadin in chemical
constitution, are capable of giving maintenance, but
fail to induce growth. A condition of stunting is
brought about, old animals retaining the characteris-

NUTRITION AND GROWTH

149

tics of well-nourished young rats. In Fig. 7 are re-
produced curves which show the failure of a rat to
present normal growth on a diet containing protein-
free milk and gliadin as the only protein. The

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frontispiece shows the photograph of this rat (B)
and as a contrast that of a rat (A) of the same
age presenting normal growth, together with a pho-
tograph of a rat (C) of the same weight as (B) but
much younger. This stimting is apparently a method

150 THE AMINO ACIDS

which may be employed for the attainment of a type
of animal infantilism. In connection with the sub-
ject of stunting it became of interest to determine
whether this condition would remain permanent under
all circumstances or whether a return to a diet con-
taining a more typical protein than gliadin would also
cause a resumption of growth. Fig. 8 shows the
slight growth of a young white rat during 276 days
of gliadin feeding. That the capacity to grow had
not been lost, but was merely inhibited, may be seen
in the second part of the curve in which milk food
replaced the gliadin. At the beginning of the milk
food diet the rat was 314 days old, an age at which
rats usually show very little growth. Fertility is not
impaired by the act of stunting, as may be seen from
the curve in Fig. 9, for this rat, after a period of 154
days with gliadin as its protein supply, was mated
and produced four young, which were suckled during
the first month of their existence by the mother who
was still maintained upon a gliadin diet. These young
rats presented normal growth curves during this
period. When a month old, three of the young ani-
mals were removed from the mother and kept upon
diets of casein, edestin, and milk food. All showed
normal curves of growth. The fourth young rat,
kept with the mother began to exhibit a failure to
grow as soon as forced to depend upon the gliadin
food mixture. Inasmuch as casein, which has been
proved to be efficient as a source of nitrogen for both
maintenance and growth, is lacking in glycocoll.

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at a normal rate after 276 days of stunting. At this time the rat was 314 day
Biological Che^nistry ^ volume 12.]

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sole protein. After 154 days this rat was paired, four young being the result of
the mating. [From the Journal of Biological Chemistry^ volume 12.]

152 THE AMINO ACIDS

whereas gliadin is deficient in glycocoU and lysine and
fails to promote growth, it is reaso^abfe to assume
that the low content of lysine in gliadm is responsible
for the failure of white rats to grow. On the other
hand, lysine is apparently not essential for mere main-
tenance. Another conclusion which may be drawn
from these experiments is that the organism is unable
to synthesize lysine, although glycocoll may be syn-
thesized with apparent ease, as has been shown in
previous pages of this book. Growth means the for-
mation of new tissues and in the absence of sufficient
lysine the construction of new tissue does not occur
readily, or at least proceed at the normal rate. The
inference that lysine is concerned with the function
of growth may be tested from another viewpoint. If
the animals fed with gliadin, lacking in lysine, show
a failure to grow the addition of lysine to gliadin
should be followed by a resumption of normal growth.
Such trials have been made by Osborne and Mendel
and the results obtained are most strikingly seen in
the following curves. [See Fig. 10.] Failure to
grow on gliadin as the sole protein is first shown in
the curves followed by a period of growth when
lysine was added to the diet. The subsequent with-
drawal of the lysine is followed in each instance by
a cessation of growth. If lysine is added again growth
is again resumed at a normal, to cease again when
lysine is taken away. These results lead to the con-
clusion that lysine is indispensable for the functions
of growth. Data collected by Osborne and Mendel

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ih& Journal of Biological Chemistry^ volume 17.]

NUTRITION AND GROWTH 155

reveal the "teleologically interesting fact . . . that
those proteins, Hke casein, lactalbumin, and egg viteUin,
which are in nature concerned with the growth of
animals, all show a relatively high content of lysine."
The experience of these investigators with zein,
which lacks glycocoU, tryptophane and lysine, has
brought to light the fact that tryptophane is undoubt-
edly essential for maintenance and emphasizes anew
the significance of lysine as a growth promoting sub-
stance. One may also assume that a little lysine is
necessary for maintenance and this is ordinarily sup-
plied in sufficient amount by the traces in gliadin or
(in the zein and tryptophane experiments) by traces
in protein-free milk protein or from the tissues them-
selves. In an earlier portion of this chapter were
pointed out in some detail the experiments of Will-
cock and Hopkins with zein, with and without addi-
tion of tryptophane. They found that zein as the
only protein in the dietary cannot maintain growth in
the young animal nor even support life. The addi-
tion of tryptophane resulted in prolonging life with-
out causing a resumption of the growth impulse.
The outcome of the work of Osborne and Mendel
with zein alone is best shown in the chart, Fig. 11.
The large number of experiments shown here yielded
concordant results and show that neither maintenance
nor growth can be secured when zein is the only pro-
tein ingested. When tryptophane is added to the zein
food mixture, maintenance of body weight follows, as
may be seen from Fig. 12. Addition of both trypto-

156

THE AMINO ACIDS

Days

Figure 12. Indispensability of Tryptophane for Main-
tenance IN Nutrition. These experiments should be contrasted
with the failure of maintenance on zein-food alone, shown in figure
11. [From the Journal of Biological Chemistry, volume 17.]

pliane and lysine results in the establishment of perfect
maintenance and growth. [See Fig. 13.] It may be
inferred from these experiments then that tryptophane
is indispensable for maintenance in nutrition and that
the animal organism does not possess the ability to
synthesize this amino acid. That lysine cannot replace
tryptophane in the establishment of the condition of

Days

Figure 13. Growth on Foods Containing Zein+Tryptophanb
+LYSINE. The growth obtained on this diet may be contrasted with
maintenance without growth in the absence of the lysine (see Figure 12)
2.ndi failure to be maintained in the absence of both lysine and tryptophans
(Figure 11), thus demonstrating the role of these amino acids in growth
and maintenance respectively. That lysine cannot replace tryptophane in
maintenance is shown by Rat 1900. [From the Journal of Biological
Chemistry^ volume 17.]

158 THE AMINO ACIDS

maintenance, may be seen from the chart, Fig. 13.
Rat 1900.

Investigation of this type into the biochemical de-
portment of the protein cleavage products will un-
doubtedly lead ultimately to the assignment of more
or less specific functions to the various amino acids,
and hence will indirectly indicate the relative efficiency
of this or that protein in bringing about a desired
result in nutrition.

References to Literature

Mendel: Nutrition and Growth : Journal of the American
Medical Association. 1915, 64, p. 1539.

Osborne and Mendel: Journal Biological Chemistry. 1914,
ir, p. 325.

Willcock and Hopkins: Journal of Physiology. 1906, 35, p. 88.

INDEX

INDEX

Absorption,
from intestine, 48.
from large intestine, 56.
from stomach, 46, 47.
of amino acids, 47, 54, 56.
of amino acids by rectum,

131.
of amino acids by tissues,

of fat, 53.

of proteoses and pep-
tones, 52, 56.

of putrefactive products,
56.

of undigested protein, 48.
Accessory foodstuffs, 146.
Acetic acid, 101.
Acetone, 101.
Acid, acetic, 101.

aspartic, 16.

caseinic, 18.

glutamic, 16.

hippuric, 108.

homogentisic, 106, 115.

isovaleric, 100.

lactic. 111, 112, 113.

nucleic, 7.

uric, 93.
Acids, amino, 12.

diamine, 21.

hydroxy, 38.

Acids, ketone, 100.
monoamine, 21.
Adrenaline, see also Epine-
phrine, 138, 141.
Alanine, 13.
amounts of, in proteins,

22, 23.
dextrose formation from,

111.
formation from glycogen,

113.
in blood, 55.
Albuminates, 9.
Albuminoids, 6.
Albumins, 4, 5.
Alcohol-soluble proteins, 4,

5.
Alkaptonuria, 114.
Amides, as foodstuffs, 131.
Amines, 40.

fate of, 57.
Amino acid, definition of,
12.
metabolism, anomalies of,
114.
Amino acids, 12.
absorption of, 54, 56, Id.
absorption of, by rectum,

131.
action of intestinal bac-
teria upon, 38.

162

INDEX

Amino acids, as foodstuffs,
126, 131.

as functional test of liver,
71.

as protein sparers, 127.

content of, in tissues, 11,
78, 79.

deficiencies of, in gliadin,
148.

deficiencies of, in zein,
155.

description of, 12.

fate of, in tissues, 76.

formation of carbohy-
drates from, 109.

formation of, from car-

. bohydrates, 113.

formation of, in gastric
digestion, 31.

formation of, in intestinal
digestion, 35.

formulas of, 12.

further fate of, 99.

in blood, 55, IZ, 75, 78.

in digestion, 28, 29.

in duodenal contents, 35.

in intermediary metabo-
lism, 78.

in maintenance and
growth, 148.

mono, 21.

quantitative yields from
proteins, 22.

relationship of different,
19.

Amino acids, relation of, to
specific dynamic action,
123.

specific role of, in nutri-
tion and growth, 136.

synthesis of, 107, 132, 134.

synthesis of, to protein,

n.

Ammonia, 93.

amounts of, in proteins,

22, 23.
in intestinal putrefaction,
39.
Ammonium salts, as food-
stuffs, 131.
Amounts of amino acids
yielded by protein^, 22.
Anabolism, 81.
Animal infantilism, 150.
Anomalies of amino acid

metabolism, 114.
Arginase, 71.
Arginine, 17.
amounts of, in proteins,

22, 23.
catabolism of, 106.
dextrose formation from,

112.
fate of, in putrefaction,

44.
in blood, 55.
urea from, 71.
Arbacin, 6.

Artificial foodstuffs, value
of, in nutrition, 143.

INDEX

163

Artificial production of

foodstuffs, 130.
Asparagine, food value of,

132.
Aspartic acid, 16.
amounts of, in proteins,

22, 23.
dextrose formation from,

112.
in blood, 55.
Bacterial digestion and

amino acids, 36.
j3-iminazolylethylamine, 43.
jS-oxybutyric acid, 102.
Blood, amino acids in, 55,
73, 75.
fate of amino acids in, 76.
non-coagulable protein,

60.
non-protein nitrogen of,

55.
proteose of, 50.
proteoses and peptones in,

59.
seromucoid in, 61.

Cadaverine, 43, 147.
Cannabin, growth with, 148.
Carbohydrate, formation of

amino acids from, 113.
formation of, from amino

acids, 109.
"Carbon moiety" of protein,

110.
Casein, as sole protein of

diet, 144.

Caseinic acid, 18.

amount in casein, 22.
Catabolism, 81.

of amino acids, 99.
Circulating protein, 84.
Classification of proteins, 3.
Clupein, 6.

Coagulated proteins, 9.
Coagulation of protein, 2.
Colloids, 2.

Conjugated proteins, 4, 7.
Creatinine, 92.
Cresol, 38.
Cystine, 15.

absence of, in gelatin, 137.

amounts of, in proteins,
22, 23.

excretion of, in cystinu-
ria, 117.
Cystinuria, 116.

Deamination, 70, 71, 72, 99,
100, 101.

Derived proteins, 4, 8.

Dextrose, formation of,
from amino acids. 111,
112.

Diacetic acid, 104.

Diamines, 43, 117.

Diamino acids, 21.

Diaminuria, 116.

Diet, variety in, 143.

Digestion, a hydrolytic pro-
cess, 29.
and amino acids, 28.

164

INDEX

Edestin, growth with, 148.
Endogenous metabolism, 95.
Enzymes, in blood after

protein injections, 51,

61.
in protein synthesis, 67,

69.
Epinephrine, see also

Adrenaline, 41.
Erepsin, 34.
Ethereal sulphates, 94.
Excelsin, growth with, 148.
Excretion of putrefactive

products, 56.
Exogenous metabolism, 95.

Fertility, and stunting, 150.
influence of gliadin on,
150.
Fibrin, 9.
Fibrinogen, 9.
Foodstuffs, amides as, 131.
amino acids as, 126, 131.
ammonium salts as, 131.
artificial production of,

130.
value of artificial, in nu-
trition, 143.

Gaduhiston, 6.

Gastric digestion, impor-
tance of, 32.
products of, 30, 31, 32.
relation of, to amino acid
formation, 32,
Gelatin, absence of certain
amino acids in, 137.

Gelatin, as a protein sparer,
138.
nutritive value of, 137.
Gliadin, 5.
deficiency of amino acids

in, 148.
influence of, on fertility,

150.
influence of, on growth,

150.
yield of lysine and glyco-
coll, 137.
Globin, 6.
Globulins, 4, 5.

growth with, 148.
Glucosamine, 7.
Glucoproteins, 7.
Glutamic acid, 16.
amounts of, in proteins,

22, 23.
dextrose formation from,

112.
in blood, 55.
in gliadin, 148.
Glutelin, growth with, 148.
Glutelins, 4, 5.
Glutenin, 5.

growth with, 148.
Glycinin, growth with, 148.
Glycocoll, 12.
absence of, in gliadins,

137, 148.
amounts of, in proteins,

22, 23.
dextrose formation from,
111.

INDEX

165

Glycocoll, in blood, 55.

synthesis of, 108.
Glycogen, 109.

formation of alanine
from, 113.
Growth, and maintenance,
143.
influence of lysine upon,

152, 155, 156.
influence of milk food

upon, 144.
influence of tryptophane

upon, 155, 156.
influence of zein upon,

139, 155.
specific role of amino
acids in nutrition and,
136.
with various proteins, 148.
Heat production, and me-
tabolism, 89.
Hemocyanin, 8.
Hemoglobins, 8.
Heredity, in alkaptonuria,
115.
in cystinuria, 117.
Heterocyclic compounds, 21.
Hippuric acid, 108.
Histamine, 43.
Histidine, 17.
amounts of, in proteins,

22, 23.
fate of, in putrefaction,

42, 43.
in blood, 55.
Histones, 6.

Homogentisic acid, 106.
relation of, to tyrosine
and phenylalanine, 115.
Hydrolysis, of protein, 11.
Hydroxy acids, 38, 39.

Indole, 39.

ethylamine, 42.
Inorganic sulphates, 93.
Intestinal digestion, 33.
relation of, to amino acid
formation, 34.
Intestinal work, influence
of, in specific dynamic
action, 112.
Isoamylamine, 42.
Isoleucine, 14.
amounts of, in proteins,
22, 23.
Isovaleric acid, 100.

Ketone acids, 100.

Lactalbumin, growth with,

148.
Lactic acid. 111, 112, 113.
Lecithins, 8.
Lecithoproteins, 8,
Leucine, 13.
amounts of, in proteins,

22, 23.
catabolism of, 100.
fate of, in putrefaction,

42.
in blood, 55.
Leucocytes, role of, in pro-
tein synthesis, 66.

166

INDEX

Liver, in amino acid metab-
olism, 71.

role of, in protein synthe-
sis, 129.
Lysine, 16.

absence of, in zein, 137.

amounts of, in proteins,
22, 23.

inability of body to syn-
thesize, 152.

in blood, 55.

influence of, on growth,
152, 155, 156.

in gliadin, 137, 148.

in maintenance, 156.

Maintenance, and growth,
143.
influence of lysine upon,

156.
influence of tryptophane

upon, 155, 156.
influence of zein upon,
155.
Metabolism, 81.
and heat production, 89.
of amino acids, 99.
of plethora, 124.
Metaproteins, 9.
Milk, and growth, 144, 145.
food, influence of, on

stunting, 150.
protein-free, 143, 144, 145,
149, 155.
Monoamino acids, 21.
Mucoids, in blood, 60, 61.

Neutral sulphur, 93.
Nitrogen, equilibrium, 97.

form needed by body, 1.

in protein, 1, 2.

in tissue formation, 96.
Norleucine, 14.
Nucleic acid, 7.
Nucleoproteins, 7.
Nutrition, specific role of
amino acids in, 136.

Occurrence, and character-
istics of proteins, 5.

Organized protein, 84.

Ornithine, 44.
dextrose formation from,
112.

Ovalbumin, growth with,
148.

Ovovitellin, growth with,
148.

Oxidative deamination, 99.

Oxyproline, 18.
amounts of, in proteins,
22, 23.

p.oxyphenylethylamine, 40.
Parenteral introduction of

protein, fate of, 49.
Peptides, 10, 26.
Peptone, action of erepsin

upon, 34.
in gastric digestion, 31.
Peptones, 10.
in intestinal putrefaction,

37.

INDEX

167

Phenol, 38.
Phenylalanine, 14.
amounts of, in proteins,

22, 23.
catabolism of, 103.
relation of, to homogen-
tisic acid, 115, 116.
Phosphoproteins, 7.
Plastein formation, 68.
Plastic foods, 84.
Polypeptides, 10, 25.
action of enzymes upon,

26.
value of, in amino acid
mixtures, 129.
Proline, 17.
amounts of, in proteins,

22, 23.
dextrose formation from,

112.
in blood, 55.
Protamines, 6.
Proteans, 8.

Protein, action of enzymes
in synthesis of, 67, 69.
as a complex polypeptide,

26.
definition of, 2.
fate of ingested, 58.
free milk, 143, 144, 145,

146, 149, 155.
tnolecular weight of, 3.
molecule, 2.

molecule, structure of, 24.
metabolism, theories of,
81.

Protein, regeneration, place

of, 62.
sparers, 127, 132, 133, 138.
synthesis, by intestinal

bacteria, 133.
synthesis, by intestine, 62.
synthesis, from amino

acids, by tissues, 78.
synthesis, role of leuco-
cytes in, 66.
synthesis, role of liver in,

129.
Proteins, and growth, 148.
as colloids, 2.
classification of, 3.
characteristics of, 5.
composition of, 2.
conjugated, 4, 7.
crystallization of, 2.
derived, 4, 8.
influence of, on plane of

polarized light, 3.
occurrence of, 5.
quantities of amino acids

yielded by, 22.
simple, 4, 5.
specific dynamic action

of, 120.
Proteose, in blood, 50.
Proteoses, 9, 10.
action of erepsin on, 34.
and peptones, in blood, 59.
in gastric digestion, 31.
in intestinal putrefaction,

37.
Ptomaines^ 117.

168

INDEX

"Pure" diets, 142.
Purine bases, 7.
Putrefaction, fate of argin-
ine in, 44.

fate of histidine in, 42,
43.

fate of leucine in, 42.

fate of tryptophane in, 39.

fate of tyrosine in, 38.

formation of ammonia in,
39.

hydroxy acids in, 38.

nature of, 36.

products of intestinal, 38.

proteoses and peptones in,

Putrescine, 43, 117.
Pyrimidine bases, 7.

Rate of blood flow, IZ.
Relationship of different

amino acids, 19.
"Residual nitrogen" of

blood, 74.
Respiratory foods, 84.

Salmin, 6.
Scombin, 6.
Scombron, 6.
Serine, 15.
amounts of, in proteins,

22, 23.
dextrose formation from,
112.
Seromucoid, 61.

Simple proteins, 4, 5.

Skatole, 39.

Specific dynamic action, 90,

120.
Starvation, amino acids in

blood during, 78.
Structure of protein mole-
cule, 24.
Stunting, 144, 149, 150.
influence of milk food

upon, 150.
Sturin, 6.
Survival period, influence

of zein upon, 139, 140,

141.
Synthesis of amino acids,

107.

Theories of fate of in-
gested protein, 58.
Theories of protein metab-
olism, 81.
Theories of protein regen-
eration, 62.
Transformations of amino

acids in body, 99.
Tryptophane, 18.
absence of, in certain pro-
teins, 24, 137.
amounts of, in proteins,

22, 23.
catabolism of, 106.
importance of, for life,

141.
importance of, in nutri-
tion, 130.

INDEX

169

Tryptophane, inability of
body to synthesize, 156.

influence of, on mainte-
nance and growth, 155,
156.

value of, in gelatin feed-
ing, 137.

value of, in zein feeding,
139, 140, 141.
Tyramine, 41.
Tyrosine, 14.

absence of, in gelatin, 23,
137.

amounts of, in proteins,
22, 23.

catabolism of, 105.

relation of, to homogen-
tisic acid, 115, 116.

value of, in gelatin feed-
ing, 137.

value of, in zein feeding,
139, 140.

Urea, 93.

formation, 70, 71, 72, 98,

99.
Uric acid, 93.
Urine, composition of, 92,

93.

Utilization, of amides, 134.
of amino acids, 127, 130.
of ammonium salts, 133,

134.
of protein, parenterally

introduced, 51.

Valine, 13.
amounts of, in proteins,
22,23.

in blood, 55.
Variety in diet, 143.
Vitellin, 8.

"Wear and tear" quota, 89.

Zein, absence of lysine and
tryptophane in, 137.

deficiencies of, in amino
acids, 155.

effects of feeding, 141.

feeding experiments with,

139, 155.

influence of, upon growth,
139.

influence of, upon growth
and maintenance, 155.

influence of, upon survi-
val periods of mice, 139,

140, 141.

ERRATA

Page 6, line 21. For Protomines, read Protamines.

Page 14, line 5. For Isoleucine. a-amino-iS-ethyl-propionic
acid, read Isoleucine . a amino methyl-ethyl propionic acid.

Page 14, line 25. For HO.C6H5.CH2.CH<cqoh,

read HO.C6H4.CH2.CH<co6h

Page 16, line 13. For CH2 read CH2.COOH

I I

CH2.COOH CH2

I 1

CH.NH2.COOH CH.NH2.COOH

Page 33, line 19. For 1906, read 1901.

Page 44, line 10. For CH2. CH2. CH2. CH . COOH

I I

NH NH2

-NH2

[2

Arginine

read CH2.CH2.CH2.CH.COOH

NH NH2

C^NH2

Arginine

Page 111, lines 16 and 24)^ r-rrr^ ^ r^ tt r^

T-, 11 o 1- n J -10 r For CeHeOs, read C6H12O6.
Page 112, Imes 7 and 18 )

Page 119, line 6. For Ergebnisse des Physiologic, read
Ergebnisse der Physiologic.

Page 121, line 12. For were metabolism, read were meta-
bolized.

Date

Doe

'?Tl(/A

DEC I 1

^'^b

9Q4L

#

f)

i
]

Cuf^^^'' '^y AT/ON,